Her2 binding molecules

Abstract

An antigen-binding molecule that binds to HER2 comprises (i) a HER2-binding moiety and (ii) at least one linker-payload moiety, wherein the antigen-binding molecule comprises (a) a DNA damage response (DDR) inhibitor moiety and (b) a DNA topoisomerase I (TOP1) inhibitor moiety.

Description

Technical Field

[0001] This disclosure relates to molecular biology, and more specifically, to antibody technology. This disclosure also relates to medical treatment methods and preventative measures. Background Technology

[0002] Cancer remains a leading cause of death worldwide. Chemotherapy has good clinical efficacy, but due to its low specificity, it has significant side effects and a low treatment index. More targeted therapies, such as monoclonal antibody therapy, have good specificity but lower response rates. Antibody-drug conjugates (ADCs) are a treatment approach that selectively delivers cytotoxic payloads to tumors using antibody target specificity and have proven increasingly effective in clinical practice.

[0003] HER2 (also known as ERBB2, neu, etc.) is a member of the epidermal growth factor receptor (EGFR) family of transmembrane receptors. Overexpression of HER2 has been observed in approximately 20% of human breast cancers, and this phenomenon is associated with aggressive tumor growth and poor clinical prognosis in patients carrying this type of tumor (Slamon et al., Science (1987) 235:177-182).

[0004] Anti-HER2 antibody-drug conjugates (ADCs) are described in Rassy et al., Breast (2022) 66: 217-226, including ado-trastuzumab emtansine (DrugBank accession number DB05773; trade name Kadcyla®) and fam-trastuzumab deruxtecan-nxki (DrugBank accession number DB14962; trade name Enhertu®). Ado-trastuzumab emtansine contains the cytotoxic agent DM1 (a thiol-containing maytansine microtubule inhibitor) conjugated to the lysine side chain of trastuzumab via an MCC linker. Ado-trastuzumab emtansine contains the DNA topoisomerase I inhibitor DS-8201a (DXd) conjugated to trastuzumab via a tetrapeptide linker that can be cleaved by cathepsins. Mosele et al., Nat Med. (2023) 29(8):2110-2120, reported the results of a phase 2 trial (DAISY trial) of trastuzumab emtansine for the treatment of metastatic breast cancer. Approximately 71% of patients (125 / 177) experienced disease progression. Currently, there remains an unmet clinical need for effective treatment of HER2-expressing cancers. Summary of the Invention

[0005] In a first aspect, this disclosure provides an antigen-binding molecule that binds to HER2, comprising (i) a HER2-binding portion and (ii) at least one linker-payload portion, wherein the antigen-binding molecule comprises (a) a DNA damage response (DDR) inhibitor portion and (b) a DNA topoisomerase I (TOP1) inhibitor portion.

[0006] In some implementations, the DDR inhibitor portion is or includes a DDR inhibitor selected from the following: ATR inhibitor, PARP inhibitor, ATM inhibitor, WEE1 inhibitor, CHK1 / 2 inhibitor, DNA-PK inhibitor, or PLK1 inhibitor.

[0007] In some embodiments, the DDR inhibitor portion is or includes a DDR inhibitor, which is an ATR inhibitor. In some embodiments, the DDR inhibitor portion is or includes bezotitab.

[0008] In some embodiments, the DDR inhibitor portion is or includes a DDR inhibitor, which is a CHK1 / 2 inhibitor. In some embodiments, the DDR inhibitor portion is or includes preceptib.

[0009] In some embodiments, the DDR inhibitor portion is or includes a DDR inhibitor, which is a WEE1 inhibitor. In some embodiments, the DDR inhibitor portion is or includes adatitab.

[0010] In some embodiments, the DDR inhibitor portion is or includes a DDR inhibitor, which is an ATM inhibitor. In some embodiments, the DDR inhibitor portion is or includes AZD0156.

[0011] In some embodiments, the DDR inhibitor portion is or comprises a DDR inhibitor, which is a DNA-PK inhibitor. In some embodiments, the DDR inhibitor portion is or comprises nidicetib.

[0012] In some implementations, the TOP1 inhibitor portion is or comprises a selection of the following TOP1 inhibitors: camptothecin or its derivatives, eczetidine, eczetidine mesylate (DX-8951f). N- Glycyl-Ecinotecan, SN-38, DXd(1), DXd(2), Irinotecan, Etatenotecan, FL118, Topotecan, Gemmatonotecan, Belotetane, Drunotecan, Belotetane, Rubitecan, Letonotecan, Diflunotecan, Carontecan, Slatetane, Naminotecan, Elotetane, DRF-1042, Demonotecan, NSC606985, Gemitecan, ZBH-1205, Genz-644282, Non-CPT1, Indonotecan, Indotecan, AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021, VIP126, ZBH-01 and LMP-744.

[0013] In some implementations, the TOP1 inhibitor portion is or comprises a TOP1 inhibitor selected from: camptothecin or a derivative thereof, ixenoclase, ixenoclase mesylate (DX-8951f). N - Glycyl-Ecinotecan, SN-38, DXd(1), DXd(2). In some embodiments, the TOP1 inhibitor portion is or contains eccinotecan.

[0014] In some embodiments, the antigen-binding molecule includes a linker-payload portion comprising (a) a DDR inhibitor portion and (b) a TOP1 inhibitor portion.

[0015] In some of these implementations, the connector-payload section includes: (a) An amino group used for conjugation to the antigen-binding portion; (b) Clicking to at least one first payload comprising a portion of the first click group, wherein the first payload comprising a portion comprising a DDR inhibitor portion; (c) Click to at least one second payload containing portion of the second click group, wherein the second payload containing portion contains a TOP1 inhibitor portion; (d) Branched groups:

[0016] Where R N Selected from H and -(C 1-5 (alkylene)-C(O)OH, where one of the CH2 units can be replaced by -O-. a indicates the connection position between the amino group and the branched group; b indicates the connection position between the at least one first click group and the branched group; c represents the connection position between the at least one second click group and the branched group.

[0017] In some implementations, the HER2 binding portion includes: (i) Heavy chain variable (VH) regions containing the following CDRs: HC-CDR1 with the amino acid sequence of SEQ ID NO:15 HC-CDR2 with the amino acid sequence of SEQ ID NO:16 HC-CDR3 having the amino acid sequence of SEQ ID NO:17; and (ii) Light chain variable (VL) regions containing the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:23 LC-CDR2 having the amino acid sequence of SEQ ID NO:24 LC-CDR3 having the amino acid sequence of SEQ ID NO:25.

[0018] In some implementations, the antigen-binding portion that binds to HER2 includes: A VH domain comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:14; and The VL domain contains an amino acid sequence that has at least 70% sequence identity with the amino acid sequence of SEQ ID NO:22.

[0019] In some implementations, the antigen-binding portion that binds to HER2 includes: A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:12; and A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:13.

[0020] This disclosure also provides compositions comprising an antigen-binding molecule according to this disclosure, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0021] This disclosure also provides antigen-binding molecules or compositions according to this disclosure for use in medical treatment or prevention methods, or for use in diagnostic or prognostic methods.

[0022] This disclosure also provides antigen-binding molecules or compositions according to this disclosure for the treatment or prevention of cancer.

[0023] This disclosure also provides antigen-binding molecules or compositions according to this disclosure for the manufacture of medicaments for the treatment or prevention of cancer.

[0024] This disclosure also provides methods for treating or preventing cancer, comprising administering to a subject a therapeutically effective amount or a preventatively effective amount of an antigen-binding molecule according to this disclosure, or a composition according to this disclosure.

[0025] In some implementations, the cancer is selected from: cancers containing cells expressing / overexpressing EGFR family members, cancers containing cells expressing / overexpressing HER2, cancers containing cells not expressing EGFR family members, cancers containing cells not expressing HER2, HER2-low expression cancers, HR-positive cancers, solid tumors, bladder cancer, breast cancer, HER2-positive breast cancer, metastatic HER2-positive breast cancer, HER2-low expression breast cancer, unresectable or metastatic HER2-low expression breast cancer, HR-positive breast cancer, triple-negative breast cancer, cervical cancer, gastric cancer, HER2-positive gastric cancer, locally advanced or metastatic HER2-positive gastric cancer, cholangiocarcinoma, colorectal cancer, gastroesophageal junction cancer, gallbladder cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, HER2-positive gastroesophageal junction adenocarcinoma, locally advanced or metastatic HER2-positive gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumors, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, kidney cancer, lung cancer, non-small cell lung cancer, and other cancers containing HER2-positive EGFR ...stromal tumors, gastrointestinal stromal tumors, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, kidney cancer, lung cancer, non-small cell lung cancer, and other cancers containing HER2-positive EGFR-positive EGFR-positive EGFR-positive stromal tumors. ERBB2 Unresectable or metastatic non-small cell lung cancer, small cell lung cancer, melanoma, neuroendocrine tumors, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibroma, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer with activating mutations.

[0026] In some implementations, the cancer is refractory or relapsed to treatment with DNA damage repair inhibitors, and / or the cancer is refractory or relapsed to treatment with DNA topoisomerase I inhibitors.

[0027] This disclosure also provides the use of the antigen-binding molecule or composition according to this disclosure for consuming or increasing the killing effect on cells expressing HER2.

[0028] This disclosure also provides an in vitro complex, optionally isolated, comprising an antigen-binding molecule according to this disclosure that binds to HER2.

[0029] describe This disclosure relates to an antigen-binding molecule comprising a HER2-binding moiety and a linker-payload moiety comprising at least two distinct payload portions.

[0030] The antigen-binding molecule disclosed herein has unexpectedly advantageous properties compared to known anti-HER2 antibody-drug conjugates.

[0031] The inventors speculate that the resistance to trastuzumab therapy observed in the DAISY trial may be due to insensitivity to DXd, and noted that 65% of patients whose disease progressed after receiving trastuzumab treatment retained HER2 expression (Mosele et al., Nat Med. (2023) 29(8):2110-2120).

[0032] DNA damage response (DDR) is a key pathway for repair and may also be an important way to resist DNA damage payloads (such as TOP1 inhibitors). Synergistic anticancer effects have previously been observed with combination therapy using DDR inhibitors and TOP1 inhibitors (Thomas et al., Cancer Cell. (2021) 39(4):566-79 e7). In embodiments where the antigen-binding molecule of this disclosure comprises a linker-payload portion comprising (i) a TOP1 inhibitor moiety and (ii) a DNA damage response (DDR) inhibitor moiety, the DDR inhibitor is considered to increase the sensitivity of cancer cells to the TOP1 inhibitor. Furthermore, many TOP1 inhibitors and DDR inhibitors are not substrates of P-glycoproteins; therefore, patients receiving antigen-binding molecules comprising such payload portions are less likely to develop P-glycoprotein-mediated resistance to such therapies.

[0033] HER2 HER2 (also known as, for example, ERBB2, neu) is a protein identified by UniProtKB: P04626. The typical isoform of human HER2 has the amino acid sequence P04626-1 (v1, 1987-08-13; SEQ ID NO:1). ERBB2 The alternative splicing mRNA encoded by the gene produces six major isoforms: isoform 1 (SEQ ID NO:1), isoform 2 (SEQ ID NO:2), isoform 3 (SEQ ID NO:3), isoform 4 (SEQ ID NO:4), isoform 5 (SEQ ID NO:5), and isoform 6 (SEQ ID NO:6). Isoform 2 differs from isoform 1 in that positions 1 through 610 are deleted. Positions 1 through 686 of SEQ ID NO:1 are deleted in isoform 3. In isoform 4, positions 1 through 23 of SEQ ID NO:1 are replaced by a shorter 8-amino acid sequence. Positions 1 through 686 of SEQ ID NO:1 are deleted in isoform 5. Isoform 6 differs from isoform 5 in that positions 633 through 648 and 844 through 1255 of SEQ ID NO:1 are deleted, and positions 771 through 883 are replaced by a different amino acid sequence.

[0034] A typical isoform of human HER2 consists of a 22-amino acid N-terminal signal peptide (SEQ ID NO:8), followed by an extracellular domain (SEQ ID NO:9), a single transmembrane domain (SEQ ID NO:10), and a C-terminal cytoplasmic domain (SEQ ID NO:11). The mature form of human HER2 isoform 1 is shown in SEQ ID NO:7.

[0035] The structure and function of HER2 are described in Iqbal et al., Mol Biol Int. (2014) 2014: 852748, the entire contents of which are incorporated herein by reference. Like other members of the EGFR family, HER2 comprises a cysteine-rich extracellular domain, a lipophilic transmembrane domain, and an intracellular domain with tyrosine kinase activity. HER2 lacks any known direct activating ligands and may exist in a constitutively active state or be activated upon heterodimerization with other EGFR family members, such as EGFR and HER3. HER2 homodimerization or heterodimerization with EGFR / HER3 induces autophosphorylation of tyrosine residues in the cytoplasmic domain, thereby initiating signal transduction via a variety of intracellular pathways, most notably the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathways. HER2-mediated signal transduction leads to cell proliferation, survival, differentiation, angiogenesis, and tissue invasion. HER2-HER3 heterodimers are particularly effective stimulators of downstream pathways (especially the PI3K / Akt pathway).

[0036] The “HER2” mentioned in this article usually refers to the typical isoform of human HER2 (i.e., isoform 1), but also includes its isoforms, fragments, variants (including mutants) and homologs (i.e. homologs from other species, such as non-human mammal species (e.g., non-human primates, such as rhesus monkeys and cynomolgus monkeys; e.g., rodents, such as rats or mice).

[0037] As used herein, a protein “fragment,” “variant,” or “homology” may optionally be characterized as having at least 60% amino acid sequence identity with a reference protein (e.g., a typical isoform of a human protein), such as ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% amino acid sequence identity. In some embodiments, the fragment / variant / isoform / homology is characterized by its ability to perform the function performed by the reference protein.

[0038] A “fragment” typically refers to a portion of a reference protein. A “variant” typically refers to a protein whose amino acid sequence, relative to the amino acid sequence of a reference protein, contains one or more amino acid substitutions, insertions, deletions, or other modifications, but still maintains a considerable degree of sequence identity with the amino acid sequence of the reference protein (e.g., at least 60%). An “isoform” typically refers to a variant of a reference protein expressed by a species of the same species as the reference protein (e.g., human HER2 isoforms 1 through 6 are isoforms of each other). A “homologous” typically refers to a variant of the reference protein produced by a different species relative to the species to which the reference protein belongs. For example, human HER2 isoform 1 (P04626-1 v1; SEQ ID NO:1) and mouse HER2 (UniProt:P70424-1 v3, 2005-09-27) are homologs. Homologous compounds include orthologs.

[0039] The length of the “fragment” (in terms of amino acid count) can be any value, although its length may optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment originates), and its maximum length may be one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein. The minimum length of the HER2 fragment can be one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1250 amino acids, and the maximum length can be one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1250 amino acids.

[0040] Isotypes, fragments, variants, or homologs may optionally be functional isotypes, fragments, variants, or homologs having, for example, the functional properties / activities of a reference HER2 (e.g., human HER2 isotype 1), as determined by analysis using appropriate functional property / activity assays. For example, isotypes, fragments, variants, or homologs of HER2 may be associated with HER3 or EGFR.

[0041] In some implementations, HER2 is a HER2 derived from mammals (e.g., primates (rhesus monkeys, cynomolgus monkeys, non-human primates, or humans) and / or rodents (e.g., rats or mice). Isotypes, fragments, variants, or homologs of HER2 may optionally be characterized as having at least 60% amino acid sequence identity with an immature or mature HER2 isotype from a particular species (e.g., humans), such as ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity.

[0042] In some embodiments, HER2 comprises or consists of an amino acid sequence having at least 60% amino acid sequence identity with any of the sequences in SEQ ID NO:1 to 7, for example, 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.

[0043] In some embodiments, the HER2 fragment comprises or consists of an amino acid sequence having at least 60% amino acid sequence identity with SEQ ID NO:7 or 9, for example, 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.

[0044] The antigen-binding portion of HER2 An "antigen-binding molecule" is a molecule that binds to a given target antigen. An antigen-binding molecule contains an antigen-binding moiety, through which it binds to its target antigen. The antigen-binding molecule disclosed herein contains an antigen-binding moiety that binds to HER2 (i.e., the HER2-binding moiety).

[0045] The antigen-binding portion may contain or be derived from antibodies (i.e., immunoglobulins (Igs)) and antigen-binding fragments of antibodies. As used herein, “antibody” includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), as well as antibody-derived antigen-binding molecules such as scFv, scFab, biantibodies, triantibodies, scFv-Fc, microantibodies, and single-domain antibodies (e.g., VhH, etc.). Antigen-binding fragments of antibodies include, for example, Fv, Fab, F(ab')2, and F(ab') fragments.

[0046] The antigen-binding portion also includes target antigen-binding aptamers, such as nucleic acid aptamers (see, for example, the review in Zhou and Rossi, Nat Rev Drug Discov. (2017) 16(3):181-202). In some implementations, the antigen-binding portion comprises or is composed of an antigen-binding peptide / peptide, such as a peptide aptamer, thioredoxin, monoclonal antibody, anticarin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affinity, nanobody (i.e., single-domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody, or fibronectin, see, for example, the review in Reverdatto et al., CurrTop Med Chem. 2015; 15(12):1082-1101, which is incorporated herein by reference in its entirety (see also, for example, Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0047] Common techniques for producing fully human antibodies include (i) phage display, in which human antibody genes are expressed in a phage display library, and (ii) antibody production in transgenic mice that have been genetically engineered to have human antibody genes (described in Park and Smolen, Advances in Protein Chemistry (2001) 56: 369-421). In short, in human antibody gene phage display technology, genes encoding the VH and VL chains are generated from "naive" human lymphocytes through PCR amplification and cloning, and assembled into a library. Using this library, these genes can be expressed as either disulfide-linked Fab fragments or single-chain Fv (scFv) fragments. The genes encoding Fab or scFv are fused to the surface capsid protein of filamentous phages. Subsequently, the library is screened using antigens to identify Fab or scFv fragments that can bind to the target. Molecular evolution or affinity maturation procedures can be used to enhance the affinity of Fab / scFv fragments. In transgenic mouse technology, mice whose endogenous mouse Ig gene loci are replaced with their human homologous sequences through homologous recombination are immunized with antigens, and monoclonal antibodies are prepared using conventional hybridoma techniques to obtain fully human monoclonal antibodies.

[0048] In some embodiments, the antigen-binding portion according to this disclosure comprises or consists of an antigen-binding region of an antibody (e.g., an antigen-binding fragment of an antibody). The antigen-binding portion may be derived from the antibody. An antibody-derived antigen-binding portion may comprise or consist of an antigen-binding region of an antibody (e.g., an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding portion may be or comprise an Fv (e.g., provided as an scFv) or Fab region of an antibody that binds to a given target antigen, or the entire antibody.

[0049] The antigen-binding portion of this disclosure can be designed and prepared using a monoclonal antibody (mAb) sequence capable of binding to a given target antigen (e.g., HER2). Antigen-binding regions of the antibody, such as variable fragments (Fv), Fab, and F(ab')2 fragments, may also be used or provided. "Antigen-binding region" refers to any fragment of the antibody capable of binding to the target specifically targeted by the antibody.

[0050] In some embodiments, the antigen-binding portion comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) capable of specifically binding to a target antigen. In some embodiments, the antigen-binding portion is or comprises the antibody's Fv (e.g., provided as an scFv). In some embodiments, the antigen-binding portion is or comprises the antibody's Fab region. In some embodiments, the antigen-binding portion is or comprises the entire antibody (i.e., comprising both the variable and constant regions).

[0051] The antigen-binding moiety may be or may contain an antigen-binding polypeptide or an antigen-binding polypeptide complex. The antigen-binding moiety may contain more than one polypeptide, which together constitute the antigen-binding moiety. The polypeptides may be bound covalently or non-covalently. In some embodiments, the polypeptides constitute a portion of a larger polypeptide containing these polypeptides (e.g., in the case where scFv contains VH and VL, or in the case where scFab contains VH-CH1 and VL-CL).

[0052] In some embodiments, the antigen-binding moiety of this disclosure comprises or is composed of a polypeptide complex formed by protein-protein interactions between the constituent peptides / polypeptides of the antigen-binding moiety. The antigen-binding moiety can refer to a non-covalent or covalent complex composed of one or more polypeptides (e.g., 2, 3, 4, 6, or 8 polypeptides), such as an IgG-like antigen-binding moiety comprising two heavy-chain polypeptides and two light-chain polypeptides.

[0053] Antibodies typically contain six complementarity-determining regions (CDRs); three are located in the heavy chain variable region (VH): HC-CDR1, HC-CDR2, and HC-CDR3, and three are located in the light chain variable region (VL): LC-CDR1, LC-CDR2, and LC-CDR3. These six CDRs collectively define the complementary sites of the antibody, i.e., the portions of the antibody that bind to the target antigen.

[0054] The VH and VL regions contain scaffold regions (FRs) located on both sides of each CDR, which provide support for the CDR. From the N end to the C end, the VH region contains the following structure: N end - [HC-FR1] - [HC-CDR1] - [HC-FR2] - [HC-CDR2] - [HC-FR3] - [HC-CDR3] - [HC-FR4] - C end; the VL region contains the following structure: N end - [LC-FR1] - [LC-CDR1] - [LC-FR2] - [LC-CDR2] - [LC-FR3] - [LC-CDR3] - [LC-FR4] - C end.

[0055] There are several different conventions for defining antibody CDR and FR, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2 described in Retter et al., Nucl. Acids Res. (2005) 33 (Supplement 1): D671-D674. The CDR and FR of the VH and VL regions of the antibody clones described herein are defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue): D413-22), which uses the IMGTV-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In a preferred embodiment, the CDR and FR of the antigen-binding molecule mentioned herein are defined according to the IMGT information system.

[0056] In some embodiments, the antigen-binding moiety according to this disclosure comprises or consists of an Fv region that binds to HER2. In some embodiments, the VH and VL regions of the Fv are provided as a single polypeptide linked by a linker sequence, i.e., a single-chain Fv (scFv).

[0057] The VL region and light chain constant region (CL region) of the antigen-binding region of the antibody, together with the VH region and heavy chain constant region 1 (CH1 region), constitute the Fab region. In some embodiments, the antigen-binding portion comprises the Fab region, which contains VH, CH1, VL, and CL (e.g., Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide containing VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide containing VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide containing VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide containing VL and 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 a single polypeptide linked to a linker region, i.e., provided as a single-chain Fab (scFab) or a single-chain CrossFab (scCrossFab).

[0058] In some embodiments, the antigen-binding portion described herein comprises or consists of an intact antibody that binds to HER2. As used herein, an "intact antibody" is an antibody whose structure is substantially similar to that of an immunoglobulin (Ig). For example, Schroeder and Cavacini describe different types of immunoglobulins and their structures in J Allergy Clin Immunol. (2010) 125(202): S41-S52, the entire contents of which are incorporated herein by reference.

[0059] Immunoglobulins G (IgG) are glycoproteins with a molecular weight of approximately 150 kDa, consisting of two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains the VH domain, followed by the heavy chain constant region, which contains three constant domains (CH1, CH2, and CH3); similarly, the light chain contains the VL domain, followed by the CL domain. Based on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chains can be classified as kappa (κ) or lambda (λ) types.

[0060] In some implementations, the antigen-binding portion comprises or consists of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM bound to HER2.

[0061] In some embodiments, the antigen-binding portion of this disclosure comprises one or more regions of an immunoglobulin heavy chain constant sequence (e.g., CH1, hinge region, CH2, CH3, etc.). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, such as human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of human IgG1 allotypes (e.g., G1m1, G1m2, G1m3, or G1m17).

[0062] In some embodiments, the antigen-binding portion comprises one or more polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:31 or 36, for example, 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. In some embodiments, the antigen-binding portion comprises one or more polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:32, such as ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity. In some embodiments, the antigen-binding portion comprises one or more polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:33 or 48, for example, 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. In some embodiments, the antigen-binding portion comprises one or more polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:34 or 37, for example, 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. In some embodiments, the antigen-binding portion comprises one or more polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:38, 39, 49 or 50, for example, 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.In some embodiments, the antigen-binding portion comprises one or more polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequences of SEQ ID NO:30, 35, 46, or 47, for example, 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.

[0063] In some embodiments, the antigen-binding portion of this disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is the human immunoglobulin κ constant sequence (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant sequence is the human immunoglobulin λ constant sequence (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0064] In some embodiments, the antigen-binding portion comprises one or more polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequences of SEQ ID NO:40, 41, 42, 43, 44, or 45, for example, ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity.

[0065] In some embodiments described herein, one or more amino acids in the amino acid sequence mentioned herein (e.g., the amino acid sequence of an antigen-binding region, such as the amino acid sequence of the CDR or VH / VL region) are substituted by another amino acid. Substitution means replacing an amino acid residue with a different “alternative” amino acid residue. The substituted amino acid residue according to this disclosure can be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that differs from the amino acid residue at the relevant position in an equivalent unsubstituted amino acid sequence, selected from: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In some embodiments, the alternative amino acid can be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than the amino acid residues described in the preceding sentence. Examples of non-naturally occurring amino acid residues include ornithine, ornithine, homoserine, aib, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202(1991) 301-336.

[0066] In some embodiments, the substitution may be biochemically conserved. In some embodiments, if the amino acid to be substituted is provided in one of rows 1 through 5 of the table below, the substituted amino acid is another different amino acid provided in the same row:

[0067] For example, in some embodiments, if the substitution of a Met residue occurs, the alternative amino acid may be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and ortholeucine.

[0068] In some embodiments, the substituted amino acid in the substitution may have the same side chain polarity as the substituted amino acid residue. In some embodiments, the substituted amino acid in the substitution may have the same side chain charge (at pH 7.4) as the substituted amino acid residue.

[0069] In other words, in some embodiments, a nonpolar amino acid is replaced by another different nonpolar amino acid. In some embodiments, a polar amino acid is replaced by another different polar amino acid. In some embodiments, an acidic polar amino acid is replaced by another different acidic polar amino acid. In some embodiments, a basic polar amino acid is replaced by another different basic polar amino acid. In some embodiments, a neutral amino acid is replaced by another different neutral amino acid. In some embodiments, a positively charged amino acid is replaced by another different positively charged amino acid. In some embodiments, a negatively charged amino acid is replaced by another different negatively charged amino acid.

[0070] In some embodiments, the substitution may be functionally conserved. 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 containing the substitution compared to an equivalent unsubstituted molecule.

[0071] The antigen-binding molecule disclosed herein includes an antigen-binding moiety that binds to HER2.

[0072] In some embodiments, the antigen-binding portion includes a CDR of the antigen-binding portion capable of binding to HER2. In some embodiments, the antigen-binding portion includes a FR of the antigen-binding portion capable of binding to HER2. In some embodiments, the antigen-binding portion includes both a CDR and a FR of an antibody capable of binding to HER2. That is, in some embodiments, the antigen-binding portion includes a VH region and a VL region of an antibody capable of binding to HER2.

[0073] In some embodiments, the antigen-binding moiety capable of binding to HER2 according to this disclosure may be or may be derived from a HER2-binding antibody selected from: trastuzumab (DrugBank accession number DB00072; which consists of a polypeptide having the amino acid sequences of SEQ ID NO:12 and SEQ ID NO:13), pertuzumab (DrugBank accession number DB06366), maggotuximab (DrugBank accession number DB14967), telmituzumab (e.g., described in Fiedler et al., ESMOOpen. (2018) 3(4): e000381), CT-P6 (e.g., described in Jeong et al., Expert Opin Biol Ther (2019) 19(10):1085-1095), PF-05280014 (e.g., described in Paik, BioDrugs (2018) 32(5):515-518), SB3 (e.g., described in Lamb, BioDrugs (2018) 32(3):293-296), ABP-980 (e.g., described in Dhillon, BioDrugs (2018) 32(5):511-514), MYL-1410 (e.g., described in Rugo et al., JAMA (2017) 317:37-47), BCD-022 (e.g., described in Alexander et al., BMC Cancer (2020) 20:783), HD201 (e.g., described in Pivot et al., Clin Ther. (2018) 40(3):396-405.e4), and HLX22 (e.g., described in Yang et al. BioDrugs. 2022; 36(3): 393-409). In some embodiments, the antigen-binding portion is trastuzumab or is derived from trastuzumab.

[0074] In some embodiments, the antigen-binding moiety can bind to the same region of DLL3 or to an overlapping region of HER2, which is the HER2 region bound by an antigen-binding molecule comprising the VH and VL sequences of the HER2-binding antibody described above. In some embodiments, the antigen-binding moiety can bind to the same region of HER2 or to an overlapping region of HER2, which is the HER2 region bound by an antigen-binding molecule comprising the VH and VL sequences of trastuzumab (i.e., an antigen-binding molecule comprising VH having the amino acid sequence SEQ ID NO:14 and VL having the amino acid sequence SEQ ID NO:22).

[0075] In some embodiments, the antigen-binding portion is capable of binding to a polypeptide comprising or consisting of an amino acid sequence of one of SEQ ID NO: 1, 7 or 9.

[0076] The binding ability of the antigen-binding moiety to a given peptide / peptide can be analyzed by methods well known to those skilled in the art, including by ELISA, immunoblotting (e.g., Western blotting), immunoprecipitation, surface plasmon resonance (SPR; see, for example, Hearty et al., Methods Mol Biol (2012) 907:411-442) or Bio-Layer Interferometry (see, for example, Lad et al., (2015) J Biomol Screen 20(4): 498-507).

[0077] In embodiments where the antigen-binding portion can bind to a peptide / peptide comprising a reference amino acid sequence, the peptide / peptide may include one or more additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments, the peptide / peptide includes, for example, 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 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 acids provided at one or both ends of the reference sequence (i.e., the N-terminus and C-terminus) correspond to the positions at the ends of the reference sequence in the HER2 amino acid sequence.

[0078] In some embodiments, the antigen-binding moiety is capable of binding to a peptide / peptide bound by an antibody comprising the VH and VL sequences of trastuzumab (i.e., an antigen-binding molecule comprising VH having the amino acid sequence SEQ ID NO:14 and VL having the amino acid sequence SEQ ID NO:22).

[0079] In some implementations, the antigen-binding portion is capable of binding to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of the HER2-binding antibody described above.

[0080] In some embodiments, the antigen-binding moiety comprises the heavy chain CDR and light chain CDR of the HER2-binding antibody described above. In some embodiments, the antigen-binding moiety comprises the VH and VL regions of the HER2-binding antibody described above. In some embodiments, the antigen-binding moiety comprises the heavy chain polypeptide (i.e., containing the VH, CH1, CH2, and CH3 region sequences) and the light chain polypeptide (i.e., containing the VL and CL region sequences) of the HER2-binding antibody described above.

[0081] In some embodiments, the antigen-binding moiety comprises the heavy chain CDR and light chain CDR of trastuzumab. In some embodiments, the antigen-binding moiety comprises the VH and VL regions of trastuzumab. In some embodiments, the antigen-binding moiety comprises the heavy chain polypeptide (i.e., containing the VH, CH1, CH2, and CH3 region sequences) and the light chain polypeptide (i.e., containing the VL and CL region sequences) of trastuzumab.

[0082] In some implementations, the antigen-binding portion includes: VH region containing the following CDRs: HC-CDR1 with the amino acid sequence of SEQ ID NO:15 HC-CDR2 with the amino acid sequence of SEQ ID NO:16 HC-CDR3, having the amino acid sequence of SEQ ID NO:17, Or variants thereof, wherein one, two, or three amino acids in HC-CDR1, and / or one, two, or three amino acids in HC-CDR2, and / or one, two, or three amino acids in HC-CDR3 are substituted with another amino acid; and VL region containing the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:23 LC-CDR2 having the amino acid sequence of SEQ ID NO:24 LC-CDR3 having the amino acid sequence of SEQ ID NO:25; Or variants thereof, wherein one, two or three amino acids in LC-CDR1, and / or one, two or three amino acids in LC-CDR2, and / or one, two or three amino acids in LC-CDR3 are replaced by another amino acid.

[0083] In some implementations, the antigen-binding portion includes: The VH region contains an amino acid sequence that has at least 60% sequence identity with the amino acid sequence of SEQ ID NO:14, for example, one of the following: ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity; and The VL region contains an amino acid sequence that has at least 60% sequence identity with the amino acid sequence of SEQ ID NO:22, for example, ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity.

[0084] In some implementations, the antigen-binding portion comprises or consists of the following: (i) One or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:12, 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; and (ii) One or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:13, 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.

[0085] In some embodiments, the antigen-binding molecule of this disclosure (e.g., its antigen-binding portion) includes an Fc region. As used herein, an "Fc region" refers to a polypeptide complex formed by the interaction between two polypeptides, each polypeptide containing a CH2-CH3 region of a constant sequence of an immunoglobulin (Ig) heavy chain.

[0086] In this article, the “CH2 domain” refers to the amino acid sequence corresponding to the CH2 domain of immunoglobulin (Ig). According to the EU numbering system described by Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85, the CH2 domain refers to the region consisting of positions 231 to 340 in the constant region of immunoglobulin. The “CH3 domain” refers to the amino acid sequence corresponding to the CH3 domain of immunoglobulin (Ig). According to the EU numbering system described by Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85, the CH3 domain refers to the region consisting of positions 341 to 447 in the constant region of immunoglobulin. The “CH2-CH3 region” refers to the amino acid sequence corresponding to the CH2 and CH3 domains of immunoglobulin (Ig). According to the EU numbering system described by Edelman et al. in Proc Natl Acad Sci USA (1969) 63(1): 78-85, the CH2-CH3 region refers to the region consisting of positions 231 to 447 in the immunoglobulin constant region.

[0087] In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region according to this disclosure correspond to the CH2 domain / CH3 domain / CH2-CH3 region of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region correspond to the CH2 domain, CH3 domain, and / or CH2-CH3 region of human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region correspond to the CH2 domain / CH3 domain / CH2-CH3 region of human IgG1 allotypes (e.g., G1m1, G1m2, G1m3, or G1m17). In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region correspond to the CH2 domain / CH3 domain / CH2-CH3 region of human IgG1 allotype G1m3.

[0088] The Fc region can interact with Fc receptors and other molecules of the immune system to produce functional effects. Fc-mediated effector functions have been reviewed, for example, by Jefferis et al., Immunol Rev 1998 163:59-76 (into the entirety by reference), and arise 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 interactions between the Fc region and Fc receptors expressed by immune cells, through the binding of the Fc region to complement protein C1q to recruit complement pathway components, and subsequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, production of cytokines and / or chemokines, and antigen processing and presentation.

[0089] In some embodiments, the antigen-binding molecule according to this disclosure comprises one or more (e.g., two) polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:38 or 39, such as ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity. In some embodiments, the antigen-binding molecule includes an Fc region containing one or more (e.g., two) polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:38 or 39, such as ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity.

[0090] Modifications to the Fc region of antibodies that affect Fc-mediated function are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73, the entire contents of which are incorporated herein by reference. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, the antigen-binding molecule of this disclosure comprises an Fc region containing modifications for increasing or decreasing Fc-mediated function compared to an antigen-binding molecule containing a corresponding unmodified Fc region. If Fc region / CH2 / CH3 is described as containing modifications “corresponding” to a reference substitution, equivalent substitutions in homologous Fc / CH2 / CH3 are considered.

[0091] In some embodiments, the antigen-binding molecule of this disclosure includes an Fc region containing a modification. In some embodiments, the antigen-binding molecule of this disclosure includes an Fc region containing a modification in one or more of the CH2 and / or CH3 regions.

[0092] In some embodiments, the Fc region contains modifications for reducing / preventing Fc-mediated functions (e.g., ADCC, ADCP, CDC). In some embodiments, the Fc region contains modifications for reducing / preventing ADCC. In some embodiments, the Fc region contains modifications for reducing / preventing CDC. In some embodiments, the Fc region contains modifications for reducing / preventing binding to Fc receptors. In some embodiments, the Fc region contains modifications for reducing / preventing binding to Fcγ receptors. In some embodiments, the Fc region contains modifications for reducing / preventing glycosylation of amino acid residues corresponding to N297.

[0093] In some embodiments, the Fc region contains a modification at an amino acid residue corresponding to N297. In some embodiments, the Fc region contains a modification corresponding to N297A, N297Q, or N297G, as described in Leabman et al., Mabs. (2013) 5:896–903. It is known that replacing "N297" with "A", "G", or "Q" can eliminate glycosylation, thereby reducing the binding of Fc to C1q and Fcγ receptors, and consequently reducing CDC and ADCC. In some embodiments, the Fc region contains a modification corresponding to N297A.

[0094] In some embodiments, the antigen-binding molecule according to this disclosure comprises one or more (e.g., two) polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:49 or 50, such as ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity. In some embodiments, the antigen-binding molecule comprises an Fc region containing one or more (e.g., two) polypeptides whose amino acid sequences have at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:49 or 50, such as ≥70%, ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity.

[0095] The antigen-binding molecule according to this disclosure may include a HER2-binding portion according to any of the embodiments described above, and a linker-payload portion as described below.

[0096] Connector - Payload Section The aspects and embodiments of this disclosure relate to antigen-binding molecules comprising a linker-payload portion. As used herein, a “linker-payload portion” means a portion comprising one or more payload portions and a linker portion for linking the payload portions to an antigen-binding region of the antigen-binding molecule.

[0097] The payload portion of this disclosure contains or consists of a cytotoxic agent. The payload portion is described in the following references: for example, Parslow et al., Biomedicines. Sep 2016; 4(3):14; Goundry and Parker, Org. Process Res. Dev. (2022) 26, 8, 2121–2123; Fu et al., Signal Transduction and Targeted Therapy (2022) 7:93; Wang et al., Acta Pharmaceutica Sinica B (2023) 13(10): 4025-4059; and Conilh et al., J. Hematol. & Oncol. (2023) 16:3, all of which are incorporated herein by reference in their entirety.

[0098] Specifically, this disclosure relates to an antigen-binding molecule comprising at least one linker-payload portion, wherein the antigen-binding molecule comprises (a) a payload portion that is a DNA damage response (DDR) inhibitor and (b) a payload portion that is a DNA topoisomerase I (TOP1) inhibitor.

[0099] For the sake of brevity, in the following text, "the payload portion as a DDR inhibitor" can be simply referred to as "DDR inhibitor portion", and similarly, "the payload portion as a TOP1 inhibitor" can be simply referred to as "TOP1 inhibitor portion".

[0100] DNA damage response (DDR) is a complex network of mechanisms for detecting and repairing DNA damage to maintain genome stability. DDR has been reviewed in publications such as Groelly et al. in Nature Reviews Cancer (2023) 23:78-94 and Molinaro et al. in Cancers (Basel). (2021) 13(15): 3819, the full contents of which are incorporated herein by reference.

[0101] The detection and repair pathways of DNA damage are mediated by proteins such as ATM (ataxia-telangiectasia mutant protein) and ATR (ataxia-telangiectasia and Rad3-associated protein). ATM is a protein kinase activated by double-strand breaks in DNA, initiating downstream signaling. ATR is activated by DNA damage and replication stress, particularly responding to single-strand breaks and stalled DNA replication forks. CHK1 and CHK2 (checkpoint kinases 1 and 2) are downstream effectors of ATM and ATR; they phosphorylate various target proteins to halt cell cycle progression and promote DNA repair. PARP (poly-ADP-ribose polymerase) is involved in repairing single-strand DNA breaks, helping to recruit repair factors and form repair complexes at DNA damage sites. DNA-PK (DNA-dependent protein kinase) helps to join broken DNA ends together, performing non-homologous end joining (NHEJ) to repair double-strand breaks. DDR is promoted through cell cycle regulation by WEE1 and PLK1 (polo-like kinase 1). WEE1 is a kinase that phosphorylates and inhibits CDKs (cyclin-dependent kinases), thereby slowing down cell cycle progression and buying more time for DNA damage repair before cell division. PLK1 regulates cell cycle checkpoints and promotes repair processes through phosphorylation of Polθ. RAD51 is an ATPase involved in DNA repair. Ubiquitin-specific proteases (USPs) regulate DDR by affecting the ubiquitination of proteins involved in DDR. Protein kinase membrane-associated tyrosine / threonine 1 (PKMYT1) regulates the cell cycle and participates in DDR-related signaling. Aurora-A may contribute to G2 DNA damage checkpoints through activation of PLK1 and CDC25B and plays an important role in the mitotic DNA damage response.

[0102] Most cancer cells are more dependent on DDR than non-cancer cells. DDR inhibitors and their application in cancer treatment are described in, for example, Cheng et al., Eur J Med Chem. (2022) 230:114109, Wang et al., FrontImmunol. (2022) 13:854730, and Choi and Lee, Int J Mol Sci. (2022) 23(3):1701, all of which are incorporated herein by reference in their entirety.

[0103] In some embodiments, the DDR inhibitor portion according to this disclosure is or includes a DDR inhibitor selected from: (a) PARP inhibitors (e.g., olaparib, rucaparib, niraparib, tarazoparib, veliparib, pamipanib, cimipanib, senaparib, SC-10914, 2X-121, AMXI-5001, JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-1002, RBN-2397, fluzopanib, NMS-03305293, AZD9574); (b) ATM inhibitors (e.g., CP-466722, KU-55933, KU-60019, KU-59403, AZ31, AZ32, AZD0156, AZD1390, XRD-0394, M4076, M3541, WSD-0628, SYH-2051, IMP-08, SP-1161, INT-6C4 / 5C4); (c) ATR inhibitors (e.g., M6620 (bezotitab), M4344 (VX-803), AZD6738 (celasitab), BAY1895344 (elimosatitab), RP3500 (carmonocetib), ATRN119, ART380, IMP9064, HRS2398, M1774, IMP9064, SC0245, LF0397, NU6027); (d) WEE1 inhibitors (e.g., adatib, Debio 0123, PD0166285, PD0407824, AZD1775, ZN-c3, IMP7068, SY4835, SCO191, IMP7068); (e) CHK1 / 2 inhibitors (e.g., CBP-501, preceptib, MK-8776, GDC-0575, SRA-737, PF-00477736, AZD7762, LY2603618 (rabuselti), LY2880070, XL884, BEBT260, MU380, NU7441, KU-5778); (f) DNA-PK inhibitors (e.g., CC-115, LY-3023414, AsiDNA, M3814 (nidicetib, peposertib), VX-984 (M9831), BR-101801, XRD-0394, SL901, XZP-6877, IMP-11, ZL-2201, BR-2006, AZD7648, NU7441); (g) PLK1 inhibitors (e.g., BI-6727 (voracetyl), PCM-075 (onvatinib), CYC140 (proxetil)); (h) Polθ inhibitors (e.g., ART4215, ART6043, neomycin, RP-6685, RP-3467); (i) RAD51 inhibitors (e.g., CYT0851); (j) Inhibitors of ubiquitin-specific proteases (USP) family enzymes (e.g., inhibitors of USP11, USP7, USP4, USP37, USP39, USP45, USP24 and / or USP1; e.g., KSQ-4279); (k) PKMYT1 inhibitors (e.g., RP6306); and (l) Aurora-A inhibitors (e.g., aristotelin, WJ05129 (JS112), JAB-2485).

[0104] In some implementations, the DDR inhibitor portion is or contains celasiltib.

[0105] In some implementations, the DDR inhibitor portion is or includes bezotitab: .

[0106] In some implementations, the DDR inhibitor portion is or includes preceptib: .

[0107] In some implementations, the DDR inhibitor portion is or includes adatitab, which can be linked in the following manner, or it can be linked at other locations: .

[0108] In some implementations, the DDR inhibitor portion is or includes AZD0156, which can be connected in the following manner, or it can be connected in other locations: .

[0109] In some implementations, the DDR inhibitor portion is or includes nidicetib: .

[0110] During DNA replication and transcription, the DNA helix is ​​subjected to enormous torsional stress, which is relieved by the action of DNA topoisomerases I and II (TOP1 and TOP2). They cut the DNA strand, causing it to unwind and then rejoin the break (see, for example, Delgado et al., Biochem J. (2018) 475(2): 373–398). DNA topoisomerase inhibitors block the rejoining step, leading to DNA breakage and cell death. DNA topoisomerase I inhibitors and their application in cancer treatment have been described 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 incorporated herein by reference in their entirety.

[0111] In some embodiments, the TOP1 inhibitor portion according to this disclosure is or includes a selection of TOP1 inhibitors: camptothecin or a derivative thereof, eczetidine, eczetidine mesylate (DX-8951f). N - Glycyl-Ecinotecan, SN-38, DXd(1), DXd(2), Irinotecan, Etatenotecan, FL118, Topotecan, Gemmatonotecan, Belotetane, Drutatecan, Belotetane, Rubitecan, Letonotecan, Diflunotecan, Carontecan, Slatetane, Namitetane, Elotetane, DRF-1042, Demonotecan, NSC606985, Gemmatonotecan ZBH-1205, Genz-644282, Non-CPT1, Indonotecan (LMP-400), Indonotecan (LMP-776), AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021, VIP126, ZBH-01, and LMP-744.

[0112] In some implementations, the TOP1 inhibitor is or contains eczema: .

[0113] In some implementations, the TOP1 inhibitor is or contains beloteccan:

[0114] In some implementations, the TOP1 inhibitor portion is or contains SN38:

[0115] In some implementations, the TOP1 inhibitor portion is or includes DXd: .

[0116] According to this disclosure, the linker portion can be any portion suitable for connecting the payload portion to the antigen-binding region of the antigen-binding molecule of this disclosure. Therefore, they typically include groups capable of connecting to the payload portion, groups connecting to the antigen-binding region of the antigen-binding molecule, and a linker core.

[0117] The connective component is described, for example, in Su et al., Acta Pharmaceutica Sinica B (2021) 11(12):3889-3907, and Fu et al., Signal Transduction and Targeted Therapy (2022) 7:93. According to this disclosure, the connector portion can be a detachable connector portion or a non-detachable portion.

[0118] Cleavable linkers typically exploit the differences between the systemic circulatory environment and the cancer cell / tumor microenvironment to release payload portions in a targeted manner. Cleavable linkers include chemically cleavable linkers (e.g., linkers cleavable by acids, GSH, or Fe(II)) and enzyme-cleavable linkers (e.g., linkers cleavable by cathepsins, glycosidases, phosphatases, or sulfatases).

[0119] In some embodiments, the linker portion according to this disclosure is a chemically cleavable linker. In some embodiments, the linker portion according to this disclosure is an enzymatically cleavable linker. In some embodiments, the linker portion is an acid-cleavable linker, such as containing a hydrazone group (e.g., a 6-maleimide propionyl hydrazone linker or a (4-(4-acetylphenoxy)butyric acid) hydrazone linker), a carbonate group, or a silyl ether group. In some embodiments, the linker portion is a GSH-cleavable linker, such as containing a disulfide bond. In some embodiments, the linker portion is an Fe(II)-cleavable linker, such as containing a 1,2,4-trioxopentane group. In some embodiments, the linker portion is a cathepsin-cleavable linker, such as containing a dipeptide (e.g., a valine-citrulline linker, a phenylalanine-lysine linker, or a valine-alanine linker), a triglycyl peptide (CX), or a cBu-Cit group. In some embodiments, the linker portion is GGFG (glycine-glycine-phenylalanine-glycine). In some embodiments, the linker portion is a linker cleavable by glucuronidase, for example, containing a β-glucuronide group. In some embodiments, the linker portion is a linker cleavable by sulfatase, for example, containing a β-galactoside group. In some embodiments, the linker portion is a linker cleavable by phosphatase, for example, containing a pyrophosphate group. In some embodiments, the linker portion is a linker cleavable by sulfatase, for example, containing an aryl sulfate group. In some embodiments, the linker portion is a photoresponsive linker, for example, containing a heptamethrin fluorophore group, an O-nitrobenzyl group, or a PC4AP group. In some embodiments, the linker portion is a bioorthogonally cleavable linker, for example, containing a dsProc group.

[0120] Non-cleavable linkers remain inert in the chemical and enzymatic environments common in vivo, and the payload portion is released after ADC treatment by lysosomal proteases. Non-cleavable linkers include those containing thioether or maleimide hexanoyl groups.

[0121] In some embodiments, the linker portion is a thioether linker. In some embodiments, the linker portion is a maleimide hexanoyl linker, for example, containing a 2-(maleimide methyl)-1,3-dioxane (MD) group or a Mal-PAB group. In some embodiments, the linker portion contains a polyethylene glycol (PEG) group and an alkyne, triazole, or piperazine group.

[0122] In some embodiments, the linker-payload portion according to this disclosure has an amino (-NH2) group for attachment to the antigen-binding portion, for example, by enzymatic conjugation. In some of these embodiments, enzymatic conjugation may be performed using microbial transglutaminase to conjugate the linker-payload portion to the antigen-binding portion.

[0123] In some embodiments, the linker portion also includes a spacer portion. Due to the large volume of the payload portion, a spacer portion is sometimes necessary. Commonly used spacer portions include p-aminobenzylcarbamate (PABC), hemiacetal, PEG, polar acylsulfonamide, polar carbamoylsulfonamide, and HydraSpace (e.g., described in Verkade et al., Antibodies (Basel) (2018) 7(1):12 and WO 2016 / 053107 A1, both of which are incorporated herein by reference in their entirety). PABC is commonly used as a spacer portion in dipeptide linkers that are cleavable by cathepsins, linkers that are cleavable by β-glucuronidase, linkers that are cleavable by β-galactosidase, and linkers that are cleavable by phosphatases. In some embodiments, p-aminobenzyl (PAB) is used as the spacer group.

[0124] In some embodiments, the antigen-binding molecule according to this disclosure includes a linker-payload portion, which in turn includes a DDR inhibitor portion and a TOP1 inhibitor portion. That is, in some embodiments, the antigen-binding molecule includes a linker-payload portion that includes (a) a DDR inhibitor portion and (b) a TOP1 inhibitor portion. In some embodiments, the DDR inhibitor portion and the TOP1 inhibitor portion are provided in the same linker-payload portion. In some embodiments, the DDR inhibitor portion and the TOP1 inhibitor portion are linked to the antigen-binding portion of the antigen-binding molecule via the same linker portion.

[0125] Methods for connecting multiple payload portions to a single connector have been described in, for example, the following literature: Yamazaki et al., Nat Commun. (2021) 12(1): 3528; Kumar et al., Bioorg Med Chem Lett (2018) 28 (23-24): 3617-3621; Levengood et al., Angew Chem Int Ed Engl (2017) 56(3): 733-737; and Wang et al., Acta Pharmaceutica Sinica B (2023) 13(10): 4025-4059, all of which are incorporated herein by reference in their entirety.

[0126] In some embodiments, the DDR inhibitor and TOP1 inhibitor payload portions are provided in the same linker-payload portion. In some embodiments, the DDR inhibitor and TOP1 inhibitor payload portions are linked to the antigen-binding portion of the antigen-binding molecule via the same linker portion.

[0127] In some implementations, the payload portions of the DDR inhibitor and TOP1 inhibitor are linked to the antigen-binding portion of the antigen-binding molecule via a branched linker portion. The linker may contain multiple branches to allow for flexibility in the DAR.

[0128] In some implementations, the payload portions of the DDR inhibitor and TOP1 inhibitor are linked to the antigen-binding portion of the antigen-binding molecule via a linker portion with increased hydrophilicity.

[0129] In some implementations, the payload portions of the DDR inhibitor and TOP1 inhibitor are linked to the antigen-binding portion of the antigen-binding molecule via hydrophilic side-chain linker portions.

[0130] In some embodiments, the DDR inhibitor portion and the TOP1 inhibitor portion are connected to a linker portion of the linker-payload portion via orthogonal functional groups. In some embodiments, the linker-payload portion includes a trifunctional linker portion that can connect the antigen-binding portion to two different payload portions. For example, methods for producing linker-payload portions comprising two different payloads are described in Yamazaki et al., Nat Commun. (2021) 12(1): 3528 and Kumar et al., Bioorg Med Chem Lett (2018) 28 (23-24): 3617-3621.

[0131] Kumar et al., Bioorg Med Chem Lett (2018) 28 (23-24): 3617-3621, describe a branched connecting part containing the following structure: (a) A group for attaching the antigen-binding moiety (a self-stabilizing N-arylmaleimide); and (b) The two orthogonal functional groups used to connect the payload portion are: (i) an alkyne group, used to introduce the loaded moiety via a copper-mediated azido-alkyne cycloaddition reaction (CuAAC), and (ii) Ketone group, used to introduce the payload moiety via an aminooxy reaction, thereby forming an oxime bond.

[0132] The branched linker portion described in Kumar et al., Bioorg Med Chem Lett (2018) 28 (23-24): 3617-3621 has the following structure:

[0133] Therefore, in some embodiments, the linker-payload portion according to this disclosure includes a linker portion comprising: (i) a portion derived from a group for attachment to an antigen-binding site (e.g., a self-stabilizing N-arylmaleimide group), (ii) a portion derived from an alkynyl group adapted to introduce a first payload portion (i.e., a divalent triazole) via CuAAC, and (iii) a portion derived from a ketone group adapted to introduce a second payload portion via an aminooxy group reaction, thereby forming an oxime bond (i.e., an oxime). According to these embodiments, in some cases, the first payload portion is the DDR inhibitor portion described herein, and the second payload portion is the TOP1 inhibitor portion described herein. In some embodiments, the first payload portion is the TOP1 inhibitor portion described herein, and the second payload portion is the DDR inhibitor portion described herein.

[0134] In some embodiments, the antigen-binding molecule according to this disclosure comprises a linker-loaded portion comprising: (i) a first load portion conjugated to the linker portion via a CuAAC reaction between an azide group and an alkyne group; and (ii) a second load portion conjugated to the linker portion via an oxime bond between an alkoxyamine or hydrazide group and a ketone group. According to these embodiments, in some cases, the first load portion is a DDR inhibitor portion as described herein, and the second load portion is a TOP1 inhibitor portion as described herein. In some embodiments, the first load portion is a TOP1 inhibitor portion as described herein, and the second load portion is a DDR inhibitor portion as described herein.

[0135] Yamazaki et al., Nat Commun. (2021) 12(1): 3528, describe a branched connecting sub-part containing the following structure: (a) A group used to link the antibody (lysine-based antibody); and (b) The two orthogonal functional groups used to connect the payload portion are: (i) One or two azide groups for introducing the loaded moiety via a strain-promoted azido-dibenzocyclooctyn (DBCO) cycloaddition reaction; and (ii) Methyltetraazine group, used to introduce the effective loading part via the trans-cyclooctene (TCO) cycloaddition reaction.

[0136] The branched linker portion described in Yamazaki et al., Nat Commun. (2021) 12(1): 3528 has the following structure:

[0137] Therefore, in some embodiments, the linker-payload portion according to this disclosure includes a linker portion comprising: (i) a portion derived from a group for linking to an antigen-binding site (e.g., a lysine-based group), (ii) one or two portions derived from an azide group adapted to introduce a first payload portion via a DBCO cycloaddition reaction, and (iii) a portion derived from a methyltetraazine group for introducing a second payload portion via a TCO cycloaddition reaction. According to these embodiments, in some cases, the first payload portion is the DDR inhibitor portion described herein, and the second payload portion is the TOP1 inhibitor portion described herein. In some embodiments, the first payload portion is the TOP1 inhibitor portion described herein, and the second payload portion is the DDR inhibitor portion described herein.

[0138] In some embodiments, the antigen-binding molecule according to this disclosure comprises a linker-load portion comprising: (i) a first load portion conjugated to the linker portion via a DBCO cycloaddition reaction between a DBCO group and an azide group; and (ii) a second load portion conjugated to the linker portion via a TCO cycloaddition reaction between a TCO group and a methyltetraazine group. According to these embodiments, in some cases, the first load portion is the DDR inhibitor portion described herein, and the second load portion is the TOP1 inhibitor portion described herein. In some embodiments, the first load portion is the TOP1 inhibitor portion described herein, and the second load portion is the DDR inhibitor portion described herein.

[0139] In some embodiments, the DDR inhibitor portion and the TOP1 inhibitor portion are linked to the linker portion of the linker-payload portion via a cysteine ​​group. For example, such a method for producing a linker-payload portion comprising two different payloads is described in Levengood et al., Angew Chem Int Ed Engl (2017) 56(3): 733-737.

[0140] Levengood et al., Angew Chem Int Ed Engl (2017) 56(3): 733-737, describe a linker-loaded moiety comprising two distinct payload portions, each payload being constructed by sequential deprotection of orthogonally protected cysteines and linked to a maleimide group (e.g., via a cleavable linker) that undergoes a Michael reaction with the deprotected cysteine. The branched linker moiety described in Levengood et al., Angew Chem Int Ed Engl (2017) 56(3): 733-737 has the following structure:

[0141] Therefore, in some embodiments, the linker-load portion according to this disclosure comprises: (i) a first load portion, which is conjugated to the linker-load portion by reducing a cysteine ​​residue with a protective disulfide group (e.g., an S-(tert-butyl) disulfide group or an S-(isopropyl) disulfide group) and subsequently introducing the first load portion via a thiol-maleimide reaction; and (ii) a second load portion, which is conjugated to the linker-load portion by reducing a cysteine ​​residue with a protective acetamide methyl group and subsequently introducing the first load portion via a thiol-maleimide reaction. According to these embodiments, in some cases, the first load portion is the DDR inhibitor portion described herein, and the second load portion is the TOP1 inhibitor portion described herein. In some embodiments, the first load portion is the TOP1 inhibitor portion described herein, and the second load portion is the DDR inhibitor portion described herein.

[0142] In some of these implementations, the connector-payload section includes: (a) An amino group used for conjugation to the antigen-binding portion; (b) Clicking to at least one first payload comprising a portion of the first click group, wherein the first payload comprising a portion comprising a DDR inhibitor portion; (c) Click to at least one second payload containing portion of the second click group, wherein the second payload containing portion contains a TOP1 inhibitor portion; (d) Branched groups:

[0143] Where R N Selected from H and -(C 1-5 (alkylene)-C(O)OH, where one of the CH2 units can be replaced by -O-. a indicates the connection position between the amino group and the branched group; b indicates the connection position between the at least one first click group and the branched group; c represents the connection position of at least one second click group to a branched group.

[0144] In some embodiments, the DDR inhibitor portion and TOP1 inhibitor portion of the antigen-binding molecule of this disclosure are linked to the antigen-binding portion of the antigen-binding molecule via different linker portions.

[0145] In some embodiments, the antigen-binding molecule according to this disclosure comprises: (a) a linker-payload portion comprising a DDR inhibitor portion, and (b) a linker-payload portion comprising a TOP1 inhibitor portion. That is, in some embodiments, the antigen-binding molecule comprises at least two linker-payload portions, one of which comprises a DDR inhibitor portion and the other comprises a TOP1 inhibitor portion.

[0146] Methods for linking multiple payload portions to an antigen-binding portion using multiple linker portions are described, for example, in the following literature: All of these are incorporated herein by reference in their entirety by way of citation.

[0147] Intl J Mol Sci (2018) 19: 2098 describes a method in which (i) a first linker-loaded moiety (specifically maleimide-Val-Cit-PAB-α-amanitin) is conjugated to a cysteine ​​residue of a polypeptide via a thiol-maleimide reaction, and (ii) a second linker-loaded moiety (specifically an azide linked to MMAE) is conjugated to the same polypeptide via CuAAC-mediated conjugation to an alkynyl group of an engineered N-propynyl-L-lysine (PrK) residue.

[0148] Therefore, in some embodiments, the antigen-binding molecule according to this disclosure comprises: (i) a first linker-load portion conjugated to the antigen-binding portion via a thiol-maleimide reaction between a cysteine ​​residue of the antigen-binding portion and a maleimide group of the linker-load portion; and (ii) a second linker-load portion conjugated via a CuAAC reaction between an azide group of the linker-load portion and an alkynyl group of an N-propynyl-L-lysine residue of the antigen-binding portion. According to such embodiments, in some cases, the first linker-load portion comprises the DDR inhibitor portion described herein, and the second linker-load portion comprises the TOP1 inhibitor portion described herein. In some embodiments, the first linker-load portion comprises the TOP1 inhibitor portion described herein, and the second linker-load portion comprises the DDR inhibitor portion described herein.

[0149] Nilchan et al., Antib. Ther. (2019) 2:71-78 describe a dual conjugation method in which (i) a first linker-load portion is conjugated to the antigen-binding portion via a selenide conjugation between a selenocysteine ​​residue of the antigen-binding portion and an iodoacetamide group of the linker-load portion, and (ii) a second linker-load portion is conjugated to the same antigen-binding portion via a reaction between a cysteine ​​residue of the antigen-binding portion and a methyl sulfone phenyloxadiazole (MSODA) group of the linker-load portion.

[0150] Therefore, in some embodiments, the antigen-binding molecule according to this disclosure comprises: (i) a first linker-loaded portion conjugated to the antigen-binding portion via a selenide conjugation between a selenocysteine ​​residue of the antigen-binding portion and an iodoacetamide group of the linker-loaded portion; and (ii) a second linker-loaded portion conjugated via a reactive conjugation between a cysteine ​​residue of the antigen-binding portion and an MSODA group of the linker-loaded portion. According to such embodiments, in some cases, the first linker-loaded portion comprises the DDR inhibitor portion described herein, and the second linker-loaded portion comprises the TOP1 inhibitor portion described herein. In some embodiments, the first linker-loaded portion comprises the TOP1 inhibitor portion described herein, and the second linker-loaded portion comprises the DDR inhibitor portion described herein.

[0151] Another aspect of this disclosure provides a DDR inhibitor portion linked to a click group, wherein the click group is adapted to conjugate a corresponding click group in a linker portion, and wherein the linker portion is conjugated or adapted to conjugate to an antigen-binding portion.

[0152] Another aspect of this disclosure provides a TOP1 inhibitor portion linked to a click group, wherein the click group is adapted to conjugate a corresponding click group in a linker portion, and wherein the linker portion is conjugated or adapted to conjugate to an antigen-binding portion.

[0153] Based on this aspect, in some implementations, the click group is selected from: (i) Azide group; (ii) Alkynes; (iii) Tetraazine or tetraazine derivative group; (iv) Cyclooctyne, cyclooctyne derivatives, or cyclooctyne analog groups; and (v) Strained olefins.

[0154] The trifunctional linker disclosed herein employs a modular design, allowing for easy modification of the number of payloads attached to the linker, thereby altering the number of antigen-binding moieties. This has proven crucial for developing clinically relevant antibody-drug conjugates. Furthermore, the use of click groups to connect the payload-containing portion to branched groups enables the conjugation of various payload types. When orthogonal click groups are used, two different payload portions can be connected, including those with different mechanisms of action and potentially complementary properties.

[0155] The trifunctional linker of this disclosure has hydrophilic branched groups, which may reduce toxicity and improve biophysical properties, stability and pharmacokinetic properties.

[0156] As described above, in some aspects, the connector-payload section may include: (a) An amino group used for conjugation to the antigen-binding portion; (b) Clicking to at least one first payload comprising a portion of the first click group, wherein the first payload comprising a portion comprising a DDR inhibitor portion; (c) Click to at least one second payload containing portion of the second click group, wherein the second payload containing portion contains a TOP1 inhibitor portion; (d) Branched groups:

[0157] Where R N Selected from H and -(C 1-5 (alkylene)-C(O)OH, where one of the CH2 units can be replaced by -O-. a indicates the connection position between the amino group and the branched group; b indicates the connection position between the at least one first click group and the branched group; c represents the connection position of at least one second click group to a branched group.

[0158] In some implementation schemes, R N It's H.

[0159] In some implementation schemes, R N Yes - (C 1-5 Alkylene)-C(O)OH, in which one of the CH2 units can be replaced by -O-.

[0160] In some implementation schemes, R N Yes - (C 1-5 alkylene)-C(O)OH.

[0161] In some implementation schemes, R NIt is -CH2CH2OCH2CH2C(O)OH.

[0162] In some implementation schemes, R N It is CH2C(O)OH.

[0163] The amino group can be linked to a branched group via a first spacer group. The first spacer group (A1) may include: (i) C 1-7 Alkylene; and / or (ii) PEG1 to 12 groups.

[0164] The formula for A1 might be: -(CH2) xa -(C2H4O) xb -(CH2) xc -, Where xa is 0 or 1, xb is 0-12, and xc is 0 to 6, and at least one of xa and xb is 1.

[0165] In some implementations, xb is 0, and xa+xc is 1 to 7, for example 5 (i.e., A1 is -(CH2)5-).

[0166] In other implementations, xb is 1 to 12, xa is 0, and xc is 0 or 1.

[0167] In other embodiments, xb is 1 to 12. In some of these embodiments, xb is 1 to 6. In some of these embodiments, xb is 1 to 3, i.e., 1, 2, or 3.

[0168] In some implementations, xb is 1 to 12, and xc is 1 to 6 or 1 to 2. In some of these implementations, xc is 1. In some of these implementations, xc is 2.

[0169] In some embodiments, xa is 0, xb is 1 to 6, and xc is 2. In some of these embodiments, Al is -(C2H4)-O-(C2H4)-. In some of these embodiments, Al is -(C2H4O)3-(C2H4)-.

[0170] The first and second click groups can be selected from any member of the following click group pairs:

[0171] Cyclooctyne, cyclooctyne derivatives and cyclooctyne analogs used in this disclosure include:

[0172]

[0173] These groups can also be called cyclic alkynes.

[0174] Tetraazines and tetraazine derivatives used in this disclosure include:

[0175] The strained olefins used in this disclosure may have the following structures:

[0176] In some embodiments, the click group may be a dibenzo-azine octylene (DIBAC) group or a 1-ethylhept-6-enoxy group:

[0177] In some implementations, the click group may be tetramethylthiocycloheptyne sulfoxide imine (TMTHSI):

[0178] According to this disclosure, the linker between the payload portion (e.g., the DDR inhibitor portion or the TOP1 inhibitor portion) and the click group can be a cleavable linker portion or a non-cleavable portion, as described above.

[0179] In some embodiments of the trifunctional linker, the first and second click groups are identical.

[0180] In other embodiments of the trifunctional linker, the first and second click groups are selected from orthogonal click group pairs.

[0181] In some embodiments of the trifunctional linker, the first and / or second linker group is an azide.

[0182] In some embodiments of the trifunctional linker, the first and / or second linker group is a tetrazine or a tetrazine derivative.

[0183] In some embodiments of the trifunctional linker, the first and / or second linker group is an alkyne (-CCH).

[0184] In some embodiments of the trifunctional linker, the first and / or second linker group is cyclooctyne or a cyclooctyne derivative.

[0185] In some embodiments of the trifunctional linker, the first and / or second linker is norbornene or a norbornene derivative.

[0186] In some embodiments of the trifunctional linker, the first and / or second linker group is methylcyclopropene (1-MCP).

[0187] In some embodiments of the trifunctional linker, the first clicker is an azide, and the second clicker is a tetrazine or a tetrazine derivative.

[0188] In some embodiments of the trifunctional linker, the first click group is an azide and the second click group is cyclooctene.

[0189] In some embodiments of the trifunctional linker, the first clicker is an azide, and the second clicker is norbornene or a norbornene derivative.

[0190] In some embodiments of the trifunctional linker, the first click group is an azide and the second click group is methylcyclopropene (1-MCP).

[0191] In some embodiments of the trifunctional linker, the first click group is an alkyne (-CCH), and the second click group is a tetrazine or a tetrazine derivative.

[0192] In some embodiments of the trifunctional linker, the first click group is an alkyne (-CCH) and the second click group is cyclooctene.

[0193] In some embodiments of the trifunctional linker, the first clicker is an alkyne (-CCH), and the second clicker is norbornene or a norbornene derivative.

[0194] In some embodiments of the trifunctional linker, the first click group is an alkyne (-CCH) and the second click group is a methylcyclopropene (1-MCP).

[0195] In some embodiments of the trifunctional linker, the first clicker is a cyclooctyne or a cyclooctyne derivative, and the second clicker is a tetrazine or a tetrazine derivative.

[0196] In some embodiments of the trifunctional linker, the first clicker is cyclooctyne or a cyclooctyne derivative, and the second clicker is cyclooctene.

[0197] In some embodiments of the trifunctional linker, the first clicker is cyclooctyne or a cyclooctyne derivative, and the second clicker is norbornene or a norbornene derivative.

[0198] In some embodiments of the trifunctional linker, the first clicker is cyclooctyne or a cyclooctyne derivative, and the second clicker is methylcyclopropene (1-MCP).

[0199] In some embodiments of the trifunctional linker, the second linker is phenyltetraazine.

[0200] In some embodiments of the trifunctional linker group, the second clicker group is selected from the following groups: , or .

[0201] In some embodiments of the trifunctional linker, the second click group is: .

[0202] At least one first click group can be connected to a branched group via a second spacer group (B1). In some embodiments, the second spacer group is branched, such that two first click groups are connected to the branched group. In other embodiments, the second spacer group is not branched, such that a single first click group is connected to the branched group.

[0203] At least one second click group can be connected to a branched group via a third spacer group (B2). In some embodiments, the third spacer group is branched, such that two second click groups are connected to the branched group. In other embodiments, the third spacer group is not branched, such that a single second click group is connected to the branched group.

[0204] In some embodiments of the trifunctional linker group, the second spacer group (B1) has the formula (B1-1): (B1-1) R L1 It is -(C2H4O) xl3 -(CH2) xd -(C(=O)) xl4 - Where xl3 is 0 to 4, xd is 0 to 3, and xl4 is 0 or 1. R NB1 It is -(C2H4O) xe1 -(CH2) xf1 -(NH) xg1 -(C(=O)CH2) xh1 - Where xe1 is 0 to 4, xf1 is 0 to 2, xg1 is 0 or 1, and xh1 is 0 or 1. R NB2 It is H or -(C2H4O) xe2 -(CH2) xf2 -(NH) xg2 -(C(=O)CH2) xh2 - Where xe2 is 0 to 4, xf2 is 0 to 2, xg2 is 0 or 1, and xh2 is 0 or 1.

[0205] In some implementations, xl3 is 0-2. In some implementations, xl3 is 0. In some implementations, xl3 is 1. In some implementations, xl3 is 2.

[0206] In some implementations, xl4 is 0. In some implementations, xl4 is 1.

[0207] In some implementations, xe1 is 2-4. In some implementations, xe1 is 2. In some implementations, xe1 is 3. In some implementations, xe1 is 4.

[0208] In some implementations, xe2 is 2-4. In some implementations, xe2 is 2. In some implementations, xe2 is 3. In some implementations, xe2 is 4.

[0209] In some of these embodiments of the trifunctional linker group, the second spacer group (B1) has the formula (B1-2): (B1-2) Where xd is 0-3 R NB1 It is -(C2H4O) xe1 -(CH2) xf1 -(NH) xg1 -(C(=O)CH2) xh1 - Where xe1 is 0 or 1, xf1 is 0 to 2, xg1 is 0 or 1, and xh1 is 0 or 1. R NB2 It is H or -(C2H4O) xe2 -(CH2) xf2 -(NH) xg2 -(C(=O)CH2) xh2 - Where xe2 is 0 or 1, xf2 is 0 to 2, xg2 is 0 or 1, and xh2 is 0 or 1.

[0210] In some implementation schemes, R NB2 It is H. In some implementations, R NB2 It is -(C2H4O) xe2 -(CH2) xf2 -(NH) xg2 -(C(=O)CH2) xh2 - In other implementations, R NB2 With R NB1 same.

[0211] In some implementations, xd is 0-2. In some implementations, xd is 0-1. In some implementations, xd is 0. In some implementations, xd is 1. In some implementations, xd is 2. In some implementations, xd is 3. In some implementations, xd is either 0 or 2.

[0212] In some implementations, xe1 is 0. In some implementations, xe1 is 1.

[0213] In some implementations, xf1 is 0-1. In some implementations, xf1 is 0. In some implementations, xf1 is 1. In some implementations, xf1 is 2. In some implementations, xf1 is either 0 or 2.

[0214] In some implementations, xg1 is 0. In some implementations, xg1 is 1.

[0215] In some implementations, xh1 is 0. In some implementations, xh1 is 1.

[0216] In some implementations, xe2 is 0. In some implementations, xe2 is 1.

[0217] In some implementations, xf2 is 0-1. In some implementations, xf2 is 0. In some implementations, xf2 is 1. In some implementations, xf2 is 2. In some implementations, xf2 is either 0 or 2.

[0218] In some implementations, xg2 is 0. In some implementations, xg2 is 1.

[0219] In some implementations, xe1 is 1, xf1 is 2, xg1 is 0, and xh1 is 0.

[0220] In some implementations, xe1 is 0, xf1 is 0, xg1 is 0, and xh1 is 1.

[0221] In some implementations, xe1 is 1, xf1 is 2, xg1 is 1, and xh1 is 1.

[0222] In some implementations, xe2 is 1, xf2 is 2, xg2 is 0, and xh2 is 0.

[0223] In some implementations, xe2 is 0, xf2 is 0, xg2 is 0, and xh2 is 1.

[0224] In some implementations, xe2 is 1, xf2 is 2, xg2 is 1, and xh2 is 1.

[0225] In some embodiments of the trifunctional linker group, the second spacer group (B1) is selected from groups containing: , , , , , , and .

[0226] In some embodiments of the trifunctional linker group, the second spacer group (B1) is selected from groups containing:

[0227]

[0228]

[0229]

[0230]

[0231] In some embodiments of the trifunctional linker group, the second spacer group (B2) has the formula (B2-1): (B2-1) R L2 It is -(C2H4O) xl5 -(CH2) xi -(C(=O)) xl6 - Where xl6 is 0 to 4, xi is 0 to 3, and xl6 is either 0 or 1. R NB3 It is -(C2H4O) xj1 -(CH2) xk1 -(NH) xl1 -(C(=O)CH2) xm1 - Where xj1 is 0 to 4, xk1 is 0 to 2, xl1 is 0 or 1, and xm1 is 0 or 1. R NB4 It is H or -(C2H4O) xj2 -(CH2) xk2 -(NH) xl2 -(C(=O)CH2) xm2 - Where xj2 is 0 to 4, xk2 is 0 to 2, xl2 is 0 or 1, and xm2 is 0 or 1.

[0232] In some implementations, xl5 is 0-2. In some implementations, xl5 is 0. In some implementations, xl5 is 1. In some implementations, xl5 is 2.

[0233] In some implementations, xl6 is 0. In some implementations, xl6 is 1.

[0234] In some implementations, xj1 is 2-4. In some implementations, xj1 is 2. In some implementations, xj1 is 3. In some implementations, xj1 is 4.

[0235] In some implementations, xj2 is 2-4. In some implementations, xj2 is 2. In some implementations, xj2 is 3. In some implementations, xj2 is 4.

[0236] In some embodiments of the trifunctional linker group, the third spacer group (B2) has the formula (B2-2): (B2-2) Where xi is 0-3, R NB3 It is -(C2H4O) xj1 -(CH2) xk1 -(NH) xl1 -(C(=O)CH2) xm1 - Where xj1 is 0 or 1, xk1 is 0 to 2, xl1 is 0 or 1, and xm1 is 0 or 1. R NB4 It is H or -(C2H4O) xj2 -(CH2) xk2 -(NH) xl2 -(C(=O)CH2) xm2 - Where xj2 is 0 or 1, xk2 is 0 to 2, xl2 is 0 or 1, and xm2 is 0 or 1.

[0237] In some implementation schemes, R NB4 It is H. In some implementations, R NB4 It is -(C2H4O) xj2 -(CH2) xk2 -(NH) xl2 -(C(=O)CH2) xm2 - In other implementations, R NB4 With R NB3 same.

[0238] In some implementations, xi is 0-2. In some implementations, xi is 0-1. In some implementations, xi is 0. In some implementations, xi is 1. In some implementations, xi is 2. In some implementations, xi is 3. In some implementations, xi is either 0 or 2.

[0239] In some implementations, xj1 is 0. In some implementations, xj1 is 1.

[0240] In some implementations, xk1 is 0-1. In some implementations, xk1 is 0. In some implementations, xk1 is 1. In some implementations, xk1 is 2. In some implementations, xk1 is either 0 or 2.

[0241] In some implementations, xl1 is 0. In some implementations, xl1 is 1.

[0242] In some implementations, xm1 is 0. In some implementations, xm1 is 1.

[0243] In some implementations, xj2 is 0. In some implementations, xj2 is 1.

[0244] In some implementations, xk2 is 0-1. In some implementations, xk2 is 0. In some implementations, xk2 is 1. In some implementations, xk2 is 2. In some implementations, xk2 is either 0 or 2.

[0245] In some implementations, xl2 is 0. In some implementations, xl2 is 1.

[0246] In some implementations, xj1 is 1, xk1 is 2, xl1 is 0, and xm1 is 0.

[0247] In some implementations, xj1 is 0, xk1 is 0, xl1 is 0, and xm1 is 1.

[0248] In some implementations, xj1 is 1, xk1 is 2, xl1 is 1, and xm1 is 1.

[0249] In some implementations, xj2 is 1, xk2 is 2, xl2 is 0, and xm2 is 0.

[0250] In some implementations, xj2 is 0, xk2 is 0, xl2 is 0, and xm2 is 1.

[0251] In some implementations, xj2 is 1, xk2 is 2, xl2 is 1, and xm2 is 1.

[0252] In some embodiments of the trifunctional linker group, the third spacer group (B2) is selected from groups containing the following: , , , , , , and .

[0253] In some embodiments of the trifunctional linker group, the third spacer group (B2) is selected from groups containing the following:

[0254]

[0255]

[0256]

[0257]

[0258] In some embodiments of the trifunctional linker group, the second spacer group (B1) is the same as the third spacer group (B2). In some embodiments of the trifunctional linker group, the second spacer group (B1) is different from the third spacer group (B2).

[0259] In some embodiments of the trifunctional linker group, the second spacer group (B1) and the third spacer group (B2), along with the nitrogen atom to which they are attached, form one of the following groups: or .

[0260] In some embodiments of the trifunctional linker group, the second spacer group (B1) and the third spacer group (B2), along with the nitrogen atom to which they are attached, form one of the following groups:

[0261]

[0262] This disclosure provides a connector that includes: (a) An amino group conjugated to the antigen-binding portion; (b) At least one first payload portion is included when clicked onto the first click group; (c) At least one second payload of the second click group includes a portion; (d) Branched groups:

[0263] Where R N Selected from H and -(C 1-5 (alkylene)-C(O)OH, where one of the CH2 units can be replaced by -O-. a indicates the connection position between the amino group and the branched group; b indicates the connection position between the at least one first click group and the branched group; c represents the connection position of at least one second click group to a branched group.

[0264] Here are some examples of clicking on a functional group:

[0265] As shown in the table above, the reaction between the first and second members of a click group pair can produce two isomer products, i.e., a mixture. This disclosure includes both isomer forms, even if only one is shown.

[0266] The amino group can be linked to the branched group via the first spacer group (A1) as defined above.

[0267] At least one first click group can be connected to a branched group via a second spacer group (B1) as defined above.

[0268] At least one second click group can be connected to a branched group via a third spacer group (B2) as defined above.

[0269] In some implementations, the linker comprises one of the following groups: , , , , , , or .

[0270] According to this disclosure, the linker between the payload portion (e.g., the DDR inhibitor portion or the TOP1 inhibitor portion) and the click group can be a cleavable linker portion or a non-cleavable portion, as described above.

[0271] In some embodiments, a functional group is present for attaching the click group. This functional group can be selected from carbonyl (C=O) and oxygen (O). When the click group is DBCO or TMTHSI, the functional group can be carbonyl. When the click group is TCO, the functional group can be oxygen.

[0272] In some implementations, the connector between the payload portion and the click portion includes:

[0273] Q is: Q X The condition is satisfied that Q is an amino acid residue, a dipeptide residue, or a tripeptide residue; X is: , Where a = 0 to 5, b = 0 to 8, c = 0 or 1, d = 0 to 5; a can be 0, 1, 2, 3, 4, or 5. In some implementations, a is 0 to 3. In some of these implementations, a is 0 or 1. In a further implementation, a is 1.

[0274] b can be 0, 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, b is 0 to 6. In some of these embodiments, b is 0 to 4, and can also be 0, 1, 2, 3, or 4. In a further embodiment, b is 3.

[0275] c can be 0 or 1. In some of these implementations, c is 1.

[0276] d can be 0, 1, 2, 3, 4, or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 0, 1, or 2. In a further embodiment, d is 2. In other further embodiments, d is 1. In other further embodiments, d is 0.

[0277] In some implementations of X, a is 0, c is 1, d is 2, and b can be 0 to 8. In some of these implementations, b is 0, 4, or 8.

[0278] In one implementation, Q is an amino acid residue. This amino acid can be a natural amino acid or a non-natural amino acid.

[0279] In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, Ser, and Trp, wherein Cit is citrulline. In one embodiment, Q is a serine derivative (see WO2018 / 234636A1).

[0280] In one embodiment, Q comprises a dipeptide residue. The amino acid in the dipeptide can be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide comprises a natural amino acid. If the linker is a linker that is unstable to cathepsins, then the dipeptide is a cathepsin-mediated cleavage site. The dipeptide is also a cathepsin recognition site.

[0281] In one implementation, Q is selected from: CO -Phe-Lys- NH , CO -Val-Ala- NH , CO -Val-Lys- NH , CO -Ala-Lys- NH , CO -Val-Cit- NH , CO -Phe-Cit- NH , CO -Leu-Cit- NH , CO -Ile-Cit- NH , CO -Phe-Arg- NH CO -Gly-Cit- NH , CO -Gly-Ala- NH ,and CO -Trp-Cit- NH ; Cit stands for citrulline.

[0282] In some of these implementations, Q is selected from: CO -Phe-Lys- NH , CO -Val-Ala- NH , CO -Val-Lys- NH , CO-Ala-Lys- NH , CO -Val-Cit- NH .

[0283] In a further implementation, Q is selected from... CO -Phe-Lys- NH , CO -Val-Cit- NH and CO -Val-Ala- NH .

[0284] In some embodiments, the linker between the payload portion (e.g., the DDR inhibitor portion or the TOP1 inhibitor portion) and the click group comprises: PABC, a dipeptide cleavable by cathepsins, and PEG2 to PEG4 (e.g., PEG3) groups. In some of these embodiments, the linker between the payload portion and the click group comprises either: .

[0285] In some embodiments, the linker between the payload portions comprises GGFG (glycine-glycine-phenylalanine-glycine). This linker can be directly connected to the payload or connected via a CH2 group. In some of these embodiments, the linker between the payload portion and the click group comprises either: .

[0286] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0287] In some implementations, the TOP1 inhibitor moiety linked to the click group has the following structure: .

[0288] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0289] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0290] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0291] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0292] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0293] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0294] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0295] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0296] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0297] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0298] In some implementations, the DDR inhibitor portion linked to the click group has the following structure: .

[0299] Functional properties of antigen-binding molecules The antigen-binding molecules described herein can be characterized by reference to certain functional properties. In some embodiments, the antigen-binding molecules described herein may possess one or more of the following properties: It binds to cells expressing HER2; Inhibits the proliferation of cells expressing HER2; Increased killing effect on cells expressing HER2; Inhibit tumor growth and / or reduce tumor size / volume (e.g., tumor size / volume of cancers expressing HER2); Extend the survival of patients with cancer (such as cancers that express HER2).

[0300] It should be understood that a particular antigen-binding molecule may exhibit more than one of the properties described in the preceding paragraph. A suitable assay method can be used to evaluate a particular antigen-binding molecule to examine the properties described in the preceding paragraph. For example, the assay method may be, for example, an in vitro assay, or a cell-based assay or a cell-free assay. In some embodiments, the assay may be, for example, an in vivo assay, i.e., performed in a non-human animal. In some embodiments, the assay may be, for example, an ex vivo assay, i.e., performed using cells / tissues / organs obtained from a subject.

[0301] If the assay is cell-based, it may involve treating cells with an antigen-binding molecule to determine whether the antigen-binding molecule exhibits one or more of the described properties. The assay may use substances labeled with detectable entities to facilitate their detection. The assay may include evaluating the described properties after treating cells individually with a range of amounts / concentrations of a specific antigen-binding molecule (e.g., a dilution series).

[0302] Analysis of such assay results may include determining the concentration at which 50% of the maximum relevant activity level is achieved. The concentration of a specific agent that achieves 50% of the maximum relevant activity level can be referred to as the "half-maximum effective concentration" (MCM) of that agent related to the relevant activity, or "ECM". 50 Depending on their characteristics, EC 50 It can also be called "half-maximum inhibitory concentration" or "IC50". 50 "" refers to the drug concentration at which 50% of the maximum inhibitory level of a specific property is observed.

[0303] In some embodiments, when HER2 is expressed on the cell surface (i.e., in or at the cell membrane), the antigen-binding molecule of this disclosure binds to HER2 in a region accessible to the antigen-binding molecule (i.e., an extracellular antigen-binding molecule). In some embodiments, the antigen-binding molecule binds to HER2 expressed on the surface of a cell expressing HER2. In some embodiments, the antigen-binding molecule binds to a cell expressing HER2.

[0304] The ability of antigen-binding molecules to bind to specific cell types can be analyzed by contacting cells with antigen-binding molecules and detecting the antigen-binding molecules bound to the cells (e.g., after a washing step to remove unbound antigen-binding molecules). The ability of antigen-binding molecules to bind to HER2-expressing cells can be analyzed using methods such as flow cytometry and immunofluorescence microscopy.

[0305] In some implementations, antigen-binding molecules inhibit the proliferation of HER2-expressing cells (e.g., HER2-expressing cancer cells). The ability of antigen-binding molecules to inhibit the proliferation of a specific cell type can be analyzed by exposing cells to antigen-binding molecules and then assessing cell proliferation (i.e., after a sufficiently long period to observe the effect on cell proliferation). Cell proliferation can be assessed, for example, by detecting changes in cell number over time, or by introducing… 3 The in vitro analysis of H-thymidine or its evaluation by CFSE dilution assay, as described, for example, in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, the full text of which is incorporated herein by reference.

[0306] In some embodiments, in a specific assay, the antigen-binding molecule of the present invention is able to inhibit the proliferation of HER2-expressing cells to less than 1-fold the proliferation level of HER2-expressing cells observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule known not to affect the proliferation of HER2-expressing cells), for example ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold.

[0307] In one embodiment, the antigen-binding molecule described herein inhibits the proliferation of cells expressing human HER2, with an IC50 value of [missing information]. 50 The value is 100 nM or lower, 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.

[0308] In some implementations, the antigen-binding molecules according to this disclosure enhance (i.e., upregulate, enhance) the killing of cells containing / expressing HER2.

[0309] In some embodiments, the antigen-binding molecule according to this disclosure can inhibit the growth of cancer containing cells that contain / express HER2 or reduce its metastasis. In some embodiments, the antigen-binding molecule can enhance (i.e., upregulate, strengthen) the killing of cells containing / expressing HER2. In some embodiments, the antigen-binding molecule can inhibit the growth of cancer cells or can inhibit the growth of tumors containing cells containing / expressing HER2. In some embodiments, the antigen-binding molecule can inhibit the metastasis of cancer / tumor containing cells containing / expressing HER2.

[0310] For example, any method reviewed in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, the entire contents of which are incorporated herein by reference. Examples of in vitro assays for cytotoxicity / cell killing include release assays, such as... 51 Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay, ATP release assay using Cell Titre Glo, and calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on detecting factors released from lysed cells.

[0311] In some embodiments, the antigen-binding molecule according to this disclosure can reduce the number / proportion of cells expressing HER2. In some embodiments, the antigen-binding molecule according to this disclosure can consume / enhance the consumption of such cells.

[0312] In some embodiments, the antigen-binding molecule of this disclosure exhibits anticancer activity. In some embodiments, the antigen-binding molecule increases the killing effect on cancer cells. In some embodiments, the antigen-binding molecule leads to a reduction in the number of cancer cells in vivo, for example, compared to appropriate control conditions. The cancer can be the cancer described herein, for example, cancer expressing / overexpressing HER2.

[0313] In some embodiments, the antigen-binding molecule according to this disclosure reduces / inhibits the growth of cancer and / or cancerous tumors. In some embodiments, the antigen-binding molecule reduces the invasion of tissues by cancer cells. In some embodiments, the antigen-binding molecule reduces cancer metastasis. In some embodiments, the antigen-binding molecule exhibits anti-cancer activity. In some embodiments, the antigen-binding molecule reduces the growth / proliferation of cancer cells. In some embodiments, the antigen-binding molecule shortens the survival time of cancer cells. In some embodiments, the antigen-binding molecule increases the killing effect on cancer cells. In some embodiments, the antigen-binding molecule of this disclosure leads to, for example, a reduction in the number of cancer cells in the body. This cancer may be a cancer containing cells expressing HER2.

[0314] The antigen-binding molecules of this disclosure can be analyzed in appropriate assays to examine the properties described in the preceding paragraph. Such assays include, for example, in vivo models.

[0315] In some implementations, administration of the antigen-binding molecule according to this disclosure may result in one or more of the following outcomes: inhibition of cancer occurrence / progression, delay / prevention of cancer occurrence, reduction / delay / prevention of tumor growth, reduction / delay / prevention of tissue invasion, reduction / delay / prevention of metastasis, reduction of the severity of one or more cancer symptoms, reduction of cancer cell number, reduction of cancer burden, reduction of tumor size / volume, and / or prolongation of survival (e.g., progression-free survival or overall survival) in patients with cancer, as determined, for example, in a suitable model.

[0316] It should be understood that the properties described above should be evaluated after a period of time sufficient to observe the therapeutic effects associated with the use of antigen-binding molecules. Tumor growth can be monitored by monitoring changes in tumor volume over time. Tumor volume can be measured (e.g., in mm). 3 The growth of tumors is assessed by measuring changes over time (in units of measurement).

[0317] In some embodiments, in a specific assay, the antigen-binding molecule of this disclosure is capable of reducing tumor size / volume (e.g., the average tumor size / volume in the treatment group in an in vivo model, such as the average tumor size / volume of cancers expressing HER2) to less than 1-fold of the tumor size / volume observed at the same time point without treatment with the antigen-binding molecule (or after treatment with an appropriate control antigen-binding molecule known not to affect tumor growth), for example ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold. In some implementations, in the relevant models, tumor size / volume is assessed for this comparison at least 5 days after administration of the first dose of the antigen-binding molecule, for example, ≥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.

[0318] In some embodiments, in a specific assay, the tumor growth inhibition level achieved by the antigen-binding molecule of this disclosure (e.g., expressed as a tumor growth inhibition percentage, calculated as a percentage of tumor growth observed when treated with an appropriate control antigen-binding molecule) is more than one time-value of the tumor growth inhibition level observed at the same time point without treatment with the antigen-binding molecule (or after treatment with an appropriate control antigen-binding molecule known not to affect tumor growth), for example, ≥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. In some implementations, in the relevant models, an assessment of tumor growth inhibition is performed for this comparison at least 5 days after administration of the first dose of the antigen-binding molecule, for example, ≥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.

[0319] In some embodiments, in a specific assay, the antigen-binding molecule of this disclosure is capable of extending the median survival of subjects with cancer (e.g., in in vivo models, such as models of cancer expressing HER2) to more than one-fold the median survival observed when not treated with the antigen-binding molecule (or after treatment with an appropriate control antigen-binding molecule known not to affect the survival of patients with cancer), for example, ≥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. For subjects in the relevant treatment group, median survival can be expressed as the number of days after the start of the experiment.

[0320] Other sequences The antigen-binding molecules and their constituent polypeptides disclosed herein may also contain other amino acids or amino acid sequences.

[0321] The polypeptide disclosed herein may comprise one or more linker sequences between amino acid sequences. Linker sequences are known to those skilled in the art, for example, as described in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence is a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences linked by the linker sequence. Flexible linkers are known to those skilled in the art, and several flexible linkers are enumerated in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences typically contain a high proportion of glycine and / or serine residues.

[0322] 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; where 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., tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises (G4S)4 or (G4S)6 or consists of them. In some embodiments, the length of the linker sequence is 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-30 amino acids.

[0323] The antigen-binding molecules and their constituent peptides disclosed herein may contain amino acid sequences to facilitate the expression, folding, transport, processing, purification, or detection of the antigen-binding molecules / peptides. For example, the antigen-binding molecules and peptides disclosed herein may also contain amino acid sequences forming a detectable moiety, as described below.

[0324] The antigen-binding molecules and their constituent polypeptides disclosed herein may also contain signal peptides (also known as leader sequences or signal sequences). Signal peptides typically consist of 5–30 hydrophobic amino acids forming an α-helix. Secretory proteins and proteins expressed on the cell surface often contain signal peptides. Signal peptides are well-known among many proteins and are documented in databases such as GenBank, UniProt, and Ensembl, and / or can be identified / predicted 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).

[0325] Signal peptides may be located at the N-terminus of a polypeptide or may be present in newly synthesized polypeptides. Signal peptides enable efficient transport of polypeptides. They are typically removed through cleavage and therefore are not included in mature polypeptides.

[0326] Signal peptides are well-known among many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro. They can also be identified / predicted 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).

[0327] Markers and conjugates In some embodiments, the antigen-binding molecules and their constituent polypeptides disclosed herein contain a detectable portion.

[0328] In some implementations, the detectable portion is a fluorescent label, phosphorescent label, luminescent label, immunodetectable label (e.g., epitope tag), radiolabel, chemical label, nucleic acid label, or enzyme label. The antigen-binding molecule or its constituent polypeptide may be covalently or non-covalently labeled with the detectable portion.

[0329] Fluorescent markers include, for example, fluorescein, rhodamine, allophycocyanin, eosin and NDB, green fluorescent protein (GFP), rare earth chelates (e.g., europium (Eu), terbium (Tb), and samarium (Sm)), tetramethylrhodamine, Texas red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radioactive markers include radioactive isotopes, such as hydrogen. 3 ,sulfur 35 ,carbon 14 ,phosphorus 32 ,iodine 123 ,iodine 125 ,iodine 126 ,iodine 131 ,iodine 133 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47,tellurium 121m ,tellurium 122m ,tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper 67 ,fluorine 18 ,yttrium 90 ,palladium 100 ,bismuth 217 and antimony 211 Luminescent markers include radioluminescent markers, chemiluminescent markers (e.g., acridine esters, luminol, isoluminol), and bioluminescent markers. Immunologically detectable markers include haptens, peptides / peptides, antibodies, receptors, and ligands, such as biotin, avidin, streptavidin, or digoxigenin. Nucleic acid markers include aptamers.

[0330] In some embodiments, the antigen-binding molecule or its constituent polypeptide contains a radionuclide. Such conjugates may be referred to as radionuclide-pharmaceutical conjugates (RDCs) or radioimmunoconjugates (RICs). In some embodiments, the antigen-binding molecule or its constituent polypeptide contains a chelating group capable of chelating a radionuclide. Radionuclides include the radioisotopes listed above, as well as other isotopes such as lutetium. 177 Actin 225 Strontium 90 .

[0331] In some embodiments, the antigen-binding molecule or its constituent polypeptide contains epitope tags such as His (e.g., 6XHis), FLAG, c-Myc, StrepTag, hemagglutinin, 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), which may optionally be located at the N-terminus or C-terminus of the antigen-binding molecule / polypeptide.

[0332] In some embodiments, the antigen-binding molecule or its constituent polypeptide contains a portion having detectable activity, such as an enzyme portion. Enzyme portions include, for example, luciferase, glucose oxidase, galactosidase (e.g., β-galactosidase), glucuronidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0333] Nucleic acid and vector This disclosure provides one or more nucleic acids that encode an antigen-binding molecule / antigen-binding polypeptide complex or a constituent polypeptide thereof according to this disclosure. In some embodiments, the nucleic acid comprises or is composed of RNA or DNA.

[0334] The antigen-binding molecule / antigen-binding polypeptide complex or its constituent polypeptides according to this disclosure can be produced intracellularly by translating RNA encoding the polypeptide. The antigen-binding molecule / antigen-binding polypeptide complex or its constituent polypeptides can be produced intracellularly through transcription of the nucleic acid encoding the polypeptide, followed by translation of the transcribed RNA.

[0335] In some implementations, nucleic acids can be one or more vectors, or contained in / accommodated within one or more vectors. As used herein, "vector" refers to a nucleic acid molecule used as a carrier for transferring exogenous nucleic acids into cells.

[0336] Therefore, this disclosure also provides one or more vectors containing nucleic acids or multiple nucleic acids according to this disclosure. The vector can facilitate the delivery of nucleic acids encoding polypeptides according to this disclosure into cells. The vector can be an expression vector containing elements required for expressing the polypeptide according to this disclosure. The vector may contain elements facilitating the integration of the nucleic acid into the genomic DNA of the cell into which it is introduced.

[0337] The nucleic acids and vectors disclosed herein can be provided in purified or isolated form, i.e., from other nucleic acids or naturally occurring biological materials.

[0338] The vector can be a vector for expressing nucleic acids in cells (i.e., an expression vector). Such vectors may include a promoter sequence operatively linked to a nucleotide sequence encoding an antigen-binding molecule or polypeptide according to the present disclosure. The vector may also include a stop codon (i.e., the 3' end of the nucleotide sequence encoding the polypeptide in the vector nucleotide sequence) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express peptides or polypeptides from vectors according to the present disclosure.

[0339] The term "operably linked" can include situations where a nucleic acid encoding a polypeptide according to this disclosure is covalently linked to a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer), thereby subjecting the expression of the nucleic acid encoding the polypeptide to the influence or regulation of the regulatory nucleic acid sequence (and thus forming an expression cassette). Therefore, if the regulatory sequence can influence the transcription of a nucleic acid sequence, then the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

[0340] Vectors covered in this disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugating plasmids (e.g., F plasmids), non-conjugating plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, such as gamma-retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, such as SFG vectors), 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), such as those described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, the entire contents of which are incorporated herein by reference. In some embodiments, the vector according to this disclosure is a lentiviral vector.

[0341] In some embodiments, the vector may be a eukaryotic vector, i.e., a vector containing elements necessary for protein expression from a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, such as one containing a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0342] According to this disclosure, the constituent polypeptides of the antigen-binding molecule / antigen-binding polypeptide complex can be encoded by different nucleic acids from a variety of nucleic acids or different vectors from a variety of vectors.

[0343] Generate antigen-binding molecules The antigen-binding molecule according to this disclosure can be prepared according to antibody-drug conjugate production methods known to those skilled in the art.

[0344] The antigen-binding moiety according to this disclosure can be prepared by chemical synthesis, such as liquid-phase or solid-phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described in Chandrudu et al., Molecules (2013), 18: 4373-4388, the entire contents of which are incorporated herein by reference.

[0345] Alternatively, the antigen-binding moiety according to this disclosure can be generated by recombinant expression. Molecular biology techniques suitable for recombinant peptide production are well known in the art, for example those presented in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146, the entire contents of which are incorporated herein by reference. Methods for recombinant antigen-binding peptide production are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, ApplMicrobiol Biotechnol. (2016) 100: 3451–3461, the entire contents of which are incorporated herein by reference.

[0346] In some cases, the antigen-binding moiety of this disclosure consists of more than one polypeptide chain. In such cases, the generation of the antigen-binding moiety may involve the transcription and translation of more than one polypeptide, followed by the association of polypeptide chains to form the antigen-binding moiety.

[0347] For recombinant production according to this disclosure, any cell suitable for expressing the polypeptide can be used. The cell can be prokaryotic or eukaryotic. In some embodiments, the cell is a prokaryotic cell, such as an archaea or bacterial cell. In some embodiments, the bacteria can be Gram-negative bacteria, such as Enterobacteriaceae, e.g., *Escherichia coli*. In some embodiments, the cell is a eukaryotic cell, such as yeast cells, plant cells, insect cells, or mammalian cells, as described above. In some cases, the cell is not a prokaryotic cell because some prokaryotic cells do not allow the same folding or post-translational modifications as eukaryotic cells. Furthermore, very high expression levels can be achieved in eukaryotes, and the protein can be more easily purified from eukaryotes using appropriate tags. Specific plasmids can also be used to enhance the secretion of the protein into the culture medium.

[0348] In some implementations, the peptide can be prepared by cell-free protein synthesis (CFPS), as described, for example, in Zemella et al. Chembiochem (2015) 16(17): 2420-2431, the entire contents of which are incorporated herein by reference.

[0349] The generation of the antigen-binding moiety may involve culturing or fermenting eukaryotic cells modified to express the target peptide. The culture or fermentation can be carried out in a bioreactor equipped with appropriate nutrients, air / oxygen, and / or growth factor supplies. The secreted protein can be collected by separating the culture medium / fermentation broth from the cells, extracting the protein contents, and isolating individual proteins to isolate the secreted peptide. Culture, fermentation, and isolation techniques are well known to those skilled in the art, for example, as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition; incorporated herein by reference). The bioreactor comprises one or more containers for culturing cells. Culture in the bioreactor can be continuous, with reactants continuously flowing into the reactor and cultured cells continuously flowing out. Alternatively, culture can be batch-processed. Environmental conditions, such as pH, oxygen, inflow and outflow rates, and agitation within the container, are monitored and controlled in the bioreactor to provide optimal conditions for the cultured cells.

[0350] After culturing cells expressing the peptide, the target peptide can be isolated. Any suitable method known in the art for isolating proteins from cells can be used. To isolate the peptide, it may be necessary to separate the cells from the nutrient medium. If the peptide is secreted from the cells, the cells can be separated from the medium containing the target secreted peptide by centrifugation. If the target peptide is aggregated within the cells, protein isolation may include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with a lysis buffer, and disruption of the cells by, for example, sonication, rapid freeze-thaw cycles, or osmotic lysis.

[0351] The target polypeptide, which may contain other protein and non-protein components, may then need to be separated from the supernatant or culture medium. A common method for separating protein components from the supernatant or culture medium is precipitation. Proteins with different solubilities will precipitate in different concentrations of precipitant (e.g., ammonium sulfate). For example, water-soluble proteins can be extracted at lower precipitant concentrations. Therefore, proteins with different solubilities can be distinguished by adding precipitant at progressively increasing concentrations. Dialysis can then be used to remove ammonium sulfate from the separated proteins. Other methods for distinguishing different proteins are known in the art, such as ion-exchange chromatography and size chromatography. These methods can be used as alternatives to precipitation or performed after precipitation.

[0352] After isolating the target polypeptide from the culture, it may be necessary or necessary to concentrate the polypeptide. Many methods for protein concentration are known in the art, such as ultrafiltration or lyophilization.

[0353] The antigen-binding polypeptide / polypeptide complex according to this disclosure may be conjugated to a linker payload portion according to this disclosure to produce an antigen-binding molecule according to this disclosure by any suitable technique known to those skilled in the art and conventionally used in the art.

[0354] General methods for conjugating antigen-binding peptides / peptide complexes with linkers-payloads are described, for example, in Chudasama et al., Nature Chemistry, (2016), 8:114-119, Baah et al., Molecules. (2021) 26(10): 2943, and Walsh et al., Chem. Soc. Rev. (2021) 50:1305-1353, all of which are incorporated herein by reference in their entirety. Methods for conjugating antigen-binding moieties to linker-payload moieties and for purifying antigen-binding molecules resulting from such conjugations are described, for example, in Beck et al., (2017) Nat Rev DrugDiscov 16: 315-337; Peters and Brown Biosci Rep (2015) 35: art:e00225; McCombs and Owen, The AAPS Journal (2015) 17: 339-351; Jackson, Org Process Res Dev (2016) 20: 852-866; and Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical, (2016) Wiley.

[0355] General methods for conjugating antigen-binding peptides / peptide complexes with linkers-payloads are described, for example, in Chudasama et al., Nature Chemistry, (2016), 8:114-119, Baah et al., Molecules. (2021) 26(10): 2943, and Walsh et al., Chem. Soc. Rev. (2021) 50:1305-1353, all of which are incorporated herein by reference in their entirety. Methods for conjugating antigen-binding moieties to linker-payload moieties and for purifying antigen-binding molecules resulting from such conjugations are described, for example, in Beck et al., (2017) Nat Rev DrugDiscov 16: 315-337; Peters and Brown Biosci Rep (2015) 35: art:e00225; McCombs and Owen, The AAPS Journal (2015) 17: 339-351; Jackson, Org Process Res Dev (2016) 20: 852-866; and Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcome to Target Cancer (2016) Wiley.

[0356] The antigen-binding portion of this disclosure can be conjugated to the linker-payload portion of this disclosure using any suitable technique known to those skilled in the art and conventionally used in the art. Such methods are described, for example, in Chudasama et al., Nature Chemistry, (2016), 8:114-119, Baah et al., Molecules. (2021) 26(10):2943, and Walsh et al., Chem. Soc. Rev. (2021) 50:1305-1353, all of which are incorporated herein by reference in their entirety.

[0357] The conjugation of the antigen-binding moiety with the linker-payload moiety and the purification of the antigen-binding molecule resulting from such conjugation can be performed, for example, as described in Beck et al., (2017) Nat Rev Drug Discov 16: 315-337; Peters and Brown Biosci Rep (2015) 35: art:e00225; McCombs and Owen, The AAPS Journal (2015) 17: 339-351; Jackson, Org Process Res Dev (2016) 20: 852-866; and Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcome to Target Cancer (2016) Wiley, all of which are incorporated herein by reference in their entirety. Other relevant disclosures related to conjugation and linkers include: Tsuchikama and An, Protein Cell. (2018) 9(1): 33-46; Khongorzul et al., Mol CancerRes (2020) 18(1): 3-19; and Drago et al., Nature Reviews Clinical Oncology (2021) 18: 327-344. All of these publications are incorporated herein by reference in their entirety.

[0358] Lysine amide coupling Lysine-based conjugation is one of the most widely used nonspecific conjugation strategies. This type of conjugation occurs on the active amine side chain of lysine residues due to their favorable nucleophilicity. Immunoglobulin scaffolds contain over 80 lysine residues, most of which are exposed on the molecular surface. Of these surface lysine residues, more than 20 have been shown to have high solvent accessibility and serve as potential ADC conjugation sites. Lysine conjugation follows two main strategies to form stable amide or amidine bonds between the protein and drug linker complex. Generally, the active ester on the drug linker complex (typically an O-succinimide reagent, such as N-hydroxysuccinimide (NHS) or sulfonyl-NHS ester) reacts with antibody lysine residues, achieving conjugation via an amide bond. On the other hand, stable amidine bonds can be generated on the antibody by reacting an imine ester compound (e.g., Traut's reagent) with antibody lysine residues.

[0359] Drug-linker moieties containing amine reactive groups are known to be coupled to antibodies in one-step and two-step processes via amide bonds. In the first step, a small bifunctional reagent containing both amine and thiol reactive functional groups reacts with an available lysine ε-amino group as a chemical adapter, leaving a free thiol reactive group on the antibody. In the second step, the drug payload or drug-linker complex is attached to the previously introduced thiol reactive group, forming an ADC. The two-step approach is often used as an alternative when the drug / drug-linker complex contains a thiol reactive module, or when introducing an amine reactive module into the drug or drug-linker complex proves difficult. Four commonly used small adapters in two-step coupling are SPDB disulfide, MCC (maleimide methylcyclohexane-1-carboxylate), sulfonyl-SPDB, and hydrazine.

[0360] Cysteine ​​coupling Cysteine ​​modification is most commonly achieved through the modification of cysteine. N The addition occurs via 1,4-conjugation of maleimides with substituted maleimides. Maleimides are particularly attractive reagents because they are readily synthesized and react rapidly with cysteine ​​under mild conditions. The resulting thiosuccinimide conjugates are inherently unstable due to their tendency to undergo reverse Michael addition. This instability can be mitigated by forcing the thiosuccinimide to hydrolyze post-conjugation, resulting in stable chemical bonds. Consequently, many “self-hydrolyzed” maleimides have been developed, in which the addition occurs via adjacent functional groups (e.g., primary amines, polyethylene glycol (PEG) and... N (-aryl)-catalyzed ring-opening reactions are the most promising. Other reagents include α-halocarbonyl compounds, palladium oxidative addition complexes, ethynylphosphonamides, vinylphosphonates, and ethynylbenzoiodooxylons.

[0361] Kang et al., Chem Sci (2021) 12, 13613-13647 (doi: 10.1039 / D1SC02973H), summarized a number of non-maleimide cysteine ​​conjugates, including the use of the following substances: (i) Alkynyl carboxylic acid derivatives; (ii) 5-Methylenepyrrolidone (5MP); (iii) 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB); (iv) Phenylexadiazole sulfone (PODS); (v) Azadibenzocyclooctyne (DBCO); (vi) Phosphite; (vii) 3-Arylpropionitrile (APN); (viii) Perfluoroaromatics; (ix) Ethynylbenzothioxanone (EBX); (x) Bicyclo[1.1.0]butane (BCB) carboxamide; and (xi) Propyleneamide.

[0362] Another possible approach is described in Cheng et al., Front. Oncol. 12:951589 (doi:10.3389 / fonc.2022.951589), in which 2-methylsulfonylpyrimidine is used instead of maleimide.

[0363] Genetic modification of the number of accessible cysteine ​​residues on the antibody surface is a method to achieve site-selective and uniform modification. For example, in THIOMAB, engineered cysteine ​​residues are inserted into the anti-MUC16 antibody via a mutation in heavy chain alanine 114 (HC-A114). Other methods involve engineered antibodies to contain cysteine ​​mutations at D265C, S239C, E269C, K326C, or A327C, or to insert additional cysteine ​​residues before and after the HC-S239, HC-A114, and LC-V205 positions.

[0364] Introducing non-natural amino acids through genetic engineering Introducing non-classical amino acid (ncAA) sites specifically into antibodies can effectively modify them at specific sites, thereby preparing homogenized ADCs.

[0365] NcAAs with unique functional groups (e.g., ketone, azide, cyclopropene, or diene functional groups) have been developed and incorporated into antibodies. These ncAAs include: p-acetylphenylalanine (pAcF), whose ketone side chain is involved in oxime linkage reactions; Nε-(1-methylcycloprop-2-encarbamoyl)-lysine (CpK), whose cyclopropene side chain is involved in IEDDA reactions; p-azidomethylphenylalanine (pAMF), whose azide side chain is involved in click reactions; spiro[2.4]hept-4,6-diene-lysine (SCpHK), whose spiro[2.4]hept-4,6-diene side chain is involved in Diels-Alder reactions; and N6-(2-azidoethoxy)-carbonyl-L-lysine (AzK), whose azide side chain is involved in click reactions.

[0366] Azide-containing ncAAs can undergo rapid CuAAC or SPAAC reactions under physiological conditions. p-Azide-phenylalanine (pAzF) can react with linkers functionalized with, for example, cyclooctynylene and dibenzylcyclooctynylene (DBCO). Cyclopropene derivatives of lysine (N... -[((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl]-l-lysine;CypK) can undergo a rapid and efficient inverse electron-demanding Diels-Alder (IEDDA) reaction with tetrazine-functionalized linkers.

[0367] ncAA containing cyclopentadiene, spiro[2.4]hept-4,6-diene-lysine (SCpHK) and cyclopentadiene-lysine (CpHK) can undergo irreversible Diels-Alder cycloaddition reactions with maleimide-modified drugs.

[0368] Enzymatic conjugation Because of their high specificity and mild reaction conditions, enzymes can be used to achieve site-selective modification of antibodies. Enzymes can directly attach payloads to specific amino acid sequences or introduce reactive functional groups onto antibodies, which can then be further functionalized with the desired payload.

[0369] transpeptidation using sorting enzymes Sorter-mediated antibody conjugation (SMAC) is another enzymatic ligation method. SMAC utilizes Staphylococcus aureus sorterase A (a transpeptidase that cleaves the amide bond between threonine and glycine residues in the LPXTG (X = any amino acid) pentapeptide motif), subsequently catalyzing the ligation of a glycine-functionalized payload to a newly generated C-terminus. The sorterase-recognized motif and a Strep II tag (used to help remove unreacted antibodies) are fused to the C-terminus of the light and heavy chains of different antibodies. A series of pentaglycine-labeled payloads can then be ligated using a sorterase-mediated conjugation reaction.

[0370] Utilizing the transpeptidation of microbial transglutaminase Bacterial transglutaminases are an efficient method for introducing payloads into antibodies in a site-specific manner. Transglutaminases derived from *Streptomyces mobaraensis* catalyze transpeptidation reactions in which a primary amine-containing linker is covalently linked to the primary amide side chain of a specific glutamine (Q295) in the deglycosylated antibody, resulting in an ADC with a defined DAR due to the conjugation of two linker payloads (one conjugation site per heavy chain). The N297Q mutation preceding this conjugation provides two additional reaction sites (leading to the conjugation of a 4-linker payload). This is an alternative version using peptide sequence-specific transglutaminases. These enzymes can recognize and utilize LLQG motifs introduced through genetic engineering, enabling site-specific antibody-drug conjugation. Another advantage of this LLQG-specific bacterial transglutaminase is the flexibility in setting conjugation sites by inserting this short peptide motif into the antibody structure. Other alternative methods allow the use of transglutaminases without deglycosylation.

[0371] DAR will depend on the number of payloads attached to each connector-payload section.

[0372] N-Glycan Engineering The Asn297 (N297) residue within the Fc domain and the N-glycan on this residue are conserved across all IgG classes, making these components attractive reaction sites for broadly applicable ADC conjugation. Aldehyde groups were introduced to the N-glycan termini using β-1,4-galactosyltransferase (GalT) and α-2,6-sialyltransferase (SialT), thereby introducing sialic acid to each N-glycan terminus, which was then converted to aldehyde groups under mild oxidative conditions using NaIO4. The resulting aldehyde groups can be used to conjugate aminooxylated payloads.

[0373] Another approach involves introducing non-natural sugars with orthogonal reaction handles into antibodies. A technique based on this strategy is GlycoConnect, where the glycan chain at Asn297 is pruned using the endoglycosidase Endo S2, followed by the introduction of an azide group using the mutant galactosyltransferase GalT (Y289L) and N-azidoacetylgalactosamine (GalNAz). The azide handle can then be used for strain-promoted click reactions with the payload.

[0374] In some implementations, the linker payload is linked at an amino terminus, which is conjugated to the antigen-binding molecule via transglutaminase.

[0375] In some embodiments, the method further includes purification / separation of antigen-binding molecules (i.e., purification / separation from unreacted precursors and / or byproducts). In some embodiments, the antigen-binding molecules can be purified / separated by chromatography (e.g., size exclusion chromatography).

[0376] This disclosure also provides an antigen-binding molecule that can be obtained or acquired by the methods of this disclosure.

[0377] Composition This disclosure provides a composition comprising an antigen-binding molecule according to this disclosure.

[0378] The antigen-binding molecules described herein can be formulated into pharmaceutical compositions or medicaments for clinical use and may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Therefore, this disclosure provides a pharmaceutical composition / medication comprising the antigen-binding molecules described herein.

[0379] The pharmaceutical compositions / medications disclosed herein may comprise one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants (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), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium dioxide).

[0380] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, substances, compositions, dosage forms, etc., that are suitable for contact with the tissues of the subject in question (e.g., human subjects) within the bounds of reasonable medical judgment without excessive toxicity, irritation, anaphylactic responses, or other problems or complications commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener in the compositions according to this disclosure must also be "acceptable," i.e., compatible with other components in the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, or sweeteners can be found in standard pharmaceutical textbooks, such as Remington's 'The Science and Practice of Pharmacy' (edited by A. Adejare), 23rd edition (2020), Academic Press.

[0381] The pharmaceutical compositions and drugs disclosed herein can be formulated for application via local, parenteral, systemic, intracavitary, intravenous, arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal routes. In some embodiments, the pharmaceutical compositions / drugs can be formulated for administration by injection or infusion, or by ingestion.

[0382] Suitable formulations may contain antigen-binding molecules provided in a sterile or isotonic medium. Pharmaceuticals and pharmaceutical compositions may be formulated into liquid forms (including gels). Liquid formulations may be formulated for administration to selected areas of the human or animal body by injection or infusion (e.g., via catheter).

[0383] In some implementations, the pharmaceutical composition / drug is formulated for injection or infusion, such as into a blood vessel, target tissue / organ, or tumor.

[0384] This disclosure also provides methods for producing pharmaceutically useful compositions, which may include one or more steps selected from the following: This produces the antigen-binding molecules described in this article; Isolate / purify the antigen-binding molecules described herein; and / or The antigen-binding molecules described herein are mixed with pharmaceutically acceptable carriers, adjuvants, excipients, or diluents.

[0385] For example, another aspect of this disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for treating a disease / condition (such as the disease / condition described herein), the method comprising formulating the pharmaceutical composition or medicament by mixing the antigen-binding molecule described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0386] Treatment or preventive application The antigen-binding molecules and compositions described herein can be used for the treatment and preventive intervention of diseases such as cancer.

[0387] It should be understood that the antigen-binding molecules and compositions disclosed herein can be used to treat / prevent any disease / condition, and such treatment / prevention can achieve therapeutic or preventive benefits by reducing the expression level or activity of HER2, or by reducing the number or activity of cells containing / expressing HER2.

[0388] For example, the disease / disorder can be one in which HER2 or cells expressing / overexpressing HER2 are pathologically involved, such as a disease / disorder in which increased HER2 levels / activity, or an increased number / proportion of cells containing / expressing HER2, is positively correlated with the occurrence, development, or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder. In some embodiments, increased HER2 levels / activity, or an increased number / proportion of cells containing / expressing HER2, may be a risk factor for the occurrence, development, or progression of the disease / disorder.

[0389] This disclosure provides an antigen-binding molecule or composition described herein for use in medical treatment or prevention methods. It also provides the antigen-binding molecule or composition described herein for use in methods of treating or preventing cancer (e.g., the cancer described herein). It further provides the use of the antigen-binding molecule or composition described herein in the preparation of a medicament for treating or preventing cancer (e.g., the cancer described herein). It also provides a method of treating or preventing cancer (e.g., the cancer described herein) in a subject, comprising administering to the subject a therapeutically or preventively effective amount of the antigen-binding molecule or composition described herein.

[0390] These methods may be effective in reducing the development or progression of cancer, alleviating cancer symptoms, or reducing cancer pathology. These methods may be effective in preventing cancer progression, such as stopping cancer from worsening or slowing its development. In some implementations, these methods can improve cancer, such as alleviating cancer symptoms or reducing certain other factors related to cancer severity / activity. In some implementations, these methods can prevent cancer from progressing to later stages (e.g., the chronic or metastatic phase).

[0391] As used herein, “cancer” can be or includes any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation), growth, or tumor. Cancer can be benign or malignant. Cancer can be primary or secondary (metastatic). A growth or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. This cancer may originate from the following tissues or cells (e.g.): adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bones, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, bile ducts, esophagus, glial cells, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, or white blood cells.

[0392] The tumor requiring treatment may be a neurological tumor or a non-neurological tumor. Neurological tumors may originate from the central or peripheral nervous system, such as gliomas, medulloblastomas, meningiomas, neurofibromas, ependymomas, schwannomas, neurofibrosarcomas, astrocytomas, and oligodendrogliomas. Non-neurological cancers / tumors may originate from any other non-neurological tissue; examples include: melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, non-small cell lung cancer (NSCLC), hematologic malignancies, and sarcomas.

[0393] In some embodiments, the cancer to be treated / prevented comprises cells expressing EGFR family members (e.g., HER2, EGFR, HER3, or HER4) and / or cells expressing EGFR family member ligands. In some embodiments, the cancer to be treated / prevented comprises cells expressing mutant or wild-type EGFR family members (e.g., HER2, EGFR, HER3, or HER4). In some embodiments, the cancer to be treated / prevented is a cancer that is EGFR family member positive. In some embodiments, the cancer comprises cells overexpressing EGFR family members and / or EGFR family member ligands. Overexpression can be determined by detecting expression levels higher than those in corresponding non-cancer or non-tumor tissues.

[0394] The mode of expression can be determined by any suitable method. Expression can be gene expression or protein expression. Gene expression can be determined, for example, by detecting mRNA encoding HER2, such as using quantitative real-time PCR (qRT-PCR). Protein expression can be determined, for example, by antibody-based methods, such as Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[0395] In some implementations, cancer is a type of cancer in which HER2 is pathologically involved. That is, in some implementations, cancer refers to cancer caused or exacerbated by HER2 expression, cancer in which HER2 expression is a risk factor, and / or cancer in which HER2 expression is positively correlated with the occurrence, development, progression, severity, or metastasis of the cancer. Cancer may be characterized by HER2 expression; for example, cancer may contain cells expressing HER2 (e.g., tumor tissue cells). Such cancers may be referred to as HER2-positive cancers. HER2-positive cancers may contain cells expressing HER2 (e.g., on the cell surface). HER2-positive cancers may overexpress HER2.

[0396] In some implementations, the cancer to be treated / prevented comprises cells carrying a genetic variant (e.g., a mutation) that, compared to comparable cells carrying a reference allele that does not contain the genetic variant (e.g., a non-mutated or "wild-type" allele), results in increased expression and / or activity of HER2 (gene and / or protein). The genetic variant may be or includes insertions, deletions, substitutions, or larger-scale translocations / rearrangements of nucleotide sequences relative to the reference allele.

[0397] Mutations known or predicted to "lead" to increased HER2 expression will result in increased HER2 gene / protein expression, or may be associated with increased HER2 gene / protein expression. Mutations known or predicted to "lead" to increased HER2 activity will result in, or may be associated with, increased HER2-mediated signaling and / or EGFR-mediated signaling. Mutations leading to increased HER2 expression and / or activity can be termed "activating" mutations.

[0398] Mutations that increase HER2 expression may result in the expression of the HER2 gene or protein, which is not expressed in equivalent cells that do not carry the mutation and / or is not encoded by the genomic nucleic acids of those equivalent cells. In other words, HER2 expression may be a result of a mutation, so "increased expression" may be due to a lack of expression.

[0399] Mutations that increase HER2 expression may result in increased expression of the HER2 gene or protein, which is expressed in equivalent cells that do not contain the mutation, and / or encoded by the genomic nucleic acids of those equivalent cells. For example, a cell may contain a mutation that results in an increased level of transcription of the nucleic acid encoding HER2 relative to the level of transcription of the nucleic acid encoding HER2 in equivalent cells that do not contain the mutation.

[0400] In some implementations, mutations that increase HER2 expression may result in increased HER2 gene expression relative to equivalent cells without the mutation. In some implementations, mutations that increase HER2 expression may result in increased HER2 protein expression relative to equivalent cells without the mutation.

[0401] In some implementations, mutations that lead to increased HER2 expression may result in an increased level of HER2 on the cell surface or on the cell surface of cells containing the mutation, relative to equivalent cells that do not contain the mutation.

[0402] Cells with increased HER2 expression levels relative to reference cells (e.g., due to mutations) can be described as “overexpressing” HER2 or having “upregulated” HER2 expression. For example, a cancer containing cells carrying mutations that lead to increased HER2 expression can be described as a cancer containing cells exhibiting overexpression / upregulated HER2 expression, relative to a mutation-free equivalent cell. In some embodiments, the mutation-free reference cell can be a non-cancer cell (e.g., having an equivalent cell type) or a cancer cell (e.g., having an equivalent cancer type).

[0403] Mutations that lead to enhanced HER2 activity may increase the level of HER2-mediated signaling relative to HER2-mediated signaling in unmutated equivalent cells.

[0404] In some embodiments, the cancer to be treated / prevented according to this disclosure is characterized, for example, by increased expression and / or activity (i.e., gene and / or protein expression) of HER2 in organs / tissues / subjects affected by the disease / condition, compared to normal organs / tissues / subjects (i.e., in the absence of the disease / condition). In some embodiments, the cells and / or tumors of the cancer to be treated / prevented are characterized, for example, by increased expression and / or activity of HER2, compared to expression and / or activity levels observed in equivalent non-cancer / non-tumor tissues.

[0405] HER2 overexpression cancer may be due to ERBB2 Gene amplification leads to HER2 overexpression.

[0406] In some implementations, the cancer to be treated / prevented according to this disclosure is ERBB2 Amplified cancer.

[0407] ERBB2 The amplification can be identified using techniques well known in the art, such as immunohistochemical analysis and in situ hybridization analysis (see, for example...). and Jeleń, Adv Clin Exp Med. (2015) 24(5):899-903). For example, it can be assessed by fluorescence in situ hybridization. ERBB2 Amplifications, such as those described in Stocker et al., PLoS One (2016) 11(7):e0159176. ERBB2 Amplified cancers may include those obtained by in situ hybridization. ERBB2 With centromere 17 ( CEP17 The ratio of ) to 2 (e.g., ≥ 4, ≥ 8).

[0408] For reviews on HER2 and its association with and role in cancer, see, for example, Oh and Bang, Nat Rev Clin Oncol (2020) 17:33-48, Hudis, NEJM (2007) 357(1):39-51, Arteaga and Engelman, Cancer Cell. (2014) 25(3): 282-303, and Yan et al., Cancer Metastasis Rev. (2015) 34(1):157-164. All of these are incorporated herein by reference.

[0409] It has been observed in a variety of cancers, including breast cancer, stomach cancer, and esophageal cancer. ERBB2Amplification (Koboldt et al., Nature. (2012) 490:61–70). Potentially activating (i.e. gain-of-function) mutations in HER2 have also been reported in cancers such as lobular breast cancer, lung cancer, gastric cancer, bladder cancer, and endometrial cancer. The following cancers show very high HER2 expression (i.e., HER2 positive; see Table 2 in Yan et al., Cancer Metastasis Rev. (2015) 34(1):157-164): bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophagogastric junction cancer, gallbladder cancer, gastric adenocarcinoma, gastrointestinal stromal tumor, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, kidney cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, melanoma, neuroendocrine tumors, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibroma, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer.

[0410] In some implementations, the cancer is selected from: cancers containing cells expressing / overexpressing EGFR family members, cancers containing cells expressing / overexpressing HER2, cancers containing cells not expressing EGFR family members, cancers containing cells not expressing HER2, HER2-low expression cancers, HR-positive cancers, solid tumors, bladder cancer, breast cancer, HER2-positive breast cancer, metastatic HER2-positive breast cancer, HER2-low expression breast cancer, unresectable or metastatic HER2-low expression breast cancer, HR-positive breast cancer, triple-negative breast cancer, cervical cancer, gastric cancer, HER2-positive gastric cancer, locally advanced or metastatic HER2-positive gastric cancer, cholangiocarcinoma, colorectal cancer, gastroesophageal junction cancer, gallbladder cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, HER2-positive gastroesophageal junction adenocarcinoma, locally advanced or metastatic HER2-positive gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumors, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, kidney cancer, lung cancer, non-small cell lung cancer, and other cancers containing HER2-positive EGFR ...stromal tumors, gastrointestinal stromal tumors, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, kidney cancer, lung cancer, non-small cell lung cancer, and other cancers containing HER2-positive EGFR-positive EGFR-positive EGFR-positive stromal tumors. ERBB2 Unresectable or metastatic non-small cell lung cancer, small cell lung cancer, melanoma, neuroendocrine tumors, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibroma, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer with activating mutations.

[0411] In some embodiments, cancer according to this disclosure refers to cancer for which trastuzumab has been approved for treatment. In some embodiments, the cancer is selected from: metastatic HER2-positive breast cancer, unresectable or metastatic HER2-low expression (i.e., IHC 1+ or IHC 2+, ISH-) breast cancer, unresectable or metastatic non-small cell lung cancer (including... ERBB2Activating mutations), as well as locally advanced or metastatic HER2-positive gastric cancer or gastroesophageal junction adenocarcinoma.

[0412] In some implementations, the cancer is selected from: cancers containing cells that do not overexpress EGFR family members (e.g., HER2, EGFR, HER3, or HER4), cancers containing cells that do not overexpress HER2, HER2-low expression (i.e., IHC 1+ or IHC 2+, ISH-) cancers, HER2-low expression breast cancers, hormone receptor (HR) positive cancers (i.e., cancers containing cells that express estrogen receptor (ER) and / or progesterone receptor (PR)), HR positive breast cancers, and triple-negative (i.e., HER2-negative, ER-negative, and PR-negative) breast cancers.

[0413] In this article, "HER2 low expression" cancer refers to cancer with an immunohistochemical (IHC) score of 1+ for HER2 expression, or cancer with an IHC score of 2+ for HER2 expression but without associated HER2 by in situ hybridization (ISH) analysis. ERBB2 Amplified cancer. IHC analysis and scoring of HER2 expression and... ERBB2 The amplified ISH analysis is described, for example, by Wolff et al., J Clin Oncol. (2018) 36(20):2105-2122, the entire contents of which are incorporated herein by reference.

[0414] In this article, " ERBB2 "Activation mutations" may: increase ERBB2 Transcription; increase ERBB2 The level of encoded RNA; reduced ERBB2 Degradation of encoded RNA; increase ERBB2 The level of the encoded protein; increase (promote). ERBB2 Normal splicing of the encoded precursor mRNA; addition of the encoded ERBB2 Translation of the mRNA encoding the protein; increase (promote) ERBB2 Normal post-translational processing of the encoded protein; increased (promoted) ERBB2 Normal transport of encoded proteins; reduction ERBB2 Degradation of encoded proteins; improvement ERBB2 The functional level of the encoded protein; and / or conferring ERBB2 Novel properties of the encoded protein.

[0415] In some implementations, the cancer is selected from: cancers containing cells that do not overexpress EGFR family members (e.g., HER2, EGFR, HER3, or HER4), cancers containing cells that do not overexpress HER2, HER2-low expression (i.e., IHC 1+ or IHC 2+, ISH-) cancers, HER2-low expression breast cancers, hormone receptor (HR) positive cancers (i.e., cancers containing cells that express estrogen receptor (ER) and / or progesterone receptor (PR)), HR positive breast cancers, and triple-negative (i.e., HER2-negative, ER-negative, and PR-negative) breast cancers.

[0416] In some implementations, the cancer may be recurrent cancer. As used herein, “recurrent” cancer refers to cancer that responds to treatment (e.g., first-line treatment for cancer) but subsequently relapses / progresses, such as after a period of remission. For example, recurrent cancer may refer to a cancer whose growth / progression was previously suppressed by treatment (e.g., first-line treatment for cancer) but subsequently resumes. Cancer that relapses with respect to a particular treatment may be described as having acquired resistance to that treatment.

[0417] In some implementations, the cancer may be refractory cancer. As used herein, "refractory" cancer refers to cancer that does not respond to treatment (e.g., first-line treatment for cancer). For example, refractory cancer may refer to cancer whose growth / progression is not inhibited by treatment (e.g., first-line treatment for cancer). In some implementations, refractory cancer may be cancer in which a subject receiving cancer treatment does not show a partial or complete response to the treatment. Cancer that is refractory to a particular treatment may be described as having inherent resistance to such treatment.

[0418] In some embodiments, the cancer is relapsed or refractory to treatment with a DNA damage response (DDR) inhibitor. In some embodiments, the cancer is refractory to treatment with a DDR inhibitor. In some embodiments, the cancer is relapsed to treatment with a DDR inhibitor. In some embodiments, the cancer has inherent resistance to treatment with a DDR inhibitor. In some embodiments, the cancer has acquired resistance to treatment with a DDR inhibitor. According to these embodiments, the DDR inhibitor may be administered in the form of an antigen-binding molecule comprising a payload portion containing or consisting of a DDR inhibitor, or the DDR inhibitor may be administered in an unconjugated form.

[0419] In this article, when cancer is described as recurrent / refractory / resistant to a specific intervention, it can be simply described as 'recurrent / refractory / resistant' to the relevant intervention.

[0420] DDR inhibitors and their application in cancer treatment are described in, for example, 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 incorporated herein by reference in their entirety. In some embodiments, the DDR inhibitors according to this disclosure are selected from: PARP inhibitors (e.g., olaparib, rucaparib, niraparib, taprazolepanib, veliparib, pamipanib, cimetabane, senaparib, SC-10914, 2X-121, AMXI-5001, JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-1002, RBN-239). 7) ATM inhibitors (e.g., CP-466722, KU-55933, KU-60019, KU-59403, AZ31, AZ32, AZD0156, AZD1390), ATR inhibitors (e.g., M6620 (bezotitab), M4344 (VX-803), AZD6738 (celasitab), BAY1895344 (elimosatitab)), WEE1 inhibitors (e.g., adatab, Debio) 0123, PD0166285, PD0407824, AZD1775), CHK1 / 2 inhibitors (e.g., CBP-501, preceptib, MK-8776, GDC-0575, SRA-737), DNA-PK inhibitors (e.g., CC-115, LY-3023414, AsiDNA, M3814 (nidisitib), M9831 (VX-984)) and PLK1 inhibitors (e.g., BI-6727 (voracetyl) and PCM-075 (onvatinib)).

[0421] In some embodiments, the cancer is a relapsed or refractory cancer to treatment with a DNA topoisomerase I (TOP1) inhibitor. In some embodiments, the cancer is refractory to treatment with a TOP1 inhibitor. In some embodiments, the cancer is relapsed to treatment with a TOP1 inhibitor. In some embodiments, the cancer has inherent resistance to treatment with a TOP1 inhibitor. In some embodiments, the cancer has acquired resistance to treatment with a TOP1 inhibitor. According to such embodiments, the TOP1 inhibitor may be administered in the form of an antigen-binding molecule comprising a payload portion containing or consisting of a TOP1 inhibitor, or the TOP1 inhibitor may be administered in a non-conjugated form.

[0422] DNA topoisomerase I inhibitors and their application in cancer treatment have been described 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 incorporated herein by reference. In some embodiments, the TOP1 inhibitors according to this disclosure are selected from: camptothecin, irinotecan, etotecan, SN-38, DX-8951f (irinotecan mesylate), DXd(1), DXd(2), irinotecan, FL118, topotecan, gemmatocarbazone, belotetane, delutec, belotetane, rubitecan, letopecan, diflutecan, calentecan, slatecan, namitecan, irinotecan, DRF-1042, demotecan, NSC606985, gemitecan, ZBH-1205, Genz-644282, non-CPT1, indonotecan (LMP-400), indonotecan (LMP-776), and LMP744.

[0423] In some embodiments, the cancer to be treated / prevented according to this disclosure is: cancer that is relapsed or refractory to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein), and cancer that is relapsed or refractory to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). In some embodiments, the cancer is: cancer that is refractory to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein), and cancer that is refractory to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). In some embodiments, the cancer is: cancer that is relapsed to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein), and cancer that is relapsed to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). In some embodiments, the cancer is: cancer that is refractory to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein), and cancer that is relapsed to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). In some embodiments, the cancer is: cancer that relapses upon treatment with a DDR inhibitor (e.g., a DDR inhibitor as described herein), and cancer that is refractory to treatment with a TOP1 inhibitor (e.g., a TOP1 inhibitor as described herein). According to such embodiments, the TOP1 inhibitor and / or DDR inhibitor may be administered in the form of an antigen-binding molecule comprising a payload portion comprising or consisting of a TOP1 inhibitor and / or DDR inhibitor, or the TOP1 inhibitor and / or DDR inhibitor may be administered in an unconjugated form.

[0424] Treating cancer according to the methods of this disclosure can achieve one or more of the following therapeutic effects: reducing the number of cancer cells in the subject; shrinking the size of cancerous tumors / lesions in the subject; inhibiting (e.g., preventing or slowing) the growth of cancer cells in the subject; inhibiting (e.g., preventing or slowing) the development / progression of cancer (e.g., progressing to an advanced stage or metastasis); reducing the severity of cancer symptoms in the subject; prolonging the subject's survival (e.g., progression-free survival or overall survival); reducing factors related to the number or activity of cancer cells in the subject; and / or reducing the cancer burden in the subject.

[0425] Subjects may be assessed according to a revised response assessment criterion: the Lugano classification (e.g., described in Cheson et al., J Clin Oncol (2014) 32: 3059-3068, incorporated herein by reference) to determine their response to treatment. In some embodiments, treatment of subjects according to the methods of this disclosure may result in one of the following outcomes: complete response, partial response, or disease stabilization.

[0426] Prevention may refer to preventing the occurrence of cancer and / or preventing the progression of cancer, such as preventing the cancer from developing into an advanced stage (e.g., metastasis).

[0427] In some embodiments, the administration of the antigen-binding molecule / composition according to this disclosure may be associated with one or more of the following: inhibiting the occurrence / progression of cancer, delaying / preventing the occurrence of cancer, reducing / delaying / preventing tumor growth, reducing / delaying / preventing tissue invasion, reducing / delaying / preventing metastasis, alleviating the severity of one or more cancer symptoms, reducing the number of cancer cells, reducing the cancer burden, shrinking the size / volume of the tumor, and / or prolonging the survival of patients with cancer (e.g., progression-free survival or overall survival).

[0428] According to various aspects of this disclosure, methods for treating and / or preventing cancer according to this disclosure may include inhibiting tumor growth, reducing tumor size / volume, and / or prolonging the survival of patients with cancer.

[0429] According to various aspects of this disclosure, methods for or including (e.g., in the context of treating / preventing cancer, such as the cancer described herein) one or more of the following are provided: It binds to cells expressing HER2; Inhibits the proliferation of cells expressing HER2; Kill cells that express HER2; Inhibit tumor growth and / or reduce tumor size / volume (e.g., tumor size / volume of cancers expressing HER2); and / or Extend the survival of patients with cancer (such as cancers that express HER2).

[0430] Also provided are antigen-binding molecules and compositions according to this disclosure for use in such methods, and the use of antigen-binding molecules and compositions according to this disclosure in the preparation of compositions (e.g., pharmaceuticals) for use in such methods. It should be understood that these methods generally involve administering an antigen-binding molecule according to this disclosure to a subject.

[0431] Similarly, after treatment or preventative intervention according to this disclosure, subjects may observe one or more of the following (e.g., compared to pre-intervention levels / numbers / proportions): Inhibits the proliferation of cells expressing HER2; Kill cells that express HER2; Inhibit tumor growth and / or reduce tumor size / volume (e.g., tumor size / volume of cancers expressing HER2); and / or Extend the survival of patients with cancer (such as cancers that express HER2).

[0432] In some implementations, the treatment / preventive interventions according to this disclosure may be described as being "related" to one or more of the effects described in the preceding paragraph. Those skilled in the art can assess these characteristics using techniques conventionally employed in the art.

[0433] The antigen-binding molecules and compositions disclosed herein are preferably administered in a "therapeuticly effective" or "preventatively effective" amount, sufficient to provide a therapeutic or preventative benefit to the subject. The actual dosage, rate of administration, and timing of administration will depend on the nature and severity of the disease / condition and the specific circumstances of the medication being administered. The development of a treatment regimen (e.g., determination of dosage) falls under the responsibility of general practitioners and other physicians, and will typically take into account the disease / condition to be treated, the individual subject's condition, the site of delivery, the method of administration, and other factors known to the physician. Examples of the aforementioned techniques and protocols can be found in Remington's *The Science and Practice of Pharmacy* (edited by A. Adejare), 23rd edition, Edition (2020), Academic Press.

[0434] The antigen-binding molecules and compositions disclosed herein can be administered via, for example, parenteral, systemic, local, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral, or transdermal routes. Administration can be by injection, infusion, or oral administration.

[0435] In some aspects and embodiments, the articles of this disclosure can be applied to target tissues / organs (e.g., tissues / organs affected by disease / condition (e.g., tissues / organs exhibiting symptoms of disease / condition)). In some aspects and embodiments, the articles of this disclosure can be administered into the bloodstream by injection or infusion (e.g., via a cannula) (i.e., intravenous / arterial administration), or subcutaneously or orally. In some aspects and embodiments, the articles of this disclosure can be applied to tumors.

[0436] In some embodiments, the treatment or preventive intervention according to this disclosure may also include the administration of another agent to treat / prevent the associated disease / condition. The administration of the antigen-binding molecules and compositions described herein may be performed alone or in combination with other treatment methods, depending on the condition to be treated (i.e., simultaneous or sequential administration). Simultaneous administration means administration together with another therapeutic agent, for example, in the form of a pharmaceutical composition containing two agents (a combination formulation), 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 means administration of one agent first, followed by the administration of another agent alone after a specific time interval. It is not required that the two agents be administered via the same route, although this is true in some embodiments. The time interval can be any time interval.

[0437] Multiple doses of antigen-binding molecules and compositions can be provided. Predetermined time intervals can be spaced between doses, which can be selected as 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. For example, a dose can be administered every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0438] Detection methods This disclosure also provides the products of this disclosure and methods for detecting, locating, or imaging HER2 or cells expressing HER2.

[0439] The antigen-binding molecules described herein can be used in methods involving the detection of the binding of antigen-binding molecules to HER2. Such methods may involve the detection of the binding complex of the antigen-binding molecule and HER2. It should be understood that HER2 can be cellularly expressed HER2, for example, inside or on the surface of cells expressing HER2.

[0440] Therefore, a method is provided that includes contacting a sample containing or suspected of containing HER2 and detecting the formation of an antigen-binding molecule and a HER2 complex. Furthermore, a method is provided that includes contacting a sample containing or suspected of containing cells expressing HER2 and detecting the formation of an antigen-binding molecule and a complex of HER2-expressing cells.

[0441] Various suitable methods are well known in the art, including immunoassays such as sandwich assays like ELISA. These methods may involve labeling an antigen-binding molecule or target, or both, with a detectable portion, such as fluorescent labeling, phosphorescent labeling, luminescent labeling, immunodetectable labeling, radioactive labeling, chemical, nucleic acid, or enzyme labeling as described herein. Detection techniques are well known to those skilled in the art, and detection techniques corresponding to the labeling agents can be selected.

[0442] Methods for detecting HER2 or HER2-expressing cells include those for diagnosing / predicting the diseases / conditions described herein.

[0443] Such methods can be performed on patient samples in vitro or after processing the patient sample. Once the sample is collected, in vitro testing can be performed without the patient's presence; therefore, this method can be performed outside of humans or animals. In some implementations, the method is performed in vivo.

[0444] Such methods may involve detecting or quantifying HER2 and / or cells expressing HER2, for example, in patient samples. If the method includes quantifying relevant factors, it may also include comparing the determined quantities to standard or reference values ​​as part of a diagnostic or prognostic assessment. Other diagnostic / prognostic tests may be used in conjunction with the tests described herein to improve the accuracy of diagnosis or prognosis, or to confirm results obtained using the tests described herein.

[0445] Detection in a sample can be used to diagnose a disease / condition (e.g., cancer), susceptibility to a disease / condition, or to provide a prognosis (prediction) for a disease / condition (e.g., the disease / condition described herein). The diagnosis or prognosis may be related to a pre-existing (previously diagnosed) disease / condition.

[0446] Samples can be taken from any tissue or bodily fluid. Samples may include or be derived from: a volume of blood; a volume of serum, which may be extracted from the individual's blood, including the fluid portion of blood obtained after removing fibrin clots and blood cells; tissue samples or biopsies; pleural effusion; cerebrospinal fluid (CSF); or cells isolated from the individual. In some embodiments, samples may be obtained or derived from tissue affected by a disease / condition (e.g., tissue exhibiting disease symptoms, or tissue involved in the pathogenesis of the disease / condition).

[0447] Subjects may be selected for diagnostic / prognostic assessment based on the presence of symptoms indicating the disease / condition described herein, or based on subjects considered to be at risk of developing the disease / condition described herein.

[0448] This disclosure also provides methods for selecting / stratifying subjects for treatment with HER2-targeting agents. In some embodiments, subjects are selected for treatment / prevention according to the methods of this disclosure, or based on the detection / quantification of HER2 or HER2-expressing cells, for example, in samples obtained from individuals, to identify subjects who will benefit from such treatment / prevention.

[0449] Subjects According to the aspects described herein, the subject can be any animal or human. Preferably, the subject is a mammal, more preferably a human. The subject can be a mammal other than a human, but preferably a human. The subject can be male or female. The subject can be a patient. The subject may have been diagnosed with a disease or condition requiring treatment (e.g., cancer, such as the cancer described herein), may be suspected of having such a disease / condition, or may be at risk of having / being infected with such a disease / condition.

[0450] In some embodiments, subjects receiving treatment according to the treatment or prevention methods of this disclosure are subjects who have cancer (such as the cancer described herein) or are at risk of developing such cancer. In embodiments according to this disclosure, subjects may be selected for treatment based on methods characterizing certain biomarkers of such diseases / conditions.

[0451] In some implementations, patients may be selected for the treatment described herein based on the detection of cancer expressing / overexpressing HER2; such detection may be performed, for example, on samples obtained from the subject (e.g., biopsy samples, such as tumor biopsy samples).

[0452] Reagent test kit This disclosure also provides a multi-component kit. A kit according to this disclosure may include all or part of the components for performing the methods described herein.

[0453] The kit may have at least one container containing a predetermined amount of the antigen-binding molecule or composition described herein.

[0454] In some aspects of this disclosure, a multi-component kit is provided. In some embodiments, the kit may contain the antigen-binding molecules or compositions described herein and may be provided in a predetermined quantity.

[0455] This kit provides the antigen-binding molecules or compositions described herein, along with instructions for administration to a patient to treat a specific disease / condition (such as the disease / condition described herein, such as cancer).

[0456] This kit provides an antigen-binding portion according to the present disclosure and a linker-payload portion according to the present disclosure. The kit may also include reagents for conjugating the antigen-binding portion and the linker-payload portion.

[0457] The kit may also include reagents, buffers, and / or standards required to perform the methods according to this disclosure. Kits according to this disclosure may include instructions for use, such as in the form of a user manual or leaflet. The user manual may contain protocols for performing one or more of the methods described herein.

[0458] Sequence identity As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in a target sequence that are identical to those in a reference sequence after alignment and, where necessary, the introduction of gaps to achieve the maximum percentage of sequence identity between sequences. To determine the percentage of sequence identity between two or more amino acid or nucleic acid sequences, pairwise and multiple sequence alignments can be performed using various methods known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. 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). When using this type of software, it is best to use the default parameters, such as open shot penalty and extension penalty.

[0459] sequence

[0460]

[0461]

[0462]

[0463]

[0464] *** This disclosure includes combinations of the described aspects and preferred features, except for combinations that are expressly not permitted or expressly avoided.

[0465] The chapter titles used in this article are for organizational purposes only and are not intended to limit the topics described.

[0466] Various aspects and embodiments of this disclosure will now be illustrated by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0467] Throughout this specification (including the claims below), unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to include the specified integer or step or group of integers or steps but not exclude any other integer or step or group of integers or steps.

[0468] It must be noted that, unless the context explicitly specifies otherwise, as used in the specification and appended claims, the singular forms “a,” “an,” and “the” include plural indicators. A range may be expressed herein as beginning “about” of a particular value and / or ending “about” of another particular value. When such a range is expressed, another embodiment includes the range from said one particular value and / or to said other particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that a specific value forms another embodiment.

[0469] The nucleic acid sequences disclosed or mentioned in this article also explicitly consider their reverse complementary sequences.

[0470] The methods described herein are preferably performed in vitro. The term "in vitro" is intended to cover procedures performed with cultured cells, while the term "in vivo" is intended to cover procedures performed with / on a complete multicellular organism.

[0471] Numerical values ​​in this document may be expressed as “about” a specific value. Similarly, ranges in this document may be expressed as “about” a specific value and / or to “about” another specific value. The term “about” in relation to numerical values ​​is optional, for example, indicating + / - 10%. For example, mentioning “about 10%” should be understood as 9% to 11%. When “about” is used in this document, the value preceding it should also be taken into account. For example, mentioning “about 10%” also specifically considers 10%. Attached Figure Description

[0472] The implementation scheme and experiments of the principle of the present invention will be discussed and explained below with reference to the accompanying drawings.

[0473] Figure 1AThe study showed the inhibitory effects of TOP1 inhibitors and ATR inhibitors, alone and in combination, on HCT-116 cells.

[0474] Figure 1B The study showed the inhibitory effects of TOP1 inhibitors and CHK1 inhibitors, alone and in combination, on HCT-116 cells.

[0475] Figure 1C The study showed the inhibitory effects of TOP1 inhibitors and ATR inhibitors, alone and in combination, on HEC-1B cells.

[0476] Figure 1D The study showed the inhibitory effects of TOP1 inhibitors and CHK1 inhibitors, alone and in combination, on HEC-1B cells.

[0477] Figure 2 Cell death of the cancer cell lines shown was demonstrated after 7 days of in vitro treatment with trastuzumab (T-(Exa+Ber)) conjugated with ixotecan and bezotib, trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with ixotecan (isotype control).

[0478] Figure 3 The study showed the change in tumor volume over time in mice with a JIMT-1 cell line-derived xenograft model of breast ductal carcinoma, which were treated with PBS (the medium), trastuzumab conjugated with eczema and bezotib (T-(Exa+Ber)), or trastuzumab-durutecan (T-DXd).

[0479] Figure 4 The changes in body weight (in grams (g) over time in mice with a JIMT-1 cell line-derived xenograft model of breast ductal carcinoma, treated with PBS (the medium), trastuzumab conjugated with eczema and bezotib (T-(Exa+Ber)), or trastuzumab-durutecan (T-DXd), respectively.

[0480] Figure 5A The binding of trastuzumab (T(naked)), trastuzumab conjugated with eczemab and bezotib (T-(Exa+Ber)), trastuzumab derutec (T-DXd), isotype-matched control antibody (isotype(naked)), or isotype-matched control antibody conjugated with eczemab (isotype(Exa)) to live BT-474, NCI-N87, JIMT-1, HEC-1-B, or HCT116 cells was shown by flow cytometry.

[0481] Figure 5BThe mean fluorescence intensity (MFI) of trastuzumab (T(naked)), trastuzumab conjugated with eczemab and bezotib (T-(Exa+Ber)), F trastuzumab derutec (T-DXd), isotype-matched control antibody (isotype (naked)), or isotype-matched control antibody conjugated with eczemab (isotype (Exa)) as determined by flow cytometry is shown.

[0482] Figure 6A The subcellular localization of trastuzumab conjugated with eczemab and bezotiab (T-(Exa+Ber)) or trastuzumab derutecan (T-DXd) in HEC-1-B cells after 0, 0.5, or 2 hours of incubation is shown, as determined by immunofluorescence microscopy.

[0483] Figure 6B The subcellular localization of trastuzumab conjugated with eczemab and bezotib (T-(Exa+Ber)) or trastuzumab derutecan (T-DXd) in NCI-N87 cells after 0, 0.5, or 2 hours of incubation is shown, as determined by immunofluorescence microscopy.

[0484] Figure 7A The study showed cell death of HEC1-B cells after 3 days of in vitro treatment with trastuzumab (T(naked)), trastuzumab conjugated with eicentec and bezotib (T-(Exa+Ber)), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eicentec (isotype control).

[0485] Figure 7B The study showed cell death in NCI-N87 cells after 3 days of in vitro treatment with trastuzumab (T(naked)), trastuzumab conjugated with eicentec and bezotib (T-(Exa+Ber)), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eicentec (isotype control).

[0486] Figure 7C The study showed cell death of HCT-116 cells after 3 days of in vitro treatment with trastuzumab (T(naked)), trastuzumab conjugated with eicentec and bezotib (T-(Exa+Ber)), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eicentec (isotype control).

[0487] Figure 7DThe study showed cell death in BT474 cells after 3 days of in vitro treatment with trastuzumab (T(naked)), trastuzumab conjugated with eicentec and bezotib (T-(Exa+Ber)), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eicentec (isotype control).

[0488] Figure 7E The study showed cell death of JIMT-1 cells after 3 days of in vitro treatment with trastuzumab (T(naked)), trastuzumab conjugated with eicentec and bezotib (T-(Exa+Ber), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eicentec (isotype control) at a concentration of 333 nM.

[0489] Figure 7F The study showed the percentage of cell death in HEC1-B cells after 3 days of in vitro treatment with trastuzumab conjugated with eczema and bezotib (T-(Exa+Ber)), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eczema (isotype control). The concentrations of the aforementioned antigen-binding molecules provided an equivalent payload concentration of 0.857 nM.

[0490] Figure 7G The study showed the percentage of cell death in NCI-N87 cells after 3 days of in vitro treatment with trastuzumab conjugated with eczema and bezotib (T-(Exa+Ber)), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eczema (isotype control). The concentrations of the aforementioned antigen-binding molecules provided an equivalent payload concentration of 0.857 nM.

[0491] Figure 7H The study showed the percentage of cell death in HCT-116 cells after 3 days of in vitro treatment with trastuzumab conjugated with eczema and bezotib (T-(Exa+Ber)), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eczema (isotype control). The concentrations of the aforementioned antigen-binding molecules provided an equivalent payload concentration of 0.857 nM.

[0492] Figure 7I The study showed the percentage of cell death in BT474 cells after 3 days of in vitro treatment with trastuzumab conjugated with eczema and bezotib (T-(Exa+Ber)), trastuzumab-drutec (T-DXd), or an isotype-matched control antibody conjugated with eczema (isotype control). The concentrations of the aforementioned antigen-binding molecules provided an equivalent payload concentration of 0.857 nM.

[0493] Figure 7J The study showed the percentage of cell death in JIMT-1 cells after 3 days of in vitro treatment with trastuzumab conjugated with eczema and bezotib (T-(Exa+Ber)), trastuzumab delutec (T-DXd), or an isotype-matched control antibody conjugated with eczema (isotype control). The concentrations of the aforementioned antigen-binding molecules provided an equivalent payload concentration of 0.857 nM.

[0494] Figure 8 The Loewe synergistic score of eczemab and bezotitab is shown (top), as well as the percentage of inhibition of eczemab and bezotitab alone and in combination in HEC1-B cells (bottom).

[0495] Figure 9 The Loewe synergistic score of ixotecan and bezotitab is shown (top), as well as the % inhibition of ixotecan and bezotitab alone and in combination in HCT-116 cells (bottom).

[0496] Figure 10 The levels of pATR, pCHK1, pH2AX, and CHK1 in JIMT-1 cells were shown, either untreated or treated with 100 nM eczema, 100 nM bezotib, or a combination of 100 nM eczema and 100 nM bezotib, respectively.

[0497] Figure 11 The levels of pATR, pCHK1, pH2AX, and CHK1 in HCT-116 cells were shown, either untreated or treated with 75 nM eczetidine, 75 nM bezotib, or a combination of 75 nM eczetidine and 75 nM bezotib, respectively.

[0498] Figure 12 The survival of Sprague-Dawley rats after administration of the mediator, eczetidine (1, 3, 10 or 30 mg / kg), bezotib (35 mg / kg), or a combination of eczetidine and bezotib (1, 3, 10 or 30 mg / kg eczetidine + 35 mg / kg bezotib) is shown.

[0499] Figure 13 The changes in body weight of Sprague-Dawley rats after administration of the mediator, eczetidine (1, 3, 10 or 30 mg / kg), bezotitab (35 mg / kg), or a combination of eczetidine and bezotitab (1, 3, 10 or 30 mg / kg eczetidine + 35 mg / kg bezotitab) are shown.

[0500] Figure 14The results show the hematological assessments of Sprague-Dawley rats after receiving the mediator, eczema (1, 3, 10 or 30 mg / kg), bezotitab (35 mg / kg), or a combination of eczema and bezotitab (1, 3, 10 or 30 mg / kg eczema + 35 mg / kg bezotitab).

[0501] Figure 15 Clinical chemistry results are shown in Sprague-Dawley rats after receiving the mediator, eczetidine (1, 3, 10 or 30 mg / kg), bezotitab (35 mg / kg), or a combination of eczetidine and bezotitab (1, 3, 10 or 30 mg / kg eczetidine + 35 mg / kg bezotitab).

[0502] Figure 16 The tumor volume in the NCI-N87 CDX model resistant to T-DXd is shown after administration of the mediator, trastuzumab derutecan (T-DXd) (3 mg / kg) or trastuzumab conjugated with eczemacon and bezotib (T-(Exa+Ber) (3 mg / kg, 9 mg / kg)).

[0503] Figure 17 The study showed inhibition of HER2-negative MDA-MB-231 cells (alone or co-cultured with HER2-positive NCI-N87 cells) upon exposure to different concentrations of trastuzumab conjugated with eczema and bezotitab (T-(Exa+Ber)) or trastuzumab derutec (T-DXd).

[0504] Figure 18 This demonstrates that megakaryocytes take up trastuzumab (T-(Exa+Ber)) (100 ng / ml) or trastuzumab derutecan (T-DXd) (100 ng / ml) conjugated with eczema and bezotib via macropinocytosis.

[0505] Figure 19 The study showed an increase in chromatographic retention time (RT) for a single payload ADC and T-(Exa+Ber) in relation to hydrophobic interactions.

[0506] Figure 20 The stability of ADCs 5 and 6 in plasma was demonstrated over 7 days.

[0507] Figure 21 The efficacy of dual-load ADCs on NCI-N87 gastric cell line was demonstrated.

[0508] Figure 22The efficacy of dual-load ADC and single-load ADC in killing NCI-N87 gastric cell line was demonstrated.

[0509] Figure 23 The efficacy of dual-load ADC and single-load ADC in killing NCI-N87 gastric cell line was demonstrated.

[0510] Figure 24 The in vivo antitumor efficacy of dual-load ADC and single-load ADC in xenograft mouse models was demonstrated.

[0511] Figure 25 The in vivo efficacy of dual-load ADCs containing bezotitab (ATRi) or preceptib (CHK1i) was demonstrated in a mouse model.

[0512] Figure 26 The in vivo efficacy of dual-payload ADCs with different target DAR ratios was demonstrated.

[0513] Figure 27 The results show the changes in body weight after treatment with dual-payload ADCs in primate studies.

[0514] Figure 28 The biochemical and hematological results following treatment with dual-payload ADCs in primate studies are shown.

[0515] Figure 29 The efficacy of dual-load ADCs with either TMTHSI or DBCO portions on the NCI-N87 gastric cell line was demonstrated.

[0516] Figure 30 The clearance rate of dual-load ADCs with TMTHSI or DBCO motifs was shown compared to unconjugated control antibodies in immunocompetent mice.

[0517] Figure 31 The therapeutic effects of dual-load ADCs with different conjugates were demonstrated.

[0518] Example Example 1 – In vitro combination of DDR inhibitor and TOP1 inhibitor To further demonstrate the benefits of combining DDR inhibitors and TOP1 inhibitors, certain inhibitors were combined in a two-dimensional proliferation assay as follows.

[0519] cell lines HCT-116 HEC-1B Test compounds Ecinotecan (MedChemExpress, #HY-13631) Celasilt (MedChemExpress, #HY-19323) Preceptib (MedChemExpress, #HY-18174) Comparison Media control (0.25% DMSO) Cellular Test reagents Cell Titre Glo 2.0, Promega #G9243 Cells were seeded into the wells of white 96-well plates at a density of 7,000 cells per well for HEC-1B cells and 3,000 cells per well for HCT-116 cells, with 150 µl of culture medium added to each well. For single-compound experiments, 25 µl of the test compound and 25 µl of culture medium were added to the corresponding wells at different concentrations. In synergistic experiments, 25 µl of compound 1 and 25 µl of another compound were added to the corresponding wells at different concentrations. The plates were then incubated at 37°C and 5% CO2 for 3 days. After incubation, 50 µl of the assay reagent was added to each well, and the plates were shaken at 600 rpm for 20 minutes. The luminescence intensity was measured using a Perkin Elmer Victor Nivo microscope, and the inhibition percentage was calculated using the following formula: Inhibition % = 100 - ((Lum) 治疗 / Lum 媒介物 )*100) Then, the Synergy Finder tool on synergyfinder.fimm.fi was used to determine the synergistic effect between the test compounds (Loewe synergistic score). A Loewe synergistic score above 10 indicates a synergistic effect, between 10 and -10 indicates an additive effect, and below -10 indicates an antagonistic effect.

[0520] A: Ecinotecan and celacitib (an ATR inhibitor) in HCT-116 cells. Figure 1A The inhibition percentages of ixotecan and celecoxib alone and in combination are shown. The Loewe synergistic score is shown in Table 1A below. IC50 of ixotecan alone... 50 The concentration was 0.626 nM, 0.080 nM when used in combination with celecoxib at a concentration of 375 nM, and 0.044 nM when used in combination with celecoxib at a concentration of 750 nM.

[0521] Table 1A

[0522] B: Ecinotecan and preceptib (a CHK1 inhibitor) in HCT-116 cells Figure 1B The inhibition percentages of ixotecan and preripram alone and in combination are shown. The Loewe synergistic effect score is shown in Table 1B below. IC50 of ixotecan alone... 50 The concentration is 0.626 nM, 0.166 nM when used in combination with 50 nM preceptib, and 0.158 nM when used in combination with 100 nM preceptib. The IC value for preceptib alone is... 50 It is 61.14 nM.

[0523] Table 1B

[0524] C: Ecinotecan and celacidis (an ATR inhibitor) in HEC-1B cells. Figure 1C The inhibition percentages of ixotecan and celecoxib alone and in combination are shown. The Loewe synergistic effect score is shown in Table 1C below. IC50 of ixotecan alone... 50 The mean value was 113.9 nM, 19.01 nM when used in combination with 0.37 µM celasiltib, and 3.719 nM when used in combination with 1.1 µM celasiltib.

[0525] IC when celasilt is used alone 50 It is 2.118 µM.

[0526] Table 1C

[0527] D: Ecinotecan and preceptib (a CHK1 inhibitor) in HEC-1B cells. Figure 1D The inhibition percentages of ixotecan and preripram alone and in combination are shown. The Loewe synergistic effect score is shown in Table 1D below. IC50 of ixotecan alone... 50 The concentration is 113.9 nM, 8.988 nM when used in combination with 1.9 nM preceptib, and 2.013 nM when used in combination with 3.8 nM preceptib. IC50 when preceptib is used alone. 50 It is 4.466 nM.

[0528] Table 1D

[0529] In further experiments, another DDR inhibitor, bezotitab (an ATR inhibitor), was combined with the TOP1 inhibitor eczema, and the following two-dimensional proliferation assay was performed.

[0530] cell lines HEC-1-B (HTB-113) HCT-116 (CCL-247) Test compounds Ecinotecan (MedChemExpress, #HY-13631) Bezotibril (Selleckchem, #S7102) Detection CellTiter-Glo® 2.0D Cell Viability Assay (Promega, #G9243) Cells were seeded in 96-well opaque white plates with 150 μl of culture medium per well and incubated at 37°C and 5% CO2 for 24 hours. HEC-1B cells were seeded at a density of 7000 cells / well, and HCT-116 cells at a density of 3000 cells / well. The next day, 25 μl of eczemab, bezotitab, or eczemab and bezotitab at different concentrations were added to the cells, and the plates were incubated at 37°C and 5% CO2 for 3 days. After incubation, 50 μl of CellTiter-Glo reagent was added to the plate, and the plates were gently shaken at 600 rpm for 25 minutes. Cell viability was measured using a PerkinElmer VictorNivo chemiluminescence assay.

[0531] The luminescence value of the well containing only culture medium (without any cells) is used to subtract the background value.

[0532] The inhibition percentage was calculated using the formula 100 - {(Lum of cells treated with the drug / Lum of cells treated with the buffer control) * 100} (Lum = luminescence). The LOEWE synergistic score was calculated using the Synergy Finder tool on synergyfinder.fimm.fi.

[0533] Test results in Figure 8 and Figure 9 As shown in the figure, a synergistic effect was observed at certain concentrations of eczema and bezotitab.

[0534] In summary, we tested the in vitro synergistic effects of ixenotrane with two clinical-stage ATR inhibitors (bezotitab and celecoxib) in the TOP1 inhibitor-insensitive cell line HEC-1B and the TOP1 inhibitor-insensitive cell line HCT-116. Bezotitab and celecoxib both showed synergistic effects with ixenotrane across a wide range of concentrations tested in the cell lines. The effects in HEC-1B suggest that the combination of a TOP1 inhibitor and a DDR inhibitor can sensitize cells that were previously less responsive to TOP1 inhibitor treatment.

[0535] General conditions Unless otherwise stated, all chemicals, raw materials, and solvents were purchased commercially. Unless otherwise stated, all chemical reactions were carried out at ambient temperature and pressure. CombiFlash was used. ® Rapid column chromatography was performed using a NEXTGEN 100 column purchased from Agela Technologies. Preparative HPLC purification was performed using an AUNO LC-2000 column (Phenomenex Luna C18, 250 × 100 mm, 10 μm, 10 nm). 1 The H NMR spectrum was recorded on a Bruker spectrometer (400 MHz). 1 ¹H NMR chemical shifts are expressed as parts per million (δ) from the low field of tetramethylsilane (with the CDCl₃ peak at 7.26 ppm as an internal standard). Mass spectrometry data were recorded on a SHIMADZU LCMS-2020 (ESI-MS) and an Agilent 1260 / G6125B (ESI-MS) column using a Kinetex. ® EVO C18 4.6×50mm, 5μm, Kinetex ® EVO C182.1*30mm, 5μm, Shim-pack Scepter C18-120 3.0×33mm 3 µm and Poroshell 120 EC C182.7μm 3.0*30mm.

[0536] Example 2 – Synthesis of Linker-Loading Molecule-1 (LP-1)

[0537] i) (1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azapentadecan-15-acyl)-L-val Aminoacyl-L-alanine (compound I1)

[0538] Compound I1 was synthesized using the standard solid-phase Fmoc chemical method.

[0539] a) Resin loading: CH2Cl2 (200 mL) was added to 2-chlorotriphenylmethyl chloride resin (6.0 mmol, 1.00 equivalent), followed by Fmoc-Ala-OH (1.0 equivalent) and DIPEA (6.0 equivalent). The mixture was stirred at 25°C for 2 hours under a N2 atmosphere. Methanol (9.5 mL) was then added to the resin, and stirring continued for 30 minutes. The resin was then filtered and washed with DMF (300 mL x 3).

[0540] b) Deprotection: Add 20% piperidine in DMF (200 mL) to the resin and stir at 25°C for 30 minutes under N2 atmosphere. Wash the resin with DMF (200 mL × 5) and filter to obtain a resin with reactive amine groups.

[0541] c) Coupling: HBTU (2.85 equivalents) and Fmoc-Val-OH (3.0 equivalents) in DMF (200 mL) were added to the resin, followed by DIEPA (6.0 equivalents). The mixture was stirred at 25°C for 30 minutes under a nitrogen atmosphere. The resin was then washed with DMF (200 mL x 3).

[0542] d) Repeat step b to remove the Fmoc protecting group. Treat the resulting resin with Fmoc-N-amide-PEG3-acid (2.0 equivalents), HATU (1.9 equivalents), and DIPEA (4.0 equivalents) in DMF. Stir the mixture at 25°C for 30 minutes under a nitrogen atmosphere. Wash the resulting resin with DMF (200 mL x 3).

[0543] e) Peptide cleavage and purification: The resin was washed with methanol (200 mL x 3) and dried under vacuum. The dried resin was treated with a cleavage buffer consisting of 20% HFIP in CH2Cl2, stirred for 30 minutes, and then filtered. The filtrate was concentrated under reduced pressure to give crude compound I1 (1.71 g), which could be used without further purification.

[0544] ii) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-(hydroxymethyl)phenyl)amino)-5-isopropyl-2-methyl 1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (compound I3)

[0545] Compound I2 (657 mg, 5.34 mmol, 2.0 equivalent) and EEDQ (1.32 g, 5.34 mmol, 2.0 equivalent) were added to a solution of compound I1 (1.60 g, 2.67 mmol, 1.0 equivalent) in CH2Cl2 (16.0 mL). The reaction mixture was stirred at 25 °C for 12 hours. LCMS analysis showed that the starting material I1 was completely consumed and the desired product mass was detected. The reaction mixture was added to 160 mL of isopropyl ether and then centrifuged to give crude compound I3 (2.00 g) as a yellow oil. The crude product was used in subsequent reactions without further purification. MS (ESI): [M+Na] + : 727.4.

[0546] iii) (9H-fluorene-9-yl)methyl((2S,5S)-5-isopropyl-2-methyl-1-((4-((((4-nitrophenoxy)) (carbonyl)oxy)methyl)phenyl)amino)-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl) Carbamate (Compound I5)

[0547] DIEPA (671 mg, 5.20 mmol, 905 μL, 2.0 equivalent) was added to a solution of compound I3 (2.00 g, 2.6 mmol, 1.0 equivalent) and compound I4 (1.58 g, 5.2 mmol, 2.0 equivalent) in DMF (20 mL). The mixture was stirred at 25 °C for 2 h. LCMS analysis showed that the desired product yield was 31%, with 2% of the starting alcohol I3. The mixture was purified directly by preparative HPLC (TFA conditions) to remove the solvent, yielding compound I5 (680 mg, 765 μmol, yield 29.4%, purity 97.9%) as a yellow solid. MS (ESI): [M+Na] + : 870.4.

[0548] iv) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-(((((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4- Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indoleazine [1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo 9,12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (compound I6)

[0549] HOBt (33.4 mg, 247 μmol, 1.1 equivalence) and DIPEA (58 mg, 450 μmol, 78.4 μL, 2.0 equivalence) were added to a solution of compound I5 (200 mg, 225 μmol, 1.00 equivalence) in DMF (3.40 mL). The mixture was stirred at 25 °C for 2 h. LCMS analysis showed that compound I5 was completely consumed, and the desired product mass was identified. The mixture was purified directly by preparative HPLC (TFA conditions) to give compound I6 (180 mg, 148 μmol, yield 66%, purity 96.3%) as a yellow solid. MS (ESI): [M+H] + 1167.7 v) 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6- (1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-Hexahydro-1H,12H-Benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminomethyl Ester acid acid (compound I7)

[0550] Triethylamine (392 mg, 3.88 mmol, 0.54 mL, 26 equivalents) was added to a solution of compound I6 (180 mg, 148 μmol, 1.0 equivalent) in DMF (1.26 mL). The mixture was stirred at 25 °C for 2 hours. LCMS analysis showed that compound I6 was completely consumed and the desired product mass was detected. Compound I7 (140 mg, crude product) was given as a brown liquid and used directly in the next step. MS (ESI): [M+H] + 945.4 vi) Synthesis of LP-1

[0551] Triethylamine (392 mg, 3.9 mmol, 0.54 mL, 26.1 equivalences) and DBCO-OSu (59.6 mg, 148 μmol, 1.0 equivalences) were added to a solution of compound I7 (140 mg, 148 μmol, 1.0 equivalences) in DMF (1.26 mL). The mixture was stirred at 25 °C for 1 hour. LC-MS showed that compound I7 was completely consumed and the desired mass was detected. The reaction mixture was added to 18.0 mL of isopropyl ether, and the crude product precipitated slowly. The crude product was obtained by centrifugation, and the liquid supernatant was discarded. The residue was purified by preparative HPLC (TFA conditions) to give LP-1 (61.0 mg, 47.5 μmol, yield 32.5%, purity 97.5%) as a yellow solid. MS (ESI): [M+Na] +: 1253.6; 1 H NMR (400 MHz, DMSO-d6 ) δ ppm9.98 (s, 1 H), 8.36 (d, J = 6.80 Hz, 1 H), 8.04 - 8.06 (m, 1 H), 7.71 - 7.78(m, 2 H), 7.64 - 7.66 (m, 1 H), 7.58 (br d, J =8.8 Hz, 3 H), 7.41 - 7.47 (m, 4H), 7.25 - 7.37 (m, 6 H), 5.44 (s, 2 H), 5.28 (br s, 2 H), 5.07 (s, 2 H),5.00 (br d, J =13.88 Hz, 1 H), 4.38 (br t, J =6.94 Hz, 1 H), 4.28 (dd, J =9.13,6.63 Hz, 1 H), 3.93 (s, 2 H), 3.43 - 3.61 (m, 12 H), 3.27 (br t, J =6.00 Hz, 4H), 3.03 - 3.11 (m, 2 H), 2.37 (s, 3 H), 2.13 - 2.26 (m, 3 H), 1.93 - 2.03(m, 2 H), 1.81 - 1.92 (m, 2 H), 1.70 - 1.80 (m, 1 H), 1.30 (d, J =7.00 Hz, 3H), 0.84 - 0.91 (m, 6 H), 0.81 (br d, J =6.75 Hz, 3 H)。

[0552] Example 3 – Synthesis of Linker-Loading Molecule-2 (LP-2)

[0553] i) 4-((17S,20S)-1-(9H-fluorene-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo-2,7, 10,13-Tetraoxa-4,16,19-Triazacotetradecane-21-amido)benzyl(4-(5-(3-amino-6-(4-(isopropyl)) Sulfonyl)phenyl)pyrazin-2-yl)isoxazo-3-yl)benzyl)methyl)carbamate (compound I8)

[0554] HOBt (25.0 mg, 185 μmol, 1.10 equivalence) and DIPEA (43.6 mg, 337 μmol, 58.8 μL, 2.00 equivalence) were added to a solution of compound I5 (150 mg, 168 μmol, 1.0 equivalence) and bezotitab (86 mg, 185 μmol, 1.1 equivalence) in DMF (1.5 mL). The mixture was stirred at 25 °C for 2 h. LCMS analysis showed that compound I5 was completely consumed and the desired mass was detected. The mixture was purified directly by preparative HPLC (TFA conditions) to give compound I8 (150 mg, 121 μmol, yield 72.2%, purity 97.1%) as a yellow solid. MS (ESI): [M+H] + 1194.6 ii) 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6- (diazaheptadecanoamide)benzyl(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isooxa (Azol-3-yl)benzyl)methyl)carbamate (compound I9) Triethylamine (327 mg, 3.23 mmol, 450 μL, 26.5 equivalents) was added to a solution of compound I8 (150 mg, 121 μmol, 1.0 equivalent) in DMF (1.0 mL). The mixture was stirred at 25 °C for 2 hours. LCMS analysis showed that compound I8 was completely consumed and the desired product mass was observed. The reaction mixture was added to 15.0 mL of isopropyl ether and centrifuged to give crude compound I9 (200 mg, crude product) as a yellow oil. MS (ESI): [M+H] + 972.5 iii) 4-((15S,18S)-1-(((E)-cyclooct-4-en-1-yl)oxy)-15-isopropyl-18-methyl-1, 13,16-trioxo-5,8,11-trioxa-2,14,17-triazanonadecan-19-amido)benzyl(4-(5-(3-amino- 6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazo-3-yl)benzyl)(methyl)carbamate (LP-2)

[0555] NMM (17.1 mg, 169 μmol, 1.0 equivalent) was added to a solution of compound I9 (190 mg, 169 μmol, 1.0 equivalent) in DMF (1.5 mL). The mixture was stirred at 0 °C for 3 h. LC-MS showed that compound I9 was completely consumed. The resulting reaction mixture was purified directly by preparative HPLC (neutral conditions) to give compound LP-2 (55.0 mg, 45.0 μmol, yield 27.6%, purity 95.6%) as a pale yellow solid. MS (ESI): [M+Na] + :1146.6; 1 H NMR (400 MHz, DMSO- d 6) : δ ppm 9.99 (s, 1H), 8.94 (s, 1 H), 8.38 (d,J=8.50 Hz, 3 H), 7.96 - 8.03 (m, 2 H), 7.93 (d, J =8.63 Hz, 2 H), 7.77 (s, 1 H), 7.53 - 7.63 (m, 2 H), 7.25 - 7.46 (m, 5 H), 7.19 (br s, 2 H), 6.86 - 6.93 (m, 1 H), 5.49 - 5.60 (m, 1 H), 5.36 - 5.46 (m,1 H), 5.07 (br s, 2 H), 4.53 (s, 2 H), 4.35 - 4.43 (m, 1 H), 4.26 - 4.32 (m,1 H), 4.14 - 4.23 (m, 1 H), 3.93 (s, 2 H), 3.43 - 3.63 (m, 10H), 2.98 - 3.11(m, 2 H), 2.88 (s, 3 H), 2.17 - 2.30 (m, 3 H), 1.93 - 2.05 (m, 1 H), 1.75 -1.92 (m, 4 H), 1.44 - 1.68 (m, 3 H), 1.26 - 1.35 (m, 3 H), 1.19 (d, J =6.75 Hz, 6 H), 0.77 - 0.93 (m, 6 H).

[0556] Example 4 – Synthesis of Linker-Loading Molecule-3 (LP-3) i) 2,5-Dioxopyrrolidine-1-yl-1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-aza Pentadecane-15-ester (compound I11)

[0557] HOSu (6.84 g, 59.4 mmol, 1.0 equivalent) and DCC (12.2 g, 59.4 mmol, 12.0 mL, 1.0 equivalent) were added to a solution of compound I10 (25.5 g, 59.4 mmol, 1.0 equivalent) in CH2Cl2 (250 mL). The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the reaction mixture was filtered and concentrated under reduced pressure to give compound I11 as a yellow oil (31.7 g, crude product), which could be used without further purification. MS (ESI): MS calculated value: 526.53, MS measured value: [M+H] + = 527.1.

[0558] ii) (1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azapentadecan-15-acyl)-L- Valyl-L-alanine (compound I12)

[0559] DIPEA (7.68 g, 59.4 mmol, 1.0 equivalence) was added to a solution of compound I11 (31.3 g, 59.4 mmol, 1.0 equivalence) and dipeptide Val-Ala-OH (11.1 g, 59.4 mmol, 1.0 equivalence) in DMF (300 mL). The mixture was stirred at 25 °C for 4 hours. LC-MS showed that compound I11 was completely consumed. The reaction mixture was concentrated, and the residue was purified by preparative HPLC to give compound I12 (23.5 g, 37.5 mmol, 63% yield in two steps) as a white solid. MS (ESI): MS calculated value: 599.67, MS measured value: [M+H] + = 600.3 iii) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-(hydroxymethyl)phenyl)amino)-5-isopropyl-2-methyl 1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (compound I13)

[0560] To a solution of compound I12 (23.5 g, 37.5 mmol, 1.0 equivalent) in CH2Cl2 (235 mL), 4-aminobenzyl alcohol (9.25 g, 75 mmol, 2.0 equivalent) and EEDQ (18.5 g, 75.0 mmol, 2.0 equivalent) were added. The mixture was stirred at 25 °C for 12 hours in the dark. After the reaction was complete, the mixture was concentrated and washed with isopropyl ether (2.35 L) to give compound I13 (34 g, crude product) as a yellow gel-like solid, which could be used without further purification. MS (ESI): MS calculated value: 704.8, MS measured value: [M+H]+ = 705.3 iv) (9H-fluorene-9-yl)methyl((2S,5S)-5-isopropyl-2-methyl-1-((4-((((4-nitrophenoxy)) (carbonyl)oxy)methyl)phenyl)amino)-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl) Carbamate (compound I14)

[0561] PNP2O (34.5 g, 113 mmol, 2.5 equivalents) and DIPEA (15 mL, 90.8 mmol, 2.0 equivalents) were added to a solution of compound I13 (34 g, 45.4 mmol, 1.0 equivalents) in DMF (320 mL). The mixture was stirred at 25 °C for 4 hours. After the reaction was complete, the reaction mixture was purified by preparative HPLC to give compound I14 (22 g, 24.6 mmol, two-step yield 63%) as a yellow solid. MS (ESI): MS calculated value: 869.35, MS measured value: [M+H] + = 870.4 v) 4-((17S,20S)-1-(9H-fluorene-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo-2,7, 10,13-Tetraoxa-4,16,19-Triazacotetradecane-21-amido)benzyl(4-(5-(3-amino-6-(4-(isopropyl)) Sulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)methyl)carbamate (compound I15)

[0562] HOBt (350 mg, 2.59 mmol, 1.1 equivalent) and DIPEA (778 μL, 4.71 mmol, 2.0 equivalent) were added to a solution of compound I14 (2.1 g, 2.35 mmol, 1.05 equivalent) in DMF (11 mL). The mixture was stirred at 28 °C for 2 h. LC-MS analysis showed that compound I14 was completely consumed. The reaction was then concentrated and ground with isopropyl ether (3 x 110 mL). The residue was further purified by preparative HPLC to give compound I15 (2.4 g, 1.97 mmol, yield 86%) as a yellow solid. MS (ESI): MS calculated value: 1193.49, MS measured value: [M+H]+ = 1194.5 vi) (E)-2-(cyclooct-4-en-1-yloxy)acetic acid (compound I16)

[0563] To a solution of compound TCO-OH (200 mg, 1.58 mmol, 1.0 equivalent) in dry THF (2.0 mL), NaH (60% mineral oil dispersion, 190 mg, 4.75 mmol, 3.0 equivalent) was added. The mixture was stirred at 25 °C under a nitrogen atmosphere for 1 hour. Then, 2-bromoacetic acid (264 mg, 1.9 mmol, 1.2 equivalent) and KI (26.3 mg, 158 μmol, 0.10 equivalent) were added. The mixture was stirred at 70 °C for 12 hours. After the reaction was complete, the reaction mixture was quenched with H2O (10 mL) and the pH was adjusted to 2 with 1N HCl. The product was extracted with CH2Cl2 (6 x 5 mL). The combined organic layers were washed with brine (3 x 5 mL), dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by preparative HPLC to give compound I16 (82 mg, 445 μmol, yield 28%) as a white solid. MS (ESI): MS calculated value: 184.11, MS measured value: [MH] - = 183.1. 1 H NMR (400 MHz, CDCl3) δ 5.77 - 5.48 (m, 1H), 5.47 - 5.25 (m, 1H), 3.87 -3.69 (m, 1H), 3.69 - 3.53 (m, 1H), 2.99 (br d, J = 8.5 Hz, 1H), 2.52 - 1.63 (m, 8H), 1.61 - 1.40 (m, 2H).

[0564] vii) 4-((2S,5S)-20-(((E)-cyclooct-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4,7,19- (trioxo-9,12,15-trioxa-3,6,18-triazaeicosanoamide)benzyl(4-(5-(3-amino-6-(4-(isopropyl))) LP-3 (2-yl)pyrazinyl)isoxazo-3-yl)benzyl)methyl)carbamate

[0565] Triethylamine (6 mL) was added to a solution of compound I15 (2.0 g) in DMF (14 mL). The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, isopropyl ether (200 mL) was added to the reaction mixture, and compound I15* (2.0 g, crude product) was collected by centrifugation. The product can be used directly in the next step without further purification. [Note: I15* has the same chemical formula as I15, where R=H].

[0566] To a solution of crude I15* (265 mg, 272 μmol, 1.0 equivalent) and compound I16 (50 mg, 271 μmol, 1.0 equivalent) in DMF (2.5 mL), HATU (154 mg, 407 μmol, 1.5 equivalent) and DIPEA (89.7 μL, 542 μmol, 2.0 equivalent) were added. The mixture was stirred at 25 °C for 2 hours. LC-MS analysis indicated that the reaction was complete. The reaction mixture was purified by preparative HPLC to give LP-3 (205 mg, 49% yield) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6)δ 10.05 - 9.97 (m, 1H), 8.95 (s, 1H), 8.46 - 8.20 (m, 3H), 8.04 - 7.88 (m,4H), 7.78 (s, 1H), 7.65 - 7.52 (m, 2H), 7.52 - 7.10 (m, 8H), 5.50 (br dd, J =3.6, 11.6 Hz, 1H), 5.34 (br dd, J = 4.0, 11.4 Hz, 1H), 5.07 (br s, 2H), 4.53(s, 2H), 4.45 - 4.20 (m, 2H), 3.97 - 3.90 (m, 2H), 3.78 - 3.64 (m, 2H), 3.63 - 3.40 (m, 9H), 3.23 (q, J = 6.0 Hz, 2H), 2.93 - 2.82 (m, 3H), 2.31 - 1.67 (m, 11H), 1.49 - 1.38 (m, 2H), 1.38 - 1.23 (m, 4H), 1.19 (d, J = 6.8 Hz, 6H), 0.94 - 0.76 (m, 6H). MS (ESI): MS calculated value: 1137.52, MS measured value: [M+H]+ = 1138.0. HPLC purity (254 nm) = 96.8% Example 5 – Synthesis of Linker-Payload Molecules LP-4, LP-5, LP-6, LP-7, LP-8 and LP-9

[0567] i) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-(chloromethyl)phenyl)amino)-5-isopropyl-2-methyl 1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (compound I17)

[0568] SOCl2 (15.2 μL) was slowly added to a solution of compound I13 (180 mg, 250 μmol, 1.0 equivalent) in CH2Cl2 (2.0 mL). The mixture was stirred at 25 °C for 2 hours. LC-MS analysis indicated that the reaction was complete. The reaction mixture was added dropwise to ice-cold isopropyl ether (20 mL), centrifuged, and the desired product I17 was obtained as a yellow oily residue (160 mg, crude product), which was used directly in the next reaction.

[0569] ii) General procedures for payload mooring (General Procedure 1A): At 25 °C, DIPEA (3.0 equivalents) and the payload molecule (1.0 equivalent) were added to a solution of compound I14 (140 μmol, 1.2 equivalents) in DMF (approximately 1.0 mL). The mixture was stirred at 25 °C for 2 hours. The reaction progress was monitored by LCMS analysis. After the reaction was complete, the reaction mixture was concentrated, and the residue could be used directly or purified by preparative HPLC.

[0570] iii) General procedures for payload mooring (General Procedure 1B): DMAP (3.0 equivalents) and triphosgene (0.8 equivalents) were added to a solution of the payload molecule (140 μmol, 1.0 equivalent) in CH2Cl2 (1.0 mL). The mixture was stirred at 25 °C for 2 min. DIPEA (2.0 equivalents) was added, followed by compound I13 (1.0 equivalent). The mixture was stirred at 25 °C for 2 h. The reaction was monitored by LCMS analysis. After the reaction was complete, the reaction mixture was poured into ice-cold isopropyl ether. The precipitate was collected and further purified by preparative HPLC.

[0571] iv) General procedures for payload mooring (General Procedure 1C): To a solution of compound I17 (approximately 110 μmol, 1.0 equivalent) in DMF (800 μL), a loading molecule (0.7 equivalent) and DIPEA (3.0 equivalent) were added. The mixture was stirred at 25 °C for 12 hours. The reaction was monitored by LCMS analysis. After the reaction was complete, the reaction mixture was poured into ice-cold isopropyl ether. The precipitated product was collected and used directly without further purification.

[0572] v) 4-((17S,20S)-1-(9H-fluorene-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo-2,7, 10,13-Tetraoxa-4,16,19-Triazacotetraane-21-amido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-) Dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-11-yl)ethyl)(iso Propyl carbamate (compound I18a)

[0573] Preparation was carried out according to standard procedure 1A: Loading weight: belotecone. I18a was obtained as a white solid (42.0 mg, yield 76%). MS (ESI): Calculated MS value: 1164.3, Measured MS value: [M+H] + = 1164.0 vi) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-(((((S)-9-((tert-butyldimethylsilyl)) )-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1, [2-b]quinoline-4-yl)oxy)carbonyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo-9, 12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (compound I18b)

[0574] Prepared according to standard procedure 1B: Payload: SN38-(OTBS). I18b was collected as a yellow solid (75 mg, 57%). MS (ESI): MS calculated value: 1236.5, MS measured value: [M+H] + = 1237.5 vii) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-((((3-(2-(3-((5-cyanopyrazin-2-yl)amino) )-1H-pyrazole-5-yl)-3-methoxyphenoxy)propyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl 2-Methyl-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (compound) Object I18c)

[0575] Preparation was performed according to standard procedure 1A: Loading weight: preretinoin. I18c was obtained as a colorless solid (150 mg, crude product), which can be used directly without further purification. MS (ESI): MS calculated value: 1095.48, MS measured value: [M+H] + =1096.1 viii) 1-(4-((17S,20S)-1-(9H-fluorene-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxane 2,7,10,13-tetraoxa-4,16,19-triazacotetradecane-21-amido)benzyl)-4-(4-((2-allyl-1- (6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino) (Phenyl)-1-methylpiperazine-1-onium (compound I18d)

[0576] Preparation was carried out according to standard procedure 1C: Active ingredient: adatirb; yielded I18d, a yellow solid (70 mg, crude). MS (ESI): MS calculated value: 1187.6, MS measured value: [M+H] + = 1188.9 ix) N-(4-((17S,20S)-1-(9H-fluorene-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo- 2,7,10,13-Tetraoxa-4,16,19-Triazacotetraane-21-amidobenzyl)-N,N-dimethyl-3-((5-(3- Methyl-2-oxo-1-(tetrahydro-2H-pyran-4-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinoline-8-yl)pyridine-2- (Compound I18e)

[0577] Preparation was carried out according to standard procedure 1C: Effective load: AZD0156; I18e was obtained as a yellow solid (70 mg, crude product). MS (ESI): MS calculated value: 1148.5, MS measured value: [M+H]+ = 1149.5 x) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-(((((S)-(2-chloro-4-fluoro-5-(7-morpholinoquinazole)) (Lin-4-yl)phenyl)(6-methoxypyridazine-3-yl)methoxy)carbonyl)oxy)methyl)phenyl)amino)-5-isopropyl-2- Methyl-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (compound) I18f)

[0578] Prepared according to standard procedure 1A: Loading weight: nidicetib; I18f (50 mg, 40%, white solid). MS (ESI): Calculated MS value: 1211.45, Measured MS value: [M+H] + = 1212.0.

[0579] xi) General procedure for deprotecting -NHFmoc (General Procedure 2): Triethylamine (approximately 0.25 mL) was added to a solution of Fmoc-protected amine (approximately 40–60 μmol, 1.0 equivalent) in DMF (approximately 0.60 mL). The reaction mixture was then stirred at 25 °C for 3 hours. After the reaction was complete, the solvent was removed, and the product was purified by preparative HPLC to obtain the desired product.

[0580] xii) 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3, 6-Diazaheptadecanoamide)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-) Pyrano[3',4':6,7]indolazino[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate (compound) I19a)

[0581] Prepared according to general procedure 2: I19a was obtained as a white solid (30 mg, yield 88%). MS (ESI): MS calculated value: 942.0, MS measured value: [M] + = 942.0.

[0582] xii) 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3, 6-Diazaheptadecanoamide)benzyl((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyrano[3',4':6,7]indolazino[1,2-b]quinolin-4-yl)carbamate (compound I19b)

[0583] Prepared according to standard procedure 2: I19b was obtained as a yellow solid (50 mg, crude product). LC (ESI): MS calculated value: 900.3, MS measured value: [M+H] + = 901.5 xiv) 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3, 6-Diazaheptadecanoamide)benzyl(3-(2-(3-((5-cyanopyrazin-2-yl)amino)-1H-pyrazol-5-yl)-3-methyl (Oxyphenoxy)propyl)carbamate (compound I19c)

[0584] Prepared according to general procedure 2: I19c was obtained as a white solid (30 mg, yield 42%). MS (ESI): MS calculated value: 873.41, MS measured value: [M+H] + = 874.0.

[0585] xv) 4-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro- 1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)-1-(4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4, 7-Dioxo-9,12,15-trioxa-3,6-diazaheptadecanoamide (benzyl)-1-methylpiperazine-1-onium (compound) I19d)

[0586] Prepared according to standard procedure 2: I19d was obtained as a yellow solid (40 mg, yield 70%). MS (ESI): MS calculated value: 965.5, MS measured value: [M+H] + = 966.5 xvi) N-(4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa- 3,6-Diazaheptadecanoamide (benzyl)-N,N-dimethyl-3-((5-(3-methyl-2-oxo-1-(tetrahydro-2H-pyran-) 4-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinoline-8-yl)pyridin-2-yl)oxy)prop-1-aminoonium (compound I19e)

[0587] Prepared according to general procedure 2: I19e was obtained as a yellow solid (40 mg, yield 31%). MS (ESI): MS calculated value: 926.5, MS measured value: [M+H] + = 927.5 xvii) 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3, 6-Diazaheptadecanoamide)benzyl((S)-(2-chloro-4-fluoro-5-(7-morpholinoquinazolin-4-yl)phenyl)(6-methoxy (Compound I19f) pyridazine-3-yl)methyl)carbonate

[0588] Preparation was carried out according to standard procedure 2: I19f was isolated as a yellow solid (40 mg, yield 98%). MS (ESI): MS calculated value: 989.39, MS measured value: [M+H] + = 990.4.

[0589] xviii) General procedure for DBCO coupling (General procedure 3A): DBCO-OSu (2.0 equivalents) and DIPEA (3.0 equivalents) were added to a solution of amine (30 μmol, 1.0 equivalent) in DMF (1.0 mL). The mixture was stirred at 25 °C for 2 hours. LC-MS analysis showed that the amine was completely converted to the desired product. The reaction mixture was concentrated under reduced pressure and then purified directly by preparative HPLC to obtain the desired product.

[0590] xix) General procedure for TCO coupling (General procedure 3B): Compounds I16 (1.0 equivalent), HATU (2.0 equivalent), and DIPEA (2.0 equivalent) were added to a solution of amine (35 μmol, 1.0 equivalent) in DMF (0.3 mL). The reaction mixture was stirred at 25 °C for 2 hours. LC-MS analysis showed that the amine was completely converted to the desired product. The reaction mixture was concentrated under reduced pressure, and the residue was then purified directly by preparative HPLC to obtain the desired product.

[0591] xx) [4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-[2-[4-(2-azatricyclic[10.4.0.04,9]+ [hexacarbon-1(12),4(9),5,7,13,15-hexaden-10-yn-2-yl)-4-oxo-butyryl]amino]ethoxy]ethoxy] [ethoxy]acetyl]amino]-3-methyl-butyryl]amino]propionyl]amino]phenyl]methyl N-[2-[(19S)-19-ethyl] 19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20] 20 One-carbon-1(21),2,4,6,8,10,15(20)-hepten-10-yl]ethyl]-N-isopropylcarbamate (LP-4)

[0592] LP-4 was synthesized from I19a using the standard procedure. Yield: 45%. 1 H NMR (400 MHz, DMSO- d 6. )δ ppm10.13 - 9.88 (m, 1H), 8.43 - 8.29 (m, 1H), 8.25 - 8.09 (m, 1H), 8.01 - 7.82(m, 1H), 7.79 - 7.54 (m, 6H), 7.53 - 7.29 (m, 10H), 6.60 - 6.40 (m, 1H), 5.51- 5.28 (m, 4H), 5.18 - 5.05 (m, 2H), 4.49 - 4.11 (m, 3H), 4.06 - 3.85 (m,2H), 3.67 - 3.39 (m, 18H), 3.11 - 3.03 (m, 4H), 2.71 - 2.65 (m, 2H), 2.02 -1.97 (m, 2H), 1.32 - 1.23 (m, 6H), 1.14 - 1.11 (m, 3H), 0.91 - 0.80 (m, 9H).MS (ESI):MS calculated: 1229.3, MS observed: [M+H] + = 1229.4. Purity (HPLC, 254nm) = 95.5% xxi) DBCO-PEG3-VA-PABC-SN38 (LP-5)

[0593] LP-5 was synthesized from I19b using the standard procedure 3A. Yield: 38%. 1 H NMR (400 MHz, DMSO-d6 )δ ppm10.16 - 10.72 (m, 1 H), 9.93 - 10.11 (m, 1 H), 8.35 - 8.46 (m, 1 H), 8.02 -8.09 (m, 1 H), 7.73 - 7.78 (m, 1 H), 7.65 - 7.69 (m, 1 H), 7.57 - 7.60 (m, 2H), 7.45 - 7.46 (m, 1 H), 7.43 - 7.44 (m, 1 H), 7.41 - 7.42 (m, 1 H), 7.35 -7.37 (m, 1 H), 7.31 - 7.34 (m, 2 H), 7.29 - 7.30 (m, 1 H), 6.93 - 6.96 (m, 1H), 5.48 - 5.55 (m, 2 H), 5.29 - 5.34 (m, 2 H), 5.27 - 5.35 (m, 2 H), 5.05 -5.11 (m, 2 H), 4.36 - 4.43 (m, 1 H), 4.27 - 4.34 (m, 1 H), 3.92 - 3.98 (m, 2H), 3.57 - 3.60 (m, 2 H), 3.53 - 3.55 (m, 2 H), 3.49 - 3.51 (m, 2 H), 3.44 -3.47 (m, 3 H), 3.44 - 3.47 (m, 3 H), 3.07 - 3.12 (m, 3 H), 2.65 - 2.71 (m, 1H), 2.31 - 2.36 (m, 1H), 2.12 - 2.28 (m, 4H), 2.06 - 2.10 (m, 1H), 1.96 -2.04 (m, 3H), 1.86 - 1.94 (m, 2H), 1.28 - 1.32 (m, 6H), 1.23 - 1.26 (m, 3H), 0.87 - 0.92 (m, 6H), 0.80 - 0.83 (m, 3H). MS (ESI): MS calculated value: 1187.4, MS measured value: [M+H] + = 1188.7. Purity (HPLC, 254 nm) = 96.2% xxii) 4-((2S,5S)-20-(((E)-cyclooct-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4,7, 19-Trioxo-9,12,15-trioxa-3,6,18-triazaeicosanoamide)benzyl(3-(2-(3-((5-cyanopyrazine- 2-yl)amino)-1H-pyrazole-5-yl)-3-methoxyphenoxy)propyl)carbamate (LP-6)

[0594] LP-6 was synthesized from I18c according to the general procedure 3B. Yield: 46%. 1 H NMR (400 MHz, DMSO-) d 6 ) δ ppm 12.31 - 12.25 (m, 1H), 10.68 (br s, 1H), 10.00 (s, 1H), 8.61 (s, 1H), 8.40(d, J = 6.8 Hz, 1H), 7.56 (br d, J = 8.4 Hz, 2H), 7.49 - 7.43 (m, 2H), 7.34 -7.21 (m, 4H), 6.91 (br d, J = 4.5 Hz, 1H), 6.74 (t, J = 8.6 Hz, 2H), 5.59 - 5.47(m, 1H), 5.39 - 5.27 (m, 1H), 4.92 (s, 2H), 4.34 (s, 2H), 4.02 (br t, J = 5.8Hz, 2H), 3.94 (s, 2H), 3.81 (s, 2H), 3.71 (d, J = 8.6 Hz, 2H), 3.59 - 3.50 (m,8H), 3.42 - 3.39 (m, 2H), 3.26 - 3.21 (m, 2H), 3.19 - 3.15 (m, 2H), 3.05 -2.99 (m, 1H), 2.33 - 2.15 (m, 4H), 2.05 - 1.97 (m, 2H), 1.90 - 1.83 (m, 4H), 1.78 - 1.70 (m, 3H), 1.48 - 1.40 (m, 2H), 1.30 (d, J = 7.1 Hz, 3H), 0.89 - 0.81 (m, 6H). MS (ESI): MS calculated value: 1039.51, MS measured value: [M+H] + = 1040.5. Purity (HPLC, 254 nm) = 96.4% xxiii) 4-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-di Hydrogen-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)-1-(4-((2S,5S)-20-(((E)-cyclooct-4-ene-1- 5-Isopropyl-2-methyl-4,7,19-trioxo-9,12,15-trioxa-3,6,18-triazaeicosanoamide (β-Benzyl)-1-methylpiperazine-1-onium (LP-7)

[0595] LP-7 was synthesized using I19d according to general procedure 3B. Yield: 37%. 1 H NMR (400 MHz, DMSO-) d 6 )δppm 10.23 - 10.57 (m, 1 H), 8.83 - 8.88 (m, 1 H), 8.69 - 8.81 (m, 1 H), 7.99- 8.06 (m, 1 H), 7.73 - 7.80 (m, 3 H), 7.58 - 7.67 (m, 3 H), 7.44 - 7.54 (m, 4 H), 6.99 - 7.04 (m, 2 H), 5.61 - 5.72 (m, 1 H), 5.49 - 5.57 (m, 1 H), 5.28- 5.37 (m, 2 H), 4.96 - 5.02 (m, 1 H), 4.78 - 4.86 (m, 1 H), 4.61 - 4.71 (m,4 H), 4.26 - 4.43 (m, 2 H), 3.92 - 3.97 (m, 2 H), 3.64 - 3.73 (m, 4 H), 3.54- 3.62 (m, 6 H), 3.50 - 3.53 (m, 4 H), 3.38 - 3.43 (m, 4 H), 3.21 - 3.25 (m,3 H), 2.99 - 3.04 (m, 4 H), 2.31 - 2.34 (m, 1 H), 2.23 - 2.29 (m, 3 H), 2.15- 2.20 (m, 1 H), 1.97 - 2.07 (m, 3 H), 1.83 - 1.92 (m, 3 H), 1.74 - 1.79 (m, 2 H), 1.46 (s, 6 H), 1.31 - 1.35 (m, 3 H), 0.81 - 0.90 (m, 6 H). MS (ESI): MS calculated value: 1131.6, MS measured value: [M+H] + = 1132.1. Purity (HPLC, 254 nm): 97.3% xxiv) N-(4-((2S,5S)-20-(((E)-cyclooct-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4, 7,19-trioxo-9,12,15-trioxa-3,6,18-triazaeicosanoamide)benzyl)-N,N-dimethyl-3-((5- (3-Methyl-2-oxo-1-(tetrahydro-2H-pyran-4-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinoline-8-yl)pyridine- 2-yl)oxy)prop-1-aminoonium (LP-8)

[0596] LP-8 was synthesized from I19e according to general procedure 3B. Yield: 32% (obtained as a mixture of quaternary ammonium salts). 1 H NMR (400 MHz, DMSO- d 6 )δ ppm 10.17 - 10.33 (m, 1 H), 8.90 - 8.95 (m, 1 H), 8.69 (d, J=2.75 Hz, 1 H), 8.41 - 8.47 (m, 2 H), 8.22 - 8.27 (m, 1 H), 8.14 -8.19 (m, 1 H), 7.95 - 8.00 (m, 1 H), 7.71 - 7.76 (m, 2 H), 7.39 - 7.50 (m, 4H), 6.97 - 7.04 (m, 1 H), 5.47 - 5.57 (m, 1 H), 5.29 - 5.38 (m, 1 H), 5.11 -5.21 (m, 1 H), 4.50 - 4.54 (m, 2 H), 4.42 - 4.46 (m, 2 H), 4.35 - 4.40 (m, 1H), 4.27 - 4.32 (m, 1 H), 4.03 - 4.09 (m, 2 H), 3.92 - 3.96 (m, 2 H), 3.69 -3.72 (m, 1 H), 3.51 - 3.58 (m, 10 H), 3.21 - 3.26 (m, 4 H), 2.97 - 3.00 (m, 6H), 2.64 - 2.77 (m, 4 H), 2.30 - 2.38 (m, 4 H), 2.24 - 2.29 (m, 2 H), 2.14 -2.21 (m, 1 H), 1.85 - 2.04 (m, 7 H), 1.69 - 1.77 (m, 2 H), 1.41 - 1.47 (m, 2 H), 1.30 - 1.34 (m, 3 H), 1.23 - 1.26 (m, 1 H), 0.87 - 0.90 (m, 3 H), 0.79 -0.83 (m, 3 H). MS (ESI): MS calculated value: 1092.6, MS measured value: [M+H] + = 1093.0. Purity (HPLC, 254 nm): 99.9% (quaternary ammonium salt mixture) xxv) (S)-(2-chloro-4-fluoro-5-(7-morpholinoquinazolin-4-yl)phenyl)(6-methoxypyridazine-3-yl)methyl 4-((2S,5S)-20-(((E)-cyclooct-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4,7,19-trioxo-9, 12,15-trioxa-3,6,18-triazaeicosanoamide (benzyl) carbonate (LP-9)

[0597] LP-9 was synthesized from I19f according to general procedure 3B. Yield: 39%. 1 H NMR (400 MHz, DMSO- d 6 )δppm 10.04 (s, 1H), 9.11 (s, 1H), 8.40 (d, J = 6.8 Hz, 1H), 7.85 (d, J = 9.3 Hz,1H), 7.83 - 7.77 (m, 2H), 7.60 - 7.55 (m, 2H), 7.53 (dd, J = 2.5, 6.6 Hz, 1H),7.47 - 7.42 (m, 2H), 7.29 (dd, J = 8.9, 12.5 Hz, 3H), 7.20 (d, J = 1.9 Hz, 1H),7.18 (s, 1H), 5.56 - 5.46 (m, 1H), 5.37 - 5.26 (m, 1H), 5.14 (s, 2H), 4.39(t, J = 7.1 Hz, 1H), 4.29 (dd, J = 6.5, 8.9 Hz, 1H), 4.01 (s, 3H), 3.94 (s, 2H), 3.80 - 3.73 (m, 4H), 3.70 (d, J = 8.8 Hz, 2H), 3.62 - 3.50 (m, 8H), 3.46 - 3.38(m, 6H), 3.26 - 3.20 (m, 2H), 3.01 (br dd, J = 3.2, 9.9 Hz, 1H), 2.31 - 2.13(m, 3H), 2.06 - 1.95 (m, 2H), 1.91 - 1.80 (m, 2H), 1.78 - 1.70 (m, 2H), 1.48- 1.39 (m, 2H), 1.30(d, J = 7.0 Hz, 3H), 1.23 (s, 1H), 0.90 - 0.80 (m, 6H). MS (ESI): MS calculated value: 1155.48, MS measured value: [M+H] +=1156.5. Purity (HPLC, 254 nm): 95.2% xxvi) N-[20-(11,12-disodehydrodibenzo[b,f]acoxine-5(6H)-yl)-17,20-dioxo-4, 7,10,13-Tetraoxa-16-azaeicosano-1-acyl]glycylglycyl-L-phenylalanyl-N-[(2- {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- Benzo[de]pyrano[3′,4′:6,7]indolazo[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycine Aminoamide (LP-10)

[0598] N-[20-(11,12-disodehydrodibenzo[b,f]acoxin-5(6H)-yl)-17,20-dioxo-4,7,10,13-tetraoxa-16-azaeicosano-1-acyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycamide is available from suppliers such as Key Organics Ltd, UK (CAS No.: 2694856-51-2).

[0599] Example 6 – Synthesis of Linker-Loading Molecule LP-11 i) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl) 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazid [1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7-trioxane 9,12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (I20)

[0600] HOBt (416 mg, 3.08 mmol, 1.1 equivalence) and DIPEA (725 mg, 5.61 mmol, 927 μL, 2.0 equivalence) were added to a solution of compound I14 (2.5 g, 2.8 mmol, 1.0 equivalence) in DMF (25 mL). The mixture was stirred at 25 °C for 2 hours. LCMS analysis indicated that the reaction was complete. Isopropyl ether (25 mL) was then added to the reaction mixture, and the precipitate was collected by centrifugation to give compound I20 (4.0 g, crude product), a brown oily substance, which was used directly in the next step. MS (ESI): MS calculated value: 1165.4, MS measured value: [M+H] + = 1166.1 ii) 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6- (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-Hexahydro-1H,12H-Benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminomethyl Ester acid acid (I20*) Triethylamine (12 mL) was added to a solution of compound I20 (4.0 g, 1.0 equivalent) in DMF (28 mL). The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, isopropyl ether (400 mL) was added to the reaction mixture, and the mixture was centrifuged to obtain the intermediate amine I20* (4 g, crude product). The crude product was characterized by LC-MS analysis and used directly in the next step. MS (ESI): MS calculated value: 943.4, MS measured value: [M+H]+ = 944.2.

[0601] iii)[4-[[(2S)-2-[[(2S)-3-methyl-2-[[2-[2-[2-[2-[(3,3,6,6-tetramethyl-1-oxo- 1λ 6 -Thioheptacyclic hepta-4-yne-1-ylide)carbamoylamino]ethoxy]ethoxy]ethoxy]acetyl]amino]butyryl] [N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-]amino]propionyl]amino]phenyl]methyl N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-] Dioxo-8-oxa-4,15-diazahexane [14.7.1.0] 2,14 .0 4,13 .0 6,11 .0 20,24 ] Carbon-24-1,6(11),12, 14,16(24),17,19-hepten-23-yl]carbamate (LP-11):

[0602] Triethylamine (23.59 μL, 37.2 μmol, 2.5 equivalents) and DMAP (10.5 mg, 85.95 μmol, 0.81 equivalents) were sequentially added to a solution of compound I20* (100 mg, 105.9 μmol, 1.0 equivalents) and compound TMTHSI-OSu (37.86 mg, 111.2 μmol, 1.05 equivalents) in DMF (1 mL). The mixture was stirred at 25 °C for 18 hours. After the reaction was complete (observed by LC-MS analysis), the solvent was removed under reduced pressure, and the residue was purified by preparative HPLC to give LP-11 (23 mg) as a yellow solid. Purity obtained by HPLC (220 nm): 95.6%, Rt = 10.720 min; MS (ESI): [(M+H)] + =1169.4, [(M+2H)] 2+ =585.5. 1 H NMR (400 MHz, DMSO- d6)δ ppm 9.99(s, 1 H), 8.37 (d, J=7.00 Hz, 1 H), 8.06 (d, J=8.63 Hz, 1 H), 7.78 (d, J=10.38 Hz, 1 H), 7.59 (d, J=8.63 Hz, 2 H), 7.44 (d, J=9.13 Hz, 1 H), 7.36 (d,J=8.00 Hz, 2 H), 7.31 (s, 1 H), 6.52 (d, J=6.63 Hz, 2 H), 5.45 (s, 2 H), 5.29(br s, 3 H), 5.08 (s, 2 H), 4.29 - 4.39 (dd, J=8.63, 6.88 Hz, 2 H), 3.95 (s, 2 H), 3.84 - 3.87 (d, 2 H), 3.42 - 3.65 (m, 10 H), 3.07 (d, J=5.25 Hz, 2 H), 2.89 (s, 1 H), 2.73 (s, 1 H), 2.38 (d, J=1.13 Hz, 4 H), 2.15 - 2.24 (m, 2 H), 1.95 - 2.04 (m, 2 H), 1.82 - 1.92 (m, 2 H), 1.28 - 1.34 (m, 12 H), 1.18 (s, 3H), 0.88 (d, J=7.13 Hz, 6 H), 0.83 (d, J=6.63 Hz, 3 H).

[0603] Example 7 – Synthetic route of connector 1

[0604] a) Compound 2 can be prepared from compound 1 by coupling reaction of tert-butyl 2-bromoacetate (e.g., using triethanolamine (TEA) in THF).

[0605]

[0606] b) Compound 4 – Boc protection Compound 4 can be prepared by treating compound 3 with Boc2O and a base (e.g., TEA) under anhydrous conditions (e.g., in acetonitrile).

[0607] c) SN2 transformation of compound 5-ol to azide Compound 5 can be prepared by treating compound 4, for example, for 48 hours with diphenylphosphohydrin (DPPA) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in a dry aprotic solvent (e.g., toluene / DMF).

[0608] d) Deprotection of compound 6-Boc Compound 6 can be prepared from compound 5 by removing the Boc group, for example using HCl in ethyl acetate.

[0609] e) Compound 7 Compound 7 can be prepared by linking compound 6 to compound 6B, for example, by using potassium carbonate in acetonitrile, for example at 60°C.

[0610] f) Compound 8 Compound 8 can be prepared from compound 7 by removing the Boc group, for example using HCl in ethyl acetate.

[0611] g) Compound 9 Compound 9 can be prepared from compound 8 by attaching 9H-fluorene-9-ylmethyl N-(2-oxoethyl)carbamate, for example, NaBH(OAc)3 used in DCE.

[0612]

[0613] h) Compound 10 Compound 10 can be prepared from compound 9 by using a urea-forming reaction with compound 2.

[0614] i) Compound 11 Compound 11 can be prepared from compound 10 by removing the Fmoc group.

[0615] j) Compound 13 Compound 13 can be prepared from compound 11 by coupling with 2-[4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl]acetic acid.

[0616] k) Connector 1 Linker 1 can be prepared from compound 13 by removing Boc and tert-butoxy groups, for example using HCl in ethyl acetate.

[0617] Example 8 – Synthesis route of connector 2

[0618] a) Compound 14 Compound 14 can be prepared from compound 9 by linking 9H-fluorene-9-ylmethyl N-(2-oxoethyl)carbamate, for example using NaBH(OAc)3 in DCE. b) Compound 15 Compound 15 can be prepared from compound 14 by coupling with 2-[4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl]acetic acid.

[0619] c) Compound 16 Compound 16 can be prepared from compound 15 by removing the Fmoc protecting group.

[0620] d) Compound 17 Compound 17 can be prepared from compound 16 by coupling with 2-[4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl]acetic acid.

[0621] e) Connector 2 Linker 2 can be prepared from compound 17 by removing Boc and tert-butoxy groups, for example using HCl in ethyl acetate.

[0622] Example 9 – Synthesis of connectors 3 and 4 i) Synthesis of the general intermediate AA_9: i (a) Methyl 3-(2-(2-oxoethoxy)ethoxy)propionate (compound AA_2)

[0623] A solution of Dess-Martin periodinane (DMP) (122 g, 288 mmol, 1.5 equivalent) in CH2Cl2 (200 mL) was added to a solution of compound AA_1 (37 g, 192 mmol, 1.0 equivalent) in CH2Cl2 (100 mL). The mixture was stirred at 25 °C for 4 hours. The reaction mixture was then poured into a saturated aqueous solution of NaHCO3 (100 mL) and extracted with CH2Cl2 (3 x 100 mL). The combined organic layers were washed with a saturated aqueous solution of Na2S2O3 (100 mL) and brine (3 x 50 mL), dried over anhydrous Na2SO4, and concentrated to obtain the residue. The residue was ground with methanol (200 mL) for 2 hours, and the filtrate was concentrated to obtain compound AA_2 (>38 g, crude product), a yellow oil. The crude product was used directly in the next step without any further purification.

[0624] i (b) 1-(tert-butyl)19-methyl 4,7,13,16-tetraoxa-10-azanonadecanedioate (compound AA_) 4)

[0625] Compounds AA3 (68 g, 291 mmol, 1.5 equivalents), AcOH (16.7 mL, 291 mmol, 1.5 equivalents), and NaBH3CN (24.4 g, 389 mmol, 2.0 equivalents) were added to a solution of compound AA2 (36.6 g, 194 mmol, 1.0 equivalents) in MeOH (500 mL). The mixture was stirred at 25 °C for 4 hours. The reaction mixture was then concentrated under reduced pressure and purified by preparative HPLC to give compound AA4 (24 g, 58.9 mmol, 30% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ ppm 3.84 (br t, J = 4.14 Hz, 2 H), 3.71 - 3.80 (m, 6 H), 3.70 (m, 4 H), 3.66 (br d, J = 2.51 Hz, 4 H), 3.62 (s, 3 H), 3.57 (d, J = 5.27 Hz, 1H), 3.49 (s, 1H) 3.47 (d, J = 4.77 Hz, 1 H), 3.30 (br s, 2 H), 2.58 - 2.65 (m, 2 H), 2.51 (m, 2 H), 1.46 (s, 9 H). MS (ESI): MS calculated value: 407, MS measured value: [M+H] + = 408.

[0626] i (c) 1-(tert-butyl)19-methyl-10-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-aza) (Compound AA_6) hexadecane-16-yl)carbamoyl)-4,7,13,16-tetraoxa-10-azanonadecanedioate

[0627] A solution of compound AA_5 (24.7 g, 84.6 mmol, 1.5 equivalents) and triethylamine (47 mL, 338 mmol, 6.0 equivalents) in CH₂Cl₂ (200 mL) was slowly added to a solution of triphosgene (12.5 g, 42.3 mol, 0.70 equivalents) in CH₂Cl₂ (600 mL). The mixture was stirred at 0 °C for 1 hour. Then, amine AA_4 (23 g, 56.4 mmol, 1.0 equivalents) in CH₂Cl₂ (100 mL) was slowly added at 0 °C for at least 10 minutes. The mixture was stirred at 0 °C for another 2 hours. The reaction mixture was poured into a saturated aqueous solution of NaHCO₃ (100 mL) and extracted with CH₂Cl₂ (3 x 100 mL). The combined organic layers were washed with brine (3 x 50 mL), dried over anhydrous Na2SO4, concentrated, and purified by preparative HPLC to give compound AA_6 (22 g, yield 54%) as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ ppm5.94 - 6.06 (m, 1 H), 5.08 (br s, 1 H), 3.75 (t, J = 6.40 Hz, 2 H), 3.70 (s, 3H), 3.58 - 3.67 (m, 22 H), 3.55 (br d, J = 4.77 Hz, 4 H), 3.47 (t, J = 5.02 Hz, 4H), 3.34 - 3.39 (m, 2 H), 3.29 - 3.34 (m, 2 H), 2.57 - 2.64 (m, 2 H), 2.48 -2.53 (m, 2 H), 1.46 (d, J = 1.51 Hz, 18 H). LCMS: Calculated MS value: 725, Measured MS value: [M+H] + =726.

[0628] i (d) 19-(2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)-2,2-dimethyl-4, 18-Dioxo-3,8,11,14,22,25-hexaoxa-5,17,19-triazaoctacosane-28-acid (compound AA_7):

[0629] LiOH·H2O (1.9 g, 45.4 mmol, 1.5 equivalent) was added to a solution of compound AA_6 (22 g, 30.3 mmol, 1.0 equivalent) in THF / H2O (1:1, 220 mL). The mixture was stirred at 0 °C for 2 hours. After the reaction was complete, the reactants were acidified to pH 3 with 1N HCl and extracted with CH2Cl2 (3 x 500 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give compound AA_7 (20 g, crude), a white solid that could be used without further purification. LCMS: MS calculated value: 711, MS measured value: [M+H] + =712.

[0630] i (e) tert-butyl-1-azido-12-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)- 22-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-yl)carbamoyl)-13-oxo 3,6,9,16,19,25,28-hepta-oxa-12,22-diazatriacontane-31-ester (compound AA_8)

[0631] Compounds AA_14 (12.9 g, 30.9 mol, 1.0 equivalent), HATU (14 g, 42.1 mmol, 1.5 equivalent), and DIPEA (6.96 g, 42.1 mmol, 1.5 equivalent) were added to a solution of compound AA_7 (20 g, 30.9 mol, 1.0 equivalent) in CH2Cl2 (200 mL). The mixture was stirred at 25 °C for 2 hours. It was then added to H2O (100 mL) and extracted with CH2Cl2 (3 x 100 mL). The combined organic phases were washed with brine (50 mL × 3), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give compound AA_8 (26 g, crude), a yellow solid, which could be used in the next step without further purification. 1 H NMR (400 MHz, CDCl3) δ ppm 6.01 - 6.16 (m, 1 H), 5.02 - 5.26 (m, 1 H), 3.76 - 3.82 (m, 4 H), 3.53 - 3.72 (m, 56 H), 3.44 - 3.52 (m, 4 H), 3.41 (br s, 4 H), 2.67 - 2.77 (m, 2 H), 2.54 - 2.61 (m, 2 H), 1.41 - 1.47 (m, 18 H).

[0632] i (f)1-Azide-12-(2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-22-((2, 2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-yl)carbamoyl)-13-oxo-3,6, 9,16,19,25,28-heptaoxa-12,22-diazatriacontane-31-acid (compound AA_9)

[0633] Compound AA_8 (26 g, 32.3 mmol, 1.0 equivalent) was stirred in a 2N HCl / 1,4-dioxane (260 mL) solution at 0 °C for 2 hours. The reaction mixture was then concentrated under reduced pressure to give an intermediate acid (25 g, crude), which was redissolved in CH₂Cl₂ (250 mL), followed by the addition of (Boc)₂O (8.55 g, 39.1 mmol, 1.5 equivalent) and triethylamine (5.51 mL, 39.1 mmol, 1.5 equivalent). The mixture was stirred at 25 °C for 2 hours. The reaction mixture was then concentrated under reduced pressure and further purified by silica gel column chromatography (CH₂Cl₂ / MeOH = 20:1) to give compound AA_9 (18.6 g, 17.5 mmol, two-step yield 58%) as a white solid. 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm 6.63 - 6.81 (m, 1 H), 6.22 - 6.31 (m, 1 H), 5.76 (s, 1 H), 3.34 - 3.61 (m, 64 H), 3.05 (s, 4 H), 2.57 - 2.62 (m, 2 H), 2.44 (t, J = 6.27 Hz, 2 H), 1.38 (s, 9 H). MS(ESI): Calculated value: 1057, Measured MS value: [M+H] + = 1058.

[0634] ii) Synthesis of intermediates AA_13 and AA_18 ii (a) tert-butyl(1-(4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)-2-oxo-6,9,12,15-tetra- Oxa-3-azaheptadecane-17-yl)carbamate (compound AA_12)

[0635] To a solution of compound AA_11 (6.7 g, 29 mmol, 1.0 equivalent) in DMF (67 mL), DIPEA (13.1 mL, 79.4 mmol, 2.73 equivalent), HATU (30.2 g, 79.4 mmol, 2.73 equivalent), and compound AA_10 (13.4 g, 39.68 mmol, 1.36 equivalent) were added. The mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure and purified by preparative HPLC to give compound AA_12 (10 g, 63% yield) as a pink solid. MS (ESI): Exact mass: 548.3, MS value: [M+H] + = 549.1 ii (b) N-(14-amino-3,6,9,12-tetraoxatetradecyl)-2-(4-(6-methyl-1,2,4,5-tetraazine- 3-yl)phenyl)acetamide (compound AA_13)

[0636] Compound AA_12 (10 g, 18.2 mmol, 1.0 equivalent) was added to 1,4-dioxane (100 mL) containing 2N HCl. The reaction mixture was stirred at 25 °C for 2 hours. The solvent was then concentrated under reduced pressure to give compound AA_13 (8.1 g, 84% yield) as a pink solid, which was ready for use without further purification. MS (ESI): Exact mass: 448.3, MS determination: [M+H] + = 449.

[0637] ii (c) tert-butylbis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamate (Compound AA_15)

[0638] To a solution of compound AA_14 (12 g, 28.6 mmol, 1.0 equivalent) in CH2Cl2 (120 mL), (Boc)2O (12.5 g, 57.2 mmol, 2.0 equivalent) and triethylamine (9.9 mL, 71.2 mmol, 2.5 equivalent) were added. The mixture was stirred at 25 °C for 15 hours. After completion, the reaction mixture was diluted with water (200 mL) and extracted with CH2Cl2 (3 x 200 mL). The combined organic layers were washed with brine (3 × 50.0 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by rapid silica gel column chromatography to give compound AA_15 (13.6 g, 87% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δppm 3.53 - 3.72 (m, 24 H), 3.32 - 3.49 (m, 8 H), 1.42 - 1.48 (m, 9 H). MS (ESI): MS calculated value: 519.6, MS measured value: [M+H] + = 520.2 ii (d) tert-butylbis(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (chemical) Compound AA_16)

[0639] Triphenylphosphine (20.6 g, 78.7 mmol, 3.0 equivalent) was added to a solution of compound AA_15 (13.6 g, 26.2 mmol, 1.0 equivalent) in THF (100 mL), and the reaction mixture was stirred at 25 °C for 3 hours. Then, H2O (100 mL) was added, and the mixture was stirred at 80 °C for another 12 hours. After completion, the reaction mixture was diluted with CH2Cl2 (500 mL) and stirred for 5 minutes. The organic layer was discarded, and the aqueous layer was concentrated and lyophilized to give compound AA_16 (12 g, crude product), a colorless oil that could be used without further purification. 1 H NMR (400 MHz, D2O) δ ppm 1.38 - 1.45 (m, 9 H), 3.09 - 3.17 (m, 4 H), 3.39 - 3.49 (m, 4 H), 3.61 - 3.73 (m, 24 H). LCMS: MS calculated value: 467.6, MS measured value: [M+H] + =468.3.

[0640] ii (e) tert-butylbis(1-(4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)-2-oxo-6,9,12-tris( Oxa-3-azatetradecane-14-yl)carbamate (compound AA_17)

[0641] Compounds AA11 (2.21 g, 9.63 mmol, 2.5 equivalence), DCC (1.98 g, 9.63 mmol, 2.5 equivalence), HOBt (1.56 g, 11.5 mmol, 3.0 equivalence), and DIPEA (3.82 mL, 23.1 mmol, 6.0 equivalence) were added to a solution of compound AA16 (1.8 g, 3.85 mmol, 1.0 equivalence) in CH2Cl2 (40 mL). The reaction mixture was stirred at room temperature for 6 hours. After stirring, the contents were diluted with CH2Cl2 (200 mL) and washed with water (400 mL). The aqueous layer was extracted with CH2Cl2 (2 x 200 mL). The combined organic layers were washed with brine (3 × 50 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by rapid silica gel column chromatography to give compound AA_17 (2.79 g, yield 81%) as a purple oil. 1 H NMR (400 MHz, CDCl3): δ ppm 8.51 - 8.57 (m, 4 H), 7.50 - 7.55 (m, 4 H), 3.65 - 3.69 (m, 4 H), 3.53 - 3.61 (m, 24 H), 3.47 (s, 8 H), 3.08 - 3.12 (m, 6 H), 1.42 - 1.46 (m, 9 H). MS (ESI): MS calculated value: 891.5, MS measured value: [M+H] + = 914.7.

[0642] ii (f) N,N'-(3,6,9,15,18,21-hexaoxa-12-azatoridecane-1,23-diyl)bis(2-(4- (6-Methyl-1,2,4,5-tetraazine-3-yl)phenyl)acetamide) (Compound AA_18)

[0643] Compound AA_17 (1.41 g, 1.59 mmol, 1.0 equivalent) was dissolved in 2N HCl / 1,4-dioxane (15 mL), and the mixture was stirred at 25 °C for 2 hours. After the reaction mixture was completed, it was concentrated under reduced pressure to give compound AA_18 (1.3 g, crude product), which was a purple oil and could be used without further purification.

[0644] iii) Synthesis of connectors 3 and 4 iii (a) tert-butyl(14-(1-azido-12-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy) (ethyl)-13-oxo-3,6,9,16,19-pentaoxa-12-azatetramono-21-yl)-41-(4-(6-methyl-1,2,4, 5-Tetraazine-3-yl)phenyl)-13,23,40-trioxo-3,6,9,17,20,27,30,33,36-nonazo-12,14,24,39- Tetraaza-tetra-tetra-alkyl)carbamate (compound AA_19)

[0645] Compounds AA_13 (3.92 g, 8.7 mmol, 1.2 equivalence), HATU (4.14 g, 10.9 mmol, 1.5 equivalence), and DIPEA (2.41 mL, 14.5 mmol, 2.0 equivalence) were added to a solution of compound AA_9 (7.7 g, 7.28 mmol, 1.0 equivalence) in CH₂Cl₂ (80 mL). The mixture was stirred at 25 °C for 30 min. The reaction mixture was then concentrated and filtered through a silica gel stopper to give compound AA_19 (10 g, crude product) as a purple solid, which was used directly in the next step. LCMS: MS calculated value: 1487, MS measured value: [M+H] + =1488.

[0646] iii (b) 10-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-N1, N1-bis(2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N19-(1-(4-(6-methyl-1,2,4,5- Tetraazine-3-yl)phenyl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecane-17-yl)-4,7,13,16-tetraoxa- 10-Zazanonadecanidamide (linker 3)

[0647] A solution of compound AA_19 (10 g, 6.7 mmol, 1.0 equivalent) in 2N HCl / 1,4-dioxane (90 mL) was stirred at 0 °C for 30 min. The reaction mixture was then concentrated under reduced pressure and purified by preparative HPLC (0.01% TFA). After lyophilization, linker 3 (3.9 g, 42%) was obtained as a purple solid. 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm7.90(s, 1 H), 7.69 - 7.86 (m, 3 H), 7.54 (d, J = 8.28 Hz, 2 H), 6.28 (br t, J =5.52 Hz, 1 H), 3.33 - 3.65 (m, 82 H), 3.11 - 3.27 (m, 6 H), 3.00 (s, 3 H), 2.98 (br s, 2 H), 2.60 (s, 2 H), 2.32 (t, J = 6.53 Hz, 2 H). MS (ESI): Calculated MS value: 1836, Measured MS value: [M+H] + = 1837. HPLC purity (254 nm) = 98.6% iii (c) tert-butyl(14-(1-azido-12-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy) (ethyl)-13-oxo-3,6,9,16,19-pentaoxa-12-azatetramono-21-yl)-38-(4-(6-methyl-1,2,4, 5-Tetraazine-3-yl)phenyl)-2,4-(1-(4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)-2-oxo-6,9,12-trioxane) (-3-azatetradecane-14-yl)-13,23,37-trioxa-3,6,9,17,20,27,30,33-octaoxa-12,14,24, 36-Tetraazaoctadecyl)carbamate (compound AA_20)

[0648] Compounds AA_18 (7.25 g, 8.75 mmol, 1.2 equivalent), HATU (4.16 g, 10.9 mmol, 1.5 equivalent), and DIPEA (2.41 mL, 14.6 mmol, 2.00 equivalent) were added to a solution of compound AA_9 (7.7 g, 7.28 mmol, 1.0 equivalent) in CH2Cl2 (77 mL). The mixture was stirred at 25 °C for 30 min. The reaction mixture was then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give compound AA_20 (14.7 g, crude product) as a purple solid, which could be used directly without further purification. 1 H NMR (400 MHz, DMSO- d 6 ) δ ppm 8.57 - 8.60 (m, 1 H), 8.39 (d, J = 8.28 Hz, 4 H), 8.23 ​​(br s, 2 H), 7.53 (d, J = 8.28 Hz, 4 H), 6.73 (br s, 1 H), 3.37 - 3.59 (m, 96 H), 3.23 (br d, J = 5.77 Hz, 4 H), 3.01 - 3.11 (m, 4 H), 2.99 (s, 6 H), 2.58 (br t, J = 6.78 Hz, 4 H), 1.35 -1.38 (m, 9 H). LCMS: MS calculated value: 1831, MS measured value: [M+H] + =1832.

[0649] iii (e) 10-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-N1, N1-bis(2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N19,N19-bis(1-(4-(6-methyl-1, 2,4,5-Tetraazine-3-yl)phenyl)-2-oxo-6,9,12-trioxa-3-azatetradecane-14-yl)-4,7,13,16-tetraoxo Z-10-azanonadecanidamide (linker 4)

[0650] A solution (150 mL) of compound AA_20 (14.7 g, 8.02 mmol, 1.0 equivalent) in 2N HCl / 1,4-dioxane was stirred at 0 °C for 30 min. The reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under reduced pressure, purified by preparative HPLC (0.01% TFA), and then lyophilized to give linker 4 (4.9 g, two-step yield 39%, purity 96.7%) as a purple solid. 1 H NMR (400 MHz, DMSO- d6 ) δ ppm 8.40 (d, J = 8.28 Hz, 4 H), 8.19- 8.29 (m, 2 H), 7.68 - 7.89 (m, 3 H), 7.54 (d, J = 8.28 Hz, 4 H), 6.16 -6.35 (m, 1 H), 3.35 - 3.61 (m, 96 H), 3.24 (br d, J = 5.77 Hz, 4 H), 3.11 -3.18 (m, 2 H), 2.99 (s, 6 H), 2.94 - 2.99 (m, 2 H), 2.59 (br t, J = 6.78 Hz, 4H). MS (ESI): MS calculated value: 1729, MS measured value: [(M+2H) / 2] 2+ = 866. HPLC purity (254 nm) = 96.7% Example 10 - Synthesis of Connector 5 i) Intermediate AA_57

[0651] To a solution of AA_56 (5.0 g, 20.6 mmol, 1.0 equivalent), FmocHN-CH2-CHO (6.07 g, 21.6 mmol, 1.05 equivalent) in DCE (75.0 mL) was added, and the mixture was stirred for 5 minutes. Then, NaBH(OAc)3 (8.71 g, 41.1 mmol, 2.0 equivalent) was added to the mixture at 0 °C. The mixture was heated to room temperature and stirred at 25 °C for 16 hours. After completion, H2O (10.0 mL) was added to the reaction mixture for quenching, and the product was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (TFA conditions) to give AA_57 (6.74 g, 10.8 mmol, yield 52.7%, TFA salt) as a colorless oil. LCMS:R t 0.88 min, MS calculated value: 508.3, MS measured value: [M+Na] + =531.4.HPLC:ES27056-40-p1cc, R t : 4.08 min, purity: 97.27%. 1 H NMR: 400 MHz, CDCl3 δ 7.76 (d, J= 7.53 Hz, 2 H), 7.62 (d, J = 7.28 Hz, 2 H), 7.37 - 7.45 (m, 2H), 7.29 - 7.37 (m, 2 H), 6.76 (br s, 1 H), 4.35 (d, J = 7.28 Hz, 2 H), 4.18 -4.26 (m, 1 H), 3.92 (br d, J = 4.27 Hz, 4 H), 3.57 - 3.69 (m, 6 H), 3.46 - 3.53 (m, 6 H), 3.38 (t, J = 4.77 Hz, 4 H).

[0652] ii) Intermediate AA_58

[0653] Diethylamine (50.0 mL) was added to a solution of AA_57 (6.74 g, 13.3 mmol, 1.00 equivalent) in MeCN (100 mL), and the mixture was stirred at 25 °C for 4 hours. After the reaction was complete, the solvent was concentrated to obtain the residue. The residue was purified by column chromatography (SiO2, dichloromethane:MeOH = 100 / 1 to 20 / 1) to give AA_58 (2.73 g, 9.53 mmol, yield 71.9%) as a yellow oil.

[0654] LCMS: R t 0.45 minutes, MS calculated value: 286.2, MS measured value: 287.1 [M+H] + HPLC: ES27056-60-p1c1, R t : 2.27 minutes, purity: 84.56%. 1 H NMR: 400 MHz, CDCl3 δ : 3.61 - 3.66 (m, 4 H),3.57 (t, J = 5.77 Hz, 4 H), 3.35 - 3.43 (m, 4 H), 2.75 - 2.84 (m, 6 H), 2.67 -2.70 (m, 2 H).

[0655] iii) Intermediate AA_59

[0656] At 0 °C, FmocHN-CH2-CHO (491 mg, 1.75 mmol, 0.50 equivalent) and NaBH(OAc)3 (1.11 g, 5.24 mmol, 1.50 equivalent) were added to a solution of AA_58 (1.0 g, 3.49 mmol, 1.0 equivalent) in DCE (30.0 mL). The mixture was allowed to slowly reach room temperature and stirred at 25 °C for 16 hours. After completion, the mixture was quenched with H2O (5.0 mL) and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative-HPLC (TFA conditions) to give AA_59 (560 mg, 841 μmol, yield 24%, TFA salt) as a yellow oil. LCMS: ES27056-83-p1a, R t : 1.07 min, MS calculated value: 551.3, MS measured value: 569.3 [M+H2O] + . 1 H NMR: 400 MHz, CDCl3 δ 7.76(d, J = 7.53 Hz, 2 H), 7.62 (br d, J = 7.53 Hz, 2 H), 7.37 - 7.44 (m, 2 H), 7.28- 7.36 (m, 2 H), 6.57 (br s, 1 H), 4.34 (br d, J = 7.03 Hz, 2 H), 4.17 - 4.25(m, 1 H), 3.79 (br d, J = 4.27 Hz, 4 H), 3.56 - 3.66 (m, 8 H), 3.38 - 3.44 (m, 6 H), 3.31 (br s, 4 H), 3.22 (br s, 2 H).

[0657] iv) Intermediate AA_60

[0658] To a solution of AA_59 (743 mg, 953 μmol, 1.00 equivalence) in dichloromethane (18.5 mL), (Boc)₂O (438 μL, 1.91 mmol, 2.00 equivalence) and triethylamine (133 μL, 953 μmol, 1.0 equivalence) were added. The mixture was stirred at 25 °C for 2 hours. After stirring, the reaction mixture was diluted with H₂O (50.0 mL) and extracted with dichloromethane (25.0 mL x 3). The combined organic layers were washed with brine (approximately 25 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography (SiO₂, dichloromethane / MeOH = 100 / 1 to 20 / 1) to give AA_60 (609 mg, 934 μmol, 98% yield) as a white solid. LCMS:, R t 0.95 min, MS calculated value: 651.4, MS measured value: 652.3 [M+H] + . 1 H NMR: 400 MHz, CDCl3 δ 7.77 (d, J = 7.53 Hz, 2H), 7.60 (br d, J = 7.28 Hz, 2 H), 7.37 - 7.45 (m, 2 H), 7.29 - 7.36 (m, 2 H), 4.54 (br s, 1 H), 4.42 (br d, J = 4.52 Hz, 1 H), 4.21 (br t, J = 6.40 Hz, 1 H), 3.37 - 3.67 (m, 10 H), 3.33 (br s, 6 H), 3.24 (br t, J = 6.27 Hz, 2 H), 2.54 -2.84 (m, 6 H), 1.41 - 1.50 (m, 9 H).

[0659] v) Intermediate AA_61

[0660] Diethylamino (4.81 mL) was added to a solution of AA_60 (609 mg, 934 μmol, 1.0 equivalent) in acetonitrile (9.0 mL). The mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the solvent was concentrated under vacuum to obtain the residue. The residue was further purified by column chromatography (SiO2, dichloromethane / MeOH = 100 / 1 to 15 / 1) to give AA_61 (328 mg, 764 μmol, yield 82%) as a milky white solid. LCMS:R t 1.03 min, MS calculated value: 429.3, MS measured value: 430.2 [M+H] + . 1 H NMR: 400 MHz, CDCl3 δ : 3.60 - 3.66 (m, 4 H), 3.57 (t, J = 5.90Hz, 4 H), 3.39 (t, J = 4.89 Hz, 4 H), 3.29 (br s, 4 H), 2.85 (t, J = 6.53 Hz, 2H), 2.80 (t, J = 5.77 Hz, 4 H), 2.74 (br s, 2 H), 1.47 (s, 9 H).

[0661] vi) Intermediate AA_62

[0662] DIPEA (200 μL, 1.15 mmol, 1.50 equivalent) was added to a solution of AA_61 (328 mg, 764 μmol, 1.0 equivalent) and Tz-OSu (375 mg, 1.15 mmol, 1.50 equivalent) in CH2Cl2 (5.0 mL). The mixture was stirred at 25 °C for 3 h. After stirring, the reaction mixture was diluted with H2O (5.0 mL) and extracted with dichloromethane (5.0 mL x 3). The combined organic layers were washed with brine (approximately 5.0 mL), dried over Na2SO4, filtered, and concentrated under vacuum to obtain the residue. The residue was then purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to give AA_62 (485 mg, 756 μmol, yield 99.0%) as a purple oil. LCMS:R t 0.87 minutes, MS calculated value: 641.4, MS measured value: 642.5 [M+H] + . 1H NMR:ES27056-100-p1b1, 400 MHz, CDCl3 δ : 8.52 - 8.60 (m, 2 H),7.49 - 7.55 (m, 2 H), 3.63 (br s, 2 H), 3.58 - 3.62 (m, 4 H), 3.55 (br s, 2H), 3.40 (br s, 4 H), 3.36 (t, J = 4.89 Hz, 4 H), 3.23 (br s, 1 H), 3.10 (s, 3H), 2.79 (br s, 2 H), 2.69 (s, 1 H), 2.65 - 2.73 (m, 1 H), 2.10 - 2.39 (m, 5H), 1.44 (s, 9H).

[0663] vii) Intermediate compound AA_63

[0664] AA_62 (485 mg, 756 μmol, 1.0 equivalent) was dissolved in 2N HCl / EtOAc (10 mL) and stirred at 25 °C for 2 hours. After the reaction was complete, the solvent was concentrated under reduced pressure to obtain the residue. The residue was further purified by preparative HPLC (TFA conditions) to give AA_63 (456 mg, 592 μmol, yield 78.4%, TFA salt), as a purple oil. LCMS: R t 0.76 minutes, MS calculated value: 541.3, MS measured value: 542.4 [M+H] + HNMR: 400 MHz, CDCl3 δ 8.53(d, J = 8.28 Hz, 2 H), 7.91 (br s, 1 H), 7.55 (d, J = 8.53 Hz, 2 H), 3.77 - 3.85 (m, 4 H), 3.57 - 3.70 (m, 10 H), 3.40 - 3.46 (m, 4 H), 3.27 - 3.37 (m, 6 H), 3.18 - 3.24 (m, 2 H), 3.10 (s, 3H).

[0665] viii) Intermediate compound AA_64

[0666] At 0 °C, a solution of R26 (225 mg, 770 μmol, 1.30 equivalence) and triethylamine (495 μL, 3.55 mmol, 6.00 equivalence) in dichloromethane (7.5 mL) was added to a solution of triphosgene (114 mg, 385 μmol, 0.65 equivalence) in dichloromethane (7.50 mL). The mixture was stirred at 0 °C for 1 hour. Then, at 0 °C, the resulting mixture was added dropwise to a solution of AA_63 (456 mg, 592 μmol, 1.0 equivalence) in dichloromethane (10.0 mL). The mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the reaction mixture was diluted with dichloromethane (approximately 25 mL). It was further washed with H2O (approximately 20 mL) and extracted with CH2Cl2 (50.0 mL x 3). The combined organic layers were washed with brine (50.0 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography (SiO₂, DCM / MeOH = 100 / 1 to 20 / 1) to give AA₆₄ (573 mg, 666 μmol) as a purple oil. LCMS:R t 0.88 minutes, MS calculated value: 859.5, MS measured value: 860.8 [M+H] + HPLC:R t : 3.48 min, purity: 93.87%. 1 H NMR: 400 MHz, CDCl3 δ 8.55 (d, J = 8.28 Hz, 2 H), 7.45 - 7.56 (m, 3 H), 7.38 (br s, 1 H), 5.08 (br s, 1 H), 3.49 - 3.72 (m, 28 H), 3.29 - 3.42 (m, 14 H), 3.20 (br s, 2 H,) 3.10 (s, 3 H), 2.71 - 2.85 (m, 6 H), 1.45 (s, 9 H).

[0667] ix) Connector 5

[0668] AA_64 (509 mg, 592 μmol, 1.00 equivalent) was dissolved in 2N HCl / EtOAc (10.0 mL) and stirred at 25 °C for 2 hours. After the reaction was complete, the solvent was concentrated under vacuum to obtain the residue. The residue was purified by preparative HPLC (TFA conditions). The eluent was washed with 1% NaHCO3 (50 mL) and extracted with dichloromethane (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give linker 5 (180 mg, 237 μmol, yield 40%) as a purple oil. LCMS: ES27056-112-p1a1, R t 0.78 minutes, MS calculated value: 759.4, MS measured value: 760.6 [M+H] + HPLC: ES27056-112-p1cc, RT: 2.18 min, purity: 95.37%. 1 H NMR: 400 MHz, CDCl3 δ 8.55 (d, J = 8.53 Hz, 2 H), 7.53 (d, J = 8.53 Hz, 3 H), 3.62 -3.67 (m, 9 H), 3.60 (d, J = 4.77 Hz, 4 H), 3.49 - 3.59 (m, 10 H), 3.32 - 3.43(m, 10 H), 3.21 (br d, J = 5.52 Hz, 2 H), 3.10 (s, 3 H), 2.87 (t, J = 5.27 Hz, 2H), 2.79 (t, J = 5.52 Hz, 4 H), 2.75 (br d, J = 4.77 Hz, 2 H).

[0669] Example 11 - Synthesis of Connector 6 i) Intermediate AA_65

[0670] AcOH (780 μL, 13.6 mmol, 6.0 equivalence) was added to a solution of AA_58 (1.3 g, 4.54 mmol, 2.0 equivalence) in THF (25 mL), followed by the addition of aldehyde 1 (1.24 g, 2.27 mmol, 1.0 equivalence) and NaBH3CN (571 mg, 9.08 mmol, 4.00 equivalence). The mixture was stirred at 45 °C for 72 hours. The mixture was then diluted with approximately 5 mL of H2O and concentrated under vacuum to obtain a residue. The residue was further purified by preparative HPLC (TFA conditions) to give AA_65 (1.06 g, 1.30 mmol, 57% yield) as a colorless oil. LCMS: ES27056-114-p2a5, R t 0.97 minutes, MS calculated value: 816.4, MS measured value: 839.6 [M+Na] + HPLC: ES27056-114-p1c, R t Time taken: 4.756 minutes; Purity: 90%. 1 H NMR: 400 MHz, CDCl3 δ : 7.70 - 7.84 (m, 4 H), 7.59 (br s, 2 H), 7.31 - 7.50 (m, 8 H), 7.21 - 7.27 (m, 2 H), 4.51 (br s, 4H), 4.12 - 4.24 (m, 2 H), 3.84 (br s, 4 H), 3.66 (br d, J = 4.02 Hz, 6 H), 3.21- 3.50 (m, 12 H), 2.68 - 3.18 (m, 6 H), 2.25 (br d, J = 12.55 Hz, 1 H).

[0671] ii) Intermediate compound AA_66

[0672] To a solution of AA_65 (393 mg, 422 μmol, 1.0 equivalence) in dichloromethane (4.00 mL), (Boc)₂O (388 μL, 1.69 mmol, 4.00 equivalence) and triethylamine (88.1 μL, 633 μmol, 1.50 equivalence) were added. The mixture was stirred at 25 °C for 4 hours, then diluted with dichloromethane (2.0 mL) and purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 100 / 1 to 20 / 1) to give AA_66 (354 mg, 386 μmol, 91% yield) as a colorless oil. LCMS: ES27056-123-p1a9, R t 1.05 minutes, MS calculated value: 916.5, MS measured value: 917.8 [M+H] + HPLC: ES27056-123-p1c, R t 5.27 minutes, purity: 92.87%. 1 H NMR: 400 MHz, CDCl3 δ :7.68 - 7.85 (m, 4 H), 7.48 - 7.63 (m, 4 H), 7.28 - 7.47 (m, 8 H), 4.29 - 4.71 (m, 5 H), 4.19 (br s, 2 H), 3.87 (br s, 2 H), 3.22 - 3.73 (m, 18 H), 2.51 -3.22 (m, 7 H), 2.04 (br d, J = 12.05 Hz, 1 H), 1.41 (br t, J = 7.28 Hz, 9 H).

[0673] ii) Intermediate compound AA_67

[0674] Dimethylamine (6.64 mL) was added to a solution of AA_66 (657 mg, 716 μmol, 1.0 equivalent) in MeCN (15.0 mL). The mixture was stirred at 25 °C for 2 hours, and then the reaction mixture was concentrated under vacuum to obtain a residue. The residue was purified by column chromatography (SiO2, dichloromethane / MeOH = 50 / 1 to 5 / 1) to give AA_67 (242 mg, 512 μmol, yield 71.5%) as a colorless oil. LCMS: , R t 1.61 minutes, MS calculated value: 472.3, MS measured value: 473.3 [M+H]+ .

[0675] HNMR: 400 MHz, CDCl3 δ 3.63 (t, J = 5.02 Hz, 4 H), 3.57 (t, J = 5.90 Hz, 4 H), 3.21 - 3.42 (m, 8 H), 2.76 - 2.89 (m, 8 H), 2.73 (br s, 4 H), 1.47 (s, 9 H).

[0676] iii) Intermediate compound AA_68

[0677] At 0 °C, DIPEA (446 μL, 2.56 mmol, 5.0 equivalent) and HATU (623 mg, 1.64 mmol, 3.2 equivalent) were added to a solution of Tz acid (methyltetraazine (CAS: 1380500-88-8)) (377 mg, 1.64 mmol, 3.2 equivalent) in dichloromethane (2.5 mL). The mixture was then added to a solution of AA_67 (242 mg, 512 μmol, 1.0 equivalent) in dichloromethane (2.5 mL). The mixture was stirred at 25 °C for 2 hours. The mixture was then diluted with H2O (approximately 2.0 mL) and extracted with dichloromethane (5.0 mL x 3). The combined organic layers were washed with brine (approximately 5.0 mL), dried over Na2SO4, filtered, and concentrated under vacuum to obtain the residue. The residue was further purified by preparative HPLC (TFA conditions) to give AA_68 (351 mg, 391 μmol, yield 76%) as a purple oil. LCMS: R t 0.94 minutes, MS calculated value: 896.5, MS measured value: 898.5 [M+H] + HPLC:R t 10.37 minutes, purity: 93.28%. ¹H NMR: 400 MHz, CDCl₃ δ: 8.38 - 8.63 (m, 4 H), 7.34 - 7.63 (m, 4 H), 3.88 (s, 1 H), 3.68 -3.83 (m, 3 H), 3.52 - 3.67 (m, 9 H), 3.29 - 3.52 (m, 12 H), 3.17 - 3.29 (m, 1H), 3.09 (s, 6H), 2.57 - 2.99 (m, 4H), 1.44 - 1.51 (m, 9H).

[0678] iv) Intermediate compounds of AA_69

[0679] AA-68 (91.6 mg, 102 μmol, 1.0 equivalent) was dissolved in 2N HCl / EtOAc (500 μL) and stirred at 25 °C for 2 hours. LC-MS showed that AA-68 was completely consumed and the desired mass peak was identified. The mixture was concentrated under vacuum to obtain the residue. The residue was purified by preparative HPLC (TFA conditions) to give AA-69 (81.0 mg, 102 μmol, yield 99.5%) as a purple oil. LCMS: ES27056-125-p3a1, R t 0.85 minutes, MS calculated value: 796.4, MS measured value: 819.8 [M+Na] + HPLC:R t 2.87 minutes, purity: approximately 100% v) Intermediate compound AA_70

[0680] At 0 °C, a solution of amine 1 (38.2 mg, 131 μmol, 1.3 equivalents) and triethylamine (83.8 μL, 602 μmol, 6.0 equivalents) in dichloromethane (1.5 mL) was added to a solution of triphosgene (19.4 mg, 65.3 μmol, 0.65 equivalents) in DCM (1.50 mL). The mixture was stirred at 0 °C for 1 hour. TLC showed that the reaction was complete. The mixture was then added to a solution of AA_69 (80.0 mg, 100 μmol, 1.0 equivalents) in dichloromethane (1.0 mL). The mixture was stirred at 25 °C for 2 hours. Afterward, the reaction mixture was diluted with dichloromethane (2.0 mL), washed with H2O (2.0 mL), and extracted with CH2Cl2 (15 mL x 3). The combined organic layers were washed with brine (approximately 15 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO₂, dichloromethane / MeOH = 100 / 1 to 20 / 1) to give AA₇₀ (118 mg, 10⁶ μmol) as a purple oil. LCMS: R t 4.47 minutes, MS calculated value: 1115.3, MS measured value: 1116.6 [M+H] + HPLC: Purity: Approximately 100%. 1 H NMR: 400 MHz, CDCl3 δ : 8.33 -8.59 (m, 4 H), 7.64 (br d, J = 8.03 Hz, 2 H), 7.38 (br s, 2 H), 3.24 - 4.08 (m,67 H), 3.10 (d, J = 2.26 Hz, 6 H), 1.40 - 1.53 (m, 21 H).

[0681] vi) Connector 6

[0682] AA_70 (111 mg, 99.5 μmol, 1.0 equivalent) was dissolved in 2N HCl / EtOAc (1.50 mL) and stirred at 25 °C for 2 hours. After the reaction was complete, the solvent was concentrated under vacuum to obtain the residue, which was then purified by preparative HPLC (TFA conditions). The eluent was washed with 1% NaHCO3 (50.0 mL) and extracted with dichloromethane (250 mL x 3). The combined organic layers were washed with brine (250 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give linker 6 (50 mg, 49.3 μmol, yield 49%) as a purple oil. LCMS: MS calculated value: 1015.1, MS measured value: 1015.5 [M+H] + HPLC: Purity: 96.74%. 1H NMR: 400 MHz, CDCl3 δ : 8.43 - 8.60 (m, 4 H), 7.35 - 7.68 (m, 5H), 3.91 (s, 1 H), 3.31 - 3.81 (m, 40 H), 3.18 - 3.30 (m, 2 H), 3.09 (s, 6H), 2.85 - 3.00 (m, 2 H), 2.71 - 2.82 (m, 6 H).

[0683] Example 12 – 4-((2S,5S,31S,34S)-18-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)-35-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl) Synthesis of carbamoyl(oxy)methyl)phenyl)amino)-5,31-diisopropyl-2,34-dimethyl-4,7,29,32,35-pentoxo-9,12,15,21,24,27-hexaoxo-3,6,18,30,33-pentazapentadecanoamide)benzyl(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isooxazol-3-yl)benzyl)(methyl)carbamoyl ester (LP-13) i) 2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]tert-butyl acetate (compound I28)

[0684] At -78°C, DMSO (9.6 mL, 68.1 mmol, 2.0 equivalents) was added to a solution of oxalyl chloride (5.9 mL, 136 mmol, 4.0 equivalents) in CH2Cl2 (90.0 mL), followed by compound I27 (9.0 g, 34.0 mmol, 1.0 equivalents), and the reaction mixture was stirred at the same temperature for 1 hour. Triethylamine (28.4 mL, 204 mmol, 6.0 equivalents) was then added at -78°C, and stirring continued for 1 hour, followed by warming to room temperature. After completion, the reaction mixture was filtered and concentrated under reduced pressure to give compound I28 (12.0 g, crude), a colorless oil that could be used without further purification. 1 H NMR (400 MHz, DMSO-) d 6 ): δ 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).

[0685] ii) 2-[2-[2-[2-[2-[2-[2-(2-tert-butoxy-2-oxoethoxy)ethoxy]ethoxy]ethylamino [Alkyl]ethoxy]ethoxy]ethoxy]tert-butyl acetate (compound I30):

[0686] To a solution of compounds I28 (12.0 g, 1.0 equivalent) and I29 (17.9 g, 681 μmol, 1.5 equivalent) in MeOH (120 mL), NaBH3CN (2.85 g, 454 mmol, 1.0 equivalent) was added. The mixture was stirred at 20–25 °C for 1 hour. After stirring, the reaction mixture was extracted with CH2Cl2 (200 mL) and H2O (100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound I30 (18.0 g, crude), as a yellow oil. MS (ESI): [M+H] + :510.6.

[0687] iii) 2-[2-[2-[2-[2-[2-[2-(2-tert-butoxy-2-oxoethoxy)ethoxy]ethoxy]ethyl- (9H-fluorene-9-ylmethoxycarbonyl)amino]ethoxy]ethoxy]ethoxy]tert-butyl acetate (compound I31):

[0688] To a solution of compound I30 (18.0 g, 1.0 equivalent) and FmocOSu (17.9 g, 681 μmol, 1.5 equivalent) in THF (90 mL) and H2O (90 mL), NaHCO3 (5.9 g, 70.6 mmol, 2.0 equivalent) was added. The mixture was stirred at 20–25 °C for 1 hour. After completion, LCMS showed that compound I30 had been completely consumed. The reaction mixture was extracted with DCM (200 mL) and H2O (100 mL). The combined organic matter was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to give compound I31 (2.1 g, 2.87 mmol, purity 98.1%; and 2.2 g, 3.01 mmol, purity 92.4%, yield 16.6%) as a white solid. Purity obtained by HPLC (220 nm): 98.1%; MS (ESI): [M+H] + 732.3; 1 H NMR (400 MHz, DMSO- d 6 ): δ 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 = 5.4Hz, 1H), 3.95 - 3.99 (m, 4H), 3.46 - 3.56 (m, 16H), 3.38 - 3.44 (m, 4H), 3.13 (br s, 4H), 1.40 - 1.42 (m, 18H).

[0689] iv) 2-[2-[2-[2-[2-[2-[2-(2-tert-butoxy-2-oxoethoxy)ethoxy]ethoxy]ethyl- (9H-fluorene-9-ylmethoxycarbonyl)amino]ethoxy]ethoxy]ethoxy]tert-butyl acetate (compound I32):

[0690] Compound I31 (2.1 g, 2.87 mmol, 1.0 equivalent) was dissolved in 50% FA / DCM (21.0 mL) and stirred at 20–25 °C for 16 hours. LCMS analysis showed that compound I31 was completely consumed. The reaction mixture was quenched by adding NaHCO3 (100 mL) at 0–5 °C. The resulting mixture was purified directly by preparative HPLC (TFA conditions) to give compound I32 (700 mg, 1.03 mmol, yield 35.9%) as a colorless oil. Purity obtained by HPLC (220 nm): 98.2%; MS (ESI): [M+H] + 676.3; 1 H NMR (400 MHz, DMSO- d 6 ): δ 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) v) (2S,5S)-18-(((9H-fluorene-9-yl)methoxy)carbonyl)-1-((4-(((((1S,9S)-9-ethyl-5- Fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]ind Dolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7- Trioxo-9,12,15,21,24,27-hexaoxa-3,6,18-triazanonadecan-29-acid (compound I33):

[0691] To solutions of compound I32 (200 mg, 295 μmol, 1.0 equivalent) and compound I26 (CAS: 2845164-91-0) (308 mg, 377 μmol, 1.3 equivalent) in DMF (2.0 mL), DIPEA (58.6 μL, 354 μmol, 1.2 equivalent), HOBt (47.9 mg, 354 μmol, 1.2 equivalent), and EDCI (67.9 mg, 354 μmol, 1.2 equivalent) were added, respectively. The mixture was stirred at 20–25 °C for 3 hours. LCMS analysis showed that compound I32 was completely consumed. The resulting mixture was directly purified by preparative HPLC (TFA conditions) to give compound I33 (900 mg, 637 μmol, yield 68.3%) as a white solid. Purity obtained by HPLC (220 nm): 97.3%; MS (ESI): [M+H] + : 1413.5. The reaction was carried out in parallel, yielding a total of 900 mg I33.

[0692] vi) (2S,5S)-18-(((9H-fluorene-9-yl)methoxy)carbonyl)-1-((4-(((((1S,9S)-9-ethyl-5- Fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]ind Dolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7- Trioxo-9,12,15,21,24,27-hexaoxa-3,6,18-triazanonadecan-29-acid (compound I34):

[0693] Compound I33 (900 mg, 637 μmol, 1.0 equivalent) was dissolved in 50% FA / DCM (9.0 mL) and stirred at 20–25 °C for 72 hours. LC-MS analysis showed that compound I33 was completely consumed. The resulting product was ground with isopropyl ether (10 mL) at 0–5 °C to give compound I34 (500 mg, crude product), a colorless oil. MS (ESI): [M+H] + :1356.8.

[0694] vii) (9H-fluorene-9-yl)methyl((2S,5S)-1-((4-((((4-(5-(3-amino-6-(4-(isopropylsulfonyl) yl)phenyl)pyrazin-2-yl)isoxazo-3-yl)benzyl)(methyl)carbamoyl)oxy)methyl)phenyl)amino)-5-iso propyl-2-methyl-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl)((2S,5S)-1- ((4-((((1S,9S)-9-ethyl-1-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-) 1H,12H-Benzo[de]pyrano[3',4':6,7]indolazo[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl (phenyl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane- 17-yl)carbamate (compound I35)

[0695] To a solution of compound I34 (500 mg, 368 μmol, 1.0 equivalent) and compound I24 (392 mg, 479 μmol, 1.3 equivalent) in DMF (5.0 mL), DIPEA (152 μL, 921 μmol, 2.5 equivalent), HOBt (74.7 mg, 552 μmol, 1.5 equivalent), and EDCI (105 mg, 552 μmol, 1.5 equivalent) were added sequentially. The mixture was stirred at 20–25 °C for 2 hours. LC-MS analysis showed that compound I34 had been consumed and the desired mass was identified. The reaction mixture was used directly in the next step without further purification. MS (ESI): [(M+2H) / 2] 2+ :1062.5.

[0696] viii) 4-((2S,5S,31S,34S)-35-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1, [2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-5,31-diisopropyl-2,34-dimethyl-4,7, 29,32,35-pentaoxo-9,12,15,21,24,27-hexaoxo-3,6,18,30,33-pentazapentadecanoamide)benzyl 4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazo-3-yl)benzyl)(methyl)amino Formate (compound I36):

[0697] Triethylamine (1.0 mL) was added to the above solution of compound I35. The reaction mixture was stirred at 20–25 °C for 4 h. LC-MS showed that compound I35 was completely consumed and the desired mass was detected. The resulting mixture was purified directly by preparative HPLC (TFA conditions) to give compound I36 (200 mg, 105 μmol, yield 28.5%) as a white solid. HPLC purity (220 nm): 92.9%; MS (ESI): [(M+2H) / 2] 2+ :950.5.

[0698] ix) 4-((2S,5S,31S,34S)-18-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl 1S,9S)-35-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy (methyl)phenyl)amino)-5,31-diisopropyl-2,34-dimethyl-4,7,29,32,35-pentoxo-9,12,15,21, 24,27-Hexaoxo-3,6,18,30,33-pentazapentadecanoamide)benzyl(4-(5-(3-amino-6-(4-(isopropyl) sulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)methyl)carbamate (LP-13)

[0699] To a solution of compound I36 (30 mg, 14.9 μmol, 1.0 equivalent) and compound MC-acid (6.2 mg, 29.3 μmol, 2.0 equivalent) in DMF (300 μL), DIPEA (6.10 μL, 37.2 μmol, 2.5 equivalent), HOBt (4.0 mg, 29.8 μmol, 2.0 equivalent), and EDCI (5.7 mg, 29.8 μmol, 2.0 equivalent) were added. The mixture was stirred at 20–25 °C for 1 hour. LC-MS showed that compound I36 was completely consumed and the desired product peak was detected. The solvent was removed under reduced pressure, and the residue was purified by preparative HPLC (AcOH conditions) to give LP-13 (13.1 mg) as a white solid. Purity obtained by HPLC (220 nm): 96.9%; MS (ESI): [(M+2H) / 2] 2+ :1047.1. 1 H NMR (400 MHz, DMSO- d 6)δ ppm 9.99 (s, 1 H), 9.91 - 10.06 (m, 1 H), 8.93 (s, 1 H), 8.88 - 8.96 (m, 1 H), 8.34 - 8.41 (m, 3 H), 8.34 - 8.41 (m, 1 H), 7.91 - 8.00 (m, 4 H), 7.74 (s, 1 H), 7.59 (br d, J = 8.6 Hz, 4 H), 7.20 - 7.50 (m, 10 H), 7.18 (br d, J =3.8 Hz, 1 H), 7.14 - 7.20 (m, 1 H), 6.97 (s, 1 H), 5.44 (s, 1 H), 5.27 (br d, J = 4.0 Hz, 2 H), 5.05 - 5.09 (m, 3 H), 4.52 (s, 2 H), 4.35 - 4.44 (m, 2 H), 4.24 - 4.32 (m, 2 H), 3.93 (s, 3 H), 3.40 - 3.61 (m, 28 H), 2.87 (s, 3 H), 2.67 (s, 2 H), 2.35 (s, 2 H), 2.36 (br s, 1 H), 2.33 (br d , J= 1.6 Hz, 3 H),2.21 - 2.28 (m, 3 H), 1.94 - 2.06 (m, 4 H), 1.69 (s, 2 H), 1.40 - 1.48 (m, 4H), 1.26 - 1.34 (m, 8 H), 1.23 (br s, 3 H), 1.19 (d, J = 6.8 Hz, 8 H), 0.85 -0.90 (m, 9 H), 0.81 (br d, J = 6.5 Hz, 6 H).

[0700] Example 13: [4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-[2-[2-[2-[2-[2-[2-[(1S)-1-[[(1S)-2-[4-[[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane[14.7.1.02,14.04,13.0]] 6,11.020,24] Ticosico-1,6(11),12,14,16(24),17,19-hepten-23-yl]carbamoyloxymethyl]aniline]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-2-oxo-ethoxy]ethoxy]ethoxy]ethyl-[2-[2-[2-[2-[(3,3,6,6-tetramethyl-1-oxo-1λ 6 Synthesis of -thionyl-hept-4-yne-1-yl)carbamoylamino]ethoxy]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ethoxy]acetyl]amino]-3-methyl-butyryl]amino]propionyl]amino]phenyl]methyl N-[[4-[5-[3-amino-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]isoxazo-3-yl]phenyl]methyl]-N-methylcarbamate (LP-12) i) 4-((2S,5S,31S,34S)-18-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-nitrogen) (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino (carboxymethyl)oxy)methyl)phenyl)amino)-5,31-diisopropyl-2,34-dimethyl-4,7,29,32,35-pentoxo- 9,12,15,21,24,27-hexaoxo-3,6,18,30,33-pentazapentadecanoamide)benzyl(4-(5-(3-amino- 6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazo-3-yl)benzyl)(methyl)carbamate (compound) I37)：

[0701] To a solution of compound I36 (85 mg, 42.2 μmol, 1.0 equivalence) and compound R1-2 (36.4 μL, 82.4 μmol, 2.0 equivalence) in DMF (1.0 mL), DIEA (17.4 μL, 105 μmol, 2.5 equivalence), HOBt (11.4 mg, 84.4 μmol, 2.0 equivalence), and EDCI (16.1 mg, 84.4 μmol, 2.0 equivalence) were added sequentially. The mixture was stirred at 20–25 °C for 1 hour. LC-MS showed that compound I36 was completely consumed and the desired mass was detected. The crude reaction mixture was used directly for the next step. MS (ESI): [(M+2H) / 2] 2+ :1155.9.

[0702] ii) 4-((2S,5S,31S,34S)-18-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetyl 1S,9S)-35-((4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy (methyl)phenyl)amino)-5,31-diisopropyl-2,34-dimethyl-4,7,29,32,35-pentoxo-9,12,15,21, 24,27-Hexaoxo-3,6,18,30,33-pentazapentadecanoamide)benzyl(4-(5-(3-amino-6-(4-(isopropyl) sulfonyl)phenyl)pyrazin-2-yl)isoxazo-3-yl)benzyl)methyl)carbamate (compound I38):

[0703] Triethylamine (0.2 mL) was added to a solution of compound I37 in DMF (0.8 mL). The mixture was stirred at 20–25 °C for 1 hour. LC-MS showed that compound I37 was completely consumed and the desired mass was detected. The resulting reaction mixture was purified directly by preparative HPLC (TFA conditions) to give compound I38 (55 mg, 26.3 μmol, yield 62%) as a white solid. HPLC purity (220 nm): 98.5%; MS (ESI): [(M+2H) / 2] 2+ :1045.1.

[0704] iii) [4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-[2-[2-[2-[2-[2-[[(1S)-1-[[(1S)-2- [4-[[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexane] Cyclo[14.7.1.02,14.04,13.06,11.020,24] 24-carbon-1,6(11),12,14,16(24),17,19-hepten- [23-yl]carbamoyloxymethyl]aniline]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl] [amino]-2-oxo-ethoxy]ethoxy]ethoxy]ethyl-[2-[2-[2-[2-[(3,3,6,6-tetramethyl-1-oxo-1 λ 6 -Thioheptan-4-yne-1-ylidene)carbamoylamino]ethoxy]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy] [4-] ethoxy] acetyl] amino]-3-methyl-butyryl] amino] propionyl] amino] phenyl] methyl N-[[4- [5-[3-amino-6-(4-isopropylsulfonylphenyl)pyrazin-2-yl]isoxazo-3-yl]phenyl]methyl]-N-methyl-amino Formate (LP-12):

[0705] Triethylamine (10.6 μL, 76.6 μmol, 4.0 equivalents), TMTHSI-OSu (39 mg, 114.6 μmol, 6.0 equivalents), and DMAP (4.68 mg, 38.3 μmol, 2.0 equivalents) were added to a solution of compound I38 (40 mg, 19.1 μmol, 1.0 equivalents) in DMF. The mixture was stirred at 20–25 °C for 2 hours and monitored by LC-MS. After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by preparative HPLC (HCOOH conditions) to give LP-12 (20 mg, 8.64 μmol, yield 45.1%) as a white solid. Purity obtained by HPLC (220 nm): 97.7%; MS (ESI): [(M+2H) / 2] 2+ :1157.8; 1 H NMR (400 MHz, DMSO- d 6 ): δ ppm 10.0 (s, 2 H), 8.93 (s, 1 H), 8.35 - 8.41 (m, 4 H), 8.05 (br d, J = 8.4 Hz, 1 H), 7.98 (br d, J = 7.4 Hz, 2 H), 7.93 (d, J = 8.5 Hz, 2 H), 7.73 - 7.78 (m, 2 H), 7.55 - 7.63(m, 4 H), 7.31 - 7.47 (m, 8 H), 7.30 (s, 1 H), 7.18 (br s, 2 H), 6.64 (br t, J = 5.2 Hz, 1 H), 6.52 (s, 1 H), 5.44 (s, 2 H), 5.27 (br d, J = 4.8 Hz, 3 H), 5.07 (br s, 4 H), 4.52 (s, 2 H), 4.39 (br t, J= 6.7 Hz, 2 H), 4.25 - 4.33 (m, 2 H), 4.16 (s, 2 H), 3.93 (s, 4 H), 3.87 (s, 1 H), 3.84 (s, 1 H), 3.36 - 3.66 (m, 42 H), 3.06 - 3.13 (m, 3 H), 2.87 (s, 3 H), 2.35 (s, 3 H), 2.33 (br s, 1H), 1.96 - 2.02 (m, 2 H), 1.83 - 1.92 (m, 2 H), 1.28 - 1.34 (m, 12 H), 1.17 -1.21 (m, 12 H), 0.85 - 0.90 (m, 9 H), 0.82 (br d, J = 6.6 Hz, 6 H).

[0706] Example 14: Synthesis of N,N-bis(2-(2-azidoethoxy)ethyl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (linker 7):

[0707] HOBt (40.2 mg, 149 μmol, 1.0 equivalent), DIPEA (57.8 mg, 447 μmol, 73.9 μL, 3.0 equivalent), and EDCl (57.1 mg, 298 μmol, 2.0 equivalent) were added to a solution of compound R1 (40 mg, 149 μmol, 1.00 equivalent) in DMF (400 μL). The mixture was stirred at 25 °C for 1 hour. LC-MS showed that the starting material had been consumed. The reaction mixture was concentrated under vacuum, and the residue was purified by preparative HPLC (TFA conditions) to give linker 7 (59 mg, 119 μmol, yield 79%, purity 98.6%) as an oil. LCMS (ESI): MS calculated value: 493.5, MS measured value: [M+H]+ = 494.2. HRMS: MS calculated value: 493.5, MS measured value: [M+H]+ = 494.1. HPLC: Rt = 8.97 min, purity: 98.6%. 1 H NMR: 400 MHz, CDCl3. δ:8.86 (s, 2 H), 3.59 - 3.70 (m, 12 H), 3.31 - 3.42 (m, 7 H), 2.56 - 2.64 (m, 4H), 1.96 - 2.05 (m, 2 H).

[0708] Example 15 – ADC Synthesis, Monomer Purity, Potency, and DAR Determination A. Synthesis The ADCs in Table C are prepared according to the general methods outlined below.

[0709] Antibodies were generated by transient expression of recombinant antibodies in CHO cells. In short, expression vectors encoding the heavy and light chains of the relevant antibodies (e.g., trastuzumab (DrugBank accession number DB00072; UNII: P188ANX8CK)) were transiently transfected into CHO cells at a 1:1 ratio. The expressed antibodies were then purified from the culture supernatant using a MabSelect SuRe Protein A column (Cytiva, #11003494).

[0710] ADC synthesis involves two steps: first, the enzymatic addition of a branched linker, followed by conjugation of the payload-containing moiety. These events occur at the glutamine-295 (Q295) site (also containing N297A substitution) within the CH2 domain of the antibody's Fc region. The first step of the process is the conjugation of the linker to the modified antibody's Q295 site, mediated by microbial transglutaminase (MTG or MTGase). After the clickable handle is attached, different combinations of DNA topoisomerase I inhibitors (TOP1I) and DNA damage response inhibitors (DDRi) are introduced via metal-free click chemistry using orthogonal strain-promoted cycloaddition reactions of azide-dibenzocyclooctylene (DBCO), tetramethylthiocycloheptyne sulfoxide imine (TMTHSI), and methyltetraazine-transcyclooctene (TCO). Generally, the TOP1i payload reacts with the azide using the DBCO group, while the DDRi payload reacts with the methyltetraazine using the TCO group in a single enzymatic reaction to obtain the desired ADC.

[0711] Material The following materials were used in the synthesis process: - MTGase enzyme: Activa® TI transglutaminase (Ajinomoto), unit activity 98.56 U / g - PBS pH 7.45 (ThermoFisher) - Activated carbon (Merck) - Sodium deoxycholate (Merck) - Propylene glycol (Merck) - DMSO (Merck) - Protein Concentrator (Millipore) - 0.22 µM syringe filter (Pall) Enzymatic conjugation: Mix the reagents listed in Table A with PBS buffer (prepared according to the manufacturer's instructions) in a sterile glass vial with a volume at least twice the reaction volume. Stir the reaction...

Claims

1. An antigen-binding molecule that binds to HER2, comprising (i) a HER2-binding moiety and (ii) at least one linker-payload moiety, wherein, The antigen-binding molecule comprises (a) a DNA damage response (DDR) inhibitor portion and (b) a DNA topoisomerase I (TOP1) inhibitor portion.

2. The antigen-binding molecule of claim 1, wherein the DDR inhibitor portion is or comprises a DDR inhibitor selected from: ATR inhibitor, PARP inhibitor, ATM inhibitor, WEE1 inhibitor, CHK1 / 2 inhibitor, DNA-PK inhibitor, PLK1 inhibitor, Polθ inhibitor, RAD51 inhibitor, USP inhibitor, PKMYT1 inhibitor, or Aurora-A inhibitor.

3. The antigen-binding molecule according to claim 1 or claim 2, wherein the DDR inhibitor portion is or comprises a DDR inhibitor, and the DDR inhibitor is (a) An ATR inhibitor, optionally wherein the DDR inhibitor portion is or contains bezotitab; (b) A CHK1 / 2 inhibitor, optionally wherein the DDR inhibitor portion is or contains preceptib; (c) WEE1 inhibitor, optionally, wherein the DDR inhibitor portion is or contains adatitab; (d) An ATM inhibitor, optionally wherein the DDR inhibitor portion is or comprises AZD0156; or (e) DNA-PK inhibitor, optionally wherein the DDR inhibitor portion is or contains nidicisib.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the TOP1 inhibitor portion is or comprises a TOP1 inhibitor selected from the following: camptothecin or a derivative thereof, ixenocarbazine, ixenocarbazine mesylate (DX-8951f), N-glycyl-ixenocarbazine, SN-38, DXd(1), DXd(2), irinotecan, etotecan, FL118, topotecan, gemmatocarbazine, belotetane, derushetane, belotetane, rubitecan, letopecan, dioxetane ... Flutecan, calentecan, slatecan, namitecan, icotin, DRF-1042, demotecan, NSC606985, gemiotecan, ZBH-1205, Genz-644282, non-CPT1, indonotecan, indotecan, AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021, VIP126, ZBH-01, and LMP-744.

5. The antigen-binding molecule according to any one of claims 1 to 4, wherein the TOP1 inhibitor portion is or comprises a TOP1 inhibitor selected from eczema, belotecane, SN-38, and DXd.

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises a linker-payload portion, the linker-payload portion comprising (a) a DDR inhibitor portion and (b) a TOP1 inhibitor portion.

7. The antigen-binding molecule of claim 6, wherein the linker-payload portion comprises: (a) An amino group used for conjugation to the antigen-binding portion; (b) Clicking to at least one first payload comprising a portion of the first click group, wherein the first payload comprising a portion comprising a DDR inhibitor portion; (c) Click to at least one second payload containing portion of the second click group, wherein the second payload containing portion contains a TOP1 inhibitor portion; (d) Branched groups: Where R N Selected from H and -(C 1-5 (alkylene)-C(O)OH, where one of the CH2 units can be replaced by -O-. a indicates the connection position between the amino group and the branched group; b indicates the connection position between the at least one first click group and the branched group; c represents the connection position between the at least one second click group and the branched group.

8. The antigen-binding molecule according to any one of claims 1 to 7, wherein the HER2-binding portion comprises: (i) Heavy chain variable (VH) regions containing the following CDRs: HC-CDR1 with the amino acid sequence of SEQ ID NO:15 HC-CDR2 with the amino acid sequence of SEQ ID NO:16 HC-CDR3 having the amino acid sequence of SEQ ID NO:17; and (ii) Light chain variable (VL) regions containing the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:23 LC-CDR2 having the amino acid sequence of SEQ ID NO:24 LC-CDR3 having the amino acid sequence of SEQ ID NO:

25.

9. The antigen-binding molecule according to any one of claims 1 to 8, wherein the antigen-binding portion binding to HER2 comprises: A VH domain comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:14; and The VL domain contains an amino acid sequence that has at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

22.

10. The antigen-binding molecule according to any one of claims 1 to 9, wherein the antigen-binding portion binding to HER2 comprises: A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:12; and A polypeptide comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

13.

11. A composition comprising an antigen-binding molecule according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

12. The antigen-binding molecule according to any one of claims 1 to 10 or the composition according to claim 11, for use in medical treatment or prevention methods, or for use in diagnostic or prognostic methods.

13. The antigen-binding molecule according to any one of claims 1 to 10 or the composition according to claim 11, for the treatment or prevention of cancer.

14. Use of the antigen-binding molecule according to any one of claims 1 to 10 or the composition according to claim 11 in the preparation of a medicament for treating or preventing cancer.

15. A method of treating or preventing cancer, comprising administering to a subject a therapeutically effective amount or a preventatively effective amount of an antigen-binding molecule according to any one of claims 1 to 10, or a composition according to claim 11.

16. The antigen-binding molecule or composition according to claim 13, the use according to claim 14, or the method according to claim 15, wherein the cancer is selected from: cancers comprising cells expressing / overexpressing EGFR family members, cancers comprising cells expressing / overexpressing HER2, cancers comprising cells not expressing EGFR family members, cancers comprising cells not expressing HER2, HER2-low expression cancer, HR-positive cancer, solid tumors, bladder cancer, breast cancer, HER2-positive breast cancer, metastatic HER2-positive breast cancer, HER2-low expression breast cancer, unresectable or metastatic HER2-low expression breast cancer, HR-positive breast cancer, triple-negative breast cancer, cervical cancer, gastric cancer, HE R2-positive gastric cancer, locally advanced or metastatic HER2-positive gastric cancer, cholangiocarcinoma, colorectal cancer, gastroesophageal junction cancer, gallbladder cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, HER2-positive gastroesophageal junction adenocarcinoma, locally advanced or metastatic HER2-positive gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, kidney cancer, lung cancer, non-small cell lung cancer, unresectable or metastatic non-small cell lung cancer containing ERBB2 activating mutations, small cell lung cancer, melanoma, neuroendocrine tumors, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibroma, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer.

17. The antigen-binding molecule or composition used according to claim 13 or claim 16, the use according to claim 14 or claim 16, or the method according to claim 15 or claim 16, 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.

18. Use of the antigen-binding molecule according to any one of claims 1 to 10 or the composition according to claim 11 for reducing or increasing the killing effect on cells expressing HER2.

19. An optionally isolated in vitro complex comprising an antigen-binding molecule that binds to HER2 according to any one of claims 1 to 10.

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