Trop2-binding molecules

Abstract

Provided here are antigen-binding molecules that bind to TROP2, comprising (i) a TROP2-binding moiety, and (ii) a linker-payload moiety, wherein the linker-payload moiety is (I), where RP1 and RP2 are (II).

Description

[0001] TROP2-Bindinq Molecules

[0002] Cross-Reference to Related Applications

[0003] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 694,509, filed September 13, 2024, and U.S. Provisional Patent Application No. 63 / 743,743, filed January 10, 2025, the disclosure of which are hereby incorporated herein by reference in their entirety.

[0004] Reference to an Electronic Sequence Listing

[0005] The content of the electronic sequence listing (366592000840seqlist.xml; Size: 65,228 bytes; and Date of Creation: August 29, 2025) is herein incorporated by reference in its entirety.

[0006] Technical Field

[0007] The present disclosure relates to molecular biology, more specifically antibody technology. The present disclosure also relates to methods of medical treatment and prophylaxis.

[0008] Background

[0009] Cancers remain the leading cause of deaths worldwide. Chemotherapies have good clinical benefits, but due to their low specificity they have very significant side effects and low therapeutic indices. More targeted therapies, such as monoclonal antibody therapies, show good specificity but response rates are smaller. Antibody-drug conjugates (ADCs) are a therapeutic modality that harness an antibody’s target specificity to selectively deliver cytotoxic payloads to tumors and are proving increasingly effective in the clinic.

[0010] TROP2 is a transmembrane glycoprotein encoded by the TACSTD2 gene. TROP2 is an intracellular calcium signal transducer. Under physiological conditions, TROP2 plays an essential role in embryonic development, placental tissue formation, embryo implantation, stem cell proliferation, organ development, and tight junction maintenance. TROP2 can also activate multiple pro-oncogenic signals including the MAPK / ERK pathway, the cyclin D / E pathway, and p-catenin signalling.

[0011] Although expression is observed in various normal tissues, overexpression of TROP2 is observed across many tumour types. TROP2 expression also has prognostic significance. A meta-analysis of 16 observational studies consisting of 2,569 solid tumor patients showed high TROP2 expression was significantly correlated with poor overall survival and poor disease-free survival (Zeng et al., Scientific Reports (2016) 6:33658).

[0012] Anti-TROP2 antibody drug conjugates (ADCs) are described in Parisi et al., Cancer Treatment Reviews (2023) 118:102572, and include sacituzumab govitecan (DrugBank Acc. No. DB12893; marketed as Trodelvy™) and datopotamab deruxtecan (DrugBank Acc. No. DB16410). Sacituzumab govitecan comprises the cytotoxic agent SN-38 (a topoisomerase I inhibitor) conjugated to a humanised antibody (RS7) via a hydrolysable linker. Datopotamab deruxtecan comprises the DNA topoisomerase I inhibitor DS-8201a (DXd) conjugated to a humanised anti-TROP2 antibody via a tetrapeptide-based cleavable linker. Despite showing promising overall response rates in some cancer indications, the majority of responders progress within one year.

[0013] There remains an unmet clinical need for the effective treatment of TROP2-expressing cancers.

[0014] Summary

[0015] In a first aspect, the present disclosure provides an antigen-binding molecule that binds to TROP2, comprising (i) a TROP2-binding moiety, and (ii) a linker-payload moiety, wherein the linker-payload moiety is:

[0016] In RPi and RP2 the group shown as: In some embodiments, the linker-payload moiety is conjugated to a glutamine residue of the TROP2- binding moiety. In some embodiments, the ratio between the linker-payload moiety and the TROP2- binding moiety is from about 1 :1 to about 2:1 . In some embodiments, the ratio between the linker-payload moiety and the TROP2-binding moiety is about 2:1 . In some embodiments, the ratio between the linkerpayload moiety and the TROP2-binding moiety is 1 :1. In some embodiments, the ratio between the linkerpayload moiety and the TROP2-binding moiety is 2:1.

[0017] In some embodiments, the TROP2-binding moiety is an antibody or an antigen-binding fragment thereof. In some embodiments, the TROP2-binding moiety comprises:

[0018] (i) a heavy chain variable (VH) region incorporating the following CDRs:

[0019] HC-CDR1 having the amino acid sequence of SEQ ID NO: 13

[0020] HC-CDR2 having the amino acid sequence of SEQ ID NO: 14

[0021] HC-CDR3 having the amino acid sequence of SEQ ID NO: 15; and

[0022] (ii) a light chain variable (VL) region incorporating the following CDRs:

[0023] LC-CDR1 having the amino acid sequence of SEQ ID NO:21 LC-CDR2 having the amino acid sequence of SEQ ID NO:22 LC-CDR3 having the amino acid sequence of SEQ ID NO:23.

[0024] In some embodiments, the antigen-binding moiety that binds to TROP2 comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:12; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0025] In some embodiments, the antigen-binding moiety that binds to TROP2 comprises: a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:10; and a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:11 .

[0026] In some embodiments, the duration of an anti-cancer response observed in subjects having a TROP2- expressing cancer following treatment with the antigen-binding molecule is greater than the duration of the anti-cancer response observed in comparable subjects following treatment with an equivalent dose of an anti-TROP2 TOP1 i ADC.

[0027] The present disclosure also provides a composition comprising an antigen-binding molecule according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0028] The present disclosure also provides an antigen-binding molecule or a composition according to the present disclosure, for use in a method of medical treatment or prophylaxis, or in a method of diagnosis or prognosis. The present disclosure also provides an antigen-binding molecule or a composition according to the present disclosure, for use in treating or preventing a cancer.

[0029] The present disclosure also provides use of an antigen-binding molecule or a composition according to the present disclosure, in the manufacture of a medicament for treating or preventing a cancer.

[0030] 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 or a composition according to the present disclosure.

[0031] In some embodiments, the cancer is selected from: a cancer comprising cells expressing / overexpressing TROP2, a solid tumour, metastatic cancer, unresectable cancer, hormone receptor-positive cancer, lung cancer, non-small cell lung cancer (NSCLC), non-small cell lung cancer adenocarcinoma (adNSCLC), squamous non-small cell lung cancer (sqNSCLC), breast cancer, hormone receptor-positive breast cancer, metastatic breast cancer, hormone receptor-positive metastatic breast cancer, HER2-positive breast cancer, HER2-positive metastatic breast cancer, HER2-negative breast cancer, metastatic HER2- negative breast cancer, unresectable HER2-negative breast cancer, hormone receptor-positive and HER2-negative breast cancer, metastatic hormone receptor-positive and HER2-negative breast cancer, unresectable hormone receptor-positive and HER2-negative breast cancer, triple-negative breast cancer (TNBC), metastatic triple negative breast cancer, unresectable triple-negative breast cancer, pancreatic cancer, prostate cancer, castration-resistant prostate cancer (CRPC), metastatic castration-resistant prostate cancer (mCRPC)), bladder cancer, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), endometrial cancer, ovarian cancer, esophageal cancer, esophageal squamous-cell carcinoma (ESCC), cervical cancer, thyroid cancer, biliary tract cancer (BTC), urinary tract cancer, urothelial cancer, metastatic urothelial cancer, and unresectable urothelial cancer.

[0032] In some embodiments, the cancer is refractory or relapsed to treatment with a DNA damage repair inhibitor, and / or wherein the cancer is refractory or relapsed to treatment with a DNA topoisomerase I inhibitor.

[0033] The present disclosure also provides use of an antigen-binding molecule or a composition according to the present disclosure, to deplete or to increase killing of cells expressing TROP2.

[0034] The present disclosure also provides an in vitro complex, optionally isolated, comprising an antigenbinding molecule according to the present disclosure bound to TROP2.

[0035] Description

[0036] The present disclosure relates to antigen-binding molecules comprising a TROP2-binding moiety and a linker-payload moiety comprising payloads derived from exatecan (a TOP1 inhibitor) and berzosertib (a DDR inhibitor, more specifically an ATR inhibitor). The antigen-binding molecules of the present disclosure are provided with unexpected and advantageous properties relative to known anti-TROP2 antibody-drug conjugates.

[0037] DNA damage response (DDR) is a key pathway for repair, and might be an important mode of resistance to DNA damaging payloads such as TOP1 inhibitors. Synergistic anticancer effects have previously been observed through combined treatment using a DDR inhibitor and a TOP1 inhibitor (Thomas et al., Cancer Cell. (2021) 39(4):566-79 e7). In embodiments wherein the antigen-binding molecule of the present disclosure comprises a linker-payload moiety comprising (i) a TOP1 inhibitor moiety and (ii) a DNA damage response (DDR) inhibitor moiety, the DDR inhibitor is thought to increase the sensitivity of the cells of the cancer to the TOP1 inhibitor. Moreover, many TOP1 inhibitors and DDR inhibitors are not substrates for P-glycoprotein, and so patients treated with antigen-binding molecules comprising such payload moieties are less likely to develop P-glycoprotein-mediated resistance to such therapy.

[0038] It is understood that topoisomerase I inhibitor, DNA topoisomerase I inhibitor, TOP1 inhibitor, and TOP1 i as used herein refer to the same class of inhibitors.

[0039] TROP2

[0040] TROP2 (trophoblast cell surface antigen 2) is the protein identified by UniProtKB: P09758. TROP2 is also known e.g. as tumor-associated calcium signal transducer 2 (TACSTD2), gastric antigen 733-1 (GA733- 1), epithelial glycoprotein-1 (EGP-1), and membrane component chromosome 1 surface marker 1 (M1 S1). Human TROP2 has the amino acid sequence of P09758 (v3, 2007-11-13; SEQ ID NO:1). Human TROP2 is encoded by the human TACSTD2 gene.

[0041] Human TROP2 comprises a 26 amino acid N-terminal signal peptide (SEQ ID NO:3), followed by an extracellular domain (SEQ ID NO:4), a single-pass transmembrane domain (SEQ ID NO:8), and a cytoplasmic domain (SEQ ID NO:9) at the C-terminus. The mature form of human TROP2 is shown in SEQ ID NO:2.

[0042] The extracellular domain comprises a cysteine-rich domain (CRD) (SEQ ID NO:5), a thyroglobulin type-1 domain (TY-1) (SEQ ID NO:6), and a cysteine-poor domain (CPD) (SEQ ID NO:7). TROP2 has 4 N- linked glycosylation sites in the extracellular domain at the residues 33, 120, 168, and 208.

[0043] TROP2 monomers may form cis- or trans-dimers. At the interface of a TROP2 cis-dimer, a loop from the TY-1 domain from one monomer interacts with the CPD pC sheet of the other monomer via hydrogen bonds. The interface of a trans-dimer is formed by interaction between the CPD pc sheets of two TROP2 monomers. TROP2 cis- or trans-dimers can form tetramers. Tetrameric assembly of two cis- or trans- dimers occurs via the CRD domains. The intracellular domain of TROP2 comprises a phosphatidylinositol 4,5-bisphosphate (PIP2)-binding motif, a conserved tyrosine site, and a serine phosphorylation site at S303. Part of TROP2 signaling is dependent on protein kinase C (PKC) and involves phosophyorylation of S303.

[0044] The structure and function of TROP2 is described e.g. in Shvartsur & Bonavida, Genes Cancer. (2015) 6(3-4): 84-105, which is hereby incorporated by reference in its entirety.

[0045] TROP2 has been implicated in numerous intracellular signaling pathways. TROP2 transduces an intracellular calcium signal. TROP2-induced signal transduction can occur without extracellular Ca2+, suggesting a mobilization of Ca2+ from internal stores. TROP2 provides crucial signals for cells with requirements for proliferation, survival, self-renewal, and invasion. TROP2 has several ligands, including claudin-1 , claudin-7, cyclin D1 , IGF-1 and PKC. TROP2 has stem cell-like qualities and regulates cell growth, transformation, regeneration, and proliferation, which explains why its overexpression can lead to tumor progression. It is expressed on the surface of many stem / progenitor cells and has a role in maintaining tight junction integrity. TROP2 is also purported to be a modulator and / or an enhancer of EpCAM-induced cell signaling. TROP2 modulation of EpCAM can cause EpCAM to proliferate and migrate into liver parenchyma.

[0046] Reference herein to ‘TROP2’ generally refers to the canonical isoform of the human TROP2 (i.e. isoform 1), but also contemplates isoforms, fragments, variants (including mutants) and homologues thereof (i.e. from other species, e.g. non-human mammalian species (e.g. a non-human primate, e.g. rhesus, cynomolgous; e.g. a rodent, e.g. rat or mouse).

[0047] As used herein, a ‘fragment’, ‘variant’ or ‘homologue’ of a protein may optionally be characterised as having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98% or >99% amino acid sequence identity to the amino acid sequence of the reference protein (e g. the canonical isoform of the human protein). In some embodiments fragments / variants / isoforms / homologues may be characterised by ability to perform a function performed by the reference protein.

[0048] A ‘fragment’ generally refers to a fraction of the reference protein. A ‘variant’ generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein. An ‘isoform’ generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein. A ‘homologue’ generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein. For example, human TROP2 (P09758 v3; SEQ ID NO:1) and mouse TROP2 (UniProt: P, v1 , 2003-03-01) are homologues of one another. Homologues include orthologues. A ‘fragment’ may be of any length (by number of amino acids), although may optionally be at least 20% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein. A fragment of TROP2 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, or 300 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, or 300 amino acids.

[0049] Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property / activity of the reference TROP2 (e.g. human TROP2), as determined by analysis by a suitable assay for the functional property / activity. For example, an isoform, fragment, variant or homologue of TROP2 may display association with IGF-1 , Claudin-1 or Claudin-7.

[0050] In some embodiments, the TROP2 is TROP2 from a mammal (e.g. a primate (rhesus, cynomolgous, nonhuman primate or human) and / or a rodent (e g. rat or murine) TROP2). Isoforms, fragments, variants or homologues of TROP2 may optionally be characterized as having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature TROP2 isoform from a given species, e.g. human.

[0051] In some embodiments, the TROP2 comprises, or consists of, an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to one of SEQ ID NO:1 or 2.

[0052] In some embodiments, a fragment of TROP2 comprises, or consists of, an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:2 or 4.

[0053] In some embodiments, a fragment of TROP2 comprises, or consists of, an amino acid sequence having at least 60% amino acid sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to one of SEQ ID NOs:5 to 7. In some embodiments, a fragment of TROP2 comprises, or consists of, an amino acid sequence having at least 60% amino acid sequence identity, e g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:67.

[0054] TROP2-binding antigen-binding moieties

[0055] An ‘antigen-binding molecule’ refers to a molecule that binds to a given target antigen. Antigen-binding molecules comprise an antigen-binding moiety through which the antigen-binding molecule binds to its target antigen. The antigen-binding molecules of the present disclosure comprise an antigen-binding moiety that binds to TROP2 ( / .e. a TROP2-binding moiety).

[0056] Antigen-binding moieties may comprise, or may be derived from, antibodies ( / .e. immunoglobulins (Igs)) and antigen-binding fragments of antibodies. As used herein, ‘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.

[0057] 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). In some embodiments, 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 (i.e. a single-domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody or 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).

[0058] Commonly employed techniques for the production of fully human antibodies include (i) phage display, in which human antibody genes are expressed in phage display libraries, and (ii) production of antibodies in transgenic mice engineered to have human antibody genes (described in Park and Smolen, Advances in Protein Chemistry (2001) 56: 369-421). Briefly, in the human antibody gene-phage display technique, genes encoding the VH and VL chains are generated by PCR amplification and cloning from ‘naive’ human lymphocytes, and assembled into a library from which they can be expressed either as disulfide- linked Fab fragments or as single-chain Fv (scFv) fragments. The Fab- or scFv-encoding genes are fused to a surface coat protein of filamentous bacteriophage and Fab or scFv capable of binding to the target of interest can then be identified by screening the library with antigen. Molecular evolution or affinity maturation procedures can be employed to enhance the affinity of the Fab / scFv fragment. In the transgenic mouse technique, 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.

[0059] In some embodiments, an antigen-binding moiety according to the present disclosure 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). In some embodiments, an antigen-binding moiety may be or comprise the Fv (e.g. provided as an scFv) or the Fab region of an antibody that binds to a given target antigen, or the whole antibody. The antigen-binding moieties of the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to a given target antigen (e.g. TROP2). 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 for which the given antibody is specific.

[0060] In some embodiments, 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. In some embodiments, the antigen-binding moiety is or comprises the Fv (e.g. provided as an scFv) of an antibody. In some embodiments, the antigen-binding moiety is or comprises the Fab region of an antibody. In some embodiments, the antigen-binding moiety is or comprises the whole antibody ( .e. comprising variable and constant regions).

[0061] 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. In some embodiments, 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).

[0062] In some embodiments, an antigen-binding moiety according to the present disclosure 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.

[0063] 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.

[0064] The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, 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.

[0065] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671 -D674. 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. In preferred embodiments, the CDRs and FRs of antigenbinding molecules referred to herein are defined according to the IMGT information system.

[0066] In some embodiments, an antigen-binding moiety according to the present disclosure comprises, or consists of, an Fv region that binds to TROP2. In some embodiments, 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).

[0067] 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. In some embodiments, the antigen-binding moiety comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g. CK or CA). In some embodiments, 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). In some embodiments, 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. In some embodiments, 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).

[0068] In some embodiments, an antigen-binding moiety described herein comprises, or consists of, a whole antibody which binds to TROP2. As used herein, ‘whole antibody’ refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.

[0069] Immunoglobulins of type G (i.e. IgG) 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. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , Ig A2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (A).

[0070] In some embodiments, the antigen-binding moiety comprises, or consists of, an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e g. lgA1 , lgA2), IgD, IgE, or IgM which binds to TROP2.

[0071] In some embodiments, 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. In some embodiments, 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 , Ig A2) , IgD, IgE or IgM, e.g. a human IgG (e.g. hlgG1 , hlgG2, hlgG3, hlgG4), hlgA (e.g. hlgA1 , hlgA2), hlgD, hlgE or hlgM. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of a human Ig G 1 allotype (e.g. G1 ml , G1 m2, G1 m3 or G1 ml 7).

[0072] In some embodiments, 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:47 or 52. In some embodiments, 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:48. In some embodiments, 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:49 or 64. In some embodiments, 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:50 or 53. In some embodiments, 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:54, 55, 65 or 66. In some embodiments, 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:46, 51 , 62 or 63.

[0073] In some embodiments, an antigen-binding moiety of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; CK). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CA), e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.

[0074] In some embodiments, 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:56, 57, 58, 59, 60, or 61. In some embodiments described herein, one or more amino acids of an amino acid sequence referred to herein (e.g. an amino acid sequence of an antigen-binding moiety, e.g. an amino acid sequence of a CDR or VH / VL region) 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 (j.e. encoded by the genetic code) which is non-identical to the amino acid residue at the relevant position of the equivalent, unsubstituted amino acid sequence, selected from: 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). In some embodiments, 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. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogues such as those described in Ellman, et al., Meth. Enzym. 202 (1991) 301 -336.

[0075] In some embodiments, a substitution may be biochemically conservative. In some embodiments, where an amino acid to be substituted is provided in one of rows 1 to 5 of the table below, the replacement amino acid of the substitution is another, non-identical amino acid provided in the same row:

[0076] By way of illustration, in some embodiments wherein substitution is of a Met residue, the replacement amino acid may be selected from Ala, Vai, Leu, He, Trp, Tyr, Phe and Norleucine.

[0077] In some embodiments, a replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, 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:

[0078] That is, in some embodiments, a nonpolar amino acid is substituted with another, non-identical nonpolar amino acid. In some embodiments, a polar amino acid is substituted with another, non-identical polar amino acid. In some embodiments, an acidic polar amino acid is substituted with another, non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is substituted with another, non- identical basic polar amino acid. In some embodiments, a neutral amino acid is substituted with another, non-identical neutral amino acid. In some embodiments, a positive amino acid is substituted with another, non-identical positive amino acid. In some embodiments, a negative amino acid is substituted with another, non-identical negative amino acid.

[0079] In some embodiments, 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.

[0080] The antigen-binding molecules of the present disclosure comprise an antigen-binding moiety that binds to TROP2.

[0081] In some embodiments, the antigen-binding moiety comprises the CDRs of an antigen-binding moiety which is capable of binding to TROP2. In some embodiments, the antigen-binding moiety comprises the FRs of an antigen-binding moiety which is capable of binding to TROP2. In some embodiments, the antigen-binding moiety comprises the CDRs and the FRs of an antibody that is capable of binding to TROP2. That is, in some embodiments the antigen-binding moiety comprises the VH region and the VL region of an antibody that is capable of binding to TROP2.

[0082] In some embodiments, an antigen-binding molecule which is capable of binding to TROP2 according to the present disclosure may be, or may be derived from, a TROP2-binding antibody selected from: hRS7 (sacituzumab) (described in e.g.WO03074566); MAAP-9001 a (datopotamab) (described in e.g. Okajima et a / ., Mol Cancer Ther. (2021) 20(12):2329-2340; PrE11 (described in e.g. Ikeda et a / ., Biochemical and Biophysical Research Communications (2015) 458(4):877-882); AR47A6.4.2 (described in e.g. Truong et al., Mol Cancer Ther. (2007) 6 (11_Supplement): PR-12); 7E6 (described in e.g. Liu et al., WO2013068946); 2EF (described in e.g. Guerra et a / ., Cancers (2023) 15(14):3721); and Hu2G10 (described in e.g. Albert et al., J. Clin. Oncol. (2022) 40:e14548).

[0083] In some embodiments the antigen-binding moiety is capable of binding the same region of TROP2, or an overlapping region of TROP2, to the region of TROP2 which is bound by an antigen-binding molecule comprising the VH and VL sequences of a TROP2-binding antibody described hereinabove. In some embodiments the antigen-binding moiety is capable of binding the same region of TROP2, or an overlapping region of TROP2, to the region of TROP2 which is bound by an antigen-binding molecule comprising the VH and VL sequences of sacituzumab (i.e. an antigen-binding molecule comprising a VH having the amino acid sequence of SEQ ID NO: 12, and a VL having the amino acid sequence of SEQ ID NO:20). In some embodiments the antigen-binding moiety is capable of binding the same region of TROP2, or an overlapping region of TROP2, to the region of TROP2 which is bound by an antigenbinding molecule comprising the VH and VL sequences of datopotamab (i.e. an antigen-binding molecule comprising a VH having the amino acid sequence of SEQ ID NO:30, and a VL having the amino acid sequence of SEQ ID NO:38).

[0084] In some embodiments, the antigen-binding moiety is capable of binding to the region of TROP2 shown in SEQ ID NO:67. In some embodiments, the antigen-binding moiety contacts the region of TROP2 shown in SEQ ID NO:67. In some embodiments, the antigen-binding moiety binds to TROP2 via contact with one or more amino acids of the region shown in SEQ ID NO:67. In some embodiments, the epitope of the antigen-binding moiety comprises or consists of the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the antigen-binding moiety binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:67.

[0085] In some embodiments, the antigen-binding moiety is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of one of SEQ ID NOs:1 , 2 or 4.

[0086] The ability of an antigen-binding moiety to bind to a given peptide / polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g. western blot), immunoprecipitation, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442) or Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507). In embodiments where the antigen binding moiety is capable of binding to a peptide / polypeptide comprising a reference amino acid sequence, the peptide / polypeptide may comprise one or more additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments the peptide / polypeptide comprises e g. 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, I Q- 20, 10-30, 10-40, 10-50, 20-30, 20-40 or 20-50 additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments the additional amino acid(s) provided at one or both ends ( / .e. the N-terminal and C-terminal ends) of the reference sequence correspond to the positions at the ends of the reference sequence in the context of the amino acid sequence of TROP2.

[0087] In some embodiments the antigen-binding moiety is capable of binding to a peptide / polypeptide which is bound by an antibody comprising the VH and VL sequences of sacituzumab ( / .e. an antigen-binding molecule comprising a VH having the amino acid sequence of SEQ ID NO: 12, and a VL having the amino acid sequence of SEQ ID NO:20). In some embodiments the antigen-binding moiety is capable of binding to a peptide / polypeptide which is bound by an antibody comprising the VH and VL sequences of datopotamab (i.e. an antigen-binding molecule comprising a VH having the amino acid sequence of SEQ ID NO:30, and a VL having the amino acid sequence of SEQ ID NO:38).

[0088] In some embodiments the antigen-binding moiety is capable of binding to a peptide / polypeptide which is bound by an antibody comprising the VH and VL sequences of a TROP2-binding antibody described hereinabove.

[0089] In some embodiments, the antigen-binding moiety comprises the heavy chain CDRs and the light chain CDRs of a TROP2-binding antibody described hereinabove. In some embodiments, the antigen-binding moiety comprises the VH and VL of a TROP2-binding antibody described hereinabove. In some embodiments, the antigen-binding moiety comprises the heavy chain polypeptide (i.e. comprising VH, CH1 , CH2 and CH3 region sequences) and light chain polypeptide (i.e. comprising VL and CL region sequences) of a TROP2-binding antibody described hereinabove.

[0090] In some embodiments, the antigen-binding moiety comprises the heavy chain CDRs and the light chain CDRs of sacituzumab. In some embodiments, the antigen-binding moiety comprises the VH and VL of sacituzumab. In some embodiments, the antigen-binding moiety comprises the heavy chain polypeptide (i.e. comprising VH, CH1 , CH2 and CH3 region sequences) and light chain polypeptide (i.e. comprising VL and CL region sequences) of sacituzumab.

[0091] In some embodiments, the antigen-binding moiety comprises: a VH region incorporating the following CDRs:

[0092] HC-CDR1 having the amino acid sequence of SEQ ID NO: 13

[0093] HC-CDR2 having the amino acid sequence of SEQ ID NO: 14

[0094] HC-CDR3 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 , and / or in which 1 or 2 or 3 amino acids in HC-CDR2, and / or in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and a VL region incorporating the following CDRs:

[0095] LC-CDR1 having the amino acid sequence of SEQ ID NO:21

[0096] LC-CDR2 having the amino acid sequence of SEQ ID NO:22

[0097] LC-CDR3 having the amino acid sequence of SEQ ID NO:23; or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 , and / or in which 1 or 2 or 3 amino acids in LC-CDR2, and / or in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0098] In some embodiments, the antigen-binding moiety comprises: a VH region comprising an amino acid sequence having at least 60% sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:12; and a VL region comprising an amino acid sequence having at least 60% sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the amino acid sequence of SEQ ID NQ:20.

[0099] In some embodiments, an antigen-binding moiety comprises, or consists of:

[0100] (i) one or more (e.g. two) polypeptides comprising, or consisting of, an amino acid sequence having at least 70% sequence identity, more preferably one of >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:10; and

[0101] (ii) one or more (e.g. two) polypeptides comprising, or consisting of, an amino acid sequence having at least 70% sequence identity, more preferably one of >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:11 .

[0102] In some embodiments, the antigen-binding moiety comprises the heavy chain CDRs and the light chain CDRs of datopotamab. In some embodiments, the antigen-binding moiety comprises the VH and VL of datopotamab. In some embodiments, the antigen-binding moiety comprises the heavy chain polypeptide (i.e. comprising VH, CH1 , CH2 and CH3 region sequences) and light chain polypeptide (i.e. comprising VL and CL region sequences) of datopotamab.

[0103] In some embodiments, the antigen-binding moiety comprises: a VH region incorporating the following CDRs:

[0104] HC-CDR1 having the amino acid sequence of SEQ ID NO:31 HC-CDR2 having the amino acid sequence of SEQ ID NO:32 HC-CDR3 having the amino acid sequence of SEQ ID NO:33, or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 , and / or in which 1 or 2 or 3 amino acids in HC-CDR2, and / or in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid; and a VL region incorporating the following CDRs:

[0105] LC-CDR1 having the amino acid sequence of SEQ ID NO:39 LC-CDR2 having the amino acid sequence of SEQ ID NO:40 LC-CDR3 having the amino acid sequence of SEQ ID NO:41 ; or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 , and / or in which 1 or 2 or 3 amino acids in LC-CDR2, and / or in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0106] In some embodiments, the antigen-binding moiety comprises: a VH region comprising an amino acid sequence having at least 60% sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:30; and a VL region comprising an amino acid sequence having at least 60% sequence identity, e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:38.

[0107] In some embodiments, an antigen-binding moiety comprises, or consists of:

[0108] (i) one or more (e.g. two) polypeptides comprising, or consisting of, an amino acid sequence having at least 70% sequence identity, more preferably one of >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:28; and

[0109] (ii) one or more (e.g. two) polypeptides comprising, or consisting of, an amino acid sequence having at least 70% sequence identity, more preferably one of >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:29.

[0110] In some embodiments, an antigen-binding molecule of the present disclosure (e.g. an antigen-binding moiety thereof) comprises an Fc region. As used herein, 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.

[0111] Herein, a ‘CH2 domain’ refers to an amino acid sequence corresponding to the CH2 domain of an immunoglobulin (Ig). The CH2 domain is the region of an Ig formed by positions 231 to 340 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85. A ‘CH3 domain’ refers to an amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). The CH3 domain is the region of an Ig formed by positions 341 to 447 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85. A ‘CH2-CH3 region’ refers to an amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). The CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85.

[0112] In some embodiments, a CH2 domain, CH3 domain and / or a CH2-CH3 region according to the present disclosure 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 , IgE or IgM. In some embodiments, 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. h IgG 1 , hlgG2, hlgG3, hlgG4), hlgA (e g. h Ig A1 , hlgA2), hlgD, h Ig E or hlgM. In some embodiments, 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). In some embodiments, 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.

[0113] 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. 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.

[0114] In some embodiments, an antigen-binding molecule according to the present disclosure 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:54 or 55. In some embodiments, the antigen-binding molecule comprises an Fc region comprising 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:54 or 55.

[0115] Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et al., Protein Cell (2018) 9(1):63-73, which is hereby incorporated by reference in its entirety. Exemplary Fc region modifications known to influence antibody effector function are summarised in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, 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. Where 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.

[0116] In some embodiments, 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.

[0117] In some embodiments, 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.

[0118] In some embodiments, the Fc region comprises modification at the amino acid residue corresponding to N297. In some embodiments, 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. In some embodiments, the Fc region comprises modification corresponding to N297A.

[0119] In some embodiments, an antigen-binding molecule according to the present disclosure 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:65 or 66. In some embodiments, the antigen-binding molecule comprises an Fc region comprising 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:65 or 66.

[0120] An antigen-binding molecule according to the present disclosure may comprise a TROP2-binding moiety according to any embodiment described hereinabove, and a linker-payload moiety as described hereinbelow. Linker-payload moieties

[0121] Aspects and embodiments of the present disclosure relate to antigen-binding molecules comprising a linker-payload moiety. As used herein, 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.

[0122] In particular, the present disclosure relates to antigen-binding molecules comprising at least one linkerpayload moiety which is:

[0123] This comprises payloads derived from exatecan (a TOP1 inhibitor) and berzosertib (a DDR inhibitor, more specifically an ATR inhibitor).

[0124] In RPi and RP2 the group shown as:

[0125] In some embodiments, the linker-payload moiety is conjugated to the antigen-binding moiety (e g. a TROP2-binding moiety) via a glutamine residue of the antigen-binding moiety. In some embodiments, the conjugation is between the amine group of the linker moiety and the glutamine at Q295 (EU numbering) of the antigen-binding moiety. In some embodiments, the ratio between the linker-payload moiety to the antigen-binding moiety is from about 1 :1 to about 2:1 . In some embodiments, the ratio between the linker-payload moiety to the antigen-binding moiety is about 2:1 . In some embodiments, the ratio between the linker-payload moiety and the TROP2-binding moiety is 1 :1 . In some embodiments, the ratio between the linker-payload moiety and the TROP2-binding moiety is 2:1 .

[0126] In some embodiments, the ratio between payloads derived from exatecan and payloads derived from berzosertib is about 1 :1. In some embodiments, the payloads are conjugated to the antigen-binding moiety via one or more click groups of the linker-payload moiety. In some embodiments, the ratio between payloads (derived from exatecan and berzosertib) and the antigen-binding moiety is from about 2:1 to about 8:1 . In some embodiments, the ratio between payloads (derived from exatecan and berzosertib) and the antigen-binding moiety is about 4:1 . In some embodiments, the ratio between payloads (derived from exatecan and berzosertib) and the antigen-binding moiety is about 8:1. In some embodiments, the ratio between payload derived from exatecan and the antigen-binding moiety is from about 2:1 to about 4:1 . In some embodiments, the ratio between payload derived from exatecan and the antigen-binding moiety is about 4:1 . In some embodiments, the ratio between payload derived from berzosertib and the antigen-binding moiety is from about 2:1 to about 4:1 . In some embodiments, the ratio between payload derived from berzosertib and the antigen-binding moiety is about 4:1.

[0127] The DNA Damage Response (DDR) is a complex network of mechanisms for detecting and repair DNA damage, in order to preserve genomic stability. The DDR is reviewed e.g. in Groelly et a / ., Nature Reviews Cancer (2023) 23:78-94 and Molinaro et a / ., Cancers (Basel). (2021) 13(15): 3819, both of which are hereby incorporated by reference in their entirety.

[0128] The detection of DNA damage and initiation of repair pathways is mediated by proteins such as ATM (Ataxia-Telangiectasia Mutated) and ATR (Ataxia-Telangiectasia and Rad3-Related). 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 (Checkpoint Kinases 1 and 2) 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) is involved in repairing single-strand DNA breaks, helping to recruit repair factors and the formation of repair complexes at the sites of DNA damage. DNA- PK (DNA-Dependent Protein Kinase) helps bring broken DNA ends together for non-homologous endjoining (NHEJ), for repairing double-strand breaks. The DDR is facilitated by cell cycle regulation through WEE1 and PLK1 (Polo-Like Kinase 1). WEE1 is a kinase that phosphorylates and inhibits DDKs (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.

[0129] Most cancerous cells have a greater dependency on the DDR than non-cancerous cells. DDR inhibitors and their use for the treatment of cancers is described e g. in Cheng et al., Eur J Med Chem. (2022) 230:114109, Wang et al., 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.

[0130] Berzosertib has the structure:

[0131] During DNA replication and transcription, significant torsional strain is placed on the DNA helix, and this is relieved through the action of DNA topoisomerases I and II (TOP1 and TOP2), which cleave the DNA strand and allow it to untwist, before resealing the breaks (see e.g. Delgado et al., Biochem J. (2018) 475(2): 373-398). 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.

[0132] Exatecan has the structure: The linker-payload moiety comprises a cleavable linker moiety, which is enzyme cleavable, more specifically cathepsin-cleavable.

[0133] The linker-payload moiety has an amino (NH2) group for linkage to the antigen-binding moiety, for example by enzymatic conjugation. In some of these embodiments, enzymatic conjugation with microbial transglutaminase may be used to conjugate the linker-payload moiety to the antigen-binding moiety.

[0134] The linker-payload moiety further comprises spacer moieties, more specifically para-aminobenzyl carbamate (PABC) and PEG groups.

[0135] The linker payload moiety also comprises a branching group:

[0136] The linker -payload moiety also comprises two groups derived from the click-pair of azide (-Na) and

[0137] Functional properties of the antigen-binding molecules

[0138] The antigen-binding molecules described herein may be characterised by reference to certain functional properties. In some embodiments, an antigen-binding molecule described herein may possess one or more of the following properties: binds to cells expressing TROP2; inhibits proliferation of TROP2-expressing cells; increases killing of cells expressing TROP2; inhibits tumor growth and / or reduces tumor size / volume (e.g. of a TROP2-expressing cancer); increases survival of subjects having a cancer (e g. a TROP2-expressing cancer). It will be appreciated that 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. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays. In some embodiments, the assays may be e.g. in vivo assays, i.e. performed in non-human animals. In some embodiments, the assays may be e.g. ex vivo assays, i.e. performed using cells / tissue / an organ obtained from a subject.

[0139] Where 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 orderto 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).

[0140] 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. The 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’. Depending on the property, the ECso 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.

[0141] In some embodiments, the antigen-binding molecule of the present disclosure binds to TROP2 in a region which is accessible to an antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when TROP2 is expressed at the cell surface (i.e. in or at the cell membrane). In some embodiments, the antigen-binding molecule binds to TROP2 expressed at the cell surface of a cell expressing TROP2. In some embodiments, the antigen-binding molecule binds to TROP2-expressing cells.

[0142] The ability of an antigen-binding molecule to bind to a given cell type can be analysed by contacting cells with the antigen-binding molecule, and detecting antigen-binding molecule bound to the cells, e.g. after a washing step to remove unbound antigen-binding molecule. The ability of an antigen-binding molecule to bind to TROP2-expressing cells can be analysed by methods such as flow cytometry and immunofluorescence microscopy.

[0143] In some embodiments, the antigen-binding molecule inhibits proliferation of TROP2-expressing cells (e.g. TROP2-expressing cancer cells). The ability of an antigen-binding molecule to inhibit proliferation of a given cell type can be analysed by contacting cells with the antigen-binding molecule, and subsequently evaluating proliferation of the cells (i.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 of3H-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. In some embodiments, the antigen-binding molecule of the present invention is capable of inhibiting proliferation of TROP2-expressing cells to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of proliferation of TROP2-expressing 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 TROP2-expressing cells), in a given assay.

[0144] In some embodiments, the antigen-binding molecule described herein inhibits proliferation of cells expressing human TROP2 with an IC$o 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.

[0145] In some embodiments, the antigen-binding molecule according to the present disclosure potentiates ( / .e. upregulates, enhances) cell killing of cells comprising / expressing TROP2.

[0146] In some embodiments, an antigen-binding molecule according to the present disclosure may inhibit growth or reduce metastasis of a cancer comprising cells comprising / expressing TROP2. In some embodiments, an antigen-binding molecule may potentiate ( / .e. upregulate, enhance) cell killing of cells comprising / expressing TROP2. In some embodiments, an antigen-binding molecule may inhibit growth of cells of a cancer, or may inhibit growth of a tumor, comprising cells comprising / expressing TROP2. In some embodiments, an antigen-binding molecule may inhibit metastasis of a cancer / tumor comprising cells comprising / expressing TROP2.

[0147] Cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (201 1), 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 the51Cr 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.

[0148] In some embodiments an antigen-binding molecule according to the present disclosure is capable of reducing the number / proportion of cells expressing TROP2. In some embodiments, an antigen-binding molecule according to the present disclosure is capable of depleting / enhancing depletion of such cells.

[0149] In some embodiments, an antigen-binding molecule of the present disclosure displays anticancer activity. In some embodiments, the antigen-binding molecule increases killing of cancer cells. In some embodiments, the antigen-binding molecule causes a reduction in the number of cancer cells in vivo, e.g. as compared to an appropriate control condition. The cancer may be a cancer as described herein, e.g. a cancer expressing / overexpressing TROP2.

[0150] In some embodiments, an antigen-binding molecule according to the present disclosure reduces / inhibits growth of a cancer and / or of a tumor of a cancer. In some embodiments, an antigen-binding molecule reduces tissue invasion by cells of a cancer. In some embodiments, an antigen-binding molecule reduces metastasis of a cancer. In some embodiments, an antigen-binding molecule displays anticancer activity. 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. In some embodiments, an antigen-binding molecule of the present disclosure causes a reduction in the number of cancer cells e.g. in vivo. The cancer may be a cancer comprising cells expressing TROP2.

[0151] An antigen-binding molecule of the present disclosure may be analysed for the properties described in the preceding paragraph in appropriate assays. Such assays include e.g. in vivo models.

[0152] In some embodiments, 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.

[0153] It will be appreciated that the properties recited in the preceding paragraph are evaluated after a period of time sufficient for an effect associated with treatment using the antigen-binding molecule to be observed. Tumor growth may be monitored by investigating tumor volume over time. Tumor growth may be evaluated by measuring tumor volume (e.g. in mm3) overtime.

[0154] In some embodiments, 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 TROP2-expressing cancer) 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 tumor size / volume observed at the same time point in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence tumor growth), in a given assay. In some embodiments, 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. In some embodiments, 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. 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, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of tumor growth inhibition observed at the same time point in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence tumor growth), in a given assay. In some embodiments, 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.

[0155] In some embodiments, 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 TROP2-expressing cancer) to 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, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the median survival observed in the absence of treatment with the antigen-binding molecule (or following treatment with an appropriate control antigen-binding molecule known not to influence survival of subjects having the cancer), in a given assay. Median survival may be expressed in days from the start of the experiment, for subjects in the relevant treatment groups.

[0156] In some embodiments, an antigen-binding molecule according to the present disclosure possesses one or more novel, similar or improved functional properties as compared to a known anti-TROP2 antibodydrug conjugate. In some embodiments, an antigen-binding molecule according to the present disclosure possesses one or more novel, similar or improved functional properties as compared to an anti-TROP2 antibody-drug conjugate comprising a topoisomerase I inhibitor payload moiety (only); such molecules may be referred to herein as anti-TROP2 TOP1 i ADCs. Anti-TROP2 TOP1 i ADCs include e.g. sacituzumab govitecan (DrugBank Acc. No. DB12893; marketed as Trodelvy™) and datopotamab deruxtecan (DrugBank Acc. No. DB16410). In some embodiments, an antigen-binding molecule possesses one or more novel, similar or improved functional properties as compared to sacituzumab govitecan (DrugBank Acc. No. DB12893; marketed as Trodelvy™). Sacituzumab govitecan is an example of an anti-TROP2 TOP1 i ADC, and comprises the cytotoxic agent SN-38 (a topoisomerase I inhibitor) conjugated to a humanised antibody (RS7) via a hydrolysable linker.

[0157] In some embodiments, an antigen-binding molecule described herein may display one or more of the following properties: similar or increased binding to cells expressing TROP2, as compared to binding to such cells displayed by an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan); similar or increased internalization by TROP2-expressing cells, as compared to internalization of an anti-TROP2 TOP1 i ADC (e g. sacituzumab govitecan) by such cells; similar or increased killing of cells expressing TROP2, as compared to killing of such cells displayed by an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan); similar or increased inhibition of tumor growth and / or reduction of tumor size / volume e.g. of a TROP2-expressing cancer), as compared to tumor growth inhibition / tumor size / volume reduction displayed by an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan); and / or similar or increased duration of an anti-cancer response (e.g. a reduction in tumor size / volume or a reduction in tumor growth inhibition) in subjects having a cancer (e.g. a TROP2-expressing cancer) following treatment with the antigen-binding molecule, as compared to the duration of the anti-cancer response observed following treatment of comparable subjects with an anti-TROP2 TOP1 i ADC (e g. sacituzumab govitecan).

[0158] In accordance with the preceding paragraph, a level / duration of a given property / outcome which is ‘similar to’ a reference level / duration 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 / duration. In some embodiments, a level / duration of a given property / outcome which is ‘increased’ relative to a reference level / duration 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 / duration.

[0159] It will be appreciated that for the purposes of such evaluations, equivalent amounts of the antigen-binding molecule and anti-TROP2 TOP1 i ADC (e g. sacituzumab govitecan) may be compared. In some embodiments, equivalent amounts of the respective payloads are compared.

[0160] In some embodiments, the antigen-binding molecule of the present disclosure binds to TROP2- expressing cells with an ECso which is similar to or less than the ECso with which an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan) binds to the relevant cells, in a given assay. In some embodiments, the ECso for binding of the antigen-binding molecule to TROP2-expressing cells 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 ECso for binding of an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan) to cells of the same type, as determined in the same assay. In some embodiments, the ECso for binding of the antigen-binding molecule to TROP2-expressing cells is 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 ECso for binding of an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan) to cells of the same type, as determined in the same assay.

[0161] In some embodiments, the antigen-binding molecule of the present disclosure displays a level of internalization by TROP2-expressing cells that is similar to or greater than the level of internalization of an anti-TROP2 TOP1 I ADC (e.g. sacituzumab govitecan) by cells of the same type, in a given assay. In some embodiments, the level of internalization of the antigen-binding molecule by TROP2-expressing cells 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 of internalization of an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan) by cells of the same type, as determined in the same assay. In some embodiments, the level of internalization of the antigen-binding molecule by TROP2-expressing cells is 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, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of internalization of an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan) by cells of the same type, as determined in the same assay.

[0162] In some embodiments, the antigen-binding molecule of the present disclosure increases the killing of cells expressing TROP2 to a level that is similar to or greater than the level of killing of the relevant cells displayed by an anti-TROP2 TOP1 i ADC (e g. sacituzumab govitecan), in a given assay. In some embodiments, the antigen-binding molecule increases the killing of TROP2-expressing cells (e.g. TROP2- expressing cancer cells) to a level that 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 of cell killing of the relevant cells displayed by an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan), as determined in the same assay. In some embodiments, the antigen-binding molecule increases the killing of TROP2-expressing cells (e.g. TROP2-expressing cancer cells) to a level that is 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, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of cell killing of the relevant cells displayed by an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan), as determined in the same assay.

[0163] In some embodiments, the antigen-binding molecule of the present disclosure reduces tumor size / volume to a level that is similar to or greater than the level of reduction in tumor size / volume observed following treatment with (an equivalent dose of) an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan), in a given assay. In some embodiments, 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 TROP2-expressing cancer) to a level that 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 tumor size / volume observed at the same time point following treatment with (an equivalent dose of) an anti-TROP2 TOP11 ADC (e.g. sacituzumab govitecan), as determined in the same assay. In some embodiments, 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 TROP2- expressing cancer) 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 tumor size / volume observed at the same time point following treatment with (an equivalent dose of) an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan), as determined in the same assay. In some embodiments, 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.

[0164] In some embodiments, the antigen-binding molecule of the present disclosure achieves a level of tumor growth inhibition that is similar to or greater than the tumor growth inhibition observed following treatment with (an equivalent dose of) an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan), in a given assay. In some embodiments, 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 >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 of tumor growth inhibition observed at the same time point following treatment with (an equivalent dose of) an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan), as determined in the same assay. In some embodiments, 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. 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, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of tumor growth inhibition observed at the same time point following treatment with (an equivalent dose of) an anti-TROP2 TOP1 i ADC (e.g. sacituzumab govitecan), as determined in the same assay. In some embodiments, 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. In some embodiments, the duration of an anti-cancer response (e.g. a reduction in tumor size / volume, or a reduction in tumor growth inhibition) observed in subjects having a cancer (e.g. a TROP2-expressing cancer) following treatment with an antigen-binding molecule according to the present disclosure is similar to or greater than the duration of the relevant anti-cancer response observed following treatment of comparable subjects with (an equivalent dose of) an anti-TROP2 TOP11 ADC (e g. sacituzumab govitecan). In some embodiments, the duration of an anti-cancer response (e.g. a reduction in tumor size / volume or a reduction in tumor growth inhibition) observed in subjects having a cancer (e.g. a TROP2-expressing cancer) following treatment with 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 duration of the relevant anti-cancer response observed following treatment of comparable subjects with an anti- TROP2 TOP1 i ADC (e.g. sacituzumab govitecan). In some embodiments, the duration of an anti-cancer response (e g. a reduction in tumor size / volume or a reduction in tumor growth inhibition) observed in subjects having a cancer (e.g. a TROP2-expressing cancer) following treatment with an antigen-binding molecule according to the present disclosure is 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, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the duration of the relevant anti-cancer response observed following treatment of comparable subjects with an anti-TROP2 TOP1 i ADC (e g. sacituzumab govitecan).

[0165] Additional sequences

[0166] The antigen-binding molecules of the present disclosure and their constituent polypeptides may additionally comprise further amino acids or sequences of amino acids.

[0167] 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 a / ., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, 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.

[0168] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm; wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1 , 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g. 1 , 2, 3, 4, 5 or 6) copies (e.g. in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (648)4 or (G4S)6. 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.

[0169] The 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. For example, 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.

[0170] 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).

[0171] 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.

[0172] Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).

[0173] Labels and conjugates

[0174] In some embodiments, the antigen-binding molecules of the present disclosure and their constituent polypeptides comprise a detectable moiety.

[0175] In some embodiments, 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.

[0176] 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. Radiolabels include radioisotopes such as Hydrogen3, Sulfur35, Carbon14, Phosphorus32, Iodine123, Iodine125, Iodine126, Iodine131, Iodine133, Bromine77, Technetium"111, Indium111, Indium113"1, Gallium67, Gallium68, Ruthenium95, Ruthenium97, Ruthenium103, Ruthenium105, Mercury207, Mercury203, Rhenium"111, Rhenium101, Rhenium105, Scandium47, Tellurium121"1, Tellurium122"1, Tellurium125"1, Thulium165, Thuliuml167, Thulium168, Copper67, Fluorine18, Yttrium90, Palladium100, Bismuth217and Antimony211. 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.

[0177] In some embodiments, an antigen-binding molecule or a constituent polypeptide thereof comprises a radionuclide. Such conjugates may be called radionuclide drug conjugates (RDCs) or radioimmunoconjugates (RICs). In some embodiments, an antigen-binding molecule or a constituent polypeptide thereof comprises a chelator group that is capable of chelating a radionuclide. Radionuclides include radioisotopes such as those listed above, or others such as Lutetium177, Actinium225and Strontium".

[0178] In some embodiments, 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.

[0179] In some embodiments, 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), glucuronidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.

[0180] Nucleic acids and vectors

[0181] The present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding an antigen-binding molecule / antigen-binding polypeptide complex or a constituent polypeptide thereof according to the present disclosure. In some embodiments, the nucleic acid(s) comprise or consist of DNA and / or RNA.

[0182] An antigen-binding molecule / antigen-binding polypeptide complex or a constituent polypeptide thereof according to the present disclosure may be produced within a cell by translation of RNA encoding the polypeptide(s). An antigen-binding molecule / antigen-binding polypeptide complex or a constituent polypeptide thereof may be produced within a cell by transcription from nucleic acid encoding the polypeptide(s), and subsequent translation of the transcribed RNA. In some embodiments, the nucleic acid(s) may be, or may be comprised / contained in, a vector, or a plurality of vectors. A ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.

[0183] Accordingly, the present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure. The vector may facilitate delivery of the nucleic acid(s) encoding a polypeptide according to the present disclosure to a cell. The vector may be an expression vector comprising elements required for expressing a polypeptide according to the present disclosure. The vector may comprise elements facilitating integration of the nucleic acid(s) into the genomic DNA of cell into which the vector is introduced.

[0184] Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

[0185] A vector may be a vector for expression of the nucleic acid in the cell (i.e. an expression vector). Such vectors may include a promoter sequence operably linked to a nucleotide sequence encoding an antigenbinding molecule or polypeptide according to the present disclosure. A vector may also include a termination codon (i.e. 3’ in the nucleotide sequence of the vector to the nucleotide sequence encoding the polypeptide(s)) and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0186] The term operably linked’ may include the situation where nucleic acid encoding a polypeptide according to the present disclosure and regulatory nucleic acid sequence(s) (e.g. a promoter and / or enhancers) are covalently linked in such a way as to place the expression of the nucleic acid encoding a polypeptide under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into the desired polypeptide(s).

[0187] Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e g. SFG vector), lentiviral vectors, adenovirus vectors, adeno- associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety. In some embodiments, a vector according to the present disclosure is a lentiviral vector. In some embodiments, the vector may be a eukaryotic vector, i.e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0188] Constituent polypeptides of an antigen-binding molecule / antigen-binding polypeptide complex according to the present disclosure may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors.

[0189] Producing the antigen-binding molecules

[0190] 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.

[0191] Antigen-binding moieties according to the present disclosure may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, 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.

[0192] Alternatively, 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 (4thEdition), 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.

[0193] In some cases, the antigen-binding moieties of the present disclosure are comprised of more than one polypeptide chain. In such cases, 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.

[0194] For recombinant production according to the present disclosure, any cell suitable for the expression of polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments, the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments, the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, 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. In some cases, 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. In addition, 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.

[0195] In some embodiments 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.

[0196] Production of antigen-binding moieties may involve culture or fermentation of a eukaryotic cell modified to express the polypeptide(s) of interest. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins can be collected by partitioning culture media / fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide(s). Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thEdition; incorporated by reference herein above). Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

[0197] Following culturing the cells that express the polypeptide(s), the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide, it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted polypeptide(s) of interest. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e g. by Bonification, rapid freeze-thaw or osmotic lysis.

[0198] It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins. Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation or may be performed subsequently to precipitation. Once the polypeptide(s) of interest have been isolated from culture it may be desired or necessary to concentrate the polypeptide(s). A number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.

[0199] Conjugation of linker to antigen-binding moiety

[0200] The linker moieties, including linker-payload moieties, of the present disclosure may be conjugated to the antigen-binding moieties by enzymatic conjugation. The use of bacterial transglutaminases is a powerful approach for site-specific incorporation of the payload into the antibody. A transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined drug-to-antibody ratio (DAR) arising from the conjugation of 2 linker-payloads (one conjugation site per heavy chain). An N297Q mutation prior to this conjugation provides two more reaction sites (resulting in the conjugation of 4-linker-payloads). An alternative version using a peptide sequence-specific transglutaminase. This enzyme recognizes and utilizes LLQG motif that is genetically incorporated, resulting in site-specific antibody-drug conjugation. Another advantage of this LLQG-specific bacterial transglutaminase is that conjugation sites can be flexibly laid by inserting this short peptide motif within the antibody structure. Further alternative approaches allow for the use of transglutaminase without deglycosylation.

[0201] In some embodiments, the linker moiety is conjugated to the antigen-binding moiety (e.g. a TROP2- binding moiety) via a glutamine residue of the antigen-binding moiety. For example, the conjugation is between the amine group of the linker moiety and the glutamine at Q295 (EU numbering) of the antigenbinding moiety. In some embodiments, the antigen-binding moiety is an antibody or an antigen-binding fragment thereof.

[0202] The DAR will depend on the number of payloads per linker-payload moieties conjugated. In some embodiments, the ratio between the linker moiety to the antigen-binding moiety is from about 1 :1 to about 2:1 . In some embodiments, the ratio between the linker moiety to the antigen-binding moiety is about 2:1 .

[0203] In some embodiments, the linker-payload moiety comprises a first payload derived from exatecan and a second payload derived from berzosertib, wherein the ratio between the first payload and the second payload is about 1 :1. In some embodiments, the antigen-binding molecule describe herein has a DAR for the first payload (e.g. exatecan) of from about 3 to about 4. In some embodiments, the antigen-binding molecule describe herein has a DAR for the second payload (e.g. berzosertib) of from about 3 to about 4. In some embodiments, the antigen-binding molecule describe herein has a DAR for the first payload (e g. exatecan) of about 4 and a DAR for the second payload (e.g. berzosertib) of about 4. In some embodiments, the antigen-binding molecule describe herein has a DAR for the first payload (e.g. exatecan) and the second payload (e.g. berzosertib) of about 8.

[0204] In some embodiments, the method further comprises purifying / isolating the antigen-binding molecule ( / .e. from unreacted precursors and / or by-products). In some embodiments, the antigen-binding molecule may be purified / isolated by chromatography, e.g. size-exclusion chromatography. The present disclosure also provides an antigen-binding molecule obtained or obtainable by the methods of the present disclosure.

[0205] Compositions

[0206] The present disclosure provides a composition comprising an antigen-binding molecule according to the present disclosure.

[0207] The antigen-binding molecules described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. Thus, the present disclosure provides a pharmaceutical composition / medicament comprising an antigen-binding molecule described herein.

[0208] The pharmaceutical compositions / medicaments of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).

[0209] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rdEdition (2020), Academic Press.

[0210] Pharmaceutical 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. In some embodiments, 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.

[0211] In some embodiments, the pharmaceutical compositions / medicament is formulated for injection or infusion, e.g. into a blood vessel, tissue / organ of interest, or a tumor.

[0212] 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.

[0213] For example, 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.

[0214] Therapeutic and prophylactic applications

[0215] The antigen-binding molecules and compositions described herein find use in therapeutic and prophylactic intervention for disease, e.g. cancers.

[0216] It will be appreciated that the antigen-binding molecules and compositions of the present disclosure may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from a reduction in the level of expression or activity of TROP2, or a reduction in the number or activity of cells comprising / expressing TROP2.

[0217] For example, the disease / condition may be a disease / condition in which TROP2, or cells expressing / overexpressing TROP2 are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of TROP2, or an increase in the number / proportion of cells comprising / expressing TROP2 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. In some embodiments, an increased level / activity of TROP2, or an increase in the number / proportion of cells comprising / expressing TROP2 may be a risk factor for the onset, development or progression of the disease / condition.

[0218] The present disclosure provides an antigen-binding molecule or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is an antigen-binding molecule or composition described herein for use in a method of treating or preventing a cancer (e.g. a cancer described herein). Also provided is the use of an antigen-binding molecule or composition described herein in the manufacture of a medicament for treating or preventing a cancer (e.g. a cancer described herein). Also provided is a method of treating or preventing a cancer (e.g. a cancer described herein) in a subject, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigenbinding molecule or composition described herein.

[0219] 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. In some embodiments, 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. In some embodiments, the methods may prevent development of the cancer to a later stage (e.g. a chronic stage or metastasis).

[0220] As used herein, 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.

[0221] Tumors to be treated may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers / tumors may originate in any other non-nervous tissue; examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.

[0222] In some embodiments the cancer is a cancer in which TROP2 is pathologically-implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of TROP2, a cancer for which expression of TROP2 is a risk factor and / or a cancer for which expression of TROP2 is positively associated with onset, development, progression, severity or metastasis of the cancer. The cancer may be characterised by expression of TROP2, e g. the cancer may comprise cells (e.g. cells of tumor tissue) expressing TROP2. Such cancers may be referred to as being positive for TROP2. A cancer which is ‘positive’ for TROP2 may be a cancer comprising cells expressing TROP2 (e.g. at the cell surface). A cancer which is ‘positive’ for TROP2 may overexpress TROP2.

[0223] 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 TROP2, 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.

[0224] In some embodiments, 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 TROP2, relative to comparable cells harboring a reference allele not comprising the genetic variant (e.g. a nonmutated, 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.

[0225] A mutation resulting in’ increased expression of TROP2 may be known or predicted to cause, or may be associated with, increased gene / protein expression of TROP2. A mutation resulting in’ increased activity of TROP2 may be known or predicted to cause, or may be associated with, increased TROP2-mediated signaling. Mutations resulting in increased expression and / or activity of TROP2 may be referred to as ‘activating’ mutations.

[0226] A mutation which causes increased expression of TROP2 may result in gene or protein expression of TROP2 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 TROP2 may be a result of the mutation, and thus ‘increased expression’ may be from no expression.

[0227] A mutation which causes increased expression of TROP2 may result in increased gene or protein expression of TROP2 which is expressed by, and / or which is encoded by genomic nucleic acid of, an equivalent cell not comprising the mutation. By way of illustration, a cell may comprise a mutation resulting in an increase in the level of transcription of nucleic acid encoding TROP2 relative to the level of transcription of nucleic acid encoding TROP2 by an equivalent cell not comprising the mutation.

[0228] In some embodiments, a mutation which causes increased expression of TROP2 may cause an increase in gene expression of TROP2 relative to an equivalent cell not comprising the mutation. In some embodiments, a mutation which causes increased expression of TROP2 may cause an increase in protein expression of TROP2 relative to an equivalent cell not comprising the mutation. In some embodiments, a mutation which causes increased expression of TROP2 may cause an increase in the level of TROP2 on or at the cell surface of a cell comprising the mutation, relative to an equivalent cell not comprising the mutation.

[0229] Cells having increased expression of TROP2 relative to the level of expression of TROP2 by a reference cell (e.g. as a result of mutation) may be described as ‘overexpressing’ TROP2, or having upregulated expression’ of TROP2. For example, a cancer comprising cells harboring a mutation resulting in increased expression of TROP2 relative to equivalent cells lacking the mutation may be described as a cancer comprising cells displaying overexpression / upregulated expression of TROP2. In some embodiments, 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).

[0230] A mutation which causes increased activity of TROP2 may result in an increase in TROP2-mediated signaling relative to the level of TROP2-mediated signaling by an equivalent cell not comprising the mutation.

[0231] It will be appreciated that a mutation which causes increased expression / activity of TROP2 may be a mutation which impacts a pathway associated with the regulation of TROP2 expression / activity. That is, a mutation may cause increased or decreased expression / activity of a component / molecule / protein / nucleic acid of a pathway that is associated with regulating TROP2 expression / activity, and this may result in increased expression / activity of TROP2.

[0232] In some embodiments, 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 TROP2 (i.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 (i.e. in the absence of the disease / condition). In some embodiments, 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 TROP2, e.g. as compared to the level of expression and / or activity observed in equivalent non- cancerous cells / non-tumor tissue.

[0233] TROP2 and its association with and role in cancer is reviewed e.g. in Qiu et al., BBA - Reviews on Cancer (2023) 1878:188902, Chmielewska & Kubiatowski, Oncology in Clinical Practice (2023) 19(6):427-432, and Lombardi et al., Cancers (2023) 15(6):1744, all of which are hereby incorporated by reference in their entirety.

[0234] Overexpression of TROP2 has been reported in a wide range of cancers. Tumor types with strong to moderate TROP2 IHC staining include: lung cancers (e.g. non-small cell lung cancers (e g. adNSCLC, sqNSCLC)), breast cancers (e.g. hormone receptor-positive (HR+) breast cancer, metastatic breast cancer, HR+ metastatic breast cancer, HER2-positive (HER2+) breast cancer, HER2+ metastatic breast cancer, triple-negative breast cancer (TNBC)), pancreatic cancers, prostate cancers (e.g. castrationresistant prostate cancer (CRPC), metastatic castration-resistant prostate cancer (mCRPC)), bladder cancers, head and neck cancers {e g. head and neck squamous cell carcinomas (HNSCC)), endometrial cancers, ovarian cancers, esophageal cancers {e.g. squamous-cell carcinoma (ESCC)), cervical cancers, thyroid cancers and biliary tract cancers (BTC). Expression of TROP2 in tumors and normal tissue is described in e.g. Dum et al., Pathobiology (2022) 89(4):245-258. Accordingly, in some embodiments, the cancer to be treated is selected from: lung cancer e.g. non-small cell lung cancer {e.g. adNSCLC, sqNSCLC)), breast cancer {e.g. hormone receptor-positive (HR+) breast cancer, metastatic breast cancer, HR+ metastatic breast cancer, HER2-positive (HER2+) breast cancer, HER2+ metastatic breast cancer, triple-negative breast cancer (TNBC)), pancreatic cancer, prostate cancer {e.g. castrationresistant prostate cancer (CRPC), metastatic castration-resistant prostate cancer (mCRPC)), bladder cancer, head and neck cancer {e g. head and neck squamous cell carcinoma (HNSCC)), endometrial cancer, ovarian cancer, esophageal cancer (e.g.esophageal squamous-cell carcinoma (ESCC)), cervical cancer, thyroid cancer, and biliary tract cancer (BTC). In some embodiments, the cancer to be treated is selected from: non-small cell lung cancer adenocarcinoma (adNSCLC), hormone receptor-positive breast cancer (HR+ BC), metastatic castration-resistant prostate cancer (mCRPC), bladder cancer, endometrial cancer, triple-negative breast cancer (TNBC), ovarian cancer, pancreatic cancer, head and neck squamous cell carcinoma (HNSCC), and esophageal squamous-cell carcinoma (ESCC).

[0235] In some embodiments, a cancer according to the present disclosure is a cancer for which sacituzumab govitecan is an approved treatment. In some embodiments, a cancer is selected from: triple-negative breast cancer {e g. metastatic or unresectable triple negative breast cancer); hormone receptor-positive {i.e. a cancer comprising cells expressing estrogen receptor (ER) and / or progesterone receptor (PR)) and HER2-negative breast cancer {e.g. metastatic or unresectable hormone receptor-positive and HER2- negative breast cancer); bladder cancer and cancers of the urinary tract {e g. urothelial cancer {e g. metastatic or unresectable urothelial cancer)).

[0236] In some embodiments, the cancer to be treated is selected from: a cancer comprising cells expressing / overexpressing TROP2, a solid tumour, metastatic cancer, unresectable cancer, hormone receptor-positive cancer, lung cancer, non-small cell lung cancer (NSCLC), non-small cell lung cancer adenocarcinoma (adNSCLC), squamous non-small cell lung cancer (sqNSCLC), breast cancer, hormone receptor-positive breast cancer, metastatic breast cancer, hormone receptor-positive metastatic breast cancer, HER2-positive breast cancer, HER2-positive metastatic breast cancer, HER2-negative breast cancer, metastatic HER2-negative breast cancer, unresectable HER2-negative breast cancer, hormone receptor-positive and HER2-negative breast cancer, metastatic hormone receptor-positive and HER2- negative breast cancer, unresectable hormone receptor-positive and HER2-negative breast cancer, triplenegative breast cancer (TNBC), metastatic triple negative breast cancer, unresectable triple-negative breast cancer, pancreatic cancer, prostate cancer, castration-resistant prostate cancer (CRPC), metastatic castration-resistant prostate cancer (mCRPC)), bladder cancer, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), endometrial cancer, ovarian cancer, esophageal cancer, esophageal squamous-cell carcinoma (ESCC), cervical cancer, thyroid cancer, biliary tract cancer (BTC), urinary tract cancer, urothelial cancer, metastatic urothelial cancer, and unresectable urothelial cancer. In some embodiments, a cancer to be treated / prevented in accordance with the present disclosure is selected from: lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, breast cancer and breast ductal carcinoma.

[0237] In some embodiments, a cancer to be treated / prevented in accordance with the present disclosure comprises / expresses / overexpresses canonical / wild-type TROP2 (e.g. TROP2 having the amino acid sequence as shown in SEQ ID NO: 1 or 2). In some embodiments, a cancer to be treated / prevented in accordance with the present disclosure does not comprise / express / overexpress TROP2 comprising a mutation (e.g. a mutation that attenuates / decreases the binding of the antigen-binding moiety to TROP2, or a mutation that reduces TROP2 plasma membrane localisation). In some embodiments, a cancer to be treated / prevented in accordance with the present disclosure does not comprise / express / overexpress TROP2 comprising a T256R mutation ( / .e. TROP2 comprising substitution of T at position 256 of SEQ ID NO:1 , or the position corresponding to position 256 of SEQ ID NO:1 , with R).

[0238] In some embodiments, 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 EGFR, relative to comparable cells harboring a reference allele not comprising the genetic variant (e g. a nonmutated, 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.

[0239] Genetic variation known or predicted to increase the expression and / or activity of EGFR / EGFR is described e.g. in Gazdar, Oncogene. (2009) 28(Suppl 1): S24-S31 , which is hereby incorporated by reference in its entirety. Activating mutations to EGFR include mutations to L858 (e g. L858R), G719 (e.g. G719S, G719A or G719C), T790 (e g. T790M), V765 (e.g. V765A), T783 (e.g. T783A), and deletion mutations in exon 19 (e.g. deletions including deletion of L747 to E749, and deletion of E746 to A750). In some embodiments, the cancer to be treated / prevented in accordance with the present disclosure comprises cells harboring: a mutation to L858 (e g. L858R), a mutation to G719 (e g. G719S, G719A or G719C), a mutation to T790 (e.g. T790M), a mutation to V765 (e.g. V765A), a mutation to T783 (e.g.

[0240] T783A), or a deletion mutation in exon 19 (e.g. deletion of L747 to E749, or deletion of E746 to A750).

[0241] In some embodiments, genetic variation resulting in an increase in the expression of, or an increase in the activity of a gene product of, EGFR is or comprises amplification of EGFR. EGFR amplification can be identified using techniques well known in the art, such as in situ hybridization and NGS. For example, EGFR amplification can be evaluated by fluorescence in situ hybridization, e g. as described in French et al., Neuro-Oncology (2019) 21 (10): 1263-1272. In some embodiments, the cancer to be treated / prevented in accordance with the present disclosure comprises cells comprising EGFR amplification.

[0242] In some embodiments, the cancer may be a relapsed cancer. As used herein, 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. For example, 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.

[0243] In some embodiments, the cancer may be a refractory cancer. As used herein, a ‘refractory’ cancer refers to a cancer which has not responded to a treatment (e.g. a first line therapy for the cancer). For example, 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). In some embodiments 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.

[0244] In some embodiments, 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.

[0245] Herein, where a cancer is described as being relapsed / refractory / resistant, etc. with respect to a given intervention, it may be simply described as being ‘relapsed / refractory / resistant to’ the relevant intervention.

[0246] DDR inhibitors and their use for the treatment of cancers is described e.g. in Cheng et al., Eur J Med Chem. (2022) 230:114109, Wang et a!., Front Immunol. (2022) 13:854730 and Choi and Lee, Int J Mol Sci. (2022) 23(3):1701 , all of which are hereby incorporated by reference in their entirety. In some embodiments, a DDR inhibitor according to the present disclosure is selected from: a PARP inhibitor (e.g. olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, simmiparib, senaparib, SC-10914, 2X- 121 , AMXI-5001 , JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-1002, RBN-2397), an ATM inhibitor (e.g. CP-466722, KU-55933, KU-60019, KU-59403, AZ31 , AZ32, AZD0156, AZD1390), an ATR inhibitor (e.g. M6620 (berzosertib), M4344 (VX-803), AZD6738 (ceralasertib), BA Y1895344 (elimusertib)), a WEE1 inhibitor (e.g. adavosertib, Debio 0123, PD0166285, PD0407824, AZD1775), a CHK1 / 2 inhibitor (e.g. CBP-501 , prexasertib, MK-8776, GDC-0575, SRA-737), a DNA-PK inhibitor (e.g. CC-115, LY-3023414, AsiDNA, M3814 (nedisertib), M9831 (VX-984)) and a PLK1 inhibitor (e.g. BI-6727 (volasertib), PCM-075 (onvansertib)).

[0247] In some embodiments, the cancer is a cancer that is relapsed or refractory with respect to treatment with a DNA topoisomerase I (TOP1) inhibitor. In some embodiments, the cancer is refractory with respect to treatment with a TOP1 inhibitor. In some embodiments, the cancer is relapsed with respect to treatment with a TOP1 inhibitor. In some embodiments, the cancer has intrinsic resistance to treatment with a TOP1 inhibitor. In some embodiments, the cancer has acquired resistance to treatment with a TOP1 inhibitor. In accordance with such embodiments, 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.

[0248] 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. In some embodiments, a TOP1 inhibitor according to the present disclosure is selected from: camptothecin, irinotecan, etirinotecan, SN-38, DX-8951f (extatecan mesylate), 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), indimrtecan (LMP-776) and LMP744.

[0249] In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, the cancer is: relapsed 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). In accordance with such embodiments, 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.

[0250] 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. In some embodiments, 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.

[0251] 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).

[0252] In some embodiments, 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).

[0253] In accordance with various aspects of the present disclosure, 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.

[0254] In accordance with various aspects of the present disclosure, 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 TROP2; inhibiting the proliferation of TROP2-expressing cells; killing cells expressing TROP2; inhibiting tumor growth and / or reducing tumor size / volume, e.g. of a TROP2-expressing cancer; and / or increasing the survival of subjects having a cancer, e.g. a TROP2-expressing cancer.

[0255] Also provided are 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.

[0256] Similarly, 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 TROP2-expressing cells; killing of cells expressing TROP2; inhibition of tumor growth and / or reduction of tumor size / volume, e.g. of a TROP2-expressing cancer; and / or increased survival of a subject having a cancer, e.g. a TROP2-expressing cancer.

[0257] In some embodiments, therapeutic / prophylactic intervention in accordance with the present disclosure may be described as being associated with’ one or more of the effects described in the preceding paragraph. The skilled person is readily able to evaluate such properties using techniques that are routinely practiced in the art.

[0258] 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), 23rdEdition (2020), Academic Press.

[0259] 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.

[0260] In some aspects and embodiments, articles of the present disclosure may be administered to a tissue / organ of interest (e.g. a tissue / organ affected by the disease / condition, e.g. a tissue / organ in which symptoms of the disease / condition manifest). In some aspects and embodiments, articles of the present disclosure may be administered to the blood (i.e. intravenous / intra-arterial administration) by injection or infusion (e.g. via cannula), or may be administered subcutaneously or orally. In some aspects and embodiments, articles of the present disclosure may be administered to a tumor.

[0261] In some embodiments, 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.

[0262] 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).

[0263] Methods of detection

[0264] The present disclosure also provides the articles of the present disclosure for use in methods for detecting, localising or imaging TROP2, or cells expressing TROP2.

[0265] The antigen-binding molecules described herein may be used in methods that involve detecting binding of the antigen-binding molecule to TROP2. Such methods may involve detection of the bound complex of the antigen-binding molecule and TROP2. It will be appreciated that the TROP2 may be TROP2 expressed by a cell, e.g. in or at the cell surface of a cell expressing TROP2.

[0266] As such, a method is provided, comprising contacting a sample containing, or suspected to contain, TROP2, and detecting the formation of a complex of the antigen-binding molecule and TROP2. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell expressing TROP2, and detecting the formation of a complex of the antigen-binding molecule and a cell expressing TROP2.

[0267] Suitable method formats are well known in the art, including immunoassays such as sandwich assays, e.g. ELISA. The methods may involve labelling the antigen-binding molecule, or target(s), or both, with a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label, radiolabel, chemical, nucleic acid or enzymatic label as described herein. Detection techniques are well known to those of skill in the art and can be selected to correspond with the labelling agent.

[0268] Methods comprising detecting TROP2, or cells expressing TROP2, include methods for diagnosing / prognosing a disease / condition described herein.

[0269] Methods of this kind may be performed in vitro on a patient sample, or following processing of a patient sample. Once the sample is collected, the patient is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body. In some embodiments, the method is performed in vivo.

[0270] Such methods may involve detecting or quantifying TROP2 and / or cells expressing TROP2, e g. in a patient sample. Where the method comprises quantifying the relevant factor, the method may further comprise comparing the determined amount against a standard or reference value as part of the diagnostic or prognostic evaluation. Other diagnostic / prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm a result obtained by using the tests described herein.

[0271] Detection in a sample may be used for the purpose of diagnosis of a disease / condition (e.g. a cancer), predisposition to a disease / condition, or for providing a prognosis (prognosticating) for a disease / condition, e g. a disease / condition described herein. The diagnosis or prognosis may relate to an existing (previously diagnosed) disease / condition.

[0272] A sample may be taken from any tissue or bodily fluid. The sample may comprise or may be derived from: a quantity of blood; a quantity of serum derived from the individual’s blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said individual. In some embodiments, the sample may be obtained or derived from a tissue or tissues which are affected by the disease / condition (e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease / condition).

[0273] A subject may be selected for diagnostic / prognostic evaluation based on the presence of symptoms indicative of a disease / condition described herein, or based on the subject being considered to be at risk of developing a disease / condition described herein.

[0274] The present disclosure also provides methods for selecting / stratifying a subject for treatment with a TROP2-targeted agent. In some embodiments a subject is selected for treatment / prevention in accordance with the methods of the present disclosure, or is identified as a subject which would benefit from such treatment / prevention, based on detection / quantification of TROP2, or cells expressing TROP2, e.g. in a sample obtained from the individual.

[0275] Subjects

[0276] 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.

[0277] In some embodiments, 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. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for certain markers of such disease / condition. In some embodiments, a patient may be selected for treatment described herein based on the detection of a cancer expressing / overexpressing TROP2, e.g. in a sample obtained from the subject (e.g. a biopsy, e.g. of a tumor).

[0278] In some embodiments, a patient may be selected for treatment described herein based on detection of a relapsed / refractory cancer subsequent to administration of a therapy for the cancer, e.g. a first line therapy for the cancer or a second line therapy for the cancer. In some embodiments, a patient may be selected for treatment described herein based on detection of a relapsed / refractory cancer subsequent to administration of the standard-of-care therapy (e.g. the first line standard of care) for the given cancer. In some embodiments, a patient may be selected for treatment described herein based on detection of a relapsed / refractory cancer subsequent to administration of a therapy for the cancer comprising a TROP2 antibody drug conjugate e.g. sacituzumab govitecan).

[0279] Kits

[0280] The present disclosure also provides kits of parts. A kit according to the present disclosure may comprise components for performing a method described herein, in whole or in part.

[0281] The kit may have at least one container having a predetermined quantity of an antigen-binding molecule or composition described herein.

[0282] In some aspects of the present disclosure a kit of parts is provided. In some embodiments, the kit may comprise an antigen-binding molecule or composition described herein, and which may be provided in a predetermined quantity.

[0283] 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).

[0284] 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.

[0285] The kit may further comprise reagents, buffers and / or standards required for execution of a method according to the present disclosure. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0286] Sequence identity

[0287] As used herein, ‘sequence identity’ refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951 -960), T-coffee (Notredame et al. 2000, J.

[0288] 5 Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such software, the default parameters, e g. for gap penalty and extension penalty, are preferably used.

[0289] 10 Sequences

[0290] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0291] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0292] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0293] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0294] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a’, ‘an’, and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about’, it will be understood that the particular value forms another embodiment.

[0295] Where a nucleic acid sequence is disclosed or referred to herein, the reverse complement thereof is also expressly contemplated.

[0296] Methods described herein may preferably be performed in vitro. The term ‘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.

[0297] 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 %.

[0298] Brief Description of the Figures

[0299] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0300] Figure 1A shows binding of ADC-1 , sacituzumab govitecan, S-N297A or Iso-Exa to live NCI-H2122, HCC827, T47D or H2030 cells, as determined by flow cytometry. EC50 (M) values are shown.

[0301] Figure 1 B shows the mean fluorescence intensity (MFI) for binding of ADC-1 , sacituzumab govitecan, S- N297A or Iso-Exa to live NCI-H2122, HCC827, T47D or H2030 cells, as determined by flow cytometry. EC50 (M) values are shown.

[0302] Figure 2A shows internalization of fluorescently-labelled ADC-1 , sacituzumab govitecan, S-N297A or Iso- Exa molecules by NCI-H2122 cells overtime, as determined using the Incucyte® S3 Live-Cell Analysis System (Sartorius).

[0303] Figure 2B shows internalization of fluorescently-labelled ADC-1 , sacituzumab govitecan, S-N297A or Iso- Exa molecules by HCC827 cells over time, as determined using the Incucyte® S3 Live-Cell Analysis System (Sartorius).

[0304] Figure 2C shows internalization of fluorescently-labelled ADC-1 , sacituzumab govitecan, S-N297A or Iso- Exa molecules by NCI-N87 cells over time, as determined using the Incucyte® S3 Live-Cell Analysis System (Sartorius).

[0305] Figure 3A shows killing of NCI-H2122 cells by ADC-1 or Iso-Exa, as determined using the CellTiter-Glo® 2D Cell Viability Assay (Promega, #G9242).

[0306] Figure 3B shows killing of HCC827 cells or T47D cells by ADC-1 or Iso-Exa, as determined using the CellTiter-Glo® 3D Cell Viability Assay (Promega, #G9682). Figure 4A shows growth of tumors in a HCC827 CDX model of non-small cell lung adenocarcinoma, following administration of a single, 3 mg / kg dose of ADC-1 or sacituzumab govitecan, or vehicle (control).

[0307] Figure 4B shows growth of tumors in a HCC827 CDX model of non-small cell lung adenocarcinoma, following administration of a single, 9 mg / kg dose of ADC-1 or sacituzumab govitecan, or vehicle (control).

[0308] Figure 5A shows growth of tumors in a NCI-H2122 CDX model of non-small cell lung adenocarcinoma, following administration of a single, 3 mg / kg dose of ADC-1 or sacituzumab govitecan, or vehicle (control).

[0309] Figure 5B shows growth of tumors in a NCI-H2122 CDX model of non-small cell lung adenocarcinoma, following administration of a single, 9 mg / kg dose of ADC-1 or sacituzumab govitecan, or vehicle (control).

[0310] Figure 6A shows the effect on body weight of nonhuman primate (NHP) after dosing with ADC-1 at 50 mg / kg and 80 mg / kg.

[0311] Figure 6B shows the effect on alanine aminotransferase (ALT) of NHP after dosing with ADC-1 at 50 mg / kg and 80 mg / kg.

[0312] Figure 6C shows the effect on alkaline phosphatase (ALP) of NHP after dosing with ADC-1 at 50 mg / kg and 80 mg / kg.

[0313] Figure 6D shows the effect on neutrophils of NHP after dosing with ADC-1 at 50 mg / kg and 80 / mg / kg. Figure 6E shows the effect on platelets of NHP after dosing with ADC-1 at 50 mg / kg and 80 / mg / kg. Figure 6F shows the effect on red blood cell count of NHP after dosing with ADC-1 at 50 mg / kg and 80 / mg / kg.

[0314] Figure 6G shows the effect on reticulocytes of NHP after dosing with ADC-1 at 50 mg / kg and 80 / mg / kg. Figure 6H shows the effect on white blood cell count of NHP after dosing with ADC-1 at 50 mg / kg and 80 / mg / kg.

[0315] Figure 7A shows the pharmacokinetic profile after dose 1 with ADC-1 at 50 mg / kg.

[0316] Figure 7B shows the pharmacokinetic profile after dose 2 with ADC-1 at 50 mg / kg.

[0317] Figure 8A shows the pharmacokinetic profile after dose 1 with ADC-1 at 80 mg / kg.

[0318] Figure 8B shows the pharmacokinetic profile after dose 2 with ADC-1 at 80 mg / kg.

[0319] Figure 9A shows the pharmacokinetic profile of free payload levels of ADC-1 at 50 mg / kg in NHP plasma.

[0320] Figure 9B shows the pharmacokinetic profile of free payload levels of ADC-1 at 80 mg / kg in NHP plasma.

[0321] General methods

[0322] All chemicals, raw materials and solvents were purchased from commercial sources, unless indicated otherwise. All chemical reactions were run under ambient conditions, unless otherwise indicated. Flash column chromatography was performed with CombiFlash® NEXTGEN 100, and the column was purchased from Agela Technologies. Prep-HPLC purifications were carried out using AUNO LC-2000, and the column is of Phenomenex Luna C18, 250 x 100 mm, 10pm, 10nm.1H NMR spectra were recorded on a Bruker spectrometer (400 MHz).1H NMR chemical shifts are expressed in parts per million (6) downfield from tetramethylsilane (with the CDCI3 peak at 7.26 ppm used as a standard). 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 3um and Poroshell 120 EC C18 2.7pm 3.0*30mm.

[0323] Synthesis of intermediate 17

[0324] a) Oxybis(ethane-2, 1-diyl)dimethanesulfonate (12)

[0325] To the solution of 2,2'-oxybis(ethan-1-ol) 11 (20 g, 188.46 mmol, 1.0 eq.) in 400 mL of CH2CI2 at 0 °C, triethylamine (1 18.8 mL, 848.07 mmol, 4.5 eq.) was added followed by the dropwise addition of methanesulfonyl chloride (32 mL, 414.62 mmol, 2.2 eq.) under nitrogen atmosphere. The reaction mixture was stirred at rt for 16 h. The reaction mixture was quenched with sulfate buffer solution and extracted with CH2CI2. Combined organic layers were washed with brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to get compound I2 as a pale yellow solid (49 g, Crude). b) 2-(2-azidoethoxy)ethyl methanesulfonate (13)

[0326] To the solution of oxybis(ethane-2,1-diyl) dimethanesulfonate 12 (20 g, 76.33 mmol, 1 .0 eq.) in 500 mL of Acetonitrile at rt was added TBAB (2.46 g, 7.63 mmol, 0.1 eq.) followed by portion wise addition of NaNs (4.96 g, 76.33 mmol, 1 .0 eq.) under nitrogen atmosphere. The reaction mixture was stirred at 70 °C for 40 h. The reaction mixture was filtered and filtrate was concentrated under reduced pressure to get crude compound. The crude product was purified by MPLC Flash Column chromatography using EtOAc in Hexane (20:80) eluent to afford the title compound 13 as pale-yellow liquid (7.8 g, 48%). c) Tert-butyl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate (15)

[0327] To a stirred solution of tert-butyl (2-aminoethyl)carbamate 14 (3.0 g, 18.73 mmol, 1.0 eq.) and 2-(2- azidoethoxy)ethyl methane sulfonate I3 (7.83 g, 37.47 mmol, 2.0 eq.) in 100 mL of acetonitrile were added K2CO3 (7.76 g, 56.19 mmol, 3.0 eq.) and KI (310 mg, 1 .87 mmol, 0.1 eq,) at rt under nitrogen atmosphere. The reaction was stirred at 100° C for 16 h. The reaction mixture was filtered and filtrate was concentrated under reduced pressure to get crude compound. The crude product was purified by MPLC Flash Column chromatography using EtOAc in Hexane (25:75) eluent to afford the title compound I5 as pale-yellow liquid (4.0 g, 55%). ES / APCI: (M+1): 387.3 d) N1,N1-bis(2-(2-azidoethoxy)ethyl)ethane-1,2-diamine dihydrochloride (16)

[0328] To the stirred solution of tert-butyl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate 15 ((0.65 g, 1 .6819 mmol,1 .0 eq.) in 10 mL CH2CI2 at 0°C 4M HCI in 1 ,4-dioxane (5 mL) was added and stirred at rt for 2 h. The reaction mixture was evaporated under reduced pressure to get crude product I6 (0.7 g) as a pale brown liquid. HRMS: (M+1): 287.2390. e) 2,5-dioxopyrrolidin-1-yl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate (17)

[0329] To the solution of N1 ,N1 -bis(2-(2-azidoethoxy)ethyl)ethane-1 ,2-diamine dihydrochloride I6 (0.7 g, 1.9484 mmol, 1.0 eq.) and bis(2,5-dioxopyrrolidin-1-yl) carbonate (0.6 g, 2.3381 mmol, 1.2 eq.) in 10 mL of THF, DIPEA (1.01 mL, 5.8452 mmol, 3.0 eq.) was added and refluxed for 1 h. The reaction mixture was evaporated under reduced pressure to get crude product. The crude product was diluted with EtOAc and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to get 0.81 g of crude pale brown liquid 17. a) tert-butyl (1-azido-6-(2-(2-azidoethoxy)ethyl)-10-oxo-3,14, 17,20-tetraoxa-6,9, 11-triazadocosan-22- yl)carbamate (113)

[0330] To the solution of 2,5-dioxopyrrolidin-1 -yl (2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)carbamate 17 (0.5 g, 0. mmol) and tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate 112 (0.410 g, 1.403 mmol) in DCM was added, DIPEA (0.64 mL, 3.509 mmol) and was added and stirred the reaction mixture at rt for 16h . The reaction mixture was diluted with DCM and washed with water. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to get crude product. The crude product was purified by MPLC Flash Column chromatography using MeOH in CH2CI2 (7:93) eluent to afford the title compound 113 as a colorless liquid (0.3 g, 42%). Confirmed by1H NMR (DMSO- 6, 400 MHz) & ESMS.: (M+1): 605.4. b) 1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(2-(bis(2-(2-azidoethoxy)ethyl)amino)ethyl)urea (LM3)

[0331] To the solution of tert-butyl (1-azido-6-(2-(2-azidoethoxy)ethyl)-10-oxo-3,14,17,20-tetraoxa-6,9,11- triazadocosan-22-yl)carbamate 113 (0.15 g, 0.248 mmol) in CH2Cl2 was added 4M HCI in 1 ,4-dioxane (1.0 mL) at 0° C, stirred at RT for 1 h. The reaction mixture was evaporated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to get crude material. The crude product was washed with ether and the residue was dissolved in CH2CI2. The organic layer was washed with saturated NaHCOs solution then aqueous layer was extracted with 2% MeOH in CH2CI2. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford LM3 (0.1 g, 80%) as pale yellow liquid.1H NMR (DMSO-d6) 400 MHz) 5 ppm 6.04 (t, J=5.6 Hz, 1 H), 5.78 (t, J=5.6 Hz, 1 H), 3.58-3.56 (m, 4H), 3.52-3.39 (m, 12H), 3.37-3.33 (m, 10H), 3.14-3.10 (m, 2H), 3.04-3.01 (m, 2H), 2.68-2.62 (m, 6H). HRMS: (M+1): 505.2753. ELSD HPLC purity: 6.615 min (99.42% purity).

[0332] Example 2 -Synthesis of LP-2

[0333] To a solution of oxalyl chloride (5.9 mL, 136 mmol, 4.0 equiv.) in CH2CI2 (90.0 mL), DMSO (9.6 mL, 68.1 mmol, 2.0 equiv.) was added at -78°C followed by the addition of tert-butyl 2-(2-(2-(2- hydroxyethoxy)ethoxy)ethoxy)acetate 119 (9.0 g, 34.0 mmol, 1.0 equiv.) and the reaction mixture was stirred at the same temperature for 1 h. Thereaftertriethylamine (28.4 mL, 204 mmol, 6.0 equiv.) was added at -78°C and stirred for further 1 h and allowed to warm to room temperature. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give compound I20 (12.0 g, crude) as a colorless oil, which was used without any further purification.1H NMR (400 MHz, DMSO-cfs): 6 ppm 9.54 (s, 1 H), 4.14 (s, 2 H), 3.55 - 3.60 (m, 2 H), 3.44 - 3.55 (m, 8 H), 1.38 (s, 9 H). ii) Tert-butyl 2-[2-[2-[2-[2-[2-[2-(2-tert-butoxy-2-oxo-ethoxy)ethoxy]ethoxy]ethylamino] ethoxy]ethoxy]ethoxy]acetate (122)

[0334] 122

[0335] To a solution of compound 120 (12.0 g, 1.0 equiv.) and tert-butyl 2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)acetate 121 (17.9 g, 681 pmol, 1.5 equiv.) in MeOH (120 mL), NaBHsCN (2.85 g, 454 mmol, 1.0 equiv.) was added. The mixture was stirred at 20-25°C for 1 h. Upon completion, the reaction mixture was extracted with CH2CI2 (200 mL) and H2O (100 mL). The combined organic layers were dried over Na2SC>4, filtered and concentrated under reduced pressure to give compound 122 (18.0 g, crude) as a yellow oil. MS (ESI): [M+H]+:510.6.

[0336]

[0337] To a solution of compound 122 (18.0 g, 1 .0 equiv.), FmocOSu (17.9 g, 681 pmol, 1 .5 equiv.) in THF (90 mL) and H2O (90 mL), NaHCCh (5.9 g, 70.6 mmol, 2.0 equiv.) was added. The mixture was stirred at 20-25°C for 1 h. Upon completion, the reaction LCMS showed compound I22 was consumed completely. The reaction was extracted with DCM (200 mL) and H2O (100 mL). The combined organics were dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiC>2, Petroleum ether / Ethyl acetate=100 / 1 to 5 / 1) to give compound I23 (2.1 g, 2.87 mmol, 98.1 % purity, and 2.2 g, 3.01 mmol, 92.4% purity. 16.6% yield) as a white solid. Purity by HPLC (220 nm): 98.1 %; MS (ESI): [M+H]+:732.3;1H NMR (400 MHz, DMSO-cfe): 5 ppm 7.88 (d, J = 7.4 Hz, 2 H), 7.63 (d, J = 7.4 Hz, 2 H), 7.37 - 7.44 (m, 2 H), 7.30 - 7.36 (m, 2 H), 4.45 (d, J = 5.6 Hz, 2 H), 4.27 (br t, J = 5.4 Hz, 1 H), 3.95 - 3.99 (m, 4 H), 3.46 - 3.56 (m, 16 H), 3.38 - 3.44 (m, 4 H), 3.13 (br s, 4 H), 1.40 - 1.42 (m, 18 H).

[0338] Compound 123 (2.1 g, 2.87 mmol, 1.0 equiv.) was dissolved in 50% FA / DCM (21.0 mL) and stirred at 20- 25 °C for 16 hrs. LCMS analysis showed compound I23 was consumed completely. The reaction mixture was quenched by adding NaHCOs (100 mL) at 0-5°C. The resultant mixture was purified directly by prep- HPLC (TFA condition) to give compound I24 (700 mg, 1.03 mmol, 35.9% yield) as a colourless oil. Purity by HPLC (220 nm): 98.2%; MS (ESI): [M+H]+:676.3;1H NMR (400 MHz, DMSO-d8): 5 ppm 7.88 (d, J = 7.5 Hz, 2 H), 7.64 (d, J = 7.4 Hz, 2 H), 7.38 - 7.43 (m, 2 H), 7.30 - 7.36 (m, 2 H), 4.45 (d, J = 5.6 Hz, 2 H), 4.23 - 4.32 (m, 1 H), 3.96 (s, 2 H), 3.92 (s, 2 H), 3.44 - 3.58 (m, 20 H), 3.12 (br s, 4 H), 1.40 (s, 9 H)

[0339] To a solution of compound 124 (200 mg, 295 pmol, 1.0 equiv.) and compound 125 (Val-Ala-PABC- Exatecan, CAS: 2845164-91-0)(308 mg, 377 pmol, 1 .3 equiv.) in DMF (2.0 mL) DIPEA (58.6 uL, 354 pmol, 1 .2 equiv ), HOBt (47.9 mg, 354 pmol, 1 .2 equiv.) and EDCI (67.9 mg, 354 pmol, 1 .2 equiv.) were added respectively. The mixture was stirred at 20-25 °C for 3 hrs. LCMS analysis showed compound I24 was consumed completely. The resultant mixture was purified directly by prep-HPLC (TFA condition) to give compound I26 (900 mg, 637 pmol, 68.3% yield) as a white solid. Purity by HPLC (220 nm): 97.3%; MS (ESI): [M+H]+:1413.5. The reactions were performed in parallel to obtain a total of 900 mg of I26.

[0340]

[0341] Compound 126 (900 mg, 637 pmol, 1.0 equiv.) was dissolved in 50% FA / DCM (9.0 mL) and stirred at 20- 25 °C for 72 hrs. LCMS analysis showed compound I26 was consumed completely. The resultant was triturated with isopropyl ether (10 mL) at 0-5 °C to give compound I27 (500 mg, crude) as a colourless oil. MS (ESI): [M+H]+:1356.8. The residue was purified by prep-HPLC (TFA condition) to give compound 130 (1.6 g, 1.81 mmol, 84.0% yield) as a yellow solid. Purity by HPLC (220 nm): 95.8%; MS (ESI): [M+Na]+: 882.8.

[0342] Compound 130 (1.5 g, 1.69 mmol, 1.0 equiv.) was first dissolved in 50% formic acid in CH2CI2 (15.0 mL) and stirred at 20-25 °C for 24 h. LC-MS analysis showed compound I30 was consumed and detected the desired mass. The residue was purified by prep-HPLC (AcOH condition) to give compound 131 (750 mg, 95.9 pmol, 84.0% yield) as a yellow solid. Purity by HPLC (220 nm): 92.5%; MS (ESI): [M+Na]+:782.8.

[0343]

[0344] 13-2

[0345] To a solution of compound I27 (500 mg, 368 pmol, 1.0 equiv.) and compound 131 (392 mg, 479 pmol, 1.3 equiv.) in DMF (5.0 mL), DIPEA (152 uL, 921 pmol, 2.5 equiv ), HOBt (74.7 mg, 552 pmol, 1.5 equiv.) and EDCI (105 mg, 552 pmol, 1 .5 equiv.) were added successively. The mixture was stirred at 20-25 °C for 2 h. LC-MS analysis showed compound 127 was consumed, and the desired mass was identified. The reaction mixture 132 was directly used to the next step without further purifications. MS (ESI): [(M+2H) / 2]2+:1062.5.

[0346] 133

[0347] To the above solution of compound 132, triethylamine (1 .0 mL) was added. The reaction mixture was stirred at 20-25 °C for 4 h. LC-MS showed compound 132 was consumed completely, and the desired mass was detected. The resultant mixture was directly purified by prep-HPLC (TFA condition) to give compound 133

[0348] (200 mg, 105 pmol, 28.5% purity) as a white solid. Purity by HPLC (220 nm): 92.9%; MS (ESI): [(M+2H) / 2]2+:950.5.

[0349] To a solution of compound 133 (85 mg, 42.2 pmol, 1.0 equiv.) and 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13- tetraoxa-4-azapentadecan-15-oic acid I34 (36.4 pL, 82.4 pmol, 2.0 equiv.) in DMF (1 .0 mL) DIEA (17.4 pL, 105 pmol, 2.5 equiv.), HOBt (11.4 mg, 84.4 pmol, 2.0 equiv.) and EDCI (16.1 mg, 84.4 pmol, 2.0 equiv.) were added successively. The mixture was stirred at 20-25 °C for 1 hr. LC-MS showed compound I33 was consumed completely and detected the desired mass. The reaction mixture 135 was directly used for the next step. MS (ESI): [(M+2H) / 2]2+:1155.9.

[0350]

[0351] To the solution of compound 135, in DMF (0.8 mL), triethylamine (0.2 mL) was added. The mixture was stirred at 20-25 °C for 1 hr. LCMS showed compound I35 was consumed completely, and detected the desired mass. The resultant reaction mixture was purified directly by prep-HPLC (TFA condition) to give compound 136 (55 mg, 26.3 pmol, 62% yield) as a white solid. Purity by HPLC (220 nm): 98.5%; MS (ESI): [(M+2H) / 2]2+: 1045.1.

[0352]

[0353] LP 2

[0354] To a solution of compound I36 (40 mg, 19.1 pmol, 1.0 equiv.) and DBCO-OSu (15.4 mg, 38.3 pmol, 2.0 equiv.) in DMF (400 pL) was added NMM (3.8 mg, 38.3 pmol, 4.2 pL, 2.0 equiv.). The mixture was stirred at 20-25 °C for 1 h and monitored by LC-MS analysis. Upon completion, the reaction mixture was concentrated and the the residue was purified by prep-HPLC (NH4HCO3 condition) to give LP-2 (15 mg) as a white solid. Purity by HPLC (220 nm): 95.6%; MS (ESI): [(M+2H) / 2]2+:1188.7.1H NMR (400 MHz, DMSO- cfe) 6 ppm 9.94 - 10.04 (m, 2 H), 8.93 (s, 1 H), 8.37 (d, J = 8.3 Hz, 4 H), 8.04 (s, 1 H), 7.91 - 8.01 (m, 4 H), 7.71 - 7.78 (m, 3 H), 7.64 - 7.69 (m, 1 H), 7.57 - 7.62 (m, 4 H), 7.23 - 7.51 (m, 16 H), 7.18 - 7.19 (m, 1 H), 6.51 (s, 1 H), 5.44 (s, 1 H), 5.27 (br s, 2 H), 5.08 (br s, 3 H), 4.52 (s, 1 H), 4.27 - 4.42 (m, 3 H), 4.15 (s, 2 H), 3.93 (br s, 3 H), 3.41 - 3.60 (m, 36 H), 3.03 - 3.14 (m, 4 H), 2.87 (s, 3 H), 2.57 - 2.62 (m, 8 H), 2.35 (s, 3 H), 2.17 - 2.26 (m, 3 H), 1.94 - 2.03 (m, 4 H), 1.30 (br d, J = 7.1 Hz, 6 H), 1.24 (s, 3 H), 1.19 (d, J = 6.8 Hz, 6 H), 0.85 - 0.90 (m, 9 H), 0.82 (br d, J = 6.6 Hz, 6 H).

[0355] Example 3 - ADC synthesis

[0356] ADC-1 comprises: (i) the antibody formed by the polypeptides having the amino acid sequences SEQ ID

[0357] NO:68 and SEQ ID NO:11 (i.e. sacituzumab comprising hlgG1 -derived Fc region including the amino acid substitution N297A (EU numbering)); (ii) the linker LM3, conjugated via MTGase-catalysed transpeptidation reaction between the glutamine at Q295 of the antibody, and the amine group of LM3; and (iii) the linker-payload LP-2, introduced via reaction between the DBCO group of LP-2 and the azide groups of LM3.

[0358] ADC-1 was prepared according to the general method outlined below.

[0359] Antibodies were made in-house from transient expression in CHO cells based on known sequences for sacituzumab.

[0360] The ADC synthesis consists of two steps, the enzymatic addition of a linker moiety, followed by the conjugation of the payload comprising moiety. These events take place at the site of glutamine-295 (Q295), in the CH2 domain on modified antibody containing a mutation of the asparagine 297 into alanine (N297A). The first step of the process is the microbial transglutaminase (MTG or MTGase)-mediated conjugation of the linker moiety onto Q295 of the modified antibody. Having attached the click group, payload comprising moieties are introduced via metal-free click chemistry.

[0361] Materials

[0362] The following materials were used in the synthesis:

[0363] MTGase Enzyme: Activa® Tl transglutaminase (Ajinomoto), unit activity 98.56 U / g

[0364] PBS pH 7.45 (ThermoFisher)

[0365] Activated carbon (Merck)

[0366] Sodium deoxycholate (Merck)

[0367] Propylene glycol (Merck)

[0368] - DMSO (Merck)

[0369] Protein concentrators (Millipore)

[0370] 0.22 pM syringe filter (Pall)

[0371] Butyl Sepharose HP column

[0372] Enzymatic conjugation

[0373] The reagents set out in table A were combined in PBS buffer (prepared according to the manufacturer’s instructions) in a sterile glass bottle with a size of at least 2 times the reaction volume. The reaction was agitated using a magnetic stirrer bar and was incubated for 17-24 h at 25°C. This reaction gives an ADC intermediate.

[0374] Table A: Concentration of MTGase reaction components

[0375] 1Mole equivalent per mole antibody

[0376] The mixture was then filtered using a 0.22 pm PES filter, and chased with 7 mL of PBS.

[0377] Purification

[0378] The ADC intermediate formed was then cleaned by hydrophobic interaction chromatography using a Butyl Sepharose HP column, to remove excess linker and residual enzyme.

[0379] Samples were spiked 1 :1 with 50 mM sodium phosphate, 1 ,5M ammonium sulphate, pH 7.4.

[0380] Approximately 40 g / L load spiked to 750 mM ammonium sulphate was applied to the column, and eluted with 20 mM sodium phosphate, 200 mM ammonium sulphate.

[0381] Click conjugation

[0382] Payload comprising moieties were conjugated to the ADC intermediate in a one-pot reaction by reaction of the members of the click-pair groups.

[0383] Conjugation of LP-2 was achieved by combining 1 mg / mL ADC intermediate with 11 mM sodium deoxycholate, 20% DMSO in PBS (3.5 mol. equivalents per conjugation site), and stirring at 150 rpm at 25°C for 17-24 h. The mixture was then filtered using a 0.22 pm PES filter, and chased three times with 7 mL of PBS.

[0384] Removal of free payload comprising moieties

[0385] Activated carbon was used to remove the free payload comprising moieties from the reaction mixture, as follows:

[0386] Step 1 : Dilute activated carbon powder into 100 mg / mL in PBS;

[0387] Step 2: Based on the amount of ADC in the reaction mix, add 1 :1 ratio of activated carbon solution (weight : weight);

[0388] Step 3: Incubate reaction mixture with activated carbon at ambient temperature (25°C) and stir at 150 rpm for 1 h.

[0389] Suspended activated carbon was removed from the mixture using a 0.22 pm PES filter, and chased three times with 7 mL of PBS. ADCs were subsequently concentrated to a final concentration of 10-15 mg / mL using Centricon® UF (11 VC) and DF (10 DV) centrifugal filters (Millipore).

[0390] Monomeric purity

[0391] Monomeric purity was determined using size exclusion chromatography.

[0392] Samples were prepared by diluting them to 1 mg / mL in PBS. The samples were analysed on a Thermo Ultimate 3000 UPLC / Waters ACQUITY H-Class PLUS Bio System equipped with a ACQUITY UPLC Protein BEH SEC Column, 200 A, 1 .7 pm, 4.6 mm X 150 mm column and a ACQUITY UPLC Protein BEH SEC Guard Column, 200 A, 1 .7 pm, 4.6 mm X 30 mm guard column.

[0393] The analysis method was as follows: o Mobile phase: 0.2M Potassium phosphate buffer, 0.2M Potassium Chloride, 15% (v / v) IPA, pH 6.8 o Flow rate: 0.35 mL / min o Run time: 15 minutes o Column temperature: Room temperature o UV detection: 280 nm o Injection load: 10 pg

[0394] The percentage purity results (% purity) were determined as

[0395] % purity = (Monomer peak area / Total peak area) x 100%. ii) Potency determination

[0396] The binding potency to the target antigen relative to a refence material was evaluated by ELISA.

[0397] The potency determination was carried out on a Molecular Devices, SpectraMax iD3 plate reader, equipped with a BioTek 405 TS plate washer.

[0398] The following reagents were used during the potency determination:

[0399] • Wash buffer: PBS with 0.05% Tween 20

[0400] • Blocking buffer: PBS with 1 % BSA

[0401] • Coating antigen: His tag target antigen, 1 ug / mL

[0402] • Secondary antibody: Anti-human IgG Fc antibody (HRP), 1 :7000 (abeam, #ab97725)

[0403] • TMB solution: 1 -Step™ turbo TMB-ELISA Substrate Solution (Thermo scientific, #34022)

[0404] • Stop solution: ELISA stop solution (Invitrogen, SS04)

[0405] The test was carried out as follows, briefly, a 96-well plate was coated with 100 pl / well of coating antigen overnight (or up to 72hrs) at 4°C. The plate was then blocked by washing the plate three times with wash buffer (200 pl / well) and then blocking the plate with blocking buffer (200 pl / well) and subsequently incubating at room temperature for 1 hour. Samples were then added to the wells by washing the plate three times with wash buffer (200 pl / well) and then adding serially diluted samples (100 pl / well) and subsequently incubating at room temperature for 1 hour. Secondary antibody addition was carried out by washing the plate three times with wash buffer (200 pl / well) and adding diluted secondary antibody (1 :7000) to each well (100 pl / well) and incubating for 1 hour at room temperature in the dark. This was followed by TMB addition, which involved adding TMB equilibrated to room temperature (100 pl / well), the plate was then incubated at room temperature for 15 minutes in the dark. The reaction was then stopped by adding stop solution equilibrated to room temperature (100 pl / well) and the plates were read at 450 nm on a plate reader.

[0406] Results were generated by preparing a 4-parameter logistic dose-response curve to compute the ECso values of the samples and reference. The relative potency of the samples was determined by ECso of reference / ECso of sample x 100%.

[0407] Drug-to-Antibody Ratio (DAR)

[0408] DAR was determined by reversed phase liquid chromatography-mass spectrometry (RPLC-MS). DAR analysis was used to determine the average number of payloads and linkers attached to the Fc region of the ADC.

[0409] Samples were prepared by reducing 5 pg of ADC in 10 mM DTT at 40°C for 30 min and injecting a 5 pL aliquot for analysis.

[0410] The samples were analysed on a Waters ACQUITY UPLC H-Class PLUS Bio System instrument equipped with a Waters TUV and Xevo® G2-XS QTof Mass Spectrometer detector and an ACQUITY UPLC Protein BEH C4 column, 300 A, 1 .7 pm, 2.1 mm X 50 mm (#186004495).

[0411] The analysis method was as follows:

[0412] Column Temperature: 70°C

[0413] Mobile phase A: Water + 0.1 % formic acid

[0414] Mobile phase B: Acetonitrile + 0.1 % formic acid

[0415] Wavelength: 280 nm

[0416] Effective gradient: linear increase from 20 to 80 % of solvent B within 1 to 3.5 min at the flow rate of 0.4 mL / min

[0417] Xevo G2-XS: MS scan from 350 to 4000 m / z, ESI positive, sensitivity mode

[0418] The results were processed according to the following method. Analyte peaks time window: input time window range which covers the whole region of the peak and expected RT (i.e. mid-point of the time window range); background subtract results 5 % from baseline. The MaxEntl deconvolution parameters were as follows: input m / z range; output mass range; TOF resolution 20,000. The mass error tolerance was 100 ppm. The amino acid modifiers were input relevant modifiers such as -Lysine C-TERM, Pyroglutamic acid E N-TERM and linker-payload; select type as variable and maximum modification of 1. After processing and inspection of the deconvoluted spectra for identified species the overall DAR for each payload (e.g. exatecan and berzosertib), was calculated according to the following equation:

[0419] DAR = 2*£(DAR of payload* relative abundance / 100)

[0420] The results of this analysis are given in table C. Table C

[0421] Example 4 - Functional characterization of ADC-1

[0422] 4.1 Analysis of binding of TROP2-specific ADCs to TROP2-expressinq cells

[0423] Binding of the following antigen-binding molecules to TROP2-expressing cells was analysed by flow cytometry:

[0424] • ADC-1 (see Example 3).

[0425] • Sacituzumab N297A (S-N297A). S-N297A is the antibody formed by the polypeptides having the amino acid sequences SEQ ID NO:68 and SEQ ID NO:11 .

[0426] • Sacituzumab govitecan (DrugBank Acc. No. DB12893; marketed as Trodelvy®; Medchem Express Cat. No. HY-132254). Sacituzumab govitecan comprises SN-38 conjugated to a humanised anti-TROP2 antibody (RS7) via a hydrolysable linker.

[0427] • Isotype-Exatecan (Iso-Exa; Synaffix Cat. No. SNFX-MW2945). This is an isotype-matched control antibody conjugated to exatecan.

[0428] Binding of ADC-1 , S-N297A, Sacituzumab govitecan and Iso-Exa to cells of the following TROP2- expressing human cancer cell lines was evaluated:

[0429] • NCI-H2122 (CRL-5985) - non-small cell lung adenocarcinoma

[0430] • HCC827 (CRL-2868) - non-small cell lung adenocarcinoma, comprising E746-A750 deletion in EGFR

[0431] • T47D (HTB-133) - breast ductal carcinoma

[0432] Binding to the cells of NCI-H2030 (CRL-5914; non-small cell lung adenocarcinoma), which do not express TROP2, was also evaluated.

[0433] Briefly, cells maintained in culture in vitro were harvested with accutase (STEMCELL technologies, Cat. No. 07920) and stained with Zombie NIR fixable live / dead stain (BioLegend, Cat. No. 423106) in PBS (at 1 :3000 in PBS) at 4°C, for 15 min. Cells were then washed twice with FACS buffer (PBS + 2mM EDTA + 1% BSA). Cells were then seeded in wells of 96-well plates at 80,000 cell / well. The indicated antigenbinding molecules were then added to wells in up to a 12-point, 10-fold dilution series, starting from a concentration of 1 pg / mL, and wells were incubated for 1 h at 4°C. Cells were subsequently washed twice with FACS buffer, and stained with Alexa Fluor™ 647-conjguated goat anti-Human IgG (H+L) Cross- Adsorbed Secondary Antibody (Thermo Fisher Scientific, Cat. No. A21445) at a 1 :1000 dilution, and incubated for 30 min at 4°C. Cells were subsequently washed twice with FACS buffer, and events were acquired using a Cytek Northern Lights flow cytometer. The data were analyzed using FlowJo software (version 10.1). The percentage of live cells that were stained with the antigen-binding molecules was determined. Graphs were plotted using GraphPad PRISM 10, fitting the data points to a four-parameter logistic model. EC50 (M) values for the % binding ( / .e. the percentage of the live cells that are positive for TROP2 as determined by staining with the relevant antigen-binding molecule) and mean fluorescence intensities (MFIs) were determined.

[0434] The results are shown in Figures 1 A and 1 B. ADC-1 , sacituzumab govitecan and S-N297A were found to display a similar ability to bind to TROP2-expressing cells. None of the molecules bound to cells of the TROP2-negative cell line NCI-H2030.

[0435] 4.2 Analysis of internalization of TROP2-specific ADCs by TROP2-expressing cells Internalization of different antigen-binding molecules by TROP2-expressing cells was analysed using the Incucyte® S3 Live-Cell Analysis System (Sartorius).

[0436] Briefly, NCI-N87 cells (CRL-5822; gastric carcinoma; 9,000 cells / well), HCC827 cells (3,000 cells / well) and NCI-H2122 cells (10,000 / well) were seeded in wells of a 96-well flat bottom plate (Corning, #3595) in 80 pL / well of culture medium, and cultured for 24 h at 37°C, 5% CO2. ADC-1 , T-DXd or Iso-Exa (see Example 4.1) were conjugated to Incucyte® Human Fabfluor-pH Antibody Labeling Dye for Antibody Internalization (Sartorius, Cat. No. 4722) in accordance with the manufacturer’s instructions. Cells were then incubated with 20 pl of freshly-prepared FabFluor-conjugates in duplicate, at a final concentration of 10 nM (HCC827 cells) or 3 nM (NCI-N87 and NCI-H2122 cells). Plates were then incubated at 37°C, 5% CO2 for 5 days (HCC827 and NCI-87 cells) or 3 days (NCI-H2122 cells), within the Incucyte® S3 Live-Cell Analysis System machine. Adherent Cell-by-Cell module was applied and 4 images / well were captured every 2 h at 20x magnification during the incubation period. The data were processed using the Cell-by- Cell analysis module in IncuCyte 2022revB software. Parameters from four images captured within each well were analyzed, and data from the mean red fluorescence intensity per cell were normalized to t = 0 using the IncuCyte 2022revB software. Graphs were plotted using GraphPad PRISM 10.

[0437] The results are shown in Figures 2A to 2C.

[0438] ADC-1 and sacituzumab govitecan were internalized rapidly by NCI-H2122 cells, and reached saturation within 7 h (Figure 2A). S-N297A was also internalized rapidly by NCI-H2122 cells and reached saturation within 7 h, although the extent of internalization was slightly lower than for ADC-1 and sacituzumab govitecan (Figure 2A). Iso-Exa was not internalized, indicating that the internalization observed for ADC- 1 , sacituzumab govitecan and S-N297A was target antigen-specific (Figure 2A).

[0439] ADC-1 and sacituzumab govitecan were both internalized by HCC827 cells, and reached saturation within 70-80 h (Figure 2B). Sacituzumab was also internalized by HCC827 cells and reached saturation within 110 h, although the extent and rate of internalization was slightly lower than for ADC-1 and sacituzumab govitecan (Figure 2B). Iso-Exa was not internalized, indicating that the internalization observed for ADC-1 , sacituzumab govitecan and S-N297A was target antigen-specific (Figure 2B). ADC-1 and sacituzumab govitecan were internalized rapidly by NCI-N87 cells, and reached saturation within 36 h (Figure 2C). S-N297A was also internalized rapidly by NCI-N87 cells and reached saturation within 32 h, although the extent and rate of internalization was slightly lower than for ADC-1 and sacituzumab govitecan (Figure 2C). Iso-Exa was not internalized, indicating that the internalization observed for ADC-1 , sacituzumab govitecan and S-N297A was target antigen-specific (Figure 2C).

[0440] 4.3 Analysis of efficacy of TROP2-specific ADCs to kill TROP2-expressing cancer cells in vitro Antigen-binding molecules were evaluated for their ability to kill TROP2-expressing cancer cell lines, NCI-H2122, T47D and HCC827, using the CellTiter-Glo® 2D Cell Viability Assay (Promega, #G9242) and the CellTiter-Glo® 3D Cell Viability Assay (Promega, #G9682).

[0441] For NCI-H2122 cells, cells were seeded at 6,000 cells / well in wells of 96-well white opaque plates, in 175 pL of cell culture medium, and then incubated for 24 h at 37°C, 5% CO2. 25 pL of samples of a dilution series (3-fold dilution series, starting from a concentration of 5.5 pg / mL) ADC-1 or Iso-Exa were subsequently added to the plates, and the wells were incubated for 5 days at 37°C, 5% CO2.

[0442] For T47D and HCC827 cells, cells seeded at 2,000 cells / well (HCC827 cells were seeded in the presence of 3% Matrigel (Corning, Cat. No. 354234)) in wells of Nunclon™ Sphera™ 96-Well, U-Shaped-Bottom Microplates, in 175 pL of cell culture medium, and then incubated for 3 days at 37“C, 5% C02 to allow spheroids to form. 25 pL of samples of a dilution series (3-fold dilution series, starting from a concentration of 5.5 pg / mL) ADC-1 or Iso-Exa were subsequently added to the plates, and the wells were incubated for 7 days.

[0443] Post-incubation, 50 pL of CellTiter-Glo reagent was added to the plates, and cells were incubated for 25 mins with gentle shaking at 600 rpm. Cell viability was analysed by measurement of luminescence using Victo Nivo (Perkin Elmer). Background signal was subtracted based on luminescence values from wells having media only (i.e. no cells). Percent inhibition of luminescence was calculated using the formula 100- {(Lum of cells treated with drug / Lum of cells treated with buffer control) *100} , Lum= Luminescence. Average (mean) luminescence values and SEMs were calculated and graphs were plotted using GraphPad PRISM 10.

[0444] The results are shown in Figures 3A and 3B. ADC-1 was found to display dose-dependent killing of the various different TROP2-expressing cancer cells, but Iso-Exa did not.

[0445] 4.4 Analysis of in vivo efficacy of TROP2-specific ADCs to treat TROP2-positive cancer Antigen-binding molecules were evaluated for their ability to inhibit the growth of tumors in (i) a HCC827 cell line-derived model of non-small cell lung adenocarcinoma, and (ii) a NCI-H2122 cell line-derived model of non-small cell lung adenocarcinoma.

[0446] HCC827 CDX model: Model: Human cell line-derived model of EGFR mutant (E746-A750 deletion), non-small cell lung adenocarcinoma

[0447] Cell line: HCC827 (CRL-2868)

[0448] Model established by: subcutaneous injection of 1 x 107cells in Matrigel into right flanks of 6-8 week old female NOD SCID mice.

[0449] Test agents: ADC-1 (cone. 7.008 mg / mL), sacituzumab govitecan (cone. 10 mg / mL)

[0450] Treatment: mice were administered a single dose of the relevant agent by intravenous (IV) injection, when average tumor volume reached 150 mm3.

[0451] Treatment groups:

[0452] Vehicle control: phosphate buffered saline (n = 7)

[0453] 3 mg / kg ADC-1 (n = 7)

[0454] 3 mg / kg sacituzumab govitecan (n = 7)

[0455] 9 mg / kg ADC-1 (n = 7)

[0456] 9 mg / kg sacituzumab govitecan (n = 7)

[0457] Tumor volumes were measured 2 times a week using a digital caliper, and calculated using the formula [L x Wx W) / 2], Tumor growth inhibition (TGI) was calculated as (1 - ([tumor volume of treatment- day 0] / [tumor volume of vehicle- day 0], Mean TGI and SEMs were calculated and graphs were plotted using GraphPad PRISM 10.

[0458] The results are shown in Figures 4A and 4B. A single dose of ADC-1 at 3 mg / kg achieved 95% TGI up to day 35 in this model, while sacituzumab govitecan achieved 18% TGI (Figure 4A). At 9 mg / kg, treatment with ADC-1 led to sustained tumor regression up to day 50 in this model. Sacituzumab govitecan showed minimal TGI <5 % at single dose of 9 mg / kg (Figure 4B). Thus, ADC-1 was found to be more potent than sacituzumab govitecan at the same concentration and dosing regimen, with prolonged duration of response up to day 50.

[0459] NCI-H2122 CDX model:

[0460] Model: Human cell line-derived model of (EGFR wildtype), non-small cell lung adenocarcinoma

[0461] Cell line: NCI-H2122 (CRL-5985)

[0462] Model established by: subcutaneous injection of 1 x 106cells in Matrigel into right flanks of 6-8 week old female NCr Nude mice.

[0463] Test agents: ADC-1 (cone. 7.008 mg / mL), sacituzumab govitecan (cone. 10 mg / mL)

[0464] Treatment: mice were administered a single dose of the relevant agent by intravenous (IV) injection, when average tumor volume reached 150 mm3.

[0465] Treatment groups:

[0466] Vehicle control: phosphate buffered saline (n = 6)

[0467] 3 mg / kg ADC-1 (n = 6)

[0468] 3 mg / kg sacituzumab govitecan (n = 6)

[0469] 9 mg / kg ADC-1 (n = 6)

[0470] 9 mg / kg sacituzumab govitecan (n = 6) Tumor volumes were measured 2 times a week using a digital caliper, and calculated using the formula [L x Wx W) / 2], Tumor growth inhibition (TGI) was calculated as (1 - ([tumor volume of treatment - day 0] I [tumor volume of vehicle - day 0], Mean TGI and SEMs were calculated and graphs were plotted using GraphPad PRISM 10.

[0471] The results are shown in Figures 5A and 5B. A single dose of ADC-1 at 3 mg / kg achieved 63% TGI in this model (Figure 5A). At 9 mg / kg ADC-1 achieved 78.2% TGI up to day 38, while sacituzumab govitecan displayed 0% TGI at this dose at day 38 post-dosing. ADC-1 was found to be significantly more effective at inhibiting tumor growth than sacituzumab govitecan, at the same concentration and under the same dosing regimen, with prolonged duration of response up to day 42.

[0472] Example 5- Nonhuman Primate (NHP) Study

[0473] ADC-1 was administered to Cynomolgus monkeys to determine toxicity and pharmacokinetics (PK) in NHP.

[0474] Toxicity

[0475] A total of 4 monkeys were randomly assigned to 2 groups (1 sex / group). ADC-1 was administered at 50 mg / kg or 80 mg / kg. A total of two doses were administered to each monkey on Days 1 and 15 followed by a 2-week recovery period after the second dose. All study animals were evaluated for body weight, biochemistry, and haematology. Gross necropsy was performed at the end of the recovery period followed by histopathology of gross lesions, if any, and all major organs.

[0476] In group 1 (50 mg / kg), in-life phase was completed. No significant change in body weight was observed. Biochemistry and haematology parameters were within the normal physiologic range. Forelimb and hindlimb skin darkening was potentially test article related but not considered adverse. In group 2 (80 mg / kg), in-life phase was completed. Forelimb and hindlimb skin darkening was observed in both NHPs. Decrease in body weight, neutrophils and white blood cell counts, along with skin ulceration and small thymus in female NHP were observed. Overall, ADC-1 was tolerated and did not result in any unanticipated toxicity in NHP up to 80 mg / kg. No significant test article related change in body weight, hematology and clinical chemistry parameters relative to pre-dose was observed in group 1 and group 2.

[0477] Results are shown in figures 6A-6H. The dotted lines in figures 6A-6H indicate the normal range in monkeys for the measured parameter.

[0478] Pharmacokinetic (PK)

[0479] Toxicokinetics of ADC-1 was assessed at 50 mg / kg and 80 mg / kg dosed at Q2Wx 2 followed by a 2- week recovery period. PK profiles of total and conjugated mAb (ADC*) were comparable with mean T1 / 2 of ~ 6 - 9 days in NHP. *ADC (conjugated mAb) was estimated via amount of payload attached to captured Ab. Both conjugated exatecan (ADC-exatecan) and berzosertib (ADC-berzosertib) were measured. A dose dependent increase in Cmaxwas observed from 50 mg / kg to 80 mg / kg. Free payload concentrations were < 1 .5 ng / ml (< 2 nM). No significant accumulation of Total Ab or ADC was observed at the tested doses.

[0480] Overall, ADC-1 was stable in NHP. At 50 mg / kg, free payload levels for ADC-1 were 500-fold lower than Trodelvy. PK profiles of total and conjugated mAb were comparable, with T1 / 2 of 6-9 days in NHP. A dose dependent increase in Cmax was observed from 50 mg / kg to 80 mg / kg. The results are shown in Tables 1 to 6 and figures 7A, 7B, 8A, 8B, 9A, and 9B. ADC-exatecan and ADC-berzosertib refer to conjugated exatecan and conjugated berzosertib, respectively. Free exatecan and free berzosertib refer to free payload exatecan and free payload berzosertib, respectively. Table t

[0481] Table 2

[0482] Table 3

[0483] Table 4

[0484] Table 5

[0485] [1] Trodelvy data was obtained from a 3 month intermittent dosing (QWX2; 4 cycles) study followed by recovery. See, NDA / BLA Multi-Disciplinary Review and Evaluation BLA 7611 15, Trodelvy, sacituzumab govitecan-hziy, Center for Drug Evaluation and Research. ADC-1 data was from a non-GLP study (Q2WX2, 2 week recovery). Dose 1 data summarized.

[0486] * For ADC-1 conjugated Ab / ADC was derived from quantification of payload attached to captured Ab / ADC. Similar profile was obtained when calculated using conjugated exatecan or berzosertib. Conjugated exatecan (ADC-exatecan) data shown.

[0487] Example 6- Nonhuman Primate (NHP) Study at 50 mg / kg and 80 mg / kg

[0488] ADC-1 was administered to Cynomolgus monkeys to determine toxicity and pharmacokinetics (PK) in NHP.

[0489] A total of 4 monkeys were randomly assigned to 2 groups (1 sex / group). ADC-1 was administered at 50 mg / kg or 80 mg / kg by intravenous slow bolus injection (over 3-5 minutes) once every two weeks for 15 days. A total of two doses were administered to each monkey on Days 1 and 15 followed by a 2-week recovery period after the second dose. Monkeys were approximately 2.5 to 5 years of age, and body weights at dosing initiation ranged from 2 to 4 kg.

[0490] Pathology

[0491] Gross necropsy was performed at the end of the recovery period followed by histopathology of gross lesions, if any, and all major organs. A complete necropsy was conducted on Study Day 30. Gross observations were noted from the carcass and musculoskeletal system, all external surfaces and orifices, cranial cavity and external surface of the brain, thoracic, abdominal, and pelvic cavities with their associated organs and tissues, gross lesions, and oral cavity was examined. Bone marrow smears from a rib were prepared for study animals euthanized at scheduled necropsy. Organs were weighed at the time of scheduled necropsy. Relative (to brain weight and to body weight) organ weights were calculated based on brain weight and terminal body weight.

[0492] For all study animals, all tissue masses and gross lesions were collected. Tissues were trimmed and fixed in 10% neutral buffered formalin (NBF) with the exception of the eyes with optic nerves, testes and epididymides, which were fixed in Modified Davidson’s solution for 24 to 72 hours. After fixation, these tissues were transferred to 10% NBF.

[0493] Representative samples of selected organs and all gross lesions and masses from all groups were removed from fixative, trimmed, processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Only the high-dose group was examined microscopically initially. From the low- dose group, slides were examined microscopically only for 1) gross lesions and masses, 2) tissues with potential compound-related effects (target organs) based on experimental findings from high-dose group. Microscopic lesions were noted and their incidences were tabulated.

[0494] Overall, no ADC-1 -related mortality occurred. Absolute and relative (to brain and body weight) thymus weights were low in females with a slight dose response. The relationship to ADC-1 was uncertain. ADC- 1 -related macroscopic changes were noted at the injection sites, and skin of the forelimbs and hindlimbs. Small thymus was noted in both females and correlated with decreased cellularity of the cortex in one of the animals; however, the relationship to ADC-1 was unknown.

[0495] ADC-1 -related microscopic changes occurred in the bone marrow (sternum), injection sites, and the skin of the forelimbs. Bone marrow findings (mild increased cellularity of the erythroid lineage) correlated with decreased hematocrit (HCT) and increased reticulocyte count (#RET) and reticulocyte percent (%RET). These findings were considered compensatory changes due to lower erythrocyte numbers at earlier time points. The bone marrow change in one of the animals (decreased cellularity of hematopoietic cells) was possibly related to ADC-1 ; however, it differed from the change noted in the other animals, and additional evidence of inflammation and hindlimb skin ulcer were noted, suggesting confounding findings. Microscopic findings in the skin of the forelimbs included mild, diffuse melanin pigment of the epithelium, mild to moderate, diffuse, subcutaneous hemorrhage, and mild to moderate subcutaneous fibrosis. Microscopic findings at the injection sites and the skin were minor and likely partially related to the dosing procedure (IV slow bolus).

[0496] In conclusion, administration of ADC-1 to Cynomolgus monkeys once every 14-days for two doses in total by intravenous slow bolus injection at dosages of 50 or 80 mg / kg / dose was well tolerated. Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) is 80 mg / kg / dose.

Claims

Claims:1 . An antigen-binding molecule that binds to TROP2, comprising (i) a TROP2-binding moiety, and (ii) a linker-payload moiety, wherein the linker-payload moiety is:

2. The antigen-binding molecule according to claim 1 , wherein the linker-payload moiety is conjugated to a glutamine residue of the TROP2-binding moiety.

3. The antigen-binding molecule according to claim 1 or claim 2, wherein the TROP2-binding moiety is an antibody or an antigen-binding fragment thereof.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the ratio between the linker-payload moiety and the TROP2-binding moiety is from about 1 :1 to about 2:1 .

5. The antigen-binding molecule according to claim 4, wherein the ratio between the linker-payload moiety and the TROP2-binding moiety is about 2:1 .

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the drug-to-antibody ratio (DAR) for exatecan is about 4.

7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the drug-to-antibody ratio (DAR) for berzosertib is about 4.

8. The antigen-binding molecule according to any one of claims 1 to 7, wherein the drug-to-antibody ratio (DAR) for exatecan and berzosertib is about 8.

9. The antigen-binding molecule according to any one of claims 1 to 8, wherein the TROP2-binding moiety comprises:(i) a heavy chain variable (VH) region incorporating the following CDRs:HC-CDR1 having the amino acid sequence of SEQ ID NO: 13HC-CDR2 having the amino acid sequence of SEQ ID NO: 14HC-CDR3 having the amino acid sequence of SEQ ID NO: 15; and(ii) a light chain variable (VL) region incorporating the following CDRs:LC-CDR1 having the amino acid sequence of SEQ ID NO:21 LC-CDR2 having the amino acid sequence of SEQ ID NO:22 LC-CDR3 having the amino acid sequence of SEQ ID NO:23.

10. The antigen-binding molecule according to any one of claims 1 to 9, wherein the antigen-binding moiety that binds to TROP2 comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:12; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NQ:20.11 . The antigen-binding molecule according to any one of claims 1 to 10, wherein the antigen-binding moiety that binds to TROP2 comprises: a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:10; and a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:11 .

12. The antigen-binding molecule according to any one of claims 1 to 11 , wherein the duration of an anticancer response observed in subjects having a TROP2-expressing cancer following treatment with the antigen-binding molecule is greater than the duration of the anti-cancer response observed in comparable subjects following treatment with an equivalent dose of an anti-TROP2 TOP1 i ADC.

13. A composition comprising an antigen-binding molecule according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

14. An antigen-binding molecule according to any one of claims 1 to 12, or a composition according to claim 13, for use in a method of medical treatment or prophylaxis, or in a method of diagnosis or prognosis.

15. An antigen-binding molecule according to any one of claims 1 to 12, or a composition according to claim 13, for use in treating or preventing a cancer.

16. Use of an antigen-binding molecule according to any one of claims 1 to 12, or a composition according to claim 13, in the manufacture of a medicament for treating or preventing a cancer.

17. 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 any one of claims 1 to 12, or a composition according to claim 13.

18. The antigen-binding molecule or composition for use according to claim 15, the use according to claim 16, or the method according to claim 17, wherein the cancer is selected from: a cancer comprising cells expressing / overexpressing TROP2, a solid tumour, metastatic cancer, unresectable cancer, hormone receptor-positive cancer, lung cancer, non-small cell lung cancer (NSCLC), non-small cell lung cancer adenocarcinoma (adNSCLC), squamous non-small cell lung cancer (sqNSCLC), breast cancer, hormone receptor-positive breast cancer, metastatic breast cancer, hormone receptor-positive metastatic breast cancer, HER2-positive breast cancer, HER2-positive metastatic breast cancer, HER2-negative breast cancer, metastatic HER2-negative breast cancer, unresectable HER2-negative breast cancer, hormone receptor-positive and HER2-negative breast cancer, metastatic hormone receptor-positive and HER2-negative breast cancer, unresectable hormone receptor-positive and HER2-negative breast cancer, triple-negative breast cancer (TNBC), metastatic triple negative breast cancer, unresectable triple-negative breast cancer, pancreatic cancer, prostate cancer, castration-resistant prostate cancer (CRPC), metastatic castration-resistant prostate cancer (mCRPC)), bladder cancer, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), endometrial cancer, ovarian cancer, esophageal cancer, esophageal squamous-cell carcinoma (ESCC), cervical cancer, thyroid cancer, biliary tract cancer (BTC), urinary tract cancer, urothelial cancer, metastatic urothelial cancer, and unresectable urothelial cancer.

19. The antigen-binding molecule or composition for use according to claim 15 or claim 18, the use according to claim 16 or claim 18, or the method according to claim 17 or claim 18, wherein the cancer is refractory or relapsed to treatment with a DNA damage repair inhibitor, and / or wherein the cancer is refractory or relapsed to treatment with a DNA topoisomerase I inhibitor.

20. Use of an antigen-binding molecule according to any one of claims 1 to 12, or a composition according to claim 13, to deplete or to increase killing of cells expressing TROP2.

21. An in vitro complex, optionally isolated, comprising an antigen-binding molecule according to any one of claims 1 to 12 bound to TROP2.

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