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WO2024170660A1 - Polythérapies pour le traitement du cancer avec des molécules de liaison thérapeutiques - Google Patents

Polythérapies pour le traitement du cancer avec des molécules de liaison thérapeutiques Download PDF

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Publication number
WO2024170660A1
WO2024170660A1 PCT/EP2024/053805 EP2024053805W WO2024170660A1 WO 2024170660 A1 WO2024170660 A1 WO 2024170660A1 EP 2024053805 W EP2024053805 W EP 2024053805W WO 2024170660 A1 WO2024170660 A1 WO 2024170660A1
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seq
amino acid
acid sequence
antibody
fra
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Marco Gymnopoulos
Jorge ZERON-MEDINA CUAIRAN
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AstraZeneca AB
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AstraZeneca AB
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Priority to IL322687A priority Critical patent/IL322687A/en
Priority to KR1020257030708A priority patent/KR20250148670A/ko
Priority to CN202480021748.1A priority patent/CN120936382A/zh
Priority to AU2024220870A priority patent/AU2024220870A1/en
Publication of WO2024170660A1 publication Critical patent/WO2024170660A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • 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
    • 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
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present disclosure relates to a method of treating cancer in a human subject in need thereof, comprising administering to the human subject: (a) an antibody-drug conjugate (ADC) comprising an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin; and (b) a PARP1 inhibitor.
  • ADC antibody-drug conjugate
  • the present disclosure also relates to a kit comprising (a) an ADC comprising an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin; and (b) a PARP1 inhibitor.
  • Cancer remains one of the most leading diseases worldwide despite years of study into the mechanisms of cancer pathogenesis and the development of numerous potential anti-cancer drugs.
  • lung cancer and ovarian cancer are the third- and fifth-most common cancer in women, respectively.
  • Chemotherapy and radiotherapy are the most common cancer treatments. Nonetheless, these therapies are linked to various negative side effects, including fatigue, nausea, and hair loss. These problems are complicated by the fact that chemotherapy treatments are frequently administered over long periods of time.
  • a number of antibody therapies for cancer have been developed and marketed, leading to a reduction in the need for conventional forms of chemotherapy for a number of cancer types.
  • monoclonal antibodies has greatly improved over this time period, there are relatively few clinically available anticancer antibodies, and even fewer that may be used to target a variety of cancer types. Furthermore, there is a need to increase the potency of therapeutic antibodies, which is generally limited by the prevalence of the target antigen’s expression and the subsequent effects on the cancer cell following antibody binding.
  • Conjugating monoclonal antibodies to cytotoxic molecules to generate antibody drug conjugates (ADCs) is a novel approach to deliver targeted therapy for malignancies.
  • ADCs such as fam-trastuzumab deruxtecan- nxki (breast and gastric cancer), brentuximab vedotin (Hodgkin lymphoma) and polatuzumab vedotin-piiq (Nonhodgkin lymphoma), respectively.
  • FRs Folate receptors
  • the FR family includes FRa, FRP, FRy and FRS.
  • FR binds with folate molecules and transports them into cells, such that the folate molecules are delivered to the folate cycle to support metabolism of nucleotides.
  • folate is important for DNA synthesis, methylation and repair (Cheung, et al., Oncotarget. 2016;7(32):52553-52574).
  • FRa is a glycosylphosphatidylinositol (GPI)-anchored membrane protein having high affinity to the active form of folate, 5-methyltetrahydrofolate (5-MTF).
  • GPI glycosylphosphatidylinositol
  • FRa is expressed by the F0LR1 gene.
  • Previous studies have shown that FRa plays a crucial role in embryogenesis (Kelemen, Int J Cancer. 2006;119(2):243-250). Folate transport in adults, however, is mainly driven by ubiquitous expression of reduced folate carriers and proton coupled folate transporters (Zhao, et al., Anna Rev Nutr. 2011;31:177-201).
  • the distribution of FRa expression in adults is usually limited to the apical surfaces of polarised epithelia, such as choroid plexus, kidney, lung, and placenta.
  • FRa which is also known as Folate Receptor 1 (FOLR1) or folate binding protein (FBP)
  • FBP folate binding protein
  • Antibodies against FRa in the art suffer from deficiencies, such as poor internalisation, short half-life, and insufficient cytotoxicity.
  • the FRa- targeting ADCs in the art employ microtubule inhibitors, which have been associated with specific toxicities in clinical trials, such as corneal inflammation (mirvetuximab soravtansine, which consists of an anti-FRa antibody M9346A conjugated via a sulfo-SPBD linker with the maytansinoid warhead DM4) (Moore et al.
  • interstitial lung disease (MORAb-202, which consists of the humanised antibody farletuzumab derived from LK26 conjugated to the eribulin warhead) (Sato, et al. (2020) ESMO Abstract https://doi.Org/10.1016/j.annonc.2020.01.026), and neuropathy and neutropenia (STRO-002, which consists of an anti-FRa antibody SP8166 conjugated to the hemiasterlin warhead) (Naumann, et al. (2021) J Clin Oncol 39 (Suppl 15/abstr 5550) https://doil0.1200/JCO.2021.39.15_suppl.5550). Recent studies have also found that cancer cells might acquire resistance to microtubule inhibitors (Ganguly, et al., Biochim Biophys Acta. 2011 Dec; 1816(2): 164-171).
  • the present disclosure provides, inter alia, methods of treating cancer comprising combination therapies of an ADC comprising an anti-FRa antibody and a PARP1 inhibitor, and kits comprising the same.
  • a method of treating cancer in a human subject in need thereof comprising administering to the human subject:
  • ADC antibody-drug conjugate
  • the cancer comprises cancer cells having heterogeneous expression of FRa and/or a low expression of FRa; optionally wherein the cancer cell has a similar FRa expression to Igrov-1 cell line.
  • the cancer is selected from ovarian cancer, lung cancer (e.g. lung adenocarcinoma), endometrial cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinomas (HNSCC), breast cancer (e.g. TNBC), cervical cancer and malignant pleural mesothelioma.
  • lung cancer e.g. lung adenocarcinoma
  • endometrial cancer pancreatic cancer
  • gastric cancer e.g. lung adenocarcinoma
  • RCC renal cell carcinoma
  • HNSCC head and neck squamous cell carcinomas
  • breast cancer e.g. TNBC
  • malignant pleural mesothelioma pleural mesothelioma.
  • said cancer may be selected from ovarian cancer and lung cancer.
  • the lung cancer is a non- small-cell lung cancer (NSCLC), optionally wherein the NSCLC is selected from squamous NSCLC, adenocarcinoma NSCLC, or a combination thereof.
  • NSCLC non- small-cell lung cancer
  • kit comprising:
  • an ADC comprising an anti-FRa antibody or antigen-binding fragment thereof linked to a cytotoxin
  • the PARP1 inhibitor is AZD5305 having the formula: or a pharmaceutically acceptable salt thereof.
  • the anti-FRa antibody or antigenbinding fragment thereof comprises:
  • a heavy chain CDR1 of SEQ ID NO: 1 SDSATWN
  • a heavy chain CDR2 of SEQ ID NO: 2 RTYYRSKWYNDYAVSVKS
  • a heavy chain CDR3 of SEQ ID NO: 3 GVGSFDY
  • a light chain CDR1 of SEQ ID NO: 4 RASQSISSWLA
  • a light chain CDR2 of SEQ ID NO: 5 KASGLES
  • a light chain CDR3 of SEQ ID NO: 6 QQYNSYSQLT
  • a heavy chain CDR1 of SEQ ID NO: 7 SEQ ID NO: 7
  • a heavy chain CDR2 of SEQ ID NO: 8 SISGRSYIYYADSVKG
  • a heavy chain CDR3 of SEQ ID NO: 9 EQQLALDY
  • a light chain CDR1 of SEQ ID NO: 10 RASQGISNFLA
  • AASSLQS AASSLQS
  • a light chain CDR3 of SEQ ID NO: 12 QQYNSYPFT
  • a heavy chain CDR1 of SEQ ID NO: 13 SNSAAWN
  • a heavy chain CDR2 of SEQ ID NO: 14 RYYRSNWYNDYTLSVKS
  • a heavy chain CDR3 of SEQ ID NO: 15 GVGRFDS
  • a light chain CDR1 of SEQ ID NO: 16 RASQSISSWLA
  • a light chain CDR2 of SEQ ID NO: 17 KSSLES
  • a light chain CDR3 of SEQ ID NO: 18 QEYKTYSIFT
  • a heavy chain CDR1 of SEQ ID NO: 19 (SYNMN), a heavy chain CDR2 of SEQ ID NO: 20 (SISSGSSYIYYADSMKG); a heavy chain CDR3 of SEQ ID NO: 21 (GMTTLTFDY); a light chain CDR1 of SEQ ID NO: 22 (RASQGISTFLA); a light chain CDR2 of SEQ ID NO: 23 (AASSLQS); and a light chain CDR3 of SEQ ID NO: 24 (QQYISYPLT);
  • a heavy chain CDR1 of SEQ ID NO: 25 (SYSMN), a heavy chain CDR2 of SEQ ID NO: 26 (SISSRSSYVYYADSVKG); a heavy chain CDR3 of SEQ ID NO: 27 (GMTTLTFDY); a light chain CDR1 of SEQ ID NO: 28 (RASQGISSFLA); a light chain CDR2 of SEQ ID NO: 29 (AASSLQS); and a light chain CDR3 of SEQ ID NO: 30 (QQYNSYPLT); or (f) a heavy chain CDR1 of SEQ ID NO: 31 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 32 (RTYYRSKWYSDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 33 (GGAPFDY); a light chain CDR1 of SEQ ID NO: 34 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 35 (KASSLES); and a light chain CDR
  • the anti-FRa antibody or antigenbinding fragment thereof comprises:
  • VH comprising an amino acid sequence that is at least 90% identical to the amnio acid sequence of SEQ ID NO: 37 and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 38;
  • VH comprising an amino acid sequence that is at least 90% identical to the amnio acid sequence of SEQ ID NO: 39 and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 40;
  • VH comprising an amino acid sequence that is at least 90% identical to the amnio acid sequence of SEQ ID NO: 41 and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 42;
  • VH comprising an amino acid sequence that is at least 90% identical to the amnio acid sequence of SEQ ID NO: 43 and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44;
  • VH comprising an amino acid sequence that is at least 90% identical to the amnio acid sequence of SEQ ID NO: 45 and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 46;
  • VH comprising an amino acid sequence that is at least 90% identical to the amnio acid sequence of SEQ ID NO: 47 and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 48.
  • the anti-FRa antibody or antigenbinding fragment thereof comprises:
  • the anti-FRa antibody or antigenbinding fragment thereof comprises:
  • the anti-FRa antibody comprises a constant heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 109 or 111 and a constant light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 110.
  • the anti-FRa antibody comprises a constant heavy chain amino acid sequence of SEQ ID NO: 109 or 111 and a constant light chain amino acid sequence of SEQ ID NO: 110.
  • the anti-FRa antibody comprises:
  • a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 57 and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 58; or (f) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 59 and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 60.
  • the anti-FRa antibody comprises:
  • the antigen-binding fragment is a Fab fragment, a Fab’ fragment, or a F(ab’)2 fragment.
  • the anti-FRa antibody or antigenbinding fragment thereof is humanised, chimeric, or fully human. In particular embodiments, the anti-FRa antibody or antigen-binding fragment thereof is fully human.
  • the anti-FRa antibody or antigenbinding fragment thereof is monoclonal, polyclonal, recombinant, or multispecific.
  • the anti-FRa antibody or antigenbinding fragment thereof is of the IgGl, IgG2, IgG3 or IgG4 type. In particular embodiments, the anti-FRa antibody or antigen-binding fragment thereof is of the IgGl type.
  • the cytotoxin is linked to the anti- FRa antibody or antigen-binding fragment thereof via a linker R L selected from:
  • G L is a linker for connecting to the anti-FRa antibody or antigen-binding fragment thereof;
  • R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound to form a cyclopropylene or cyclobutylene group; and e is 0 or 1 ; or
  • R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound to form a cyclopropylene or cyclobutylene group.
  • G L is
  • R L is
  • the cytotoxin is selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof.
  • the cytotoxin is a topoisomerase I inhibitor.
  • the topoisomerase I inhibitor is represented by formula (I): and salts and solvates thereof; wherein R L is defined above.
  • the topoisomerase I inhibitor is:
  • the topoisomerase I inhibitor is:
  • the drug to antibody ratio is in the range of about 1 to 20, optionally wherein the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In some embodiments of any aspect of the disclosure, the DAR is about 8 or about 4.
  • the DAR is about 8.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3
  • GVGSFDY a light chain CDR1 of SEQ ID NO: 4
  • RASQSISSWLA a light chain CDR2 of SEQ ID NO: 5
  • KASGLES a light chain CDR3 of SEQ ID NO: 6
  • the anti-FRa antibody or antigenbinding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38;
  • the ADC and the PARP1 inhibitor are administered separately or sequentially.
  • the ADC and the PARP1 inhibitor are administered together.
  • a method of treating cancer in a human subject in need thereof comprising administering to the human subject:
  • ADC antibody-drug conjugate
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); aheavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38;
  • SUBSTITUTE SHEET (RULE 26) or a pharmaceutically acceptable salt thereof.
  • kit comprising:
  • ADC antibody-drug conjugate
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); aheavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38;
  • cytotoxin is topoisomerase I inhibitor SG3932
  • Figure 1 shows the binding of (A) AB 1370026; (B) AB 1370035; (C) AB 1370049; (D) AB1370083; (E) AB1370096; and (F) AB1370117 to human FRa, cyno FRa, mouse FRa, human FRP or human FRy by HTRF assay.
  • Figure 2 shows the High Content Profiler multiparametric analysis of antibody internalisation into KB cells with intensity on the vertical axis plotted against sample concentration on the horizontal axis.
  • Antibodies with an uptake above a cut-off determined by the positive control samples (circles) were shortlisted and are shown as grey diamonds, with those below the cut-off as white diamonds.
  • the data points for the 6 exemplary antibodies, run in duplicate in the assay, are indicated by black diamonds and labelled with sample name. At least one sample per exemplary antibody was above the cut-off.
  • Figure 3 shows the 6 exemplary IgGs in HTRF epitope competition assays with (A) Comparator 1 IgG; (B) Comparator 2 IgG; and (C) Comparator 3 IgG.
  • Figure 4 shows the results of the AC-SINS self-interaction assay. Antibodies were assayed for their propensity to self-associate in HSA (black columns) buffer. The negative control antibody exhibited low levels of self-interaction, whereas the positive control antibody exhibits high levels of self-interaction in HSA, as expected. The threshold for flagging an antibody at risk (> 5 nm) is indicated by the dotted horizontal line.
  • Figure 5 shows the internalisation of anti-FRa antibodies into (A) Jeg-3 cell (with medium FRa expression) and (B) KB cell (with high FRa expression) over time measured by increase in fluorescence on CX7 instrument.
  • Figure 6 shows the chromatograms of AB1370049-SG3932 DAR8 obtained from (A) UHPLC-RP at 214 nm (in reduced form); (B) UHPLC-RP at 330 nm (in reduced form); (C) UHPLC-SEC at 280 nm; and (D) UHPLC-HIC at 214 nm.
  • Figure 7 shows the chromatograms of AB1370049-SG3932 DAR4 obtained from (A) UHPLC-RP at 214 nm (in reduced form); (B) UHPLC-RP at 330 nm (in reduced form); (C) UHPLC-SEC at 280 nm; and (D) UHPLC-HIC at 214 nm.
  • Figure 8 shows the progress of chemical transformations to a series of DAR8 ADCs when exposed to various sera. The chemical processes observed are (A) deconjugation; and (B) maleimide hydrolysis.
  • Figure 9 shows the 6-day cytotoxicity assay with lead DAR8 ADCs on (A) KB cell (with high FRa expression); (B) Jeg-3 cell (with medium-high FRa expression); and (C) Igrov- 1 cell (with medium FRa expression).
  • Figure 10 shows the bystander killing activity of AB1370049-SG3932 DAR8.
  • FRa- positive and FRa-negative KB cells were treated alone or in a 1:1 ratio with 1 nM AB 1370049- SG3932 DAR8 ADC for 6 days. Cytotoxic activity was measured after 6 days with flow cytometry.
  • Figure 11 shows the KB xenograft study with lead DAR8 ADCs.
  • single intravenous dose of (A) 1.25 mg/kg or (B) 5 mg/kg were administered.
  • Figure 12 shows the OVCAR-3 xenograft study with lead DAR8 ADCs.
  • single intravenous dose of (A) 1.25 mg/kg or (B) 5 mg/kg were administered.
  • Figure 13 shows the IGROV-1 xenograft study with AB1370049-SG3932 DAR8. At day 33, single intravenous dose of 5 mg/kg was administered.
  • Figure 14 shows KB xenograft study with lead DAR4 ADCs. At day 7, single intravenous dose of 5 mg/kg was administered.
  • Figure 15 shows (A) OVCAR-3 and (B) CaCo-2 xenograft study with single dose administration of AB1370049-SG3932 DAR8 or FRa-DM4 ADC at 1.25, 2.5 and 5 mg/kg.
  • Figure 16 shows the median percent tumour growth resulting from a single administration of (A) 5 mg/kg AB1370049-SG3932 DAR8 in 51 PDX models; and (B) 2.5 mg/kg AB1370049-SG3932 DAR8 in 39 PDX models.
  • Figure 17 shows the PDX model study with single dose administration of AB 1370049- SG3932 DAR8 at 2.5 mg/kg or 5 mg/kg for (A) ovarian PDX model CTG-0711; (B) NSCLC PDX model CTG-2367; and (C) endometrial PDX model CTG-2268.
  • Figure 18 shows the measured cell viability signals on (A) progenitor cells in megakaryocytic lineage; (B) progenitor cells in myeloid lineage; (C) progenitor cells in erythroid lineage; (D) expanded and differentiated cells in megakaryocytic lineage; (E) expanded and differentiated cells in myeloid lineage; and (F) expanded and differentiated cells in erythroid lineage.
  • X axis represents drug concentrations and Y axis represent percent of viability (values are average of triplicates -/+SD).
  • AB1370049-SG3932 DAR8 does not show exacerbated toxicity over a non-targeting ADC control, in primary CD34 + bone marrow-derived hematopoietic stem progenitor cells induced to differentiate into erythroid, myeloid, or megakaryocytic lineages.
  • Figure 19 shows the mean (+SD) unconjugated mAh vs AB1370049-SG3932 DAR8 concentrations-time profiles in cynomolgus monkeys.
  • PK profiles at 15 and 25 mg/kg plasma samples were collected and processed using the immuno capture LC-MS/MS assay and noncompartmental PK.
  • Total mAh is a measure of intact antibody (in the case of the ADC includes ADC or deconjugated mAb).
  • Total ADC is a measure of intact ADC only.
  • Figure 20 shows the results of a 6-day cytotoxicity assay on (A) KB cells treated with 0.000457 to 3 nM of AZD5335, 123.45 to 10000 nM of AZD5305, or a combination of both; (B) IGROV-1 cells treated with 0.00152 to 10 nM of AZD5335, 123.45 to 10000 nM of AZD5305, or a combination of both; (C) OVCAR-3 cells treated with 0.00152 to 50 nM of AZD5335, 0.0247 to 2 nM of AZD5305, or a combination of both; and (D) SKOV-3 cells treated with 0.1 nM to 700 nM of AZD5335, 30 nM to 30 pM of AZD5305.
  • the data points with AZD5305 only are included in the graph for comparison, and they do not correspond to the concentration provided on the x-axis.
  • Figure 21 shows the Synergy Matrix heat map for AZD5335 and AZD5305 in (A) KB cells; (B) IGROV-1 cells; (C) OVCAR-3 cells and (D) SKOV-3 cells.
  • Synergy was calculated using Bliss Model analysis. Higher positive scores indicated higher synergy. Score 0 means additive and negative values mean antagonism. The darker the black colouration, the greater the synergy score.
  • FIG 22 shows the OVCAR-3 CDX model study with monotherapy using AZD5335 or AZD5305 or combination therapy using AZD5335 and AZD5305.
  • OVCAR-3 xenograft tumours were grown in female NSG mice until reached a volume of -175 mm 3 . Mice were then randomised and dosed on Day 33. Tumour volumes were measured twice a week.
  • NIP228- SG3932 abbreviated to “Nip228”
  • AZD5305 abbreviated to “PARPSel” alone showed very weak to no anti-tumour activity.
  • TGI Modest tumour growth inhibition
  • FIG. 23 shows the CTG3718 PDX model study with monotherapy using AZD5335 or AZD5305 or combination therapy using AZD5335 and AZD5305.
  • CTG3718 PDX tumours were grown in female NSG mice until tumours reached a volume of -180 mm 3 . Mice were then randomised and dosed on Day 26. Tumour volumes were measured twice a week.
  • AZD5305 also abbreviated as “5305” was inactive.
  • Modest tumour growth inhibition (TGI) was observed with AZD5335 (also abbreviated as “5335”) as a single agent but the combination with 3.5 mg/kg AZD5335 + AZD5305 yielded significant tumour growth inhibition.
  • NIP228-SG3932 isotype ADC (also abbreviated as “NIP228”) did not demonstrate any TGI, though a small level of tumour growth inhibition was observed with the group combined with AZD5305. Values are mean ⁇ SEM tumour volumes for n:4 animals per group.
  • Figure 24 shows the OV2022F PDX model study with monotherapy using AZD5335 or AZD5305 or combination therapy using AZD5335 and AZD5305.
  • OV2022F xenograft tumours were grown in female NSG mice until reached a volume of -180 mm 3 . Mice were then randomised and dosed on Day 34. Tumour volumes were measured twice a week. NIP228- SG3932 (abbreviated to “Nip228”) and AZD5305 (“PARPSel”) alone showed some antitumour activity.
  • TGI Modest tumour growth inhibition
  • any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
  • reference to “an agent” includes a plurality of such agents and reference to “the agent” includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth.
  • “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. In particular embodiments, the term “about” shall be understood herein as plus or minus ( ⁇ ) 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, ⁇ 0.1%, of the numerical value of the number with which it is being used. Embodiments described herein as "comprising" one or more features may also be considered as disclosure of the corresponding embodiments “consisting of” such features.
  • amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”.
  • amino acid sequence is synonymous with the term “peptide”.
  • protein and polypeptide are used interchangeably herein.
  • the conventional one-letter and three-letter codes for amino acid residues may be used.
  • the 3 -letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
  • the inventors have developed an exemplary array of anti-FRa antibodies with a high affinity and specific binding to FRa on cancer cells (e.g. does not specifically bind to other FR family members such as FRP and FRy).
  • the inventors performed a thorough assessment of antibody developability, checking propensity for reversible- self association, internalisation, non-specific binding and hydrophobicity and the stability of the mAbs to thermal and photo stressors. Additionally, the inventors employed an in vivo mouse PK study for a focused panel of mAbs to remove any that exhibited poor in vivo half-life and increased clearance.
  • ADA anti-drug antibody
  • an in silico immunogenicity assessment was conducted to remove any antibodies from consideration with increased predicted risk of ADA response.
  • the inventors identified a panel of 6 antibodies with similar, beneficial properties. In some embodiments, these antibodies are included in the ADCs described in the present disclosure.
  • the ADCs of the present disclosure encompass the antibodies or antigen-binding fragments defined herein having the recited CDR sequences or variable heavy and variable light chain sequences (reference antibodies), as well as functional variants thereof.
  • a functional variant binds to the same target antigen as the reference antibody, and may exhibit the same antigen cross-reactivity as the reference antibody.
  • the functional variants may have a different affinity for the target antigen when compared to the reference antibody, or it may be substantially the same affinity.
  • a functional antibody variant may comprise a functional variant of a CDR.
  • the term “functional variant” is used in the context of a CDR sequence, this means that the CDR has at most 2, or at most 1 amino acid differences when compared to a corresponding reference CDR sequence, and when combined with the remaining 5 CDRs (or variants thereof) enables the variant antibody to bind to the same target antigen as the reference antibody, and in particular embodiments exhibit the same antigen cross -reactivity as the reference antibody.
  • a functional variant may be referred to as a “variant antibody”.
  • Tables 1-5 show the CDR sequences, the VH and VL sequences, the heavy chain and light chain sequences, the FR sequences and the constant domain sequences, respectively, of the constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 and AB 1370117. In the event of any discrepancy, the sequences in the Tables take precedence.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises the 6 CDRs of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 or AB 1370117 of Table 2, wherein the CDRs are determined by Kabat, Chothia, or IMGT.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); wherein any one or more of said CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to said sequences.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT).
  • SDSATWN heavy chain CDR1 of SEQ ID NO: 1
  • RTYYRSKWYNDYAVSVKS a heavy chain CDR3 of SEQ ID NO: 3
  • GVGSFDY GVGSFDY
  • RASQSISSWLA light chain CDR1 of SEQ ID NO: 4
  • KASGLES KASGLES
  • a light chain CDR3 of SEQ ID NO: 6 QQYNSYSQLT
  • the anti-FRa antibody, or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 38.
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38.
  • the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 49 and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 50.
  • the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 49 and a light chain amino acid sequence of SEQ ID NO: 50.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 7 (SYAMS); a heavy chain CDR2 of SEQ ID NO: 8 (SISSGRSYIYYADSVKG); a heavy chain CDR3 of SEQ ID NO: 9 (EMQQLALDY); a light chain CDR1 of SEQ ID NO: 10 (RASQGISNFLA); a light chain CDR2 of SEQ ID NO: 11 (AASSLQS); and a light chain CDR3 of SEQ ID NO: 12 (QQYNSYPFT); wherein any one or more of said CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to said sequences.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 7 (SYAMS); a heavy chain CDR2 of SEQ ID NO: 8 (SISSGRSYIYYADSVKG); a heavy chain CDR3 of SEQ ID NO: 9 (EMQQLALDY); a light chain CDR1 of SEQ ID NO: 10 (RASQGISNFLA); a light chain CDR2 of SEQ ID NO: 11 (AASSLQS); and a light chain CDR3 of SEQ ID NO: 12 (QQYNSYPFT).
  • the anti-FRa antibody, or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 39 and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 40.
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40.
  • the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 51 and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 52.
  • the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 51 and a light chain amino acid sequence of SEQ ID NO: 52.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 13 (SNSAAWN); a heavy chain CDR2 of SEQ ID NO: 14 (RTYYRSNWYNDYTLSVKS); a heavy chain CDR3 of SEQ ID NO: 15 (GVGRFDS); a light chain CDR1 of SEQ ID NO: 16 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 17 (KASSLES); and a light chain CDR3 of SEQ ID NO: 18 (QEYKTYSIFT); wherein any one or more of said CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to said sequences.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 13 (SNSAAWN); a heavy chain CDR2 of SEQ ID NO: 14 (RTYYRSNWYNDYTLSVKS); a heavy chain CDR3 of SEQ ID NO: 15 (GVGRFDS); a light chain CDR1 of SEQ ID NO: 16 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 17 (KASSLES); and a light chain CDR3 of SEQ ID NO: 18 (QEYKTYSIFT).
  • the anti-FRa antibody, or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 41 and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 42.
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 41 and a VL of SEQ ID NO: 42.
  • the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 53 and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 54.
  • the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 53 and a light chain amino acid sequence of SEQ ID NO: 54.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 19 (SYNMN); a heavy chain CDR2 of SEQ ID NO: 20 (SISSGSSYIYYADSMKG); a heavy chain CDR3 of SEQ ID NO: 21 (GMTTLTFDY); a light chain CDR1 of SEQ ID NO: 22 (RASQGISTFLA); a light chain CDR2 of SEQ ID NO: 23 (AASSLQS); and a light chain CDR3 of SEQ ID NO: 24 (QQYISYPLT); wherein any one or more of said CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to said sequences.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 19 (SYNMN); a heavy chain CDR2 of SEQ ID NO: 20 (SISSGSSYIYYADSMKG); a heavy chain CDR3 of SEQ ID NO: 21 (GMTTLTFDY); a light chain CDR1 of SEQ ID NO: 22 (RASQGISTFLA); a light chain CDR2 of SEQ ID NO: 23 (AASSLQS); and a light chain CDR3 of SEQ ID NO: 24 (QQYISYPLT).
  • SYNMN heavy chain CDR1 of SEQ ID NO: 19
  • 20 SISSGSSYIYYADSMKG
  • a heavy chain CDR3 of SEQ ID NO: 21 GMTTLTFDY
  • RASQGISTFLA light chain CDR1 of SEQ ID NO: 22
  • AASSLQS AASSLQS
  • QQYISYPLT a light chain CDR3 of
  • the anti-FRa antibody, or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 43 and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 44.
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 43 and a VL of SEQ ID NO: 44.
  • the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 55 and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 56.
  • the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 55 and a light chain amino acid sequence of SEQ ID NO: 56.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 25 (SYSMN); a heavy chain CDR2 of SEQ ID NO: 26 (SISSRSSYVYYADSVKG); a heavy chain CDR3 of SEQ ID NO: 27 (GMTTLTFDY); a light chain CDR1 of SEQ ID NO: 28 (RASQGISSFLA); a light chain CDR2 of SEQ ID NO: 29 (AASSLQS); and a light chain CDR3 of SEQ ID NO: 30 (QQYNSYPLT); wherein any one or more of said CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to said sequences.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 25 (SYSMN); a heavy chain CDR2 of SEQ ID NO: 26 (SISSRSSYVYYADSVKG); a heavy chain CDR3 of SEQ ID NO: 27 (GMTTLTFDY); a light chain CDR1 of SEQ ID NO: 28 (RASQGISSFLA); a light chain CDR2 of SEQ ID NO: 29 (AASSLQS); and a light chain CDR3 of SEQ ID NO: 30 (QQYNSYPLT).
  • the anti-FRa antibody, or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 45 and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 46.
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 46.
  • the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 57 and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 58.
  • the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 57 and a light chain amino acid sequence of SEQ ID NO: 58.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 31 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 32 (RTYYRSKWYSDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 33 (GGAPFDY); a light chain CDR1 of SEQ ID NO: 34 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 35 (KASSLES); and a light chain CDR3 of SEQ ID NO: 36 (QQYNSYSMYT); wherein any one or more of said CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to said sequences.
  • the anti-FRa antibody, or antigen-binding fragment comprises: a heavy chain CDR1 of SEQ ID NO: 31 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 32 (RTYYRSKWYSDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 33 (GGAPFDY); a light chain CDR1 of SEQ ID NO: 34 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 35 (KASSLES); and a light chain CDR3 of SEQ ID NO: 36 (QQYNSYSMYT).
  • the anti-FRa antibody, or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 47 and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 48.
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 47 and a VL of SEQ ID NO: 48.
  • the anti-FRa antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 59 and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 60.
  • the anti-FRa antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 59 and a light chain amino acid sequence of SEQ ID NO: 60.
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises heavy chain VH FR1, VH FR2, VH FR3, and/or VH FR4 that is at least 80%, 85%, 90% or 95% identical, or identical to reference heavy chain VH FR1, VH FR2, VH FR3, and/or VH FR4, respectively of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 or AB 1370117 described in Table 4, wherein the antibody or fragment is capable of binding FRa alone (e.g. in the form of a single chain antibody fragment).
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises light chain VL FR1, VL FR2, VL FR3, and/or VL FR4 that are at least 80%, 85%, 90% or 95% identical, or identical to reference light chain VL FR1, VL FR2, VL FR3, and/or VL FR4, respectively of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 or AB 1370117 described in Table 4, wherein the antibody or fragment is capable of binding FRa alone (e.g. in the form of a single chain antibody fragment).
  • the anti-FRa antibody, or antigen-binding fragment thereof comprises (a) light chain VL FR1, VL FR2, VL FR3, and VL FR4 that are at least 80%, 85%, 90% or 95% identical, or identical to reference light chain VL FR1, VL FR2, VL FR3, and VL FR4, respectively, of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 or AB 1370117 as described in Table 4; and (b) heavy chain VH FR1, VH FR2, VH FR3, and VH FR4 that are at least 80%, 85%, 90% or 95% identical, or identical to reference heavy chain VH FR1, VH FR2, VH FR3, and VH FR4, respectively, of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095
  • the anti-FRa antibody comprises a constant heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 109 and a constant light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 110.
  • the anti-FRa antibody comprises a constant heavy chain amino acid sequence of SEQ ID NO: 109 and a constant light chain amino acid sequence of SEQ ID NO: 110.
  • the anti-FRa antigen-binding fragment comprises a constant heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 111 and a constant light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 110.
  • the anti-FRa antigen-binding fragment comprises a constant heavy chain amino acid sequence of SEQ ID NO: 111 and a constant light chain amino acid sequence of SEQ ID NO: 110.
  • Table 3 FRa antibody heavy chain and light chain sequences
  • Table 4 FRa antibody FR regions
  • Antibodies in the ADCs of the disclosure may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions.
  • amino acid residues at nonconserved positions are substituted with conservative or non-conservative residues.
  • conservative amino acid replacements are contemplated.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine).
  • amino acid substitution is considered to be conservative.
  • the inclusion of conservatively modified variants in the antibodies in the ADCs of the disclosure does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.
  • Non-conservative amino acid substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Vai), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Vai, His, He or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).
  • an electropositive side chain e.g., Arg, His or Lys
  • an electronegative residue e.g., Glu or As
  • non-standard amino acids such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues.
  • the antibodies in the ADCs of the present disclosure can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine.
  • Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins.
  • an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
  • Methods for synthesising amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3 -azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • the desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3 -azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine.
  • the non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification.
  • Essential amino acids in the antibodies in the ADCs of the present disclosure can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the antibodies in the ADCs of the present disclosure.
  • related components e.g. the translocation or
  • the “percent sequence identity” between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimise alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.
  • sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D.
  • Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E.
  • Percent sequence identity can be determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimise the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).
  • variable domains in both the heavy and light chains of an antibody or antigen-binding fragment thereof in the ADC are altered by at least partial replacement of one or more CDRs and/or by partial framework region replacement and sequence changing.
  • the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and in certain embodiments from an antibody from a different species. It is not necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen-binding capacity of one variable domain to another. Rather, it is only necessary to transfer those residues that are necessary to maintain the activity of the antigen-binding site.
  • the antibody or antigen-binding fragment thereof in the ADC can include, in addition to a VH and a VL, a heavy chain constant region or fragment thereof.
  • the heavy chain constant region is a human heavy chain constant region, e.g., a human IgG constant region, e.g., a human IgGl constant region.
  • a residue is inserted to the heavy chain constant region of the antibody in the ADC for site-specific conjugation e.g., for conjugating the cytotoxin.
  • a cysteine residue may be inserted between amino acid S239 and V240 in the CH2 region of IgGl, which may be referred to as “a 239 insertion” or “239i.”
  • the antibodies in the ADCs disclosed herein can be modified to comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI, CH2 or CH3) and/or to the light chain constant domain (CL).
  • a modified constant region wherein one or more domains are partially or entirely deleted are contemplated.
  • a modified antibody in the ADC will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ACH2 constructs).
  • the omitted constant region domain can be replaced by a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility typically imparted by the absent constant region.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody.
  • the constant region modifications moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin.
  • Yet other modifications of the constant region can be used to eliminate disulphide linkages or oligosaccharide moieties that allow for enhanced localisation due to increased antigen specificity or antibody flexibility.
  • the antibody or antigen-binding fragment thereof in the ADC has no antibody-dependent cellular cytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity (CDC) activity.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the antibody or antigen-binding fragment thereof in the ADC can be engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibodies or fragments thereof.
  • a peptide spacer can be inserted between the hinge region and the modified CH2 and/or CH3 domains.
  • compatible constructs can be expressed in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer.
  • Such a spacer can be added, for instance, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible.
  • Amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct.
  • any spacer added to the construct can be relatively non-immunogenic, or even omitted altogether, so as to maintain the desired biochemical qualities of the modified antibodies.
  • an antibody or antigen-binding fragment thereof in the ADC can be modified by the partial deletion or substitution of a few or even a single amino acid in a constant region.
  • the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding and thereby increase tumour localisation.
  • one or more constant region domains that control the effector function e.g., complement C1Q binding
  • Such partial deletions of the constant regions can improve selected characteristics of the antibody or antigen-binding fragment thereof in the ADC e.g., serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
  • the constant regions of the antibody and antigen-binding fragment thereof in the ADC can be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it is possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody or antigen - binding fragment thereof in the ADC.
  • a conserved binding site e.g., Fc binding
  • a heavy chain constant region or fragment thereof e.g., a human IgG constant region or fragment thereof
  • the IgG constant domain can contain one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385- 389, and 428-436, wherein the amino acid position numbering is according to the EU index as set forth in Kabat.
  • the IgG constant domain can contain one or more of a substitution of the amino acid at Kabat position 252 with Tyrosine (Y), Phenylalanine (F), Tryptophan (W), or Threonine (T), a substitution of the amino acid at Kabat position 254 with Threonine (T), a substitution of the amino acid at Kabat position 256 with Serine (S), Arginine
  • R Glutamine
  • Q Glutamic acid
  • E Aspartic acid
  • T Threonine
  • L Leucine
  • P Proline
  • the anti-FRa antibodies or antigenbinding fragments thereof in the ADC comprise a YTE mutant.
  • YTE refers to a mutation in IgGl Fc that results in an increase in the binding to human FcRn and improves the serum half-life of the antibody having the mutation.
  • a YTE mutant comprises a combination of three mutations, M252Y/S254T/T256E (EU numbering Kabat et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, D.C.), introduced into the heavy chain of an IgGl. See U.S. Patent No. 7,658,921, which is incorporated by reference herein.
  • the YTE mutant has been shown to increase the serum half-life of antibodies approximately four-times as compared to wild-type versions of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006); Robbie et al., (2013) Antimicrob. Agents Chemother. 57, 6147-6153). See also U.S. Patent No. 7,083,784, which is hereby incorporated by reference in its entirety.
  • the anti-FRa antibody or antigen-binding fragment thereof in the ADC comprises:
  • E at the N-terminus (e.g. position 1) of the VH; or Q at the N-terminus (e.g. position 1) of the VH.
  • polynucleotides encoding the anti-FRa antibodies or antigen-binding fragments thereof of the ADCs of the disclosure may be any of the nucleotide sequences in Tables 6-7. In the event of any discrepancy, the sequences in the Tables take precedence.
  • the polynucleotide encoding the anti-FRa antibodies or antigenbinding fragments thereof in the ADCs of the disclosure comprises a sequence encoding (a) VL that is at least 80%, 85%, 90% or 95% identical, or identical to reference VL nucleotide sequence of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 or AB 1370117 as described in Table 6; and (b) VH that is at least 80%, 85%, 90% or 95% identical, or identical to reference VH nucleotide sequence of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 or AB 1370117 as described in Table 6.
  • the polynucleotide encoding the anti-FRa antibodies in the ADCs of the disclosure comprises a sequence encoding (a) a light chain that is at least 80%, 85%, 90% or 95% identical, or identical to reference light chain nucleotide sequence of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 or AB 1370117 as described in Table 7; and (b) a heavy chain that is at least 80%, 85%, 90% or 95% identical, or identical to reference heavy chain nucleotide sequence of any one of constructs AB 1370049, AB 1370026, AB 1370035, AB 1370083, AB 1370095 or AB 1370117 as described in Table 7.
  • Table 6 FRa antibody VH and VL nucleotide sequences
  • Table 7 FRa antibody heavy and light chain nucleotide sequences
  • the polynucleotide sequence(s) include sequences that have been removed from their naturally occurring environment, recombinant or cloned (e.g. DNA) isolates, and chemically synthesised analogues or analogues biologically synthesised by heterologous systems.
  • the polynucleotide sequence(s) may be prepared by any means known in the art. For example, large amounts of the sequence(s) may be produced by replication and/or expression in a suitable host cell.
  • the natural or synthetic DNA fragments coding for a desired fragment will typically be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell.
  • DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured bacterial, insect, mammalian, plant or other eukaryotic cell lines.
  • the polynucleotide sequence(s) may also be produced by chemical synthesis, e.g. a polynucleotide by the phosphoramidite method or the tri-ester method and may be performed on commercial automated oligonucleotide synthesisers.
  • a double- stranded (e.g. DNA) fragment may be obtained from the single stranded product of chemical synthesis either by synthesising the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Variants of a polynucleotide are also described herein. Polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both.
  • a polynucleotide variant comprises an alteration that produces silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide.
  • a polynucleotide variant is produced by a silent substitution due to the degeneracy of the genetic code.
  • a polynucleotide variant can be produced for a variety of reasons, e.g., to optimise codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coll).
  • antibody refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen.
  • the antibodies in the ADCs of the present disclosure are generally isolated or recombinant.
  • isolated when used herein refers to a polypeptide, e.g., an antibody, that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated antibody will be prepared by at least one purification step. Thus, an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. For instance, an isolated antibody that specifically binds to FRa is substantially free of antibodies that specifically bind antigens other than FRa.
  • an antibody comprises at least two “light chains” (LC) and two “heavy chains” (HC).
  • the light chains and heavy chains of such antibodies are polypeptides consisting of several domains.
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as “VH”) and a heavy chain constant region (abbreviated herein as “CH”).
  • the heavy chain constant region comprises the heavy chain constant domains CHI, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM).
  • Each light chain comprises a light chain variable domain (abbreviated herein as “VL”) and a light chain constant domain (abbreviated herein as “CL”).
  • the antibody in the ADC is a full-length antibody.
  • An “intact” or “full-length” antibody, as used herein, refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulphide bonds.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • the variable regions VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) (also known as hypervariable regions), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the VH or VL chain of the antibody in the ADC can further include all or part of a heavy or light chain constant region.
  • binding between an antibody and its target antigen or epitope is mediated by the CDRs.
  • epitope refers to a target protein region (e.g. polypeptide) capable of binding to (e.g. being bound by) an antibody or antigen-binding fragment in the ADC of the disclosure.
  • the CDRs are the main determinants of antigen specificity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al.
  • the sequence of a CDR may be identified by reference to any number system known in the art, for example, the Kabat system (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); the Chothia system (Chothia &, Lesk, “Canonical Structures for the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196, 901-917 (1987)); or the IMGT system (Lefranc et al., “IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig superfamily V-like domains,” Dev. Comp. Immunol. 27, 55-77 (2003)) (see Table 8).
  • Kabat system Kabat system
  • Chothia system Chothia &, Lesk, “Canonical Structures for the Hypervariable Regions of Immunoglobulins,” J. Mol.
  • Table 8 CDR definitions
  • the “constant domains” (or “constant regions”) of the heavy chain and of the light chain are not involved directly in binding of an antibody to a target, but exhibit various effector functions.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • IgA immunoglobulin G
  • IgD immunoglobulin G
  • IgE immunoglobulin M
  • IgM immunoglobulin M
  • IgG molecules interact with multiple classes of cellular receptors.
  • IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIII.
  • FcyR Fey receptors
  • binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody - coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the anti-FRa antibodies or antigen -binding fragments thereof in the ADCs are IgG isotype.
  • the anti-FRa antibodies or antigen-binding fragments in the ADCs can be any IgG subclass, for example IgGl, IgG2, IgG3, or IgG4 isotype.
  • the anti-FRa antibodies or antigen-binding fragments thereof in the ADCs are based on an IgGl isotype.
  • the use of a wildtype human IgGl molecule that is close to a natural IgG could reduce developability and other risks.
  • the present inventors have devised ADCs using a human IgGl mAb structure, which it is believed, without being bound by theory, will be less immunogenic than other anti-FRa ADCs being developed such as IMGN151.
  • Fc region refers to the portion of a native immunoglobulin that is formed by two Fc chains.
  • Each “Fc chain” comprises a constant domain CH2 and a constant domain CH3.
  • Each Fc chain may also comprise a hinge region.
  • a native Fc region is homodimeric.
  • the Fc region may be heterodimeric because it may contain modifications to enforce Fc heterodimerisation.
  • the Fc region contains the carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor), and has no antigen binding activity.
  • Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein.
  • Human IgGl, IgG2, IgG3, and IgG4 heavy chain sequences can be obtained in a variety of sequence databases, including the UniProt database (www.uniprot.org) under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN) respectively.
  • the anti-FRa antibodies in the ADCs of the disclosure are monoclonal antibodies.
  • a “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants.
  • the term “monoclonal antibody” can encompass both full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site.
  • “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.
  • the anti-FRa antibodies in the ADCs of the disclosure are isolated monoclonal antibodies.
  • the antibody in the ADC is a fully human monoclonal antibody.
  • methods of the disclosure may employ ADCs having polyclonal antibodies.
  • the anti-FRa antibodies and antigen-binding fragments thereof in the ADCs of the disclosure may be derived from any species by recombinant means.
  • the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof.
  • non-human derived antibodies or antigen-binding fragments in the ADCs may be genetically or structurally altered to be less immunogenic upon administration to the human patient.
  • human or humanised antibodies especially as recombinant human or humanised antibodies.
  • human antibody means an antibody produced in a human or an antibody having an amino acid sequence corresponding to an antibody produced in a human made using any technique known in the art.
  • a human antibody may include intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.
  • a human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo).
  • a human antibody can be made in a human cell (through recombinant expression), a non-human animal, or a prokaryotic or eukaryotic cell that can express functionally rearranged human immunoglobulin (such as heavy and light chain) genes.
  • a linker peptide that is not found in native human antibodies can be included in a single chain human antibody.
  • an Fv may have a linker peptide, such as two to about eight glycine or other amino acid residues, that joins the heavy chain’s variable region and the light chain’s variable region. These linker peptides are considered to be of human origin.
  • Human antibodies can be produced using a variety of techniques, including phage display techniques that use antibody libraries derived from human immunoglobulin sequences.
  • Transgenic mice that are unable to express functional indigenous immunoglobulins but can express human immunoglobulin genes can also be used to make human antibodies (see, for example, PCT Publication Nos. WO 1998/24893; WO 1992/01047; WO 1996/34096; WO 1996/33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, each of which is incorporated by reference herein). Human antibodies can also be directly prepared using various techniques known in the art.
  • Immortalised human B lymphocytes immunised in vitro or isolated from an immunised individual that produce an antibody directed against a target antigen can be generated. See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147 (l):86-95 (1991); U.S. Patent 5,750,373.
  • humanised antibody refers to antibodies in which the framework or CDRs have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin.
  • a murine CDR may be grafted into the framework region of a human antibody to prepare the “humanised antibody.” See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270.
  • “humanised antibodies” are those in which the constant region has been additionally modified or changed from that of the original antibody to generate desirable properties.
  • Humanised antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanised and engineered products using three-dimensional models of the parental, engineered, and humanised sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen, such as FRa.
  • FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • Humanised antibodies can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimise antibody specificity, affinity, and/or capability.
  • humanised antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • Humanised antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanised antibodies are described in U.S. Pat. Nos. 5,225,539 or 5,639,641, each of which is incorporated by reference herein.
  • chimeric antibody refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present disclosure are those in which the constant region has been modified or changed from that of the original antibody to generate desirable properties. Such chimeric antibodies are also referred to as “class-switched antibodies”. Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions.
  • the antibody in the ADC of the disclosure is a full-length antibody described above.
  • the antibody can be an antigen-binding fragment.
  • the term “antigen-binding fragment” as used herein incudes any naturally -occurring or artificially- constructed configuration of an antigen-binding polypeptide comprising one, two or three light chain CDRs, and/or one, two or three heavy chain CDRs, wherein the polypeptide is capable of binding to the antigen.
  • the antigen-binding fragment in the ADC of the disclosure is a Fab fragment.
  • the antibody in the ADC according to the disclosure can also be a Fab', an Fv, an scFv, an Fd, a V NAR domain, an IgNAR, an intrabody, an IgG CH2, a minibody, a singledomain antibody, an Fcab, an scFv-Fc, F(ab')2, a di-scFv, a bi-specific T-cell engager (BiTE®), a F(ab')3, a tetrabody, a triabody, a diabody, a DVD-Ig, an (scFv)2, a mAb2 or a DARPin.
  • Fab fragment and “Fab” are used interchangeably herein and contain a single light chain (e.g. a constant domain CL and a VL) and a single heavy chain (e.g. a constant domain CHI and a VH).
  • the heavy chain of a Fab fragment is not capable of forming a disulphide bond with another heavy chain.
  • a “Fab 1 fragment” contains a single light chain and a single heavy chain but in addition to the CHI and the VH, a “Fab 1 fragment” contains the region of the heavy chain between the CHI and CH2 domains that is required for the formation of an inter-chain disulphide bond. Thus, two “Fab 1 fragments” can associate via the formation of a disulphide bond to form a F(ab')2 molecule.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains. Each chain includes a portion of the constant region necessary for the formation of an inter-chain disulphide bond between two heavy chains.
  • Fv fragment contains only the variable regions of the heavy and light chain. It contains no constant regions.
  • a “single-domain antibody” is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).
  • a “single-chain Fv” (“scFv”) is antibody fragment containing the VH and VL domain of an antibody, linked together to form a single chain.
  • a polypeptide linker is commonly used to connect the VH and VL domains of the scFv.
  • a “tandem scFv”, also known as a TandAb®, is a single-chain Fv molecule formed by covalent bonding of two scFvs in a tandem orientation with a flexible peptide linker.
  • a “bi-specific T cell engager” (BiTE®) is a fusion protein consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumour cell antigen.
  • a “diabody” is a small bivalent and bispecific antibody fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH-VL polypeptide linker
  • a “DARPin” is a bispecific ankyrin repeat molecule. DARPins are derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins.
  • a DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences.
  • a DARPin is composed of 4 to 6 modules. Because each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of binders is done by ribosome display, which is completely cell-free and is described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.
  • the antibody or antigen-binding fragment thereof in the ADC can be further modified to contain additional chemical moieties not normally part of a protein.
  • Those derivatised moieties can improve the solubility, the biological half-life or absorption of the antibody or antigen-binding fragment thereof in the ADC.
  • the moieties can also reduce or eliminate any desirable side effects of the antibodies or antigen-binding fragments thereof in the ADCs. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).
  • the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure specifically bind to FRa.
  • the term “specifically binding to FRa” refers to an antibody that is capable of binding to the defined target with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting FRa.
  • an antibody specifically binding to FRa does not bind to other antigens, or does not bind to other antigens with sufficient affinity to produce a physiological effect.
  • the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure specifically bind to human FRa (UniProt ID: P15328) and/or cynomolgus monkey FRa (UniProt ID: A0A2K5U044).
  • the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure specifically bind to human FRa.
  • the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure specifically bind to human FRa and cynomolgus monkey FRa.
  • the FRa has a sequence of SEQ ID NO: 112 or SEQ ID NO: 113. In particular embodiments, the FRa has a sequence of SEQ ID NO: 112.
  • SEQ ID NO: 112 Human FRa protein (predicted mature, secreted polypeptide)
  • SEQ ID NO: 113 Cyno FRa protein (predicted mature, secreted polypeptide)
  • the antibody or antigen-binding fragment thereof in the ADC does not bind to one or more selected from a mouse FRa (UniProt ID: P35846), rat FRa (UniProt ID: G3V8M6), human FRp (UniProt ID: P14207), human FRy (UniProt ID: P41439), or a combination thereof.
  • the term “does not bind” means that the antibody or antigen-binding fragment thereof in the ADC of the disclosure does not substantially bind to one of more of said molecules (e.g. mouse FRa, rat FRa, human FRP, human FRy, or a combination thereof).
  • the term “substantially no” when used in the context of binding herein may mean less than 5%, 2%, 1%, 0.5% or 0.1% of cells expressing one or more of said molecules in a cell culture become bound by the antibody or antigen-binding fragment thereof in the ADC of the disclosure (upon contact therewith).
  • the term “substantially no” when used in the context of binding herein may mean no such cells become bound.
  • Binding affinity generally 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). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • the antibody or antigen-binding fragment in the ADC of the disclosure binds to FRa molecule with sufficient affinity such that the antibody is useful as a therapeutic agent or a diagnostic reagent in targeting FRa.
  • the anti-FRa antibody or antigen-binding fragment thereof in the ADC binds to human FRa with a KD of about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less.
  • the anti-FRa antibody or antigen-binding fragment thereof in the ADC binds to human FRa with a KD of about 0.5 to about 50 nM, about 0.5 to about 40 nM, about 0.5 to about 30 nM, about 0.5 to about 20 nM, about 1 to about 50 nM, about 1 to about 40 nM, about 1 to about 30 nM, about 1 to about 20 nM, about 2 to about 50 nM, about 2 to about 40 nM, about 2 to about 30 nM, about 2 to about 20 nM, about 5 to about 50 nM, about 5 to about 40 nM, about 5 to about 30 nM, about 5 to about 20 nM, about 10 to about 50 nM, about 10 to about 40 nM, about 10 to about 30 nM or about 10 to about 20 nM.
  • the anti-FRa antibody or antigen-binding fragment thereof in the ADC binds to cyno FRa with a KD of about 100 nM or less, about 80 nM or less, about 60 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less.
  • the anti-FRa antibody or antigen-binding fragment thereof in the ADC binds to cyno FRa with a KD of about 1 to about 100 nM, about 1 to about 80 nM, about 1 to about 60 nM, about 1 to about 40 nM, about 2 to about 100 nM, about 2 to about 80 nM, about 2 to about 60 nM, about 2 to about 40 nM, about 5 to about 100 nM, about 5 to about 80 nM, about 5 to about 60 nM, about 5 to about 40 nM, about 10 to about 100 nM, about 10 to about 80 nM, about 10 to about 60 nM, about 10 to about 40 nM, about 20 to about 100 nM, about 20 to about 80 nM, about 20 to about 60 nM, about 20 to about 40 nM, about 30 to about 100 nM, about 30 to about 80 nM, about 30 to about 60 nM, or about 30 to about 40 nM, about
  • the affinity or avidity of an antibody or antigen-binding fragment thereof for an antigen can be determined experimentally using any suitable method well known in the art, e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., KINEXA® or BIACORETM analysis).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • kinetics e.g., KINEXA® or BIACORETM analysis.
  • Direct binding assays as well as competitive binding assay formats can be readily employed. (See, e.g., Berzofsky el al., Antibody-Antigen Interactions, In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein.)
  • the binding affinity of the anti-FRa antibodies, or antigen-binding fragments thereof, in the ADCs of the disclosure may be determined using a FRa binding affinity assay as described herein.
  • the binding affinity of the anti-FRa antibody, or antigen-binding fragment thereof, in the ADC of the disclosure is determined by Biacore, e.g. Biacore T200 at 25°C.
  • Biacore e.g. Biacore T200 at 25°C.
  • the affinity of the recombinant human FRa ECD for the anti-FRa antibody, or antigen-binding fragment thereof may be measured using the Biacore T200 at 25°C, for example using the following protocol.
  • Protein A is covalently immobilised to a CM5 chip surface using standard amine coupling techniques at a concentration of 50 pg/ml in 10 mM Sodium acetate pH 4.0.
  • the antibody, or antigen-binding fragment thereof, is captured onto the Protein A surface in HBS-EP+ buffer pH 7.4 at 10 pl/min to enable FRa ECD binding.
  • the FRa ECD is serially diluted (0.4 nM-100 nM human FRa ECD; 0.8 nM-200 nM cyno FRa ECD; 30 nM-4000 nM mouse FRa ECD and rat FRa ECD) in HBS-EP+ buffer pH 7.4 and flowed over the chip at 50 pl/min, with 2 minutes association and 8 minutes dissociation.
  • the chip surface is fully regenerated with pulses of 3 M MgCh to remove captured antibody, or antigen-binding fragment thereof, together with any bound FRa ECD.
  • Multiple buffer-only injections are made under the same conditions to allow for double reference subtraction of the final sensorgram sets, which are analysed using Biacore T200 Evaluation Software.
  • the binding affinity of the anti-FRa antibody, or antigen-binding fragment thereof, in the ADC of the disclosure may be determined by Octet, e.g. Octet red.
  • the binding affinity of the anti-FRa antibody may be assayed by Octet red at 25 °C , for example using the following protocol.
  • the binding assays are performed on Octet RED384 (ForteBio) at 25 °C in assay buffer containing PBS, 0.1% v/v BSA (Sigma, A9576), 0.01% v/v Tween-20 (Sigma, P9416) (pH 7.4) using tilted bottom black 384-well plates (ForteBio, 18-5076).
  • Assays are set up using either protein A or anti human capture biosensors (AHC) (ForteBio, 18-5089) according to the manufacturer’s instructions.
  • 10 pg/ml of anti -rat FRa IgG (Sino Biological, 81073-RP01) is coated onto protein A biosensors (ForteBio, NC9490476) and 10 pg/ml of test human IgG is loaded onto anti human capture biosensors (AHC) (ForteBio, 18-5089) for 180 seconds.
  • Association is measured by incubating loaded biosensors with 500 nM human FRa (in house) or 500 nM rat FRa (Sino Biological, 81073-R08H). Dissociation is measured following transfer into assay buffer. Data are analysed using the Octet data analysis software version 7.0.
  • the antibody or antigen-binding fragment thereof in the ADC of the disclosure can be produced by transfecting a host cell with one or more vectors comprising polynucleotides encoding the respective antibodies or fragments, culturing the host cell under conditions that allow synthesis of said antibody or antigen-binding fragment molecule; and recovering said antibody or antigen-binding fragment molecule from said culture.
  • the antibody or antigen-binding fragment thereof (e.g. as monoclonal antibodies) in the ADC can be made using recombinant DNA methods as described in U.S. Patent No. 4,816,567, which is incorporated by reference herein.
  • the polynucleotides encoding a monoclonal antibody are isolated from mature B -cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures.
  • the isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E.
  • monoclonal antibodies are generated by the host cells.
  • recombinant monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described in McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991).
  • Affinity maturation strategies and chain shuffling strategies are known in the art and can be employed to generate high affinity human antibodies or antigen-binding fragments thereof. See Marks et al., BioTechnology 10:779-783 (1992), incorporated by reference in its entirety.
  • anti-FRa antibody fragments in the ADCs are produced recombinantly.
  • Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments.
  • anti-FRa antibody fragments can also be isolated from the antibody phage libraries discussed above.
  • the anti-FRa antibody fragments in the ADCs can also be linear antibodies as described in U.S. Patent No. 5,641,870, which is incorporated by reference herein. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • Techniques can be adapted for the production of single-chain antibodies specific to FRa. (see, e.g., U.S. Pat. No. 4,946,778).
  • methods can be adapted for the construction of Fab expression libraries to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for FRa, or derivatives, fragments, analogs or homologs thereof. See, e.g., Huse et al., Science 246:1275-1281 (1989).
  • Antibody fragments can be produced by techniques known in the art including, but not limited to: F(ab')2 fragment produced by pepsin digestion of an antibody molecule; Fab fragment generated by reducing the disulphide bridges of an F(ab')2 fragment; Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent; or Fv fragments.
  • ADC Antibody-drug conjugates
  • the “antibody-drug conjugate” (ADC) of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof as described herein, wherein the anti-FRa antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.
  • the cytotoxin (also referred to as a cytotoxic agent) can be any molecule known in the art that inhibits or prevents the function of cells and/or causes destruction of cells (cell death), and/or exerts anti-neoplastic/anti-proliferative effects.
  • a number of classes of cytotoxic agents are known to have potential utility in ADC molecules.
  • Suitable cytotoxic agents for the present disclosure include, but are not limited to, topoisomerase I inhibitors (TOPOi), amanitins, auristatins, daunomycins, doxorubicins, duocarmycins, dolastatins, enediynes, lexitropsins, taxanes, puromycins, maytansinoids, vinca alkaloids, tubulysins and pyrrolobenzodiazepines (PBDs).
  • TOPOi topoisomerase I inhibitors
  • cytotoxic agents examples include AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1, vinblastine, methotrexate, and netropsin, and derivatives and analogs thereof. Additional disclosure regarding cytotoxins suitable for use in ADCs can be found, for example, in International Patent Application Publication Nos. WO 2015/155345 and WO 2015/157592, incorporated by reference herein in their entirety.
  • the cytotoxic agent is typically linked to, or “loaded onto” the antibody or antigenbinding fragment.
  • the agent loading (p) is the average number of agent(s) per antibody or antigen-binding fragment. It will be understood by the person skilled in the art that more than one of said agent(s) (e.g. TOPOi) may be conjugated to the antibody or antigen-binding fragment thereof.
  • the average number of cytotoxic agents per antibody (or antigenbinding fragment thereof) is in the range of about 1 to 20. In some embodiments the range is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In some embodiments, there is one agent per antibody (or antigen-binding fragment thereof). In some embodiments, the number of agents per antibody (or antigen-binding fragment thereof) can be expressed as a ratio of agent (z.e., drug) to antibody. This ratio is referred to as the Drug to Antibody Ratio (DAR).
  • the DAR is the average number of drugs (z.e., cytotoxic agents) linked to each antibody. In some embodiments of the present disclosure, the DAR is in the range of about 1 to 20.
  • the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10.
  • the DAR is about 4 (e.g., 3.8-4.2) or about 8 (e.g., 7.6-8.4).
  • the DAR is about 8 (e.g., 7.6-8.4).
  • the antibody or antigen-binding fragment thereof in the ADC of the disclosure is conjugated to one or more cytotoxin selected from a topoisomerase I inhibitor, tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof.
  • the antibody or antigen-binding fragment thereof may be conjugated to one or more cytotoxin selected from the group consisting of topoisomerase I inhibitor SG3932 (also known as AZ14170133), SG4010, SG4057 or SG4052 (the structures of which are provided below), or a combination thereof.
  • the antibody or antigen-binding fragment thereof may be conjugated to a topoisomerase I inhibitor, for instance the topoisomerase I inhibitor SG3932.
  • the antibody or antigen-binding fragment thereof in the ADC of the disclosure is not conjugated to, or the anti-FRa ADC of the disclosure does not comprise a microtubule inhibitor such as a tubulin inhibitor (e.g. maytansinoids, auristatins).
  • a microtubule inhibitor such as a tubulin inhibitor (e.g. maytansinoids, auristatins).
  • Microtubule inhibitor class of molecules suffer from potentially difficult-to-treat toxicities that limit dosing.
  • the present disclosure demonstrates, using in vivo and in vitro models, the stability and efficacy of anti-FRa mAbs when conjugated to a TOPOi payload and deployed as ADCs.
  • the anti-FRa ADC of the disclosure comprises a topoisomerase I inhibitor.
  • Topoisomerase inhibitors are chemical compounds that block the action of topoisomerase (topoisomerase I and II), which is a type of enzyme that controls the changes in DNA structure by catalysing the breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. Topoisomerase I inhibitors are advantageous as they mediate highly effective tumour cell killing with fewer toxicities to the patient.
  • alternative payloads such as microtubule inhibitor that have generally been used to-date for the development of anti-FRa ADCs are known to have toxicity problems (Hinrichs, et al. AAPS J. 2015 Sep; 17(5): 1055-1064).
  • a less hydrophobic linker with a less potent warhead e.g. TOPOi
  • bystander activity may be achieved by increasing the potency and/or improving warhead permeability through increased hydrophobicity, this may result in increased toxicity due to nonspecific uptake.
  • topoisomerase I inhibitor A general example of a suitable topoisomerase I inhibitor is represented by the following compound:
  • A* Said compound is denoted as A*, and may be referred to as a “Drug Unit” herein.
  • the compound may be provided with a linker for connecting (preferably conjugating) to an antibody or antigen-binding fragment in the ADC of the disclosure.
  • the linker is attached (e.g. conjugated) in a cleavable manner to an amino residue, for example, an amino acid of an antibody or antigen -binding fragment in the ADC of the disclosure.
  • topoisomerase I inhibitor is represented by the following compound, with the formula “I”: and salts and solvates thereof, wherein R L is defined above.
  • D L is a topoisomerase I inhibitor having a linker (e.g. Drug Linker unit) that is of formula III: and salts and solvates thereof, wherein R LL is defined above.
  • the compound of formula I is of the formula I p : and salts and solvates thereof, wherein R LP is a linker for connection to an antibody or antigenbinding fragment thereof in the ADC of the disclosure, wherein said linker is selected from: (Ia p ): wherein
  • Q xp is such that Q p is an amino-acid residue, a dipeptide residue or a tripeptide residue;
  • X p is: G L is defined above;
  • aP may be 0, 1, 2, 3, 4 or 5. In some embodiments, aP is 0 to 3. In some of these embodiments, aP is 0 or 1. In further embodiments, aP is 0. bP may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b is 0 to 12. In some of these embodiments, bP is 0 to 8, and may be 0, 2, 4 or 8. cP may be 0 or 1. dP may be 0, 1, 2, 3, 4 or 5. In some embodiments, dP is 0 to 3. In some of these embodiments, dP is 1 or 2. In further embodiments, dP is 2.
  • aP is 0, cP is 1 and dP is 2, and bP may be from 0 to 8. In some of these embodiments, bP is 0, 4 or 8.
  • the ADC of formula IV is of the formula IV P :
  • R LLP is a linker connected to the antibody or antigen-binding fragment thereof, wherein said linker is selected from
  • the compound of formula I is of the formula I p2 : and salts and solvates thereof, wherein R LP2 is a linker for connection to an antibody or antigenbinding fragment thereof in the ADC of the disclosure, wherein said linker is selected from: (Ia P2 ): wherein
  • Q x is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;
  • GL is a linker for connecting to an antibody or antigen-binding fragment thereof in the ADC of the disclosure
  • aP2 may be 0, 1, 2, 3, 4 or 5. In some embodiments, aP2 is 0 to 3. In some of these embodiments, aP2 is 0 or 1. In further embodiments, aP2 is 0. blP2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, blP2 is 0 to 12. In some of these embodiments, blP2 is 0 to 8, and may be 0, 2, 3, 4, 5 or 8.
  • b2P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b2P2 is 0 to 12. In some of these embodiments, b2P2 is 0 to 8, and may be 0, 2, 3, 4, 5 or 8.
  • cP2 may be 0 or 1.
  • dP2 may be 0, 1, 2, 3, 4 or 5.
  • dP2 is 0 to 3.
  • dP2 is 1 or 2.
  • dP2 is 2.
  • dP2 is 5.
  • aP2 is 0, blP2 is 0, cP2 is 1 and dP2 is 2, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5 or 8. In some embodiments of X P2 , aP2 is 1, b2P2 is 0, cP2 is 0 and dP2 is 0, and blP2 may be from 0 to 8. In some of these embodiments, blP2 is 0, 2, 3, 4, 5 or 8. In some embodiments of X P2 , aP2 is 0, blP2 is 0, cP2 is 0 and dP2 is 1, and b2P2 may be from 0 to 8.
  • b2P2 is 0, 2, 3, 4, 5 or 8.
  • blP2 is 0, b2P2 is 0, cP2 is 0 and one of aP2 and dP2 is 0.
  • the other of aP2 and d is from 1 to 5. In some of these embodiments, the other of aP2 and d is 1. In other of these embodiments, the other of aP2 and dP2 is 5.
  • the ADC of formula IV is of the formula IV P2 :
  • R LLP2 is a linker connected to the antibody or antigen-binding fragment thereof, wherein said linker is selected from
  • topoisomerase I inhibitors include those having the following formulas:
  • an antibody or antigen-binding fragment thereof in the ADC of the disclosure is conjugated to a topoisomerase I inhibitor having the following formula:
  • topoisomerase I inhibitors Synthetic methods of making topoisomerase I inhibitors are described in, for example, WO 2020/200880, which is incorporated by reference herein.
  • topoisomerase I inhibitors are preferred as outlined above, it should be noted that any suitable agent (e.g. drug/ cytotoxin) may be linked to an antibody or antigen -binding fragment thereof in the ADC of the disclosure. Examples of other suitable agents are outlined below.
  • Tubulysin and pyrrolobenzodiapezine are examples of other suitable agents.
  • the cytotoxin is a tubulysin or tubulysin derivative. In some embodiments, the cytotoxin is Tubulysin A, having the following chemical structure:
  • Tubulysins are members of a class of natural products isolated from myxobacterial species. As cytoskeleton-interacting agents, tubulysins are mitotic poisons that inhibit tubulin polymerisation and lead to cell cycle arrest and apoptosis. As used herein, the term “tubulysin” refers both collectively and individually to the naturally occurring tubulysins and analogs and derivatives of tubulysins. Illustrative examples of tubulysins are disclosed, for example, in W02004005326A2, W02012019123A1, WO2009134279A1, W02009055562A1,
  • the cytotoxin may be a pyrrolobenzodiazepine (PBD) or a PBD derivative.
  • PBD pyrrolobenzodiazepine
  • PBD derivative a PBD derivative.
  • PBD translocates to the nucleus where it crosslinks DNA, preventing replication during mitosis, damaging DNA by inducing single strand breaks, and subsequently leading to apoptosis.
  • Some PBDs have the ability to recognise and bind to specific sequences of DNA; the preferred sequence is PuGPu.
  • PBDs are of the general structure:
  • PBDs differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring.
  • All of the known natural products have an (S)-configuration at the chiral Cl la position which provides them with a right-handed twist when viewed from the C ring towards the A ring.
  • PBD anti-tumour antibiotic anthramycin
  • Family members include abbeymycin, chicamycin, DC-81, mazethramycin, neothramycins A and B, porothramycin, prothracarcin, sibanomicin (DC- 102), sibiromycin and tomamycin.
  • PBDs and ADCs comprising them are also described in WO 2015/155345 and WO 2015/157592, incorporated in their entirety herein by reference.
  • the antibody or antigen-binding fragment may be conjugated to the cytotoxin by a linker.
  • Linker or “Spacer” as used herein means a divalent chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody or antigenbinding fragment thereof to a cytotoxin to form an ADC.
  • the linker or spacer is a peptide spacer.
  • the linker or spacer is a non-peptide (e.g. chemical) spacer. Suitable linkers have two reactive termini, one for antibody conjugation and the other for cytotoxin conjugation.
  • one or both of the reactive termini will be absent or incomplete (such as being only the carbonyl of the carboxylic acid).
  • the linker is attached (e.g. conjugated) in a cleavable manner to an amino residue, for example, an amino acid of an antibody or antigen-binding fragment in the ADC described herein.
  • the linker is cleavable under intracellular circumstances, allowing the drug unit to be released from the antibody in the intracellular environment.
  • the linker unit may not be cleavable.
  • the drug is released, for example, by antibody degradation.
  • non-cleavable payloads require complete mAb digestion in the lysosome and the resulting drug-containing product may be too polar, e.g. for achieving bystander effect.
  • the ADC is preferably stable and intact before being transported or delivered into a cell, i.e. the antibody should be attached to the drug moiety.
  • the linkers are stable, but inside the cell, they can be cleaved at a high rate.
  • An effective linker will: (i) maintain the antibody's specific binding properties; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e. not cleaved, until the conjugate has been delivered or transported to its targeted site; and (iv) maintain the cytotoxic moiety's cell-killing or cytostatic effect.
  • Standard analytical methods such as mass spectroscopy, HPLC, and the separation/analysis technique LC/MS can be used to assess the stability of the ADC.
  • the linkers may be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulphide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).
  • Linkers hydrolysable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, or the like. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661).
  • Linkers cleavable under reducing conditions include, for example, a disulphide.
  • disulphide linkers are known in the art, including, for example, those that can be formed using SATA (N- succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl- alpha-methyl-alpha-(2-pyridyl-dithio)toluene) (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987)).
  • SATA N- succinimidyl-S-acetyl
  • the linker is susceptible to enzymatic hydrolysis.
  • Such linkers may be advantageous in particular embodiments over pH sensitive cleavable linkers, which may not be stable enough and cleave prematurely before reaching the target cell, and thus potential off-target toxicity may be observed.
  • the enzymatically cleavable linker can be, e.g., a peptide-containing linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • intracellular proteolytic release of the therapeutic drug is that the agent is usually attenuated when conjugated, and the conjugates' serum stabilities are usually high.
  • the peptidyl linker is at least two amino acids long or at least three amino acids long.
  • Exemplary amino acid linkers include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Peptides comprising the amino acids valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine are examples of appropriate peptides.
  • Natural amino acids, minor amino acids, and non- naturally occurring amino acid analogs, such as citrulline are all examples of amino acid residues that make up an amino acid linker component.
  • Exemplary dipeptides include valinecitrulline (VC or Val-Cit) and alanine-phenylalanine (AF or Ala-Phe).
  • Exemplary tripeptides include glycine-valine-citrulline (Gly- Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly).
  • the linker includes a dipeptide such as Val-Cit, Ala-Vai, or Phe-Lys, Val- Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit.
  • the linker comprises PEG.
  • a stable protease-cleavable linker containing PEG can limit payload hydrophobicity and be able to selectively cleave and release the free drug inside target cancer cells.
  • a less hydrophobic nature of the linker as described herein can enable high loading of the drug onto the antibody or antigen-binding fragment (e.g. DAR8) without aggregation, which would be significantly higher than mirvetuximab soravtansine (DAR3-4) or derivatives thereof, such as IMGN151 (DAR3.5). This could allow the ADC to deliver a significantly higher concentration of cytotoxin payload to the target cancer cells via binding to FRa on the cancer cells.
  • the linker comprises maleimide.
  • maleimide in the linker may allow the generation of DAR8 and DAR4 ADCs by making use of the native interchain disulphides in the antibodies. This is advantageous over the conjugation of surface amines from lysine residues which could result in a mixture of DAR species and batch-to-batch variability. There may also be reproducibility issues that affect ADC efficacy if conjugation sites interfere with antigen binding. Moreover, other conjugation methods e.g. azide-alkyne click chemistry involving an engineered antibody may not easily achieve a DAR of more than 4.
  • the anti-FRa antibody or antigen-binding fragment thereof in the ADC of the disclosure is linked to a cytotoxin via a linker R L selected from:
  • G L is a linker for connecting to an antibody or antigen-binding fragment thereof in the ADC of the disclosure
  • R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or 1 ; or
  • G L , X, Q x e.g. within the linker of la described above
  • the linker of lb will be outlined.
  • Ar represents a C5-6 arylene group, e.g. phenylene, and X represents Cl -4 alkyl.
  • G L is selected from G L l and G L1 ' 2 .
  • G L is G L l .
  • a 0 to 5
  • d 0 to 5
  • a may be 0, 1, 2, 3, 4 or 5.
  • a is 0 to 3.
  • a is 0 or 1.
  • a is 0.
  • bl may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
  • bl is 0 to 12.
  • bl is 0 to 8, and may be 0, 2, 3, 4, 5 or 8.
  • b2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
  • b2 is 0 to 12.
  • b2 is 0 to 8, and may be 0, 2, 3, 4, 5 or 8.
  • cl may be 0 or 1.
  • c2 may be 0 or 1.
  • d may be 0, 1, 2, 3, 4 or 5.
  • d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2. In further embodiments, d is 5.
  • a is 0, bl is 0, cl is 1, c2 is 0 and d is 2, and b2 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5 or 8. In some embodiments of X, a is 1, b2 is 0, cl is 0, c2 is 0 and d is 0, and bl may be from 0 to 8. In some of these embodiments, bl is 0, 2, 3, 4, 5 or 8. In some embodiments of X, a is 0, bl is 0, cl is 0, c2 is 0 and d is 1, and b2 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5 or 8.
  • bl is 0, b2 is 0, cl is 0, c2 is 0 and one of a and d is 0.
  • the other of a and d is from 1 to 5. In some of these embodiments, the other of a and d is 1. In other of these embodiments, the other of a and d is 5.
  • a is 1, b2 is 0, cl is 0, c2 is 1, d is 2, and bl may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5 or 8.
  • Q is an amino acid residue.
  • the amino acid may be a natural amino acid or a non-natural amino acid.
  • Q may be selected from: Phe, Lys, Vai, Ala, Cit, Leu, He, Arg, and Trp, where Cit is citrulline.
  • Q comprises a dipeptide residue.
  • the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids.
  • the dipeptide comprises natural amino acids.
  • the linker is a cathepsin labile linker
  • the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
  • Q is selected from:
  • Q is selected from:
  • dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
  • Q is a tripeptide residue.
  • the amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids.
  • the tripeptide comprises natural amino acids.
  • the linker is a cathepsin labile linker
  • the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin.
  • Q is a tetrapeptide residue.
  • the amino acids in the tetrapeptide may be any combination of natural amino acids and non-natural amino acids.
  • the tetrapeptide comprises natural amino acids.
  • the linker is a cathepsin labile linker
  • the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide then is a recognition site for cathepsin.
  • Tetrapeptide linkers of particular interest are:
  • the tetrapeptide is:
  • NH - represents the N-terminus
  • the C-terminus binds to the NH of the “Drug Unit” (e.g. A* as discussed below).
  • Glu represents the residue of glutamic acid, i.e. aGlu represents the residue of glutamic acid when bound via the a-chain, i.e.
  • the amino acid side chain is chemically protected, where appropriate.
  • the side chain protecting group may be a group as discussed above.
  • Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
  • R L1 and R L2 may be independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group.
  • both R L1 and R L2 are H. In some embodiments, R L1 is H and R L2 is methyl. In some embodiments, both R L1 and R L2 are methyl.
  • R L1 and R L2 together with the carbon atom to which they are bound form a cyclopropylene group. In some embodiments, R L1 and R L2 together with the carbon atom to which they are bound form a cyclobutylene group.
  • e is 0. In other embodiments, e is 1 and the nitro group may be in any available position of the ring. In some of these embodiments, it is in the ortho position. In others of these embodiments, it is in the para position.
  • R L is selected from:
  • an antibody-drug conjugate of the disclosure may be of the general formula IV :
  • L is an antibody or antigenbinding fragment thereof in the ADC of the disclosure
  • D L is a “Drug Unit” (e.g. cytotoxin such as TOPOi) having a linker R LL connected to the antibody or antigen-binding fragment thereof in the ADC of the disclosure, wherein the linker is preferably selected from
  • the drug loading is represented by p, the number of “Drug Units” e.g. cytotoxin such as TOPOi) per antibody or antigen-binding fragment thereof.
  • Drug loading may range from 1 to 20 Drug units (D) per antibody or antigen-binding fragment thereof.
  • p represents the average drug loading of the conjugates in the composition, and p ranges from 1 to 20. In some embodiments, the range of p is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10; preferably wherein p is 8.
  • G LL may be selected from: where Ar represents a C5-6 arylene group, e.g. phenylene and X represents C1-4 alkyl.
  • G LL is selected from G LL l and G LL1 ' 2 . In some of these embodiments, G LL is G LL l .
  • R LL is a group derived from the R L groups above. It will be recognised by one of skill in the art that any one or more of the chemical groups, moieties and features disclosed herein may be combined in multiple ways to form linkers useful for conjugation of the antibodies and cytotoxins as disclosed herein.
  • the enantiomerically enriched form has an enantiomeric ratio greater than 60:40, 70:30; 80:20 or 90:10. In further embodiments, the enantiomeric ratio is greater than 95:5, 97:3 or 99:1.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor, represented by the following compound with the formula “I”:
  • R L is defined above; wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor, represented by the following compound with the formula “I”:
  • R L is defined above; wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor SG3932
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor SG3932 wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor SG4010
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor SG4010 wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor SG4057 wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor SG4057 wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor SG4052 wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8. In some embodiments, the anti-FRa ADC of the disclosure comprises an anti-FRa antibody or antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor SG4052
  • SUBSTITUTE SHEET (RULE 26) wherein the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% thereof,
  • cytotoxin is a topoisomerase I inhibitor represented by the following compound, with the formula “I”:
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% thereof,
  • cytotoxin is a topoisomerase I inhibitor represented by the following compound, with the formula “I”:
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% thereof,
  • the cytotoxin is a topoisomerase I inhibitor SG3932
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% thereof,
  • the cytotoxin is a topoisomerase I inhibitor SG3932
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 100
  • SUBSTITUTE SHEET (RULE 26) (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 38, optionally wherein the anti-FRa antibody or antigen-bind
  • the cytotoxin is a topoisomerase I inhibitor SG4010
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical,
  • SUBSTITUTE SHEET (RULE 26) at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 38, optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 49 and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 50;
  • the cytotoxin is a topoisomerase I inhibitor SG4010
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% thereof,
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% thereof,
  • the cytotoxin is a topoisomerase I inhibitor SG4057
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% thereof,
  • the cytotoxin is a topoisomerase I inhibitor SG4052
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 8.
  • the anti-FRa antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light
  • SUBSTITUTE SHEET (RULE 26) chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37 and a VL with an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 38, optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical,
  • the cytotoxin is a topoisomerase I inhibitor SG4052
  • the DAR is in the range of about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In particular embodiments, the DAR is about 4.
  • Internalisation can be a useful property of an ADC.
  • internalisation allows the delivery of payloads to a cell.
  • the inventors have shown that antibodies and ADCs described herein demonstrated rapid internalisation and lysosome trafficking.
  • the anti-FRa ADC of the disclosure binds to FRa on the surface of a cell, and is internalised into the cell.
  • the internalisation of the anti- FRa ADC of the disclosure into a FRa-expressing cell is saturated within about 4 hours or less, about 5 hours or less, about 6 hours or less, about 7 hours or less, about 8 hours or less, about 9 hours or less, about 10 hours or less, about 11 hours or less, or about 12 hours or less.
  • the anti-FRa ADC of the present disclosure inhibits or suppresses proliferation (e.g. of a tumour) by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or about 100% (in particular embodiments, at least 40%) relative to a level of inhibition or suppression in the absence of the anti-FRa ADC.
  • Cellular proliferation can be assayed using art-recognised techniques which measure rate of cell division, and/or the fraction of cells within a cell population undergoing cell division, and/or rate of cell loss from a cell population due to terminal differentiation or cell death (e.g., thymidine incorporation).
  • the anti-FRa ADC of the present disclosure exerts cytotoxicity towards a cell expressing FRa at an EC50 value of about 1000 ng/ml or less, about 500 ng/ml or less, about 400 ng/ml or less, about 300 ng/ml or less, about 290 ng/ml or less, about 280 ng/ml or less, about 270 ng/ml or less, about 260 ng/ml or less, or about 250 ng/ml or less.
  • the anti-FRa ADC of the present disclosure exerts cytotoxicity towards a cell expressing FRa at an IC50 value of about 100 pg/ml or less, about 50 pg/ml or less, about 25 pg/ml or less, about 10 pg/ml or less, about 5 pg/ml or less, about 2.5 pg/ml or less, about 1 pg/ml or less, about 0.75 pg/ml or less, about 0.5 pg/ml or less, about 0.25 pg/ml or less, about 0.1 pg/ml or less, about 0.075 pg/ml or less, about 0.05 pg/ml or less, about 0.025 pg/ml or less, about 0.01 pg/ml or less.
  • the anti-FRa ADC of the present disclosure inhibits or suppresses proliferation of a cell population having heterogeneous expression of FRa and/or a low expression of FRa. In some embodiments, the anti-FRa ADC of the present disclosure inhibits or suppresses proliferation of a cell population having a medium expression of FRa (e.g. Jeg-3, OVCAR-3 cell line or cells with a similar or equivalent level of FRa expression), medium-high expression of FRa (e.g. Igrov-1 cell line or cells with a similar or equivalent level of FRa expression), high expression of FRa (e.g. KB cell line or cells with a similar or equivalent level of FRa expression).
  • FRa medium expression of FRa
  • medium-high expression of FRa e.g. Igrov-1 cell line or cells with a similar or equivalent level of FRa expression
  • high expression of FRa e.g. KB cell line or cells with a similar or equivalent level of FRa expression.
  • the ADCs of the present disclosure can be made in a variety of ways, using known organic chemistry reactions, conditions, and reagents, such as: (1) reacting a reactive substituent of an antibody or antigen-binding fragment with a bivalent linker reagent, then reacting with a cytotoxin, such as a topoisomerase I inhibitor; or (2) reacting a reactive substituent of a cytotoxin, such as a topoisomerase I inhibitor, with a bivalent linker reagent, then reacting with a reactive substituent of an antibody or antigen-binding fragment thereof.
  • Reactive substituents that may be present within an antibody, or antigen-binding fragment thereof, as disclosed herein include, without limitation, nucleophilic groups such as (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • nucleophilic groups such as (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • Reactive substituents that may be present within an antibody, or antigen-binding fragment thereof, as disclosed herein include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids.
  • the reactive substituents present within an antibody, or antigen-binding fragment thereof as disclosed herein include amine or thiol moieties.
  • Certain antibodies have cysteine bridges, which are reducible interchain disulphides. By treating antibodies with a reducing agent (such as DL-dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP)), they can be made reactive for conjugation with linker reagents. Each cysteine bridge will theoretically result in the formation of two reactive thiol nucleophiles.
  • a reducing agent such as DL-dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP)
  • the reaction of lysines with 2 -iminothiolane can be used to introduce additional nucleophilic groups into antibodies.
  • One, two, three, four, or more cysteine residues can be used to insert reactive thiol groups into an antibody (or fragment thereof) (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).
  • Engineering antibodies with reactive cysteine amino acids is described in U.S. Pat. No. 7,521,541, which is incorporated by reference herein.
  • the antibody or antigen-binding fragment thereof can have one or more carbohydrate groups that can be chemically changed to contain one or more sulphydryl groups.
  • the ADC is then formed by conjugation through the sulphur atom of the sulphydryl group.
  • the antibody may contain one or more carbohydrate groups that can be oxidised to produce an aldehyde (-CHO) group (see, for example, Laguzza et al., J. Med. Chem. 1989, 32(3), 548-55). Conjugation through the corresponding aldehyde results in the formation of the ADC. Further protocols for the modification of proteins for the attachment or association of cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002). Methods for the conjugation of linker-drug moieties to cell- targeted proteins such as antibodies, immunoglobulins or fragments thereof are found, for example, in U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163; W02005/037992; W02005/081711; and W02006/034488, each of which is incorporated by reference herein.
  • the antibody or antigen-binding fragment thereof is stochastically conjugated to a cytotoxin, such as a topoisomerase I inhibitor, for example, by partial reduction of the antibody or fragment, followed by reaction with a desired agent, with or without a linker moiety attached.
  • a cytotoxin such as a topoisomerase I inhibitor
  • the antibody or fragment may be reduced using DTT or other reducing agent to perform a similar reduction e.g. TCEP.
  • the agent with or without a linker moiety attached can then be added at a molar excess to the reduced antibody or fragment in the presence of DMSO.
  • a quenching agent such as N-acetyl-L-cysteine may be added to quench unreacted agent.
  • the reaction mixture may then be purified (by e.g. TFF, SEC-FPLC, CHT, spin filter centrifugation) and buffer-exchanged into PBS or other relevant formulation buffer.
  • a cytotoxin is conjugated to an antibody or antigen-binding fragment thereof by site-specific conjugation.
  • site-specific conjugation of therapeutic moieties to antibodies using reactive amino acid residues at specific positions yields homogeneous preparations of an ADC with uniform stoichiometry.
  • the site-specific conjugation can be through a cysteine residue or a non-natural amino acid.
  • the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof through at least one cysteine residue.
  • Cysteine amino acids may be engineered at reactive sites in an antibody (or antigen-binding fragment thereof) and which preferably do not form intrachain or intermolecular disulphide linkages (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; W02009/052249).
  • the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof through a cysteine substitution of at least one of positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339, 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442, 443 and 446, wherein the numbering corresponds to the EU index in Kabat.
  • the specific Kabat positions are 239, 442, or both.
  • the specific positions are Kabat position 442, an amino acid insertion between Kabat positions 239 and 240, or both.
  • the cytotoxin is conjugated to the antibody or antigenbinding fragment thereof through a thiol-maleimide linkage.
  • the amino acid side chain is a sulphydryl side chain.
  • the resulting product may be a mixture of ADCs with a distribution of agent units attached to an antibody, e.g. 1, 2, 3, etc.
  • Liquid chromatography methods such as hydrophobic interaction (HIC) may separate compounds in the mixture by agent loading value.
  • Preparations of an ADC with a single agent loading value (p) may be isolated.
  • the average number of cytotoxic agents per antibody (or antigen-binding fragment) in preparations of ADCs from conjugation reactions may be characterised by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis.
  • the quantitative distribution of ADC in terms of p may also be determined.
  • ELISA the averaged value of p in a particular preparation of an ADC may be determined (Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Sanderson etal. (2005) Clin. Cancer Res. 11:843-852).
  • separation, purification, and characterisation of homogeneous ADC, where p is a certain value from antibody with other agents may be achieved by means such as reverse phase HPLC, electrophoresis, TFF, SEC-FPLC, CHT, spin filter centrifugation. Such techniques are also applicable to other types of conjugates.
  • C5-6 arylene The term “C5-6 arylene”, as used herein, pertains to a divalent moiety obtained by removing two hydrogen atoms from an aromatic ring atom of an aromatic compound.
  • the prefixes denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms.
  • the ring atoms may be all carbon atoms, as in “carboarylene groups”, in which case the group is phenylene (Ce).
  • the ring atoms may include one or more heteroatoms, as in “heteroarylene groups”.
  • heteroarylene groups include, but are not limited to, those derived from: Ni: pyrrole (azole) (Cs), pyridine (azine) (Ce);
  • Si thiophene (thiole) (Cs);
  • N1O1 oxazole (Cs), isoxazole (Cs), isoxazine (Ce);
  • N2O1 oxadiazole (furazan) (Cs);
  • N3O1 oxatriazole (Cs);
  • N1S1 thiazole (C5), isothiazole (Cs);
  • N2 imidazole (1,3-diazole) (Cs), pyrazole (1,2-diazole) (Cs), pyridazine (1,2-diazine) (Ce), pyrimidine (1,3-diazine) (Ce) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (Ce); and N3: triazole (Cs), triazine (Ce).
  • Ci-4 alkyl refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
  • alkyl includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
  • saturated alkyl groups include, but are not limited to, methyl (Ci), ethyl (C2), propyl (C3) and butyl (C4).
  • saturated linear alkyl groups include, but are not limited to, methyl (Ci), ethyl (C2), n-propyl (C3) and n-butyl (C4).
  • saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec -butyl (C4) and tert-butyl (C4).
  • C2-4 Alkenyl The term “C2-4 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
  • C2-4 alkynyl The term “C2-4 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.
  • C3-4 cycloalkyl refers to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
  • cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C3) and cyclobutane (C4); and unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3) and cyclobutene (C4).
  • a corresponding salt of the active compound/ agent for example, a pharmaceutically-acceptable salt.
  • a pharmaceutically-acceptable salt examples are discussed in Berge, etal., J. Pharm. Sci., 66, 1-19 (1977).
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Al +3 .
  • suitable organic cations include, but are not limited to, ammonium ion (z.e. NH4 + ) and substituted ammonium ions (e.g. NH3R + , NH2R2 + , NHR3 + , NR4 + ).
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH3)4 + .
  • a salt may be formed with a suitable anion.
  • a suitable inorganic compound e.g. -NH2 may be -NH3 +
  • a salt may be formed with a suitable anion.
  • Ill anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric, nitrous, phosphoric, and phosphorous.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulphonic, cinnamic, citric, edetic, ethanedisulphonic, ethanesulphonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxy maleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulphonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulphonic, propionic, pyruvic, salicylic, stearic, succinic, sulphanilic, tartaric, toluenesulphonic, trifluoroacetic acid and valeric.
  • solvate is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a monohydrate, a di-hydrate, a tri-hydrate, etc.
  • Certain compounds/ agents of the disclosure may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z- forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and P-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).
  • a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.
  • Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
  • the inhibitor of PARP1 ((Poly(ADP-Ribose) Polymerase 1) is AZD5305.
  • the language “inhibit,” “inhibition” or “inhibiting” includes a decrease in the baseline activity of a biological activity or process.
  • AZD5305" refers to a compound with the chemical name of 5-[4-[(7-ethyl- 6-oxo-5H-l,5-naphthyridin-3-yl)methyl]piperazin-l-yl]-N-methyl-pyridine-2-carboxamide and structure shown below:
  • the PARP1 inhibitor is a free base of AZD5305. In some embodiments, the PARP1 inhibitor is a pharmaceutically acceptable salt of AZD5305. In some embodiments, the PARP1 inhibitor is a crystalline AZD5305. In some embodiments, the PARP1 inhibitor is a crystalline Form A AZD5305.
  • the PARP1 inhibitors also refer to pharmaceutically acceptable salts thereof.
  • the ADC and/or the PARP1 inhibitor of the disclosure can be administered to the subject as a pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition comprising: (i) the ADC of the disclosure; (ii) the PARP1 inhibitor of the disclosure; and (iii) a pharmaceutically acceptable excipient.
  • composition refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.
  • Such composition can be sterile, and can comprise a pharmaceutically acceptable carrier, such as physiological saline.
  • Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), a stabilising agent e.g., human albumin), a preservative (e.g., benzyl alcohol), and absorption promoter to enhance bioavailability, and/or other conventional solubilising or dispersing agents.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognised pharmacopeia for use in animals, and more particularly in humans.
  • compositions according to the present disclosure may comprise, in addition to the active ingredient (the ADC and the PARP1 inhibitor of the disclosure), a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.
  • the anti-FRa antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38;
  • the PARP1 inhibitor is AZD5305 having the formula: or a pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition of the disclosure can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
  • a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
  • excipients examples include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as any combination thereof.
  • isotonic agents such as sugars, polyalcohols, or sodium chloride in the composition.
  • a pharmaceutical composition of the disclosure may be comprised within one or more formulation selected from a capsule, a tablet, an aqueous suspension, a solution, a nasal aerosol, a lyophilised powder which can be reconstituted to make a suspension or solution before use, or a combination thereof.
  • the pharmaceutical composition comprises more than one type of ADC and/or PARP1 inhibitor of the disclosure.
  • a pharmaceutical composition may comprise a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant e.g., polysorbate), optionally a stabiliser agent (e.g., human albumin), etc.
  • a buffer e.g., acetate, phosphate or citrate buffer
  • a surfactant e.g., polysorbate
  • a stabiliser agent e.g., human albumin
  • compositions disclosed herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating a disorder or one or more symptoms thereof, and/or in research.
  • the pharmaceutical compositions disclosed herein may be suitable for veterinary uses or pharmaceutical uses in humans.
  • compositions of the disclosure can be administered to a patient by any appropriate systemic or local route of administration.
  • administration may be oral, buccal, sublingual, ophthalmic, intranasal, intratracheal, pulmonary, topical, transdermal, urogenital, rectal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular or intratumoural.
  • compositions of the disclosure can be formulated for administration by any appropriate means, for example by epidermal or transdermal patches, ointments, lotions, creams, or gels; by nebulisers, vaporisers, or inhalers; by injection or infusion; or in the form of capsules, tablets, liquid solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders.
  • the most suitable route for administration in any given case will depend on the physical and mental condition of the subject, the nature and severity of the disease, and the desired properties of the formulation.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • a capsule may comprise a solid carrier such a gelatin.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • the ADC of the disclosure and the PARP1 inhibitor of the disclosure are formulated in two separate pharmaceutical compositions. Accordingly, in some embodiments, the ADC of the disclosure and the PARP1 inhibitor of the disclosure are administered separately or sequentially.
  • kits comprising an ADC of the disclosure and a PARP1 inhibitor of the disclosure. There is further embraced use of said kit in the methods of the present disclosure.
  • a therapeutic combination for treating cancer in a human subject in need thereof wherein the therapeutic combination comprises the ADC of the disclosure and the PARP1 inhibitor of the disclosure.
  • an article of manufacture comprising:
  • an article of manufacture comprising:
  • a method for manufacturing a medicament comprising:
  • the ADC can have any antibody or antigen binding fragment thereof as described herein. In some embodiments of any aspects of the disclosure, the ADC can have any linker as described herein. In some embodiments of any aspects of the disclosure, the ADC can have any cytotoxic agent as described herein.
  • the anti-FRa antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38;
  • the PARP1 inhibitor is AZD5305 having the formula: or a pharmaceutically acceptable salt thereof.
  • the kit comprises an isolated (e.g., purified) ADC as described herein. In some embodiments, the kit comprises one or more containers. In some embodiments, the kit comprises all of the components necessary and/or sufficient to administer the combination of ADC and PARP1 inhibitor to a subject. In some embodiments, the kit comprises all of the instructions necessary to administer the combination of ADC and PARP1 inhibitor to a subject.
  • the kit comprises one or more containers filled with one or more of the ADCs and/or PARP1 inhibitors of the disclosure.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects 118
  • SUBSTITUTE SHEET (RULE 26) approval by the agency of manufacture, use or sale for human administration.
  • instructions on how to employ the provided pharmaceutical composition in the treatment of cancer may also be included or be made available to a patient or a medical service provider.
  • cancer such as ovarian cancer, lung cancer (e.g. lung adenocarcinoma or NSCLC), endometrial cancer, breast cancer (e.g. TNBC), cervical cancer, pancreatic cancer, gastric cancer, renal cell carcinoma, colorectal cancer, head and neck squamous cell carcinomas (HNSCC) and malignant pleural mesothelioma
  • HNSCC head and neck squamous cell carcinomas
  • malignant pleural mesothelioma may also be included or be made available to a patient or a medical service provider.
  • the kit may provide the antigen or antigen-binding fragment and a cytotoxin that is not conjugated to the antibody or antigen-binding fragment, but is in a form suitable for conjugation thereto; optionally wherein the kit is further provided with instructions and/or reagents for conjugating the cytotoxin to the antibody or antigen-binding fragment.
  • the kit comprises all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.
  • the present disclosure encompasses therapies which involve administering an anti-FRa antibody-drug conjugate (ADC) of the disclosure in combination with a PARP1 inhibitor, pharmaceutical compositions comprising an anti-FRa ADC of the disclosure and a PARP1 inhibitor, or therapeutic combinations comprising an anti-FRa ADC of the disclosure and a PARP1 inhibitor to a subject, for preventing, treating, or ameliorating symptoms associated with a disease, disorder, or infection.
  • ADC anti-FRa antibody-drug conjugate
  • a subject is successfully “treated” for a disease or disorder (preferably cancer), according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder (preferably cancer).
  • a disease or disorder preferably cancer
  • a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the disclosure.
  • the terms “subject”, “individual” and “patient” are used interchangeably herein to refer to a mammalian subject.
  • the “subject” is a human, domestic animals, farm animals, sports animals, and zoo animals, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc.
  • the subject is a cynomolgus monkey (Macacafascicularis').
  • the subject is a human.
  • the subject may not have been previously diagnosed as having cancer.
  • the subject may have been previously diagnosed as having cancer.
  • the subject may also be one who exhibits disease risk factors, or one who is asymptomatic for cancer.
  • the subject may also be one who is suffering from or is at risk of developing cancer.
  • the subject has been previously administered a cancer therapy.
  • a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of an anti-FRa ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure; a pharmaceutical composition of the disclosure; or a therapeutic combination of the disclosure.
  • an anti-FRa ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure; a pharmaceutical composition of the disclosure; or a therapeutic combination of the disclosure, for use in therapy, for example for treating a disease or disorder (e.g. cancer).
  • a disease or disorder e.g. cancer
  • an anti-FRa ADC of the disclosure for use in therapy, for example for treating a disease or disorder (e.g. cancer), wherein the treatment comprises the administration of the anti-FRa ADC and a PARP1 inhibitor of the disclosure to a subject.
  • a disease or disorder e.g. cancer
  • a PARP1 inhibitor of the disclosure for use in therapy, for example for treating a disease or disorder (e.g. cancer), wherein the treatment comprises the administration of an anti-FRa ADC of the disclosure and the PARP1 inhibitor to a subject.
  • a disease or disorder e.g. cancer
  • a method for preventing the onset of a disease or disorder comprising administering to a subject a therapeutically effective amount of an anti- FRa ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure; a pharmaceutical composition of the disclosure; or a therapeutic combination of the disclosure.
  • an anti-FRa ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure; a pharmaceutical composition of the disclosure; or a therapeutic combination of the disclosure, for use in a method for preventing the onset of a disease or disorder (e.g. cancer).
  • a disease or disorder e.g. cancer
  • an anti-FRa ADC of the disclosure for use in a method for preventing the onset of a disease or disorder (e.g. cancer), wherein the method comprises the administration of the anti-FRa ADC and a PARP1 inhibitor of the disclosure to a subject.
  • a PARP1 inhibitor of the disclosure for use in a method for preventing the onset of a disease or disorder (e.g. cancer), wherein the method comprises the administration of an anti-FRa ADC of the disclosure and the PARP1 inhibitor to a subject.
  • terapéuticaally effective amount is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.
  • a method for treating a cancer comprising administering an anti-FRa ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure; a pharmaceutical composition of the disclosure; or a therapeutic combination of the disclosure to a subject.
  • an anti-FRa ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure; a pharmaceutical composition of the disclosure; or a therapeutic combination of the disclosure, for use in treating a cancer.
  • an anti-FRa ADC of the disclosure for use in treating a cancer, wherein the treatment comprises the administration of the anti-FRa ADC and a PARP1 inhibitor of the disclosure to a subject.
  • a PARP1 inhibitor of the disclosure for use in treating a cancer, wherein the treatment comprises the administration of an anti-FRa ADC of the disclosure and the PARP1 inhibitor to a subject.
  • the subject is a human subject in need thereof.
  • the cancer is associated with FRa expression.
  • a cancer referred to herein may comprise a cancerous cell that expresses FRa. Said cancerous cell may be comprised within a tumour.
  • the cancer is a tumour or other mass of malignant cells comprising a cancer cell which expresses FRa.
  • the cancer comprises cancer cells with heterogeneous expression of FRa and/or a low expression of FRa.
  • the FRa molecule is expressed in the cancer cell at a level similar to the level of expression in a non-cancer cell.
  • the FRa molecule is expressed in the cancer cell at a level lower than the level of expression in a non-cancer cell.
  • the cancer further comprises a cancer cell that does not express FRa.
  • the cancer is selected from ovarian cancer, lung cancer (e.g. lung adenocarcinoma), endometrial cancer, breast cancer (e.g. TNBC), cervical cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinomas (HNSCC) and malignant pleural mesothelioma.
  • lung cancer e.g. lung adenocarcinoma
  • endometrial cancer e.g. TNBC
  • breast cancer e.g. TNBC
  • cervical cancer pancreatic cancer
  • gastric cancer e.g., renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinomas (HNSCC) and malignant pleural mesothelioma
  • RNC renal cell carcinoma
  • HNSCC head and neck squamous cell carcinomas
  • malignant pleural mesothelioma pleural mesothelioma
  • cancers include, but are not limited to, benign, pre -malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. ovarian carcinoma, lung cancer, nonsmall cell lung cancer (squamous cell carcinoma or adenocarcinoma), endometrial cancer, pancreatic cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinomas, malignant pleural mesothelioma, breast carcinoma (e.g. TNBC), and kidney cancer. Any type of cell may be treated, including but not limited to, lung, gastrointestinal, breast (mammary), ovarian, kidney (renal) and pancreas.
  • neoplasms and tumours e.g., histocytoma, glioma, astrocyoma, osteoma
  • cancers
  • the cancer is homologous recombination deficient (HRD) cancer.
  • the cancer comprises one or more cells having a mutation in an HRD gene selected from BRCA E BRCA2. ATM, BRI PE BARD1, CDK12. CH EKE CHEK2. EANCL. PALB2, PPP2R2A, RAD51B, RAD51 C, RAD51D, and RAD54L. Mutation in an HRD gene may also be termed a mutation in a gene involved with homologous recombination repair (HRRm+).
  • the mutated HRD gene is selected from BRCA E BRCA2. and ATM.
  • the mutated HRD gene is BRCA1.
  • the mutated HRD gene is BRCA2.
  • the mutated HRD gene is ATM.
  • a method for depleting a population of FRa-positive cells in a subject comprising administering a therapeutically effective amount of an anti-FRa ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure; a pharmaceutical composition of the disclosure; or a therapeutic combination of the disclosure to a subject.
  • an anti-FRa ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure; a pharmaceutical composition of the disclosure; or a therapeutic combination of the disclosure for use in a method of depleting a population of FRa- positive cells in a subject.
  • an anti-FRa ADC of the disclosure for use in a method for depleting a population of FRa-positive cells in a subject, wherein the method comprises the administration of the anti-FRa ADC and a PARP1 inhibitor of the disclosure to a subject.
  • a PARP1 inhibitor of the disclosure for use in a method for depleting a population of FRa-positive cells in a subject, wherein the method comprises the administration of an anti-FRa ADC of the disclosure and the PARP1 inhibitor to a subject.
  • the FRa-positive cells have heterogeneous expression of FRa and/or a low expression of FRa.
  • the ADC and the PARP1 inhibitor are administered separately or sequentially. In some embodiments of any of aspect of the disclosure, the ADC and the PARP1 inhibitor are administered together.
  • the anti-FRa antibody or antigen-binding fragment thereof of the ADC comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN), a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), optionally wherein the anti-FRa antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38;
  • the PARP1 inhibitor is AZD5305 having the formula: or a pharmaceutically acceptable salt thereof.
  • the combination of the anti-FRa ADC and the PARP1 inhibitor has a synergistic effect, e.g. in treating cancer.
  • a hybridoma campaign was undertaken using a humanised transgenic mouse to obtain high affinity, fully human antibodies binding to folate receptor alpha (FRa).
  • FRa folate receptor alpha
  • the human FRa gene was inserted into the pDEST12.2 OriP vector.
  • the sequence corresponding to the soluble region of the folate receptor was further cloned with an N-terminal CD33 leader and C-terminal Avi and His6 tags.
  • the sequence responsible for GPI anchoring was removed, resulting in a soluble construct.
  • the human FRa protein was expressed and purified using standard methods. Briefly, plasmid DNA was prepared and transfected into an in-house suspension-adapted CHO cell line using PEI-mediated delivery with cells at a density of 4 x 10 6 cells/ml at the point of transfection. Cells were cultured at 34°C, 5% CO2, 140 rpm, 70% humidity for 7 days. Conditioned medium was harvested and purified using a 5ml HisTrap excel column (Cytiva) for affinity capture, followed by polishing on a HiLoad Superdex 75 16/600 pg column (Cytiva) equilibrated in DPBS.
  • Fractions were analysed by SDS-PAGE for purity, were pooled, and had their concentration determined via UV absorbance, and were snap frozen in liquid nitrogen prior to storage at -80°C.
  • the protein was incubated with recombinant BirA enzyme, ATP and biotin and subsequently purified on size exclusion chromatography as descried above.
  • Antibodies were expressed as described for the target antigens and purified using Protein A chromatography.
  • RIMMS repetitive immunisation multiple site
  • Tail vein bleeds were obtained from mice before immunisation, on day 13 after the first immunisation, and on day 20 after second immunisation.
  • the IgG titres to human FRa were determined by serum ELISA.
  • the serum IgG titres to human FRa and a negative protein control were determined by ELISA in 96-well microtitre plates using standard techniques. Antibodies were detected using an HRP labelled polyclonal goat anti-mouse IgG specific secondary antibody (Jackson Immunolabs), and the assay was developed using TMB substrate (Sigma) followed by the addition of 0.5 M sulphuric acid to stop the reaction. The plates were then read using a PerkinEhner EnVision 2103 multilabel plate reader.
  • the serum titration curves for human FRa and the negative protein control were plotted and the respective area under the curves (AUC) were calculated.
  • lymph nodes were aseptically harvested, and cells were isolated by mechanical disruption and then counted. These cells were mixed with SP2/0 myeloma cells and fused using an electrofusion apparatus. The resultant fusions were mixed with a methylcellulose-based semi-solid media and plated out into OmniTray plates. The cells in semi-solid media were cultured for 13 days at 37°C in a 5% CO2 incubator. During this incubation period, clonal colonies are formed from a single progenitor hybridoma cell. These colonies secrete IgG that is trapped in the vicinity of the colony by the FITC conjugated anti- IgG present in the semi-solid media.
  • the resultant immune complex formation can be observed around the cell as a fluorescent ‘halo’ when visualised by ClonePix FL colony picker (Molecular Devices). These haloed colonies are then picked into 96 well microtitre plates. After 3-5 days in culture, the supernatants of the picked colonies were harvested and screened for human FRa binding.
  • mRNA Messenger RNA
  • dT magnetic oligo
  • PCR amplification was performed using poly-C and constant region VH or VL primers specific to all mouse IgG subclasses. PCR amplicons were sequenced by Sanger sequencing.
  • mice IgGs of all subclasses were purified from overgrown cell culture supernatants on ProPlus resin (Phynexus) using Perkin Elmer Minitrack.
  • the captured mouse IgGs were eluted with 100 mM HEPES, 140 mM NaCl pH 3.0 and then neutralised with an equal volume of 200 mM HEPES pH 8.0.
  • the purified IgGs were quantified using an absorbance reading at 280 nm in UV-Star 384 well plate.
  • Mouse hybridoma IgG clones were molecularly reformatted to generate constructs expressing mouse VH and VL domains and the relevant mouse IgG constant domains for each hybridoma essentially as described by Persic et al., Gene 187:9-18, 1997.
  • the VH domain was cloned into the relevant vector containing the mouse heavy chain constant domains and regulatory elements to express whole IgGl heavy chain in mammalian cells.
  • the VL domain was cloned into a vector for the expression of the appropriate mouse light chain (lambda or kappa) constant domains and regulatory elements to express whole IgG light chain in mammalian cells.
  • IgGs mammalian suspension CHO cells were transiently transfected with the heavy and light chain IgG vectors. IgGs were expressed and secreted into the medium. IgGs were purified from clarified supernatants using MabSelect SuRe chromatography columns (GE Healthcare Lifesciences Cat no: 11003493 for 1ml columns; 11003495 for 5ml columns) and the AktaXpressTM purification system from GE Healthcare Lifesciences. The eluted material was buffer exchanged into PBS using PD-10 desalting columns (GE Healthcare Lifesciences; Cat no: 17085101).
  • the concentration of IgG was determined spectrophotometric ally using extinction coefficients based on the amino acid sequences of the IgGs (Pace et al., Protein Sci. 4:2411-2423, 1995), and the purified IgGs were analysed for purity using SDS-PAGE and HP-SEC analysis.
  • a number of approaches are available for mAb discovery, including phage display, immunisation and the use of a binding profile (which would involve identifying mAbs that compete with folate binding to FRa).
  • a dual approach was used to generate anti-FRa antibodies through immunisation.
  • the first route involved immunisation of a human transgenic mouse (z.e. Del-1) which contains full human VH and Vk domains in the Ig locus. This ensured that a high diversity of mAbs would result and would additionally be fully human and ready for development without requiring humanisation.
  • the second route involved immunisation of non- transgenic mice (z.e. CD-I), in which humanisation of the mAbs would be required.
  • the generated antibodies were characterised for strength of binding to target, ortholog and paralog specificity.
  • Table 9 indicates the species the protein is from, the vector into which the construct was cloned, the signal peptide and epitope tags fused to the protein.
  • Table 10 further shows the sequence for each insert. In each case, the sequence corresponding to the soluble region of each folate receptor was cloned with an N-terminal CD33 leader and C-terminal Avi and His6 tags. The sequence responsible for GPI anchoring was removed, resulting in a soluble construct.
  • HTRF assays were performed in 384-well white shallow well non-binding plates (Corning, 4513) in assay buffer containing phosphate-buffered saline (PBS) (Life Technologies, 14190), 0.1% v/v bovine serum albumin (BSA) (Sigma, A9576), and 0.4 M potassium fluoride (VWR International, 26820).
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • VWR International 26820
  • Time-resolved fluorescence at 590 and 665 nm was measured following excitation at 320 nm on an Envision (PerkinElmer) plate reader after the indicated incubation periods. Ratio values of (665 nm emission/590 nm emission) x 10,000 were used to calculate % Delta F according to the following equation:
  • the negative control ratio was derived from nonspecific binding (NSB) control wells. Curves were analysed using GraphPad Prism software using a four-parameter logistic curvefitting equation.
  • Species binding assays were performed on the Octet RED384 (ForteBio) at 25 °C in assay buffer containing PBS, 0.1% v/v BSA (Sigma, A9576), 0.01% v/v Tween-20 (Sigma, P9416) (pH 7.4) using tilted bottom black 384-well plates (ForteBio, 18-5076). Assays were set up using either protein A or anti human capture biosensors (AHC) (ForteBio, 18-5089) according to the manufacturer’s instructions.
  • AHC anti human capture biosensors
  • the 2586 hybridoma supernatants were tested for binding to human FRa, cyno FRa, mouse FRa, human FRP or human FRy in a HTRF assay format. This experiment was performed to isolate antibodies that were cross reactive to cyno FRa, while ensuring no binding was observed to the paralogs: FRP and FRy.
  • sequence similarity of human folate receptor to paralogs and orthologs multiple sequence alignments were determined using the Clustal Omega vl .2.2 algorithm. Sequence identities for human FRa to paralogs (Table 11) and orthologs (Table 12) are presented below.
  • Table 12 Percentage sequence identity of mature human, cyno, mouse and rat FRa
  • a binder was defined as an IgG that had an assay signal of >30% delta F.
  • a total of 129 IgG showed binding only to human and cyno FRa, 9 IgG showed binding only to human, cyno and mouse FRa, and a further 30 IgG showed binding only to human, cyno or mouse FRa.
  • the data for the 6 lead IgG is shown in Table 13.
  • the 168 hybridoma IgG that were identified in the hybridoma supernatant screen were made as Phynexus purified IgG. These were tested in the antigen binding assay at 4 dilutions of the IgG.
  • 115 IgG were identified as IgG that are human and cyno FRa cross reactive and do not bind human FRP and FRy.
  • the data for the 6 lead IgG is shown in Figures 1A-1F.
  • the 6 lead IgGs also showed binding to human FRa but no binding to rat FRa.
  • the generated antibodies were characterised for their rate of internalisation.
  • PhyNexus purified IgG samples from the Hybridoma outputs were evaluated in an internalisation assay. Frozen stocks of KB cells were plated overnight before the addition of IgG samples. The assay was run using a fixed acquisition method with 2.5-hour incubation before being fixed. The assay was run with pHrodo Green-labelled detection reagents and Cell Mask Red (Thermo Fisher Scientific).
  • Frozen vials of KB cells were thawed, diluted in medium, centrifuged and resuspended in fresh medium prior to counting. Cells were plated at 10,000 cells/well in MEM + NEAA + 10% FCS (Thermo Fisher Scientific) and prepared plates were incubated overnight at 37°C, 5% CO2 in a humidified incubator.
  • Antibodies and secondary detection reagents were prepared and pre-incubated for 30 min at room temperature. Medium was removed from cells and the premixed antibody and detection reagent was added. Plates were incubated at 37°C for 2.5 hours. Following incubation a 30pl/well 7.2% formaldehyde (Thermo Fisher Scientific) + Hoechst (Thermo Fisher Scientific) diluted 1:5000 solution was added to yield 3.7% formaldehyde fix and a 1:10000 dilution of nuclear stain.
  • a high-throughput internalisation assay was run to assay the rate of antibody internalisation into KB cells.
  • Phynexus purified antibodies were labelled, added to KB cells and the cells fixed after a 2.5-hour time course.
  • Image analysis was performed using Columbus and outputs were analysed within High Content Profiler in Spotfire, allowing multiparametric analysis of the acquired image data and the development of a ‘Hit List’ containing mAbs exhibiting an enhanced rate of uptake (Figure 2). All 6 lead antibodies demonstrated internalisation above the cut-off determined by the positive control samples.
  • the aim of this experiment was to epitope bin the generated anti-FRa antibodies, in particular the 6 exemplary antibodies.
  • HTRF time resolved fluorescence
  • Comparator Antibody 1 and Comparator Antibody 2 assays 0.5 nM biotinylated human FRaand 0.5 nM DyLight 650 labelled Comparator Antibody 1 or Comparator Antibody 2 was used.
  • Comparator Antibody 3 assay 1.25 nM biotinylated human FRaand 1.25 nM DyLight 650 labelled Comparator Antibody 3 was used. Assay plates were incubated for 3-4 h at room temperature prior to fluorescence measurement.
  • the 115 human/cyno folate receptor-specific hybridoma IgG were profiled in the three HTRF epitope competition assays to determine epitope diversity. Eight IgG inhibited in all assays, 41 in the Comparator Antibody 1 and Comparator Antibody 2 assays, 39 in the Comparator Antibody 2 and Comparator Antibody 3 assays, 12 in the Comparator Antibody 1 and Comparator Antibody 3 assays, 4 in the Comparator Antibody 1 assay only, 6 in the Comparator Antibody 3 assay only, and 5 IgG did not inhibit in any assays.
  • the 6 exemplary antibodies were reformatted, expressed and purified as human IgGl and profiled in the HTRF epitope competition assays to confirm potency.
  • all the lead exemplary antibodies could inhibit in the Comparator Antibody 2 competition assay.
  • AB 1370117 and AB 1370035 inhibited in all 3 epitope competition assays.
  • AB 1370026, AB 1370083 and AB 1370095 inhibited in only the Comparator Antibody 1 and Comparator Antibody 2 epitope competition assays.
  • AB 1370049 inhibited in only the Comparator Antibody 2 and Comparator Antibody 3 competition assays (see Table 15 and Figure 3).
  • Table 15 Summary of IC50 profiling in the HTRF epitope competition assays
  • the 6 antibodies were profiled by epitope competition assays. A range of behaviour and epitope diversity was observed, with some molecules competing with all three comparator antibodies, and others competing with only a subset.
  • AB 1370049 showed no competition with Comparator Antibody 1, as well as demonstrating the weakest competition with Comparator Antibody 2, while competing with Comparator Antibody 3.
  • Antibody affinity to the human and cyno FRa proteins was measured with a Biacore T200 surface plasmon resonance system (Cytiva) at 25°C.
  • Protein A was covalently immobilised to a CM5 chip surface using standard amine coupling techniques at a concentration of 50 pg/ml in 10 mM Sodium acetate, pH 4.0.
  • the antibody was captured onto the Protein A surface in HBS-EP+ buffer, pH 7.4 at 10 pl/min to enable FRa ECD binding.
  • the FRa ECD was serially diluted (0.4 nM-100 nM human FRa ECD; 0.8 nM-200 nM cyno FRa ECD; 30 nM-4000 nM mouse FRa ECD and rat FRa ECD) in HBS-EP+ buffer, pH 7.4 and flowed over the chip at 50 pl/min, with 2 minutes association and 8 minutes dissociation.
  • the chip surface was fully regenerated with pulses of 3 M MgCh to remove captured antibody together with any bound FRa ECD.
  • Multiple buffer-only injections were made under the same conditions to allow for double reference subtraction of the final sensorgram sets, which were analysed using Biacore T200 Evaluation Software to derive equilibrium dissociation constants.
  • Antibody affinity of the panel of antibodies to human and cyno FRa proteins was measured by SPR. All antibodies bound both human and cyno proteins.
  • the affinity to human FRa was in the range of 1-16 nM, and to cyno FRa 1-37 nM.
  • Kinetic binding parameters of the 6 exemplary antibodies are summarised in Table 16 below.
  • the 6 antibodies all had dissociation constants in the low nM range and in all cases the affinity for binding to cyno FRa was within 2.5-fold of that for binding to human FRa. Thus, all 6 exemplary antibodies appear suitable from an affinity perspective for further development as a therapeutic.
  • Generating a therapeutic antibody that is developable and aligned to manufacturing requirements requires a thorough assessment of an antibody’s physicochemical properties.
  • a panel of the generated antibodies were assessed for their expression titre, stability, and tendency towards reversible self-association.
  • Hydrophobic interaction chromatography (HIC) HPLC UHPLC-HIC analysis was performed on a Shimadzu Prominence system using a Sepax Proteomix HIC Butyl-NP5 5 pm non-porous 4.6 x 35 mm column eluting with a gradient of 1.5 M to 0 M ammonium sulphate and 0 to 20% acetonitrile in 25 mM sodium phosphate pH 7.40 on a ⁇ 1 mg/ml sample of each lead mAh as a 1:1 dilution with 1.5 M ammonium sulphate in 25 mM sodium phosphate pH 7.40 buffer. The more hydrophobic the species the later it is eluted and hence a higher retention time.
  • AC-SINS Affinity-capture self-interaction nanoparticle spectroscopy
  • Antibodies for analysis were prepared at a final concentration of 45 pg/ml in a solution containing 12 pl capture nanoparticles and 20 mM histidine, 120 mM sucrose, 80 mM arginine, pH 6 (HSA) buffer to a total volume of 120 pl in a 96-well plate. Samples were incubated at room temperature for 20 minutes and then duplicate 50 pl aliquots were transferred to a 384-well polystyrene plate (Nunc 384-well transparent polystyrene plate, Thermo Scientific). Sample absorbance was measured in a plate reader and the redshift of the wavelength was determined for each sample as compared to that for buffer only control wells. A shift of >5 nm is flagged as at risk of self- association.
  • Antibodies were assayed for non-specific binding to baculovirus particles by ELISA as described by Hotzel et al (Hotzel et al 2012 mAbs 4:6, 753-760). Preparations of each antibody were made at either 100 nM or 10 nM in PBS (Gibco 14190-086) + 0.5% BSA (Sigma A9576) and used in duplicates in the ELISA assay on 96-well Nunc Maxisorp F plates coated overnight at 4°C with 50 pl/well of either 1% Baculovirus extract in 50mM sodium carbonate (BV plate) or with 50mM sodium carbonate (blank plate).
  • the HRP substrate - TMB (SureBlue Reserve, KPL 53-00-03) was then added at 50 pl/well and following the colour change, the reaction was stopped by adding 50 pl/well of 0.5M sulphuric acid. Absorbance was measured at 450nm. A BV score is calculated by averaging the 450 nm absorbance at 10 nM and 100 nM concentration for each antibody sample, then dividing by the secondary only control sample. A BV score > 5 may indicate a risk of increased clearance due to non-specific binding.
  • IgG were diluted to 1 mg/ml in PBS then incubated at 4°C or 45°C for 2 weeks then filtered using filter spin columns (Millipore, UFC30HVNB).
  • High performance- size exclusion chromatography HP-SEC was performed by loading 70 pl of IgG onto a TSKgel G3000SWXL; 5 pm, 7.8 mm x 300 mm column using a flow rate of 1 ml/min and 0.1 M sodium phosphate dibasic anhydrous and 0.1 M sodium sulphate, pH 6.8 as the isocratic running buffer. Larger molecules are excluded from the pores of the size exclusion column to a greater extent than smaller molecules, and therefore elute earlier.
  • Peaks eluting earlier than the monomer peak are recorded as aggregates. Peaks eluting after the monomer peak (excluding the buffer-related peak) are recorded as fragments.
  • antibodies were profiled in the HTRF epitope competition assay for changes in potency.
  • HIC retention times correspond to increased hydrophobicity, which may indicate a risk of aggregation and increased clearance due to non-specific uptake.
  • a more hydrophobic mAb might result in a more hydrophobic ADC, which could result in aggregation during conjugation, instability as an ADC, and potentially more non-specific uptake into normal tissues and thus toxicity.
  • the panel of generated antibodies showed a retention time in the range of about 2.0-2.8 min by HPLC-HIC.
  • the 6 exemplary antibodies exhibited acceptable low retention times as compared to the panel of antibodies tested (see Table 17).
  • AC-SINS Affinity -capture self-interaction nanoparticle spectroscopy
  • Antibodies were also tested for their tendency to self-interact by Affinity Capture SelfInteraction Nanoparticle Spectroscopy (AC-SINS). This behaviour underlies, or is associated with, undesirable properties such as reversible self-association, aggregation, viscosity, opalescence, and phase separation.
  • AC-SINS By monitoring the wavelength of peak absorbance (plasmon wavelength) of gold particles coated with antibody, AC-SINS indirectly measures the propensity of a protein to self-interact in an environment mimicking high protein concentration.
  • the redshifts for the exemplary antibodies indicated negligible levels of self-interaction in HSA buffer for all test antibodies, whilst the negative and positive control antibodies behaved as expected (Figure 4).
  • the antibodies were assayed for the level of binding to baculovirus particles in an ELISA format. In all cases, the antibodies displayed negligible levels of non-specific binding all of which were below the cut-off assay threshold of 5. Advantageously, this suggests a low risk of poor clearance in vivo.
  • Plasma antibody concentrations were determined by the universal ELISA IgG assay.
  • AUCiast area under the plasma concentration versus time curve to the last measurable timepoint
  • the exemplary anti-FRa antibodies exhibited a range of pharmacokinetic properties in wild-type SCID mice, with AB 1370035 demonstrating the longest half-life in this model, closely followed by AB 1370049, which also gave the lowest clearance alongside AB 1370117.
  • Antibody concentrations were measured with an immuno-capture LC-MS/MS assay. Briefly, a polyclonal anti-human antibody was conjugated to magnetic beads. Then 25 pl of plasma sample was diluted in TBS and incubated together with the magnetic beads. After capturing, the magnetic beads were washed multiple times before digestion with trypsin under the presence of internal standards. The digestion was quenched with the addition of acid. An aliquot of the trypsin digested liquid content was then transferred to the injection plate for antibody analysis by LC-MS/MS.
  • the signature tryptic peptide on the human antibody Fc region (VVSVLTVLHQDWLNGK) was used to calculate the concentration of total antibody in the selected matrix.
  • the tryptic digest from the immuno -affinity enriched samples was separated using reversed phase chromatography (RPLC) followed with detection using multiple reaction monitoring (MRM) for the signature peptide.
  • the internal standard used in this experiment is the isotopically labeled peptide.
  • the peak area ratio of the analyte against the internal standards was used to calculate against the standard curve created by spiking the ADC reference material in the desired matrix.
  • the standard curves and QCs are prepared by spiking the target compound (the ADC reference material) at different levels into the same matrix as the sample matrix.
  • the quantification range covers 100 ng/ml- 12,000 ng/ml, with the dilution QC covering up to 50- fold dilution.
  • the standard curve was fitted with a liner regression, with weighting of l/x2.
  • AUCiast area under the plasma concentration versus time curve to the last measurable timepoint
  • AB 1370049 exhibited similar pharmacokinetic properties in hFcRn Tg32 and wild-type SCID mice.
  • the hFcRn Tg32 mouse is a transgenic mouse model, where the mouse FcRn has been knocked out and the human FcRn has been knocked in. As such, it represents a particularly appropriate model to predict the human pharmacokinetics of AB 1370049. Based on the hFcRn Tg32 mouse pharmacokinetics, the human clearance of AB 1370049 will likely be low and the half-life will be similar to other clinically used antibodies and is therefore expected to enable convenient dosing.
  • KB and Jeg-3 cells were stained with cell trace violet (Thermo scientific) and plated in a 96-well plate and incubated overnight. Monoclonal antibodies were incubated at RT with 50 nM of Fab-pHast human (ATS bio, Carlsbad) prior to addition to the plated cells. Cell images (20x) were taken and automatically analysed for fluorescence area every 30 min for up to 48 h in Celllnsight CX7 High-Content Screening Platform (Thermo Scientific).
  • the reduction mixture was heated at +37 °C for 2 hours (or until full reduction was observed by UHPLC) in an incubated orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, excess reducing agent was removed via spin filter centrifugation into PBS + 1 mM EDTA using a 15 ml Amicon Ultracell 50 kDa MWCO spin filter.
  • SG3932 was added as a DMSO solution (12-24 molar equivalent/antibody, 4.8-9.0 micromoles, in 2.0-2.3 ml DMSO) to 18-19 ml of this reduced antibody solution (37.0-46.9 mg, 247-313 nanomoles) at 2.0-2.5 mg/ml for a 10% (v/v) final DMSO concentration.
  • the lead antibodies were all amenable for DAR8 conjugation, showing high conjugation efficiency with DAR > 7.5, no loss of DAR during purification, no aggregation/fragmentation during manufacture and > 98% monomeric purity. Accordingly, the ADCs of the disclosure are able to specifically deliver a significantly higher concentration of cytotoxin payload to the target cancer cells via binding to FRa on the cancer cells.
  • the DAR8 ADCs are also homogeneous and provide the advantage of reproducibility and limited batch- to-batch variation during manufacture. This would allow the delivery of a higher number of less potent drugs (e.g. TOPOi) to the target cancer cell while maintaining tolerability.
  • the relatively low hydrophobicity of the generated ADCs could result in less non-specific uptake by normal tissues thus potentially leading to improved tolerability compared to comparator ADCs delivering more hydrophobic drugs, such as mirvetuximab soravtansine or IMGN151.
  • the exemplary lead ADCs were then further assessed for their thermal and serum stability, in vivo mouse PK, as well as in vitro and in vivo efficacy (see Examples below).
  • DAR4 ADCs for lead mAbs with SG3932 payload for in vitro and in vivo assessment of activity and in comparison with DAR8 ADCs for lead selection.
  • the reduction mixture was heated at +37 °C for 3 hours in an incubated orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, SG3932 was added as a DMSO solution (7 molar equivalent/antibody, 2.91- 4.12 micromoles, in 1.02-1.33 ml DMSO) to this reduced antibody solution (23-30 mg, 153- 200 nanomoles) for a 10% (v/v) final DMSO concentration.
  • the lead antibodies were all amenable for tuning TCEP equivalents for stochastic DAR4 conjugation, showing no DAR loss during purification, no aggregation/fragmentation during manufacture, > 99% monomeric purity and high yields.
  • the exemplary lead ADCs were then further assessed for their in vivo mouse PK, as well as in vivo efficacy (see Examples below).
  • ADCs (1.00 mg/ml, diluting as required with PBS) were spiked into the sera (mouse (ab7486, abeam), rat (C13SDZ, BioRad), IgG depleted human (DHP-2001, Access Biologicals), and cyno (custom order, BioIVT), 20x dilution of ADC in serum).
  • the resulting mixtures (1.8 ml) were aliquoted (200 pl) into 96 well plates (Sterile NuncTM). The plates were sealed with AeraSealTM film, covered with microplate lids (NuncTM), and incubated in a 5% CO2 incubator at 37°C. Sample aliquots were removed at various timepoints (0, 1, 3, and 7 days) and the samples stored at -80°C prior to analysis.
  • the resins were then washed (3 x 300 pl per sample) with PBS, the ADCs eluted by incubating the resins in IgG Elution Buffer (25 min, 50 pl per sample), and the solutions of eluted ADCs filtered using a MultiScreenHTS HV Filter Plate (0.45 pm, clear, non-sterile).
  • Samples were submitted for analysis on an LC-MS system consisting of a UltiMate 3000 HPLC stack fitted with a Variable Wavelength Detector (VWD), coupled to an Exactive Plus EMR Orbitrap Mass Spectrometer (Thermo Scientific). Samples (78 pl) were injected onto a MAbPac RP column (2.1 x 50 mm, 4 pm, Thermo Scientific, 088648) and separated on a segmented gradient (acetonitrile in water with 0.03% trifluoracetic acid, 5-25% over 30 s, then 25-60% over 2 min). The analytes were detected at 280 nm on the VWD, and on the mass spectrometer (MS) in EMR Full MS mode.
  • VWD Variable Wavelength Detector
  • MS Exactive Plus EMR Orbitrap Mass Spectrometer
  • MS tune file parameters Sheath gas flow rate: 35, Auxiliary gas flow rate: 10, Sweep gas flow rate: 0, Spray voltage: 3.5 kV, Capillary temperature: 300°C, S-lens RF level: 200, Auxiliary gas heater temperature: 300.
  • MS method parameters Polarity: positive, In-source CID: 20.0 eV, Microscans: 10, Resolution 17500, AGC target: 3e6, Maximum IT: auto, Scan range: 1000-4000 m/z.
  • the mass spectra were deconvoluted using Thermo BioPharma Finder. Peaks in the deconvoluted spectra deemed to be chemically significant had their percentage height values extracted, so that the DAR and the percentage of significant chemical modifications for each sample could be determined. Comparison of DAR values of a given ADC in a given serum over time allowed for the percentage deconjugation for each sample to be determined.
  • the peak area ratio of the analyte against the internal standards was used to calculate against the standard curve created by spiking the ADC reference material in the desired matrix.
  • two signature tryptic peptides on the AB1370049-SG3932 DAR8 antibody were used to calculate the concentration of total antibody in the selected matrix: heavy chain (VVSVLTVLHQDWLNGK) and light chain (DSTYSLSSTLTLSK).
  • the papain released warhead was used to calculate the concentration.
  • the isotopically labelled peptides or warhead were used as the internal standard.
  • Total ADC concentrations were measured with an immuno-capture LC-MS/MS assay. Briefly, a polyclonal anti-human antibody was conjugated to magnetic beads. Then 25 pl of plasma sample was diluted in TBS and incubated together with the magnetic beads. After capturing, the magnetic beads were washed multiple times before digested with trypsin. An aliquot of the trypsin digested supernatant was subjected to enzymatic digestion overnight with Papain under the presence of an internal standard. The digested samples were quenched with the addition of an acid before analysis by LC-MS/MS.
  • the papain released warhead was used to calculate the concentration of ADC.
  • the internal standard used in this experiment is the isotopically labeled warhead.
  • the papain digest from the immuno-affinity enriched samples was separated using reversed phase chromatography (RPLC) followed with detection using multiple reaction monitoring (MRM) for the released warhead.
  • RPLC reversed phase chromatography
  • MRM multiple reaction monitoring
  • the peak area ratio of the analyte against the internal standards was used to calculate against the standard curve created by spiking the ADC reference material in the desired matrix.
  • the standard curves and QCs were prepared by spiking the target compound (the ADC reference material) at different levels into the same matrix as the sample matrix.
  • the quantification range covers 100 ng/ml- 12,000 ng/ml, with the dilution QC covering up to 50-fold dilution.
  • the standard curve was fitted with a linear regression, with weighting of l/x2.
  • AUCiast area under the plasma concentration versus time curve to the last measurable timepoint
  • the exemplary anti-FRa ADCs exhibited a range of pharmacokinetic properties in wildtype SCID mice.
  • the SCID mouse pharmacokinetics of all exemplary antibodies exhibited a low plasma clearance and a long plasma half-life in SCID mice.
  • AB1370049-SG3932 DAR4 or AB1370049-SG3932 DAR8 concentrations were measured with an immuno -capture LC-MS/MS assay. Briefly, a polyclonal anti-human antibody was conjugated to magnetic beads. Then 25pl of plasma sample was diluted in TBS and incubated together with the magnetic beads. After capturing, the magnetic beads were washed multiple times before digested with trypsin. An aliquot of the trypsin digested supernatant was subjected to enzymatic digestion overnight with Papain under the presence of an internal standard. The digested samples were quenched with the addition of an acid before analysis by LC-MS/MS.
  • the papain released warhead was used to calculate the concentration of DAR4 or DAR8 ADC.
  • the internal standard used in this experiment is the isotopically labeled warhead.
  • the papain digest from the immuno-affinity enriched samples was separated using reversed phase chromatography (RPLC) followed with detection using multiple reaction monitoring (MRM) for the released warhead.
  • MRM multiple reaction monitoring
  • the peak area ratio of the analyte against the internal standards was used to calculate against the standard curve created by spiking the DAR4 or DAR8 ADC reference material in the desired matrix.
  • the standard curves and QCs are prepared by spiking DAR4 or DAR8 ADC (the ADC reference material) at different levels into the same matrix as the sample matrix.
  • the quantification range covers 100 ng/ml- 12,000 ng/ml, with the dilution QC covering up to 50- fold dilution.
  • the standard curve was fitted with a linear regression, with weighting of l/x2.
  • AUCiast area under the plasma concentration versus time curve to the last measurable timepoint
  • AB1370049-SG3932 DAR8 exhibited a slightly higher clearance and lower half-life in hFcRn Tg32 than in wild-type SCID mice.
  • the hFcRn Tg32 mouse is a transgenic mouse model, where the mouse FcRn has been knocked out and the human FcRn has been knocked in. As such, it represents an appropriate model to predict the human pharmacokinetics of AB1370049-SG3932 DAR8. Based on the hFcRn Tg32 mouse pharmacokinetics, the human clearance of AB 1370049-SG3932 DAR8 will likely be low and the half-life will be in common with other clinically used ADCs and should enable convenient dosing.
  • FRa expression Cancer cell lines with various levels of FRa expression were obtained from ATCC (American Tissue Culture collection). KB cells are a cervical cancer cell line with HeLa contaminant, while IGROV-1 is an ovarian cancer cell line and JEG-3 is a choriocarcinoma cell line. Cells were plated in duplicates onto 96-well plates. After an incubation for 24 h, cells were treated with a range of 0-66.66 nM (0-10 pg/ml) of Lead ADCs or the non-targeting ADC control NIP228 for 6 days. On day 6 the cells were then incubated for 10 min with CellTiter- Glo reagent, and luminescence was measured using a 96-well plate reader. Background luminescence was measured in medium without cells and subtracted from experimental values. The IC50 values were calculated on Graph Pad Prism.
  • AB1370049-SG3932 DAR8 cannot only induce apoptosis and eventually cell death in homogenously FRa-expressing tumours, but also in heterogeneously expressed tumours as shown in co-culture experiments in which 50% of cells have FRa expressed and 50% not.
  • ADC gets internalised in FRa-positive cells and free warhead is released and kills the targeted cancer cell.
  • the free warhead can also diffuse into neighbouring cancer cells that do not express FRa and can kill them as well.
  • EXAMPLE 18 In vivo anti-cancer activity of the exemplary ADCs in cell-derived xenograft models
  • mice were inoculated subcutaneously into female CB17-SCID mice (Charles River Laboratories). When tumours reached approximately 150-200 mm 3 , mice were randomly assigned into groups.
  • Each of the 6 lead DAR8 ADCs was administered intravenously as single dose (day 7) at 0.3125 mg/kg, 0.625 mg/kg, 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg and 10 mg/kg and DAR4 ADCs at 0.625 mg/kg, 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg and 20 mg/kg with corresponding dose levels of Isotype control NIP228.
  • mice For Caco-2 xenograft model, 5 x 10 6 cells/mouse in 50% Matrigel were inoculated subcutaneously into female athymic nude mice (Harlon Laboratories). When tumours reached approximately 150 mm 3 , mice were randomly assigned into groups.
  • AB1370049-SG3932 DAR8 was administered intravenously as single dose (day 17) at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, corresponding isotype control NIP228 at 1.25 mg/kg, 5 mg/kg and FRa-DM4 ADC (“biosimilar to mirvetuximab soravtansine”, with a DAR of about 3) at 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg, with corresponding isotype control NIP228 at 1.25 mg/kg and 5 mg/kg.
  • FRa-DM4 ADC biosimilar to mirvetuximab soravtansine
  • mice Severe Combined Immunodeficiency mice
  • Control animals received a 100 ul intravenous dose of vehicle, whilst treated animals received one intravenous dose of AB1370049-SG3932 DAR8 at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, dosed at 4 ml/kg, corresponding isotype control NIP228 at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg and FRa-DM4 ADC (“biosimilar to mirvetuximab soravtansine”, with a DAR of about 3) at 5 mg/kg, with corresponding isotype control NIP228 at 5 mg/kg.
  • AB1370049-SG3932 DAR8 at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, dosed at 4 ml/kg
  • corresponding isotype control NIP228 at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg and FRa-DM4 ADC (“biosimilar to mirvetuximab soravtansine”, with a DAR of about 3) at
  • AB1370049-SG3932 DAR8 was administered as single dose at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, and FRa-DM4 ADC (“biosimilar to mirvetuximab soravtansine”, with a DAR of about 3) at 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg.
  • the exemplary lead ADCs all demonstrated robust anti-tumour activity in a variety of different xenograft models, and at different doses.
  • the anti-tumour activity was similar or better than the comparator molecule, FRa-DM4.
  • tumours were selected for investigation. 6-8 week old female Athymic Nude-Foxnlnu stock mice were implanted subcutaneously with tumour tissue fragments. When sufficient stock animals reached 1000 - 1500 mm 3 , tumours were harvested and fragments were implanted into pre-study animals. When tumours reached an average volume of 150-300 mm 3 animals were randomised by tumour volume into 5 groups. Group 1 received no treatment, Group 2 and Group 4 received 5 mg/kg or 2.5 mg/kg of AB1370049-SG3932 DAR8 respectively. Group 3 and 5 received the isotype control antibody NIP228-SG3932 at 5 mg/kg or 2.5 mg/kg respectively. All dosed animals received one dose intravenously on Day 0.
  • tumour volume [length (mm) x width (mm) 2 x 0.52, where the length and width are the longest and shortest diameters of the tumour, respectively.
  • the study endpoint in all models was performed when the mean tumour volume of the control group reached 1200 mm 3 or if the maximum tumour volume wasn’t reached, a maximum of 60 days. If the 1200 mm 3 volume occurred before Day 28, treatment groups were measured up to Day 28. After randomisation of the animals onto study, 3 animals per model were used to collect tumour samples. Tumours were collected at approximately 400-600 mm 3 , with half the tumour being processed for immunohistochemistry analysis and the other half processed for genomic analysis.
  • tumour fragments were subcutaneously implanted using trocars into female NOD-SCID mice (Envigo). When tumours reached approximately 150-200 mm 3 , mice were randomly assigned into groups.
  • AB1370049-SG3932 DAR8 was administered intravenously as a single dose at 2.5 mg/kg, 5 mg/kg with corresponding dose levels of Isotype control NIP228.
  • the response at the end tumour volume (FTV) from initial tumour volume (ITV) was calculated at the time that resulted in the greatest decrease from the initial tumour volume.
  • the antitumour response was calculated for each tumour bearing mouse using the formula:
  • Tumour Growth (%) [(FTV-ITV)/ITV] x 100
  • Tumour Growth (%) was calculated for each animal in a study group and the group response was calculated as the median response of all treated mice.
  • the median percent tumour growth resulting from a single administration of 5.0 mg/kg AB1370049-SG3932 DAR8 or NIP228-SG3932 in 51 PDX models is summarised in Table 23 and Figure 16A.
  • Tumour growth inhibition was observed after a single dose of 5.0 mg/kg AB1370049-SG3932 DAR8, with 54% of models (29 of 54) exhibiting a reduction in tumour volume from baseline of 30% or greater.
  • a single dose of 5.0 mg/kg NIP228-SG3932 resulted in a reduction in tumour volume from baseline of 30% or greater in 22% (11 of 49) of the models in which it was tested.
  • 5.0 mg/kg AB1370049-SG3932 DAR8 resulted in a reduction in tumour volume from baseline of 30% or greater in 78.3% (18 of 23: 95% CI 58% - 91%) of the models tested.
  • 5.0 mg/kg of NIP228- SG3932 resulted in a reduction of tumour volume from baseline of 30% or greater in 43.5% (10 of 23: 95% CI 26% - 63%) of the models tested.
  • Table 23 The results of assessment of the in vivo anti-tumour response to AB 1370049-SG3932 DAR8 (5.0 mg/kg) in the 54 models (30 graduates models and 24 in-house models)
  • the median percent tumour growth resulting from a single administration of 2.5 mg/kg AB1370049-SG3932 DAR8 or NIP228-SG3932 in 39 PDX models is summarised in Table 24 and Figure 16B.
  • Specific tumour growth inhibition was observed after a single intravenous dose of 2.5 mg/kg AB1370049-SG3932 DAR8 in 49% of tested PDX models (19 out of 39) while 51% of models were not responding (20 out of 39).
  • Only two models out of 37 tested responded to the same dose of non-targeting ADC NIP228-SG3932 DAR8.
  • Activity of nontargeting ADC is dose-dependent as less models responded to NIP228-SG3932 DAR8 (5%) at the lower dose concentration.
  • Table 24 The results of assessment of the in vivo anti-tumour response to AB 1370049-SG3932
  • AB1370049-SG3932 DAR8 has demonstrated an anti-tumour response in a number of models across the 4 indications ovarian cancer, NSCLC, CRC and endometrial cancer.
  • Figures 17A-17C show the representative studies from the models CTG-0711 (ovarian cancer), CTG- 2367 (NSCLC) and CTG-2268 (endometrial).
  • AB 1370049-SG3932 DAR8 is highly efficacious in FRa-expressing tumours with high, medium and medium-low expression-levels. There is in general a positive correlation between antitumour- activity of AB 1370049-SG3932 DAR8 and FRa expression levels.
  • HSPC primary haematopoietic stem and progenitor cells
  • Cell Expand media capable of supporting erythroid cell differentiation (Preferred Cell Systems, SEC-BFU1-40H), myeloid cell differentiation (Preferred Cell Systems, SEC-GM1-40H), or megakaryocytic cell differentiation (Stem Cell Technologies, 09707), at a concentration of 5000 cells/ml for erythroid and myeloid cells, or 15000 cells/ml for megakaryocytic cells.
  • Cells (100 pl) were plated into triplicate white walled, clear bottomed 96 well tissue culture plates (Coming) with the addition of exemplary ADC (e.g., AB1370049-SG3932 DAR8) or NIP228 isotype control ADC (200, 66.66, 22.22, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03 and 0 pg/ml) and cultured for 5 days in a humidified incubator at 37°C, with 5% CO2.
  • exemplary ADC e.g., AB1370049-SG3932 DAR8
  • NIP228 isotype control ADC
  • exemplary ADC e.g., AB1370049-SG3932 DAR8
  • non-FRa-targeting NIP228 control ADC or unconjugated mAb (NIP228and AB 1370049 respectively)
  • concentrations 200, 66.66, 22.22, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03 and 0 pg/ml for 5 days in a humidified incubator at 37°C, with 5% CO2.
  • myeloid cell differentiation or megakaryocytic cell differentiation cells were respectively seeded at 10000 cells/ml and 15000 cells/ml for 5 days, prior to be spun down and seeded in fresh media respectively at 10000 cells/ml and 30000 cells/ml for an additional 5 days prior to be plated in myeloid differentiation media or megakaryocytic at 20000 cells/ml and 30000 cells/ml for 4 days in presence of AB1370049-SG3932 DAR8 (200, 66.66, 22.22, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03 and 0 pg/ml).
  • Viability was determined using CellTiter-Glo 2.0 from Promega (using an optimised volume of 10 pl/well), with luminescence detected using an Envision plate reader (Perkin Elmer). Relative Luminescence signal was normalised in GraphPad software (Prism) to percentage of control with controls equalling 100 and maximum cell death equalling 0.
  • HSPC haematopoietic stem and progenitor cells
  • AB1370049-SG3932 DAR8 exhibits similar toxicity levels to a non-FRa-targeting ADC molecule carrying the same payload ( Figures 18A-18F). This observation is seen in primary CD34 + bone marrow cells differentiated into any lineage, regardless of their level of differentiation. Similar results were observed with other exemplary ADCs of the disclosure, including mAbs AB 1370095, AB 1370026 and AB 1370117 when conjugated to the same TOPOi payload. When dosed in similar culture conditions as described for AB 1370049- SG3932 DAR8, the unconjugated mAb (AB 1370049) did not induce any significant decrease of ATP levels in HSPCs. In addition, HSPC treated by AB 1370049 conjugated with the same TOPOi payload at DAR4 showed comparable level of toxicity to HSPC treated with non-FRa- targeting ADC DAR4.
  • Plasma Pharmacokinetic (PK) analyses of AB1370049-SG3932 DAR8 in cynomolgus monkeys was carried out including peak and total exposure, clearance, and half-life. PK samples were collected from cynomolgus monkeys across various dose levels for the lead ADC candidate and unconjugated antibody. Non-compartmental analysis was performed to estimate PK parameters.
  • Total ADC and the total antibody concentrations were measured with an immunocapture LC-MS/MS assay. Briefly, a polyclonal anti-human antibody was conjugated to magnetic beads. Then 40 pl of plasma sample was diluted in TBS and incubated together with the magnetic beads. After capturing, the magnetic beads were washed multiple times before digestion with trypsin under the presence of internal standards. The digestion was quenched with the addition of acid. An aliquot of the trypsin digested liquid content was then transferred to the injection plate for the total Ab analysis.
  • ADC assay an aliquot of the trypsin digested supernatant was subjected to enzymatic digestion overnight with Papain under the presence of an internal standard. The digested samples were quenched with the addition of acid before analysis by LC-MS/MS. The papain released warhead was used to calculate the concentration. The internal standard used in this experiment is the isotopically labeled warhead.
  • the papain digest from the immuno-affinity enriched samples was separated using reversed phase chromatography (RPLC) followed with detection using multiple reaction monitoring (MRM) for the released warhead.
  • RPLC reversed phase chromatography
  • MRM multiple reaction monitoring
  • the signature tryptic peptide on the human antibody Fc region (VVSVLTVLHQDWLNGK) was used to calculate the concentration of total antibody in the selected matrix.
  • the tryptic digest from the immuno-affinity enriched samples was separated using reversed phase chromatography (RPLC) followed with detection using multiple reaction monitoring (MRM) for the signature peptide.
  • the internal standard used in this experiment is the isotopically labeled peptide.
  • the peak area ratio of the analyte against the internal standards was used to calculate against the standard curve created by spiking the ADC reference material in the desired matrix.
  • the standard curves and QCs were prepared by spiking the target compound (the ADC reference material) at different levels into the same matrix as the sample matrix.
  • the quantification range is from 100- 15000 ng/ml, with the dilution factor up to 100 fold.
  • the standard curve was fitted with a linear regression, with weighting of l/x2, the accuracy and precision of the assay is within 20% for all levels, except for at LLOQ (25%).
  • Unconjugated Warhead Assay The unconjugated warhead assay was performed by precipitation procedure.
  • the internal standard used in this experiment is the isotopically labeled warhead.
  • the sample was precipitated with a buffer containing high percentage of organic solvent with spiked in internal standard.
  • the supernatant was then dried under nitrogen before reconstitution to the appropriate buffer for injection to LCMS.
  • the sample was then separated using reversed phase chromatography (RPLC) followed with detection using multiple reaction monitoring (MRM) for the unconjugated warhead.
  • RPLC reversed phase chromatography
  • MRM multiple reaction monitoring
  • the standard curves and the QCs were prepared by spiking the unconjugated warhead at different levels into the same matrix as the sample matrix.
  • the quantification range of the assay is from 0.059 - 29.5 ng/ml.
  • the standard curve was fitted with a linear regression. The accuracy and precision of the assay is within 15% for all levels, except for at LLOQ (20%).
  • the exposure of the unconjugated antibody is higher in comparison to dose matched ADC with slower clearance and a longer half-life over the ADC as may be expected for the addition of eight cytotoxic warheads.
  • the PK of the ADC is well within acceptable criteria for an ADC in cynomolgus monkeys.
  • Table 25 Mean NCA PK parameters unconjugated mAb and conjugated mAb in Monkeys following a single administration
  • the DAR8 ADC (AB1370049-SG3932 DAR8) was taken forward into dose range finding study in monkeys where dose-dependent exposure was seen with consistently long halflife and slow clearance, as previously indicated in the mouse PK studies.
  • AB 1370049-SG3932 DAR8 demonstrated a longer half-life and slower clearance than the most advanced FRa ADCs in the art (Olga, et al. Cancer Res August 152020 (80) (16 Supplement) 2890; DOI: 10.1158/1538-7445. AM2020-2890; WO2020223221A1).
  • the toxicity profile of the ADC as described in the present disclosure could result in more tolerable therapy and potential clinical superiority.
  • PARP1 limits the cytotoxicity of TOPOi by enhancing the excision and repair of topoisomerase I cleavage complexes. Therefore, this example aims to assess the cytotoxic activity of the combination of AZD5335 (AB1370049-SG3932 DAR8) with a PARP1 inhibitor (AZD5305) in cancer cell lines expressing different levels of FRa.
  • KB cells ( ⁇ 37 million FRa / cell) are a cervical cancer cell line with HeLa contaminant, while IGROV-1 ( ⁇ 1.3 million FRa / cell), OVCAR-3 (-180,000 FRa / cell) and SKOV-3 (402,000 FRa / cell) cells are ovarian cancer cell lines. All cell lines were obtained from ATCC.
  • KB cells were grown in MEM medium (ThermoFisher) with 10% FBS (ThermoFisher) at 37°C with 5% CO2 in a humidified incubator.
  • IGROV-1 cells were grown in RPMI-1640 medium (ThermoFisher) with 10% FBS at 37°C with 5% CO2 in a humidified incubator.
  • OVCAR-3 cells were grown in RPMI-1640 medium with 20% FBS at 37°C with 5% CO2 in a humidified incubator.
  • SKOV-3 cells were grown in McCoy’s 5a medium with 10% FBS at 37 °C with 5% CO2 in a humidified incubator.
  • KB, IGROV-1, OVCAR-3 and SKOV-3 cells were seeded at a density of 350 cells/well in 20 pl of complete growth media in white polystyrene tissue-culture treated 384-well plates (Corning, Corning, NY, USA). After 24 hours, 4x stocks of AZD5335 and AZD5305 were prepared in complete growth medium (KB cells: MEM medium; IGROV-1 and OVCAR-3 cells: RPMI-1640 medium; SKOV-3 cells: McCoy’s 5a medium) containing 10% heat inactivated FBS (Thermo Fisher Scientific).
  • the cells were treated with 10 pl of a suboptimal dose of AZD5335 (3 nM to 0.5 pM for KB, 10 nM to 1.5 pM for IGROV-1, 50 nM to 32pM for OVCAR-3, and 700 nM to 0.1 nM for SKOV-3) or AZD5305 (10 pM to 123 nM for KB and IGROV-1, 2 nM to 0.02 nM for OVCAR-3, and 30 pM to 30 nM for SKOV-3) or a combination of AZD5335 and AZD5305.
  • the final volume was adjusted to 40 pl per well. Cells were then incubated in a humidified atmosphere with 5% CO2 at 37°C for 6 days.
  • AZD5335 was determined by generating half-maximal inhibitory concentration (IC50) values using a nonlinear regression model [log inhibitor vs. response] in GraphPad Prism, version 8.1 (GraphPad, San Diego, CA USA) and presented as percent cell viability relative to mock-treated control cells (media alone) using the formula [(Treated cells)/(Mock-treated control cells)] x 100.
  • the Bliss synergy model was used to estimate the two-drug combination effect and pharmacologic synergy (Bliss score > 0) or antagonistic (Bliss score ⁇ 0) response of AZD5335 in combination with AZD5305, determined using the Combenefit Software (Cancer Research UK Cambridge Institute) (Veroli et al. Bioinformatics. 2016 Sep 15; 32(18): 2866-2868).
  • HSA Highest Single Agent
  • Berenbaum 1989 was also used to estimate the two-drug combination effect and pharmacologic synergy. This model quantifies the higher of the two single compound effects at their corresponding concentrations. Combined effect is compared with the effect of each single agent at the concentration used in the combination. Excess over the highest single agent effect (i.e. a positive HSA score) indicates cooperativity. HSA does not require the compounds to affect the same target. Synergy scores were calculated using the software Genedata Screener®.

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Abstract

L'invention concerne des méthodes de traitement du cancer comprenant des polythérapies faisant intervenir (i) un ADC comprenant un anticorps anti-FRα, et (ii) un inhibiteur de PARP1, et des kits les comprenant.
PCT/EP2024/053805 2023-02-16 2024-02-15 Polythérapies pour le traitement du cancer avec des molécules de liaison thérapeutiques Ceased WO2024170660A1 (fr)

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