WO2025083071A2 - Conjugués anticorps-médicament - Google Patents

Conjugués anticorps-médicament Download PDF

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Publication number
WO2025083071A2
WO2025083071A2 PCT/EP2024/079226 EP2024079226W WO2025083071A2 WO 2025083071 A2 WO2025083071 A2 WO 2025083071A2 EP 2024079226 W EP2024079226 W EP 2024079226W WO 2025083071 A2 WO2025083071 A2 WO 2025083071A2
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antigen
moiety
inhibitor
binding molecule
group
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WO2025083071A3 (fr
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
Gary KHOO
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Hummingbird Bioscience Holdings Ltd
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Hummingbird Bioscience Holdings Ltd
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment

Definitions

  • the present disclosure relates to molecular biology, more specifically antibody technology.
  • the present disclosure also relates to methods of medical treatment and prophylaxis, as well as branched moieties and their use in antibody drug-conjugates.
  • ADCs Antibody-drug conjugates
  • chemotherapeutic regimens consist of a combination of drugs.
  • Co-delivery of small molecules can overcome resistance, generate additive or synergistic effects, and enhance therapeutic efficacy.
  • the present disclosure provides an antigen-binding molecule that binds to a target antigen, comprising (i) a target antigen-binding moiety, and (ii) at least one linker-payload moiety, wherein the antigen-binding 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 which is an ATR inhibitor. In some embodiments, 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 an ATM inhibitor. In some embodiments, the DDR inhibitor moiety is, or comprises, AZD0156.
  • 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, P1003,
  • 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 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 present disclosure provides a linker between:
  • an antigen-binding moiety comprising a moiety derived from a compound of the second aspect.
  • the linker 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 present disclosure provides a conjugate comprising:
  • conjugate 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.
  • modified antigen-binding 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 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 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 the use of 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 a cancer comprising cells expressing/overexpressing the target antigen.
  • the cancer is a hematologic cancer.
  • the cancer is selected from: a myeloid hematologic cancer, lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, AIDS-related lymphoma, cutaneous T cell lymphoma, mycosis fungicides, primary central nervous system lymphoma, Sezary syndrome, Waldenstrom macroglobulinemia, leukemia, T cell leukemia, B cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, acute promyelocytic leukemia, chronic promyelocytic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, myeloma, multiple myeloma, myelodysplastic
  • the cancer is refractory or relapsed to treatment with a DDR inhibitor, and/or wherein the cancer is refractory or relapsed to treatment with a TOP1 inhibitor.
  • the present disclosure also provides 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 the target antigen.
  • the present disclosure also provides an in vitro complex, optionally isolated, comprising an antigenbinding molecule according to the present disclosure bound to the target antigen.
  • the antigen-binding molecules of the present disclosure are provided with unexpected and advantageous properties relative to known antibody-drug conjugates.
  • DNA damage repair is a key mode of resistance to Topoisomerase I (TOP1) DNA damaging agents, and the combination of both payloads into a single antibody drug conjugate is expected to show improved, possibly synergistic, effects.
  • the dual payload ADC is also expected to possess advantageous properties compared to combining a chemotherapeutic DDR inhibitor with a TOP1 -inhibiting ADC, in reducing the overlapping toxicities of the chemotherapeutic DDR inhibitor and a TOP1 inhibiting ADC, such as neutropenia and thrombocytopenia.
  • the dual DDR inhibitor, TOP1 inhibitor approach is also expected to mitigate against resistance to TOP1 inhibitors, which is sometimes observed on treatment with ADCs comprising a TOP1 inhibitor payload.
  • ADCs comprising a TOP1 inhibitor payload.
  • Such resistance is described in Mosele, et al., Nat Med (2023) 29(8) 2110-2120 (PMID37488289); Zhang, et al., Br J Cancer (2021) 125(10) 1333-1340 (PMID34294893); Muai, et al., Mol Cell (2018) 69(3) 371-384 (PMID29395061).
  • DDR inhibitor and TOP1 inhibitor combinations have also been shown preclinically and clinically to resensitize tumors to TOP1 inhibitors, restoring responsiveness to validated TOP1 inhibitor therapy.
  • Josse etal., Cancer Res (2014) 74(23): 6968-6979 describes berzosertib (an ATR inhibitor) potentiating the effect of irinotecan in CRC CDX model, COLO205; Coussy et al., Sci Trans Med (2020) 12 (531): eaax2625 describes berzosertib (an ATR inhibitor) increasing sensitivity to irinotecan in SLFN11- negative, TNMC patient-derived xenograft tumors with BRCAness; Thomas et al., Cancer Cell (2021) 39(4): 566-579.
  • 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.
  • 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’.
  • 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 end- joining
  • WEE1 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 Pol0.
  • 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 eta!., 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 (azenosertib), IMP7068, SY4835, SCO191 , IMP7068;
  • 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 CYC140
  • 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. In some embodiments, the DDR inhibitor moiety is, or comprises, berzosertib:
  • the DDR inhibitor moiety is, or comprises, prexasertib:
  • 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:
  • the DDR inhibitor moiety is not, or does not comprise, veliparib. In some embodiments, where the DDR inhibitor moiety is or comprises a PARP inhibitor, the PARP inhibitor is not veliparib. In some embodiments, the DDR inhibitor moiety is not, or does not comprise, a PARP inhibitor.
  • 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, FL1 18, 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, K
  • the TOP1 inhibitor moiety is not, or does not comprise, PBX-7016 described in WO 2023/249473 A1 (the structure of which is shown in Example 3 of WO 2023/249473 A1).
  • the camptothecin derivative is not PBX-7016 described in WO 2023/249473 A1 .
  • the TOP1 inhibitor moiety is not, or does not comprise, a camptothecin derivative described in WO 2023/249473 A1 .
  • 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.
  • the antigen-binding molecule of the present disclosure does not comprise veliparib, and does not comprise PBX-7016 described in WO 2023/249473 A1.
  • 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.
  • Payload comprising moieties
  • a payload comprising moiety according to the present disclosure may comprise a payload, and a linker moiety between the payload and a click group, which click group is selected from the click group pairs described below.
  • the linker moiety may be a cleavable linker moiety or a non-cleavable moiety.
  • 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 due to the bulky nature of payload moieties. Commonly employed spacer moieties include paraaminobenzyl 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 paraaminobenzyl 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., Antibod
  • 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.
  • a payload moiety according to the present disclosure may comprise or consist of a cytotoxic agent.
  • cytotoxic agent such 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 et al., 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.
  • a payload moiety according to the present disclosure may comprise or consist of a non-cytotoxic agent.
  • payload moieties are described e.g. in Liu et al., Expert Opinion on Biological Therapy. 2016 16 (5), 591- 593 (doi:10.1517/14712598.2016.1161753); Yu et al., (2016). Next Horizons: ADCs Beyond Oncology. In: Damelin, M. (eds) Innovations for Next-Generation Antibody-Drug Conjugates. Cancer Drug Discovery and Development (doi: 10.1007/978-3-319-78154-9_14); McPherson & Hobson, in Methods in Molecular Biology.
  • a payload moiety according to the disclosure may comprise or consist of a immunological agent, such as: non-steroidal agents, e.g. PDE4 inhibitors, LXR agonists, tyrosine-kinase inhibitors, bisphosphonates; steroidal agents, e.g. glucocorticoid receptor modulators (GRM); immunostimulatory agents, e.g.
  • TLR7/8 agonists TLR4 agonists, TLR9 agonists, STING agonists, kinesin spindle kinase (KSP) inhibitors.
  • Other immunological agents include kinase inhibitors, growth factor inhibitors (e.g.
  • EGFR EGFR , PDGF, VEGF inhibitors
  • Calcineurin inhibitors CRAC inhibitors , PARP1 antagonists, PPARy agonists, Kv1.3 antagonists, PP2A agonists, MYD88 inhibitors, BCL-2 inhibitors, Adenosine A2A receptor (A2ar) agonists, calcium-activated potassium channel (Kca3.1) agonists, TGF- R1 inhibitors, TGF-R2 inhibitors, GLi 1 inhibitors, tankyrase (TNKS) antagonists, Traf2 and Nck- interacting kinase (TNIK) antagonists , imides, and vitamin D receptor (VDR) agonists.
  • A2A receptor A2ar
  • Ca3.1 calcium-activated potassium channel
  • TGF- R1 inhibitors TGF-R2 inhibitors
  • GLi 1 inhibitors GLi 1 inhibitors
  • tankyrase (TNKS) antagonists Traf2 and Nck- interacting kinase (TNIK
  • a payload moiety according to the disclosure may comprise or consist of a HDAC inhibitor, a NAMPT inhibitor or a DHFR agent.
  • a payload moiety according to the disclosure may comprise or consist of an antibiotic, an amantin, or a matrix metalloproteinase (MMP).
  • MMP matrix metalloproteinase
  • a payload moiety according to the disclosure may comprise or consist of an amyloid beta plaque-degrading agent. In some embodiments, a payload moiety may comprise or consist of a tau protein aggregate-degrading agent.
  • a payload moiety according to the disclosure may comprise or consist of an atherosclerotic plaque-stabilising agent.
  • a payload moiety according to the disclosure may comprise or consist of an adipose tissue-degrading agent.
  • a payload moiety may comprise or consist of an insulin resistance-modulating agent or a metabolic pathway-activating agent.
  • a payload moiety according to the disclosure may comprise or consist of a clotting factor. In some embodiments, a payload moiety may comprise or consist of an erythropoiesis-stimulating agent.
  • a payload moiety according to the disclosure may comprise or consist of an extracellular matrix-degrading agent.
  • a payload moiety according to the present disclosure may comprise or consist of a microtubule-targeting agent, a DNA-targeting agent, an RNA-targeting agent, an immune systemactivating agent, an apoptosis-promoting agent, a metabolism-inhibiting agent and a proteasome inhibiting agent.
  • a payload moiety according to the present disclosure comprises, or consists of, a microtubule-targeting agent.
  • Microtubules play important roles in maintaining proper cellular morphology, signal transduction, organelle transportation, cell motility and cell division. Microtubules are formed of tubulin, and agents that disrupt the tubulin polymerization dynamics, resulting in cell cycle arrest and apoptosis.
  • Tubulin inhibitors have a stronger toxicity to rapidly-dividing cancerous cells than slower- growing, non-cancerous cells.
  • Microtubule-targeting agents include tubulin polymerization enhancers (e.g. auristatins, taxanes), and tubulin polymerization inhibitors (e.g. maytansinoids, colchicine).
  • a payload moiety according to the present disclosure comprises, or consists of, a maytansinoid, e.g. maytansine or a derivative thereof, e.g. mytansine, DM1 (mertansine) or DM4 (ravtansine).
  • a payload moiety comprises, or consists of colchicine or a derivative thereof.
  • a payload moiety comprises, or consists of, an auristatin, e.g. a dolastatin 10 derivative, e.g. monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin PE, auristatin PYE, PF-06380101 , auristatin F-hydroxypropylamide (AF-HPA) or azastatin.
  • a payload moiety comprises, or consists of, a halichondrin B derivative, e.g. eribulin.
  • a payload moiety comprises, or consists of, a tubulysin or a derivative thereof, e.g.
  • a payload moiety comprises, or consists of, a cryptophycin or a derivative thereof, e.g. cryptophycin-1 , cryptophycin-52, cryptophycin-55 or cryptophycin-55gly.
  • a payload moiety comprises, or consists of, an EG5 (/.e. kinesin/KSP/KIF11) inhibitor, e.g. ispinesib (SB715992) or a derivative thereof, or filanesib (ARRY-520) or a derivative thereof.
  • a payload moiety comprises, or consists of, a taxane, e.g.
  • a payload moiety comprises, or consists of vinca alkaloid, e.g. vinblastine, vincristine, vindesine, vinorelbine or vinflunine.
  • a payload moiety comprises, or consists of hemiasterlin or a derivative thereof, e.g. hemiasterlin, hemisterlin A or HTI-286.
  • a payload moiety according to the present disclosure comprises, or consists of, a DNA-targeting agent.
  • DNA-targeting agents include agents that directly or indirectly destroy DNA through introducing/promoting the formation of single- and/or double-strand breaks (e.g. enediynes, topoisomerase inhibitors), DNA alkylating agents (e.g. pyrrolo[2,1-c][1 ,4] benzodiazepines, indolinobenzodiazpines, duocarmycins), and DNA crosslinking agents (e.g. mitomycin C).
  • a payload moiety according to the present disclosure comprises, or consists of, an enediyne, e.g.
  • a payload moiety comprises, or consists of, a topoisomerase inhibitor, e.g. a TOP1 or TOP2 inhibitor, e.g. camptothecin or a derivative thereof, e.g. SN- 38, exatecan, exatecan mesylate (DX-8951f), A/-glycyl-exatecan or deruxtecan (Dxd); e.g. an anthracycline, e.g.
  • a payload moiety comprises, or consists of, a pyrrolo[2,1-c][1 ,4] benzodiazepine (PBD) dimer, or a derivative thereof, e.g. a PDB, KMR-28-39, SJG-136 SGD-1882 or SG3199 dimer.
  • a payload moiety comprises, or consists of, indolinobenzodiazpine (IGN; monoimine) or a derivative thereof.
  • a payload moiety comprises, or consists of, a pyridinobenzodiazepine (PDD) dimer, or a derivative thereof, e.g. a PDD or FGX5-67 dimer.
  • a payload moiety comprises, or consists of, a duocarmycin or a derivative thereof, e.g. duocarmycin A, CC1065, duocarmycin SA, DUBA, seco-DIBA or seco-CBI.
  • a payload moiety comprises, or consists of, mitomycin C.
  • a payload moiety according to the present disclosure comprises, or consists of, a RNA-targeting agent.
  • Small molecule inhibitors that target RNA can kill both dividing and dormant tumor cells.
  • RNA-targeting agents include RNA splicing inhibitors (e.g. thailanstatin and derivatives thereof) and RNA polymerase II inhibitors (e.g. amatoxins, RNA polymerase ll-IN-2).
  • a payload moiety according to the present disclosure comprises, or consists of, thailanstatin or a derivative thereof, e.g. thailanstatin A, thailanstatin B, thailanstatin C or FR901464.
  • a payload moiety comprises, or consists of, an amatoxin, e.g. a-amanitin or p-amanitin. In some embodiments, a payload moiety comprises, or consists of, RNA polymerase ll-IN-2.
  • a payload moiety according to the present disclosure comprises, or consists of, an immune system-activating agent.
  • Immune-stimulating antibody conjugates employ small molecule-based engagement of the innate and/or adaptive immune systems.
  • a variety of immune- modulating payloads are in development, including Toll-like receptor (TLR) agonists, stimulator of interferon genes (STING) agonists and glucocorticoid receptor modulators (GRMs).
  • TLR Toll-like receptor
  • STING stimulator of interferon genes
  • GRMs glucocorticoid receptor modulators
  • a payload moiety according to the present disclosure comprises, or consists of, a TLR agonist, e.g. an agonist of TLR7, TLR8 or TLR9.
  • a payload moiety comprises, or consists of, a STING agonist, e.g. a cyclic dinucleotide (CDN; e.g. 2,3 cGAMP; 3,3 cGAMP; c-di-GMP or c-di-AMP) or a benzimidazole.
  • a payload moiety comprises, or consists of, a glucocorticoid receptor modulator, e.g. dexamethasone or a derivative thereof.
  • a payload moiety according to the present disclosure comprises, or consists of, an apoptosis-promoting agent.
  • Anti-apoptotic proteins such as Bcl-xL can play important roles in tumorigenesis, metastasis and drug resistance.
  • a payload moiety according to the present disclosure comprises, or consists of, a Bcl-xL inhibitor.
  • a payload moiety comprises, or consists of, ABT-737.
  • a payload moiety according to the present disclosure comprises, or consists of, a metabolism-inhibiting agent.
  • Metabolism-inhibiting agents such as niacinamide phosphate ribose transferase (NAMPT) inhibitors control the concentration of NAD+ within cells, inducing energy crisis and cell death, and antifolate antimetabolites such as methotrexate, that inhibit dihydrofolate reductase (DHFR) and thereby DNA synthesis.
  • NAMPT niacinamide phosphate ribose transferase
  • a payload moiety according to the present disclosure comprises, or consists of, a NAMPT inhibitor, e.g. FK-866 or A-1293201 .
  • a payload moiety according to the present disclosure comprises, or consists of, a DHFR inhibitor, e.g. methotrexate of a derivative thereof.
  • a payload moiety according to the present disclosure comprises, or consists of, a proteasome-inhibiting agent.
  • Proteasome-inhibiting agents include carmaphycins.
  • a payload moiety according to the present disclosure comprises, or consists of, a carmaphycin or a derivative thereof, e.g. carmaphycin A or carmaphycin B.
  • 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 antigen-binding 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.
  • a linkerpayload moiety comprising two different payloads is described e.g. in Levengood, etal., Angew Chem Int Ed Engl (2017) 56(3): 733-737.
  • 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-buty I) 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-buty I) 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.
  • the present disclosure provides a tri-functional linker moiety 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.
  • 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-).
  • xb is from 1 to 12
  • xa is 0
  • 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.
  • 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-(C 2 H4)-. In some of these embodiments, A1 is -(C2H4 ⁇ D)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-
  • DIBAC dibenzoazacyclooctyne
  • the first and second click groups are the same.
  • 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.
  • 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):
  • 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): (B1-2) where xd is 0-3,
  • R NB2 iS H Or -(C2H 4 O)xe2-(CH2)xf2-(NH)x S 2-(C( O)CH2)xh2- where xe2 is 0 or 1 , xf2 is 0 to 2, xg2 is 0 or 1 , and xh2 is 0 or 1 .
  • 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
  • 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 TOP1 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:
  • 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:
  • 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:
  • the DDR inhibitor moiety linked to click group has the structure:
  • the antigen-binding molecule of the present disclosure does not comprise, or consist of, the structure of ‘Trastuzumab-Veliparib(4)-25-6(4)’ as described in WO 2023/249473 A1 , e.g. as described in Example 7 of WO 2023/249473 A1 .
  • Antigen-binding molecules and antigen-binding moieties are provided.
  • an ‘antigen-binding molecule’ refers to a molecule that binds to a given target antigen.
  • Antigen-binding molecules comprise one or more antigen-binding moieties through which the antigen-binding molecule binds to its target antigen(s).
  • aspects and embodiments of the present disclosure relate to antigen-binding molecules comprising a target antigen-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).
  • 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 disu Ifide-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.
  • mAbs monoclonal antibodies
  • Antigen-binding regions of antibodies such as variable fragment (Fv), Fab and F(ab’)2 fragments may also be used/provided.
  • An ‘antigen-binding region’ is any fragment of an antibody that binds to the target antigen for which the given antibody is specific.
  • aspects and embodiments of the present disclosure relate to antigen-binding molecules that bind (/.e. through an antigen-binding moiety) to a given target.
  • the target antigen for a target antigen-binding moiety may be any molecule.
  • a target antigen may be a peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof.
  • a target antigen may be expressed at the cell surface of a cell expressing the target antigen.
  • a target antigen is a disease-associated antigen.
  • a ‘disease-associated antigen’ refers to an antigen whose presence is indicative of a given disease/disease state, or an antigen for which an elevated level of the antigen is positively-correlated with a given disease/disease state.
  • the disease-associated antigen may be an antigen whose expression is associated with the development, progression or severity of symptoms of a given disease.
  • the disease-associated antigen may be associated with the cause or pathology of the disease, or may be expressed abnormally as a consequence of the disease.
  • a disease-associated antigen may be an antigen of an infectious agent or pathogen, a cancer-associated antigen, an autoimmune disease-associated antigen, a neurological disease-associated antigen, a cardiovascular disease-associated antigen, a metabolic disease- associated antigen, a hematologic disease-associated antigen or a fibrotic disease-associated antigen.
  • the disease-associated antigen is an antigen of a pathogen.
  • the pathogen may be prokaryotic (bacteria), eukaryotic (e.g. protozoan, helminth, fungus), virus or prion.
  • the pathogen is an intracellular pathogen.
  • the pathogen is a virus, e.g. a virus as described hereinabove.
  • the pathogen is a bacterium.
  • the target antigen is a cancer-associated antigen.
  • a cancer-associated antigen is an antigen whose expression or overexpression is associated with cancer.
  • the cancer-associated antigen is a receptor molecule, e.g. a cell surface receptor.
  • the cancer-associated antigen is a cell signalling molecule, e.g. a cytokine, chemokine, interferon, interleukin or lymphokine.
  • the cancer-associated antigen is a growth factor or a hormone.
  • the cancer-associated antigen is a viral antigen.
  • a cancer cell antigen may be abnormally expressed by a cancer cell (e.g.
  • the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell.
  • a cancer cell antigen may be capable of eliciting an immune response.
  • the antigen is expressed at the cell surface of the cancer cell (/.e. the cancer cell antigen is a cancer cell surface antigen).
  • the part of the antigen which is bound by an antigen-binding molecule described herein is displayed on the external surface of the cancer cell (/.e. is extracellular).
  • the cancer cell antigen may be a cancer-associated antigen.
  • the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer.
  • the cancer- associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer.
  • the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and/or protein level) by cells of a cancer, e.g. as compared to the level of expression by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type).
  • the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type).
  • the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.
  • Cancer-associated antigens include oncofetal antigens: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: fibroblast activation protein (FAP), B-cell maturation antigen (BCMA), CD19, HER2/neu, BING-4, calcium- activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1 , survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1/2, MC1 R, prostate specific antigen; mutated antigens: p-catenin, BRCA1/2, CDK4, CML66, Fibronectin, M
  • cancer cell antigens include heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B.
  • HSP70 heat-shock protein 70
  • HRP90 heat-shock protein 90
  • GFP78 glucose-regulated protein 78
  • vimentin nucleolin
  • FAPP feto-acinar pancreatic protein
  • ALPPL-2 alkaline phosphatase placental-like 2
  • siglec-5 siglec-5
  • stress-induced phosphoprotein 1 TRF1
  • PTK7 protein tyrosine kinase 7
  • cyclophilin B cyclophilin B.
  • the target antigen is an immune cell surface molecule.
  • An immune cell surface molecule is any molecule which is expressed in or at the cell membrane of an immune cell.
  • the part of the immune cell surface molecule which is bound by the antigen-binding moiety is on the external surface of the immune cell (/.e. is extracellular).
  • the immune cell surface molecule may be expressed at the cell surface of any immune cell.
  • the immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte.
  • the lymphocyte may be e.g.
  • the immune cell may express a CD3 polypeptide (e.g. CD3y CD3e CD3 or CD35), a TCR polypeptide (TCRa or TCRp), CD27, CD28, CD4 or CD8.
  • the immune cell is a T cell, e.g. a CD3+ T cell.
  • the T cell is a CD3+, CD4+ T cell.
  • the T cell is a CD3+, CD8+ T cell.
  • the T cell is a T helper cell (TH cell).
  • the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).
  • CTL cytotoxic T lymphocyte
  • the immune cell is a T cell or an NK cell.
  • an immune cell surface molecule may be a CD3-TCR complex polypeptide, e.g. TCRa, TCRp, TCRy, TCR5, TRAC, TRBC1 , TRBC2, TRGC1 , TRGC2, TRDC, CD3e, CD35, CD3y, CD3 or CD3r
  • an immune cell surface molecule is CD3, CD8, CD4 or CD28.
  • an immune cell surface molecule is a checkpoint molecule (e.g. PD-1 , CTLA-4, LAG-3, TIM-3, VISTA, TIGIT or BTLA), or a ligand for a checkpoint molecule (e.g.
  • the immune cell surface molecule is a costimulatory molecule (e.g. CD28, 0X40, 4-1 BB, ICOS or CD27), or a ligand for a costimulatory molecule (e.g. CD86, CD80, OX40L 4-1 BBL, ICOSL or CD70).
  • a costimulatory molecule e.g. CD28, 0X40, 4-1 BB, ICOS or CD27
  • a ligand for a costimulatory molecule e.g. CD86, CD80, OX40L 4-1 BBL, ICOSL or CD70.
  • 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 moieties 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 the relevant target antigen.
  • 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 VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1) region of an antigen-binding region of an antibody together constitute the Fab region.
  • 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 the relevant target antigen.
  • whole antibody refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig).
  • Ig immunoglobulin
  • 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. Depending on the heavy chain, immunoglobulins may be classed as IgG (e.g.
  • 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 the relevant target antigen.
  • IgG e.g. lgG1 , lgG2, lgG3, lgG4
  • IgA e.g. lgA1 , lgA2
  • IgD IgE
  • IgM which binds to the relevant target antigen
  • 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:2 or 7.
  • 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 antigenbinding 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 NO:11 , 12, 13, 14, 15 or 16.
  • 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:
  • a nonpolar amino acid is substituted with another, non-identical nonpolar amino acid.
  • 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.
  • multispecific antigen-binding molecules By ‘multispecific’ it is meant that the antigen-binding molecule binds to more than one target antigen (e.g. one of 1 , 2, 3, 4, 5, 6 or more target antigens).
  • the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two, different antigen-binding moieties. In some embodiments, the antigen-binding molecule comprises at least two antigen-binding moieties, wherein each antigen-binding moiety binds to a different target antigen.
  • the antigen-binding molecule comprises: (i) an antigen-binding polypeptide (e.g. a scFv, scFab, polypeptide aptamer or VhH) or and an antigen-binding polypeptide complex (e.g. a Fv, Fab or whole antibody) that binds to a first antigen, and (ii) an antigen-binding polypeptide (e.g. a scFv, scFab, polypeptide aptamer or VhH) or and an antigen-binding polypeptide complex (e.g. a Fv, Fab or whole antibody) that binds to an antigen other than the first antigen.
  • an antigen-binding polypeptide e.g. a scFv, scFab, polypeptide aptamer or VhH
  • an antigen-binding polypeptide complex e.g. a Fv, Fab or whole antibody
  • the antigen-binding molecule comprises: (i) an antigen-binding moiety comprising the VH and VL of an antibody that binds to a first antigen, and (ii) an antigen-binding moiety comprising the VH and VL of an antibody that binds to an antigen other than the first antigen.
  • Multispecific antigen-binding molecules may be provided in any suitable format, such as those formats described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182- 212, which is hereby incorporated by reference in its entirety.
  • Multispecific antigen-binding molecule formats include those shown in Figure 2 of Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212: antibody conjugates, e.g. lgG2, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, KA-body common HC; CH1/CL fusion proteins, e.g.
  • scFv2-CH1/CL, VHH2-CH1/CL ‘variable domain only’ bispecific antigen-binding molecules, e.g. tandem scFv (taFV), triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple heads, tandem dAbA/HH, tetravalent dAb.VHH;
  • Non-lg fusion proteins e.g.
  • scFv2-albumin scDb-albumin
  • taFv-albumin taFv-toxin
  • miniantibody DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g.
  • Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-lg fusion proteins, e.g. DNL-Fabs, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g.
  • DAF two-in one-IgG
  • bispecific antigenbinding polypeptides/polypeptide complexes comprising at least two antigen-binding moieties.
  • Methods for producing bispecific antigenbinding polypeptides/polypeptide complexes include chemically crosslinking antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:l V:2.13:2.13.1 — 2.13.16, which is hereby incorporated by reference in its entirety.
  • SPDP A/-succinimidyl-3-(-2-pyridyldithio)-propionate
  • bispecific antigen-binding polypeptides/polypeptide complexes include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001 . Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1 — 2.13.16.
  • Antigen-binding polypeptides/polypeptide complexes according to the present disclosure may also 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.
  • the multispecific (e.g. bispecific) antigen-binding polypeptides/polypeptide complexes may be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigen-binding polypeptide/polypeptide complex, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber- Schwarz), or French, How to make bispecific antibodies, Methods Mol. Med. 2000; 40:333-339, the entire contents of both of which are hereby incorporated by reference.
  • a DNA construct encoding the light and heavy chain variable domains for the two antigenbinding moieties, and including sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques.
  • a recombinant bispecific antigen-binding polypeptide/polypeptide complex can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e.g. a mammalian host cell), and expressed recombinant bispecific polypeptide/polypeptide complex can then optionally be purified.
  • 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.
  • 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:9 or 10.
  • 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:9 or 10.
  • 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 C1 q 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:20 or 21.
  • 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 NQ:20 or 21 .
  • Functional properties of the antigen-binding molecules 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:20 or 21 .
  • an antigen-binding molecule described herein may possess one or more of the following properties: binds to cells expressing the target antigen for the antigen-binding molecule; inhibits proliferation of cells expressing the target antigen for the antigen-binding molecule; increases killing of cells expressing the target antigen for the antigen-binding molecule; inhibits proliferation and/or increases killing of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule; inhibits tumor growth and/or reduces tumor size/volume (e.g. of a cancer expressing the target antigen for the antigen-binding molecule); increases survival of subjects having a cancer (e.g. a cancer expressing the target antigen for the antigen-binding molecule).
  • 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 its target antigen in a region which is accessible to an antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when the relevant antigen is expressed at the cell surface (i.e. in or at the cell membrane). In some embodiments, the antigen-binding molecule binds to its target antigen when it is expressed at the cell surface. In some embodiments, the antigen-binding molecule binds to cells expressing its target antigen.
  • an antigen-binding molecule i.e., an extracellular antigen-binding molecule
  • the ability of an antigen-binding molecule to bind to a given cell type can be analyzed 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 cells expressing a given target antigen can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.
  • the antigen-binding molecule inhibits proliferation of cells expressing the target antigen for the antigen-binding molecule.
  • the ability of an antigen-binding molecule to inhibit proliferation of a given cell type can be analyzed 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 cells expressing the target antigen for the antigen-binding molecule 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 the same cells observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule known not to influence proliferation of cells expressing the relevant target antigen), in a given assay.
  • the antigen-binding molecule described herein inhibits proliferation of cells expressing the target antigen for the antigen-binding molecule 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 inhibits proliferation of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule.
  • ‘in proximity to’ a cell expressing the target antigen for the antigen-binding molecule refers to the region/area within 100 pm of the cell expressing the target antigen.
  • Cells in proximity to a cell expressing the target antigen for the antigen-binding molecule may also be referred to as surrounding cells or bystander cells. Such cells may or may not express the target antigen for the antigen-binding molecule themselves.
  • cells in proximity to a cell expressing the target antigen for the antigen-binding molecule may be within 100 pm (e.g. within 90 pm, 80 pm, 70 pm, 60 pm, 50 pm, 40 pm, 30 pm, 20 pm, or 10 pm) of the cell expressing the target antigen.
  • the antigen-binding molecule of the present invention is capable of inhibiting proliferation of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule to less than 1 times, e.g.
  • the antigen-binding molecule does not inhibit proliferation of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigenbinding molecule.
  • the antigen-binding molecule according to the present disclosure potentiates (/.e. upregulates, enhances) cell killing of cells comprising/expressing the target antigen for the antigenbinding molecule.
  • an antigen-binding molecule according to the present disclosure may inhibit growth or reduce metastasis of a cancer comprising cells comprising/expressing the target antigen for the antigen-binding molecule.
  • an antigen-binding molecule may potentiate (/.e. upregulate, enhance) cell killing of cells comprising/expressing the target antigen for the antigen-binding molecule.
  • an antigen-binding molecule may potentiate (/.e. upregulate, enhance) cell killing of cells (e.g. cells that do not comprise/express the target antigen) in proximity to a cell comprising/expressing the target antigen for the antigen-binding molecule.
  • an antigen-binding molecule does not potentiate (/.e. upregulate, enhance) cell killing of cells (e.g. cells that do not comprise/express the target antigen) in proximity to a cell comprising/expressing the target antigen for the antigen-binding molecule.
  • an antigen-binding molecule may inhibit growth of cells of a cancer, or may inhibit growth of a tumor, comprising cells comprising/expressing the target antigen for the antigen-binding molecule.
  • an antigen-binding molecule may inhibit metastasis of a cancer/tumor comprising cells comprising/expressing the target antigen for the antigenbinding molecule.
  • an antigen-binding molecule according to the present disclosure may inhibit growth or reduce metastasis of a cancer comprising (i) cells comprising/expressing the target antigen for the antigen-binding molecule, and (ii) cells not comprising/expressing the target antigen for the antigenbinding molecule.
  • an antigen-binding molecule may inhibit growth of cells of a cancer, or may inhibit growth of a tumor, comprising (i) cells comprising/expressing the target antigen for the antigen-binding molecule, and (ii) cells not comprising/expressing the target antigen for the antigenbinding molecule.
  • an antigen-binding molecule may inhibit metastasis of a cancer/tumor comprising (i) cells comprising/expressing the target antigen for the antigen-binding molecule and (ii) cells not comprising/expressing the target antigen for the antigen-binding molecule.
  • Cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, hereby incorporated by reference in its entirety.
  • Examples of in vitro assays of 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.
  • 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.
  • release assays
  • an antigen-binding molecule according to the present disclosure is capable of reducing the number/proportion of cells expressing the target antigen for the antigen-binding molecule. In some embodiments, an antigen-binding molecule according to the present disclosure is capable of depleting/enhancing depletion of such cells.
  • an antigen-binding molecule of the present disclosure displays anticancer activity.
  • the antigen-binding molecule increases killing of cancer cells.
  • 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 the target antigen for the antigen-binding molecule.
  • an antigen-binding molecule 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. In some embodiments, 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.
  • 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 the target antigen for the antigenbinding molecule.
  • the cancer may be a cancer comprising cells expressing the target antigen for the antigen-binding molecule and cells that do not express the target antigen for the antigen-binding molecule.
  • An antigen-binding molecule of the present disclosure may be analyzed 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 cancer expressing the target antigen for the antigen-binding molecule) 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 cancer expressing the target antigen for the antigen-binding molecule) to greater than 1 times, e.g.
  • a cancer expressing the target antigen for the antigen-binding molecule may comprise cells comprising/expressing the target antigen and may further comprise cells which do not comprise/express the target antigen.
  • an antigen-binding molecule according to the present disclosure possesses one or more novel, similar or improved functional properties as compared to: (i) an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only a TOP1 inhibitor moiety (/.e. not also comprising a DDR inhibitor moiety), and/or (ii) an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only a DDR inhibitor moiety (/.e. not also comprising a TOP1 inhibitor moiety).
  • an antigen-binding molecule possesses one or more novel, similar or improved functional properties as compared to: (i) an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only the same TOP1 inhibitor moiety as the antigen-binding molecule (/.e. not comprising the DDR inhibitor moiety of the antigen-binding molecule), and/or (ii) an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only the same DDR inhibitor moiety as the antigen-binding molecule (/.e. not comprising the TOP1 inhibitor moiety of the antigen-binding molecule).
  • an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only the same TOP1 inhibitor moiety as the antigen-binding molecule (/.e. not comprising the DDR inhibitor moiety of the antigen-binding molecule)’ is referred to simply as ‘an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety’
  • ‘an antigen-binding molecule comprising the same target antigen-binding moiety, and comprising only the same DDR inhibitor moiety as the antigen-binding molecule (/.e. not comprising the TOP1 inhibitor moiety of the antigen-binding molecule)’ is referred to simply as ‘an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety’.
  • an antigen-binding molecule according to the present disclosure may display one or more of the following: similar binding to cells expressing the relevant target antigen for the antigen-binding molecule, as compared to an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety; increased killing of cells expressing the relevant target antigen for the antigen-binding molecule, as compared to killing of such cells displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety; similar killing of cells (e.g.
  • tumor size/volume e.g. of a cancer expressing the relevant target antigen for the antigen-binding molecule
  • tumor growth inhibition/reduction in tumor size/volume displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety e.g. of a cancer expressing the relevant target antigen for the antigen-binding molecule
  • similar internalization into cells expressing the relevant target antigen for the antigen-binding molecule as compared to internalization displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety
  • similar toxicological properties as compared to those of an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety.
  • a level of a given property/outcome which is ‘similar to’ a reference level may be >0.5 times and ⁇ 2 times, e.g. one of >0.55 times and ⁇ 1 .9 times, >0.6 times and ⁇ 1 .8 times, >0.65 times and ⁇ 1 .7 times, >0.7 times and ⁇ 1 .6 times, >0.75 times and ⁇ 1 .5 times, >0.8 times and ⁇ 1.4 times, >0.85 times and ⁇ 1 .3 times, >0.9 times and ⁇ 1.2 times or >0.95 times and ⁇ 1 .1 times the reference level.
  • a level of a given property/outcome which is ‘increased’ relative to a reference level may be greater than 1 times, e.g. one of >1.01 times, >1.02 times, >1.03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times or >5 times the reference level.
  • a level of a given property/outcome which is ‘reduced’ relative to a reference level may be 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 reference level.
  • the antigen-binding molecule of the present disclosure binds to cells expressing the relevant target antigen for the antigen-binding molecule with an EC50 which is similar to the EC50 with which an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety binds to the relevant cells, in a given assay.
  • the EC50 for binding of the antigen-binding molecule to cells expressing the relevant target antigen is >0.5 times and ⁇ 2 times, e.g.
  • the antigen-binding molecule of the present disclosure increases the killing of cells expressing the relevant target antigen for the antigen-binding molecule to a level that is greater than the level of killing of the same cells displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, in a given assay.
  • the antigen-binding molecule increases the killing of cells expressing the relevant target antigen for the antigen-binding molecule (e.g. cancer cells expressing the relevant target antigen for the antigen-binding molecule) to a level that is greater than 1 times, e.g.
  • the antigen-binding molecule of the present disclosure kills cells expressing the relevant target antigen for the antigen-binding molecule with an EC50 which is less than the EC50 with which an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety kills the relevant cells, in a given assay.
  • the EC50 of the antigen-binding molecule of the present disclosure for killing cells expressing the relevant target antigen is less than 1 times, e.g.
  • the antigen-binding molecule of the present disclosure kills cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule with an EC50 which is similar to the EC50 with which an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety kills the relevant cells, in a given assay.
  • the EC50 of the antigen-binding molecule of the present disclosure for killing cells e.g.
  • cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule is >0.5 times and ⁇ 2 times, e.g. one of >0.55 times and ⁇ 1 .9 times, >0.6 times and ⁇ 1 .8 times, >0.65 times and ⁇ 1 .7 times, >0.7 times and ⁇ 1 .6 times, >0.75 times and ⁇ 1 .5 times, >0.8 times and ⁇ 1 .4 times, >0.85 times and ⁇ 1 .3 times, >0.9 times and ⁇ 1 .2 times or >0.95 times and ⁇ 1.1 times the EC50 for killing cells of the same type for an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.
  • the antigen-binding molecule of the present disclosure reduces tumor size/volume to a level that is greater than the level of reduction in tumor size/volume observed following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, in a given assay.
  • the antigen-binding molecule 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 cancer expressing the relevant target antigen for the antigen-binding molecule) 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 relevant antigen-binding molecule, in the relevant model.
  • the antigen-binding molecule of the present disclosure achieves a level of tumor growth inhibition that is greater than the tumor growth inhibition observed following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, in a given assay.
  • the antigen-binding molecule 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 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 relevant antigen-binding molecule, in the relevant model.
  • the antigen-binding molecule of the present disclosure achieves a synergistic level of killing of cells expressing the relevant target antigen for the antigen-binding molecule, as compared to the level of cell killing achieved individually by equivalent antigen-binding molecules comprising only the TOP1 inhibitor or DDR inhibitor moiety. That is, in some embodiments, the antigen-binding molecule of the present disclosure achieves a level of cell killing that is synergistic (/.e. super-additive), relative to what is observed when equivalent antigen-binding molecules comprising only the TOP1 inhibitor or DDR inhibitor moiety are used alone.
  • the antigen-binding molecule of the present disclosure achieves a synergistic level of tumor growth inhibition and/or a synergistic reduction in tumor size/volume, as compared to the level of achieved individually by equivalent antigen-binding molecules comprising only the TOP1 inhibitor or DDR inhibitor moiety. That is, in some embodiments, the antigen-binding molecule of the present disclosure achieves a level of tumor growth inhibition and/or a reduction of tumor size/volume that is synergistic (/.e. super-additive), relative to what is observed when equivalent antigen-binding molecules comprising only the TOP1 inhibitor or DDR inhibitor moiety are used alone.
  • a ‘synergistic’ or ‘super-additive’ level of a relevant effect refers to a level of the effect which is greater than the sum of the effects observed for the individual comparator molecules (/.e. an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety, and an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety), when used alone.
  • Dose-response curves may be plotted, and evaluated in order to determine whether the antigen-binding molecule of the disclosure achieves a synergistic level of the relevant effect relative to the equivalent antigen-binding molecules comprising only the TOP1 or DDR inhibitor moiety.
  • the antigen-binding molecule of the present disclosure displays similar internalization into cells expressing the relevant target antigen for the antigen-binding molecule, as compared to internalization into cells of the same type displayed by an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety.
  • the antigen-binding molecule is internalized into cells expressing the relevant target antigen for the antigen-binding molecule to a level that is >0.5 times and ⁇ 2 times, e.g.
  • >0.55 times and ⁇ 1.9 times >0.6 times and ⁇ 1 .8 times, >0.65 times and ⁇ 1 .7 times, >0.7 times and ⁇ 1 .6 times, >0.75 times and ⁇ 1 .5 times, >0.8 times and ⁇ 1 .4 times, >0.85 times and ⁇ 1.3 times, >0.9 times and ⁇ 1 .2 times or >0.95 times and ⁇ 1 .1 times the level of internalization of an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety into the relevant cells, as determined in the same assay.
  • evaluation of internalization for the purposes of such comparison is performed after incubation of the relevant antigen-binding molecule with the relevant cells for more than 5 min, e.g. one of >30 min, >1 h, >1 .5 h, >2 h or >2.5 h.
  • the antigen-binding molecule of the present disclosure displays similar toxicity to subjects administered the antigen-binding molecule as compared to subjects administered an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety.
  • the antigen-binding molecule has a similar toxicological profile as compared to an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety.
  • the number/proportion of red blood cells/white blood cells/lymphocytes/monocytes/neutrophils/platelets in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and ⁇ 2 times, e.g.
  • the hemoglobin concentration/hematocrit percentage/mean corpuscular volume, mean corpuscular hemoglobin/mean corpuscular hemoglobin concentration in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and ⁇ 2 times, e.g.
  • >0.55 times and ⁇ 1.9 times >0.6 times and ⁇ 1 .8 times, >0.65 times and ⁇ 1 .7 times, >0.7 times and ⁇ 1 .6 times, >0.75 times and ⁇ 1 .5 times, >0.8 times and ⁇ 1 .4 times, >0.85 times and ⁇ 1.3 times, >0.9 times and ⁇ 1 .2 times or >0.95 times and ⁇ 1 .1 times the level observed following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.
  • the level of a correlate of hepatic function e.g. alkaline phosphatase, alanine aminotransferase, albumin, total protein
  • a correlate of hepatic function e.g. alkaline phosphatase, alanine aminotransferase, albumin, total protein
  • the level of a correlate of renal function e.g. blood urea nitrogen and/or creatinine
  • the level of a correlate of renal function is >0.5 times and ⁇ 2 times, e.g.
  • the level of sodium/potassium/phosphate/calcium in the peripheral blood of a subject following administration of an antigen-binding molecule according to the present disclosure is >0.5 times and ⁇ 2 times, e.g. one of >0.55 times and ⁇ 1.9 times, >0.6 times and ⁇ 1 .8 times, >0.65 times and ⁇ 1 .7 times, >0.7 times and ⁇ 1 .6 times, >0.75 times and ⁇ 1 .5 times, >0.8 times and ⁇ 1 .4 times, >0.85 times and ⁇ 1.3 times, >0.9 times and ⁇ 1 .2 times or >0.95 times and ⁇ 1 .1 times the level observed in the peripheral blood of a subject following treatment with an equivalent antigen-binding molecule only comprising the TOP1 inhibitor moiety and/or an equivalent antigen-binding molecule only comprising the DDR inhibitor moiety, as determined in the same assay.
  • 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.
  • 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 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), glucoronidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.
  • 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 of which 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
  • 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
  • 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 diseases, e.g. cancers.
  • 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 the target antigen for the antigen-binding molecule, or a reduction in the number or activity of cells comprising/expressing the target antigen for the antigen-binding molecule.
  • the disease/condition may be a disease/condition in which the target antigen for the antigen-binding molecule, or cells expressing/overexpressing the target antigen for the antigen-binding molecule are pathologically-implicated, e.g. a disease/condition in which an increased level/activity of the target antigen for the antigen-binding molecule, or an increase in the number/proportion of cells comprising/expressing the target antigen for the antigen-binding molecule 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 the target antigen for the antigen-binding molecule, or an increase in the number/proportion of cells comprising/expressing the target antigen for the antigen-binding molecule 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.
  • the disease/condition is a cancer.
  • 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).
  • 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.
  • 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 antigen-binding molecule or composition 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, endometrial cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.
  • NHL Non-Hodgkin’s lymphoma
  • CML chronic myelogenous leukemia
  • AML acute myeloid leukemia
  • MDS myelodys
  • the cancer is breast cancer, lung cancer (e.g. small cell lung cancer), gastric cancer or endometrial cancer.
  • lung cancer e.g. small cell lung cancer
  • gastric cancer e.g. gastric cancer or endometrial cancer.
  • the cancer is breast ductal carcinoma, small cell lung carcinoma, gastric carcinoma or endometrial carcinoma.
  • the cancer is a hematologic cancer.
  • the cancer is a myeloid hematologic cancer, lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, AIDS-related lymphoma, cutaneous T cell lymphoma, mycosis fungicides, primary central nervous system lymphoma, Sezary syndrome, Waldenstrom macroglobulinemia, leukemia, T cell leukemia, B cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, acute promyelocytic leukemia, chronic promyelocytic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, myeloma, multiple myeloma, myelodysplastic syndrome, or a myeloproliferative disorder.
  • the cancer to be treated/prevented comprises cells expressing the target antigen for the antigen-binding molecule. In some embodiments, the cancer to be treated/prevented is a cancer which is positive for the target antigen for the antigen-binding molecule. In some embodiments, the cancer comprises cells that overexpress the target antigen for the antigen-binding molecule.
  • 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.
  • the cancer to be treated/prevented is a cancer which is heterogenous for expression of the target antigen for the antigen-binding molecule. In some embodiments, the cancer to be treated/prevented comprises a population of cells heterogenous for expression of the target antigen for the antigen-binding molecule.
  • the cancer to be treated/prevented comprises cells that do not express the target antigen for the antigen-binding molecule. In some embodiments, the cancer to be treated/prevented comprises cells that are negative for the target antigen for the antigen-binding molecule. In some embodiments, the cancer to be treated/prevented comprises cells expressing the target antigen for the antigen-binding molecule and cells that do not express the target antigen for the antigen-binding molecule. In some embodiments the cancer to be treated/prevented comprises cells which are positive for the target antigen for the antigen-binding molecule and cells which are negative for the target antigen for the antigen-binding molecule.
  • 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 the target antigen for the antigen-binding molecule, 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 the target antigen for the antigen-binding molecule is pathologically-implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of the target antigen for the antigen-binding molecule, a cancer for which expression of the target antigen for the antigen-binding molecule is a risk factor and/or a cancer for which expression of the target antigen for the antigen-binding molecule is positively associated with onset, development, progression, severity or metastasis of the cancer.
  • the cancer may be characterised by expression of the target antigen for the antigen-binding molecule, e.g. the cancer may comprise cells (e.g.
  • a cancer which is ‘positive’ for the target antigen for the antigen-binding molecule may be a cancer comprising cells expressing the target antigen for the antigen-binding molecule (e.g. at the cell surface).
  • a cancer which is ‘positive’ for the target antigen for the antigen-binding molecule may overexpress the target antigen for the antigen-binding molecule.
  • 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 the target antigen for the antigen-binding molecule, 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 the target antigen for the antigen-binding molecule may be known or predicted to cause, or may be associated with, increased gene/protein expression of the target antigen for the antigen-binding molecule. Mutations resulting in increased expression and/or activity of the target antigen for the antigen-binding molecule may be referred to as ‘activating’ mutations.
  • a mutation which causes increased expression of the target antigen for the antigen-binding molecule may result in gene or protein expression of the target antigen for the antigen-binding molecule 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 the target antigen for the antigen-binding molecule may be a result of the mutation, and thus ‘increased expression’ may be from no expression.
  • a mutation which causes increased expression of the target antigen for the antigen-binding molecule may cause an increase in gene expression of the gene encoding the relevant antigen relative to an equivalent cell not comprising the mutation. In some embodiments, a mutation which causes increased expression of the target antigen for the antigen-binding molecule may cause an increase in protein expression of the relevant protein relative to an equivalent cell not comprising the mutation.
  • a cancer comprising cells harboring a mutation resulting in increased expression of the target antigen for the antigen-binding molecule relative to equivalent cells lacking the mutation may be described as a cancer comprising cells displaying overexpression/upregulated expression of the target antigen for the antigen-binding molecule.
  • 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 cancer to be treated/prevented in accordance with the present disclosure may be characterised by an increase in the expression and/or activity of the target antigen for the antigenbinding molecule (/.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 the target antigen for the antigenbinding molecule, e.g. as compared to the level of expression and/or activity observed in equivalent non- cancerous cells/non-tumor tissue.
  • 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, fluzoparib, NMS-03305293, AZD9574), 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), RP3500 (camonsertib), ATRN119, ART380, IMP9064, HRS2398, M1774, IMP9064, SC0245, LF0397, NU6027
  • a WEE1 inhibitor e.g.
  • adavosertib Debio 0123, PD0166285, PD0407824, AZD1775 ZN-c3 (azenosertib), 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, CC-115, MU380, NU7441 , KU-5778), a DNA-PK inhibitor (e.g.
  • a RAD51 inhibitor e.g. CYT0851
  • USP inhibitor e.g. an inhibitor of USP11 , USP7, USP4, USP37, USP39, USP45, USP24 and/or USP1 ; e.g. KSQ-4279
  • PKMYT1 inhibitor e.g. RP6306
  • an Aurora-A inhibitor
  • 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.
  • Example 4 herein demonstrates that antigen-binding molecules comprising a DDRi inhibitor moiety and a TOP1 inhibitor moiety according to the present disclosure are useful to kill cancer cells that are resistant/insensitive to treatment with the same antibody conjugated only to a TOP1 inhibitor moiety.
  • Example 11 herein demonstrates that antigen-binding molecules comprising a DDRi inhibitor moiety and a TOP1 inhibitor moiety according to the present disclosure are useful to inhibit tumour growth in a cellline derived mouse model of cancer resistant/insensitive to treatment with the same antibody conjugated only to a TOP1 inhibitor moiety.
  • Example 5 herein demonstrates that treatment with an antigen-binding molecule comprising a DDRi inhibitor moiety and a TOP1 inhibitor moiety according to the present disclosure achieves much more potent inhibition of tumor growth in a cell line-derived mouse model of breast ductal carcinoma, as compared to treatment with the same antibody conjugated only to a TOP1 inhibitor moiety.
  • 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), LMP744, AZ14170132, SHR9265, Ed-04, KL610023, A1 .9, ZD06519, P1003, P1021 , VIP
  • 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 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
  • a TOP1 inhibitor e.g. a TOP1 inhibitor as described herein
  • 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.
  • 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).
  • Antigen-binding molecules according to the present disclosure may be employed for the treatment/prevention of diseases/conditions other than cancers.
  • Antibody-drug conjugates are useful for the treatment/prevention of diseases/conditions including infectious diseases, autoimmune diseases, neurological diseases, cardiovascular diseases, metabolic diseases, hematological diseases and fibrotic diseases; see e.g. Mike Ward, “Exploring the potential of ADCs beyond oncology”, Drug Target Review, published 6 August 2024.
  • the disease/condition to be treated/prevented in accordance with the present disclosure is an infectious disease/condition, e.g. a disease/condition caused by infection with a pathogen (e.g. a disease/condition caused by bacterial, viral, fungal, or parasitic infection).
  • a pathogen e.g. a disease/condition caused by bacterial, viral, fungal, or parasitic infection
  • the target antigen of the target antigen-binding moiety may be an antigen of a pathogen as described herein. It will be appreciated that in aspects and embodiments wherein the disease/condition to be treated/prevented is a disease/condition caused by infection with a pathogen (e.g.
  • the target antigen of the target antigen-binding moiety of the antigen-binding molecule of the present disclosure may be an antigen of the relevant pathogen (/.e. an antigen of the relevant bacterium, virus, fungus, or parasite).
  • an infectious disease/condition according to the present disclosure is a disease/condition caused by/associated with bacterial infection, e.g. infection with Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio chloerae, Staphylococcus spp., Streptococcus spp.
  • the infectious disease is a disease caused by/associated with a virus, e.g.
  • the infectious disease is a disease caused by/associated with fungal infection, e.g. infection with Alternaria sp, Aspergillus sp, Candida sp and Histoplasma sp.
  • the fungal infection may be fungal sepsis or histoplasmosis.
  • the infectious disease is a disease caused by/associated with parasitic infection, e.g.
  • the autoimmune disease/condition is selected from: diabetes mellitus type 1 , celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, arthritis (e.g. rheumatoid arthritis), and systemic lupus erythematosus.
  • the autoimmune disease/condition is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis or diabetes mellitus type 1 .
  • the disease/condition to be treated/prevented in accordance with the present disclosure is a neurological disease/condition.
  • the target antigen of the target antigen-binding moiety may be a neurological disease-associated antigen.
  • the target antigen of the target antigen-binding moiety of the antigen-binding molecule of the present disclosure may be an antigen associated with the relevant neurological disease/condition.
  • the target antigen may be an antigen expressed by pathological effectors of the relevant neurological disease/condition, or an antigen otherwise implicated in the pathology of the neurological disease/condition.
  • antigen-binding molecules according to the present disclosure comprising payload moieties having cytotoxic activity can be employed to kill/increase killing of pathological effectors of the relevant neurological disease/condition (e.g. immune cells).
  • antigen-binding molecules according to the present disclosure comprising appropriate payload moieties may be employed to increase degradation of plaques (e.g. amyloid beta plaques) or protein aggregates (e.g. tau protein aggregates) that are implicated in the pathology of the disease/condition.
  • the disease/condition to be treated/prevented in accordance with the present disclosure is a cardiovascular disease/condition.
  • the target antigen of the target antigen-binding moiety may be a cardiovascular disease-associated antigen.
  • the target antigen of the target antigen-binding moiety of the antigen-binding molecule of the present disclosure may be an antigen associated with the relevant cardiovascular disease/condition.
  • the target antigen may be an antigen expressed by pathological effectors of the relevant cardiovascular disease/condition, or an antigen otherwise implicated in the pathology of the cardiovascular disease/condition.
  • antigen-binding molecules according to the present disclosure comprising payload moieties having cytotoxic activity can be employed to kill/increase killing of pathological effectors of cardiovascular remodelling/fibrosis.
  • antigen-binding molecules according to the present disclosure comprising appropriate payload moieties may be employed to stabilise atherosclerotic plaques.
  • the cardiovascular disease/condition is selected from: atherosclerosis, heart failure (e.g. heart failure with preserved ejection fraction (HFpEF) or heart failure with reduced ejection fraction (HFrEF)), varicose veins, cerebral infarcts, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), fibrosis of the atrium, atrial fibrillation, fibrosis of the ventricle, ventricular fibrillation, myocardial fibrosis, interstitial fibrosis, replacement fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrotic vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), arterial stiffness, chronic pulmonary hypertension and AIDS-associated pulmonary hypertension.
  • the cardiovascular disease/condition is atherosclerosis or heart failure.
  • the disease/condition to be treated/prevented in accordance with the present disclosure is a metabolic disease/condition.
  • the target antigen of the target antigen-binding moiety may be a metabolic disease-associated antigen.
  • the target antigen of the target antigen-binding moiety of the antigenbinding molecule of the present disclosure may be an antigen associated with the relevant metabolic disease/condition.
  • the target antigen may be an antigen expressed by pathological effectors of the relevant metabolic disease/condition, or an antigen otherwise implicated in the pathology of the metabolic disease/condition.
  • antigen-binding molecules according to the present disclosure comprising payload moieties having cytotoxic activity can be employed to kill/increase killing of pathological effectors of metabolic disease (e.g. inflammatory immune cells) or to increase degradation of adipose cells/tissue.
  • antigen-binding molecules according to the present disclosure comprising appropriate payload moieties may be employed to modulate insulin resistance and/or activate metabolic pathways.
  • the metabolic disease/condition is selected from: diabetes mellitus, obesity, prediabetes, metabolic syndrome, pregnancy-associated hyperglycemia (/.e. gestational diabetes), hyperglycaemia, amyloidosis, hypertension, and hypercholesterolemia.
  • the metabolic disease/condition is diabetes mellitus or obesity.
  • the disease/condition to be treated/prevented in accordance with the present disclosure is a hematologic disease/condition.
  • the target antigen of the target antigen-binding moiety may be a hematologic disease-associated antigen. It will be appreciated that in aspects and embodiments wherein the disease/condition to be treated/prevented is a hematologic disease/condition, the target antigen of the target antigen-binding moiety of the antigen-binding molecule of the present disclosure may be an antigen associated with the relevant hematologic disease/condition.
  • the target antigen may be an antigen expressed by pathological effectors of the relevant hematologic disease/condition, or an antigen otherwise implicated in the pathology of the hematologic disease/condition.
  • antigen-binding molecules comprising appropriate payload moieties may be employed to deliver clotting factors (/.e. for the treatment of bleeding disorders) or erythropoiesis-stimulating agents (/.e. for the treatment of anemias).
  • the hematologic disease/condition is selected from: a bleeding disorder, (e.g. hemophilia or von Willebrand disease) or an anemia (e.g. a nutritional, hemolytic, or aplastic anemia).
  • the hematologic disease/condition is hemophilia or an anemia.
  • the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by fibrosis (/.e. a fibrotic disease/condition).
  • the target antigen of the target antigen-binding moiety may be a fibrotic disease-associated antigen.
  • the target antigen of the target antigen-binding moiety of the antigen-binding molecule of the present disclosure may be an antigen associated with the relevant fibrotic disease/condition.
  • the target antigen may be an antigen expressed by pathological effectors of the relevant fibrotic disease/condition, or an antigen otherwise implicated in the pathology of the fibrotic disease/condition.
  • antigen-binding molecules according to the present disclosure comprising payload moieties having cytotoxic activity can be employed to kill/increase killing of pathological effectors of fibrosis (e.g. myofibroblasts).
  • antigen-binding molecules according to the present disclosure comprising appropriate payload moieties may be employed to increase degradation of extracellular matrix.
  • a disease/condition characterised by fibrosis is selected from: pulmonary fibrosis (e.g. idiopathic pulmonary fibrosis), asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), liver fibrosis, chronic liver disease, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver cirrhosis, primary biliary cirrhosis (PBC), progressive massive fibrosis, cardiovascular fibrosis, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), fibrosis of the atrium, atrial fibrillation, fibrosis of the ventricle, ventricular fibrillation, myocardial fibrosis, systemic sclerosis, scleroderma, kidney fibrosis, chronic kidney disease (CKD), Alport's syndrome, diabetic nephropathy, chronic glomerulonephritis
  • 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 the target antigen for the antigen-binding molecule; inhibiting the proliferation of cells expressing the target antigen for the antigen-binding molecule; killing cells expressing the target antigen for the antigen-binding molecule; inhibiting proliferation and/or increasing killing of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule; inhibiting tumor growth and/or reducing tumor size/volume, e.g. of a cancer expressing the target antigen for the antigen-binding molecule; and/or increasing the survival of subjects having a cancer, e.g. a cancer expressing the target antigen for the antigen-binding molecule.
  • 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 cells expressing the target antigen for the antigen-binding molecule; killing of cells expressing the target antigen for the antigen-binding molecule; inhibition of proliferation and/or increased killing of cells (e.g. cells that do not express the target antigen) in proximity to a cell expressing the target antigen for the antigen-binding molecule; inhibition of tumor growth and/or reduction of tumor size/volume, e.g. of a cancer expressing the target antigen for the antigen-binding molecule; and/or increased survival of a subject having a cancer, e.g. a cancer expressing the target antigen for the antigen-binding molecule.
  • inhibition of proliferation of cells expressing the target antigen for the antigen-binding molecule e.g. compared to the level/number/proportion etc.
  • 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 affected by the 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 affected by the 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 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 the target antigen for the antigen-binding molecule, 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.
  • 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.
  • the present disclosure provides a linker-payload molecule comprising at least a first payload and at least a second payload for conjugation to an antigen-binding moiety, wherein the linker for conjugation to the antibody comprises a moiety derived from a compound of the second aspect.
  • linker-payload molecule comprises:
  • the linker-payload molecule comprising one of the following groups:
  • the present disclosure also provides a modified antigen-binding molecule comprising a moiety derived from a compound of the second aspect.
  • modified antigen-binding 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 modified antigen-binding molecule comprises one of the following groups:
  • 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.
  • 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 3A 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 3B 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 4A shows tumor volume over time, for mice having a human small cell lung carcinoma cell line- derived xenograft model of small cell lung carcinoma (cell line SHP77), and treated with PBS (vehicle) or an antibody targeting a tumor associated antigen expressed by cells of the human small cell lung carcinoma cell line, conjugated to both exatecan and berzosertib (TAA (Exa+Ber)).
  • PBS vehicle
  • TAA berzosertib
  • Figure 4B shows bodyweight in grams (g) over time, for mice having a human small cell lung carcinoma cell line-derived xenograft model of small cell lung carcinoma (cell line SHP77), and treated with PBS (vehicle) or an antibody targeting a tumor associated antigen expressed by cells of the human small cell lung carcinoma cell line, conjugated to both exatecan and berzosertib (TAA (Exa+Ber)).
  • PBS vehicle
  • TAA berzosertib
  • 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 8A shows binding of anti-TAA antibody (TAA (naked)), anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)), anti-TAA antibody conjugated to exatecan only (TAA (Exa)), isotype-matched control antibody (Isotype (Naked)) or isotype-matched control antibody conjugated to exatecan (Isotype (Exa)) to live SHP-77 cells, as determined by flow cytometry.
  • Figure 8B shows the mean fluorescence intensity (MFI) for anti-TAA antibody (TAA (naked)), anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)), anti-TAA antibody conjugated to exatecan only (TAA (Exa)), isotype-matched control antibody (Isotype (Naked)) or isotype-matched control antibody conjugated to exatecan (Isotype (Exa)) bound to live SHP-77 cells, as determined by flow cytometry.
  • MFI mean fluorescence intensity
  • Figure 8C shows binding of anti-TAA antibody (TAA (naked)), anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)), anti-TAA antibody conjugated to exatecan only (TAA (Exa)), isotype-matched control antibody (Isotype (Naked)) or isotype-matched control antibody conjugated to exatecan (Isotype (Exa)) to live NCI-H82 cells, as determined by flow cytometry.
  • Figure 8D shows the mean fluorescence intensity (MFI) for anti-TAA antibody (TAA (naked)), anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)), anti-TAA antibody conjugated to exatecan only (TAA (Exa)), isotype-matched control antibody (Isotype (Naked)) or isotype-matched control antibody conjugated to exatecan (Isotype (Exa)) bound to live NCI-H82 cells, as determined by flow cytometry.
  • MFI mean fluorescence intensity
  • Figure 9A shows subcellular localization of anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)) or anti-TAA antibody conjugated to exatecan only (TAA (Exa)), within NCI- H82 cells after incubation for Oh, 0.5h or 2h, as determined by immunofluorescence microscopy.
  • Figure 9B shows subcellular localization of anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)) or anti-TAA antibody conjugated to exatecan only (TAA (Exa)), within TAA overexpressing HEK293 cells after incubation for Oh, 0.5h or 2h, as determined by immunofluorescence microscopy.
  • Figure 10 shows % change in bodyweight over time following administration to mice of anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)) or anti-TAA antibody conjugated to exatecan only (TAA (Exa)), at 3 mg/kg bodyweight, 10 mg/kg bodyweight, 30 mg/kg bodyweight or 60 mg/kg bodyweight, or following administration of vehicle only (Vehicle).
  • Figure 11A shows the level of the indicated red blood cell indices and anaemic parameters in the blood of mice following administration of two doses of anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)) or anti-TAA antibody conjugated to exatecan only (TAA (Exa)), at 3 mg/kg bodyweight, 10 mg/kg bodyweight, 30 mg/kg bodyweight or 60 mg/kg bodyweight, or vehicle only (Vechicle).
  • Figure 11B shows the level of the indicated white blood cell indices and platelets in the blood of mice following administration of two doses of anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)) or anti-TAA antibody conjugated to exatecan only (TAA (Exa)), at 3 mg/kg bodyweight, 10 mg/kg bodyweight, 30 mg/kg bodyweight or 60 mg/kg bodyweight, or vehicle only (Vechicle).
  • Figure 12A shows the level of the indicated liver, kidney and pancreatic indices in the blood of mice following administration of two doses of anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)) or anti-TAA antibody conjugated to exatecan only (TAA (Exa)), at 3 mg/kg bodyweight, 10 mg/kg bodyweight, 30 mg/kg bodyweight or 60 mg/kg bodyweight, or vehicle only (Vechicle).
  • Figure 12B shows the level of the indicated electrolytes in the blood of mice following administration of two doses of anti-TAA antibody conjugated to both exatecan and berzosertib (TAA (Exa+Ber)) or anti- TAA antibody conjugated to exatecan only (TAA (Exa)), at 3 mg/kg bodyweight, 10 mg/kg bodyweight, 30 mg/kg bodyweight or 60 mg/kg bodyweight, or vehicle only (Vechicle).
  • Figure 13A 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 13B 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 14A 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 14B 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 15A 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 15B 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 16A 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 16B 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 17 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 18 shows antibody concentration in immunocompetent BALB/c (top panel) and immunocompromised NSG (bottom panel) mice following administration of 10 mg/kg pertuzumab conjugated to both exatecan and berzosertib (‘Conjugated Ab (dual payload ADC)’) or 10 mg/kg pertuzumab (‘Unconjugated (naked) Ab’).
  • Figure 19 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).
  • Figure 20 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 21 shows % increase in hydrophobic interaction chromatography retention time (RT) for a single payload ADC and T-(Exa+Ber).
  • Figure 22 shows the stability of ADCs 5 and 6 in plasma over 7 days.
  • Figure 23 shows the efficacy of dual payload ADCs on NCI-N87 gastric cell line cells.
  • Figure 24 shows the efficacy of dual and single payload ADCs in killing NCI-N87 gastric cell line cells.
  • Figure 25 shows the efficacy of dual and single payload ADCs in killing NCI-N87 gastric cell line cells.
  • Figure 26 shows the in vivo anti-tumor efficacy of dual and single payload ADCs in a xenograft mouse model.
  • Figure 27 shows the in vivo efficacy of dual payload ADCs with berzosertib (ATRi) or prexasertib (CHK1i) in a mouse model.
  • Figure 28 shows the in vivo efficacy of dual payload ADCs with different target DAR ratios.
  • Figure 29A shows the weight change results in a primate study following treatment with a dual payload ADC.
  • Figure 29B shows the biochemistry and haemotology results in a primate study following treatment with a dual payload ADC.
  • Figure 30 shows the efficacy of dual payload ADCs with TMTHSI or DBCO moieties on NCI-N87 gastric cell lines.
  • Figure 31 shows the clearance of dual payload ADCs with TMTHSI or DBCO moieties compared to the unconjugated control antibody in immunocompetent mice.
  • Figure 32 shows efficacy of dual payload ADCs with different conjugations.
  • Example 1 In vitro combination of DDR inhibitors and TOP1 inhibitors
  • 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, 50 pl 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.
  • 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 HCT-116 (CCL-247)
  • 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-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).
  • 1 H NMR (400 MHz, DMSO-cfc) 5 ppm 10.17 - 10.33 (m, 1 H), 8.90 - 8.95 (m, 1 H), 8.69 (d, J 2.75 Hz, 1 H), 8.41 - 8.47 (m, 2 H), 8.22 - 8.27 (m, 1 H), 8.14 - 8.19 (m, 1 H), 7.95 - 8.00 (m, 1 H), 7.71 - 7.76 (m, 2 H), 7.39 - 7.50 (m, 4 H), 6.97 - 7.04 (m, 1 H), 5.47 - 5.57 (m, 1 H), 5.29 - 5.38 (m, 1 H), 5.11 - 5.21 (m, 1 H), 4.50 - 4.54 (m, 2 H), 4.42 - 4.46 (m, 2 H), 4.35 - 4.40 (m, 1
  • 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 (Boc)2O 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 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.
  • 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 3 A solution of compound AA_19 (10 g, 6.7 mmol, 1 .0 eq.) in 2N HCI in 1 ,4-dioxane (90 mL) was stirred at 0 °C for 30 min. Then the reaction mixture was concentrated under reduced pressure and purified by preparative HPLC (0.01 % TFA) to afford linker 3 (3.9 g, 42%) as a purple solid after lyophilization.
  • 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.

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Abstract

L'invention concerne une molécule de liaison à l'antigène qui se lie à un antigène cible, comprenant (i) un fragment de liaison à l'antigène cible, et (ii) au moins un fragment lieur-charge utile, la molécule de liaison à l'antigène comprenant (a) un fragment inhibiteur de réponse à l'endommagement de l'ADN (DDR), et (b) un fragment inhibiteur d'ADN topoisomérase 1 (TOP1).
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IL299368A (en) * 2020-06-24 2023-02-01 Astrazeneca Uk Ltd A combination of an antibody-drug conjugate and an ATR inhibitor
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