WO2023178451A1 - Anticorps anti-récepteur alpha du folate et procédés d'utilisation - Google Patents

Anticorps anti-récepteur alpha du folate et procédés d'utilisation Download PDF

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WO2023178451A1
WO2023178451A1 PCT/CA2023/050405 CA2023050405W WO2023178451A1 WO 2023178451 A1 WO2023178451 A1 WO 2023178451A1 CA 2023050405 W CA2023050405 W CA 2023050405W WO 2023178451 A1 WO2023178451 A1 WO 2023178451A1
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amino acid
seq
set forth
acid sequence
nos
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PCT/CA2023/050405
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English (en)
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James R. RICH
Rupert H. DAVIES
Stuart Daniel Barnscher
Dunja UROSEV
Sukhbir Singh Kang
Peter Wing Yiu Chan
Samir DAS
Andrea HERNANDEZ ROJAS
Robert William Gene
Ada G. H. YOUNG
Samuel Oliver LAWN
Danny Chui
Duncan Browman
Brandon Clavette
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Zymeworks Bc Inc.
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Publication of WO2023178451A1 publication Critical patent/WO2023178451A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A61K47/6877—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 being a hybrid immunoglobulin the antibody being an immunoglobulin containing regions, domains or residues from different species
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • 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
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    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
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    • 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
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    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/524—CH2 domain
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    • C07K2317/526—CH3 domain
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/77—Internalization into the cell
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Definitions

  • the present disclosure relates to the field of antibody therapeutics and, in particular, to antibodies targeting human folate receptor alpha (hFR ⁇ ).
  • FR ⁇ Folate receptor alpha
  • GPI glycosyl-phosphatidylinositol
  • FOLR1 glycosyl-phosphatidylinositol
  • FOLR2 glycosyl-phosphatidylinositol
  • FOLR3 FRy
  • F0LR4 FR6
  • FR ⁇ has been identified as a highly relevant cancer therapy target as it is overexpressed in a variety of cancers including ovarian cancer, triple- negative breast cancer (TNBC), endometrial cancer, mesothelioma and lung cancer, with minimal expression in non-malignant tissues.
  • TNBC triple- negative breast cancer
  • TNBC triple- negative breast cancer
  • mesothelioma mesothelioma
  • lung cancer with minimal expression in non-malignant tissues.
  • anti-FR ⁇ antibodies and methods of use are anti-FR ⁇ antibodies and methods of use.
  • One aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that specifically binds to human folate receptor alpha (hFR ⁇ ), wherein the antibody construct competes for binding to hFR ⁇ with a reference antibody that specifically binds to an epitope within hFR ⁇ comprising amino acid residues E120, D121, R123, T124, S125 and Y126 of SEQ ID NO: 15.
  • Another aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that specifically binds to an epitope within human folate receptor alpha (hFR ⁇ ) comprising amino acid residues El 20, D121, R123, T124, S125 and Y126 of SEQ ID NO: 15.
  • hFR ⁇ human folate receptor alpha
  • Another aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that specifically binds to human folate receptor alpha (hFR ⁇ ), the antigen- binding domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 3, 4 and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 6, 7 and 8.
  • hFR ⁇ human folate receptor alpha
  • Another aspect of the present disclosure relates to an antibody construct comprising two antigen-binding domains operably linked to an IgG Fc region, wherein each of the anti gen -binding domains specifically binds to human folate receptor alpha (hFR ⁇ ) and comprises:
  • Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding the anti-FR ⁇ antibody construct as described herein.
  • Another aspect of the present disclosure relates to an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding the anti-FR ⁇ antibody construct as described herein.
  • Another aspect of the present disclosure relates to a host cell comprising the expression vector or set of expression vectors.
  • Another aspect of the present disclosure relates to an antibody-drug conjugate comprising the anti-FR ⁇ antibody construct as described herein conjugated to one or more drug moi eties.
  • Another aspect of the present disclosure relates to an antibody-drug conjugate having general Formula I:
  • A is an anti-FR ⁇ antibody construct as described herein;
  • L is a linker
  • D is a drug moiety; m is between 1 and about 8, and n is 1 and about 12.
  • Another aspect of the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-FR ⁇ antibody construct as described herein or an antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier or diluent.
  • Another aspect of the present disclosure relates to an anti-FR ⁇ antibody construct as described herein or an antibody-drug conjugate as described herein for use in therapy, for example, in the treatment of cancer.
  • Another aspect of the present disclosure relates to a use of an anti-FR ⁇ antibody construct as described herein or an antibody-drug conjugate as described herein in the manufacture of a medicament for the treatment of cancer.
  • Another aspect of the present disclosure relates to a method of inhibiting the growth of FR ⁇ -positive tumor cells comprising contacting the cells with an anti-FR ⁇ antibody construct as described herein or an antibody-drug conjugate as described herein.
  • Another aspect of the present disclosure relates to a method of treating a subject having a cancer comprising administering to the subject an effective amount of an anti-FR ⁇ antibody construct as described herein or an antibody-drug conjugate as described herein.
  • Fig. 1A shows the sequence of the rabbit heavy chain variable domain CDRs of the chimeric antibody v23924 ported onto a human VH framework (IGHV3-23*01) (SEQ ID NO: 155)
  • Fig. IB shows the sequence of the rabbit light chain variable domain CDRs of chimeric antibody v23924 ported onto a human VL framework (IGKVI-39*01) (SEQ ID NO: 156).
  • the CDRs were assigned with the AbM definition and are marked in bold italic font.
  • Fig. 2A-D show the profiles of purified parental chimeric variant v23924 and purified representative humanized variant v30384 as analyzed by electrophoresis and UPLC-SEC.
  • Fig. 2A & C show the profiles from electrophoresis under non-reducing (NR) and reducing (R) conditions after preparative SEC purification (post prep-SEC) or after Protein A purification (post-pA) of parental chimeric variant v23924 (2A) and purified representative humanized variant v30384 (2C),
  • Fig. 2B & D show the UPLC-SEC profiles of parental chimeric variant v23924 after preparative SEC purification (2B) and purified representative humanized variant v30384 after Protein A purification (2D).
  • Fig. 3A & B depict the bio-layer interferometry (BLI) sensorgrams of parental chimeric variant v23924 (3A) and purified representative humanized variant v30384 (3B).
  • FIG. 4A-D depict the intact LC/MS profiles for representative humanized variants v30384 (4A, with an expanded view of the main peak in 4B) and v31422 (4C, with an expanded view of the main peak in 4D).
  • FIG. 5A & B shows the receptor -mediated internalization capabilities of the chimeric antibody v23924, a representative humanized variant, v30384, and the FR ⁇ -targeting antibodies mirvetuximab and farletuzumab at various concentrations in the FR ⁇ -expressing cell line IGROV- 1 as determined by flow cytometry after a 6-hour incubation (5A) and a 24-hour incubation (5B).
  • the anti -RS V antibody, palivizumab was included as a negative control.
  • FIG. 6A & B show the receptor -mediated internalization capabilities of the chimeric antibody v23924, a representative humanized variant, v30384, and the FR ⁇ -targeting antibodies mirvetuximab and farletuzumab at various concentrations in the FR ⁇ -expressing cell line OVCAR-3 as determined by flow cytometry after a 6-hour incubation (6A) and a 24-hour incubation (6B).
  • the anti-RSV antibody, palivizumab was included as a negative control.
  • Fig. 7 shows the coverage of the hFR ⁇ sequence (SEQ ID NO: 15) by peptides generated by pepsin digestion of hFR ⁇ . Each bar below the sequence represents a peptide.
  • Fig. 8A & B show a summary plot (8A) and a differential plot (8B) of the hydrogen/deuterium exchange mass spectrometry (HDX-MS) kinetics of the peptides generated by pepsin digestion of hFR ⁇ : hFOLRl (hFR ⁇ ) vs. hFOLRl-v23924 complex.
  • Fig. 9A-C show the amide deuteration level of peptide 119-126 (WEDCRTSY) (SEQ ID NO: 152) after hydrogen/deuterium exchange mass spectrometry (HDX-MS) for Ih: hFOLRl (9 A) vs. hFOLRl -v23924 complex (9B), and the differential plot (9C).
  • FIG. 10A & B show the receptor-mediated internalization capabilities of a parental humanized variant, v30384, and a representative affinity matured variant, v35356, in FR ⁇ - expressing cell lines IGROV-1 (10A) and JEG-3 (10B) as determined by flow cytometry after 5h and 24h incubation periods.
  • Palivizumab was included as a non-targeted control.
  • FIG. 11A-D show the intracellular payload delivery capabilities of representative ADCs in the cell-lines JEG-3 (11 A), Caov-3 (11B), H2110 (11C) and HEC-l-A (11D) as assessed by mass spectrometry.
  • ADCs were humanized antibody variant v30384 and affinity matured variant v35356 each conjugated to drug-linker DL1.
  • Fig. 12A-H show the in vivo anti-tumor activities of the chimeric anti-FR ⁇ antibody v23924 conjugated to drug-linker DL1 or DL7 assessed in the xenograft models: CTG-0848 PDX (12A), OV90 CDX (12B), OVCAR-3 CDX (12C), LXFA737 PDX (12D), JEG3 CDX (12E), HCC1954 CDX (12F), SKOV3 CDX (12G) and KB CDX (12H).
  • Control ADCs were vl7717 (mirvetuximab Fab with HetFc) conjugated to drug-linker DL1 or DL6.
  • Fig. 13 shows the in vivo anti -tumor activities of ADCs comprising the chimeric antibody v23924 or the humanized variants v30384 or v30399 each conjugated to drug-linker DL1, administered at 4 mg/kg or 9 mg/kg, in the mid/high-level FR ⁇ expressing OVCAR3 ovarian cancer model.
  • Fig. 14A-E show the in vivo anti -tumor activities of an ADC comprising the humanized variant v30384 conjugated to drug-linker DL1 or DL8, administered at the dosages shown, as assessed in the xenograft models: H2110 CDX (14A), SKOV3 CDX (14B & 14C), IGROV-1 CDX (14D) and LXFA737 PDX (14E).
  • Control ADCs were vl7717 (mirvetuximab Fab with HetFc) conjugated to drug-linker DL6; vl7716 (mirvetuximab Fab with HomoFc) conjugated to drug-linker DL6, and for the H2110 CDX, v31629 (farletuzumab) conjugated to drug -linker DL4.
  • Fig. 15A-D show the in vivo anti -tumor activities of an ADC comprising the humanized variantv30384 conjugated to drug-linker DL5, administered at the dosages shown, in the xenograft models: OV90 (15A), H2110 (15B) and OVCAR-3 (15C & 15D).
  • Fig. 16A-H show the results of pharmacokinetic analysis indicating concentrations of serum IgG or ADC over time in serum taken from animals treated with ADCs comprising various anti-FR ⁇ antibodies; chimeric anti-FR ⁇ antibody v23924 conjugated to drug-linker DL1 in OV90 model (16A), OVCAR-3 model (16B), LXFA737 model (16C), JEG3 model (16D) or SKOV-3 model (16E); humanized variant v30384 conjugated to drug-linker DL1 in H2110 model (16F), and humanized variant v30384 conjugated to drug-linker DL5 in OV90 model (16G) or H2100 model (16H).
  • Fig. 17 presents a table showing the CDR sequences of representative anti-FR ⁇ antibodies as defined by IMGT, Chothia, Kabat, Contact and AbM definitions.
  • Fig. 18 presents a table showing the VH and VL sequences of representative anti-FR ⁇ antibodies.
  • FIG. 19 shows cell growth inhibition (cytotoxicity) capabilities of an ADC comprising humanized antibody v30384 conjugated to drug-linker DL1 and an ADC comprising the affinity- matured variant v35356 conjugated to drug-linker DL1 in the cell lines: KB-HeLa (19A), IGROV- 1 (19B), JEG-3 (19C), SKOV-3 (19D) and MDA-MB-468 (19E).
  • Fig. 20A-D shows penetration of the anti-FR ⁇ humanized antibody variant v36675 in JEG-3 cell spheroids compared to mirvetuximab and negative control, palivizumab, at 4 hours (20A), 24 hours (20B), 48 hours (20C), and 96 hours (20D).
  • FIG. 21A & B show fixed cell confirmation screen images from a screen for specific off- target binding interactions using Retrogenix Cell Microarray Technology for the anti-FR ⁇ humanized antibody variant v36675 at 20 pg/mL (21A) and control antibody (rituximab biosimilar) at Ipg/mL (21B).
  • Fig. 22 shows competition binding between the chimeric anti-FR ⁇ antibody v23294 and the anti-FR ⁇ antibodies mirvetuximab and farletuzumab assessed in H2110 cells.
  • Fig. 23 shows the receptor -mediated internalization capabilities of a humanized variant, v30384 compared to the biparatopic anti-FR ⁇ antibody B5327A (v36264) and the anti-FR ⁇ antibody mirvetuximab (vl7716) in the FR ⁇ -expressing cell line IGROV-1 as determined by flow cytometry after a 5h incubation period.
  • Fig. 24 shows penetration of the anti-FR ⁇ humanized antibody variant v36675 in JEG-3 cell spheroids compared to the biparatopic anti-FR ⁇ antibody B5327A (v36264) and the anti-FR ⁇ antibody mirvetuximab (vl 7716) at 96 hours.
  • the present disclosure relates to antibody constructs that bind human folate receptor alpha (FR ⁇ ; also referred to herein as FOLR1), but do not show significant binding to folate receptor beta (FOLR2), gamma (FOLR3) or delta (FOLR4).
  • FOLR1 human folate receptor alpha
  • FOLR2 folate receptor beta
  • FOLR3 folate receptor beta
  • FOLR4 folate receptor beta
  • FOLR4 gamma
  • delta delta
  • the present disclosure also relates to antibody-drug conjugates (ADCs) comprising an anti-FR ⁇ antibody construct as described herein conjugated to a drug, such as a cytotoxin or an immune modulator.
  • ADCs antibody-drug conjugates
  • the anti-FR ⁇ antibody constructs and ADCs of the present disclosure may find use, for example, as therapeutics or diagnostics.
  • Certain aspects of the present disclosure relate to therapeutic methods and uses of the anti-FR ⁇ antibody constructs and ADCs, for example, in the treatment of cancer.
  • Some aspects relate to diagnostic methods and uses of the anti-FR ⁇ antibody constructs and ADCs, for example, in the diagnosis or analysis of cancer.
  • the term “about” refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • the use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”
  • compositions, use or method denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions.
  • Consisting of when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps.
  • a composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
  • a “complementarity determining region” or “CDR” is an amino acid sequence that contributes to antigen-binding specificity and affinity. “Framework” regions (FR) can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen -binding region and an antigen. From N-terminus to C-terminus, both the light chain variable region (VL) and the heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the three heavy chain CDRs are referred to herein as HCDR1 , HCDR2, and HCDR3 , and the three light chain CDRs are referred to as LCDR1 , LCDR2, and LCDR3.
  • CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
  • the three heavy chain CDRs and the three light chain CDRs are required to bind antigen.
  • antigen -binding may also occur through a combination of a minimum of one or more CDRs selected from the VH and/or VL domains, for example HCDR3.
  • CDR sequences are in common use, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), by Chothia et al. (1987, J Mol Biol, 196:901-917), as well as the IMGT, AbM (University of Bath) and Contact (MacCallum, et al, 1996, J Mol Biol, 262(5):732-745) definitions.
  • CDR definitions according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below.
  • VH includes the disclosure of the associated (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems.
  • VL includes the disclosure of the associated (inherent) light chain CDRs (LCDRs) as defined by any of the known numbering systems.
  • sequences refers to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (for example, about 80%, about 85%, about 90%, about 95%, or about 98% identity, over a specified region) when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using one of the commonly used sequence comparison algorithms as known to persons of ordinary skill in the art or by manual alignment and visual inspection. For sequence comparison, typically test sequences are compared to a designated reference sequence.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window” refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions which may be, for example, from about 10 to 600 contiguous amino acid or nucleotide positions, or from about 10 to about 200, or from about 10 to about 150 contiguous amino acid or nucleotide positions over which a test sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl.
  • BLAST and BLAST 2.0 algorithms are described in Altschul etal, 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the website for the National Center for Biotechnology Information (NCBI).
  • the term “subject,” as used herein, refers to an animal, in some embodiments a mammal, which is the object of treatment, observation or experiment.
  • the animal may be a human, a non- human primate, a companion animal (for example, dog, cat, or the like), farm animal (for example, cow, sheep, pig, horse, or the like) or a laboratory animal (for example, rat, mouse, guinea pig, non-human primate, or the like).
  • the subject is a human.
  • the present disclosure relates to antibody constructs that specifically bind to human FR ⁇ (hFR ⁇ ).
  • antibody construct refers to a polypeptide or a set of polypeptides that comprises one or more anti gen -binding domains, where each of the one or more antigen-binding domains specifically binds to an epitope or antigen.
  • each of the antigen -binding domains may bind the same epitope or antigen (i.e. the antibody construct is monospecific) or they may bind to different epitopes or antigens (i.e. the antibody construct is bispecific or multispecific).
  • the antibody construct may further comprise a scaffold and the one or more antigen -binding domains can be fused or covalently attached to the scaffold, optionally via a linker, as described herein.
  • the anti-FR ⁇ antibody construct comprises at least one antigen-binding domain that specifically binds to hFR ⁇ .
  • specifically binds to hFR ⁇ , it is meant that the antibody construct binds to hFR ⁇ but does not exhibit significant binding to any of human folate receptor beta (FOLR2), gamma (FOLR3) or delta (FOLR4).
  • the anti-FR ⁇ antibody constructs of the present disclosure may be capable of binding to an FR ⁇ from one or more non -human species.
  • the anti-FR ⁇ antibody constructs of the present disclosure are capable of binding to cynomolgus monkey FR ⁇ .
  • Human FR ⁇ is also known as “human folate receptor 1” or “FOLR1.”
  • the protein sequences of hFR ⁇ from various sources are known in the art and readily available from publicly accessible databases, such as GenBank or UniProtKB. Examples of hFR ⁇ sequences include for example those provided under NCBI reference numbers Pl 5328, AAX29268.1, AAX37119.1, NP_057937.1 and NP_057936.1.
  • An exemplary hFR ⁇ protein sequence is provided in Table 2 as SEQ ID NO: 1 (NCBI Reference Sequence: NP 057936.1).
  • An exemplary cynomolgus monkey FR ⁇ protein sequence is also provided in Table 2 (SEQ ID NO: 2; NCBI Reference Sequence: XP_005579002.2).
  • Table 2 Human and Cynomolgus Monkey FR ⁇ Protein Sequences
  • Specific binding of an antigen-binding domain to a target antigen or epitope may be measured, for example, through an enzyme-linked immunosorbent assay (ELISA), a surface plasmon resonance (SPR) technique (employing, for example, a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry or a traditional binding assay (Heeley, 2002, Endocr Res, 28:217-229).
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • specific binding may be defined as the extent of binding to a non-target protein (such as FOLR2, FOLR3 or FOLR4) being less than about 10% of the binding to hFR ⁇ as measured by ELISA or flow cytometry, for example.
  • specific binding of an antibody construct for FR ⁇ may be defined by a dissociation constant (KD) of ⁇ 1 pM, for example, ⁇ 500 nM, ⁇ 250 nM, ⁇ 100 nM, ⁇ 50 nM, or ⁇ 10 nM.
  • KD dissociation constant
  • specific binding of an antibody construct for a particular antigen or an epitope may be defined by a dissociation constant (KD) of 10' 6 M or less, for example, 10' 7 M or less, or 10' 8 M or less.
  • specific binding of an antibody construct for a particular antigen or an epitope may be defined by a dissociation constant (KD) between IO' 6 M and 10' 9 M, for example, between 10' 7 M and 10' 9 M.
  • the anti -FR ⁇ antibody constructs of the present disclosure show higher internalization into FR ⁇ -expressing cells than the reference antibodies mirvetuximab (huMovl9 or huFR107) and farletuzumab (MORAb-003).
  • Antibody internalization may be measured using art-known methods, for example, by a direct internalization method according to the protocol detailed in Schmidt, M. etal., 2008, Cancer Immunol. Immunother., 57:1879-1890, or using commercially available fluorescent dyes such as the pH Ab Dyes (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red Dyes (ThermoFisher Scientific Corporation, Waltham, MA) and Incucyte® Fabfluor-pH Antibody Labeling Reagent (Sartorius AG, Gottingen, Germany), and analysis techniques such as microscopy, FACS, high content imaging or other plate-based assays.
  • pH Ab Dyes Promega Corporation, Madison, WI
  • pHrodo iFL and Deep Red Dyes ThermoFisher Scientific Corporation, Waltham, MA
  • Incucyte® Fabfluor-pH Antibody Labeling Reagent Sescopy, FACS, high content imaging or other plate-based assays.
  • the anti -FR ⁇ antibody construct is considered to demonstrate a higher internalization into FR ⁇ -expressing cells than a corresponding reference antibody (mirvetuximab or farletuzumab) when the amount of anti -FR ⁇ antibody construct internalized into the FR ⁇ -expressing cells is at least 1.2 times greater than the amount of reference antibody internalized into the same FR ⁇ -expressing cells under the same test conditions.
  • the amount of internalized antibody is determined using an appropriate fluorescent dye and high content imaging.
  • the amount of internalized antibody is determined in cells that express FR ⁇ at a high level.
  • the amount of internalized antibody is determined in IGROV-1 cells or cells that express FR ⁇ at a similar level to IGROV-1 cells. In some embodiments, the amount of internalized antibody is determined after a 6-hour incubation period. In some embodiments, the amount of internalized antibody is determined after a 24-hour incubation period.
  • the anti -FR ⁇ antibody construct is considered to demonstrate a higher internalization into FR ⁇ -expressing cells than a corresponding reference antibody (mirvetuximab or farletuzumab) when the amount of anti -FR ⁇ antibody construct internalized into the FR ⁇ -expressing cells is at least 1.3 times greater, at least 1.4 times greater, at least 1.5 times greater, 1.6 times greater, 1.7 times greater, 1.8 times greater, 1.9 times greater, or 2.0 times greater, than the amount of reference antibody internalized into the same FR ⁇ -expressing cells under the same test conditions.
  • the amount of internalized antibody is determined using an appropriate fluorescent dye and high content imaging.
  • the amount of internalized antibody is determined in cells that express FR ⁇ at a high level. In some embodiments, the amount of internalized antibody is determined in IGROV-1 cells or cells that express FR ⁇ at a similar level to IGROV-1 cells. In some embodiments, the amount of internalized antibody is determined after a 6-hour incubation period. In some embodiments, the amount of internalized antibody is determined after a 24-hour incubation period.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise at least one antigen- binding domain that is capable of binding to hFR ⁇ .
  • the at least one antigen-binding domain capable of binding to hFR ⁇ typically is an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment.
  • an antigen-binding antibody fragment include, but are not limited to, a Fab fragment, a Fab’ fragment, a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb).
  • a “Fab fragment” contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI) along with the variable domains of the light and heavy chains (VL and VH, respectively).
  • Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region.
  • a Fab fragment may also be a single-chain Fab molecule, i.e. a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain.
  • the C-terminus of the Fab light chain may be connected to theN-terminus of the Fab heavy chain in the single-chain Fab molecule.
  • An “scFv” includes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody in a single polypeptide chain.
  • the scFv may optionally further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form a desired structure for antigen binding.
  • an scFv may include a VL connected from its C- terminus to the N-terminus of a VH by a polypeptide linker.
  • an scFv may comprise a VH connected through its C-terminus to the N-terminus of a VL by a polypeptide linker (see review in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer -Verlag, New York, pp. 269-315 (1994)).
  • An “sdAb” format refers to a single immunoglobulin domain.
  • the sdAb may be, for example, of camelid origin. Camelid antibodies lack light chains and their antigen-binding sites consist of a single domain, termed a “VHH.”
  • An sdAb comprises three CDR/hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and easy to express, for example, as a fusion with the Fc chain of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1): 13-22).
  • each additional antigen-binding domain may independently be an immunoglobulin-based domain, such as an antigen-binding antibody fragment, or a non- immunoglobulin-based domain, such as a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule capable of specifically binding to its target, for example, a natural or engineered ligand.
  • immunoglobulin-based domain such as an antigen-binding antibody fragment
  • a non- immunoglobulin-based domain such as a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule capable of specifically binding to its target, for example, a natural or engineered ligand.
  • Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, fynomers, affimers, alphabodies, DARPins and avimers.
  • the present disclosure describes herein the identification of an antibody that specifically binds hFR ⁇ (variant v23924), as well as representative humanized versions of this antibody (variants v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425 and v31426) and representative affinity-matured versions of this antibody (variants v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 and v36675) (see Examples and Sequence Tables). Epitope mapping using the hFR ⁇ sequence shown in Fig.
  • SEQ ID NO: 15 determined that the epitope within the hFR ⁇ protein bound by variant v23924 comprises the amino acid residues E120, D121, R123, T124, S125 and Y126 of SEQ ID NO: 15 (see Example 13).
  • the at least one antigen-binding domain that binds hFR ⁇ comprised by the anti-FR ⁇ antibody constructs of the present disclosure binds an epitope within the hFR ⁇ protein that compri ses the amino acid residues El 20, D 121 , R123 , T 124, S 125 and Y126 of SEQ ID NO: 15.
  • the hFR ⁇ epitope bound by the anti-FR ⁇ antibody constructs is a non-linear (or discontinuous) epitope comprising the amino acid residues E120, D121, R123, T124, S125 and Y126 of SEQ ID NO: 15.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen -binding domain that competes for binding to hFR ⁇ with an antibody that binds to an epitope within the hFR ⁇ protein comprising the amino acid residues E120, D121, R123, T124, S125 and Y126. In certain embodiments, the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain that competes for binding to hFR ⁇ with antibody v23924 described herein.
  • the antibody that binds to an epitope within the hFR ⁇ protein comprising the amino acid residues E120, D121, R123, T124, S125 and Y126 or antibody v23924 (the reference antibody) is first allowed to bind to hFR ⁇ under saturating conditions and then the ability of the test antibody construct to bind to hFR ⁇ is measured.
  • test antibody construct is able to bind to hFR ⁇ at the same time as the reference antibody, then the test antibody construct is considered to bind to a different epitope than the reference antibody. Conversely, if the test antibody construct is not able to bind to hFR ⁇ at the same time as the reference antibody, then the test antibody construct is considered to bind to the same epitope, to an overlapping epitope, or to an epitope that is in close proximity to the epitope bound by the reference antibody.
  • Competition assays may also be run in which the binding order of the reference and test antibodies is reversed, that is, the test antibody is first allowed to bind to hFR ⁇ under saturating conditions and then the ability of the reference antibody construct to bind to hFR ⁇ is measured.
  • Such competition assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), bio-layer interferometry, flow cytometry and the like.
  • An “antibody that competes with” a reference antibody refers to an antibody that blocks binding of the reference antibody to its epitope in a competition assay by 50% or more.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise at least one antigen-binding domain that specifically binds to hFR ⁇ , where the antigen- binding domain comprises a set of CDRs based on the CDRs of antibody variant v23924 described herein.
  • the CDR sequences of the antibody v23924 and representative humanized or affinity- matured versions of this antibody are shown in Fig. 17.
  • Analysis of the CDR sequences from the parental and affinity-matured anti-FR ⁇ antibodies identified a minimal amino acid sequence present in each CDR as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems. These amino acid sequences are represented by the minimal consensus CDR sequences provided in Table 3. Extended versions of these CDR consensus sequences based on CDR sequences defined by the AbM numbering system are shown in Table 4.
  • the anti- FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 3, 4 and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 6, 7 and 8.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen -binding domain having:
  • X 4 is W and X 5 is Y; an LCDR2 amino acid sequence as set forth in SEQ ID NO: 7, and an LCDR3 amino acid sequence as set forth in SEQ ID NO: 8, where X 6 is S, X 7 is N, X 8 is V and X 9 is D, or X 6 is W, X 7 is H, X 8 is I and X 9 is L.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 9, 10 and 11, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 12, 13 and 14.
  • HCDR1, HCDR2 and HCDR3 comprising the sequences as set forth in SEQ ID NOs: 9, 10 and 11
  • LCDR1, LCDR2 and LCDR3 comprising the sequences as set forth in SEQ ID NOs: 12, 13 and 14.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen -binding domain having:
  • an LCDR1 amino acid sequence as set forth in SEQ ID NO: 12 where X 15 is R or Q, X 16 is G and X 17 is D, or X 15 is R, X 16 is W and X 17 is Y; an LCDR2 amino acid sequence as set forth in SEQ ID NO: 13, and an LCDR3 amino acid sequence as set forth in SEQ ID NO: 14, where X 18 is S, X 19 is N, X 20 is V and X 21 is D, or X 18 is W, X 19 is H, X 20 is I and X 21 is L.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen -binding domain having:
  • a LCDR1 amino acid sequence selected from the LCDR1 amino acid sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675; aLCDR2 amino acid sequence selected from the LCDR2 amino acid sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35354,
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) selected from the heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675, as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) selected from the light chain CDR amino acid sequences (LCDR1, LCD
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675, as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an anti gen -binding domain comprising the CDR sequences of the VH domain of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising the CDR sequences of the VL domain of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675.
  • VH and VL sequences of v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 and v36675 are provided in Fig. 18.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675.
  • a VH amino acid sequence selected from the VH amino acid sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen -binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising a VH amino acid sequence and a VL amino acid sequence selected from the VH and VL amino acid sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen- binding domain that comprises a set of CDRs (i.e.
  • heavy chain HCDR1, HCDR2 and HCDR3, and light chain LCDR1 , LCDR2 and LCDR3) that have 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100% sequence identity to a set of CDRs of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675, where the % sequence identity is calculated across all six CDRs and where the antigen-binding domain retains the ability to bind hFR ⁇ .
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a variant of the set of CDR sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675, where the variant comprises between 1 and 10 amino acid substitutions across the set of CDRs (i.e.
  • the CDRs may be modified by up to 10 amino acid substitutions with any combination of the six CDRs being modified), and where the antigen-binding domain retains the ability to bind hFR ⁇ .
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a variant of the set of CDR sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675, where the variant comprises between 1 and 7 amino acid substitutions, between 1 and 5 amino acid substitutions, between 1 and 4 amino acid substitutions, between 1 and 3 amino acid substitutions, between 1 and 2 amino acid substitutions, or 1
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an anti gen -binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675, where the antigen-binding domain retains the ability to bind hFR ⁇ .
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen- binding domain that comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675, where the antigen-binding domain retains the ability to bind hFR ⁇ .
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen -binding domain having:
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an anti gen -binding domain comprising the CDR sequences of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133 or 136.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising the CDR sequences of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 39, 64, 119, 124 or 130.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen -binding domain having:
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen -binding domain having:
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences as set forth in any one of SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133 or 136.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an anti gen -binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences as set forth in any one of SEQ ID NOs: 39, 64, 119, 124 or 130.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences as set forth in any one of SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133 or 136, and a VL amino acid sequence selected from the VL amino acid sequences as set forth in any one of SEQ ID NOs: 39, 64, 119, 124 or 130.
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising:
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise an antigen-binding domain comprising:
  • the anti-FR ⁇ antibody constructs of the present disclosure may have various formats.
  • the minimal component of the anti-FR ⁇ antibody construct is an antigen-binding domain that binds to hFR ⁇ .
  • the anti-FR ⁇ antibody constructs may further optionally comprise one or more additional antigen-binding domains and/or a scaffold.
  • each additional antigen- binding domain may bind to the same epitope within hFR ⁇ , may bind to a different epitope within hFR ⁇ , or may bind to a different antigen.
  • the anti-FR ⁇ antibody construct may be, for example, monospecific, biparatopic, bispecific or multispecific.
  • the anti-FR ⁇ antibody construct comprises at least one antigen- binding domain that binds to hFR ⁇ and a scaffold, where the antigen -binding domain is operably linked to the scaffold.
  • operably linked means that the components described are in a relationship permitting them to function in their intended manner. Examples of suitable scaffolds are described below.
  • the anti-FR ⁇ antibody construct comprises two anti gen -binding domains optionally operably linked to a scaffold.
  • the anti-FR ⁇ antibody construct may comprise three or four antigen-binding domains and optionally a scaffold.
  • at least a first antigen-binding domain is operably linked to the scaffold and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold or to the first antigen-binding domain or, when more than two antigen- binding domains are present, to another antigen-binding domain.
  • Anti-FR ⁇ antibody constructs that lack a scaffold may comprise a single anti gen -binding domain in an appropriate format, such as an sdAb, or they may comprise two or more antigen- binding domains optionally operably linked by one or more linkers.
  • the antigen-binding domains may be in the form of scFvs, Fabs, sdAbs, or a combination thereof.
  • scFvs as the antigen -binding domains
  • formats such as a tandem scFv ((scFv)2 or taFv) may be constructed, in which the scFvs are connected together by a flexible linker.
  • scFvs may also be used to construct diabody formats, which comprise two scFvs connected by a short linker (usually about 5 amino acids in length). The restricted length of the linker results in dimerization of the scFvs in a head-to-tail manner.
  • the scFvs may be further stabilized by inclusion of an interdomain disulfide bond.
  • a disulfide bond may be introduced between VL and VH through introduction of an additional cysteine residue in each chain (for example, at position 44 in VH and position 100 in VL) (see, for example, Fitzgerald etal., 1997, Protein Engineering, 10: 1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct having a DART format (see, for example, Johnson et al., 2010, J Mol. Biol., 399:436-449).
  • formats comprising two sdAbs, such as VHs or VHHs, connected together through a suitable linker may be employed in some embodiments.
  • Other examples of anti-FR ⁇ antibody construct formats that lack a scaffold include those based on Fab fragments, for example, Fab? and F(ab’)2 formats, in which the Fab fragments are connected through a linker or an IgG hinge region.
  • an scFv or a sdAb may be fused to the C- terminus of either or both of the light and heavy chain of a Fab fragment resulting in a bivalent (Fab-scFv/sdAb) construct.
  • the anti-FR ⁇ antibody construct may be in an antibody format that is based on an immunoglobulin (Ig).
  • the anti-FR ⁇ antibody construct may be based on an IgG class immunoglobulin, for example, an IgGl, IgG2, IgG3 or IgG4 immunoglobulin.
  • the anti-FR ⁇ antibody construct may be based on an IgGl immunoglobulin.
  • an anti-FR ⁇ antibody construct when an anti-FR ⁇ antibody construct is based on a specified immunoglobulin isotype, it is meant that the anti-FR ⁇ antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype.
  • an anti-FR ⁇ antibody construct based on a given Ig isotype may comprise at least one antigen-binding domain operably linked to an Ig scaffold, where the scaffold comprises an Fc region from the given isotype and optionally an Ig hinge region from the same or a different isotype.
  • anti-FR ⁇ antibody constructs may also comprise hybrids of isotypes and/or subclasses in some embodiments. It is also to be understood that the Fc region and/or hinge region may optionally be modified to impart one or more desirable functional properties as is known in the art.
  • the anti-FR ⁇ antibody constructs may be derived from two or more immunoglobulins that are from different species, for example, the anti-FR ⁇ antibody construct may be a chimeric antibody or a humanized antibody.
  • a “chimeric antibody” typically comprises at least one variable domain from a non- human antibody, such as a rabbit or rodent (for example, murine) antibody, and at least one constant domain from a human antibody.
  • the human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison etal., 1984, Proc. Natl. Acad. Sci. USA, 81 :6851 -55, and U.S. Patent No. 4,816,567.
  • humanized antibody is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDR) of the recipient are replaced by residues from a hypervariable region (CDR) of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate, having the desired specificity and affinity for a target antigen.
  • CDR grafting This technique for creating humanized antibodies is often referred to as “CDR grafting.”
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues, or the humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody.
  • a variable domain in a humanized antibody will comprise all or substantially all of the hypervariable regions from a non-human immunoglobulin and all or substantially all of the FRs from a human immunoglobulin sequence.
  • Humanized antibodies are described in more detail in Jones, et al., 1986, Nature, 321 :522-525; Riechmann, et al., 1988, Nature, 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol, 2:593-596, for example.
  • a number of approaches are known in the art for selecting the most appropriate human frameworks in which to graft the non-human CDRs.
  • Early approaches used a limited subset of well -characterised human antibodies, irrespective of the sequence identity to the non-human antibody providing the CDRs (the “fixed frameworks” approach).
  • More recent approaches have employed variable regions with high amino acid sequence identity to the variable regions of the non-human antibody providing the CDRs (“homology matching” or “best-fit” approach).
  • An alternative approach is to select fragments of the framework sequences within each light or heavy chain variable region from several different human antibodies. CDR-grafting may in some cases result in a partial or complete loss of affinity of the grafted molecule for its target antigen.
  • SDRs specificity-determining residues
  • the anti-FR ⁇ antibody construct of the present disclosure comprises humanized antibody sequences, for example, one or more humanized variable domains.
  • the anti-FR ⁇ antibody construct can be a humanized antibody.
  • humanized antibodies based on the anti-FR ⁇ antibody v23924 are described herein (v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425 and v31426; see Examples and Sequence Tables).
  • the anti-FR ⁇ antibody constructs of the present disclosure comprise one or more antigen-binding domains operably linked to a scaffold.
  • the antigen-binding domain(s) may be in one or a combination of the forms described above (for example, scFvs, Fabs and/or sdAbs).
  • Suitable scaffolds include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogues and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer -forming “zipper” peptides derived from Jun and Fos, IgG CHI and CL domains or barnase-barstar toxins), cytokines, chemokines or growth factors.
  • Other examples include antibodies based on the DOCK-AND-LOCKTM (DNLTM) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, for example, Chang, et al., 2007, Clin. Cancer Res., 13 :5586s-5591 s).
  • a scaffold may be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. Where the scaffold is a polypeptide, each antigen-binding domain of the anti-FR ⁇ antibody construct may be linked to either the N- or C-terminus of the polypeptide scaffold.
  • Anti-FR ⁇ antibody constructs comprising a polypeptide scaffold in which one or more of the anti gen -binding domains are linked to a region other than the N- or C-terminus, for example, via the side chain of an amino acid with or without a linker, are also contemplated in certain embodiments.
  • the antigen-binding domain(s) may be linked to the scaffold by genetic fusion or chemical conjugation.
  • the antigen-binding domain(s) are linked to the scaffold by genetic fusion.
  • the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.
  • a number of protein domains are known in the art that comprise selective pairs of two different polypeptides and may be used to form a scaffold.
  • An example is leucine zipper domains such as Fos and Jun that selectively pair together (Kostelny, etal., JImmunol, 148: 1547-53 (1992); Wranik, etal., J. Biol. Chem., 287: 43331-43339 (2012)).
  • protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogues and derivatives, toxins, cytokines, chemokines and growth factors.
  • the use of protein scaffolds in combination with anti gen -binding moieties has been described (see, for example, Muller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11 :582-593; Vallera et al., 2005, Clin. Cancer Res., 11 :3879-3888; Song et al., 2006, Biotech. AppL Biochem., 45: 147-154, and U.S. Patent Application Publication No. 2009/0285816).
  • Antigen-binding moieties such as scFvs, diabodies or single chain diabodies to albumin has been shown to improve the serum half-life of the antigen-binding moi eties (Muller et al., ibid.).
  • Antigen-binding moi eties may be fused at the N- and/or C-termini of albumin, optionally via a linker.
  • albumin in the form of heteromultimers that comprise two transporter polypeptides obtained by segmentation of an albumin protein such that the transporter polypeptides self-assemble to form quasi-native albumin have been described (see International Patent Application Publication Nos. WO 2012/116453 and WO 2014/012082).
  • the heteromultimer includes four termini and thus can be fused to up to four different antigen-binding moi eties, optionally via linkers.
  • the anti-FR ⁇ antibody construct may comprise a protein scaffold.
  • the anti-FR ⁇ antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, an albumin or an albumin analogue or derivative.
  • the anti-FR ⁇ antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, for example, an IgG Fc region.
  • Fc region refers to a C -terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • the anti-FR ⁇ antibody constructs of the present disclosure may comprise a scaffold that is based on an immunoglobulin Fc region.
  • the Fc region may be dimeric and composed of two Fc polypeptides or alternatively, the Fc region may be composed of a single polypeptide.
  • an “Fc polypeptide” in the context of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain that is capable of stable self-association.
  • first Fc polypeptide and second Fc polypeptide may be used interchangeably provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.
  • An Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain.
  • an Fc polypeptide of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence.
  • the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.
  • the anti-FR ⁇ antibody construct may comprise a scaffold that is based on an IgG Fc region. In some embodiments, the anti-FR ⁇ antibody construct may comprise a scaffold that is based on a human IgG Fc region. In some embodiments, the anti-FR ⁇ antibody construct may comprise a scaffold based on an IgGl Fc region. In some embodiments, the anti- FR ⁇ antibody construct may comprise a scaffold based on a human IgGl Fc region.
  • the anti-FR ⁇ antibody construct may comprise a scaffold based on an IgG Fc region, which is a homodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the amino acid sequences of the first and second Fc polypeptides are the same.
  • the anti-FR ⁇ antibody construct may comprise a scaffold based on an IgG Fc region, which is a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the amino acid sequences of the first and second Fc polypeptides are different.
  • the anti-FR ⁇ antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications.
  • the anti-FR ⁇ antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications.
  • the anti-FR ⁇ antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain is an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications.
  • an “asymmetric amino acid modification” refers to a modification, such as a substitution or an insertion, in which an amino acid at a specific position on a first CH3 or CH2 sequence is different to the amino acid on a second CH3 or CH2 sequence at the same position.
  • asymmetric amino acid modifications can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids at the same respective position on each of the first and second CH3 or CH2 sequences.
  • Each of the first and second CH3 or CH2 sequences of a heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.
  • the anti-FR ⁇ antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, where the modified CH3 domain comprises one or more amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc.
  • the amino acid modifications are asymmetric amino acid modifications.
  • Amino acid modifications that may be made to the CH3 domain of an Fc in order to promote formation of a heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96/027011 (“knobs into holes”), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 (“electrostatic steering”), Davis et al., 2010, Prot Eng Des Sei, 23(4):195-202 (strand exchange engineered domain (SEED) technology) and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab-arm exchange).
  • the anti-FR ⁇ antibody construct may comprise a scaffold based on a modified Fc region as described in International Publication No. WO 2012/058768 or WO 2013/063702.
  • Table 5 provides the amino acid sequence of the human IgGl Fc sequence (SEQ ID NO: 16), corresponding to amino acids 231 to 447 of the full-length human IgGl heavy chain.
  • the CH3 sequence comprises amino acids 341-447 of the full-length human IgGl heavy chain.
  • CH3 domain amino acid modifications that promote formation of a heterodimeric Fc as described in in International Patent Application Publication Nos. WO 2012/058768 and WO 2013/063702.
  • the anti-FR ⁇ antibody construct may comprise a heterodimeric
  • Fc scaffold having a modified CH3 domain comprising the modifications of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 5.
  • the anti-FR ⁇ antibody construct may comprise a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications. Modifications in the CH2 domain can affect the binding of Fc receptors (FcRs) to the Fc, such as receptors of the FcyRI, FcyRII and FcyRIII subclasses.
  • FcRs Fc receptors
  • the anti-FR ⁇ antibody construct comprises a scaffold based on an IgG Fc having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcyRI, FcyRII and FcyRIII receptors.
  • a number of amino acid modifications to the CH2 domain that selectively alter the affinity of the Fc for different Fey receptors are known in the art. Amino acid modifications that result in increased binding and amino acid modifications that result in decreased binding can each be useful in certain indications. For example, increasing binding affinity of an Fc for FcyRIIIa (an activating receptor) may result in increased antibody dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of the target cell. Decreased binding to FcyRIIb (an inhibitory receptor) likewise may be beneficial in some circumstances. In certain indications, a decrease in, or elimination of, ADCC and complement -mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains comprising amino acid modifications that result in increased binding to FcyRIIb or amino acid modifications that decrease or eliminate binding of the Fc region to all of the Fey receptors (“knock-out” variants) may be useful.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • CDC complement -mediated cytotoxicity
  • amino acid modifications to the CH2 domain that alter binding of the Fc by Fey receptors include, but are not limited to, the following: S298A/E333A/K334A and S298A/E333A/K334A/K326A (increased affinity for FcyRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1 -2): 132-41); F243L/R292P/Y300L/V305I/P396L (increased affinity for FcyRIIIa) (Stavenhagen, etal. 2007, Cancer Res 67(18):8882-90);
  • F243L/R292P/Y300L/L235V/P396L (increased affinity for FcyRIIIa) (Nordstrom JL, etal., 2011, Breast Cancer Res, 13(6):R123); F243L (increased affinity for FcyRIIIa) (Stewart, et al., 2011, Protein Eng Des Sei., 24(9):671-8); S298A/E333A/K334A (increased affinity for FcyRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604); S239D/I332E/A330L and S239D/I332E (increased affinity for FcyRIIIa) (Lazar, etal., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D/S267E and S267E/L328F (increased affinity for FcyRII
  • the anti-FR ⁇ antibody construct comprises a scaffold based on an IgG Fc having a modified CH2 domain, in which the modified CH2 domain comprises one or more amino acid modifications that result in decreased or eliminated binding of the Fc region to all of the Fey receptors (i.e. a “knock-out” variant).
  • mutants that may be introduced into the hinge or CH2 domain to produce a “knock-out” variant include the amino acid modifications L234A/L235A, and L234A/L235A/ D265S.
  • the anti-FR ⁇ antibody constructs described herein may comprise a scaffold based on an IgG Fc in which native glycosylation has been modified.
  • glycosylation of an Fc may be modified to increase or decrease effector function.
  • mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine or histidine i.e. N297A, Q, K or H
  • results in an aglycoslated Fc that lacks all effector function results in an aglycoslated Fc that lacks all effector function (Bolt et al, 1993, Eur. J. Immunol, 23:403-411; Tao & Morrison, 1989, J. Immunol, 143 :2595- 2601).
  • glycosylation variants include those with bisected oligosaccharides, for example, variants in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc).
  • GlcNAc N-acetylglucosamine
  • Such glycosylation variants may have reduced fiicosylation and/or improved ADCC function (see, for example, International Publication No. WO 2003/011878, U.S. Patent No. 6,602,684 and US Patent Application Publication No. US 2005/0123546).
  • Useful glycosylation variants also include those having at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, for example, International Publication Nos. WO 1997/030087, WO 1998/58964 and WO 1999/22764).
  • the anti-FR ⁇ antibody constructs have the format of a full-size antibody (FSA).
  • the anti-FR ⁇ antibody constructs have the format of an IgG FSA, for example, an IgGl FSA.
  • the anti-FR ⁇ antibody construct is a FSA comprising a first heavy chain sequence (Hl), a second heavy chain sequence (H2), a first light chain sequence (LI) and a second light chain sequence (L2).
  • the anti- FR ⁇ antibody construct is a monospecific FSA with a homodimeric Fc and comprises Hl, H2, LI and L2 sequence, where Hl and H2 have the same amino acid sequence, and LI and L2 have the same amino acid sequence.
  • the anti-FR ⁇ antibody construct is a monospecific FSA with a heterodimeric Fc and comprises Hl, H2, LI and L2 sequences, where Hl and H2 have different amino acid sequences, and LI and L2 have the same amino acid sequence.
  • the anti-FR ⁇ antibody construct is a bispecific or biparatopic FSA with a heterodimeric F c and comprises H 1 , H2, L 1 and L2 sequences, where H 1 and H2 have different amino acid sequences, and LI and L2 have different amino acid sequences.
  • the anti-FR ⁇ antibody construct is a FSA having a set of H 1 , H2, LI and L2 sequences comprising the Hl , H2, LI and L2 amino acid sequences as set forth in Tables A & B for any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, V31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675.
  • anti-FR ⁇ antibody constructs that are FSAs having a set of Hl , H2, LI and L2 sequences comprising the Hl, H2, LI and L2 amino acid sequences as set forth in Tables A & B for any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 or v36675, in which one or both of the Hl and H2 sequences comprise a C-terminal
  • anti-FR ⁇ antibody constructs described herein may be produced using standard recombinant methods known in the art (see, for example, U.S. Patent No. 4,816,567 and “Antibodies : A Laboratory Manual,” 2 nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).
  • a polynucleotide or set of polynucleotides encoding the anti-FR ⁇ antibody construct is generated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • Polynucleotide(s) encoding the anti-FR ⁇ antibody construct may be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New Y ork, 1994 & update, and “Antibodies: A Laboratory Manual ” 2 nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).
  • the number of polynucleotides required for expression of the anti-FR ⁇ antibody construct will be dependent on the format of the construct, including whether or not the antibody construct comprises a scaffold.
  • the format of the construct including whether or not the antibody construct comprises a scaffold.
  • two polynucleotides each encoding one polypeptide chain will be required
  • three polynucleotides each encoding one polypeptide chain will be required.
  • multiple polynucleotides may be incorporated into one vector or into more than one vector.
  • the polynucleotide or set of polynucleotides is incorporated into an expression vector or vectors together with one or more regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide.
  • regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals.
  • the expression vector may optionally further contain heterologous nucleic acid sequences that facilitate expression or purification of the expressed protein.
  • the expression vector may be an extrachromosomal vector or an integrating vector.
  • Suitable host cells for cloning or expression of the anti-FR ⁇ antibody constructs include various prokaryotic or eukaryotic cells as known in the art.
  • Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells and yeast cells (such as Saccharomyces or Pichia cells).
  • Prokaryotic host cells include, for example, E. coli, A. salmonicida or B. subtilis cells.
  • the anti-FR ⁇ antibody construct may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, as described for example in U.S. Patent Nos.
  • Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, in particular fungi and yeast strains whose glycosylation pathways have been “humanized” resulting in the production of an antibody construct with a partially or fully human glycosylation pattern (see, for example, Gerngross, 2004, Nat. Biotech. 22:1409- 1414, and Li et al., 2006, Nat. Biotech. 24:210-215).
  • Suitable host cells for the expression of glycosylated anti-FR ⁇ antibody constructs are usually eukaryotic cells.
  • U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PL ANTIBODIESTM technology for producing antigen -binding constructs in transgenic plants.
  • Mammalian cell lines adapted to grow in suspension may be particularly useful for expression of antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, for example, Graham etal, 1977, J.
  • MRC 5 cells including FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR CHO cells, see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NS0 and Sp2/0).
  • CHO Chinese hamster ovary
  • myeloma cell lines such as Y0, NS0 and Sp2/0.
  • Exemplary mammalian host cell lines suitable for production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003).
  • the host cell may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line.
  • the host cell may be a mammalian HEK293T, CHO, HeLa, NS0 or COS cell line, or a cell line derived from any one of these cell lines.
  • the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.
  • the host cells comprising the expression vector(s) encoding the anti-FR ⁇ antibody construct may be cultured using routine methods to produce the anti-FR ⁇ antibody construct.
  • host cells comprising the expression vector(s) encoding the anti-FR ⁇ antibody construct may be used therapeutically or prophylactically to deliver the anti- FR ⁇ antibody construct to a subject, or polynucleotides or expression vectors may be administered to a cell from a subject ex vivo and the cell then returned to the body of the subject.
  • the anti-FR ⁇ antibody constructs are purified after expression.
  • Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3 rd Ed., Scopes, Springer -Verlag, NY, 1994).
  • Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse -phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC.
  • Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
  • the bacterial proteins A and G bind to the Fc region.
  • the bacterial protein L binds to the Fab region of some antibodies. Purification may also be enabled by a particular fusion partner. For example, antibodies may be purified using glutathione resin if a GST fusion is employed, Ni +2 affinity chromatography if a His-tag is employed or immobilized anti -flag antibody if a flag-tag is used. The degree of purification necessary will vary depending on the use of the anti-FR ⁇ antibody constructs. In some instances, no purification may be necessary.
  • the anti-FR ⁇ antibody constructs are substantially pure.
  • the term “substantially pure” (or “substantially purified”) when used in reference to an anti-FR ⁇ antibody construct described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, such as a native cell, or a host cell in the case of recombinantly produced construct.
  • an anti-FR ⁇ antibody construct that is substantially pure is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.
  • Certain embodiments of the present disclosure relate to a method of making an anti-FR ⁇ antibody construct comprising culturing a host cell into which one or more polynucleotides encoding the anti-FR ⁇ antibody construct, or one or more expression vectors encoding the anti- FR ⁇ antibody construct, have been introduced, under conditions suitable for expression of the anti- FR ⁇ antibody construct, and optionally recovering the anti-FR ⁇ antibody construct from the host cell (or from host cell culture medium).
  • the anti-FR ⁇ antibody constructs described herein may comprise one or more post-translational modifications. Such post -translational modifications may occur in vivo, or they be conducted in vitro after isolation of the anti-FR ⁇ antibody construct from the host cell.
  • Post-translational modifications include various modifications as are known in the art (see, for example, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1 -12, 1983; Seifter et al, 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Ann. N.Y. Acad. Sci., 663:48-62).
  • the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.
  • post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4.
  • post-translational modifications include, for example, addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O- linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moi eties to the amino acid backbone, and addition or deletion of an N-terminal methionine residue resulting from prokaryotic host cell expression.
  • Post -translational modifications may also include modification with a detectable label, such as an enzymatic, fluorescent, luminescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase and acetylcholinesterase.
  • suitable prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.
  • luminescent materials include luminol, and bioluminescent materials such as luciferase, luciferin and aequorin.
  • suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon and fluorine.
  • post-translational modifications include acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • cDNA messenger RNA
  • recombinant polynucleotides include plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide that “encodes” a given polypeptide is a polynucleotide that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • inventions of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an anti-FR ⁇ antibody construct described herein.
  • the polynucleotide(s) may be comprised by a single vector or by more than one vector.
  • the polynucleotides are comprised by a multi ci str onic vector.
  • Certain embodiments of the present disclosure relate to host cells comprising polynucleotide(s) encoding an anti-FR ⁇ antibody construct described herein or one or more vectors comprising the polynucleotide(s).
  • the host cell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g. Y0, NS0, Sp20 cell).
  • ADCs antibody-drug conjugates
  • ADCs comprising an anti-FR ⁇ antibody construct as described herein conjugated to one or more drug moi eties, such as cytotoxins or immune modulators.
  • the anti-FR ⁇ antibody construct is conjugated to a drug moiety via a linker, which may be a cleavable or non-cleavable linker.
  • the anti-FR ⁇ antibody construct may be conjugated to a single drug molecule, or it may be conjugated to multiple drug molecules.
  • the number of drug molecules conjugated to a single anti-FR ⁇ antibody construct is defined by the drug-to-antibody ratio (DAR).
  • the DAR is in the range of from about 1 to about 12, or from about 2 to about 12, or from about 2 to about 8.
  • the ADCs comprising an anti-FR ⁇ antibody construct have the general Formula I:
  • A is an anti-FR ⁇ antibody construct as described herein; L is a linker; D is a drug moiety; m is between 1 and about 8, and n is between 1 and about 12.
  • m is between 1 and 6. In some embodiments, m is 1 or 2. In some embodiments, n is between about 1 and about 8, for example, between about 2 and about 8.
  • cytotoxic or immunomodulatory ADC payloads may be employed as the drug moiety in the ADCs comprising the anti-FR ⁇ antibody constructs.
  • examples include, but are not limited to, maytansinoids and maytansinoid analogues, benzodiazepines and pyrrol Whyzodiazepines, duocarmycins such as CC-1065 and analogues thereof, calicheamicins and calicheamicin analogues, auristatins and auristatin analogues, hemiasterlins and hemiasterlin analogues, tubulysins and tubulysin analogues, amatoxins and amatoxin analogues, camptothecins and camptothecin analogues, eribulin, TLR agonists (such as agonists of TLR7 and/or TLR8) and STING agonists.
  • TLR agonists such as agonists of TLR7 and/or TLR
  • the drug moiety comprised by the ADCs of the present disclosure is an auristatin or auristatin analogue, a hemiasterlin or a hemiasterlin analogue, a camptothecin or camptothecin analogue, or eribulin.
  • the drug moiety is linked to the anti- FR ⁇ antibody construct by a linker.
  • Linkers are bifunctional or multifunctional moieties capable of linking one or more drug molecules to the antibody construct.
  • the linker may be bifimctional (or monovalent) such that it links a single drug molecule to a single site on the antibody construct.
  • the linker may be multifunctional (or polyvalent) such that it links more than one drug molecule to a single site on the antibody construct. Multifunctional linkers may also be used to link one drug molecule to more than one site on the antibody construct in some embodiments.
  • Attachment of a linker to an anti-FR ⁇ antibody construct can be accomplished in a variety of ways, such as through surface lysines, reductive-coupling to oxidized carbohydrates, or through cysteine residues liberated by reducing interchain disulfide linkages.
  • attachment of a linker to an anti-FR ⁇ antibody construct may be achieved by modification of the antibody construct to include additional cysteine residues (see, for example, U.S. Patent Nos.
  • Linkers typically include a functional group capable of reacting with the target group or groups on the antigen binding construct and one or more functional groups capable of reacting with a target group on the drug moiety.
  • Suitable functional groups are known in the art and include those described, for example, in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press).
  • Non-limiting examples of functional groups for reacting with free cysteines or thiols include mal eimide, haloacetamide, haloacetyl, activated esters such as succinimide esters, 4- nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.
  • Also useful in this context are “self- stabilizing” maleimides such as those described in Lyon et al., 2014, Nat. Biotechnol, 32:1059- 1062.
  • Non-limiting examples of functional groups for reacting with surface lysines and amines include activated esters (such as N-hydroxy succinamide (NHS) esters and sulfo-NHS esters), imido esters (such as Traut’s reagent), isothiocyanates, aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)).
  • activated esters such as N-hydroxy succinamide (NHS) esters and sulfo-NHS esters
  • imido esters such as Traut’s reagent
  • isothiocyanates such as aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)).
  • DTPA diethylenetriaminepentaacetic anhydride
  • TSTU succinimido-l,l,3,3-tetra-methyluronium tetrafluoroborate
  • PyBOP benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate
  • Non-limiting examples of functional groups capable of reacting with an electrophilic group on the antibody construct or drug moiety include hydrazide, oxime, amino, hydrazine, thiosemi carbazone, hydrazine carboxylate and arylhydrazide.
  • a linker that includes a functional group that allows for bridging of two interchain cysteines on the antibody binding construct may be used, such as a ThioBridgeTM linker (Badescu et al., 2014, Bioconjug. Chem., 25:1124-1136), a dithiomal eimide (DTM) linker (Behrens et al, 2015, Mol. Pharm., 12:3986-3998), a dithioaryl(TCEP)pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802) or a dibromopyridazinedione-based linker (Maruani et al, 2015, Nat. Commun., 6:6645).
  • a ThioBridgeTM linker Bodescu et al., 2014, Bioconjug. Chem., 25:1124-1136
  • DTM dithiomal eimide
  • linkers for linking drugs to antibodies are known in the art, including hydrazone-, disulfide- and peptide-based linkers.
  • Linkers may be cleavable or non-cleavable.
  • a cleavable linker is typically susceptible to cleavage under intracellular conditions, for example, through lysosomal processes. Examples include linkers that are protease -sensitive, acid-sensitive or reduction-sensitive.
  • Non-cleavable linkers by contrast, rely on the degradation of the antibody in the cell, which typically results in the release of an amino acid-linker-drug moiety.
  • cleavable linker that may be useful in certain embodiments is a peptide- containing linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease.
  • Examples include dipeptide-containing linkers, such as those comprising the dipeptides Val-Cit, Phe-Lys, Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp- Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met- (D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, MesLys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys or Met-(D)Lys; tripeptide-containing linkers such as those comprising the tripeptides Met-C
  • Additional useful cleavable linkers include disulfide-containing linkers and linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers.
  • disulfide-containing linkers include, but are not limited to, N-succinimydyl-4-(2 -pyridyl di thio) butanoate (SPDB) and N-succinimydyl-4-(2-pyridyldithio)-2-sulfo butanoate (sulfo-SPDB).
  • Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group.
  • Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.
  • a further example of a cleavable linker is a linker comprising a ⁇ -glucuronide, which is cleavable by P-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf etal., 2002, Curr. Pharm. Des., 8: 1391-1403).
  • Cleavable linkers may optionally further comprise one or more additional functionalities such as self-immolative/self-elimination groups, stretchers or hydrophilic moi eties.
  • Self-immolative and self-elimination groups that find use in linkers include, for example, /?-aminobenzyl (PAB) and /?-aminobenzyloxycarbonyl (PABC) groups, methylated ethylene diamine (MED) and hemi-aminal groups.
  • PAB /?-aminobenzyl
  • PABC /?-aminobenzyloxycarbonyl
  • MED methylated ethylene diamine
  • hemi-aminal groups hemi-aminal groups.
  • Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PABC or PABE group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U.S. Patent No. 7,375,078.
  • linker may include one or more self- immolative/self-elimination groups, for example, a PABC group, a PABE group, or a combination of a PABC or PABE group and an MED.
  • Stretchers that find use in linkers for ADCs include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide.
  • Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) stretchers.
  • PEG and mPEG stretchers also function as hydrophilic moieties and may be particularly useful with hydrophobic drugs, although their use in linkers with other drugs is also contemplated in some embodiments.
  • a stretcher may have one of the following structures: wherein:
  • R is H or C1-C6 alkyl; t is an integer between 2 and 10, and u is an integer between 1 and 10.
  • linker, L is a cleavable linker having Formula II:
  • Z is a linking group that joins the linker to a target group on the anti-FR ⁇ antibody construct, A;
  • Str is a stretcher
  • AA1 and AA2 are each independently an amino acid, wherein AA1-[AA2] r forms a protease cleavage site;
  • X is a self-immolative group; q is 0 or 1; r is 1, 2 or 3; s is 0, 1 or 2;
  • # is the point of attachment to the anti-FR ⁇ antibody construct, A, and
  • % is the point of attachment to the drug moiety, D.
  • Z is where # is the point of attachment to A, and * is the point of attachment to the remainder of the linker.
  • Str is selected from: wherein:
  • R is H or C1-C6 alkyl; t is an integer between 2 and 10, and u is an integer between 1 and 10.
  • ADCs of Formula I may comprise a disulfide-containing linker.
  • linker, L is a cleavable linker having Formula III: wherein:
  • Z is a linking group that joins the linker to a target group on the anti-FR ⁇ antibody construct, A;
  • Q is -(CH2) P - or -(CH2CH2O) q -, wherein p and q are each independently an integer between 1 and 10; each R is independently H or C1-C6 alkyl; n is 1, 2 or 3;
  • # is the point of attachment to the anti-FR ⁇ antibody construct, A, and
  • % is the point of attachment to the drug moiety, D.
  • ADCs of Formula I may comprise a P-glucuronide-containing linker.
  • non-cleavable linkers are known in the art for linking drugs to antibodies and may be useful in the ADCs of the present disclosure in certain embodiments.
  • non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with the antibody, as well as a maleimido- or haloacetyl -based moiety for reaction with the drug, or vice versa.
  • An example of such a non-cleavable linker is based on sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l -carboxylate (sulfo-SMCC).
  • Sulfo- SMCC conjugation typically occurs via a mal eimide group which reacts with sulfhydryls (thiols, — SH), while the sulfo-NHS ester is reactive toward primary amines.
  • linkers include those based on N-succinimidyl 4- (maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)- cy cl ohexane-1 -carboxy -(6 -amidocaproate) (“long chain” SMCC or LC-SMCC), K- maleimidoundecanoic acid N-succinimidyl ester (KMUA), y-maleimidobutyric acid N- succinimidyl ester (GMBS), 8-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m- mal ei mi mim,
  • ADCs comprising an anti-FR ⁇ antibody construct as described herein may be prepared by one of several routes known in the art, employing standard organic chemistry reactions, conditions, and reagents (see, for example, Bioconjugate Techniques (G.T.
  • conjugation may be achieved by (1) reaction of a functional group of an antibody construct with a bivalent linker reagent, to form antibody -linker intermediate A-L, via a covalent bond, followed by reaction with an activated drug moiety D; or (2) reaction of a functional group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with a functional group of an antibody construct.
  • Conjugation methods (1) and (2) may be employed with a variety of antibody constructs, drug moi eties, and linkers to prepare the ADCs described here.
  • linkers, linker components and drugs are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, for example, March’ s Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki etal., 2002, J. Org. Chem., 67: 1866-1872; Frisch et al., 1997 , Bioconj. Chem., 7 : 180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press), and Antibody-Drug Conjugates: Methods in Molecular Biology (Ducry (Ed.), 2013, Springer)).
  • drug- linkers suitable for reaction with a selected antibody construct are also available commercially, for example, drug- linkers comprising DM1, DM4, MMAE, MMAF or Duocarmycin SA are available from Creative BioLabs (Shirley, NY).
  • drug- linkers comprising DM1, DM4, MMAE, MMAF or Duocarmycin SA are available from Creative BioLabs (Shirley, NY).
  • Various antibody drug conjugation services are also available commercially from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL) and Piramal Pharma Solutions (Grangemouth, UK).
  • the ADCs once prepared, may be purified by standard techniques such as chromatography (for example, HPLC, size-exclusion, adsorption, ion exchange and/or affinity capture), dialysis and/or tangential flow filtration.
  • chromatography for example, HPLC, size-exclusion, adsorption, ion exchange and/or affinity capture
  • dialysis dialysis and/or tangential flow filtration.
  • Certain aspects of the present disclosure relate to the therapeutic or diagnostic use of the anti-FR ⁇ antibody constructs and ADCs.
  • FR ⁇ is overexpressed in a wide variety of cancers and certain embodiments of the present disclosure thus relate to the methods of using the anti-FR ⁇ antibody constructs and ADCs in the treatment or diagnosis of an FR ⁇ -positive cancer.
  • Certain embodiments relate to methods of inhibiting the growth of FR ⁇ -positive tumor cells comprising contacting the cells with an anti-FR ⁇ antibody construct or ADC described herein.
  • the cells may be in vitro or in vivo.
  • the anti-FR ⁇ antibody constructs and ADCs may be used in methods of treating an FR ⁇ -positive cancer or tumor in a subject.
  • Cancers that overexpress FR ⁇ are typically solid tumors. Examples include, but are not limited to, ovarian cancer, endometrial cancer, lung cancers (such as non -small cell lung cancer (NSCLC)), mesothelioma, breast cancer (including triple negative breast cancer (TNBC)), colorectal cancer, biliary tract cancer, pancreatic cancer and esophageal cancer.
  • lung cancers such as non -small cell lung cancer (NSCLC)
  • mesothelioma such as non -small cell lung cancer (NSCLC)
  • breast cancer including triple negative breast cancer (TNBC)
  • colorectal cancer colorectal cancer
  • biliary tract cancer pancreatic cancer and esophageal cancer.
  • Certain embodiments of the present disclosure relate to methods of treating a FR ⁇ -positive cancer with an anti-FR ⁇ antibody construct or ADC as described herein, where the cancer is ovarian cancer, endometrial cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), mesothelioma, breast cancer, colorectal cancer, biliary tract cancer, pancreatic or esophageal cancer.
  • the anti-FR ⁇ antibody construct or ADC as described herein may be useful in treating triple negative breast cancer (TNBC).
  • Treatment of an FR ⁇ -positive cancer may result in one or more of alleviation of symptoms, shrinking the size of the tumor, inhibiting growth of the tumor, diminishing one or more direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, improving survival, increasing progression-free survival, remission and/or improving prognosis.
  • the anti-FR ⁇ antibody constructs or ADCs when used in the treatment of cancer, may be administered systemically to the subject to be treated, for example, by bolus injection or continuous infusion into the subject’s bloodstream. In certain embodiments, when used in the treatment of cancer, the anti-FR ⁇ antibody constructs or ADCs may be administered to the subject locally at the site to be treated.
  • the anti-FR ⁇ antibody constructs or ADCs may be used alone or in combination with one or more known chemotherapeutic or immunotherapeutic agents typically used in the treatment of cancer. Combinations of the anti-FR ⁇ antibody constructs or ADCs with standard chemotherapeutics or immunotherapeutics may act to improve the efficacy of the chemotherapeutic or immunotherapeutic and, therefore, may improve standard cancer therapies. This application can be important in the treatment of drug-resistant cancers which are not responsive to standard treatment.
  • the anti-FR ⁇ antibody construct or ADC may be administered prior to, or after, administration of the chemotherapeutic or immunotherapeutic agents, or they may be administered concomitantly.
  • the dosage of the the anti-FR ⁇ antibody construct or ADC to be administered is not subject to defined limits, but it will a therapeutically effective amount.
  • a “therapeutically effective amount” refers to that amount of an anti-FR ⁇ antibody construct or ADC described herein which, when administered to a subject, is sufficient to effect a treatment of the particular indication.
  • a therapeutically effective amount of anti-FR ⁇ antibody construct or ADC in respect of cancer treatment may, for example, have one or more of the following effects: reduce the number of cancer cells, reduce the tumor size, inhibit cancer cell infiltration into peripheral organs, inhibit tumor metastasis, inhibit tumor growth; increase survival time and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • efficacy may alternatively be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • Certain embodiments relate to methods of detecting the presence of FR ⁇ in a biological sample, such as a sample comprising cells or tissue, using an anti-FR ⁇ antibody construct described herein.
  • the biological sample may have been taken from a patient, for example, a patient known or suspected to have a cancer.
  • Some embodiments relate to methods of detecting the presence of FR ⁇ in a biological sample that comprise contacting the sample with an anti-FR ⁇ antibody construct described herein.
  • Certain embodiments relate to methods of diagnosing a disorder associated with increased expression of FR ⁇ , such as a cancer, using an anti-FR ⁇ antibody construct described herein.
  • the method of diagnosis may be an in vivo method in which the anti-FR ⁇ antibody construct is administered to the subject, or it may be an in vitro method in which a sample taken from the subject is contacted with the anti-FR ⁇ antibody construct.
  • administration may be systemic or local.
  • the anti-FR ⁇ antibody construct may be labelled with a detectable label, such as a fluorescent, luminescent, chromophoric, chemiluminescent, radioactive or enzymatic label as is known in the art.
  • a detectable label such as a fluorescent, luminescent, chromophoric, chemiluminescent, radioactive or enzymatic label as is known in the art.
  • the anti-FR ⁇ antibody constructs and ADCs may be provided in the form of pharmaceutical compositions comprising the anti-FR ⁇ antibody construct or ADC and a pharmaceutically acceptable carrier or diluent.
  • the compositions may be prepared by known procedures using well-known and readily available ingredients.
  • compositions may be formulated for administration to a subject by, for example, parenteral, oral (including, for example, buccal or sublingual), topical, rectal or vaginal routes, or by inhalation or spray.
  • Parenteral oral
  • parenteral including, for example, buccal or sublingual
  • Topical rectal or vaginal routes, or by inhalation or spray.
  • parenteral administration may be subcutaneous injection, or intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal or intrathecal injection or infusion.
  • the pharmaceutical composition will typically be formulated in a format suitable for administration to the subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution.
  • Pharmaceutical compositions may be provided as unit dosage formulations.
  • compositions comprising the anti-FR ⁇ antibody constructs or ADCs may be formulated for parenteral administration by infusion or in a unit dosage injectable form, for example as lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed.
  • examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3 -pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • compositions comprising the anti-FR ⁇ antibody constructs or ADCs may be in the form of a sterile injectable aqueous or oleaginous solution or suspension.
  • a sterile injectable aqueous or oleaginous solution or suspension Such suspensions may be formulated using suitable dispersing or wetting agents and/or suspending agents that are known in the art.
  • the sterile injectable solution or suspension may comprise the anti-FR ⁇ antibody construct or ADC in a non-toxic parentally acceptable diluent or solvent.
  • Acceptable diluents and solvents that may be employed include, for example, 1,3 - butanediol, water, Ringer’s solution or isotonic sodium chloride solution.
  • sterile, fixed oils may be employed as a solvent or suspending medium.
  • various bland fixed oils may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Adjuvants such as local anesthetics, preservatives and/or buffering agents may also be included in the inj ectable solution or suspension.
  • compositions comprising the anti-FR ⁇ antibody constructs or ADCs may be formulated for intravenous administration to a subject, for example a human.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and/or a local anaesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”),' Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).
  • kits comprising an anti-FR ⁇ antibody construct or ADC as described herein.
  • the kit typically will comprise a container holding the anti-FR ⁇ antibody construct or ADC and a label and/or package insert on or associated with the container.
  • the label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the label or package insert may further include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration.
  • the container may have a sterile access port.
  • the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle.
  • the kit may optionally comprise one or more additional containers comprising other components of the kit.
  • a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution or dextrose solution
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline Ringer’s solution or dextrose solution
  • dextrose solution other buffers or diluents.
  • Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • one or more components of the kit may be lyophilized or provided in a dry form, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component s).
  • the kit may further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.
  • Biological Assays Expression levels of FR ⁇ in the cell lines and CDX models was assessed in-house using a research level IHC assay and assigned a relative expression level (high/mid/low or strong/moderate/weak). PDX models were assessed similarly using archival tumor samples.
  • Antibodies that specifically bind folate receptor alpha (FR ⁇ ) were generated by immunizing rabbits with human FR ⁇ antigen, isolated and sequenced as described below.
  • Antibodies to FR ⁇ were raised in rabbits immunized with soluble HIS-tagged human folate receptor 1 antigen (FR ⁇ -HIS) (ACROBiosy stems, Newark, DE; Cat# FO1-H82E2). Briefly, two New Zealand White rabbits were immunized with a primary boost consisting of 200 pg of FR ⁇ -HIS antigen mixed with Alum (5 mg/injection)/CpG (10 pg/inj ection) administered subcutaneously at 3 sites (0.333 mL/site) along the rabbit's dorsal body.
  • FR ⁇ -HIS human folate receptor 1 antigen
  • Anti-human FR ⁇ antibody titers were determined by flow cytometry using CHO cells expressing human FR ⁇ . Briefly, CHO cells were transiently transfected with a pTT5-based expression plasmid (National Research Council of Canada) encoding human FR ⁇ according to manufacturer’ s instructions for LipofectamineTM 2000 (Thermo Fisher Scientific Corp., Waltham, MA). A dilution of immunized rabbit sera starting at 1 :400 and serially diluted 1 :2 over 11 points was incubated with 50,000 CHO cells transiently expressing human FR ⁇ for 30 minutes.
  • pTT5-based expression plasmid National Research Council of Canada
  • LipofectamineTM 2000 Thermo Fisher Scientific Corp., Waltham, MA
  • Immunized rabbits with desired titers above 100,000 were sacrificed, and the spleens harvested.
  • the lymphoid cells were dissociated by grinding in FACS buffer (PBS, 2% v/v FBS) to release the cells from the tissues.
  • the cells were pelleted and then suspended for 1 minute in 5 mL of Pharm LyseTM (Becton, Dickinson & Co., Franklin Lakes, NJ) to lyse red blood cells. Equal volume of FACS buffer was added to neutralize the Pharm LyseTM and the resultant lymphocyte sample was pelleted and resuspended in FACS buffer.
  • the lymphocyte suspension was then stained with goat anti -rabbit IgG Alexa Fluor -647 (Jackson Immuno Research Labs, West Grove, PA) to identify IgG+ B cells. After 30 minutes of staining, IgG+ B cells were sorted on a FACSAriaTM (Becton, Dickinson & Co., Franklin Lakes, NJ) and counted.
  • IgG+ B cells were sorted on a FACSAriaTM (Becton, Dickinson & Co., Franklin Lakes, NJ) and counted.
  • Selected Lymphocyte Antibody Method (SLAM) (Babcook et al., 1996, Proc Natl Acad Sci USA, 93(15):7843— 7848), B cells were plated at different densities ranging from single cell up to 50 cells in a 384 well plate, expanded in culture for 7 days and the supernatants harvested for detection of anti -human FR ⁇ antibodies. The 384 well plates were stored at -80°C.
  • a subsequent PCR reaction was then performed on these unique sequences using V- segment family and J-segment family-specific primers.
  • the resulting amplicons were cloned into pTT5-based expression plasmids (National Research Council of Canada).
  • Unique heavy chain sequences and light chain sequences emerging from a single well sample were co-expressed in HEK293-6E cells (National Research Council of Canada) in all possible combinations to determine the correct heavy and light chain pairing.
  • Antibodies produced were assayed for binding to antigen that was transiently expressed on HEK293 cells.
  • Coding sequences for antibody variable regions were cloned in frame into a huIgGl expression and a huCK expression vector (based on the pTT 5 vector).
  • the huIgGl constant region starts at alanine Kabat-118 and huCK constant region starts at arginine Kabat-108.
  • the activities of the resultant recombinant chimeric antibodies were confirmed in specificity binding assays and were found comparable to the parental ones.
  • EXAMPLE 2 HUMANIZATION OF ANTI-FR ⁇ ANTIBODY
  • the CDR sequences of v23924 are provided in Table 2.1, and the VH and VL sequences are provided in Table 2.2. Humanization was conducted as described below.
  • Monoclonal antibody (mAb) variants were then assembled such that in cycle one, each of the humanized heavy chains was paired with each of the humanized light chains to provide 30 humanized variants and in cycle two, additional humanized heavy chains were paired with select humanized light chains to give an additional 15 humanized variants, for a total of 45 humanized variants to be evaluated experimentally.
  • Aversion of v23924 that contained a HetFc instead of a HomoFc was also produced (variant v30618). All constructs included two identical kappa light chains.
  • the two identical full-length heavy chains comprised by the HomoFc region contained the human CHl-hinge-CH2-CH3 domain sequence of IGHGl *01 (SEQ ID NO: 146; see Table
  • heterodimeric full-length heavy chains comprised by the HetFc region contained the human CHl -hinge-CH2-CH3 domain sequence of IGHGl *01 with the following mutations in the Fc region:
  • HetFc-A T350V_L351Y_F405A_Y407V
  • HetFc-B T350V T366L K392L T394W
  • Each of the humanized VH domain sequences from cycle one was appended to the human CHl-hinge-CH2-CH3 (HetFc-A and HetFc-B) domain sequence of IGHGl *01 to provide twelve humanized full heavy chain sequences (six humanized VH x2).
  • Rabbit VH and each of the additional five humanized VH domain sequences from cycle two were appended to the human CHl-hinge-CH2-CH3 domain sequence of IGHGl *01, to provide the parental rabbit-human chimeric full heavy chain sequence and five additional humanized full heavy chain sequences.
  • Each of the VL domain sequences was appended to the human kappa CL sequence of IGKC*01 to provide five humanized light chain sequences.
  • Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 150) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842)) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into a pTT5 vector to produce heavy chain expression vectors.
  • Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA.
  • the heavy and light chains of each of the humanized antibody variants were expressed in 200 ml cultures of CHO-3E7 cells. Briefly, CHO-3E7 cells, at a density of 1.7-2 x 10 6 cells /ml, viability >95%, were cultured at 37°C in FreeStyleTM F17 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 4 mM glutamine (GE Life Sciences, Marlborough, MA) and 0.1% Pluronic® F-68 (Gibco/ Thermo Fisher Scientific, Waltham, MA).
  • a total volume of 200 ml CHO-3E7 cells + lx antibiotic/antimycotics (GE Life Sciences, Marlborough, MA) was transfected with a total of 200 ug DNA (100 ug of antibody DNA and 100 ug of GFP/AKT/stuffer DNA) using PEI -MAX® (Polyscience, Inc., Philadelphia, PA) at a DNA:PEI ratio of 1 :4 (w/w).
  • Protein A purification was performed in batch mode or using ImL HiTrapTM MabSelectTM SuReTM columns (Cytiva, Marlborough, MA).
  • batch mode clarified supernatant samples were incubated in batch with mAb Select SuReTM resin (GE Healthcare, Chicago, IL) cleaned-in-place (CIP’d) with NaOH and equilibrated in Dulbecco’s PBS (DPBS). Resin was poured into CIP’d columns, the columns were washed with DPBS.
  • protein was eluted with 100 mM sodium citrate buffer pH 3.0.
  • eluted fractions were pH adjusted by adding 10% (v/v) IM HEPES (pH -10.6-10.7) to yield a final pH of 6-7.
  • Samples were buffer exchanged into DPBS. Protein was quantitated based on absorbance at 280nm (A280 nm).
  • Parental rabbit-human antibody chimera variants (v23924 and v30618) were further purified by preparatory SEC chromatography on a Superdex 200 Increase 10/30 column (GE Healthcare, Chicago, IL) in DPBS mobile phase following Protein A purification.
  • Fig. 2 A and 2C show the Caliper electrophoresis results for the parental chimeric antibody v23924 and a representative humanized variant, v30384.
  • NR non-reducing
  • R reducing
  • UPLC-SEC was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 pm particles) (Waters LTD, Mississauga, ON) set to 30°C and mounted on a Waters Acquity UPLCTM H-Class Bio system with a photodiode array (PDA) detector.
  • the mobile phase was Dulbecco’s phosphate buffered saline (DPBS) with 0.02% Tween 20 pH 7.4 and the flow rate was 0.4 ml/min. Total run time for each injection was 7 min with a total mobile phase volume of 2.8 mL.
  • Fig. 2B and 2D show the UPLC-SEC profiles for the parental chimeric antibody v23924 (post SEC purification) and for a representative humanized antibody v30384 (post Protein A purification, respectively.
  • the UPLC-SEC profile for the representative humanized antibody sample reflected high species homogeneity, comparable to the parental chimeric antibody sample.
  • the samples from the remaining humanized antibody variants had similar profiles to that shown for the representative humanized antibody sample.
  • hFR ⁇ antigen binding was assessed using the Octet® RED96 system (ForteBio, Fremont, CA) by cycling through the following steps: loading of antibodies (0.9 pg/mL) onto anti-human IgG Fc capture (AHC) biosensors over 200s; stabilization of baseline for 60s; association to recombinant His-tagged human FR ⁇ (ACROBiosystems, Newark, DE) at multiple relevant concentrations spanning the expected KD for 400-500s; recordation of dissociation for 500-1000s; and regeneration performed by cycling 3 times between 10 mM glycine pH 1.5 (15s) and the assay buffer (Is) before proceeding to the next antibody.
  • AHC anti-human IgG Fc capture
  • the assay buffer used was KB buffer (kinetics buffer, composed of PBS pH 7.4, 0.1 % BSA, 0.02 % Tween 20, 0.05% sodium azide) supplemented with 0.06% Tween 20 and in some instances also with 1% BSA.
  • the experiment was conducted at 30°C with a shake speed of 1000 rpm.
  • HetFc heterodimeric Fc region
  • HomoFc homodimeric Fc region (see Example 2)
  • 2 n 2
  • the avidity of antigen binding for parental and selected humanized variants in FSA format was assessed by surface plasmon resonance (SPR) as described below.
  • SPR surface plasmon resonance
  • the SPR assay for determination of hFR ⁇ affinity and avidity of the parental chimeric antibody (v23924) and the humanized variants was carried out on a BiacoreTM T200 SPR system with PBS-T (PBS + 0.05% (v/v) Tween 20) running buffer (with 0.5 M EDTA stock solution added to 3.4 rnM final concentration) at a temperature of 25°C.
  • CM5 Series S sensor chip, BiacoreTM amine coupling kit (NHS, EDC and 1 M ethanolamine), and 10 mM sodium acetate buffers were purchased from GE Healthcare Life Science (Mississauga, ON, Canada).
  • PBS running buffer with 0.05% Tween20 (PBST) was purchased from Teknova Inc. (Hollister, CA).
  • Recombinant human FR ⁇ was purchased from ACRObio systems (Newark, DE).
  • OAA one armed antibody
  • FSA full size antibody
  • OAA antibody samples are equivalents of respective FSA samples and were produced in the similar manner to FSA samples described in Example 2.
  • Example 3 The apparent purity of the humanized antibody variants from Example 3 was assessed using mass spectrometry after Protein A purification (Example 2) and non-denaturating deglycosylation.
  • the antibody variant samples contained Fc N-linked glycans only
  • the samples were treated with N-glycosidase F (PNGase-F) only.
  • PNGase-F N-glycosidase F
  • the purified samples were de-glycosylated with PNGaseF as follows: 0.1U PNGaseF/pg of antibody in 50mM Tris-HCl pH 7.0, overnight incubation at 37°C, final protein concentration of 0.48 mg/mL. After deglycosylation, the samples were stored at 4°C prior to LC-MS analysis.
  • the deglycosylated protein samples were analyzed by intact LC-MS using an Agilent 1100 HPLC system coupled to an LTQ-OrbitrapTM XL mass spectrometer (ThermoFisher, Waltham, MA) (tuned for optimal detection of larger proteins (>50kDa)) via an Ion Max electrospray source.
  • the samples were injected onto a 2.1 x 30 mm Poros R2 reverse phase column (Applied Biosystems Corp., Waltham, MA) and resolved using a 0.1% formic acid aq/acetonitrile (degassed) linear gradient consisting of increasing concentration (20-90%) of acetonitrile.
  • the column was heated to 82.5°C and solvents were heated pre-column to 80°C to improve protein peak shape.
  • the cone voltage (source fragmentation setting) was approximately 40 V
  • the FT resolution setting was 7,500
  • the scan range was m/z 400-4,000.
  • the LC-MS system was evaluated for IgG sample analysis using a deglycosylated IgG standard (Waters IgG standard) as well as a deglycosylated mAb standard mix (25:75 half:full sized antibody).
  • Example 2 which results in the presence of some half-antibodies.
  • Fig. 4 depicts LC/MS profile for two representative humanized variants, v30384 and v31422. In the LC/MS profiles of all samples, a side peak of ⁇ +266Da was observed, which is likely an artifact of the analysis.
  • EXAMPLE 5 THERMAL STABILITY OF HUMANIZED ANTI-FR ⁇ ANTIBODIES
  • the thermal stability of the humanized antibody variants was assessed by differential scanning calorimetry (DSC) as described below.
  • DSC differential scanning calorimetry
  • 400 ⁇ L of purified samples primarily at concentrations of 0.4 mg/mL in PBS were used for DSC analysis with a VP -Capillary DSC (GE Healthcare, Chicago, IL).
  • VP -Capillary DSC GE Healthcare, Chicago, IL
  • 5 buffer blank injections were performed to stabilize the baseline, and a buffer injection was placed before each sample injection for referencing.
  • Each sample was scanned from 20°C to 100°C at a 60°C/hr rate, with low feedback, 8 sec filter, 3 min pre-scan thermostat, and 70 psi nitrogen pressure.
  • the resulting thermograms were referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA) to determine melting temperature (Tm) as an indicator of thermal stability.
  • Fab Tm values determined for the humanized variants are shown in Table 5.1. All humanized variants exhibited increased thermal stability compared to the parental antibody, v23924 (Fab Tm of ⁇ 72 °C), with Fab Tm values ranging from ⁇ 81-84°C. Of the humanized variants, v30394 showed the highest thermal stability.
  • the isoelectric point of the humanized antibody variants was determined by capillary isoelectric focusing (cIEF) as described below.
  • cIEF was carried out using CE-UV Agilent 7100 Capillary Electrophoresis (CE) system. 5ug (or maximal 2.5uL) of sample was applied to the capillary (ampholytes range of 3.0-10.0). pl markers mix, 4.1, 4.22, 5.5, 7.0 and 10.0 for system suitability tests and 4.1 and 10.0 for sample analyses were used.
  • the Agilent 7100 CE system equipped with an external water bath set to 6°C, the detector filter assembly (280nm) and 9 bar external pressure were used for all CE runs.
  • the neutral coated capillary (fluorocarbon) was cut at both ends at a distance of 8.5 cm and 24.5 cm from the detection window, respectively, equipped with a green alignment interface and fitted into the Agilent capillary cassette.
  • capillaries were conditioned as follows: high pressure flush at 3.5 bar with 350 mM acetic acid for 5 minutes, with water for 2 minutes and with cIEF gel for 5 minutes.
  • capillaries were conditioned as follows: high pressure flush at 3.5 bar with 4.3 M urea solution for 3 minutes and with water for 2 minutes. Samples were inj ected by applying 2 bar high pressure for 100 seconds, followed by a water dip of both inlet and outlet electrode.
  • Focusing was performed for 10 minutes at 25 kV with 200 mM phosphoric acid as anolyte and 300 mM NaOH as catholyte. Using chemical mobilization, the outlet vial was exchanged for 350 mM acetic acid and 30 kV was applied for 30 minutes. After each run, a high pressure flush at 3.5 bar with water was performed for 2 minutes. Manual integration for peaks RT and electropherograms were obtained using Agilent OpenLAB Intelligent Reporting A.01.06. I l l software. Raw data (signal vs retention time) were exported to a CVS file and main isoform pl, pl range and pl at the center of mass were calculated (based on the internal pl markers) in Microsoft Excel.
  • EXAMPLE 7 CHROMATOGRAPHIC ANALYSIS OF ANTI-FR ⁇ ANTIBODIES
  • HIC hydrophobic interaction chromatography
  • SEC size exclusion chromatography
  • Analytical SEC was performed using an Agilent Infinity II 1260 HPLC with Advance Bio SEC column (300 A, 2.7 pm, 7.8 x 150 mm) equilibrated with 5 column volumes of Mobile Phase A (150 mM ISfePO4, pH 6.95) at room temperature. Typically, 20-30 pg of sample at 2-3 mg/mL concentration was eluted isostatically for 7 mins at 1 mL/min and absorbance was monitored at A280. Chromatograms were integrated to provide complete, baseline-to-baseline integration of each peak, with reasonably placed separation between partially resolved peaks.
  • the peak corresponding to the major component for IgG was reported as the monomer based on the SEC profile of control trastuzumab. Any peak occurring prior to 3.3 min was designated as high molecular weight species (HMWS), and any peak occurring after 3.3 min was designated as low molecular weight species (LMWS), excluding solvent peaks (over 5.2 min). 7.3 Results
  • HIC retention time HIC-RT
  • SEC monomer % SEC monomer % of parental and humanized variants are provided in Table 7.2.
  • HIC and SEC showed favourable biophysical behaviour of all humanized variants.
  • Parental chimeric antibody v23924 eluted at 6.5 mins on HIC gradient, whereas all humanized variants eluted between 6.0-6.7 mins.
  • SEC profiles showed >90% monomer for all humanized variants, with variant v30389 having the lowest monomer % (94%). All these variants had ⁇ 5% HMWS and ⁇ 5% LMWS.
  • Selected humanized variants (v30389, v30394, v30399, v31423 and v31424) underwent a 40°C stability study for a duration of 14 days at ⁇ lmg/ml sample concentration in PBS pH 7.4 buffer. Samples were characterized at timepoints 0, 5, 7, 11 and 14 days. An acid stability study was performed upon buffer exchange of the sample into acetate buffer of pH 3.6 at varied sample concentrations for a duration of Aligina, with characterization at timepoints 0, 15, 30 and 60 min. Additionally, freeze-thaw (from -80°C to room temperature) constituting three cycles of 30 min per cycle was performed at ⁇ lmg/ml sample concentration in PBS pH7.4.
  • EXAMPLE 9 FUNCTIONAL CHARACTERIZATION OF ANTI-FR ⁇ ANTIBODIES - FR ⁇ SPECIFICITY
  • FRq, FOLR2 and FOLR4 binding Briefly, HEK293-6e cells were transfected for ⁇ 24 hours to transiently express human FR ⁇ (Cat. No. 13420), FOLR2 (Cat. No. 13481) and FOLR4 (Cat. No. 13483) (all from GenScript Biotech, Piscataway, NJ), 1 ug DNA per 1 million cells. Following transfection, 50,000 cells were seeded in V-bottom 96-well plates and incubated with 50 nM primary antibody for 45 min under standard culturing conditions. Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No.
  • FOLR3 binding ELISA 96-well plate was coated with commercial purified FOLR3 protein (R&D Systems, Inc., Minneapolis, MN; Cat. No. 5319-FR) for 1 hr at 37°C. The plate was blocked with 1% milk in PBS, pH 7.4, for 1 hr at RT. Following blocking, primary antibodies were added at 7 nM for 1 hr at RT. HRP -conjugated secondary antibody (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-035-098) was then added at 0.4 pg/ml, for 1 hr at RT. Plates were developed using tetramethylbenzidine (TMB) andHCl was used to stop reaction. Absorbance was read at 450 nm using a SynergyTM Hl microplate reader (BioTek Instruments, Winooski, VT).
  • TMB tetramethylbenzidine
  • Anti-FR ⁇ , anti-FOLR2, anti-FOLR3 and anti-FOLR4 control antibodies showed expected binding to the respective target proteins by flow cytometry or ELISA.
  • flow cytometry live singlet cell population was gated using FlowJoTM v8 software (BD Biosciences, Franklin Lake, NJ), and AF647 GeoMean and % positive binding was determined in this population for each antibody.
  • % positive binding of anti-FOLR3 and v23924 antibodies was determined using raw absorbance values compared to negative control absorbance signal. v23924 showed expected binding to human FR ⁇ , and did not exhibit binding cross reactivity to F0LR2, F0LR3 or F0LR4, indicating FR ⁇ specificity.
  • EXAMPLE 10 FUNCTIONAL CHARACTERIZATION OF ANTI-FR ⁇ ANTIBODIES - BINDING TO CYNOMOLGUS FR ⁇ [00273] The cross-reactivity of parental chimeric antibody v23924 to human and cynomolgus monkey FR ⁇ was assessed by flow cytometry using transfected CHO-S cells as described below. Palivizumab (anti -RS V) (v22277) was used as a negative control.
  • CHO-S cells were transfected for ⁇ 24 hours to transiently express human or cynomolgus monkey FR ⁇ , 1 ug DNA per 1 million cells. Following transfection, cells were seeded at 50,000 cells/well in V-bottom 96-well plates and treated with antibody for 24 hours at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti -human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4°C for 30 min. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessaTM Cell Analyzer (BD Biosciences, Franklin Lake, NJ).
  • EXAMPLE 11 FUNCTIONAL CHARACTERIZATION OF ANTI-FR ⁇ ANTIBODIES - CELLULAR BINDING
  • EXAMPLE 12 FUNCTIONAL CHARACTERIZATION OF ANTI-FR ⁇ ANTIBODIES - INTERNALIZATION
  • the receptor-mediated internalization capabilities of the parental chimeric antibody v23924 and a representative humanized variant, v30384, in FR ⁇ -expressing cell lines (IGROV-1 and OVCAR-3) were determined by high content imaging as described below.
  • the FR ⁇ -targeting antibodies mirvetuximab and farletuzumab were used as positive controls, and palivizumab (anti- RSV) (v22277) was used as a negative control.
  • antibodies were fluorescently labeled by coupling to an anti -human IgG Fc Fab fragment pHAb dye conjugate (Promega Corporation, Madison, WI; Cat. No. G9841) ( ⁇ 3 dye molecules per Fab fragment) at a 5: 1 molar excess for 24 hours at 4°C.
  • Cells were seeded and incubated overnight at 37°C in 5% CO2 in 96-well plates. Coupled antibodies were added to cells the following day and incubated at 37°C for 6-24 hours to allow for internalization. Following incubation, cells were stained with Dye Cycle Violet (ThermoFisher Scientific Corporation, Waltham, MA; Cat. No.
  • Fig. 5 A & 5B IGROV-1 cells
  • Fig. 6A & 6B OFVCAR-3 cells
  • Chimeric antibody v23924 and the humanized variant v30384 showed comparable levels of internalization in both IGROV-1 cells (high FR ⁇ ) and OVCAR-3 cells (moderate FR ⁇ ).
  • the chimeric antibody v23924 and the humanized variant v30384 showed increased internalization compared to mirvetuximab and farletuzumab positive controls across all tested concentrations (25 to 1 nM) and time points (6 and 24 hours).
  • humanized variant v30384 showed 3.3 and 6.1- fold increase in internalized fluorescence compared to mirvetuximab and farletuzumab, respectively, at 25 nM, and 2.6 and 19.1 -fold increase in internalized fluorescence compared to mirvetuximab and farletuzumab, respectively, at 5 nM (Fig. 5 A).
  • humanized variant v30384 showed 2.1 and 3.9-fold increase in internalized fluorescence compared to mirvetuximab and farletuzumab, respectively, at 25 nM, and 1.9 and 4.7-fold increase in internalized fluorescence compared to mirvetuximab and farletuzumab, respectively, at 5 nM (Fig. 5B).
  • High resolution epitope mapping of the parental chimeric antibody v23924 on human FR ⁇ antigen (hFR ⁇ ) was conducted by hydrogen/deuterium exchange mass spectrometry (HDX- MS) at NovoAb Bioanalytics Inc. (Victoria, BC. Canada) as described below.
  • Lyophilized hFR ⁇ was purchased from ACROBiosystems (Newark, DE; Catalogue no: FO1-H5229) and dissolved to a concentration of 2.5 mg/ml.
  • the antigen-antibody complex was prepared by mixing hFR ⁇ with the parental chimeric antibody v23924 at a molar ratio of 2:1. All samples had a pH of 7.4 and were clear (no precipitation was observed).
  • hFR ⁇ at a concentration of 10 pM was reduced with 100 mM of tri s-(2-carboxy ethyl) phosphine (TCEP) in the presence of 2 M guanidine at pH 2.4, and then digested with pepsin at an enzyme- to-protein molar ratio of 1 : 1.
  • HDX was initiated by mixing the protein samples with D2O buffer at a ratio of 2:8 (v/v). The resulting solutions were incubated at 26°C, and aliquots were taken at 20 s, 7 min, 1 h and 4 h, and instantly quenched by adding a 200 mM TCEP solution containing 4 M guanidine.
  • the mobile phase was 0.1% formic acid (A) and 100% acetonitrile/0.1% formic acid (B), and the peptides were separated by a 13 -minute gradient.
  • the MS survey scan was carried out within m/z 300-1600 range, with a mass resolution of 120,000 FWHM.
  • the Orbitrap detector was calibrated to be ⁇ 3 ppm error by using Calibration Mix (Calmix; ThermoFisher Scientific Corporation, Waltham, MA).
  • ETD electron transfer dissociation
  • fluoranthene radical anions were introduced into the ion trap over 50 ms.
  • Collision induced dissociation (CID) and ETD fragment ions were detected in the Orbitrap using a scan range of 150-2000 m/z.
  • the mass shift of the peptides and the deuteration status of individual amides were determined based on their centroid m/z values before and after H/D exchange. All HDX data were normalized to 100% D2O content (80% D exchange- in buffer for all the time points). Percent deuterium incorporation values were obtained by comparing the number of acquired deuterium to the total number of amide hydrogens contained in each peptide. The amide level deuteration information was calculated based on the deuterium uptake of the ETD fragments.
  • peptides have the same deuterium uptake behavior before and after antibody binding (Fig. 8A), suggesting the binding site (epitope) of the v23924 antibody is quite localized.
  • Fig. 8B three peptides (numbers 14, 15 and 16) showed significant lowering in deuterium uptake after v23924 binding, indicating they are in the epitope region.
  • the sequences ofthese peptides are WEDCRTSY (118-126) (SEQ ID NO: 151), WEDCRTSY (119- 126) (SEQ ID NO:152), and WEDCRTSYTCKSNWHKGWNWTSGF (119-142) (SEQ ID NO: 153), respectively.
  • the deuteration level of each amino acid was calculated and compared between hFR ⁇ and the v23924 complex (Fig. 9). Based on the results of the difference plot, the epitope residues were determined to be E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15 (i.e. the epitope sequence is: EDRTSY; SEQ ID NO: 154).
  • the humanized antibody v30384 (see Example 3) was affinity matured using the HuTargTM system (Innovative Targeting Solutions, Vancouver, BC, Canada). Genetic recombination was applied to the variable regions of the humanized variant v30384, and high affinity mutants were identified using next-generation sequencing (NGS).
  • NGS next-generation sequencing
  • variable domains were interspersed with RAG1/2 recombination signal sequences (RSS). These variable domains were synthesized at Integrated DNA Technologies, Inc. (Coralville, IA) and cloned into plasmid E951 (Innovative Targeting Solutions, Vancouver, BC, Canada).
  • HuTargTM cells were subjected to multiple rounds of FACS-based sorting on a BD FACSAriaTM Flow Cytometer (BD Biosciences, Franklin Lakes, NJ), with each round using a reduced amount of biotinylated soluble HIS-tagged FR ⁇ antigen (FR ⁇ -HIS; ACROBiosystems Newark, DE; cat# FO 1 -H82E2), detected with streptavidin conjugated to Al exaFluor -647 (Thermo Fisher Scientific Corp., Waltham, MA; cat# SI 1223).
  • HuTargTM cells that exhibited increased binding to biotinylated FR ⁇ -HIS were sorted directly to RNAzol RT (Sigma-Aldrich, St. Louis, MI; cat# R4533) in preparation for next-generation sequencing.
  • RNA from cells lysed in RNAzol was isolated as per manufacturer’s instructions. RNA was then digested with ezDNaseTM (Thermo Fisher Scientific Corp., Waltham, MA; cat# 11766051), and cDNA transcribed using SuperscriptTM IV (Thermo Fisher Scientific Corp., Waltham, MA; cat# 18090010) and a gene-specific primer. VH and VK domains were targeted for PCR amplification, and molecularly barcoded with NEBNext® UltraTM DNA Library Prep Kit (New England Biolabs, Ipswich, MA; cat# E7370L).
  • DNA sequences encoding mutated VH and VK domains were synthesized as “MiniGenes” (Integrated Technologies, Inc., Coralville, IA) and cloned into expression vectors to provide expression plasmids coding for complete human IgGl heavy chains and human kappa light chains, respectively.
  • Expression plasmids were matrixed with one another to pair every heavy chain plasmid with every light chain plasmid. This matrix was recombinantly expressed in Expi293TM cells (Thermo Fisher Scientific Corp., Waltham, MA; cat# A14635) according to manufacturer’s instructions to create 64 samples.
  • Protein G particles (Spherotech Inc., Lake Forest, IL) were coated with the humanized variant v30384 or affinity matured antibodies at a normalized concentration. Soluble human FR ⁇ was diluted to a limiting antigen concentration and incubated with antibody coated beads. FR ⁇ antigen binding and antibody capture was detected using AlexaFluor-647 conjugated streptavidin and AlexaFluor-488 conjugated goat anti-human IgG Fey (both from Jackson Laboratories, Bar Harbor, ME), respectively. Samples were analyzed by flow cytometry on a BD LSRFortessaTM Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). Geometric mean for FR ⁇ binding and antibody capture was analyzed for each sample. Antibody capture was normalized to FR ⁇ binding to affinity rank humanized variant v30384 against affinity matured antibodies.
  • Single point affinity ranking was performed by measuring the ratio of antibody captured on beads to the amount of antigen captured by antibody.
  • Variant v30384 binding was minimal (3x over background) using a human FR ⁇ concentration of 1.9 nM, while the majority of mutated variants exhibited higher binding ratios. Of 64 mutated variants, 3 variants showed a 4 -fold increase in binding ratio, and 10 showed an equivalent binding ratio.
  • EXAMPLE 15 ASSESSMENT OF AFFINITY OF AFFINITY MATURED ANTIBODIES FOR FR ⁇
  • Example 14 Ten of the affinity matured variants described in Example 14 were produced in full size antibody (FSA) format at WuXi Biologies (Hong Kong) Limited, China, via transient transfection in CHO-K1 cells and affinity capture purification with a subsequent polishing step (where necessary) involving primarily preparatory SEC or CEX chromatography, yielding greater than 97% sample purity by HPLC-SEC.
  • FSA format was similar to that of parental humanized variant v30384 except these variants comprised a HomoFc rather than HetFc.
  • the ten affinity matured variants were characterized for binding to hFR ⁇ using the Octet® RED96 system as described in Example 3.
  • EXAMPLE 16 CHROMATOGRAPHIC ANALYSIS OF AFFINITY MATURED ANTIBODIES
  • Example 7 The results are shown in Table 16.1. Affinity maturation of the antibody resulted in changes to the hydrophobicity/hydrophilicity as demonstrated by the variable HIC-RT. Antibody monomer content was above 97% in all cases and did not correlate with HIC-RT.
  • antibodies were fluorescently labeled by coupling to Fab-AF488 anti -Human IgG Fc labelling reagent (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-547-008) at a 1 : 1 molar ratio for 24 hours at 4°C.
  • Cells were seeded and incubated overnight at 37°C in 5% CO2 in 48-well plates. Coupled antibodies were added to cells the following day and incubated at 37°C for 24 hours to allow for internalization. Following incubation, cells were dissociated, washed and surface AF488 fluorescence was quenched using an anti-488 antibody at 10 OnM incubated at 4°C for 45 min.
  • AF488 fluorescence was analyzed by flow cytometry for all samples on a BD LSRFortessaTM Cell Analyzer (BD Biosciences, Franklin Lake, NJ), with 1,000 minimum events collected per well.
  • AF488/FITC-A GeoMean in live cell population was plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
  • Fig. 10 The results are shown in Fig. 10.
  • Parental humanized variant v30384 and the affinity matured variant v35356 showed comparable internalization in IGROV-1 (Fig. 10(A)) and JEG-3 (Fig. 10(B)) cells, when administered at 20 nM, at both 5 hour and 24 hour exposure.
  • ADCs Antibody-drug conjugates
  • DARs drug-to-antibody ratios
  • drug-linker DL1 (10 eq, 87 ⁇ L) as 20 rnM DMSO stock solution was added.
  • the conjugation reaction was mixed thoroughly by pipetting and reaction was allowed to proceed on ice for up to 1 h followed by quenching of excess drug-linker with 20 mM aqueous stock of N-acetyl cysteine (9 eq, 78 ⁇ L) for 30 mins prior to purification of the ADC from small molecules.
  • v23924 was also conjugated to drug-linkers DL2 and DL3 at DAR 4 using same conjugation procedure.
  • Fully reduced antibody was conjugated to 20 mM DMSO stock of drug-linker DL1 (18 eq, 94 ⁇ L) on ice for up to 1 h followed by quenching of excess drug-linker with 20 mM aqueous stock of N-acetyl cysteine (12 eq, 63 ⁇ L) for 30 mins prior to purification of the ADC from small molecules.
  • Fully reduced antibody was conjugated to 10 mM DMSO stock of drug-linker DL5 (15 eq, 208 ⁇ L) in presence of 10% DMSO (vol/vol) for up to 2 h at RT with continuous stirring in the dark followed by quenching of excess drug-linker with 10 mM aqueous stock of N-acetyl cysteine (20 eq, 274 ⁇ L) for 30 mins prior to purification of the ADC from small molecules.
  • EXAMPLE 20 PREPARATION OF ANTIBODY-DRUG CONJUGATES - LYSINE CONJUGATION
  • ADCs Antibody-drug conjugates
  • Table 20.1 Antibody-drug conjugates
  • DL6 A solution (207 ⁇ L) of the humanized antibody v30384 (1 mg) was reacted with 10 mM DMSO stock of drug-linker DL6 (14 eq, 9.7 ⁇ L) in PBS, pH 7.4. The conjugation reaction was mixed thoroughly by pipetting and the reaction allowed to proceed for up to 17 h at room temperature.
  • DL8 A solution (5.9 mL) of the humanized antibody v30384 (30 mg) was reacted with 20 mM DMSO stock of drug-linker DL8 (9.5 eq, 99 ⁇ L) in PBS, pH 7.4. The conjugation reaction was mixed thoroughly by pipetting and the reaction allowed to proceed for up to 18 hrs at room temperature .
  • Table 20.2 Drug-Linkers used in the Preparation of Lysine-Conjugated ADCs
  • ADCs prepared as described in Examples 19 and 20 were purified using an appropriate size 40 kD ZebaTM Spin Desalting Column (Thermo Fisher Scientific, Waltham, MA) pre- equilibrated with PBS, pH 7.4, or 10 mM Na-Acetate, pH 5.5. ADCs produced at >lmg scale, were sterile filtered (0.22 pm).
  • the purified ADCs were stored at 4°C and analyzed for total protein content using absorbance at 280 nm or using a bicinchonic acid (BCA) assay with reference to a standard curve generated from trastuzumab 1 mg/mL.
  • ADCs were also characterized by HPLC -HIC, SEC, CE- SDS and RP -HPLC -MS as described below.
  • the average DAR and DAR distribution for the ADCs were derived from HIC and LC-MS data. Endotoxin levels were assessed using the ToxinSensorTM Single Test Kit (Genescript BioTech, Piscataway, NJ; Cat# L00450) with a threshold set at 0.5 EU/mg. Residual free drug and drug-linker levels (%FD) were assessed by RP-HPLC-MS and calculated based on the following equation with a threshold set at 1 mol%DAR:
  • ADC samples were deglycosylated using Endo S for 1 hour at RT and injected onto an Agilent 1290 Infinity II LC coupled with an Agilent 6545 Quadrupole Time of Flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA). Protein species were separated using a PLRP-S column (1000 A, 8 uM, 50 x 2.1 mm) at a flow rate of 0.3 ml/min using the gradient shown in Table 21.2. Buffer A: 0.1% formic acid (FA), 0.025% trifluoracetic acid (TFA) and 10% isopropyl alcohol (IP A) in water. Buffer B : 0.1 % FA and 10% IPA in acetonitrile (ACN). Table 21.2: RP-HPLC-MS Gradient
  • MS source conditions are shown in Table 21.3 and the acquisition parameters were as follows: [00319] Mode'. MS; Mass Range'. 500 to 7000 m/z; Acquisition Rate. 1 spectra/s and 1000 ms/spectrum, 3354 transients/ spectrum.
  • Analytical SEC was performed using an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) with Advance Bio SEC column (300 A, 2.7 pm, 7.8 * 150 mm; Agilent Technologies) equilibrated with 5 column volumes of Buffer (150 mMNa2PC>4, pH 6.95) at room temperature. Typically, 20-30 pg of sample at 2-3 mg/mL concentration was eluted isostatically for 7 mins at 1 mL/min and absorbance monitored at A280. Chromatograms were integrated to provide complete, baseline-to-baseline integration of each peak, with reasonably placed separation between partially resolved peaks.
  • ND not determined 3 Small scale preparation, %FD and endotoxin not determined
  • ADCs comprising the humanized variant v30384 or the affinity-matured variant v35356 each conjugated to drug-linker DL1 (see Example 19) were determined in a panel of FR ⁇ -expressing cell lines as described below.
  • cells were seeded in 384-well plates at 1,000 cells/well density and treated with a titration of test article, generated in complete cell growth medium. Treated cells were incubated for 4 days under standard culturing conditions (37°C/5% CO2). After incubation, CellTiter-Glo® reagent (Promega Corporation, Madison, WI; Cat. No. G7570) was spiked in all wells and luminescence corresponding to ATP present in each well was measured using a SynergyTM Hl plate reader (BioTek Instruments, Winooski, VT).
  • % cytotoxicity values were calculated using ATP measurement RLU values (Relative Light Units) and plotted against test article concentration using GraphPad Prism 9 software (GraphPad Software, San Diego, CA).
  • EXAMPLE 23 INTRACELLULAR PAYLOAD RELEASE AND QUANTITATION
  • the intracellular payload delivery capabilities of representative ADCs in the cell -lines JEG-3 (high FR ⁇ ), Caov-3 (moderate FR ⁇ ), HEC-l-A (moderate FR ⁇ ), and H2110 (moderate/low FR ⁇ ) were assessed using mass spectrometry as described below.
  • the ADCs tested comprised the humanized variant v30384 or the affinity -matured variant v35356 each conjugated to drug-linker DL1 (see Example 19).
  • EXAMPLE 24 IN VIVO EFFICACY STUDIES - CHIMERIC ANTI-FR ⁇ ANTIBODY ADCs
  • tumor fragments were implanted subcutaneously to female nude mice.
  • mean tumor volume reached -100-250 mm 3
  • tumor cell suspensions (1 x10 7 cells in 0.1 ml 50% Matrigel®) were implanted subcutaneously into female CB.17 SCID mice.
  • mean tumor volume reached 100- 150 mm 3
  • tumor cell suspensions (1 x10 7 cells in 0.1 ml 50% Matrigel®) were implanted subcutaneously into NOD/SCID mice.
  • mean tumor volume reached -100 mm 3
  • tumor cell suspensions (5 x10 6 cells in 0.1 ml 50% Matrigel®) were implanted subcutaneously into nude mice.
  • mean tumor volume reached -150 mm 3
  • EXAMPLE 25 IN VIVO EFFICACY STUDIES - CHIMERIC AND HUMANIZED ANTI- FR ⁇ ANTIBODY ADCs
  • EXAMPLE 26 IN VIVO EFFICACY STUDIES - HUMANIZED ANTI-FR ⁇ ANTIBODY ADCs #1
  • control ADCs were v!7717 (mirvetuximab Fab with HetFc) conjugated to drug- linker DL6 (vl7717-DL6) and vl7716 (mirvetuximab Fab with HomoFc) conjugated to drug- linker DL6 (vl7716-DL6).
  • v31629 farletuzumab conjugated to drug- linker DL4 (v31629-DL4) was also included as a control.
  • v31629 farletuzumab conjugated to drug- linker DL4 (v31629-DL4) was also included as a control.
  • a linear mixed effects model was fit to log-transformed tumor volumes, followed by F-test for the null hypothesis that mean growth rates are equal and post -hoc pairwise comparisons.
  • tumor cell suspensions (1 x 10 7 cells in 0.1 ml 50% Matrigel®) were implanted subcutaneously into nude mice.
  • tumor cell suspensions (1 x10 7 cells in 0.1 ml 50% Matrigel®) were implanted subcutaneously into female nude mice.
  • mean tumor volume reached -100- 150 mm 3
  • v30384-DLl and V17716-DL6 both resulted in minor to moderate tumor growth inhibition across shallow dose responses, but these were not statistically significant (Fig. 14A).
  • v31629-DL4 dosed at 2.5 mg/kg resulted in a minor inhibition of tumor growth rate comparable to V17716-DL6 and v30384-DLl.
  • v30384-DL8 and V17716-DL6 resulted in a significant inhibition of tumor growth rate of 154% and 313%, respectively (p ⁇ 0.01).
  • V17716-DL6 resulted in significantly greater tumor growth rate inhibition compared to v30384- DL8 at both 4 mg/kg (132% and 21%) and 8 mg/kg (313% and 154%) dose levels (p ⁇ 0.01) (Fig. 14E).
  • EXAMPLE 27 IN VIVO EFFICACY STUDIES - HUMANIZED ANTI-FR ⁇ ANTIBODY ADCs #2
  • tumor cell suspensions (1 x10 7 cells in 0.1 ml 50% Matrigel®) were implanted subcutaneously into CB.17 SCID mice.
  • mean tumor volume reached -150 mm 3
  • v30384-DL5 resulted in a dose response across 1, 3 and 10 mg/kg (25%, 47% and 185% inhibition of tumor growth rate, respectively, significant (p ⁇ 0.01) at 3 and 10 mg/kg dose levels) (Fig. 15A).
  • v30384-DL5 resulted in a dose response across 0.3, 1, 3 and 10 mg/kg dose levels (Fig. 15B).
  • v30384-DL5 resulted in a dose response across 0.25, 0.75, 1.5 and 3 mg/kg dose levels, with the higher dose groups leading to sustained regressions with regrowth at -5 weeks post dose (Fig. 15C).
  • v30384-DL5 was dosed at 6 mg/kg, leading to lasting regressions (Fig. 15D).
  • EXAMPLE 28 PHARMACOKINETICS STUDIES
  • Test article total antibody and/or intact ADC concentrations were measured from mouse serum by 384 well plate ELISA.
  • a rabbit anti-Compound 1 capture antibody was used for ELISA of DL6 intact ADCs.
  • a mouse anti-DMl/4 capture antibody was used for ELISA of DL6 intact ADCs (Levena Biopharma, San Diego, CA).
  • Serum samples were applied after blocking, followed by application of detection antibody: goat anti-human IgG F(ab’)2 conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research Laboratories, West Grove, PA). Absorbance at 450 nm was measured after application of 3,3 ',5,5 '-tetramethylbenzidine (TMB) and HC1 quenching. Concentrations were determined with reference to standards (GraphPad Prism software (GraphPad Software, San Diego, CA)).
  • HRP horseradish peroxidase
  • v30384-DL5 demonstrated proportional PK across 1, 3 and 10 mg/kg doses in the OV90 model (Fig.l6G). Inthe H2110 model, v30384-DL5 demonstrated prolonged exposure at 10 mg/kg and greater clearance at lower doses (Fig. 16H).
  • EXAMPLE 29 PENETRATION OF ANTI-FR ⁇ ANTIBODIES IN MULTICELLULAR TUMOR SPHEROIDS
  • FR ⁇ -expressing cell line spheroids The ability of anti-FR ⁇ antibodies to penetrate FR ⁇ -expressing cell line spheroids was assessed according to the method described below.
  • Spheroids provide a three-dimensional organization of cells with layers of distinct cell populations and the formation of different gradients from the outer to the inner regions. Cell signaling is more complex in spheroids than in two- dimensional cell cultures. As a result of these features, spheroids have the potential to recapitulate drug resistance and metabolic adaptation.
  • variant v36675 The spheroid penetration capability of humanized variant v36675 was compared to mirvetuximab (vl7716) and non-FR ⁇ targeting control palivizumab (anti-RSV) (v22277).
  • Variant v36675 is the same as variant v30384 but comprises HomoFc rather than HetFc.
  • the cell line used was high FR ⁇ -expressing JEG-3 (placental choriocarcinoma).
  • Antibodies were fluorescently labeled by coupling to a Fab fragment AF488 conjugate targeting anti-Human IgG Fc (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109- 547-008) at a 1 :1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Cat. No. 10010-023), for 24 hours at 4°C.
  • JEG-3 cells were detached from culture vessels with Try ⁇ LETM Express Enzyme (IX) (Thermo Fisher Scientific, Waltham, MA) and counted using the Cellaca® MX high-throughput automated cell counter (Nexcelom Bioscience LLC, Lawrence, MA). Cells were diluted in complete growth medium (Minimum Essential Media supplemented with 10% Fetal Bovine Serum (both from Thermo Fisher Scientific, Waltham, MA)) and seeded at 3,000 cells/well into 96-well CellCarrier Spheroid ultra-low attachment plates (Perkin Elmer, Waltham, MA), centrifuged, and incubated for 3 days under standard culturing conditions to allow for spheroid formation and growth.
  • IX Try ⁇ LETM Express Enzyme
  • Fab-AF488 coupled antibodies were added to spheroids at a final concentration of 25 nM and incubated under standard culturing conditions for 4-96 hours. Following incubation, excess antibody was removed by adding 100 ⁇ L complete growth medium and removing 100 ⁇ L medium from the well, to a total three washes. Spheroids were treated with a solution of 1 pM Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA) and 100 nM anti- Alexa Fluor 488 antibody (Thermo Fisher Scientific, Waltham, MA; Cat. No. A-11094), and incubated at 37°C/5% CO2 for 2 hours.
  • 1 pM Hoechst 33342 Thermo Fisher Scientific, Waltham, MA
  • 100 nM anti- Alexa Fluor 488 antibody Thermo Fisher Scientific, Waltham, MA; Cat. No. A-11094
  • Imaging was performed using an Operetta CLSTM high content analysis system (Perkin Elmer, Waltham, MA) with confocal acquisition and 10x magnification air objective. A Z stack of 15 planes separated by 15 pm was acquired and the slice with greatest diameter representing the spheroid center slice was selected for 2D analysis. Image analysis was performed using Harmony® 4.5 software (Perkin Elmer, Waltham, MA). Briefly, spheroid identification was performed by applying a mask around Hoechst 33342 positive objects to one spheroid per well. The spheroid region was divided into subregions of concentric bands, each representing 10% area of the spheroid region. Mean AF488 fluorescence within each subregion band was quantified, corrected by subtracting the inner 10% mean AF488 fluorescence, and plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
  • results are summarized in Table 29.1 and Fig. 20.
  • the humanized antibody variant v36675 showed a greater degree of penetration than mirvetuximab into JEG-3 spheroids by AF488 intensity at each subregion band and by distance from spheroid edge to which AF488 fluorescence was detectable at all time points assessed.
  • Non-binding control palivizumab showed lesser AF488 signal throughout the spheroid compared to both anti-FR ⁇ antibodies at all time points assessed.
  • EXAMPLE 30 SPECIFICITY ASSESSMENT OF ANTI-FR ⁇ ANTIBODIES
  • Retrogenix Cell Microarray Technology (Charles River Laboratories, Wilmington, MA) was used to screen for any specific off-target binding interactions of the following anti-FR ⁇ antibodies: humanized variant v36675 and affinity matured variant v35356.
  • Retrogenix Cell Microarray Technology identifies interactions both with cell surface receptors and secreted proteins by screening test ligands for binding against a library of cDNA clones representing over 6,300 human proteins. These proteins include plasma membrane monomers, heterodimers (formed by co-expression of the separate subunits) and secreted proteins (expressed with an inert cell membrane tether). Each cDNA is spotted in duplicate onto specialized slides and overlaid with HEK293 cells. These cells become reverse-transfected, resulting in clusters of cells each overexpressing a different, individual protein (or heterodimeric complex).
  • test antibodies for the library screen i.e. the concentration at which a low level of background binding to fixed untransfected HEK293 cells and strong binding to cells over-expressing FR ⁇ was observed.
  • concentration at which a low level of background binding to fixed untransfected HEK293 cells and strong binding to cells over-expressing FR ⁇ was observed In the library screen phase, the test antibodies were screened as a pool against fixed HEK293 cells, individually expressing the -6,300 human proteins.
  • confirmation screen phase each library hit was re-expressed and re-tested with each test antibody individually using both fixed and live HEK293 cells.
  • slides were individually spotted with expression vectors encoding both ZsGreenl (for assessing transfection efficiency) and (1) in the pre-screen phase: human FR ⁇ or control receptors (EGFR and CD20); (2) in the library screen phase: the above described protein library individually arrayed in duplicate across a number of microarray slides (“slide sets”) with two replicate slides screened for each of the slide sets; or (3) in the confirmation screen phase: protein hits identified in the library screen or control receptors (EGFR and CD20) arrayed in duplicate.
  • each of the selected antibodies at 2, 5 or 20 pg/mL, control antibody (Rituximab biosimilar, which binds CD20) at Ipg/mL or PBS were added to the slides after cell fixation.
  • control antibody Rituximab biosimilar, which binds CD20
  • a pool of the two antibodies (variant v36675 at 20 pg/mL and variant v35356 at 5 pg/mL) was added to each slide after cell fixation.
  • Binding was detected using an Al exaFluor® 647 labelled anti-human IgG H+L (AF647 anti-hlgG H+L), followed by fluorescence imaging. Fluorescent images were analysed and quantitated (for transfection) using ImageQuantTM software (Version 8.2; GE Healthcare, Chicago, IL). A protein hit was defined as a duplicate spot showing an increased signal compared to background levels and was identified by visual inspection using the images gridded on the ImageQuantTM software. Hits were classified as strong, medium, weak or very weak by visual inspection by two experienced scientists based on the intensity of the duplicate spots.
  • antibodies were fluorescently labelled with Alexa Fluor 647 (AF647; ThermoFisher Scientific, Waltham, MA; Cat. No. A20006) according to manufacturer’s specifications prior to cell treatment.
  • Tumor cells were seeded at 50,000 cells/well in V-bottom 96-well plates and treated with unlabelled antibodies for 1 hour at 4°C to prevent internalization. Following incubation, cells were washed and fluorescently labelled antibodies were added for 1 hour at 4°C. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessaTM Cell Analyzer (BD Biosciences, Franklin Lake, NJ), with 1,000 minimum events collected per well.
  • AF647/APC-A GeoMean fluorescence signal geometric mean, proportional to anti-Human AF647 binding
  • % competition binding relative to untreated control and data was plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).
  • Chimeric antibody v23924 was found to compete with farletuzumab for FR ⁇ binding in the H2110 cell line, yielding close to 100% competition binding in both binding orientations (primary antibody farletuzumab and secondary fluorescently labelled antibody variant v23924, and primary antibody variant v23924 and secondary fluorescently labelled antibody farletuzumab).
  • Chimeric antibody v23924 was found not to compete for FR ⁇ binding with mirvetuximab in the H2110 cell line. These antibodies showed low levels of % competition in both binding orientations. In contrast, farletuzumab showed competition binding with mirvetuximab.
  • Chimeric antibody v23924 thus demonstrates a binding profile that is distinct from both mirvetuximab and farletuzumab.
  • EXAMPLE 32 ADDITIONAL FUNCTIONAL CHARACTERIZATION OF ANTI-FR ⁇ ANTIBODIES
  • Antibodies were fluorescently labeled by coupling to an anti -human IgG Fc Fab fragment AF488 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-547-008) at a 1 :1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Cat. No. 10010-023) for
  • Quenched AF488 fluorescence was detected by flow cytometry on a BD LSRFortessaTM Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well.
  • the AF488/FITC-A GeoMean fluorescence signal geometric mean, proportional to anti -Human Fab AF488 labelling
  • the humanized antibody variant v30384 showed comparable internalized fluorescence to the biparatopic antibody v36264 (1.1 -fold vs. v30384), while mirvetuximab (vl7716) showed a markedly lower degree of internalized fluorescence (0.5-fold vs. v30384).
  • v30384-DLl was similar in potency to V17716-DL1 (EC50 0.18 and 0.17 nM, respectively), while the biparatopic ADC, v36264-DLl, demonstrated slightly lower potency (0.35 nM).

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Abstract

Constructions d'anticorps qui se lient au récepteur alpha du folate humain (FRα ou FOLR1) et conjugués anticorps-médicament (ADC) comprenant une construction d'anticorps anti-FRα conjuguée à un médicament, tel qu'une cytotoxine ou un modulateur immunitaire, et leur utilisation en tant qu'agents thérapeutiques ou diagnostiques, par exemple, dans le traitement ou le diagnostic du cancer.
PCT/CA2023/050405 2022-03-25 2023-03-24 Anticorps anti-récepteur alpha du folate et procédés d'utilisation WO2023178451A1 (fr)

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FURUUCHI KEIJI, RYBINSKI KATHERINE, FULMER JAMES, MORIYAMA TOMOYUKI, DROZDOWSKI BRIAN, SOTO ALLIS, FERNANDO SHAWN, WILSON KERRIANN: "Antibody‐drug conjugate MORAb‐202 exhibits long‐lasting antitumor efficacy in TNBC PDx models", CANCER SCIENCE, JAPANESE CANCER ASSOCIATION, TOKYO, JP, vol. 112, no. 6, 1 June 2021 (2021-06-01), JP , pages 2467 - 2480, XP093094218, ISSN: 1347-9032, DOI: 10.1111/cas.14898 *

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