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WO2025083069A1 - Molécules de liaison à her2 - Google Patents

Molécules de liaison à her2 Download PDF

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Publication number
WO2025083069A1
WO2025083069A1 PCT/EP2024/079224 EP2024079224W WO2025083069A1 WO 2025083069 A1 WO2025083069 A1 WO 2025083069A1 EP 2024079224 W EP2024079224 W EP 2024079224W WO 2025083069 A1 WO2025083069 A1 WO 2025083069A1
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Prior art keywords
cancer
moiety
antigen
inhibitor
her2
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Inventor
Dipti THAKKAR
Bhushan DHARMADHIKARI
Shalini PALIWAL
Benjamin AYERS
Rajavel Srinivasan
Lisa OOI
Kevin Fongching CHAU
Roberto Tirado MAGALLANES
Debleena RAY
Jerome Douglas BOYD-KIRKUP
Piers INGRAM
Xiu Li SIM
Gary KHOO
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Richard Ian Clegg
Hummingbird Bioscience Holdings Ltd
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Richard Ian Clegg
Hummingbird Bioscience Holdings Ltd
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Publication of WO2025083069A1 publication Critical patent/WO2025083069A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives

Definitions

  • the present disclosure relates to molecular biology, more specifically antibody technology.
  • the present disclosure also relates to methods of medical treatment and prophylaxis.
  • ADCs Antibody-drug conjugates
  • HER2 also known e.g. as ERBB2, neu
  • EGFR epidermal growth factor receptor
  • Overexpression of HER2 is observed in approximately 20% of human breast cancers, and is implicated in the aggressive growth and poor clinical outcomes in patients having such tumors (Slamon et al. Science (1987) 235:177-182).
  • Anti-HER2 antibody drug conjugates are described in Rassy etal., Breast (2022) 66: 217-226, and include ado-trastuzumab emtansine (DrugBank Acc. No. DB05773; marketed as Kadcyla®)) and fam-trastuzumab deruxtecan-nxki (DrugBank Acc. No. DB14962; marketed as Enhertu®).
  • Ado- trastuzumab emtansine comprises the cytotoxic agent DM1 (a thiol-containing maytansinoid microtubule inhibitor) conjugated to trastuzumab at lysine side chains via an MCC linker.
  • Fam-trastuzumab deruxtecan-nxki comprises the DNA topoisomerase I inhibitor DS-8201a (DXd) conjugated to trastuzumab via a cathepsin-cleavable tetrapeptide linker.
  • Mosele et al. Nat Med. (2023) 29(8):2110-2120 reports the results of a phase 2 trial (DAISY trial) relating to the use of fam-trastuzumab deruxtecan-nxki to treat metastatic breast cancer. Disease progression was observed in -71% of patients (125/177). There remains an unmet clinical need for the effective treatment of HER2-ex pressing cancers.
  • the present disclosure provides an antigen-binding molecule that binds to HER2, comprising (i) a HER2-binding moiety, and (ii) at least one linker-payload moiety, wherein the antigenbinding molecule comprises (a) DNA damage response (DDR) inhibitor moiety, and (b) a DNA topoisomerase I (TOP1) inhibitor moiety.
  • DDR DNA damage response
  • TOP1 DNA topoisomerase I
  • the DDR inhibitor moiety is, or comprises, a DDR inhibitor selected from: an ATR inhibitor, a PARP inhibitor, an ATM inhibitor, a WEE1 inhibitor, a CHK1/2 inhibitor, a DNA-PK inhibitor, or a PLK1 inhibitor.
  • the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is an ATR inhibitor.
  • the DDR inhibitor moiety is, or comprises, berzosertib.
  • the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is a CHK1/2 inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, prexasertib.
  • the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is a WEE1 inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, adavosertib.
  • the DDR inhibitor moiety is, or comprises, a DDR inhibitor which is a DNA-PK inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, nedisertib.
  • the TOP1 inhibitor moiety is, or comprises, a TOP1 inhibitor selected from: camptothecin or a derivative thereof, exatecan, exatecan mesylate (DX-8951f), A/-glycyl-exatecan, SN-38, DXd(1), DXd(2), irinotecan, etirinotecan, FL118, topotecan, gimatecan, belotecan, deruxtecan, belotecan, rubitecan, lurtotecan, diflomotecan, karenitecan, silatecan, namitecan, elomotecan, DRF-1042, delimotecan, NSC606985, chimmitecan, ZBH-1205, Genz-644282, non-CPT1 , indotecan, indimitecan, AZ14170132, SHR9265, Ed-04, KL610023, A1 .9, ZD06519,
  • the TOP1 inhibitor moiety is, or comprises, a TOP1 inhibitor selected from: camptothecin or a derivative thereof, exatecan, exatecan mesylate (DX-8951f), A/-glycyl-exatecan, SN-38, DXd(1), DXd(2).
  • the TOP1 inhibitor moiety is, or comprises exatecan.
  • the antigen-binding molecule comprises a linker-payload moiety comprising (a) a DDR inhibitor moiety, and (b) a TOP1 inhibitor moiety.
  • the linker-payload moiety comprises:
  • R N is selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the branching group; b indicates where the at least one first click group is linked to the branching group; c indicates where the at least one second click group is linked to the branching group.
  • the HER2-binding moiety comprises:
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:15
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:17;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:23
  • LC-CDR2 having the amino acid sequence of SEQ ID NO:24
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:25.
  • the antigen-binding moiety that binds to HER2 comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:14; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:22.
  • the antigen-binding moiety that binds to HER2 comprises: a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:12; and a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:13.
  • the present disclosure also provides a composition comprising an antigen-binding molecule according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the present disclosure also provides an antigen-binding molecule according to the present disclosure, or a composition according to the present disclosure, for use in a method of medical treatment or prophylaxis, or in a method of diagnosis or prognosis.
  • the present disclosure also provides an antigen-binding molecule according to the present disclosure, or a composition according to the present disclosure, for use in treating or preventing a cancer.
  • the present disclosure also provides an antigen-binding molecule according to the present disclosure, or a composition according to the present disclosure, in the manufacture of a medicament for treating or preventing a cancer.
  • the present disclosure also provides a method of treating or preventing a cancer, comprising administering to a subject a therapeutically- or prophylactically-effective amount of an antigen-binding molecule according to the present disclosure, or a composition according to the present disclosure.
  • the cancer is selected from: a cancer comprising cells expressing/overexpressing an EGFR family member, a cancer comprising cells expressing/overexpressing HER2, a cancer comprising cells that do not overexpress an EGFR family member, a cancer comprising cells that do not overexpress HER2, a HER2-low cancer, a HR-positive cancer, a solid tumor, bladder cancer, breast cancer, HER2-positive breast cancer, metastatic HER2-positive breast cancer, HER2-low breast cancer, unresectable or metastatic HER2-low breast cancer, HR-positive breast cancer, triple-negative breast cancer, cervical cancer, gastric cancer, HER2-positive gastric cancer, locally-advanced or metastatic HER2-positive gastric cancer, cholangiocarcinoma, colorectal cancer, esophageal esophagogastric junction cancer, gallbladder cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, HER2-positive gas
  • the cancer is refractory or relapsed to treatment with a DNA damage repair inhibitor, and/or wherein the cancer is refractory or relapsed to treatment with a DNA topoisomerase I inhibitor.
  • the present disclosure also the use of an antigen-binding molecule according to the present disclosure, or a composition according to the present disclosure, to deplete or increase killing of cells expressing HER2.
  • the present disclosure also provides an in vitro complex, optionally isolated, comprising an antigenbinding molecule according to the present disclosure bound to HER2.
  • the present disclosure relates to antigen-binding molecules comprising a HER2-binding moiety and a linker-payload moiety comprising at least two different payload moieties.
  • the antigen-binding molecules of the present disclosure are provided with unexpected and advantageous properties relative to known anti-HER2 antibody-drug conjugates.
  • the inventors hypothesize that the resistance to fam-trastuzumab deruxtecan-nxki therapy observed in the DAISY trial might be due to insensitivity to DXd, noting that 65% of patients that progressed on treatment with fam-trastuzumab deruxtecan-nxki retained HER2 expression (Mosele et al. Nat Med. (2023) 29(8):2110-2120).
  • DNA damage response is a key pathway for repair, and might be an important mode of resistance to DNA damaging payloads such as TOP1 inhibitors. Synergistic anticancer effects have previously been observed through combined treatment using a DDR inhibitor and a TOP1 inhibitor (Thomas et al., Cancer Cell. (2021) 39(4):566-79 e7).
  • the antigen-binding molecule of the present disclosure comprises a linker-payload moiety comprising (i) a TOP1 inhibitor moiety and (ii) a DNA damage response (DDR) inhibitor moiety
  • the DDR inhibitor is thought to increase the sensitivity of the cells of the cancer to the TOP1 inhibitor.
  • TOP1 inhibitors and DDR inhibitors are not substrates for P-glycoprotein, and so patients treated with antigen-binding molecules comprising such payload moieties are less likely to develop P-glycoprotein-mediated resistance to such therapy.
  • HER2 (also known e.g. as ERBB2, neu) is the protein identified by UniProtKB: P04626.
  • the canonical isoform of human HER2 has the amino acid sequence of P04626-1 (v1 , 1987-08-13; SEQ ID NO:1).
  • Alternative splicing mRNA encoded by the human ERBB2 gene yields six main isoforms: isoform 1 (SEQ ID NO:1), isoform 2 (SEQ ID NO:2), isoform 3 (SEQ ID NO:3), isoform 4 (SEQ ID NO:4), isoform 5 (SEQ ID NO:5) and isoform 6 (SEQ ID NO:6).
  • Isoform 2 differs from isoform 1 in that positions 1 to 610 are absent. Positions 1 to 686 of SEQ ID NO:1 are absent from isoform 3. In isoform 4, positions 1 to 23 of SEQ ID NO:1 are replaced with a shorter, 8 amino acid sequence. Positions 1 to 686 of SEQ ID NO:1 are absent from isoform 5. Isoform 6 differs in that positions 633 to 648 and 844 to 1255 of SEQ ID NO:1 are absent, and positions 771 to 883 are replaced with a different sequence of amino acids.
  • the canonical isoform of human HER2 comprises a 22 amino acid N-terminal signal peptide (SEQ ID NO:8), followed by an extracellular domain (SEQ ID NO:9), a single-pass transmembrane domain (SEQ ID NQ:10) and a cytoplasmic domain (SEQ ID NO:11) at the C-terminus.
  • SEQ ID NO:8 22 amino acid N-terminal signal peptide
  • SEQ ID NO:9 extracellular domain
  • SEQ ID NQ:10 single-pass transmembrane domain
  • SEQ ID NO:11 cytoplasmic domain
  • HER2 comprises a cysteine-rich extracellular region, a lipophilic transmembrane domain and an intracellular domain with tyrosine kinase activity.
  • HER2 lacks any recognized direct activating ligand, and may either exist in a constitutively active state, or become activated upon forming heterodimers with other EGFR family members, such as EGFR and HER3.
  • HER2 homodimerization or heterodimerization with EGFR/HER3 induces autophosphorylation of tyrosine residues of the cytoplasmic domain, initiating a signaling through various different intracellular pathways, most notably the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathways.
  • MAPK mitogen-activated protein kinase
  • PI3K phosphatidylinositol-4,5-bisphosphate 3-kinase
  • HER2-mediated signaling leads to cell proliferation, survival, differentiation, angiogenesis, and tissue invasion.
  • the HER2- HER3 heterodimer is a particularly potent stimulator of downstream pathways, particularly the PI3K/Akt pathway.
  • HER2 generally refers to the canonical isoform of the human HER2 (/.e. isoform 1), but also contemplates isoforms, fragments, variants (including mutants) and homologues thereof (/.e. from other species, e.g. non-human mammalian species (e.g. a non-human primate, e.g. rhesus, cynomolgous; e.g. a rodent, e.g. rat or mouse).
  • non-human mammalian species e.g. a non-human primate, e.g. rhesus, cynomolgous; e.g. a rodent, e.g. rat or mouse.
  • a ‘fragment’, ‘variant’ or ‘homologue’ of a protein may optionally be characterised as having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98% or >99% amino acid sequence identity to the amino acid sequence of the reference protein (e.g. the canonical isoform of the human protein).
  • fragments/variants/ isoforms/homologues may be characterised by ability to perform a function performed by the reference protein.
  • a ‘fragment’ generally refers to a fraction of the reference protein.
  • a ‘variant’ generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein.
  • An ‘isoform’ generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein (e.g. human HER2 isoforms 1 to 6 are all isoforms of one another).
  • a ‘homologue’ generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein.
  • human HER2 isoform 1 P04626-1 v1 ; SEQ ID NO:1
  • mouse HER2 UniProt: P70424-1 v3, 2005-09-27
  • Homologues include orthologues.
  • a ‘fragment’ may be of any length (by number of amino acids), although may optionally be at least 20% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.
  • a fragment of HER2 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200 or 1250 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1250 amino acids.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference HER2 (e.g. human HER2 isoform 1), as determined by analysis by a suitable assay for the functional property/activity.
  • HER2 e.g. human HER2 isoform 1
  • an isoform, fragment, variant or homologue of HER2 may display association with HER3 or EGFR.
  • the HER2 is HER2 from a mammal (e.g. a primate (rhesus, cynomolgous, non- human primate or human) and/or a rodent (e.g. rat or murine) HER2).
  • a mammal e.g. a primate (rhesus, cynomolgous, non- human primate or human
  • rodent e.g. rat or murine
  • Isoforms, fragments, variants or homologues of HER2 may optionally be characterized as having at least 60% amino acid sequence identity, e.g.
  • the HER2 comprises, or consists of, an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to one of SEQ ID NOs:1 to 7.
  • a fragment of HER2 comprises, or consists of, an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:7 or 9.
  • an ‘antigen-binding molecule’ refers to a molecule that binds to a given target antigen.
  • Antigen-binding molecules comprise an antigen-binding moiety through which the antigen-binding molecule binds to its target antigen.
  • the antigen-binding molecules of the present disclosure comprise an antigen-binding moiety that binds to HER2 (/.e. a HER2-binding moiety).
  • Antigen-binding moieties may comprise, or may be derived from, antibodies (/.e. immunoglobulins (Igs)) and antigen-binding fragments of antibodies.
  • antibodies include monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived antigen-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH, etc.).
  • Antigen-binding fragments of antibodies include e.g. Fv, Fab, F(ab’)2 and F(ab’) fragments.
  • Antigen-binding moieties also include target antigen-binding aptamers, e.g. a nucleic acid aptamers (reviewed, for example, in Zhou and Rossi, Nat Rev Drug Discov. (2017) 16(3):181-202).
  • an antigen-binding moiety comprises or consists of an antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (/.e.
  • sdAb single-domain antibody
  • ArmRP armadillo repeat protein
  • OBody fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).
  • phage display in which human antibody genes are expressed in phage display libraries
  • production of antibodies in transgenic mice engineered to have human antibody genes (described in Park and Smolen, Advances in Protein Chemistry (2001) 56: 369-421).
  • genes encoding the VH and VL chains are generated by PCR amplification and cloning from ‘naive’ human lymphocytes, and assembled into a library from which they can be expressed either as disulfide- linked Fab fragments or as single-chain Fv (scFv) fragments.
  • the Fab- or scFv-encoding genes are fused to a surface coat protein of filamentous bacteriophage and Fab or scFv capable of binding to the target of interest can then be identified by screening the library with antigen. Molecular evolution or affinity maturation procedures can be employed to enhance the affinity of the Fab/scFv fragment.
  • mice in which the endogenous murine Ig gene loci have been replaced by homologous recombination with their human homologues are immunised with antigen, and monoclonal antibody is prepared by conventional hybridoma technology, to yield a fully human monoclonal antibody.
  • an antigen-binding moiety comprises, or consists of, the antigen-binding region of an antibody (e.g. an antigen-binding fragment of an antibody).
  • Antigen-binding moieties may be derived from antibodies.
  • Antibody-derived antigen-binding moieties may comprise, or consist of, the antigen-binding region of an antibody (e.g. an antigen-binding fragment of an antibody).
  • an antigen-binding moiety may be or comprise the Fv (e.g. provided as an scFv) or the Fab region of an antibody that binds to a given target antigen, or the whole antibody.
  • the antigen-binding moieties of the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to a given target antigen (e.g. HER2).
  • mAbs monoclonal antibodies
  • Antigen-binding regions of antibodies such as variable fragment (Fv), Fab and F(ab’)2 fragments may also be used/provided.
  • Fv variable fragment
  • Fab fragment antigen-binding region
  • An ‘antigen-binding region’ is any fragment of an antibody that binds to the target for which the given antibody is specific.
  • an antigen-binding moiety comprises the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen.
  • the antigen-binding moiety is or comprises the Fv (e.g. provided as an scFv) of an antibody.
  • the antigen-binding moiety is or comprises the Fab region of an antibody.
  • the antigen-binding moiety is or comprises the whole antibody (/.e. comprising variable and constant regions).
  • An antigen-binding moiety may be, or may comprise, an antigen-binding polypeptide, or an antigenbinding polypeptide complex.
  • An antigen-binding moiety may comprise more than one polypeptide which together form an antigen-binding moiety.
  • the polypeptides may associate covalently or non-covalently.
  • the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).
  • an antigen-binding moiety comprises, or consists of, a polypeptide complex formed by proteimprotein interaction between constituent peptides/polypeptides of the antigen-binding moiety.
  • An antigen-binding moiety may refer to a non- covalent or covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8 polypeptides), e.g. an IgG-like antigen-binding moiety comprising two heavy chain polypeptides and two light chain polypeptides.
  • Antibodies generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1 , HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1 , LC-CDR2, and LC-CDR3.
  • the six CDRs together define the paratope of the antibody, which is the part of the antibody that binds to the target antigen.
  • VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs.
  • FRs framework regions
  • VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.
  • the CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.
  • the CDRs and FRs of antigenbinding molecules referred to herein are defined according to the IMGT information system.
  • an antigen-binding moiety comprises, or consists of, an Fv region that binds to HER2.
  • the VH and VL regions of the Fv are provided as single polypeptide joined by a linker sequence, i.e. a single chain Fv (scFv).
  • the antigen-binding moiety comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g. CK or CA).
  • the Fab region comprises a polypeptide comprising a VH and a CH1 (e.g. a VH- CH1 fusion polypeptide), and a polypeptide comprising a VL and a CL (e.g. a VL-CL fusion polypeptide).
  • the Fab region comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH1 (e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region.
  • the VH, CH1 , VL and CL regions of the Fab or CrossFab are provided as single polypeptide joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).
  • an antigen-binding moiety described herein comprises, or consists of, a whole antibody which binds to HER2.
  • whole antibody refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.
  • Immunoglobulins of type G are -150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chains comprise a VL followed by a CL.
  • immunoglobulins may be classed as IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM.
  • the light chain may be kappa (K) or lambda (A).
  • the antigen-binding moiety comprises, or consists of, an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM which binds to HER2.
  • IgG e.g. lgG1 , lgG2, lgG3, lgG4
  • IgA e.g. lgA1 , lgA2
  • IgD IgE
  • IgM which binds to HER2.
  • an antigen-binding moiety of the present disclosure comprises one or more regions (e.g. CH1 , hinge, CH2, CH3, etc.) of an immunoglobulin heavy chain constant sequence.
  • the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. Ig A1 , lgA2), IgD, IgE or IgM, e.g. a human IgG (e.g.
  • the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of a human lgG1 allotype (e.g. G1 m1 , G1 m2, G1 m3 or G1 m17).
  • an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:31 or 36.
  • an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g.
  • an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g.
  • an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g.
  • an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g.
  • an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g.
  • an antigen-binding moiety of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence.
  • the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; CK).
  • the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CA), e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.
  • an antigen-binding moiety comprises one or more polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NQ:40, 41 , 42, 43, 44 or 45.
  • one or more amino acids of an amino acid sequence referred to herein are substituted with another amino acid.
  • a substitution comprises substitution of an amino acid residue with a non-identical ‘replacement’ amino acid residue.
  • a replacement amino acid residue of a substitution according to the present disclosure may be a naturally-occurring amino acid residue (/.e.
  • alanine Ala
  • arginine Arg
  • asparagine Asn
  • aspartic acid Asp
  • cysteine Cys
  • glutamine Gin
  • glutamic acid Glu
  • glycine Gly
  • histidine His
  • isoleucine lie: leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Vai).
  • a replacement amino acid may be a non-naturally occurring amino acid residue - i.e. an amino acid residue other than those recited in the preceding sentence.
  • non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogues such as those described in Ellman, etal., Meth. Enzym. 202 (1991) 301-336.
  • a substitution may be biochemically conservative.
  • the replacement amino acid of the substitution is another, non-identical amino acid provided in the same row:
  • the replacement amino acid may be selected from Ala, Vai, Leu, lie, Trp, Tyr, Phe and Norleucine.
  • a replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces.
  • a replacement amino acid in a substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces: That is, in some embodiments, a nonpolar amino acid is substituted with another, non-identical nonpolar amino acid. In some embodiments, a polar amino acid is substituted with another, non-identical polar amino acid.
  • an acidic polar amino acid is substituted with another, non-identical acidic polar amino acid.
  • a basic polar amino acid is substituted with another, non- identical basic polar amino acid.
  • a neutral amino acid is substituted with another, non-identical neutral amino acid.
  • a positive amino acid is substituted with another, non-identical positive amino acid.
  • a negative amino acid is substituted with another, non-identical negative amino acid.
  • substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target binding) of the antigen-binding moiety comprising the substitution as compared to the equivalent unsubstituted molecule.
  • the antigen-binding molecules of the present disclosure comprise an antigen-binding moiety that binds to HER2.
  • the antigen-binding moiety comprises the CDRs of an antigen-binding moiety which is capable of binding to HER2. In some embodiments, the antigen-binding moiety comprises the FRs of an antigen-binding moiety which is capable of binding to HER2. In some embodiments, the antigen-binding moiety comprises the CDRs and the FRs of an antibody that is capable of binding to HER2. That is, in some embodiments the antigen-binding moiety comprises the VH region and the VL region of an antibody that is capable of binding to HER2.
  • an antigen-binding moiety which is capable of binding to HER2 according to the present disclosure may be, or may be derived from, a HER2-binding antibody selected from: trastuzumab (DrugBank Acc. No. DB00072; which is formed of the polypeptides having the amino acid sequences of SEQ ID NO:12 and SEQ ID NO:13), pertuzumab (DrugBank Acc. No. DB06366), margetuximab (DrugBank Acc. No. DB14967), timigutuzumab (described e.g. in Fiedler et al., ESMO Open. (2016) 3(4): e000381), CT-P6 (described e.g.
  • the antigen-binding moiety is, or is derived from, trastuzumab.
  • the antigen-binding moiety is capable of binding the same region of DLL3, or an overlapping region of HER2, to the region of HER2 which is bound by an antigen-binding molecule comprising the VH and VL sequences of a HER2-binding antibody described hereinabove. In some embodiments the antigen-binding moiety is capable of binding the same region of HER2, or an overlapping region of HER2, to the region of HER2 which is bound by an antigen-binding molecule comprising the VH and VL sequences of trastuzumab (/.e.
  • an antigen-binding molecule comprising a VH having the amino acid sequence of SEQ ID NO:14, and a VL having the amino acid sequence of SEQ ID NO:22).
  • the antigen-binding moiety is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of one of SEQ ID NOs:1 , 7 or 9.
  • an antigen-binding moiety to bind to a given peptide/polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g. western blot), immunoprecipitation, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442) or Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507).
  • ELISA immunoblot
  • SPR Surface Plasmon Resonance
  • Bio-Layer Interferometry see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507.
  • the peptide/polypeptide may comprise one or more additional amino acids at one or both ends of the reference amino acid sequence.
  • the peptide/polypeptide comprises e.g. 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, IQ- 20, 10-30, 10-40, 10-50, 20-30, 20-40 or 20-50 additional amino acids at one or both ends of the reference amino acid sequence.
  • the additional amino acid(s) provided at one or both ends (/.e. the N-terminal and C-terminal ends) of the reference sequence correspond to the positions at the ends of the reference sequence in the context of the amino acid sequence of HER2.
  • the antigen-binding moiety is capable of binding to a peptide/polypeptide which is bound by an antibody comprising the VH and VL sequences of trastuzumab (/.e. an antigen-binding molecule comprising a VH having the amino acid sequence of SEQ ID NO:14, and a VL having the amino acid sequence of SEQ ID NO:22).
  • the antigen-binding moiety is capable of binding to a peptide/polypeptide which is bound by an antibody comprising the VH and VL sequences of a HER2-binding antibody described hereinabove.
  • the antigen-binding moiety comprises the heavy chain CDRs and the light chain CDRs of a HER2-binding antibody described hereinabove. In some embodiments, the antigen-binding moiety comprises the VH and VL of a HER2-binding antibody described hereinabove. In some embodiments, the antigen-binding moiety comprises the heavy chain polypeptide (/.e. comprising VH, CH1 , CH2 and CH3 region sequences) and light chain polypeptide (/.e. comprising VL and CL region sequences) of a HER2-binding antibody described hereinabove.
  • the antigen-binding moiety comprises the heavy chain CDRs and the light chain CDRs of trastuzumab. In some embodiments, the antigen-binding moiety comprises the VH and VL of trastuzumab. In some embodiments, the antigen-binding moiety comprises the heavy chain polypeptide (/.e. comprising VH, CH1 , CH2 and CH3 region sequences) and light chain polypeptide (/.e. comprising VL and CL region sequences) of trastuzumab.
  • the antigen-binding moiety comprises: a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:15
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:16
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:17, or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 , and/or in which 1 or 2 or 3 amino acids in HC-CDR2, and/or in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and a VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:25; or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 , and/or in which 1 or 2 or 3 amino acids in LC-CDR2, and/or in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.
  • the antigen-binding moiety comprises: a VH region comprising an amino acid sequence having at least 60% sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:14; and a VL region comprising an amino acid sequence having at least 60% sequence identity, e.g.
  • an antigen-binding moiety comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70% sequence identity, more preferably one of >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:12;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70% sequence identity, more preferably one of >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:13.
  • an antigen-binding molecule of the present disclosure (e.g. an antigen-binding moiety thereof) comprises an Fc region.
  • an ‘Fc region’ refers to a polypeptide complex formed by interaction between two polypeptides, each polypeptide comprising the CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence.
  • a ‘CH2 domain’ refers to an amino acid sequence corresponding to the CH2 domain of an immunoglobulin (Ig).
  • the CH2 domain is the region of an Ig formed by positions 231 to 340 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85.
  • a ‘CH3 domain’ refers to an amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig).
  • the CH3 domain is the region of an Ig formed by positions 341 to 447 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85.
  • a ‘CH2-CH3 region’ refers to an amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig).
  • the CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85.
  • a CH2 domain, CH3 domain and/or a CH2-CH3 region corresponds to the CH2 domain/CH3 domain/CH2-CH3 region of an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, Ig E or IgM.
  • the CH2 domain, CH3 domain and/or a CH2-CH3 region corresponds to the CH2 domain/CH3 domain/CH2-CH3 region of a human IgG (e.g.
  • the CH2 domain, CH3 domain and/or a CH2-CH3 region corresponds to the CH2 domain/CH3 domain/CH2-CH3 region of a human lgG1 allotype (e.g. G1 m1 , G1 m2, G1 m3 or G1 m17).
  • the CH2 domain, CH3 domain and/or a CH2-CH3 region corresponds to the CH2 domain/CH3 domain/CH2-CH3 region of human lgG1 allotype G1 m3.
  • Fc regions provide for interaction with Fc receptors and other molecules of the immune system to bring about functional effects.
  • Fc-mediated effector functions are reviewed e.g. in Jefferis et al., Immunol Rev 1998 163:59-76 (hereby incorporated by reference in its entirety), and are brought about through Fc- mediated recruitment and activation of immune cells (e.g. macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells and T cells) through interaction between the Fc region and Fc receptors expressed by the immune cells, recruitment of complement pathway components through binding of the Fc region to complement protein C1q, and consequent activation of the complement cascade.
  • immune cells e.g. macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells and T cells
  • Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and/or chemokine production, and antigen processing and presentation.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • MAC membrane attack complex
  • cell degranulation cell degranulation
  • cytokine and/or chemokine production and antigen processing and presentation.
  • an antigen-binding molecule comprises one or more (e.g. two) polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:38 or 39.
  • the antigen-binding molecule comprises an Fc region comprising one or more (e.g.
  • polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:38 or 39.
  • Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et al., Protein Cell (2016) 9(1):63-73, which is hereby incorporated by reference in its entirety.
  • the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification to increase or reduce an Fc-mediated function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.
  • an Fc region/CH2/CH3 is described as comprising modification(s) ‘corresponding to’ reference substitution(s), equivalent substitution(s) in the homologous Fc/CH2/CH3 are contemplated.
  • the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification in one or more of the CH2 and/or CH3 regions.
  • the Fc region comprises modification to reduce/prevent an Fc-mediated function (e.g. ADCC, ADCP, CDC). In some embodiments, the Fc region comprises modification to reduce/prevent ADCC. In some embodiments, the Fc region comprises modification to reduce/prevent CDC. In some embodiments, the Fc region comprises modification to reduce/prevent binding to an Fc receptor. In some embodiments, the Fc region comprises modification to reduce/prevent binding to an Fey receptor. In some embodiments, the Fc region comprises modification to reduce/prevent glycosylation of the amino acid residue corresponding to N297.
  • an Fc-mediated function e.g. ADCC, ADCP, CDC.
  • the Fc region comprises modification to reduce/prevent ADCC.
  • the Fc region comprises modification to reduce/prevent CDC.
  • the Fc region comprises modification to reduce/prevent binding to an Fc receptor.
  • the Fc region comprises modification to reduce/prevent binding to an Fey receptor.
  • the Fc region comprises modification to reduce/prevent
  • the Fc region comprises modification at the amino acid residue corresponding to N297.
  • the Fc region comprises modification corresponding to N297A or N297Q or N297G as described in Leabman et al., Mabs. (2013) 5:896-903. Substitution of ‘N297’ with ‘A’, ‘G’ or ‘Q’ is known to eliminate glycosylation, and thereby reduce Fc binding to C1q and Fey receptors, and thus also reducing CDC and ADCC.
  • the Fc region comprises modification corresponding to N297A.
  • an antigen-binding molecule comprises one or more (e.g. two) polypeptides comprising an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:49 or 50.
  • the antigen-binding molecule comprises an Fc region comprising one or more (e.g.
  • An antigen-binding molecule may comprise a HER2-binding moiety according to any embodiment described hereinabove, and a linker-payload moiety as described hereinbelow.
  • a linker-payload moiety refers to a moiety comprising one or more payload moieties, and a linker moiety for linking the payload moiety(/ies) to the antigen-binding region of the antigen-binding molecule.
  • a payload moiety according to the present disclosure comprises or consist of a cytotoxic agent.
  • Payload moieties are described e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3):14, Goundry and Parker, Org. Process Res. Dev. (2022) 26, 8, 2121-2123, Fu et al., Signal Transduction and Targeted Therapy (2022) 7:93, Wang etal., Acta Pharmaceutica Sinica B (2023) 13 (10): 4025-4059 and Conilh et al., J. Hematol. & Oncol. (2023) 16:3, all of which are hereby incorporated by reference in their entirety.
  • the present disclosure relates to antigen-binding molecules comprising at least one linkerpayload moiety, wherein the antigen-binding molecule comprises (a) a payload moiety which is a DNA damage response (DDR) inhibitor, and (b) a payload moiety which is a DNA topoisomerase I (TOP1) inhibitor.
  • DDR DNA damage response
  • TOP1 DNA topoisomerase I
  • a payload moiety which is a DDR inhibitor may be referred to simply as a ‘DDR inhibitor moiety’
  • a payload moiety which is a TOP1 inhibitor may be referred to simply as a ‘TOP1 inhibitor moiety’.
  • the DNA Damage Response is a complex network of mechanisms for detecting and repair DNA damage, in order to preserve genomic stability.
  • the DDR is reviewed e.g. in Groelly et al., Nature Reviews Cancer (2023) 23:78-94 and Molinaro et al., Cancers (Basel). (2021) 13(15): 3819, both of which are hereby incorporated by reference in their entirety.
  • ATM is a protein kinase activated by double-strand breaks in DNA, and which initiates downstream signaling.
  • ATR is activated by DNA damage and replication stress, and in particular responds to single-strand breaks and stalled DNA replication forks.
  • CHK1 and CHK2 are downstream effectors of ATM and ATR, and phosphorylate various target proteins to stop cell cycle progression, and facilitate DNA repair.
  • PARP Poly ADP-Ribose Polymerase
  • DNA- PK DNA-Dependent Protein Kinase
  • NHEJ non-homologous endjoining
  • WEE1 WEE1
  • PLK1 Poly-Like Kinase 1
  • WEE1 is a kinase that phosphorylates and inhibits CDKs (Cyclin- Dependent Kinases), thereby delaying cell cycle progression and allowing more time for DNA damage repair prior to cell division.
  • PLK1 regulates the cell cycle checkpoint and promotes repair processes, through phosphorylation of PolO.
  • RAD51 is an ATPase involved in DNA repair.
  • Ubiquitin-specific proteases USPs
  • USPs Ubiquitin-specific proteases
  • PLMYT1 Protein kinase membrane associated tyrosine/threonine 1
  • Aurora-A may contribute to the G2 DNA damage checkpoint through PLK1 and CDC25B activation, and is important in the mitotic DNA damage response.
  • DDR inhibitors and their use for the treatment of cancers is described e.g. in Cheng et al., Eur J Med Chem. (2022) 230:114109, Wang et a!., Front Immunol. (2022) 13:854730 and Choi and Lee, Int J Mol Sci. (2022) 23(3):1701 , all of which are hereby incorporated by reference in their entirety.
  • a DDR inhibitor moiety according to the present disclosure is, or comprises, a DDR inhibitor selected from:
  • a PARP inhibitor e.g. olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, simmiparib, senaparib, SC-10914, 2X-121 , AMXI-5001 , JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-1002, RBN-2397, fluzoparib, NMS-03305293, AZD9574);
  • a PARP inhibitor e.g. olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, simmiparib, senaparib, SC-10914, 2X-121 , AMXI-5001 , JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, S
  • an ATM inhibitor e.g. CP-466722, KU-55933, KU-60019, KU-59403, AZ31 , AZ32, AZD0156, AZD1390, XRD-0394, M4076, M3541 , WSD-0628, SYH-2051 , IMP-08, SP-1161 , INT-6C4/5C4;
  • an ATR inhibitor e.g. M6620 (berzosertib), M4344 (VX-803), AZD6738 (ceralasertib), BAY1895344 (elimusertib), RP3500 (camonsertib), ATRN119, ART380, IMP9064, HRS2398, M1774, IMP9064, SC0245, LF0397, NU6027);
  • a WEE1 inhibitor e.g. adavosertib, Debio 0123, PD0166285, PD0407824, AZD1775, ZN-c3, IMP7068, SY4835, SCO191 , IMP7068;
  • a CHK1/2 inhibitor e.g. CBP-501 , prexasertib, MK-8776, GDC-0575, SRA-737, PF-00477736, AZD7762, LY2603618 (rabusertib), LY2880070,, XL884, BEBT260, MU380, NU7441 , KU-5778;
  • a DNA-PK inhibitor e.g. CC-115, LY-3023414, AsiDNA, M3814 (nedisertib, peposertib), VX-984 (M9831), BR-101801 , XRD-0394, SL901 , XZP-6877, IMP-11 , ZL-2201 , BR-2006, AZD7648, NU7441;
  • a PLK1 inhibitor e.g. BI-6727 (volasertib), PCM-075 (onvansertib) , CYC140 (plogosertib)
  • BI-6727 volatileib
  • PCM-075 volatileib
  • CYC140 plogosertib
  • a PolO inhibitor e.g. ART4215, ART6043, novobiocin, RP-6685, RP-3467;
  • a RAD51 inhibitor e.g. CYT0851
  • an inhibitor of a ubiquitin-specific protease (USP) family enzyme e.g. an inhibitor of USP11 , USP7, USP4, USP37, USP39, USP45, USP24 and/or USP1 ; e.g. KSQ-4279;
  • an Aurora-A inhibitor e.g. alisertib, WJ05129 (JS112), JAB-2485.
  • the DDR inhibitor moiety is, or comprises, ceralasertib.
  • the DDR inhibitor moiety is, or comprises, berzosertib:
  • the DDR inhibitor moiety is, or comprises, prexasertib: In some embodiments, the DDR inhibitor moiety is, or comprises, adavosertib, which can be linked as follows, as well as through other positions:
  • the DDR inhibitor moiety is, or comprises, AZD0156, which can be linked as follows, as well as through other positions:
  • the DDR inhibitor moiety is, or comprises, nedisertib:
  • DNA topoisomerases I and II TOP1 and TOP2
  • DNA topoisomerase inhibitors block the resealing step, resulting in DNA fragmentation and cell death.
  • DNA topoisomerase I inhibitors and their use for the treatment of cancers is described e.g. in Pommier, Chem Rev. (2009) 109(7): 2894-2902, Li et al., Am J Cancer Res. (2017) 7(12): 2350-2394 and Thomas and Pommier, Clin Cancer Res. (2019) 25(22): 6581-6589, all of which are hereby incorporated by reference in their entirety.
  • a TOP1 inhibitor moiety is, or comprises, a TOP1 inhibitor selected from: camptothecin or a derivative thereof, exatecan, exatecan mesylate (DX- 8951f), A/-glycyl-exatecan, SN-38, DXd(1), DXd(2), irinotecan, etirinotecan, FL118, topotecan, gimatecan, belotecan, deruxtecan, belotecan, rubitecan, lurtotecan, diflomotecan, karenitecan, Silat can, namitecan, elomotecan, DRF-1042, delimotecan, NSC606985, chimmitecan, ZBH-1205, Genz-644282, non-CPT1 , indotecan (LMP-400), indimitecan (LMP-776) , AZ14170132, SHR9265, Ed-04
  • the TOP1 inhibitor moiety is, or comprises, exatecan:
  • the TOP1 inhibitor moiety is, or comprises, belotecan:
  • the TOP1 inhibitor moiety is, or comprises, SN38:
  • the TOP1 inhibitor moiety is, or comprises, DXd:
  • a linker moiety according to the present disclosure may be any moiety suitable for linking the payload moiety to the antigen-binding region of the antigen-binding molecule of the present disclosure.
  • they generally comprise a group enabling connection to the payload moiety, a group connecting conjugation to the antigen-binding region of the antigen-binding molecule, and a linker core.
  • Linker moieties are described e.g. in Su et al., Acta Pharmaceutica Sinica B (2021) 11 (12): 3889-3907, Fu et al., Signal Transduction and Targeted Therapy (2022) 7:93,
  • a linker moiety according to the present disclosure may be a cleavable linker moiety or a non-cleavable moiety.
  • Cleavable linkers typically utilise differences between the environment of systemic circulation and that in cancer cells/the tumor microenvironment to release the payload moiety in a targeted manner.
  • Cleavable linkers include chemical cleavage linkers (e.g. acid-cleavable linkers, GSH-cleavable linkers, Fe(ll)- cleavable linkers) and enzyme cleavage linkers (e.g. cathepsin-cleavable linkers, glycosidase-cleavable linkers, phosphatase-cleavable linkers, sulfatase-cleavable linkers).
  • a linker moiety according to the present disclosure is a chemical cleavage linker. In some embodiments, a linker moiety according to the present disclosure is an enzyme cleavage linker. In some embodiments, a linker moiety is an acid-cleavable linker, e.g. comprising a hydrazone group (e.g. a 6-maleimidocaproylhydrazone linker or a (4-(4-acetylphenoxy)butanoic acid) hydrazaone linker), a carbonate group or a silyl ether group. In some embodiments, a linker moiety is a GSH-cleavable linker, e.g. comprising a disulfide group.
  • a linker moiety is a Fe(ll)-cleavable linker, e.g. comprising a 1 ,2,4-trioxolane group.
  • a linker moiety is a cathepsin-cleavable linker, e.g. comprising a dipeptide (e.g. a valine-citrulline linker, a phenylalanine-lysine linker or a valinealanine linker), a triglycyl peptide (CX) or a cBu-Cit group.
  • the linker moiety is GGFG (Glycine-Glycine-Phenylalanine-Glycine).
  • a linker moiety is a glucuronidase-cleavable linker, e.g. comprising a p-glucuronide group.
  • a linker moiety is a glycosidase-cleavable linker, e.g. comprising a p-galactoside group.
  • a linker moiety is a phosphatase-cleavable linker, e.g. comprising a pyrophosphate group.
  • a linker moiety is a sulfatase-cleavable linker, e.g. comprising an arylsulfate group.
  • a linker moiety is a photo-responsive linker, e.g. comprising a heptamethine cyanine fluorophore group, an O-nitrobenzyl group or a PC4AP group.
  • a linker moiety is a biorthogonal cleavable linker, e.g. comprising a dsProc group.
  • Non-cleavable linkers remain inert in common chemical and enzymatic environments in the body, with the payload moiety being released following processing of the ADC by cellular lysosomal proteases.
  • Non- cleavable linkers include linkers comprising thioether or maleimidocaproyl groups.
  • a linker moiety is a thioether linker.
  • a linker moiety is a maleimidocaproyl linker, e.g. comprising a 2-(maleimidomethyl)-1 ,3-dioxane (MD) group or a Mal-PAB group.
  • a linker moiety comprises a polyethylene glycol (PEG) group and an alkyne, triazole or piperazine group.
  • a linker-payload moiety according to the present disclosure has an amino (-NH2) group for linkage to the antigen-binding moiety, for example by enzymatic conjugation.
  • enzymatic conjugation with microbial transglutaminase may be used to conjugate the linker-payload moiety to the antigen-binding moiety.
  • a linker moiety further comprises a spacer moiety.
  • Spacer moieties are sometimes required sue to the bulky nature of payload moieties. Commonly employed spacer moieties include para- aminobenzyl carbamate (PABC), hemiaminal groups, PEG groups, polar acyl sulfamide groups, polar carbamoyl sulfamide groups and HydraSpace (described e.g. in Verkade et al., Antibodies (Basel) (2016) 7(1):12 and WO 2016/053107 A1 , both of which are hereby incorporated by reference in their entirety).
  • PABC para- aminobenzyl carbamate
  • hemiaminal groups hemiaminal groups
  • PEG groups polar acyl sulfamide groups
  • polar carbamoyl sulfamide groups polar carbamoyl sulfamide groups
  • HydraSpace described e.g. in Verkade et al., Antibodies
  • PABC is commonly employed as a spacer moiety in cathepsin-cleavable dipeptide linkers, p- glucuronidase-cleavable linkers, p-galactosidase-cleavable linkers and phosphatase cleavable linkers.
  • para-aminobenzyl (PAB) is used as a spacer group.
  • an antigen-binding molecule comprises a linker-payload moiety, which in turn comprises both a DDR inhibitor moiety and a TOP1 inhibitor moiety. That is, in some embodiments, the antigen-binding molecule comprises a linker-payload moiety comprising: (a) a DDR inhibitor moiety, and (b) a TOP1 inhibitor moiety. In some embodiments, the DDR inhibitor moiety and TOP1 inhibitor moieties are provided in the same linker-payload moiety. In some embodiments, the DDR inhibitor moiety and the TOP1 inhibitor moiety are connected to the antigenbinding moiety of the antigen-binding molecule via the same linker moiety.
  • the DDR inhibitor and TOP1 inhibitor payload moieties are provided in the same linker-payload moiety. In some embodiments, the DDR inhibitor and TOP1 inhibitor payload moieties are connected to the antigen-binding moiety of the antigen-binding molecule through the same linker moiety.
  • the DDR inhibitor and TOP1 inhibitor payload moieties are connected to the antigen-binding moiety of the antigen-binding molecule through a branched linker moiety.
  • the linker may comprise of multiple branches to allow for DAR flexibility.
  • the DDR inhibitor and TOP1 inhibitor payload moieties are connected to the antigen-binding moiety of the antigen-binding molecule through a linker moiety that has increased hydrophilicity.
  • the DDR inhibitor and TOP1 inhibitor payload moieties are connected to the antigen-binding moiety of the antigen-binding molecule through a branched hydrophilic linker moiety.
  • the DDR inhibitor moiety and the TOP1 inhibitor moiety are connected to the linker moiety of the linker-payload moiety via orthogonal functional groups.
  • the linker-payload moiety comprises a trifunctional linker moiety providing for linkage of an antigenObinding moiety to two different payload moieties.
  • 3621 has the following structure:
  • a linker-payload moiety comprises a linker moiety comprising: (i) a moiety derived from a group for connection to the antigen-binding moiety (e.g. a self-stabilizing N-aryl maleimide group), (ii) a moiety derived from an alkyne group suitable for incorporating a first payload moiety via CuAAC (i.e. a divalent triazole), and (iii) a moiety derived from a ketone group suitable for incorporating a second payload moiety via aminooxy reaction resulting in oxime linkage (i.e. an oxime) .
  • a linker moiety comprising: (i) a moiety derived from a group for connection to the antigen-binding moiety (e.g. a self-stabilizing N-aryl maleimide group), (ii) a moiety derived from an alkyne group suitable for incorporating a first payload moiety via CuAAC (i.e.
  • the first payload moiety is a DDR inhibitor moiety as described herein, and the second payload moiety is a TOP1 inhibitor moiety as described herein.
  • the first payload moiety is a TOP1 inhibitor moiety as described herein, and the second payload moiety is a DDR inhibitor moiety as described herein.
  • an antigen-binding molecule comprises a linker-payload moiety comprising: (i) a first payload moiety conjugated to the linker moiety via a CuAAC reaction between an azide group and an alkyne group, and (ii) a second payload moiety conjugated to the linker moiety oxime linkage between an alkoxyamine or hydrazide group, and a ketone group.
  • the first payload moiety is a DDR inhibitor moiety as described herein
  • the second payload moiety is a TOP1 inhibitor moiety as described herein.
  • the first payload moiety is a TOP1 inhibitor moiety as described herein
  • the second payload moiety is a DDR inhibitor moiety as described herein.
  • a linker-payload moiety comprises a linker moiety comprising: (i) a moiety derived from a group for connection to the antigen-binding moiety (e.g. a lysine-based group), (ii) one or two moieties derived from azide groups suitable for incorporating a first payload moiety via DBCO cycloaddition, and (iii) a moiety derived from a methyltetrazine group for incorporation of a second payload moiety via TCO cycloaddition.
  • a linker moiety comprising: (i) a moiety derived from a group for connection to the antigen-binding moiety (e.g. a lysine-based group), (ii) one or two moieties derived from azide groups suitable for incorporating a first payload moiety via DBCO cycloaddition, and (iii) a moiety derived from a methyltetrazine group for incorporation
  • the first payload moiety is a DDR inhibitor moiety as described herein, and the second payload moiety is a TOP1 inhibitor moiety as described herein.
  • the first payload moiety is a TOP1 inhibitor moiety as described herein, and the second payload moiety is a DDR inhibitor moiety as described herein.
  • an antigen-binding molecule comprises a linker-payload moiety comprising: (i) a first payload moiety conjugated to the linker moiety via a DBCO cycloaddition reaction between a DBCO group and an azide group; and (ii) a second payload moiety conjugated to the linker moiety via a TCO cycloaddition reaction between a TCO group and a methyltetrazine group.
  • the first payload moiety is a DDR inhibitor moiety as described herein
  • the second payload moiety is a TOP1 inhibitor moiety as described herein.
  • the first payload moiety is a TOP1 inhibitor moiety as described herein
  • the second payload moiety is a DDR inhibitor moiety as described herein.
  • the DDR inhibitor moiety and the TOP1 inhibitor moiety are connected to a linker moiety of the linker-payload moiety via cysteine groups.
  • Levengood, et al., Angew Chem Int Ed Engl (2017) 56(3): 733-737 describes a linker-payload moiety comprising two different payload moieties, constructed by sequential deprotection of orthogonally- protected cysteines. Each payload is connected to a maleimide group (for example with a cleavable linker), and the maleimide undergoes a Michael reaction with the deprotected cysteines.
  • the branched linker moiety described in Levengood, et al., Angew Chem Int Ed Engl (2017) 56(3): 733-737 has the following structure:
  • a linker-payload moiety comprises: (i) a first payload moiety conjugated to the linker-payload moiety via reduction of a cysteine residue bearing a protecting disulfide group (e.g. a S-(tert-butyl) disulfide group or S-(isopropyl) disulfide group), and subsequent incorporation of the first payload moiety via thiol-maleimide reaction; and (ii) a second payload moiety conjugated to the linker-payload moiety via reduction of a cysteine residue bearing a protecting acetamidomethyl group, and subsequent incorporation of the first payload moiety via thiol-maleimide reaction.
  • a protecting disulfide group e.g. a S-(tert-butyl) disulfide group or S-(isopropyl) disulfide group
  • the first payload moiety is a DDR inhibitor moiety as described herein, and the second payload moiety is a TOP1 inhibitor moiety as described herein.
  • the first payload moiety is a TOP1 inhibitor moiety as described herein, and the second payload moiety is a DDR inhibitor moiety as described herein.
  • the linker-payload moiety comprises:
  • R N is selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the branching group; b indicates where the at least one first click group is linked to the branching group; c indicates where the at least one second click group is linked to the branching group.
  • the DDR inhibitor moiety and the TOP1 inhibitor moiety of the antigen-binding molecule of the present disclosure are connected to the antigen-binding moiety of the antigen-binding molecule via different linker moieties.
  • an antigen-binding molecule comprises: (a) a linker-payload moiety comprising a DDR inhibitor moiety, and (b) a linker-payload moiety comprising a TOP1 inhibitor moiety. That is, in some embodiments, the antigen-binding molecule comprises at least two linker-payload moieties, wherein one of the linker-payload moieties comprises a DDR inhibitor moiety, and wherein another of the linker-payload moieties comprises a TOP1 inhibitor moiety.
  • an antigen-binding molecule comprises: (i) a first linker-payload moiety conjugated to the antigen-binding moiety via thiol-maleimide reaction between a cysteine residue of the antigen-binding moiety and a maleimide group of the linkerpayload moiety, and (ii) a second linker-payload moiety conjugated via CuAAC reaction between an azide group of the linker-payload moiety to the alkyne group of an N-propargyl-L-lysine residue of the antigenbinding moiety.
  • the first linker-payload moiety comprises a DDR inhibitor moiety as described herein
  • the second linker-payload moiety comprises a TOP1 inhibitor moiety as described herein
  • the first linker-payload moiety comprises a TOP1 inhibitor moiety as described herein
  • the second linker-payload moiety comprises a DDR inhibitor moiety as described herein.
  • Nilchan et al., Antib. Ther. (2019) 2:71-78 describes a dual conjugation approach in which (i) a first linkerpayload moiety is conjugated to an antigen-binding moiety via selenoether conjugation between a selenocysteine residue of the antigen-binding moiety and an iodoacetamide group of the linker-payload moiety, and in which (ii) a second linker-payload moiety is conjugated to the same antigen-binding moiety via reaction between a cysteine residue of the antigen-binding moiety and a methylsulfone phenyloxadiazole (MSODA) group of the linker-payload moiety.
  • MSODA methylsulfone phenyloxadiazole
  • an antigen-binding molecule comprises: (i) a first linker-payload moiety conjugated to the antigen-binding moiety via selenoether conjugation between a selenocysteine residue of the antigen-binding moiety and an iodoacetamide group of the linker-payload moiety, and (ii) a second linker-payload moiety conjugated via reaction between a cysteine residue of the antigen-binding moiety and a MSODA group of the linker-payload moiety.
  • the first linker-payload moiety comprises a DDR inhibitor moiety as described herein
  • the second linker-payload moiety comprises a TOP1 inhibitor moiety as described herein
  • the first linker-payload moiety comprises a TOP1 inhibitor moiety as described herein
  • the second linker-payload moiety comprises a DDR inhibitor moiety as described herein.
  • a further aspect of the present disclosure provides a DDR inhibitor moiety linked to click group, wherein the click group is suitable for conjugation to a corresponding click group in a linker moiety, and wherein the linker moiety is conjugated, or is suitable for conjugation, to an antigen-binding moiety.
  • a further aspect of the present disclosure provides a TOP1 inhibitor moiety linked to click group, wherein the click group is suitable for conjugation to a corresponding click group in a linker moiety, and wherein the linker moiety is conjugated, or is suitable for conjugation, to an antigen-binding moiety.
  • the click group is selected from:
  • the tri-functional linking groups of the present disclosure are of a modular design which allows the number of payloads attached to the linker and thus the antigen-binding moieties to be readily varied, which has been shown to be important in the development of clinically relevant antibody drug conjugates. Furthermore, the use of click groups to attach payload containing moieties to the branching group allows for a wide range of payload types to be conjugated. Where orthogonal click moieties are used, two different payload moieties can be connected, including those with different, and possibly complimentary, modes of action.
  • the tri-functional linking groups of the present disclosure have a hydrophilic branching group which may lead to a reduction in toxicities and an improvement in biophysical, stability and pharmacokinetic properties.
  • linker-payload moiety may comprise:
  • R N is selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the branching group; b indicates where the at least one first click group is linked to the branching group; c indicates where the at least one second click group is linked to the branching group.
  • R N is H.
  • R N is -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-.
  • R N is -(C1-5 alkylene)-C(O)OH.
  • R N is -CH2CH2OCH2CH2C(O)OH.
  • R N is CH2C(O)OH.
  • the amino group may be linked to the branching group by a first spacer group.
  • the first spacer group is
  • (A1) may comprise:
  • A1 may be of the formula:
  • xb is 0, and xa+xc are from 1 to 7, such as 5 (i.e. A1 is -(CH2)5-). In other embodiments, xb is from 1 to 12, xa is 0 and xc is 0 or 1 .
  • xb is from 1 to 12. In some of these embodiments, xb is from 1 to 6. In some of these embodiments, xb is from 1 to 3, i.e. 1 , 2 or 3. In some embodiments where xb is 1 to 12, xc is 1 to 6, or 1 to 2. In some of these embodiments, xc is 1 . In some of these embodiments, xc is 2.
  • xa is 0, xb is 1 to 6 and xc is 2.
  • A1 is -(C2H4)-O-(C2H4)-. In some of these embodiments, A1 is -(C2H4O)3-(C2H4)-.
  • the first and second click groups may be selected from either member of the following click-group pairs:
  • Cyclooctyne, cyclooctyne derivatives and cyclooctyne analogues for use in the present disclosure include:
  • These groups can alternatively be called cyclic alkynes.
  • Strained alkenes for use in the present disclosure may have the structure:
  • the click group may be a dibenzoazacyclooctyne (DIBAC) group or a 1-ethylhept-6-
  • DIBAC dibenzoazacyclooctyne
  • the click group may be Tetramethylthiocycloheptyne sulfoximine (TMTHSI):
  • the link between the payload moiety (e.g. the DDR inhibitor moiety or the TOP1 inhibitor moiety) and the click group according to the present disclosure may be a cleavable linker moiety or a non-cleavable moiety, e.g. as described hereinabove.
  • the first and second click groups are the same. In other embodiments of the tri-functional linking group, the first and second click groups are selected from orthogonal click-group pairs.
  • the first and/or second click group is azide.
  • the first and/or second click group is tetrazine or a tetrazine derivative.
  • the first and/or second click group is an alkyne (- CCH).
  • the first and/or second click group is cyclooctyne or a cyclooctyne derivative.
  • the first and/or second click group is norbonene or a norbonene derivative.
  • the first and/or second click group is methylcyclopropene (1-MCP).
  • the first click group is azide and the second click group is tetrazine or a tetrazine derivative.
  • the first click group is azide and the second click group is cyclooctene.
  • the first click group is azide and the second click group is norbonene or a norbonene derivative. In some embodiments of the tri-functional linking group, the first click group is azide and the second click group is methylcyclopropene (1-MCP).
  • the first click group is alkyne (-CCH) and the second click group is tetrazine or a tetrazine derivative.
  • the first click group is alkyne (-CCH) and the second click group is cyclooctene.
  • the first click group is alkyne (-CCH) and the second click group is norbonene or a norbonene derivative.
  • the first click group is alkyne (-CCH) and the second click group is methylcyclopropene (1-MCP).
  • the first click group is cyclooctyne or a cyclooctyne derivative and the second click group is tetrazine or a tetrazine derivative.
  • the first click group is cyclooctyne or a cyclooctyne derivative and the second click group is cyclooctene.
  • the first click group is cyclooctyne or a cyclooctyne derivative and the second click group is norbonene or a norbonene derivative.
  • the first click group is cyclooctyne or a cyclooctyne derivative and the second click group is methylcyclopropene (1-MCP).
  • the second click group is phenyl-tetrazine.
  • the second click group is selected from the following groups: In some embodiments of the tri-functional linking group, the second click group is:
  • the at least one first click group may be linked to the branching group by a second spacer group (B1).
  • the second spacer group is branched, such that two first click groups are linked to the branching group.
  • the second spacer group is not branched, such that a single first click group is linked to the branching group.
  • the at least one second click group may be linked to the branching group by a third spacer group (B2).
  • the third spacer group is branched, such that two second click groups are linked to the branching group.
  • the third spacer group is not branched, such that a single second click group is linked to the branching group.
  • the second spacer group (B1) is of formula (B1-1): (B1-1)
  • R NB2 iS H Or -(C2H 4 O)xe2-(CH2)xf2-(NH)x S 2-(C( O)CH2)xh2- where xe2 is 0 to 4, xf2 is 0 to 2, xg2 is 0 or 1 , and xh2 is 0 or 1 .
  • xl3 is 0-2. In some embodiments, xl3 is 0. In some embodiments, xl3 is 1 . In some embodiments, xl3 is 2.
  • xl4 is 0. In some embodiments, xl4 is 1 . In some embodiments, xe1 is 2-4. In some embodiments, xe1 is 2. In some embodiments, xe1 is 3. In some embodiments, xe1 is 4.
  • xe2 is 2-4. In some embodiments, xe2 is 2. In some embodiments, xe2 is 3. In some embodiments, xe2 is 4.
  • the second spacer group (B1) is of formula (B1-2):
  • xd is 0-2. In some embodiments, xd is 0-1 . In some embodiments, xd is 0. In some embodiments, xd is 1 . In some embodiments, xd is 2. In some embodiments, xd is 3. In some embodiments, xd is 0 or 2.
  • xe1 is 0. In some embodiments, xe1 is 1.
  • xf1 is 0-1. In some embodiments, xf1 is 0. In some embodiments, xf1 is 1. In some embodiments, xf1 is 2. In some embodiments, xf1 is 0 or 2.
  • xg1 is 0. In some embodiments, xg1 is 1.
  • xh1 is 0. In some embodiments, xh1 is 1.
  • xe2 is 0. In some embodiments, xe2 is 1 .
  • xf2 is 0-1 . In some embodiments, xf2 is 0. In some embodiments, xf2 is 1 . In some embodiments, xf2 is 2. In some embodiments, xf2 is 0 or 2.
  • xg2 is 0. In some embodiments, xg2 is 1 . In some embodiments, xe1 is 1 , xf1 is 2, xg1 is 0, and xh1 is 0.
  • xe1 is 0, xf1 is 0, xg1 is 0, and xh1 is 1 .
  • xe1 is 1
  • xf1 is 2
  • xg1 is 1
  • xh1 is 1
  • xe2 is 1
  • xf2 is 2
  • xe2 is 0, xf2 is 0, xg2 is 0, and xh2 is 1 .
  • xe2 is 1
  • xf2 is 2
  • xg2 is 1
  • xh2 is 1 .
  • the second spacer group (B1) is selected from the groups containing: In some embodiments of the tri-functional linking group, the second spacer group (B1) is selected from the groups containing:
  • the second spacer group (B2) is of formula (B2-1):
  • xl5 is 0-2. In some embodiments, xl5 is 0. In some embodiments, xl5 is 1 . In some embodiments, xl5 is 2.
  • xl6 is 0. In some embodiments, xl6 is 1 .
  • xj1 is 2-4. In some embodiments, xj1 is 2. In some embodiments, xj1 is 3. In some embodiments, xj 1 is 4.
  • xj2 is 2-4. In some embodiments, xj2 is 2. In some embodiments, xj2 is 3. In some embodiments, xj2 is 4.
  • the third spacer group (B2) is of formula (B2-2):
  • xi is 0-2. In some embodiments, xi is 0-1 . In some embodiments, xi is 0. In some embodiments, xi is 1 . In some embodiments, xi is 2. In some embodiments, xi is 3. In some embodiments, xi is 0 or 2.
  • xj1 is 0. In some embodiments, xj1 is 1.
  • xk1 is 0-1. In some embodiments, xk1 is 0. In some embodiments, xk1 is 1. In some embodiments, xk1 is 2. In some embodiments, xk1 is 0 or 2.
  • xl1 is 0. In some embodiments, xl1 is 1.
  • xml is 0. In some embodiments, xml is 1. In some embodiments, xj2 is 0. In some embodiments, xj2 is 1 .
  • xk2 is 0-1 . In some embodiments, xk2 is 0. In some embodiments, xk2 is 1 . In some embodiments, xk2 is 2. In some embodiments, xk2 is 0 or 2.
  • xl2 is 0. In some embodiments, xl2 is 1 .
  • xj1 is 1 , xk1 is 2, x 11 is 0, and xml is 0. In some embodiments, xj 1 is 0, xk1 is 0, x 11 is 0, and xml is 1 . In some embodiments, xj1 is 1 , xk1 is 2, x 11 is 1 , and xml is 1 .
  • xj2 is 1 , xk2 is 2, x I2 is 0, and xm2 is 0. In some embodiments, xj2 is 0, xk2 is 0, x I2 is 0, and xm2 is 1 . In some embodiments, xj2 is 1 , xk2 is 2, x I2 is 1 , and xm2 is 1 .
  • the third spacer group (B2) is selected from the
  • the third spacer group (B2) is selected from the groups containing: In some embodiments of the tn-functional linking group, the second spacer group (B1) is the same as the third spacer group (B2). In some embodiments of the tri-functional linking group, the second spacer group (B1) is different to the third spacer group (B2).
  • the second spacer group (B1) and the third spacer group (B2) together with the nitrogen atom to which they are attached form one of the flowing groups:
  • the second spacer group (B1) and the third spacer group (B2) together with the nitrogen atom to which they are attached form one of the flowing groups:
  • the present disclosure provides a linker comprising:
  • R N is selected from H and -(C1-5 alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates where the amino group is linked to the branching group; b indicates where the at least one first click group is linked to the branching group; c indicates where the at least one second click group is linked to the branching group.
  • the reaction between the first and second members of the click group pairs can result in two isomeric products, i.e. a mixture.
  • the present disclosure includes both isomeric forms when only one is shown.
  • the amino group may be linked to the branching group by a first spacer group (A1) as defined above.
  • the at least one first click group may be linked to the branching group by a second spacer group (B1) as defined above.
  • the at least one second click group may be linked to the branching group by a third spacer group (B2) as defined above.
  • the linker comprises one of the following groups:
  • the link between the payload moiety (e.g. the DDR inhibitor moiety or the TOP1 inhibitor moiety) and the click group according to the present disclosure may be a cleavable linker moiety or a non-cleavable moiety, e.g. as described hereinabove.
  • the linker the link between the payload moiety and the click moiety comprises:
  • Q x is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue
  • a is 0, c is 1 and d is 2, and b may be from 0 to 8. In some of these embodiments, b is 0, 4 or 8.
  • Q is an amino acid residue.
  • the amino acid may a natural amino acids or a nonnatural amino acid.
  • Q is selected from: Phe, Lys, Vai, Ala, Cit, Leu, lie, Arg, Ser, and Trp, where Cit is citrulline.
  • Q is a serine derivative (see WO2018/234636A1).
  • Q comprises a dipeptide residue.
  • the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids.
  • the dipeptide comprises natural amino acids.
  • the linker is a cathepsin labile linker
  • the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
  • Q is selected from:
  • Q is selected from:
  • Q is selected from C0 -Phe-Lys- NH , co -Val-Cit- NH and C0 -Val-Ala- NH .
  • the link between the payload moiety (e.g. the DDR inhibitor moiety or the TOP1 inhibitor moiety) and the click group comprises: PABC, a cathepsin-cleavable dipeptide, and a PEG2 to PEG4 (e.g. a PEG3) group.
  • the link between the payload moiety and the click group comprises, or is:
  • the link between the payload moiety comprises GGFG (Glycine-Glycine- Phenylalanine-Glycine). This may be directly linked to the payload or linked via a CH2 group.
  • the link between the payload moiety and the click group comprises, or is:
  • the DDR inhibitor moiety linked to click group has the structure:
  • the TOP1 inhibitor moiety linked to click group has the structure:
  • the DDR inhibitor moiety linked to click group has the structure: P-3
  • the DDR inhibitor moiety linked to click group has the structure:
  • the DDR inhibitor moiety linked to click group has the structure: In some embodiments, the DDR inhibitor moiety linked to click group has the structure:
  • the DDR inhibitor moiety linked to click group has the structure: In some embodiments, the DDR inhibitor moiety linked to click group has the structure:
  • the DDR inhibitor moiety linked to click group has the structure:
  • the DDR inhibitor moiety linked to click group has the structure: In some embodiments, the DDR inhibitor moiety linked to click group has the structure:
  • the DDR inhibitor moiety linked to click group has the structure:
  • the DDR inhibitor moiety linked to click group has the structure:
  • an antigen-binding molecule described herein may possess one or more of the following properties: binds to cells expressing HER2; inhibits proliferation of HER2-expressing cells; increases killing of cells expressing HER2; inhibits tumor growth and/or reduces tumor size/volume (e.g. of a HER2-ex pressing cancer); increases survival of subjects having a cancer (e.g. a HER2-expressing cancer).
  • a given antigen-binding molecule may display more than one of the properties recited in the preceding paragraph.
  • a given antigen-binding molecule may be evaluated for the properties recited in the preceding paragraph using suitable assays.
  • the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays.
  • the assays may be e.g. in vivo assays, i.e. performed in non-human animals.
  • the assays may be e.g. ex vivo assays, i.e. performed using cells/tissue/an organ obtained from a subject.
  • assays are cell-based assays, they may comprise treating cells with an antigen-binding molecule in order to determine whether the antigen-binding molecule displays one or more of the recited properties.
  • Assays may employ species labelled with detectable entities in order to facilitate their detection.
  • Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities/concentrations of a given antigen-binding molecule (e.g. a dilution series).
  • Analysis of the results of such assays may comprise determining the concentration at which 50% of the maximal level of the relevant activity is attained.
  • concentration of a given agent at which 50% of the maximal level of the relevant activity is attained may be referred to as the ‘half-maximal effective concentration’ of the agent in relation to the relevant activity, which may also be referred to as the ‘EC50’.
  • the EC50 may also be referred to as the ‘half-maximal inhibitory concentration’ or ‘IC50’, this being the concentration of the agent at which 50% of the maximal level of inhibition of a given property is observed.
  • the antigen-binding molecule of the present disclosure binds to HER2 in a region which is accessible to an antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when HER2 is expressed at the cell surface (i.e. in or at the cell membrane). In some embodiments, the antigen-binding molecule binds to HER2 expressed at the cell surface of a cell expressing HER2. In some embodiments, the antigen-binding molecule binds to HER2-ex pressing cells.
  • the ability of an antigen-binding molecule to bind to a given cell type can be analysed by contacting cells with the antigen-binding molecule, and detecting antigen-binding molecule bound to the cells, e.g. after a washing step to remove unbound antigen-binding molecule.
  • the ability of an antigen-binding molecule to bind to HER2-ex pressing cells can be analysed by methods such as flow cytometry and immunofluorescence microscopy.
  • the antigen-binding molecule inhibits proliferation of HE R2-ex pressing cells (e.g. HER2-expressing cancer cells).
  • the ability of an antigen-binding molecule to inhibit proliferation of a given cell type can be analysed by contacting cells with the antigen-binding molecule, and subsequently evaluating proliferation of the cells (/.e. after a period of time sufficient for an effect on cell proliferation to be observed).
  • Cell proliferation can be evaluated e.g. by detecting changes in number of cells over time, or by in vitro analysis of incorporation of 3 H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety.
  • the antigen-binding molecule of the present invention is capable of inhibiting proliferation of HER2-expressing cells to less than 1 times, e.g. ⁇ 0.99 times, ⁇ 0.95 times, ⁇ 0.9 times, ⁇ 0.85 times, ⁇ 0.8 times, ⁇ 0.75 times, ⁇ 0.7 times, ⁇ 0.65 times, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times, ⁇ 0.25 times, ⁇ 0.2 times, ⁇ 0.15 times, ⁇ 0.1 times, ⁇ 0.05 times, or ⁇ 0.01 times the level of proliferation of HER2-expressing cells observed in the absence of the antigenbinding molecule (or in the presence of an appropriate control antigen-binding molecule known not to influence proliferation of HER2-expressing cells), in a given assay.
  • the antigen-binding molecule described herein inhibits proliferation of cells expressing human HER2 with an IC50 of 100 nM or less, preferably one of ⁇ 50 nM, ⁇ 40 nM, ⁇ 30 nM, ⁇ 20 nM, ⁇ 10 nM, ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM or ⁇ 500 pM.
  • the antigen-binding molecule according to the present disclosure potentiates (/.e. upregulates, enhances) cell killing of cells comprising/expressing HER2.
  • an antigen-binding molecule according to the present disclosure may inhibit growth or reduce metastasis of a cancer comprising cells comprising/expressing HER2.
  • an antigen-binding molecule may potentiate (/.e. upregulate, enhance) cell killing of cells comprising/expressing HER2.
  • an antigen-binding molecule may inhibit growth of cells of a cancer, or may inhibit growth of a tumor, comprising cells comprising/expressing HER2.
  • an antigen-binding molecule may inhibit metastasis of a cancer/tumor comprising cells comprising/expressing HER2.
  • cytotoxicity/cell killing assays include release assays such as the 51 Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) release assay, ATP release assay using Cell Titre Gio, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells.
  • LDH lactate dehydrogenase
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
  • ATP release assay using Cell Titre Gio ATP release assay using Cell Titre Gio
  • calcein-AM calcein-acetoxymethyl
  • an antigen-binding molecule according to the present disclosure is capable of reducing the number/proportion of cells expressing HER2. In some embodiments, an antigen-binding molecule according to the present disclosure is capable of depleting/enhancing depletion of such cells. In some embodiments, an antigen-binding molecule of the present disclosure displays anticancer activity. In some embodiments, the antigen-binding molecule increases killing of cancer cells. In some embodiments, the antigen-binding molecule causes a reduction in the number of cancer cells in vivo, e.g. as compared to an appropriate control condition.
  • the cancer may be a cancer as described herein, e.g. a cancer expressing/overexpressing HER2.
  • an antigen-binding molecule according to the present disclosure reduces/inhibits growth of a cancer and/or of a tumor of a cancer. In some embodiments, an antigen-binding molecule reduces tissue invasion by cells of a cancer. In some embodiments, an antigen-binding molecule reduces metastasis of a cancer. In some embodiments, an antigen-binding molecule displays anticancer activity.
  • an antigen-binding molecule reduces the growth/proliferation of cancer cells. In some embodiments, an antigen-binding molecule reduces the survival of cancer cells. In some embodiments, an antigen-binding molecule increases the killing of cancer cells. In some embodiments, an antigen-binding molecule of the present disclosure causes a reduction in the number of cancer cells e.g. in vivo.
  • the cancer may be a cancer comprising cells expressing HER2.
  • An antigen-binding molecule of the present disclosure may be analysed for the properties described in the preceding paragraph in appropriate assays.
  • assays include e.g. in vivo models.
  • administration of an antigen-binding molecule according to the present disclosure may cause one or more of: inhibition of the development/progression of the cancer, a delay to/prevention of onset of the cancer, a reduction in/delay to/prevention of tumor growth, a reduction in/delay to/prevention of tissue invasion, a reduction in/delay to/prevention of metastasis, a reduction in the severity of one or more symptoms of the cancer, a reduction in the number of cancer cells, a reduction in the cancer burden, a reduction in tumor size/volume, and/or an increase in survival of subjects having the cancer (e.g. progression free survival or overall survival), e.g. as determined in an appropriate model.
  • inhibition of the development/progression of the cancer e.g. progression free survival or overall survival
  • Tumor growth may be monitored by investigating tumor volume over time. Tumor growth may be evaluated by measuring tumor volume (e.g. in mm 3 ) over time.
  • an antigen-binding molecule of the present disclosure is capable of reducing tumor size/volume (e.g. the mean tumor size/volume for the treatment group in an in vivo model, e.g. of a HER2-expressing cancer) to less than 1 times, e.g.
  • evaluation of tumor size/volume for the purposes of such comparison is performed after more than 5 days, e.g. one of >10 days, >15 days, >20 days, >25 days, >30 days, >35 days, >40 days, >35 days, >50 days, >55 days, >60 days, >65 days, >70 days, >75 days, >80 days, >85 days, >90 days, >95 days or >100 days following administration of the first dose of the antigen-binding molecule, in the relevant model.
  • an antigen-binding molecule of the present disclosure achieves a level of tumor growth inhibition (e.g. expressed as % tumor growth inhibition, e.g. calculated relative to tumor growth observed on treatment with an appropriate control antigen-binding molecule) which is greater than 1 times, e.g.
  • evaluation of tumor growth inhibition for the purposes of such comparison is performed after more than 5 days, e.g. one of >10 days, >15 days, >20 days, >25 days, >30 days, >35 days, >40 days, >35 days, >50 days, >55 days, >60 days, >65 days, >70 days, >75 days, >80 days, >85 days, >90 days, >95 days or >100 days following administration of the first dose of the antigen-binding molecule, in the relevant model.
  • an antigen-binding molecule of the present disclosure is capable of increasing median survival of subjects having a cancer (e.g. in an in vivo model, e.g. of a HER2-ex pressing cancer) to greater than 1 times, e.g.
  • antigen-binding molecules of the present disclosure and their constituent polypeptides may additionally comprise further amino acids or sequences of amino acids.
  • the polypeptides of the present disclosure may comprise one or more linker sequences between sequences of amino acids.
  • Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety.
  • a linker sequence may be a flexible linker sequence.
  • Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence.
  • Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.
  • the linker sequence comprises or consists of (G4S)4 or (G4S)e. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.
  • antigen-binding molecules of the present disclosure and their constituent polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide.
  • antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a sequence of amino acids forming a detectable moiety, e.g. as described hereinbelow.
  • the antigen-binding molecules of the present disclosure and their constituent polypeptides may additionally comprise a signal peptide (also known as a leader sequence or signal sequence).
  • Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.
  • Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).
  • the signal peptide may be present at the N-terminus of the polypeptide, and may be present in the newly synthesised polypeptide.
  • the signal peptide provides for efficient trafficking of the polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature polypeptide.
  • Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).
  • SignalP Protein et al., 2011 Nature Methods 8: 785-786
  • Signal-BLAST Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176.
  • the antigen-binding molecules of the present disclosure and their constituent polypeptides comprise a detectable moiety.
  • a detectable moiety is a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label.
  • An antigen-binding molecule or a constituent polypeptide thereof may be covalently or non-covalently labelled with the detectable moiety.
  • Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5.
  • fluorescein e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB
  • GFP green fluorescent protein
  • Eu europium
  • Tb terbium
  • Sm samarium
  • tetramethyl rhodamine Texas Red
  • 4-methyl umbelliferone 7-amino-4-methyl coumarin
  • Cy3 Cy5
  • Radiolabels include radioisotopes such as Hydrogen 3 , Sulfur 35 , Carbon 14 , Phosphorus 32 , Iodine 123 , Iodine 125 , Iodine 126 , Iodine 131 , Iodine 133 , Bromine 77 , Technetium 99m , Indium 111 , lndium 113m , Gallium 67 , Gallium 68 , Ruthenium 95 , Ruthenium 97 , Ruthenium 103 , Ruthenium 105 , Mercury 207 , Mercury 203 , Rhenium 99m , Rhenium 101 , Rhenium 105 , Scandium 47 , Tellurium 121m , Tellurium 122m , Tellurium 125m , Thulium 165 , Thuliuml 167 , Thulium 168 , Copper 67 , Fluorine 18 , Yttrium 90 , Palladium 100 , Bismuth 217
  • Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels.
  • Immuno-detectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin.
  • Nucleic acid labels include aptamers.
  • an antigen-binding molecule or a constituent polypeptide thereof comprises a radionuclide.
  • Such conjugates may be called radionuclide drug conjugates (RDCs) or radioimmunoconjugates (RICs).
  • RDCs radionuclide drug conjugates
  • RICs radioimmunoconjugates
  • an antigen-binding molecule or a constituent polypeptide thereof comprises a chelator group that is capable of chelating a radionuclide.
  • Radionuclides include radioisotopes such as those listed above, or others such as Lutetium 177 , Actinium 225 and Strontium 90 .
  • an antigen-binding molecule or a constituent polypeptide thereof comprises an epitope tag, e.g. a His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol), optionally at the N- or C- terminus of the antigen-binding molecule/polypeptide.
  • an epitope tag e.g. a His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-
  • an antigen-binding molecule or a constituent polypeptide thereof polypeptide comprises a moiety having a detectable activity, e.g. an enzymatic moiety.
  • Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. beta-galactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.
  • the present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding an antigen-binding molecule/antigen-binding polypeptide complex or a constituent polypeptide thereof according to the present disclosure.
  • the nucleic acid(s) comprise or consist of DNA and/or RNA.
  • An antigen-binding molecule/antigen-binding polypeptide complex or a constituent polypeptide thereof according to the present disclosure may be produced within a cell by translation of RNA encoding the polypeptide(s).
  • An antigen-binding molecule/antigen-binding polypeptide complex or a constituent polypeptide thereof may be produced within a cell by transcription from nucleic acid encoding the polypeptide(s), and subsequent translation of the transcribed RNA.
  • the nucleic acid(s) may be, or may be comprised/contained in, a vector, or a plurality of vectors.
  • a ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.
  • the present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure.
  • the vector may facilitate delivery of the nucleic acid(s) encoding a polypeptide according to the present disclosure to a cell.
  • the vector may be an expression vector comprising elements required for expressing a polypeptide according to the present disclosure.
  • the vector may comprise elements facilitating integration of the nucleic acid(s) into the genomic DNA of cell into which the vector is introduced.
  • Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.
  • a vector may be a vector for expression of the nucleic acid in the cell (i.e. an expression vector).
  • Such vectors may include a promoter sequence operably linked to a nucleotide sequence encoding an antigenbinding molecule or polypeptide according to the present disclosure.
  • a vector may also include a termination codon (i.e. 3’ in the nucleotide sequence of the vector to the nucleotide sequence encoding the polypeptide(s)) and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.
  • operably linked may include the situation where nucleic acid encoding a polypeptide according to the present disclosure and regulatory nucleic acid sequence(s) (e.g. a promoter and/or enhancers) are covalently linked in such a way as to place the expression of the nucleic acid encoding a polypeptide under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette).
  • regulatory nucleic acid sequence(s) e.g. a promoter and/or enhancers
  • a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence.
  • the resulting transcript(s) may then be translated into the desired polypeptide(s).
  • Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g.
  • plasmids e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes
  • viral vectors e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g.
  • a vector according to the present disclosure is a lentiviral vector.
  • the vector may be a eukaryotic vector, i.e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell.
  • the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
  • CMV cytomegalovirus
  • Constituent polypeptides of an antigen-binding molecule/antigen-binding polypeptide complex according to the present disclosure may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors.
  • Antigen-binding molecules according to the present disclosure may be prepared according to methods for the production of antibody-drug conjugates known to the skilled person.
  • Antigen-binding moieties according to the present disclosure may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis.
  • peptides/polypeptides can be synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.
  • antigen-binding moieties according to the present disclosure may be produced by recombinant expression.
  • Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety.
  • Methods for the recombinant production of antigen-binding polypeptides are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461 , both ofwhich are hereby incorporated by reference in their entirety.
  • the antigen-binding moieties of the present disclosure are comprised of more than one polypeptide chain.
  • production of the antigen-binding moiety may comprise transcription and translation of more than one polypeptide, and subsequent association of the polypeptide chains to form the antigen-binding moiety.
  • any cell suitable for the expression of polypeptides may be used.
  • the cell may be a prokaryote or eukaryote.
  • the cell is a prokaryotic cell, such as a cell of archaea or bacteria.
  • the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli.
  • the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. a cell described hereinabove.
  • the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells.
  • very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags.
  • Specific plasmids may also be utilised which enhance secretion of the protein into the media.
  • polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g. according to a system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431 , which is hereby incorporated by reference in its entirety.
  • CFPS cell-free-protein synthesis
  • Production of antigen-binding moieties may involve culture or fermentation of a eukaryotic cell modified to express the polypeptide(s) of interest.
  • the culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air/oxygen and/or growth factors.
  • Secreted proteins can be collected by partitioning culture media/fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide(s).
  • Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th Edition; incorporated by reference herein above).
  • Bioreactors include one or more vessels in which cells may be cultured.
  • Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches.
  • the bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.
  • the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide, it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted polypeptide(s) of interest. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by Bonification, rapid freeze-thaw or osmotic lysis.
  • polypeptide(s) of interest may be isolated from the supernatant or culture medium, which may contain other protein and non-protein components.
  • a common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins. Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation or may be performed subsequently to precipitation.
  • polypeptide(s) of interest may be desired or necessary to concentrate the polypeptide(s).
  • a number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.
  • Antigen-binding polypeptides/polypeptide complexes according to the present disclosure may be conjugated to linker-payload moieties according to the present disclosure for the production of antigenbinding molecules according to the present disclosure by any suitable techniques, which are well known to the skilled person and routinely employed in the art.
  • Antigen-binding moieties according to the present disclosure may be conjugated to linker-payload moieties according to the present disclosure by any suitable techniques, which are well known to the skilled person and routinely employed in the art. Such methods are described e.g. in Chudasama et al., Nature Chemistry, (2016), 8:114-119, Baah etal., Molecules. (2021) 26(10): 2943, and Walsh et al., Chem. Soc. Rev. (2021) 50:1305-1353, all of which are hereby incorporated by reference in their entirety.
  • Conjugation of antigen-binding moieties and linker-payload moieties and the purification of antigenbinding molecules produced by such conjugation can be performed e.g. as described in in Beck et al., (2017) Nat Rev Drug Discov 16: 315-337; Peters and Brown Biosci Rep (2015) 35: art:e00225; McCombs and Owen, The AAPS Journal (2015) 17: 339-351 ; Jackson, Org Process Res Dev (2016) 20: 852-866; or Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcome to Target Cancer, (2016) Wiley, all of which are hereby incorporated by reference in their entirety.
  • Lysine-based conjugation is one of the most widely used non-specific conjugation strategies. Such conjugation occurs on reactive amine side chains of lysine residues due to their good nucleophilicity. Immunoglobulin scaffolds contains over 80 lysine residues, most of which are exposed on the surface of the molecule. Among the surface lysine residues, more than 20 have been shown as highly solvent- accessible and can serve as potential ADC conjugation sites. Lysine conjugation follows two main strategies that result in the formation of a stable amide or amidine bond between the protein and the drug-linker complex.
  • activated esters on the drug-linker complexes often O- succinimide reagents such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS esters, react with the antibody lysine residues and achieve the conjugation via amide bonds.
  • O- succinimide reagents such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS esters
  • NHS N-hydroxysuccinimidyl
  • sulfo-NHS esters react with the antibody lysine residues and achieve the conjugation via amide bonds.
  • stable amidine bonds can be generated on an antibody by the reaction of imido ester compounds, such as Traut’s reagent, with antibody lysine residues.
  • a one-step conjugation of a drug-linker moiety containing an amine-reactive group to the antibody via amide bonds is known, as well as two-step conjugation, where in the first step, a small bi-functional reagent containing both an amine- and a thiol-reactive functional groups is reacted with the available lysine e-amino groups to serve as a chemical adaptor, leaving free thiol-reactive groups on the antibody. In the second step, the payload drugs or drug-linker complexes are attached to the thiol-reactive groups introduced previously to form the ADC.
  • the two-step approach is often used when the drug/drug-linker complex contains a thiol-reactive module or as an alternative route when introducing an amine-reactive module into the drug or drug-linker complex is proven to be difficult.
  • SPDB disulfide MCC (maleimidomethyl cyclohexane-1 -carboxylate), sulfo- SPDB, and Hydrazine.
  • Cysteine modification occurs most commonly by 1 ,4-conjugate addition to A/-substituted maleimides.
  • Maleimides are particularly attractive reagents due to their synthetic accessibility and rapid reaction rates with cysteine under mild conditions.
  • the resulting thiosuccinimide conjugates are inherently unstable, due to their propensity towards retro-Michael addition. This instability can be mitigated by forcing postconjugation hydrolysis of the thiosuccinimide, creating a stable chemical linkage. Accordingly, a number of “self-hydrolysing” maleimides have now been developed, with ring-opening catalysed by adjacent functional groups such as primary amine, polyethylene glycol (PEG) and A/-aryl amongst the most promising.
  • PEG polyethylene glycol
  • reagents including a-halocarbonyls, palladium oxidative-addition complexes, ethynylphosphonamidates, vinylphosphonites and ethynylbenziodoxolones.
  • Genetic modification of the number of accessible cysteine residues on an antibody surface is a method to achieve site-selective and homogeneous modification.
  • the engineered cysteine is installed on an anti-MUC16 antibody by mutation of heavy chain alanine 114 (HC-A114).
  • ncAAs non-canonical amino acids
  • Such ncAAs include p-acetylphenylalanine (pAcF), which has a ketone side chain which can participate in oxime ligation reactions; Ne-(1- methylcycloprop-2-enecarboxamido)-lysine (CpK), which has a cyclopropene side chain which can participate in IEDDA reactions; para-azidomethyl phenylalanine (pAMF), which has a an azide side chain which can undergo click reactions; spiro[2.4]hepta-4,6-diene-lysine (SCpHK), which has a spiro[2.4]hepta-4,6-diene side chain which can participate in Diels-Alder reactions; and N6-(2- azidoethoxy)-carbonyl-L-lysine (
  • Azide-containing ncAAs can undergo rapid CuAAC or SPAAC reactions under physiological conditions, para-azidophenylalanine (pAzF) can undergo reactions with, for example, cyclooctyne-functionalised linkers and dibenzylcyclooctyne (DBCO)-functionalised linkers.
  • pAzF para-azidophenylalanine
  • DBCO dibenzylcyclooctyne
  • a cyclopropene derivative of lysine N e-[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l-lysine; CypK
  • I EDDA inverse-electron demand Diels-Alder
  • Cyclopentadiene-containing ncAAs, spiro[2.4]hepta-4,6-diene-lysine (SCpHK) and cyclopentadiene-lysine (CpHK), can undergo irreversible Diels-Alder cycloadditions with maleimide-modified drugs.
  • Enzymes can be used to achieve site-selective antibody modification due to their high specificity and mild reaction conditions. Enzymes can either directly attach a payload to a specific amino acid sequence or introduce a reactive functionality on the antibody that can be further functionalised with the desired pay load.
  • Sortase-mediated antibody conjugation (SMAC) technology is an additional enzymatic ligation approach.
  • the sortase recognition motif and a Strep II tag which is used to aid removal of unreacted antibody, were fused to the light and heavy chain C-terminus of different antibodies. Sortase-mediated conjugation can then be used to attach a series of penta-glycine tagged payloads.
  • transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined DAR arising from the conjugation of 2 linker-payloads (one conjugation site per heavy chain).
  • An N297Q mutation prior to this conjugation provides two more reaction sites (resulting in the conjugation of 4-linker- payloads).
  • An alternative version using a peptide sequence-specific transglutaminase is a powerful approach for site-specific incorporation of the payload into the antibody.
  • This enzyme recognizes and utilizes LLQG motif that is genetically incorporated, resulting in site-specific antibody-drug conjugation.
  • Another advantage of this LLQG-specific bacterial transglutaminase is that conjugation sites can be flexibly laid by inserting this short peptide motif within the antibody structure. Further alternative approaches allow for the use of transglutaminase without deglycosylation.
  • the DAR will depend on the number of payloads per linker-payload moieties conjugated.
  • Asn297 (N297) within the Fc domain and the N-glycan on this residue are conserved in all IgG classes, making these components attractive reaction sites for broadly applicable ADC conjugation.
  • Incorporation of an aldehyde group on the N-glycan terminus using p-1 ,4-galactosyltransferase (GalT) and a-2,6- sialyltransferase (SialT) introduce a sialic acid on each N-glycan terminus, which is subsequently converted into an aldehyde group using NalC under mild oxidation conditions. The aldehyde groups generated can then used to conjugate aminooxyfunctionalized payloads.
  • Another approach is to incorporate non-natural saccharides possessing orthogonal reaction handles into the antibody.
  • a technology based on this strategy is the GlycoConnect in which the glycan chain at Asn297 is trimmed using the endoglycosidase Endo S2 and then azide groups are introduced using a mutant galactosyl transferase GalT(Y289L) and N-azidoacetylgalactosamine (GalNAz).
  • the azide handles can be used for a strain-promoted click reaction with payloads.
  • the linker-payload terminates in an amino group which is conjugated to the antigen-binding molecule using transglutaminase.
  • the method further comprises purifying/isolating the antigen-binding molecule (/.e. from unreacted precursors and/or by-products).
  • the antigen-binding molecule may be purified/isolated by chromatography, e.g. size-exclusion chromatography.
  • the present disclosure also provides an antigen-binding molecule obtained or obtainable by the methods of the present disclosure.
  • the present disclosure provides a composition comprising an antigen-binding molecule according to the present disclosure.
  • the antigen-binding molecules described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.
  • a pharmaceutical composition/medicament comprising an antigen-binding molecule described herein.
  • compositions/medicaments of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents/excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g.
  • pharmaceutically-acceptable carriers e.g. liposomes, micelles, microspheres, nanoparticles
  • diluents/excipients e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium
  • vitamin A vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium
  • lubricants e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin
  • binders e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol
  • solubilisers e.g., surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).
  • pharmaceutically-acceptable refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation.
  • Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23 rd Edition (2020), Academic Press.
  • compositions and medicaments of the present disclosure may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration.
  • a pharmaceutical composition/medicament may be formulated for administration by injection or infusion, or administration by ingestion.
  • Suitable formulations may comprise the antigen-binding molecule provided in a sterile or isotonic medium.
  • Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form.
  • Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.
  • the pharmaceutical compositions/medicament is formulated for injection or infusion, e.g. into a blood vessel, tissue/organ of interest, or a tumor.
  • the present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing an antigen-binding molecule described herein; isolating/purifying an antigen-binding molecule described herein; and/or mixing an antigen-binding molecule described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.
  • a further aspect of the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease/condition (e.g. a disease/condition described herein), the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.
  • a disease/condition e.g. a disease/condition described herein
  • the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.
  • antigen-binding molecules and compositions described herein find use in therapeutic and prophylactic intervention for disease, e.g. cancers. It will be appreciated that the antigen-binding molecules and compositions of the present disclosure may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from a reduction in the level of expression or activity of HER2, or a reduction in the number or activity of cells comprising/expressing HER2.
  • the disease/condition may be a disease/condition in which HER2, or cells expressing/overexpressing HER2 are pathologically-implicated, e.g. a disease/condition in which an increased level/activity of HER2, or an increase in the number/proportion of cells comprising/expressing HER2 is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition.
  • an increased level/activity of HER2, or an increase in the number/proportion of cells comprising/expressing HER2 may be a risk factor for the onset, development or progression of the disease/condition.
  • the present disclosure provides an antigen-binding molecule or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is an antigen-binding molecule or composition described herein for use in a method of treating or preventing a cancer (e.g. a cancer described herein). Also provided is the use of an antigen-binding molecule or composition described herein in the manufacture of a medicament for treating or preventing a cancer (e.g. a cancer described herein). Also provided is a method of treating or preventing a cancer (e.g. a cancer described herein) in a subject, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigenbinding molecule or composition described herein.
  • a cancer e.g. a cancer described herein
  • the methods may be effective to reduce the development or progression of a cancer, alleviation of the symptoms of a cancer or reduction in the pathology of a cancer.
  • the methods may be effective to prevent progression of the cancer, e.g. to prevent worsening of, or to slow the rate of development of, the cancer.
  • the methods may lead to an improvement in the cancer, e.g. a reduction in the symptoms of the cancer or reduction in some other correlate of the severity/activity of the cancer.
  • the methods may prevent development of the cancer to a later stage (e.g. a chronic stage or metastasis).
  • a ‘cancer’ may be or comprise any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor.
  • the cancer may be benign or malignant.
  • the cancer may be primary or secondary (metastatic).
  • a neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue.
  • the cancer may be of tissues/cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • renal epithelia gallbladder, biliary tract, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.
  • Tumors to be treated may be nervous or non-nervous system tumors.
  • Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma.
  • Non-nervous system cancers/tumors may originate in any other non-nervous tissue; examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.
  • NHL Non-Hodgkin’s lymphoma
  • CML chronic myelogenous leukemia
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • CTCL
  • the cancer to be treated/prevented comprises cells expressing an EGFR family member (e.g. HER2, EGFR, HER3 or HER4), and/or cells expressing a ligand for an EGFR family member.
  • the cancer to be treated/prevented comprises cells expressing a mutant or wildtype version of an EGFR family member (e.g. HER2, EGFR, HER3 or HER4).
  • the cancer to be treated/prevented is a cancer which is positive for an EGFR family member.
  • the cancer comprises cells that overexpress an EGFR family member and/or a ligand for an EGFR family member. Overexpression can be determined by detection of a level of expression which is greater than the level of expression by equivalent non-cancerous cells/non-tumor tissue.
  • Expression may be determined by any suitable means.
  • Expression may be gene expression or protein expression.
  • Gene expression can be determined e.g. by detection of mRNA encoding HER2, for example by quantitative real-time PCR (qRT-PCR).
  • Protein expression can be determined e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.
  • the cancer is a cancer in which HER2 is pathologically-implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of HER2, a cancer for which expression of HER2 is a risk factor and/or a cancer for which expression of HER2 is positively associated with onset, development, progression, severity or metastasis of the cancer.
  • the cancer may be characterised by expression of HER2, e.g. the cancer may comprise cells (e.g. cells of tumor tissue) expressing HER2. Such cancers may be referred to as being positive for HER2.
  • a cancer which is ‘positive’ for HER2 may be a cancer comprising cells expressing HER2 (e.g. at the cell surface).
  • a cancer which is ‘positive’ for HER2 may overexpress HER2.
  • the cancer to be treated/prevented comprises cells harboring a genetic variant (e.g. a mutation) which causes increased (gene and/or protein) expression and/or activity of HER2, relative to comparable cells harboring a reference allele not comprising the genetic variant (e.g. a non- mutated, or ‘wildtype’ allele).
  • the genetic variant may be or comprise insertion, deletion, substitution to, or larger-scale translocation/rearrangement of, the nucleotide sequence relative to the reference allele.
  • a mutation ‘resulting in’ increased expression of HER2 may be known or predicted to cause, or may be associated with, increased gene/protein expression of HER2.
  • a mutation ‘resulting in’ increased activity of HER2 may be known or predicted to cause, or may be associated with, increased HER2-mediated signaling and/or EGFR-mediated signaling. Mutations resulting in increased expression and/or activity of HER2 may be referred to as ‘activating’ mutations.
  • a mutation which causes increased expression of HER2 may result in gene or protein expression of HER2 which is not expressed by, and/or not encoded by genomic nucleic acid of, an equivalent cell not harboring the mutation. That is, the expression of HER2 may be a result of the mutation, and thus ‘increased expression’ may be from no expression.
  • a mutation which causes increased expression of HER2 may result in increased gene or protein expression of HER2 which is expressed by, and/or which is encoded by genomic nucleic acid of, an equivalent cell not comprising the mutation.
  • a cell may comprise a mutation resulting in an increase in the level of transcription of nucleic acid encoding HER2 relative to the level of transcription of nucleic acid encoding HER2 by an equivalent cell not comprising the mutation.
  • a mutation which causes increased expression of HER2 may cause an increase in gene expression of HER2 relative to an equivalent cell not comprising the mutation. In some embodiments, a mutation which causes increased expression of HER2 may cause an increase in protein expression of HER2 relative to an equivalent cell not comprising the mutation.
  • a mutation which causes increased expression of HER2 may cause an increase in the level of HER2 on or at the cell surface of a cell comprising the mutation, relative to an equivalent cell not comprising the mutation.
  • Cells having increased expression of HER2 relative to the level of expression of HER2 by a reference cell may be described as ‘overexpressing’ HER2, or having ‘upregulated expression’ of HER2.
  • a cancer comprising cells harboring a mutation resulting in increased expression of HER2 relative to equivalent cells lacking the mutation may be described as a cancer comprising cells displaying overexpression/upregulated expression of HER2.
  • the reference cell lacking the mutation may be a non-cancerous cell (e.g. of equivalent cell type) or a cancerous cell (e.g. of equivalent cancer type).
  • a mutation which causes increased activity of HER2 may result in an increase in HER2-mediated signaling relative to the level of HER2-mediated signaling by an equivalent cell not comprising the mutation.
  • a cancer to be treated/prevented in accordance with the present disclosure may be characterised by an increase in the expression and/or activity of HER2 (/.e. gene and/or protein expression) in an organ/tissue/subject affected by the disease/condition e.g. as compared to normal organ/tissue/subject (/.e. in the absence of the disease/condition).
  • cells and/or a tumor of a cancer to be treated/prevented may be characterised by an increase in the expression and/or activity of HER2, e.g. as compared to the level of expression and/or activity observed in equivalent non- cancerous cells/non-tumor tissue.
  • a HER2-overexpressing cancer may overexpress HER2 as a consequence of amplification of the ERBB2 gene.
  • a cancer to be treated/prevented in accordance with the present disclosure is a ERBB2-amplified cancer.
  • ERBB2 amplification can be identified using techniques well known in the art, such as by immunohistochemical analysis, and analysis by in situ hybridisation (see e.g. Wesota and Jeleri, Adv Clin Exp Med. (2015) 24(5):899-903).
  • ERBB2 amplification can be evaluated by fluorescence in situ hybridisation, e.g. as described in Stocker et al., PLoS One (2016) 11 (7): e0159176.
  • ERBB2- amplified cancers may comprise a ratio of ERBB2 to centromere 17 (CEP17) >2 (e.g. >4, >8), as determined by in situ hybridisation.
  • HER2 and its association with and role in cancer is reviewed e.g. in Oh and Bang, Nat Rev Clin Oncol (2020) 17:33-48, Hudis, NEJM (2007) 357(1):39-51 , Arteaga and Engelman, Cancer Cell. (2014) 25(3): 282-303 and Yan et al., Cancer Metastasis Rev. (2015) 34(1):157-164 all of which are hereby incorporated by reference in their entirety.
  • ERBB2 amplification has been observed in various cancers including breast cancer, gastric cancer and esophageal cancer (Koboldt et al., Nature. (2012) 490:61-70). Potentially activating (/.e. gain-of-function) mutations in HER2 have also been reported in cancers such as lobular breast cancer, lung cancer, gastric cancer, bladder cancer and endometrial cancer. Very high proportions of the following cancers express HER2 (/.e. are HER2-positive; see Table 2 of Yan etal., Cancer Metastasis Rev.
  • bladder cancer bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal esophagogastric junction cancer, gallbladder cancer, gastric adenocarcinoma, gastrointestinal stromal tumor, glioblastoma multiforme, glioma, head and neck carcinoma, hepatocellular carcinoma, intestinal cancer, kidney cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, melanoma, neuroendocrine tumor, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibrous tumor, testicular cancer, thymic cancer, thyroid cancer and uterine cancer.
  • a cancer is selected from: a cancer comprising cells expressing/overexpressing an EGFR family member, a cancer comprising cells expressing/overexpressing HER2, a cancer comprising cells that do not overexpress an EGFR family member, a cancer comprising cells that do not overexpress HER2, a HER2-low cancer, a HR-positive cancer, a solid tumor, bladder cancer, breast cancer, HER2-positive breast cancer, metastatic HER2-positive breast cancer, HER2-low breast cancer, unresectable or metastatic HER2-low breast cancer, HR-positive breast cancer, triple-negative breast cancer, cervical cancer, gastric cancer, HER2-positive gastric cancer, locally-advanced or metastatic HER2-positive gastric cancer, cholangiocarcinoma, colorectal cancer, esophageal esophagogastric junction cancer, gallbladder cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, HER2-positive
  • a cancer according to the present disclosure is a cancer for which famtrastuzumab deruxtecan-nxki is an approved treatment.
  • a cancer is selected from: metastatic HER2-positive breast cancer, unresectable or metastatic HER2-low (/.e. IHC 1+ or IHC 2+, ISH-) breast cancer, unresectable or metastatic non-small cell lung cancer comprising activating mutation to ERBB2, and locally-advanced or metastatic HER2-positive gastric or gastroesophageal junction adenocarcinoma.
  • a cancer is selected from: a cancer comprising cells that do not overexpress an EGFR family member (e.g. HER2, EGFR, HER3 or HER4), a cancer comprising cells that do not overexpress HER2, a HER2-low (/.e. IHC 1+ or IHC 2+, ISH-) cancer, HER2-low breast cancer, a hormone receptor (HR)-positive cancer (/.e. a cancer comprising cells expressing estrogen receptor (ER) and/or progesterone receptor (PR)), HR-positive breast cancer and triple-negative (/.e. HER2-negative, ER-negative and PR-negative) breast cancer.
  • an EGFR family member e.g. HER2, EGFR, HER3 or HER4
  • a cancer comprising cells that do not overexpress HER2 e.g. HER2, EGFR, HER3 or HER4
  • a cancer comprising cells that do not overexpress HER2 e.g. HER2,
  • a ‘HER2-low’ cancer refers to a cancer having an immunohistochemical (IHC) score for HER2 expression of 1+, or a cancer having an IHC score for HER2 expression of 2+ provided the cancer does not comprise ERBB2 amplification as determined by in situ hybridization (ISH) analysis.
  • IHC analysis and scoring of HER2 expression and ISH analysis of ERBB2 amplification is described e.g. in Wolff et al. , J Clin Oncol. (2016) 36(20):2105-2122, which is hereby incorporated by reference in its entirety.
  • an ‘activating mutation to ERBB2’ may: increase transcription of ERBB2', increase the level of RNA encoded by ERBB2', decrease degradation of RNA encoded by ERBB2', increase the level of a protein encoded by ERBB2', increase (facilitate) normal splicing of pre-mRNA encoded by ERBB2', increase translation of mRNA encoding a protein encoded by ERBB2', increase (facilitate) normal post- translational processing of a protein encoded by ERBB2', increase (facilitate) normal trafficking of a protein encoded by ERBB2', decrease degradation of a protein encoded by ERBB2', increase the level of a function of a protein encoded by ERBB2', and/or confer a protein encoded by ERBB2 with a novel property.
  • a cancer is selected from: a cancer comprising cells that do not overexpress an EGFR family member (e.g. HER2, EGFR, HER3 or HER4), a cancer comprising cells that do not overexpress HER2, a HER2-low (/.e. IHC 1+ or IHC 2+, ISH-) cancer, HER2-low breast cancer, a hormone receptor (HR)-positive cancer (/.e. a cancer comprising cells expressing estrogen receptor (ER) and/or progesterone receptor (PR)), HR-positive breast cancer and triple-negative (/.e. HER2-negative, ER-negative and PR-negative) breast cancer.
  • an EGFR family member e.g. HER2, EGFR, HER3 or HER4
  • a cancer comprising cells that do not overexpress HER2 e.g. HER2, EGFR, HER3 or HER4
  • a cancer comprising cells that do not overexpress HER2 e.g. HER2,
  • the cancer may be a relapsed cancer.
  • a ‘relapsed’ cancer refers to a cancer which responded to a treatment (e.g. a first line therapy for the cancer), but which has subsequently re-emerged/progressed, e.g. after a period of remission.
  • a relapsed cancer may be a cancer whose growth/progression was inhibited by a treatment (e.g. a first line therapy for the cancer), and which has subsequently grown/progressed.
  • a cancer that is relapsed with respect to given treatment may be described as having acquired resistance to such treatment.
  • the cancer may be a refractory cancer.
  • a ‘refractory’ cancer refers to a cancer which has not responded to a treatment (e.g. a first line therapy for the cancer).
  • a refractory cancer may be a cancer whose growth/progression was not inhibited by a treatment (e.g. a first line therapy for the cancer).
  • a refractory cancer may be a cancer for which a subject receiving treatment for the cancer did not display a partial or complete response to the treatment.
  • a cancer that is refractory with respect to given treatment may be described as having intrinsic resistance to such treatment.
  • the cancer is a cancer that is relapsed or refractory with respect to treatment with a DNA damage response (DDR) inhibitor. In some embodiments, the cancer is refractory with respect to treatment with a DDR inhibitor. In some embodiments, the cancer is relapsed with respect to treatment with a DDR inhibitor. In some embodiments, the cancer has intrinsic resistance to treatment with a DDR inhibitor. In some embodiments, the cancer has acquired resistance to treatment with a DDR inhibitor. In accordance with such embodiments, the DDR inhibitor may have been administered in the form of an antigen-binding molecule comprising a payload moiety comprising or consisting of the DDR inhibitor, or may have been administered in unconjugated form.
  • DDR DNA damage response
  • a DDR inhibitor according to the present disclosure is selected from: a PARP inhibitor (e.g.
  • olaparib rucaparib, niraparib, talazoparib, veliparib, pamiparib, simmiparib, senaparib, SC-10914, 2X- 121 , AMXI-5001 , JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-1002, RBN-2397), an ATM inhibitor (e.g. CP-466722, KU-55933, KU-60019, KU-59403, AZ31 , AZ32, AZD0156, AZD1390), an ATR inhibitor (e.g.
  • M6620 berzosertib
  • M4344 VX-803
  • AZD6738 ceralasertib
  • BAY1895344 elimusertib
  • WEE1 inhibitor e.g. adavosertib, Debio 0123, PD0166285, PD0407824, AZD1775
  • CHK1/2 inhibitor e.g. CBP-501 , prexasertib, MK-8776, GDC-0575, SRA-737
  • DNA-PK inhibitor e.g.
  • CC-115 LY-3023414, AsiDNA, M3814 (nedisertib), M9831 (VX-984)) and a PLK1 inhibitor (e.g. BI-6727 (volasertib), PCM-075 (onvansertib)).
  • a PLK1 inhibitor e.g. BI-6727 (volasertib), PCM-075 (onvansertib)
  • the cancer is a cancer that is relapsed or refractory with respect to treatment with a DNA topoisomerase I (TOP1) inhibitor.
  • TOP1 DNA topoisomerase I
  • the cancer is refractory with respect to treatment with a TOP1 inhibitor.
  • the cancer is relapsed with respect to treatment with a TOP1 inhibitor.
  • the cancer has intrinsic resistance to treatment with a TOP1 inhibitor.
  • the cancer has acquired resistance to treatment with a TOP1 inhibitor.
  • the TOP1 inhibitor may have been administered in the form of an antigen-binding molecule comprising a payload moiety comprising or consisting of the TOP1 inhibitor, or may have been administered in unconjugated form.
  • DNA topoisomerase I inhibitors and their use for the treatment of cancers is described e.g. in Pommier, Chem Rev. (2009) 109(7): 2894-2902, Li et al., Am J Cancer Res. (2017) 7(12): 2350-2394 and Thomas and Pommier, Clin Cancer Res. (2019) 25(22): 6581-6589, all of which are hereby incorporated by reference in their entirety.
  • a TOP1 inhibitor according to the present disclosure is selected from: camptothecin, irinotecan, etirinotecan, SN-38, DX-8951f (extatecan mesylate), DXd(1), DXd(2), exatecan, FL118, topotecan, gimatecan, belotecan, deruxtecan, belotecan, rubitecan, lurtotecan, diflomotecan, karenitecan, silatecan, namitecan, elomotecan, DRF-1042, delimotecan, NSC606985, chimmitecan, ZBH-1205, Genz-644282, non-CPT1 , indotecan (LMP-400), indimitecan (LMP-776) and LMP744.
  • the cancer to be treated/prevented in accordance with the present disclosure is a cancer that is: relapsed or refractory with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and relapsed or refractory with respect to treatment with a TOP1 inhibitor (e.g. a TOP1 inhibitor as described herein).
  • a DDR inhibitor e.g. a DDR inhibitor as described herein
  • a TOP1 inhibitor e.g. a TOP1 inhibitor as described herein
  • the cancer is: refractory with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and refractory with respect to treatment with a TOP1 inhibitor (e.g. a TOP1 inhibitor as described herein).
  • the cancer is: relapsed with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and relapsed with respect to treatment with a TOP1 inhibitor (e.g. a TOP1 inhibitor as described herein).
  • the cancer is: refractory with respect to treatment with a DDR inhibitor (e.g. a DDR inhibitor as described herein), and relapsed with respect to treatment with a T0P1 inhibitor (e.g. a T0P1 inhibitor as described herein).
  • the cancer is: relapsed with respect to treatment with a DDR inhibitor (e.g.
  • the TOP1 inhibitor and/or DDR inhibitor may have been administered in the form of an antigen-binding molecule comprising a payload moiety comprising or consisting of the TOP1 inhibitor/DDR inhibitor, or may have been administered in unconjugated form.
  • Treatment of a cancer in accordance with the methods of the present disclosure achieves one or more of the following treatment effects: reduces the number of cancer cells in the subject, reduces the size of a cancerous tumor/lesion in the subject, inhibits (e.g. prevents or slows) growth of cancer cells in the subject, inhibits (e.g. prevents or slows) growth of a cancerous tumor/lesion in the subject, inhibits (e.g. prevents or slows) the development/progression of a cancer (e.g. to a later stage, or metastasis), reduces the severity of symptoms of a cancer in the subject, increases survival of the subject (e.g. progression free survival or overall survival), reduces a correlate of the number or activity of cancer cells in the subject, and/or reduces cancer burden in the subject.
  • reduces the number of cancer cells in the subject reduces the size of a cancerous tumor/lesion in the subject, inhibits (e.g. prevents or slows) growth of cancer cells in the subject, inhibits (e
  • Subjects may be evaluated in accordance with the Revised Criteria for Response Assessment: The Lugano Classification (described e.g. in Cheson et al., J Clin Oncol (2014) 32: 3059-3068, incorporated by reference hereinabove) in order to determine their response to treatment.
  • treatment of a subject in accordance with the methods of the present disclosure achieves one of the following: complete response, partial response, or stable disease.
  • Prevention may refer to prevention of development of a cancer, and/or prevention of worsening of a cancer, e.g. prevention of progression of a cancer, e.g. to a later stage (e.g. metastasis).
  • administration of an antigen-binding molecule/composition according to the present disclosure may be associated with one or more of: inhibition of the development/progression of the cancer, a delay to/prevention of onset of the cancer, a reduction in/delay to/prevention of tumor growth, a reduction in/delay to/prevention of tissue invasion, a reduction in/delay to/prevention of metastasis, a reduction in the severity of one or more symptoms of the cancer, a reduction in the number of cancer cells, a reduction in the cancer burden, a reduction in tumor size/volume, and/or an increase in survival of subjects having the cancer (e.g. progression free survival or overall survival).
  • inhibition of the development/progression of the cancer e.g. progression free survival or overall survival.
  • a method of treating and/or preventing a cancer according to the present disclosure may comprise inhibiting the growth of a tumor, reducing the size/volume of a tumor and/or increasing the survival of a subject having the cancer.
  • methods are provided which are for, or which comprise (e.g. in the context of treatment/prevention of a cancer, e.g. a cancer described herein), one or more of the following: binding to cells expressing HER2; inhibiting the proliferation of HER2-expressing cells; killing cells expressing HER2; inhibiting tumor growth and/or reducing tumor size/volume, e.g. of a HER2-ex pressing cancer; and/or increasing the survival of subjects having a cancer, e.g. a HER2-ex pressing cancer.
  • antigen-binding molecules and compositions according to the present disclosure for use in such methods, and the use of antigen-binding molecules and compositions according to the present disclosure in manufacture of compositions (e.g. medicaments) for use in such methods. It will be appreciated that the methods typically comprise administering an antigen-binding molecule according to the present disclosure to a subject.
  • one or more of the following may be observed in a subject following therapeutic or prophylactic intervention in accordance with the present disclosure (e.g. compared to the level/number/proportion etc. prior to intervention): inhibition of proliferation of HER2-expressing cells; killing of cells expressing HER2; inhibition of tumor growth and/or reduction of tumor size/volume, e.g. of a HER2-expressing cancer; and/or increased survival of a subject having a cancer, e.g. a HER2-expressing cancer.
  • therapeutic/prophylactic intervention in accordance with the present disclosure may be described as being ‘associated with’ one or more of the effects described in the preceding paragraph.
  • the skilled person is readily able to evaluate such properties using techniques that are routinely practiced in the art.
  • Administration of the antigen-binding molecules and compositions of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject.
  • the actual amount administered, and rate and timecourse of administration will depend on the nature and severity of the disease/condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23 rd Edition (2020), Academic Press.
  • Administration of the antigen-binding molecules and compositions of the present disclosure may be e.g. parenteral, systemic, topical, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral or transdermal.
  • Administration may be by injection, infusion or ingestion.
  • articles of the present disclosure may be administered to a tissue/organ of interest (e.g. a tissue/organ affected by the disease/condition, e.g. a tissue/organ in which symptoms of the disease/condition manifest).
  • a tissue/organ of interest e.g. a tissue/organ affected by the disease/condition, e.g. a tissue/organ in which symptoms of the disease/condition manifest.
  • articles of the present disclosure may be administered to the blood (/.e. intravenous/intra-arterial administration) by injection or infusion (e.g. via cannula), or may be administered subcutaneously or orally.
  • articles of the present disclosure may be administered to a tumor.
  • therapeutic or prophylactic intervention according to the present disclosure may further comprise administering another agent for the treatment/prevention of the relevant disease/condition.
  • Administration of antigen-binding molecules and compositions described herein may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • Simultaneous administration refers to administration with another therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other (e.g. within 1 , 4, 6, 8 or 12 hours) and optionally via the same route of administration (e.g. to the same tissue, artery, vein or other blood vessel).
  • Sequential administration refers to administration of one agent followed after a given time interval by separate administration of another agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments.
  • the time interval may be any time interval.
  • Multiple doses of the antigen-binding molecules and compositions may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).
  • the present disclosure also provides the articles of the present disclosure for use in methods for detecting, localising or imaging HER2, or cells expressing HER2.
  • the antigen-binding molecules described herein may be used in methods that involve detecting binding of the antigen-binding molecule to HER2. Such methods may involve detection of the bound complex of the antigen-binding molecule and HER2. It will be appreciated that the HER2 may be HER2 expressed by a cell, e.g. in or at the cell surface of a cell expressing HER2.
  • a method comprising contacting a sample containing, or suspected to contain, HER2, and detecting the formation of a complex of the antigen-binding molecule and HER2. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell expressing HER2, and detecting the formation of a complex of the antigen-binding molecule and a cell expressing HER2.
  • Suitable method formats are well known in the art, including immunoassays such as sandwich assays, e.g. ELISA.
  • the methods may involve labelling the antigen-binding molecule, or target(s), or both, with a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label, radiolabel, chemical, nucleic acid or enzymatic label as described herein.
  • Detection techniques are well known to those of skill in the art and can be selected to correspond with the labelling agent.
  • Methods comprising detecting HER2, or cells expressing HER2, include methods for diagnosing/prognosing a disease/condition described herein.
  • Methods of this kind may be performed in vitro on a patient sample, or following processing of a patient sample. Once the sample is collected, the patient is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body. In some embodiments, the method is performed in vivo.
  • Such methods may involve detecting or quantifying HER2 and/or cells expressing HER2, e.g. in a patient sample. Where the method comprises quantifying the relevant factor, the method may further comprise comparing the determined amount against a standard or reference value as part of the diagnostic or prognostic evaluation. Other diagnostic/prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm a result obtained by using the tests described herein.
  • Detection in a sample may be used for the purpose of diagnosis of a disease/condition (e.g. a cancer), predisposition to a disease/condition, or for providing a prognosis (prognosticating) for a disease/condition, e.g. a disease/condition described herein.
  • the diagnosis or prognosis may relate to an existing (previously diagnosed) disease/condition.
  • a sample may be taken from any tissue or bodily fluid.
  • the sample may comprise or may be derived from: a quantity of blood; a quantity of serum derived from the individual’s blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said individual.
  • the sample may be obtained or derived from a tissue or tissues which are affected by the disease/condition (e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease/condition).
  • a subject may be selected for diagnostic/prognostic evaluation based on the presence of symptoms indicative of a disease/condition described herein, or based on the subject being considered to be at risk of developing a disease/condition described herein.
  • the present disclosure also provides methods for selecting/stratifying a subject for treatment with a HER2-targeted agent.
  • a subject is selected for treatment/prevention in accordance with the methods of the present disclosure, or is identified as a subject which would benefit from such treatment/prevention, based on detection/quantification of HER2, or cells expressing HER2, e.g. in a sample obtained from the individual.
  • the subject in accordance with aspects described herein may be any animal or human.
  • the subject is preferably mammalian, more preferably human.
  • the subject may be a non-human mammal, but is more preferably human.
  • the subject may be male or female.
  • the subject may be a patient.
  • a subject may have been diagnosed with a disease or condition requiring treatment (e.g. a cancer, e.g. a cancer described herein), may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition.
  • a disease or condition requiring treatment e.g. a cancer, e.g. a cancer described herein
  • the subject to be treated according to a therapeutic or prophylactic method of the present disclosure herein is a subject having, or at risk of developing, a cancer, e.g. a cancer described herein.
  • a subject may be selected for treatment according to the methods based on characterisation for certain markers of such disease/condition.
  • a patient may be selected for treatment described herein based on the detection of a cancer ex pressing/ove rex pressing HER2, e.g. in a sample obtained from the subject (e.g. a biopsy, e.g. of a tumor).
  • a sample obtained from the subject e.g. a biopsy, e.g. of a tumor.
  • kits of parts may comprise components for performing a method described herein, in whole or in part.
  • the kit may have at least one container having a predetermined quantity of an antigen-binding molecule or composition described herein.
  • kits of parts may comprise an antigen-binding molecule or composition described herein, and which may be provided in a predetermined quantity.
  • the kit may provide an antigen-binding molecule or composition described herein together with instructions for administration to a patient in order to treat a specified disease/condition (e.g. a disease/condition described herein, e.g. a cancer).
  • a specified disease/condition e.g. a disease/condition described herein, e.g. a cancer.
  • the kit may provide an antigen-binding moiety according to the disclosure, and a linker-payload moiety according to the present disclosure.
  • the kit may further comprise reagents for conjugating the antigenbinding moiety and the linker-payload moiety.
  • the kit may further comprise reagents, buffers and/or standards required for execution of a method according to the present disclosure.
  • Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet.
  • the instructions may include a protocol for performing any one or more of the methods described herein.
  • sequence identity refers to the percent of nucleotides/amino acid residues in a subject sequence that are identical to nucleotides/amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol.
  • the present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
  • nucleic acid sequence is disclosed or referred to herein, the reverse complement thereof is also expressly contemplated.
  • in vitro is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with/on intact multi-cellular organisms.
  • Values may be expressed herein as ‘about’ a particular value. Similarly, ranges may be expressed herein as from ‘about’ a particular value, and/or to ‘about’ another particular value.
  • the term ‘about’ in relation to a numerical value is optional, and means for example +/- 10 %. By way of illustration, reference e.g. to ‘about 10 %’ is to be construed as 9 % to 11 %. In instances herein where ‘about’ is recited, the value it precedes is also specifically contemplated. By way of illustration, reference e.g. to ‘about 10 %’ also specifically contemplates 10 %.
  • Figure 1A shows % inhibition for a TOP1 inhibitor and an ATR inhibitor alone and in combination in HCT- 116 cells.
  • Figure 1B shows % inhibition for a TOP1 inhibitor and a CHK1 inhibitor alone and in combination in HCT- 116 cells.
  • Figure 1C shows % inhibition for a TOP1 inhibitor and an ATR inhibitor alone and in combination in HEC- 1 B cells.
  • Figure 1D shows % inhibition for a TOP1 inhibitor and a CHK1 inhibitor alone and in combination in HEC- 1 B cells.
  • Figure 2 shows % cell death following treatment in vitro of cells of the indicated cancer cell lines for 7 days with trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), Trastuzumab deruxtecan (T-DXd) or isotype-matched control antibody conjugated to exatecan (Isotype Control).
  • Figure 3 shows tumor volume over time, for mice having a JIMT-1 cell line-derived xenograft model of breast ductal carcinoma, and treated with PBS (vehicle), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)) or trastuzumab deruxtecan (T-DXd)).
  • PBS vehicle
  • trastuzumab conjugated to both exatecan and berzosertib T-(Exa+Ber)
  • trastuzumab deruxtecan T-DXd
  • Figure 4 shows bodyweight in grams (g) over time, for mice having a JIMT-1 cell line-derived xenograft model of breast ductal carcinoma, and treated with PBS (vehicle), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)) or trastuzumab deruxtecan (T-DXd)
  • PBS vehicle
  • trastuzumab conjugated to both exatecan and berzosertib T-(Exa+Ber)
  • trastuzumab deruxtecan T-DXd
  • Figure 5A shows binding of trastuzumab (T (naked), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), Trastuzumab deruxtecan (T-DXd), isotype-matched control antibody (Isotype (Naked)) or isotype-matched control antibody conjugated to exatecan (Isotype (Exa)) to live BT-474, NCI- N87, JIMT-1 , HEC-1-B or HCT116 cells, as determined by flow cytometry.
  • Figure 5B shows the mean fluorescence intensity (MFI) for trastuzumab (T (naked), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), F trastuzumab deruxtecan (T-DXd), isotype- matched control antibody (Isotype (Naked)) or isotype-matched control antibody conjugated to exatecan (Isotype (Exa)) bound to live BT-474, NCI-N87, JIMT-1 , HEC-1-B or HCT116 cells, as determined by flow cytometry.
  • MFI mean fluorescence intensity
  • Figure 6A shows subcellular localization of trastuzumab conjugated to both exatecan and berzosertib (T- (Exa+Ber)) or trastuzumab deruxtecan (T-DXd) within HEC-1-B cells after incubation for Oh, 0.5h or 2h, as determined by immunofluorescence microscopy.
  • Figure 6B shows subcellular localization of trastuzumab conjugated to both exatecan and berzosertib (T- (Exa+Ber)) or trastuzumab deruxtecan (T-DXd) within NCI-N87 cells after incubation for Oh, 0.5h or 2h, as determined by immunofluorescence microscopy.
  • Figure 7A shows % cell death following treatment in vitro of HEC1-B cells for 3 days with trastuzumab (T (naked)), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype-matched control antibody conjugated to exatecan (Isotype Control), at a concentration of 333 nM.
  • Figure 7B shows % cell death following treatment in vitro of NCI-N87 cells for 3 days with trastuzumab (T (naked)), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype-matched control antibody conjugated to exatecan (Isotype Control), at a concentration of 333 nM.
  • Figure 7C shows % cell death following treatment in vitro of HCT-116 cells for 3 days with trastuzumab (T (naked)), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype-matched control antibody conjugated to exatecan (Isotype Control), at a concentration of 333 nM.
  • trastuzumab T (naked)
  • trastuzumab conjugated to both exatecan and berzosertib T-(Exa+Ber)
  • trastuzumab deruxtecan T-DXd
  • Isotype Control isotype-matched control antibody conjugated to exatecan
  • Figure 7D shows % cell death following treatment in vitro of BT474 cells for 3 days with trastuzumab (T (naked)), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype-matched control antibody conjugated to exatecan (Isotype Control), at a concentration of 333 nM.
  • Figure 7E shows % cell death following treatment in vitro of JIMT-1 cells for 3 days with trastuzumab (T (naked)), trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber trastuzumab deruxtecan (T-DXd) or isotype-matched control antibody conjugated to exatecan (Isotype Control), at a concentration of 333 nM.
  • Figure 7F shows % cell death following treatment in vitro of HEC1-B cells for 3 days with trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype- matched control antibody conjugated to exatecan (Isotype Control), at concentrations of the antigenbinding molecules providing equivalent 0.857 nM payload concentration.
  • Figure 7G shows % cell death following treatment in vitro of NCI-N87 cells for 3 days with trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype- matched control antibody conjugated to exatecan (Isotype Control), at concentrations of the antigenbinding molecules providing equivalent 0.857 nM payload concentration.
  • Figure 7H shows % cell death following treatment in vitro of HCT-116 cells for 3 days with trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype- matched control antibody conjugated to exatecan (Isotype Control), at concentrations of the antigenbinding molecules providing equivalent 0.857 nM payload concentration.
  • Figure 7I shows % cell death following treatment in vitro of BT474 cells for 3 days with trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype- matched control antibody conjugated to exatecan (Isotype Control), at concentrations of the antigenbinding molecules providing equivalent 0.857 nM payload concentration.
  • Figure 7J shows % cell death following treatment in vitro of JIMT-1 cells for 3 days with trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd) or isotype- matched control antibody conjugated to exatecan (Isotype Control), at concentrations of the antigenbinding molecules providing equivalent 0.857 nM payload concentration.
  • Figure 8 shows the Loewe synergy score for exatecan and berzosertib (upper panel) and the % inhibition for exatecan and berzosertib alone and in combination (lower panel) in HEC1-B cells.
  • Figure 9 shows the Loewe synergy score for exatecan and berzosertib (upper panel) and the % inhibition for exatecan and berzosertib alone and in combination (lower panel) in HCT-116 cells.
  • Figure 10 shows the level of pATR, pCHK1 , pH2AX and CHK1 in JIMT-1 cells untreated or treated with 100 nM exatecan, 100 nM berzosertib, or 100 nM exatecan and 100 nM berzosertib in combination.
  • Figure 11 shows the level of pATR, pCHK1 , pH2AX and CHK1 in HCT-116 cells untreated or treated with 75 nM exatecan, 75 nM berzosertib, or 75 nM exatecan and 75 nM berzosertib in combination.
  • Figure 12 shows survival of Sprague-Dawley rats following administration of vehicle, exatecan (1 , 3, 10 or 30 mg/kg), berzosertib (35 mg/kg) or exatecan and berzosertib in combination (1 , 3, 10 or 30 mg/kg exatecan + 35 mg/kg berzosertib).
  • Figure 13 shows body weight change in Sprague-Dawley rats following administration of vehicle, exatecan (1 , 3, 10 or 30 mg/kg), berzosertib (35 mg/kg) or exatecan and berzosertib in combination (1 , 3, 10 or 30 mg/kg exatecan + 35 mg/kg berzosertib).
  • Figure 14 shows results of hematology assessments in Sprague-Dawley rats following administration of vehicle, exatecan (1 , 3, 10 or 30 mg/kg), berzosertib (35 mg/kg) or exatecan and berzosertib in combination (1 , 3, 10 or 30 mg/kg exatecan + 35 mg/kg berzosertib).
  • Figure 15 shows results of clinical chemistry assessments in Sprague-Dawley rats following administration of vehicle, exatecan (1 , 3, 10 or 30 mg/kg), berzosertib (35 mg/kg) or exatecan and berzosertib in combination (1 , 3, 10 or 30 mg/kg exatecan + 35 mg/kg berzosertib).
  • Figure 16 shows tumour volume in a T-DXd resistant NCI-N87 CDX model following administration of vehicle, Trastuzumab deruxtecan (T-DXd) (3 mg/kg) or trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber) (3 mg/kg, 9 mg/kg).
  • T-DXd Trastuzumab deruxtecan
  • T-(Exa+Ber trastuzumab conjugated to both exatecan and berzosertib
  • Figure 17 shows % inhibition of HER2-negative MDA-MB-231 cells alone or co-cultured with HER2- positive NCI-N87 cells and exposed to varying concentrations of trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)) or Trastuzumab deruxtecan (T-DXd).
  • FIG 18 shows megakaryocyte uptake via macropinocytosis of trastuzumab conjugated to both exatecan and berzosertib (T-(Exa+Ber)) (100 ng/ml) or trastuzumab deruxtecan (T-DXd) (100 ng/ml).
  • Figure 19 shows % increase in hydrophobic interaction chromatography retention time (RT) for a single payload ADC and T-(Exa+Ber).
  • Figure 20 shows the stability of ADCs 5 and 6 in plasma over 7 days.
  • Figure 21 shows the efficacy of dual payload ADCs on NCI-N87 gastric cell line cells.
  • Figure 22 shows the efficacy of dual and single payload ADCs in killing NCI-N87 gastric cell line cells.
  • Figure 23 shows the efficacy of dual and single payload ADCs in killing NCI-N87 gastric cell line cells.
  • Figure 24 shows the in vivo anti-tumor efficacy of dual and single payload ADCs in a xenograft mouse model.
  • Figure 25 shows the in vivo efficacy of dual payload ADCs with berzosertib (ATRi) or prexasertib (CHK1i) in a mouse model.
  • Figure 26 shows the in vivo efficacy of dual payload ADCs with different target DAR ratios.
  • Figure 27 shows the weight change results in a primate study following treatment with a dual payload ADC.
  • Figure 28 shows the biochemistry and haemotology results in a primate study following treatment with a dual payload ADC.
  • Figure 29 shows the efficacy of dual payload ADCs with TMTHSI or DBCO moieties on NCI-N87 gastric cell lines.
  • Figure 30 shows the clearance of dual payload ADCs with TMTHSI or DBCO moieties compared to the unconjugated control antibody in immunocompetent mice.
  • Figure 31 shows efficacy of dual payload ADCs with different conjugations.
  • the cells were seeded in wells of a white 96-well plate at a density of 7,000 cells per well for HEC-1 B cells, and 3,000 cells per well for HCT-116 cells, with 150 pl of media in each well.
  • 25 pl of the test compound and 25 pl of media were added to the respective wells at varying concentrations.
  • 25 pl of compound 1 and 25 pl of another compound were added to the respective wells at different concentrations.
  • the plates were then incubated for 3 days at 37°C with 5% CO2. Following incubation, 50pl of the detection reagent was added per well and shaken at 600 rpm for 20 minutes. The resulting luminescence was measured using Perkin Elmer Victor Nivo, and percent inhibition was calculated using the following equation:
  • Fig 1 A shows the % inhibition for exatecan and ceralasertib alone and in combination.
  • the Loewe synergy score is shown in table 1 A below.
  • the IC50 for exatecan alone was 0.626 nM, when combined with ceralasertib at 375 nM was 0.080 nM and when combined with ceralasertib at 750 nM was 0.044 nM.
  • Fig 1 B shows the % inhibition for exatecan and prexasertib alone and in combination.
  • the Loewe synergy score is shown in table 1 B below.
  • the IC50 for exatecan alone was 0.626 nM, when combined with prexasertib at 50 nM was 0.166 nM and when combined with prexasertib at 100 nM was 0.158 nM.
  • the IC50 for prexasertib alone was 61 .14 nM.
  • Fig 1 C shows the % inhibition for exatecan and ceralasertib alone and in combination.
  • the Loewe synergy score is shown in table 1 C below.
  • the IC50 for exatecan alone was 1 13.9 nM, when combined with ceralasertib at 0.37 pM was 19.01 nM and when combined with ceralasertib at 1 .1 pM was 3.719 nM.
  • the IC50 for ceralasertib alone was 2.118 pM.
  • Table 1C D Exatecan and prexasertib (a CHK1 inhibitor) in HEC-1 B cells
  • Fig 1 D shows the % inhibition for exatecan and prexasertib alone and in combination.
  • the Loewe synergy score is shown in table 1 D below.
  • the IC50 for exatecan alone was 1 13.9 nM, when combined with prexasertib at 1 .9 nM was 8.988 nM and when combined with prexasertib at 3.8 nM was 2.013 nM.
  • the IC50 for prexasertib alone was 4.466 nM.
  • HEC-1-B HEC-1-B (HTB-113)
  • the cells were seeded in 96-well white opaque plates in 150 pl of media, and incubated at 37°C with 5% CO2 for 24 hours.
  • HEC-1 B was seeded at 7000 cells/well and HCT-116 was seeded at 3000 cells/well
  • 25 pl of exatecan, berzosertib, or exatecan and berzosertib were added to cells at varying concentrations and incubated for 3 days at 37°C with 5% CO2.
  • 50 pl of CellTiter-Glo reagent was added to the plates and incubated for 25 mins with gentle shaking at 600 rpm.
  • Cell viability was measured via Luminescence using Victor Nivo, PerkinElmer.
  • exatecan was tested with two clinical stage ATR inhibitors (berzosertib and ceralasertib) for in vitro synergy in TOP1 inhibitor-low sensitivity cell line HEC-1 B and TOP1 inhibitor-high sensitivity cell line HCT-116. Both berzosertib and ceralasertib show synergy with exatecan across a wide range of concentrations tested in the cell lines.
  • the effects in HEC-1 B demonstrate the TOP1 inhibitor and DDR inhibitor combination can sensitize inherently -less sensitive cells to TOP1 inhibitor therapy.
  • Mass Spectrometric data were recorded on SHIMADZU LCMS-2020 (ESI-MS) and Agilent 1260 ⁇ G6125B (ESIMS), and the column is of Kinetex® EVO C18 4.6x50mm, 5pm, Kinetex® EVO C18 2.1*30mm, 5pm, Shim-pack Scepter C18-120 3.0x33mm 3 pm and Poroshell 120 EC C18 2.7pm 3.0*30mm.
  • step b Repeat step b to deprotect Fmoc group. Treat the resulting resin with Fmoc-N-amido-PEG3-acid (2.0 equiv.), HATU (1.9 equiv.) and DIPEA (4.0 equiv.) in DMF. The mixture was agitated under N2 atmosphere at 25 °C for 30 min. The resulting resin was washed with DMF (200 mL x 3). e) Peptide cleavage and purification: The resin was washed with methanol (200 mL x 3) and dried under vacuum. The dried resin was treated with the cleavage buffer consisting of 20% HFIP in CH2CI2 and stirred for 30 min and filtered.
  • TCO-OH 116 To a solution of compound TCO-OH (200 mg, 1.58 mmol, 1.0 eq.) in dry THF (2.0 mL) was added NaH (60% dispersion in mineral oil, 190 mg, 4.75 mmol, 3.0 eq.). The mixture was stirred at 25 °C for 1 h under N2 atmosphere. Then 2-bromoacetic acid (264 mg, 1.9 mmol, 1.2 eq.) and KI (26.3 mg, 158 pmol, 0.10 eq.) were added. The mixture was stirred at 70 °C for 12 h. Upon completion, the reaction mixture was quenched with H2O (10 mL), and adjusted to pH 2 using 1 N HCI.
  • LP-4 was synthesized according to General Procedure 3A from 119a. Yield: 45%.
  • LP-5 was synthesized according to General Procedure 3A from 119b. Yield: 38%.
  • LP-6 was synthesized according to General Procedure 3B from 118c. Yield: 46%.
  • LP-7 was synthesized according to General Procedure 3B using 119d . Yield: 37%.
  • LP-8 was synthesized according to General Procedure 3B from I19e. Yield: 32% (obtained as a mixture of quaternary salts).
  • LP-9 was synthesized according to General Procedure 3B from 119f. Yield: 39%.
  • Triethylamine (23.59 pL, 37.2 pmol, 2.5 equiv.) and DMAP (10.5 mg, 85.95 pmol, 0.81 equiv.) were added successively to a solution of compound I20* (100 mg, 105.9 pmol, 1 .0 equiv.) and compound TMTHSI-OSu (37.86 mg, 111 .2 pmol, 1 .05 equiv.) in DMF (1 mL). The mixture was stirred at 25 °C for 18 h. Upon completion (as observed by LC-MS analysis), the solvent was removed under reduced pressure and the residue was purified by prep-HPLC to give LP-11 (23 mg) as a yellow solid.
  • Example 7 Synthetic route to Linker 1 a)
  • Compound 2 may be made from compound 1 by coupling tert-butyl 2-bromoacetate, for example using triethanolamine (TEA) in THF.
  • TEA triethanolamine
  • Compound 4 may be made from compound 3 by treatment with BOC2O and a base (e.g. TEA) in anhydrous conditions, such as in acetonitrile.
  • a base e.g. TEA
  • Compound 5 may be made from compound 4 by treating with diphenylphosphoryl azide (DPPA) and 1 ,8- diazabicyclo[5.4.0]undec-7-ene (DBU) in a dry aprotic solvent (such as toluene/DMF), for example, for 48 hours.
  • DPPA diphenylphosphoryl azide
  • DBU 1 ,8- diazabicyclo[5.4.0]undec-7-ene
  • a dry aprotic solvent such as toluene/DMF
  • Compound 6 may be made from compound 5 by removal of the Boc group, for example using HCI in ethyl acetate.
  • Compound 7 may be made from compound 5 by removal of the Boc group, for example using HCI in ethyl acetate.
  • Compound 7 may be made from compound 6 by linking compound 6B, for example using potassium carbonate in acetonitrile, such as at 60°C. f) Compound 8
  • Compound 8 may be made from compound 7 by removal of the Boc group, for example using HCI in ethyl acetate.
  • Compound 9 may be made from compound 7 by removal of the Boc group, for example using HCI in ethyl acetate.
  • Compound 9 may be made from compound 8 by linking 9H-fluoren-9-ylmethyl N-(2-oxoethyl)carbamate, for example using NaBH(OAc)3 in DCE.
  • Compound 10 may be made from compound 9 by using a urea formation reaction with Compound 2. i) Compound 11
  • Compound 11 may be made from compound 10 by removal of the Fmoc group.
  • Compound 13 may be made from compound 11 by coupling 2-[4-(6-methyl-1 ,2,4,5-tetrazin-3- yl)phenyl]acetic acid. k) Linker 1
  • Linker 1 may be made from compound 13 by removal of the Boc and t-Butoxy groups, for example using HCI in ethyl acetate.
  • Compound 14 may be made from compound by linking 9H-fluoren-9-ylmethyl N-(2-oxoethyl)carbamate, for example using NaBH(OAc)3 in DCE b) Compound 15
  • Compound 15 may be made from compound 14 by coupling 2-[4-(6-methyl-1 ,2,4,5-tetrazin-3- yl)phenyl]acetic acid. c) Compound 16
  • Compound 16 may be made from compound 15 by removal of the Fmoc protecting group d) Compound 17
  • Compound 17 may be made from compound 16 by coupling 2-[4-(6-methyl-1 ,2,4,5-tetrazin-3- yl)phenyl]acetic acid. e) Linker 2
  • Linker 2 may be made from compound 17 by removal of the Boc and t-Butoxy groups, for example using HCI in ethyl acetate.
  • Linker 4 A solution of compound AA_20 (14.7 g, 8.02 mmol, 1 .0 eq.) in 2N HCI in 1 ,4-dioxane (150 mL) was stirred at 0 °C for 30 min. The reaction was monitored by LCMS analysis. Upon completion, the reaction mixture was concentrated under reduced pressure, purified by preparative HPLC (0.01 % TFA), and lyophilized to obtain linker 4 (4.9 g, 39% yield for two steps, 96.7% purity) as a purple solid.
  • AA_62 (485 mg, 756 pmol, 1 .0 eq) was dissolved in 2N HCI/EtOAc (10 mL), and stirred at 25 °C for 2 h. Upon completion of the reaction, the solvent was concentrated under reduced pressure to give a residue. The residue was further purified by prep-HPLC (TFA condition) to give AA_63 (456 mg, 592 pmol, 78.4% yield, TFA salt) as purple oil.
  • AA_64 (509 mg, 592 pmol, 1 .00 eq) was dissolved in 2N HCI/EtOAc (10.0 mL), and stirred at 25 °C for 2 h. Upon completion of the reaction, the solvent was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (TFA condition). The elute was washed with 1 % NaHCOs (50 mL) and extracted with dichloromethane (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuum to give Linker 5 (180 mg, 237 pmol, 40% yield) as purple oil.
  • AA-68 (91 .6 mg, 102 pmol, 1 .0 eq) was dissolved in 2N HCI/EtOAc (500 pL), and stirred at 25 °C for 2 h.
  • LC-MS showed AA_68 was consumed completely, and identified the desired mass peak.
  • the mixture was concentrated under vacuum to give a residue.
  • the residue was purified by prep-HPLC (TFA condition) to give AA_69 (81.0 mg, 102 pmol, 99.5% yield) as purple oil.
  • AA_70 (111 mg, 99.5 pmol, 1 .0 eq) was dissolved in 2N HCI/EtOAc (1 .50 mL), and stirred at 25 °C for 2 h. Upon completion of the reaction, the solvent was concentrated under vacuum to give a residue which was purified by prep-HPLC (TFA condition). The eluate was washed with 1 % NaHCOs (50.0 mL) and extracted with dichloromethane (250 mL x 3). The combined organic layers were washed with brine (250 mL), dried over Na2SO4, filtered and concentrated under vacuum to give Linker 6 (50 mg, 49.3 pmol, 49% yield) as purple oil.
  • Example 13 Synthesis of [4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-[2-[2-[2-[2-[2-[[(1 S)-1 -[[(1 S)-2-[4-[[(10S,23S)- 10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1 ,6(11 ),12,14,16(24), 17,19-heptaen-23- yl]carbamoyloxymethyl]anilino]-1 -methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-2-oxo- ethoxy]ethoxy]ethyl-[2-[2-[2-[2-[2-[(3,3,6,
  • the ADCs in table C were prepared according to the general method outlined below.
  • Antibodies were produced recombinantly by transient expression in CHO cells. Briefly, expression vectors encoding antibody heavy and light chains of the relevant antibody (e.g. trastuzumab (DrugBank Acc. No: DB00072; UNI I : P188ANX8CK)) were transiently transfected into CHO cells at a 1 :1 ratio. Expressed antibodies were subsequently purified from the culture supernatant using MabSelect SuRe Protein A column (Cytiva, #11003494).
  • trastuzumab DrugBank Acc. No: DB00072; UNI I : P188ANX8CK
  • the ADC synthesis consists of two steps, the enzymatic addition of a branched linker, followed by the conjugation of the payload-containing moieties. These events take place at the site of glutamine-295 (Q295), within the CH2 domain of the Fc region of the antibody (which additionally comprises the substitution N297A).
  • the first step of the process is the microbial transglutaminase (MTG or MTGase)- mediated conjugation of an linker onto Q295 of the modified antibody.
  • the reagents set out in table A were combined in PBS buffer (prepared according to the manufacturer’s instructions) in a sterile glass bottle with a size of at least 2 times the reaction volume. The reaction was agitated using a magnetic stirrer bar and was incubated for 22 hours at room temperature. This reaction gives an ADC intermediate.
  • the ADC intermediate formed is then cleaned using protein A chromatography via bind-elute mode to remove excess linker and residual enzyme at room temperature.
  • the eluted ADC intermediate was neutralized using 2M Tris at pH 7.4.
  • TOP1 i and DDRi payloads were conjugated to the ADC intermediate in a one-pot reaction in a sequential addition manner. Specifically, TOP1 inhibitors which links to a strained alkyne click moiety is added first onto the ADC intermediate at the site of azide, via a typical SPAAC (Strain-promoted azide-alkyne cycloaddition) reaction. The reaction is set on a roller machine at room temperature, following overnight incubation, DDR inhibitor which links to a TCO click moiety is added onto the ADC intermediate at the site of tetrazine, via a typical IEDDA (Inverse-Electron-Demand Diels-Alder) reaction. The reaction was stopped 2-4 hours after adding linker-payload.
  • SPAAC Stra-promoted azide-alkyne cycloaddition
  • conjugation of the first payload was achieved by combining 1-3 mg/mL ADC intermediate with ca. 11 mM sodium deoxycholate, 0.28-28 wt.% propylene glycol in TBS pH 7.5. To the solution was added 7-14 mol. equivalents per antibody of TOP1 inhibitor or linker-payload. The reaction is set on a roller machine at room temperature for at least 12 hours.
  • Conjugation of the second payload was achieved by adding 7-10 mol. equivalents per antibody of linkerpayload or TOP1 inhibitor. The mixture was then incubated for at least 2-4 hours at room temperature to allow for the click reaction to occur.
  • Activated carbon was used to remove the free drug from the reaction mixture.
  • the reaction was carried out as follows:
  • Step 1 Dilute activated carbon powder into 100 mg/mL in PBS;
  • Step 2 Based on the amount of ADC in the reaction mix, add 1 :1 ratio of activated carbon solution (weight : weight);
  • Step 3 Incubate reaction mixture with activated carbon at ambient temperature (25°C) and rotate for 1 hour.
  • Suspended activated carbon was removed from the mixture using a 0.22 pm PES filter and concentrated using protein concentrator with 50 kDa MWCO. The sample was filtered with 0.22 pm PES filter again before using it in subsequent examples.
  • the ADC is optionally further purified using HIC purification.
  • Samples were prepared by diluting them to 1 mg/mL in PBS.
  • the samples were analysed on a Thermo Ultimate 3000 UPLC I Waters ACQUITY H-Class PLUS Bio System equipped with a ACQUITY UPLC Protein BEH SEC Column, 200 A, 1 .7 pm, 4.6 mm X 150 mm column and a ACQUITY UPLC Protein BEH SEC Guard Column, 200 A, 1 .7 pm, 4.6 mm X 30 mm guard column.
  • the analysis method was as follows:
  • the binding potency to the target antigen relative to a refence material was evaluated by ELISA.
  • the potency determination was carried out on a Molecular Devices, SpectraMax iD3 plate reader, equipped with a BioTek 405 TS plate washer.
  • Blocking buffer PBS with 1% BSA
  • Coating antigen His tag target antigen, 1 ug/mL Secondary antibody: Anti-human IgG Fc antibody (HRP), 1 :7000 (abeam, #ab97725)
  • TMB solution 1-StepTM turbo TMB-ELISA Substrate Solution (Thermo scientific, #34022) Stop solution: ELISA stop solution (Invitrogen, SS04)
  • test was carried out as follows, briefly, a 96-well plate was coated with 100 pl/well of coating antigen overnight (or up to 72hrs) at 4°C. The plate was then blocked by washing the plate three times with wash buffer (200 pl/well) and then blocking the plate with blocking buffer (200 pl/well) and subsequently incubating at room temperature for 1 hour. Samples were then added to the wells by washing the plate three times with wash buffer (200 pl/well) and then adding serially diluted samples (100 pl/well) and subsequently incubating at room temperature for 1 hour.
  • Results were generated by preparing a 4-parameter logistic dose-response curve to compute the EC50 values of the samples and reference.
  • the relative potency of the samples was determined by EC50 of reference/ECso of sample x 100%.
  • DAR was determined by reversed phase liquid chromatography-mass spectrometry (RPLC-MS). DAR analysis was used to determine the average number of payloads and linkers attached to the Fc region of the ADC.
  • Samples were prepared by reducing 5 pg of ADC in 10 mM DTT at 40 °C for 30 min and injecting a 5 pL aliquot for analysis.
  • the analysis method was as follows:
  • Wavelength 280 nm
  • Effective gradient linear increase from 20 to 80 % of solvent B within 1 to 3.5 min at the flow rate of 0.4 mL/min
  • Xevo G2-XS MS scan from 350 to 4000 m/z, ESI positive, sensitivity mode
  • the results were processed according to the following method.
  • Analyte peaks time window input time window range which covers the whole region of the peak and expected RT (i.e. mid-point of the time window range); background subtract results 5 % from baseline.
  • the MaxEntl deconvolution parameters were as follows: input m/z range; output mass range; TOF resolution 20,000. The mass error tolerance was 100 ppm.
  • the amino acid modifiers were input relevant modifiers such as -Lysine C-TERM, Pyroglutamic acid E N-TERM and linker-payload; select type as variable and maximum modification of 1 .
  • the overall DAR for each payload e.g. exatecan and berzosertib
  • T-N297A is trastuzumab provided in N297A format (/.e. comprising the substitution N297A at position 297 (EU numbering) of the CH2 domains of the Fc region).
  • CP-1 is: , known as m-PEG4-DBCO (CAS: 2228857-36-9), available for example from BroadPharm as BP-24030.
  • CP-2 is: , known as m-PEG4-TCO, available for example from BroadPharm as BP-27872.
  • This method was used to capture antibody specific ADCs from a complex matrix of plasma for DAR analysis, f
  • ADCs 5 and 6 were synthesised as described in example 15 above.
  • Sample preparation was carried out as follows. Briefly, immunocapture was performed by adding 12 pL of biotinylated HER2 antigen (250 pg/mL) to 50 pL of plasma incubated ADC (0.1 mg/mL). Subsequently, 50 pL of streptavidin beads (washed with DPBS) were added and the mixture was vortexed, finally the “Sample-Antigen-Beads complex” was rotated at 4 °C for 2 hours at a rotation rate of approximately 20 rpm.
  • On bead reduction was carried out by washing the incubated complex three times with cold DPBS, followed by separating and transferring the flowthrough in microtubes with a magnetic stand for troubleshooting. 50 pl of 25 mM DTT was then added to the beads and they were incubated at 40°C for 30 min. The eluant was then collected on a magnetic stand and 5 pL of sample was injected for analysis.
  • NCI-N87 Cells of the HER2-expressing cell line NCI-N87 (CRL-5822; gastric carcinoma) were seeded in 96-well white opaque plates in 175 pl of cell culture media, and incubated at 37°C, 5% CO2 for 24 h. NCI-N87 were seeded at a density of 9000 cells/well.
  • the agents were added to the cells in culture in aliquots of 25 pl., (from 0.33 uM, 10 points of 3-fold serial dilution).
  • the cells were then incubated for 5 days at 37°C and 5% CO2 Post-incubation, 50 pl of CellTiter-Glo reagent was added to the plates and incubated for 25 mins with gentle agitation at 600 rpm. Cell viability was measured via luminescence using Victor Nivo, PerkinElmer (2 replicates).
  • ADC6 is referred to as DBCO and ADC23 is referred to as TMTHSI in figure 29.

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Abstract

L'invention concerne une molécule de liaison à l'antigène qui se lie à HER2, comprenant (i) une fraction de liaison à HER2, et (ii) au moins une fraction lieur-charge utile, la molécule de liaison à l'antigène comprenant : (a) une fraction d'inhibiteur de réponse aux dommages à l'ADN (DDR), et (b) une fraction d'inhibiteur d'ADN topoisomérase I (TOP1).
PCT/EP2024/079224 2023-10-16 2024-10-16 Molécules de liaison à her2 Pending WO2025083069A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2016053107A1 (fr) 2014-10-03 2016-04-07 Synaffix B.V. Lieur de type sulfamide, conjugués de celui-ci et procédés de préparation
WO2018234636A1 (fr) 2017-06-21 2018-12-27 Glykos Finland Oy Lieurs hydrophiles et conjugués de ceux-ci
WO2021260579A1 (fr) * 2020-06-24 2021-12-30 Astrazeneca Uk Limited Association d'un conjugué anticorps-médicament et d'un inhibiteur d'atr
WO2022187370A1 (fr) * 2021-03-03 2022-09-09 R.P. Scherer Technologies, Llc Lieurs ramifiés pour conjugués anticorps-médicament et leurs méthodes d'utilisation

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