HER2 binding molecules

Abstract

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 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 methods of medical treatment and prevention. [Background technology]

[0002] Cancer remains the leading cause of death worldwide. While chemotherapy has good clinical efficacy, its low specificity results in very serious side effects and a low treatment index. More targeted therapies, such as monoclonal antibody therapy, show good specificity but have lower response rates. Antibody-drug conjugates (ADCs) are a form of therapy that utilizes the target specificity of antibodies to selectively deliver cytotoxic payloads to tumors, and they are increasingly proving effective in clinical practice.

[0003] HER2 (also known as ERBB2 or neu) is a member of the epidermal growth factor receptor (EGFR) family of transmembrane receptors. Overexpression of HER2 is observed in approximately 20% of human breast cancers and is associated with malignant growth and poor clinical outcomes in patients with such tumors (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 and include ad-trastuzumab emtansine (DrugBank accession number: DB05773; marketed as Kadcyla®) and fam-trastuzumab deruxtecan-nxki (DrugBank accession number: DB14962; marketed as Enhertu®). Ad-trastuzumab emtansine contains the cytotoxic agent DM1 (a microtubule inhibitor that is a thiol-containing mytansinoid) conjugated to trastuzumab via an MCC linker at the lysine side chain. Fam-trastuzumab deruxtecan-nxki contains the DNA topoisomerase I inhibitor DS-8201a (DXd) conjugated to trastuzumab via a cathepsin-cleavable tetrapeptide linker. Mosele et al. Nat Med. (2023) 29(8):2110-2120 reports the results of a phase 2 trial (DAISY trial) on the use of fam-trastuzumab deruxtecan-nxki to treat metastatic breast cancer. Disease progression was observed in approximately 71% of patients (125 / 177). There remains an unmet clinical need for effective treatments for HER2-expressing cancers. [Overview of the project]

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

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

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

[0008] In some embodiments, the DDR inhibitor portion is a DDR inhibitor that is a CHK1 / 2 inhibitor, or includes one. In some embodiments, the DDR inhibitor portion is prexacertib, or includes one.

[0009] In some embodiments, the DDR inhibitor portion is a DDR inhibitor that is a WEE1 inhibitor, or includes one. In some embodiments, the DDR inhibitor portion is adavocertib, or includes one.

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

[0011] In some embodiments, the DDR inhibitor moiety is a DDR inhibitor that is a DNA-PK inhibitor, or comprises one. In some embodiments, the DDR inhibitor moiety is nedisertib, or comprises one.

[0012] In some embodiments, the TOP1 inhibitor moiety is or comprises a TOP1 inhibitor selected from the following: camptothecin or its derivatives, exatecan, exatecan mesylate (DX-8951f), N-glycyl-exatecan, SN-38, DXd(1), DXd(2), irinotecan, etirinotecan, FL118, topotecan, gimatecan, berotecan, deruxtecan, berotecan, rubitecan, raltothecan, diflomothecan, and kale. Karenitecan, Shiratecan, Namitecan, Eromotecan, DRF-1042, Delimotecan, NSC606985, Kimitecan, ZBH-1205, Genz-644282, Non-CPT1, Indotecan, Injimitecan, AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021, VIP126, ZBH-01, and LMP-744.

[0013] In some embodiments, the TOP1 inhibitor moiety is or comprises a TOP1 inhibitor selected from the following: camptothecin or its derivatives, exatecan, exatecan mesylate (DX-8951f), N-glycyl-exatecan, SN-38, DXd(1), DXd(2). In some embodiments, the TOP1 inhibitor moiety is or comprises exatecan.

[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 embodiments, the linker-payload portion is (a) an amino group for conjugation to the antigen-binding site, (b) A portion comprising at least one first payload clicked to a first click base, wherein the portion comprising the first payload comprises a DDR inhibitor portion, (c) A portion comprising at least one second payload clicked to a second click base, wherein the portion comprising the second payload comprises a TOP1 inhibitor portion, (d) Branched group: [ka] And in the formula, R N However, H and -(C 1~5 Selected from alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates the location where the amino group is linked to the branched group. b indicates a location where at least one first click group is attached to the branching group. c includes a branching group, where c indicates a location to which at least one second click group is attached.

[0016] In some embodiments, the HER2 binding portion is (i) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 15, HC-CDR2 having the amino acid sequence of SEQ ID NO: 16, HC-CDR3 having the amino acid sequence of SEQ ID NO: 17 A heavy chain variable (VH) region incorporating, and (ii) 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, It includes a light chain variable (VL) region incorporating the following.

[0017] In some embodiments, the antigen-binding moiety that binds to HER2 is A VH region containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 14, and It includes a VL region containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 22.

[0018] In some embodiments, the antigen-binding moiety that binds to HER2 is A polypeptide comprising or consisting thereof, which has at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 12, and The present invention includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 13, or a polypeptide comprising such a sequence.

[0019] This disclosure also provides compositions comprising antigen-binding molecules and pharmaceutically acceptable carriers, diluents, excipients, or auxiliaries.

[0020] This disclosure also provides antigen-binding molecules or compositions according to this disclosure for use in methods of medical treatment or prevention, or in methods of diagnosis or prognosis.

[0021] This disclosure also provides antigen-binding molecules or compositions according to this disclosure for use in the treatment or prevention of cancer.

[0022] This disclosure also provides antigen-binding molecules or compositions according to this disclosure in the manufacture of agents for treating or preventing cancer.

[0023] The Disclosure also provides a method for treating or preventing cancer, which involves administering a therapeutically or prophylactically effective amount of an antigen-binding molecule or composition according to the Disclosure to a target.

[0024] In some embodiments, cancer is selected from: cancers containing cells expressing / overexpressing EGFR family members, cancers containing cells expressing / overexpressing HER2, cancers containing cells not overexpressing EGFR family members, cancers containing cells not overexpressing HER2, HER2-low-expressing cancers, HR-positive cancers, solid tumors, bladder cancer, breast cancer, HER2-positive breast cancer, metastatic HER2-positive breast cancer, HER2-low-expressing breast cancer, unresectable or metastatic HER2-low-expressing breast cancer, HR-positive breast cancer, triple-negative breast cancer, cervical cancer, gastric cancer, HER2-positive gastric cancer, locally advanced or metastatic HER2-positive gastric cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer / gastric junction cancer, gallbladder cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, HER2-positive gastric cancer / gastric junction adenocarcinoma, locally advanced or metastatic HER2-positive gastric cancer / gastric junction adenocarcinoma, gastrointestinal stromal tumors, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, intestinal cancer, kidney cancer, lung cancer, non-small cell lung cancer, unresectable or metastatic non-small cell lung cancer with ERBB2 activating mutations, small cell lung cancer, melanoma, neuroendocrine tumors, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibrous tumor, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer.

[0025] In some embodiments, the cancer is resistant to or recurrent with treatment with DNA damage repair inhibitors, and / or hereby resistant to or recurrent with treatment with DNA topoisomerase I inhibitors.

[0026] The disclosure also provides the use of antigen-binding molecules or compositions according to the disclosure for eliminating or enhancing the killing of cells expressing HER2.

[0027] This disclosure also provides an optionally isolated in vitro complex containing the antigen-binding molecule of this disclosure bound to HER2.

[0028] Next, embodiments and experiments illustrating the principle of the present invention will be described with reference to the attached figures. [Brief explanation of the drawing]

[0029] [Figure 1A] This paper shows the percentage inhibition of TOP1 inhibitors and ATR inhibitors in HCT-116 cells, both individually and in combination. [Figure 1B] This paper shows the percentage inhibition of TOP1 inhibitors and CHK1 inhibitors in HCT-116 cells, both individually and in combination. [Figure 1C] This paper shows the percentage inhibition of TOP1 inhibitors and ATR inhibitors in HEC-1B cells, both individually and in combination. [Figure 1D] This paper shows the percentage inhibition of TOP1 inhibitors and CHK1 inhibitors in HEC-1B cells, both individually and in combination. [Figure 2] This is the percentage of cell death in cancer cell lines after 7 days of in vitro treatment with trastuzumab conjugated with both exatecan and bezocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated with exatecan (isotype control). [Figure 3] This represents the tumor volume over time in mice with a JIMT-1 cell line-derived ductal carcinoma xenograft model treated with trastuzumab conjugated to both PBS (vehicle), exatecan, and bezocertib (T-(Exa+Ber)), or trastuzumab deruxtecan (T-DXd)). [Figure 4] This shows the time course of gram (g) body weight of mice having a ductal carcinoma xenograft model derived from the JIMT-1 cell line and treated with trastuzumab conjugated to both PBS (vehicle), exatecan, and bezocertib (T-(Exa+Ber)), or trastuzumab deruxtecan (T-DXd)). [Figure 5A]This shows the binding of trastuzumab (T(unworn)), trastuzumab (T-(Exa+Ber)) conjugated to both exatecan and bezocertib, trastuzumab deruxtecan (T-DXd), isotype-matched control antibody (isotype(unworn)), or isotype-matched control antibody (isotype(Exa)) conjugated to exatecan to BT-474, NCI-N87, JIMT-1, HEC-1B, or HCT116 living cells, as measured by flow cytometry. [Figure 5B] This shows the mean fluorescence intensity (MFI) of trastuzumab (T(unworn)) conjugated to BT-474, NCI-N87, JIMT-1, HEC-1B, or HCT116 live cells, trastuzumab (T-(Exa+Ber)) conjugated to both exatecan and berzocertib, F-trastuzumab deruxtecan (T-DXd), isotype-matched control antibody (isotype(unworn)), or isotype-matched control antibody (isotype(Exa)) conjugated to exatecan, as measured by flow cytometry. [Figure 6A] This shows the intracellular localization of trastuzumab conjugated to both exatecan and bezocertib (T-(Exa+Ber)) or trastuzumab deruxtecan (T-DXd) in HEC-1B cells after 0, 0.5, or 2 hours of incubation, as measured by immunofluorescence microscopy. [Figure 6B] This shows the intracellular localization of trastuzumab conjugated to both exatecan and bezocertib (T-(Exa+Ber)) or trastuzumab deruxtecan (T-DXd) in NCI-N87 cells after 0, 0.5, or 2-hour incubation, as measured by immunofluorescence microscopy. [Figure 7A]This shows the percentage of HEC1-B cells treated in vitro for 3 days with trastuzumab (T(unworn)), trastuzumab conjugated with both exatecan and bezocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated with exatecan (isotype control). [Figure 7B] This shows the percentage of NCI-N87 cells treated in vitro for 3 days with trastuzumab (T(unworn)), trastuzumab conjugated with both exatecan and bezocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated with exatecan (isotype control). [Figure 7C] This shows the percentage of HCT-116 cells treated in vitro for 3 days with trastuzumab (T(unworn)), trastuzumab (T-(Exa+Ber)) conjugated with both exatecan and belzocertib, trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated with exatecan (isotype control). [Figure 7D] This shows the percentage of BT474 cells treated in vitro for 3 days with trastuzumab (T(unworn)), trastuzumab conjugated with both exatecan and bezocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated with exatecan (isotype control). [Figure 7E] This shows the percentage of JIMT-1 cells treated in vitro for 3 days with trastuzumab (T(unworn)), trastuzumab conjugated with both exatecan and berzocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated with exatecan (isotype control). [Figure 7F] This shows the percentage of HEC1-B cells treated in vitro for 3 days with trastuzumab conjugated to both exatecan and bezocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated to exatecan (isotype control) at an antigen-binding molecule concentration resulting in a payload equivalent concentration of 0.857 nM. [Figure 7G] This shows the percentage of NCI-N87 cells treated in vitro for 3 days with trastuzumab conjugated to both exatecan and belzocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated to exatecan (isotype control) at an antigen-binding molecule concentration resulting in a payload equivalent concentration of 0.857 nM. [Figure 7H] This shows the percentage of HCT-116 cells treated in vitro for 3 days with trastuzumab conjugated to both exatecan and belzocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated to exatecan (isotype control) at an antigen-binding molecule concentration resulting in a payload equivalent concentration of 0.857 nM. [Figure 7I] This shows the percentage of BT474 cells treated in vitro for 3 days with trastuzumab conjugated to both exatecan and bezocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated to exatecan (isotype control) at an antigen-binding molecule concentration resulting in a payload equivalent concentration of 0.857 nM. [Figure 7J]This shows the percentage of JIMT-1 cells treated in vitro for 3 days with trastuzumab conjugated to both exatecan and belzocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated to exatecan (isotype control) at an antigen-binding molecule concentration resulting in a payload equivalent concentration of 0.857 nM. [Figure 8] The Loewe synergy scores of exatecan and belzocertib in HEC1-B cells (upper panel), as well as the percentage inhibition by exatecan and belzocertib alone and in combination (lower panel), are shown. [Figure 9] The Loewe synergy scores of exatecan and belzocertib in HCT-116 cells (upper panel), as well as the % inhibition by exatecan and belzocertib alone and in combination (lower panel), are shown. [Figure 10] These are the levels of pATR, pCHK1, pH2AX, and CHK1 in untreated JIMT-1 cells, or cells treated with 100 nM exatecan, 100 nM belzocertib, or a combination of 100 nM exatecan and 100 nM belzocertib. [Figure 11] These are the levels of pATR, pCHK1, pH2AX, and CHK1 in untreated HCT-116 cells, or cells treated with 75 nM exatecan, 75 nM belzocertib, or a combination of 75 nM exatecan and 75 nM belzocertib. [Figure 12] This refers to the survival rate of Sprague Dolly rats after administration of a vehicle, exatecan (1, 3, 10, or 30 mg / kg), belzocertib (35 mg / kg), or a combination of exatecan and belzocertib (1, 3, 10, or 30 mg / kg of exatecan + 35 mg / kg of belzocertib). [Figure 13]This shows the change in body weight of Sprague Dolly rats after administration of vehicle, exatecan (1, 3, 10, or 30 mg / kg), belzocertib (35 mg / kg), or a combination of exatecan and belzocertib (1, 3, 10, or 30 mg / kg of exatecan + 35 mg / kg of belzocertib). [Figure 14] The results of hematological tests in Sprague Dolly rats after administration of vehicle, exatecan (1, 3, 10, or 30 mg / kg), belzocertib (35 mg / kg), or a combination of exatecan and belzocertib (1, 3, 10, or 30 mg / kg of exatecan + 35 mg / kg of belzocertib) are shown. [Figure 15] The results of clinical chemistry tests in Sprague Dolly rats after administration of vehicle, exatecan (1, 3, 10, or 30 mg / kg), belzocertib (35 mg / kg), or a combination of exatecan and belzocertib (1, 3, 10, or 30 mg / kg of exatecan + 35 mg / kg of belzocertib) are shown. [Figure 16] This shows tumor volume in T-DXd-resistant NCI-N87 CDX models after administration of vehicle, trastuzumab deruxtecan (T-DXd) (3 mg / kg), or trastuzumab (T-(Exa+Ber) (3 mg / kg, 9 mg / kg) conjugated with both exatecan and belzocertib). [Figure 17] This shows % inhibition of HER2-negative MDA-MB-231 cells exposed to trastuzumab (T-(Exa+Ber)) or trastuzumab deruxtecan (T-DXd) conjugated to both exatecan and berzocertib at different concentrations, either alone or co-cultured with HER2-positive NCI-N87 cells. [Figure 18] This involves macropinocytosis of trastuzumab (T-(Exa+Ber)) (100 ng / ml) or trastuzumab deruxtecan (T-DXd) (100 ng / ml) conjugated with both exatecan and bezocertib, leading to uptake by megakaryocytes. [Figure 19]This represents the percentage increase in hydrophobic interaction chromatography retention time (RT) in single-payload ADC and T-(Exa+Ber). [Figure 20] This describes the plasma stability of ADC5 and ADC6 over a 7-day period. [Figure 21] This describes the efficacy of dual-payload ADCs against cells from the NCI-N87 gastric cancer cell line. [Figure 22] The efficacy of dual and single-payload ADCs in cell killing of the NCI-N87 gastric cancer cell line. [Figure 23] The efficacy of dual and single-payload ADCs in cell killing of the NCI-N87 gastric cancer cell line. [Figure 24] This study demonstrates the in vivo antitumor efficacy of dual and single-payload ADCs in xenograft mouse models. [Figure 25] This study describes the in vivo efficacy of dual-payload ADCs containing belzocertib (ATRi) or plexacertib (CHK1i) in mouse models. [Figure 26] This demonstrates the in vivo effectiveness of dual-payload ADCs with different target DAR ratios. [Figure 27] These are the results of weight changes after treatment with dual-payload ADC in primate studies. [Figure 28] These are the results of biochemical and hematological tests after treatment with dual-payload ADC in primate studies. [Figure 29] The efficacy of dual-payload ADCs with TMTHSI or DBCO moieties against NCI-N87 gastric cancer cell lines. [Figure 30] This is the clearance of dual-payload ADCs with TMTHSI or DBCO moieties compared to non-conjugated control antibodies in immunocompetent mice. [Figure 31] This demonstrates the effectiveness of a dual-payload ADC with different conjugations. [Modes for carrying out the invention]

[0030] This disclosure relates to an antigen-binding molecule comprising a linker-payload portion including a HER2-binding portion and at least two different payload portions.

[0031] The antigen-binding molecule of this disclosure is provided, which has unexpected and advantageous properties compared to known anti-HER2 antibody-drug conjugates.

[0032] The inventors noted that 65% of patients whose disease progressed during treatment with fam-trastuzumab deruxtecan-nxki maintained HER2 expression, and hypothesized that the resistance to fam-trastuzumab deruxtecan-nxki therapy observed in the DAISY trial may be due to insensitivity to DXd (Mosele et al. Nat Med. (2023) 29(8):2110-2120).

[0033] The DNA damage response (DDR) is a crucial repair pathway and may be an important mode of resistance to DNA damage payloads such as TOP1 inhibitors. Previously, synergistic anticancer effects have been observed with combination treatment using DDR inhibitors and TOP1 inhibitors (Thomas et al., Cancer Cell. (2021) 39(4):566-79 e7). In embodiments of the antigen-binding molecule of this disclosure that include a linker-payload moiety comprising (i) a TOP1 inhibitor moiety and (ii) a DNA damage response (DDR) inhibitor moiety, the DDR inhibitor is thought to enhance the sensitivity of cancer cells to TOP1 inhibitors. Furthermore, since many TOP1 inhibitors and DDR inhibitors are not substrates of P-glycoprotein, patients treated with antigen-binding molecules containing such payload moieties are less likely to develop P-glycoprotein-mediated resistance to such therapies.

[0034] HER2 HER2 (e.g., ERBB2, also known as neu) is a protein identified by UniProtKB:P04626. The standard isoform of human HER2 has the amino acid sequence P04626-1 (v1, 1987-08-13; SEQ ID NO: 1). Alternative splicing of mRNA encoded by the human ERBB2 gene results in the following six main isoforms: isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2), isoform 3 (SEQ ID NO: 3), isoform 4 (SEQ ID NO: 4), isoform 5 (SEQ ID NO: 5), and isoform 6 (SEQ ID NO: 6). Isoform 2 differs from isoform 1 in that positions 1-610 are absent. Positions 1-686 of SEQ ID NO: 1 are absent in isoform 3. In isoform 4, positions 1-23 of SEQ ID NO: 1 are replaced with a shorter 8-amino acid sequence. Positions 1-686 of SEQ ID NO: 1 are absent in isoform 5. Isoform 6 differs from SEQ ID NO: 1 in that positions 633-648 and 844-1255 are absent, and positions 771-883 are replaced with a different amino acid sequence.

[0035] The standard isoform of human HER2 includes a 22-amino acid N-terminal signal peptide (SEQ ID NO: 8), followed by an extracellular domain (SEQ ID NO: 9), a single-pass transmembrane domain (SEQ ID NO: 10), and a C-terminal cytoplasmic domain (SEQ ID NO: 11). Mature human HER2 isoform 1 is shown in SEQ ID NO: 7.

[0036] The structure and function of HER2 are described, for example, in Iqbal et al., Mol Biol Int. (2014) 2014:852748 (which is incorporated herein by reference in its entirety). Like other EGFR family members, HER2 comprises a cysteine-rich extracellular domain, a hydrophobic transmembrane domain, and an intracellular domain with tyrosine kinase activity. HER2 has no known direct activating ligand and may exist constitutively active or be activated by heterodimerization with EGFR and other EGFR family members such as HER3. HER2 homodimerization or heterodimerization with EGFR / HER3 induces autophosphorylation of tyrosine residues in the cytoplasmic domain, thereby triggering signaling through various distinct intracellular pathways, particularly the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K) pathway. HER2-mediated signaling leads to cell proliferation, survival, differentiation, angiogenesis, and tissue invasion. HER2-HER3 heterodimers are particularly potent stimulants of downstream pathways, especially the PI3K / Akt pathway.

[0037] In this specification, references to "HER2" generally refer to the standard isoform of human HER2 (i.e., isoform 1), but also intend to include its isoforms, fragments, variants (including mutants), and homologs (i.e., those derived from other species, e.g., non-human mammalian species (e.g., non-human primates, e.g., rhesus monkeys, cynomolgus macaques; e.g., rodents, e.g., rats or mice)).

[0038] As used herein, a protein “fragment,” “variant,” or “homolog” may optionally be characterized by having at least 60% amino acid sequence identity to the amino acid sequence of a reference protein (e.g., a standard isoform of a human protein), for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity. In some embodiments, the fragment / variant / isoform / homolog may be characterized by the ability to perform the function exhibited by the reference protein.

[0039] A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein that has an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of the reference protein, but retains a large degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein expressed by the same species as the reference protein (e.g., human HER2 isoforms 1-6 are all isoforms of each other). A "homolog" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. 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 of each other. Homologs include orthologues.

[0040] The “fragment” can be of any length (length by the number of amino acids), but can be at least 20% of the length of the reference protein (i.e., the protein from which the fragment originates), and can have one maximum length of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein. A HER2 fragment may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1250 amino acids, and a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1250 amino acids.

[0041] Isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs possessing the same functional properties / activities as reference HER2 (e.g., human HER2 isoform 1), as can be measured by a suitable assay for functional properties / activity. For example, an isoform, fragment, variant, or homolog of HER2 may exhibit association with HER3 or EGFR.

[0042] In some embodiments, HER2 is mammalian HER2 (e.g., primate (rhesus macaque, crab-eating macaque, non-human primate, or human) and / or rodent (e.g., rat or mouse) HER2). An isoform, fragment, variant, or homolog of HER2 may optionally be characterized by having at least 60% of the amino acid sequence identity of an immature or mature HER2 isoform of a given species, e.g., human, of which 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of the amino acid sequence identity of an immature or mature HER2 isoform of a given species, e.g., human.

[0043] In some embodiments, HER2 includes or comprises an amino acid sequence having at least 60% amino acid sequence identity to one of SEQ ID NOs: 1 to 7, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0044] In some embodiments, the HER2 fragment comprises or consists of an amino acid sequence having at least 60% amino acid sequence identity to SEQ ID NO: 7 or 9, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity.

[0045] HER2 binding antigen binding moiety An "antigen-binding molecule" refers to a molecule that binds to a given target antigen. An antigen-binding molecule includes an antigen-binding portion, which binds to its target antigen. The antigen-binding molecules of this disclosure include an antigen-binding portion that binds to HER2 (i.e., a HER2-binding portion).

[0046] The antigen-binding moiety may include or be derived from antibodies (i.e., immunoglobulins (Ig)) and antigen-binding fragments of antibodies. As used herein, “antibody” includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as antibody-derived antigen-binding molecules, such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, and single-domain antibodies (e.g., VhH, etc.). Examples of antibody antigen-binding fragments include Fv, Fab, F(ab')2, and F(ab') fragments.

[0047] Antigen-binding sites include target antigen-binding aptamers, such as nucleic acid aptamers (for example, as outlined in Zhou and Rossi, Nat Rev Drug Discov. (2017) 16(3):181-202). In some embodiments, the antigen-binding moiety includes or comprises antigen-binding peptides / polypeptides, such as peptide aptamers, thioredoxin, monobodies, antikalin, Kunitz domains, avimers, Nottin, finomers, atrimers, DARPin, afibodies, nanobodies (i.e., single-domain antibodies (sdAb), affilins, armadillo repeat proteins (ArmRP), OBody, or fibronectin, as outlined, for example, in Reverdatto et al., Curr Top 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).

[0048] Commonly used techniques for producing fully human antibodies include (i) phage display, in which human antibody genes are expressed in phage display libraries, and (ii) antibody production in transgenic mice genetically engineered to possess human antibody genes (described in Park and Smolen, Advances in Protein Chemistry (2001) 56:369-421). In summary, in human antibody gene-phage display techniques, genes encoding VH and VL chains are generated from "naive" human lymphocytes by PCR amplification and cloning, assembled into a library, and expressed from it as either disulfide-bonded Fab fragments or single-chain Fv (scFv) fragments. The genes encoding Fab or scFv are fused to the surface coat protein of filamentous bacteriophages, and then Fab or scFv capable of binding to targets of interest can be identified by screening the library with antigens. Molecular evolution or affinity maturation methods may be used to enhance the affinity of Fab / scFv fragments. In transgenic mouse technology, to obtain fully human monoclonal antibodies, mice in which the endogenous mouse Ig gene locus has been replaced with its human homolog by homologous recombination are immunized with an antigen, and the monoclonal antibodies are prepared using conventional hybridoma technology.

[0049] In some embodiments, the antigen-binding portion according to this disclosure includes or consists of the antigen-binding region of an antibody (e.g., an antigen-binding fragment of an antibody). The antigen-binding portion may be derived from an antibody. The antibody-derived antigen-binding portion may include or consist of the 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 consist of the Fv (e.g., provided as scFv) or Fab region of an antibody that binds to a given target antigen, or a full-length antibody.

[0050] The antigen-binding regions of this disclosure may be designed and prepared using sequences of monoclonal antibodies (mAbs) capable of binding to a given target antigen (e.g., HER2). Antigen-binding regions of antibodies, such as variable fragments (scFv), Fab, and F(ab')2 fragments, may also be used / provided. The “antigen-binding region” is any fragment of a given antibody that binds to a target on which the antibody exhibits specificity.

[0051] In some embodiments, the antigen-binding portion includes a variable heavy chain region (VH) and a variable light chain region (VL) of the antibody that can specifically bind to a target antigen. In some embodiments, the antigen-binding portion is or includes the Fv of the antibody (e.g., provided as scFv). In some embodiments, the antigen-binding portion is or includes the Fab region of the antibody. In some embodiments, the antigen-binding portion is or includes the full-length antibody (i.e., including variable and constant regions).

[0052] The antigen-binding moiety may be or may comprise an antigen-binding polypeptide or an antigen-binding polypeptide complex. The antigen-binding moiety may comprise two or more polypeptides that together form the antigen-binding moiety. Polypeptides may associate covalently or non-covalently. In some embodiments, polypeptides constitute part of a larger polypeptide containing them (for example, in the case of scFv containing VH and VL, or in the case of scFab containing VH-CH1 and VL-CL).

[0053] In some embodiments, the antigen-binding moiety according to this disclosure comprises or consists of a polypeptide complex formed by protein-protein interactions between the constituent peptides / polypeptides of the antigen-binding moiety. The antigen-binding moiety may refer to an IgG-like antigen-binding moiety comprising a non-covalent or covalent complex of two or more polypeptides (e.g., 2, 3, 4, 6, or 8 polypeptides), such as two heavy-chain polypeptides and two light-chain polypeptides.

[0054] Antibodies typically contain six complementarity-determining regions (CDRs): three in the heavy chain variable (VH) region (HC-CDR1, HC-CDR2, and HC-CDR3), and three in the light chain variable (VL) region (LC-CDR1, LC-CDR2, and LC-CDR3). The six CDRs together define the paratope of the antibody, which is the portion of the antibody that binds to the target antigen.

[0055] The VH region and VL region contain framework regions (FRs) on both sides of each CDR that provide a scaffold for the CDR. The VH region includes the following structure from the N-terminus to the C-terminus: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and the VL region includes the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0056] Several different rules for defining antibody CDR and FR, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 thVBASE2 exists as described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and Retter et al., Nucleic Acids Res. (2005) 33 (suppl 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). This uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In preferred embodiments, the CDR and FR of the antigen-binding molecule referred to herein are defined according to the IMGT information system.

[0057] In some embodiments, the antigen-binding molecule according to this disclosure includes or comprises an Fv region that binds to HER2. In some embodiments, the VH and VL regions of Fv are provided as a single polypeptide linked by a linker sequence, i.e., a single-chain Fv (scFv).

[0058] The VL and light chain constant (CL) regions of the antigen-binding region of an antibody, as well as the VH region and heavy chain constant 1 (CH1) region, together constitute a Fab region. In some embodiments, the antigen-binding molecule includes a Fab region comprising VH, CH1, VL, and CL (e.g., Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising VL and CH1 (e.g., a VL-CH1 fusion polypeptide), i.e., 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 by a linker region, i.e., as a single-chain Fab (scFab) or single-chain CrossFab (scCrossFab).

[0059] In some embodiments, the antigen-binding molecules described herein include or consist of a full-length antibody that binds to HER2. As used herein, “full-length antibody” refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different types of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41–S52 (which is incorporated herein by reference in its entirety).

[0060] Type G immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa containing two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains a heavy chain constant region consisting of VH followed by three constant domains (CH1, CH2, and CH3), and similarly the light chain contains VL followed by CL. Depending 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 chain can be kappa (κ) or lambda (λ).

[0061] In some embodiments, the antigen-binding molecule includes or comprises IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that bind to HER2.

[0062] In some embodiments, the antigen-binding moiety of the Disclosure comprises one or more regions of an immunoglobulin heavy chain constant region sequence (e.g., CH1, hinge, CH2, CH3, etc.). In some embodiments, the immunoglobulin heavy chain constant region sequence is or is derived from the heavy chain constant region sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, for example, 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 region sequence is or is derived from the heavy chain constant region sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0063] In some embodiments, the antigen-binding moiety comprises one or more polypeptides having an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 31 or 36, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity. In some embodiments, the antigen-binding moiety comprises one or more polypeptides having an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 32, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity. In some embodiments, the antigen-binding moiety comprises one or more polypeptides containing an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 33 or 48, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity. In some embodiments, the antigen-binding moiety comprises one or more polypeptides containing an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 34 or 37, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity.In some embodiments, the antigen-binding moiety comprises one or more polypeptides containing an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 38, 39, 49, or 50, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity. In some embodiments, the antigen-binding moiety comprises one or more polypeptides having an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NOs. 30, 35, 46, or 47, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity.

[0064] In some embodiments, the antigen-binding moiety of the present disclosure comprises one or more regions of an immunoglobulin light chain constant region sequence. In some embodiments, the immunoglobulin light chain constant region sequence is the human immunoglobulin κ constant region (IGKC; Cκ). In some embodiments, the immunoglobulin light chain constant region sequence is the human immunoglobulin λ constant region (IGLC; Cλ), for example, IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0065] In some embodiments, the antigen-binding moiety comprises one or more polypeptides containing an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NOs. 40, 41, 42, 43, 44, or 45, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity.

[0066] In some embodiments described herein, one or more amino acids in the amino acid sequences referred herein (e.g., amino acid sequences of antigen-binding molecules, e.g., amino acid sequences of CDR or VH / VL regions) are substituted with another amino acid sequence. Substitutions include substitutions of amino acid residues at non-identical "substituted" amino acid residues. The substituted amino acid residues in the substitutions according to this disclosure may be naturally occurring amino acid residues (i.e., those encoded by the genetic code) 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 (Tr), tyrosine (Tyr), and valine (Val), which are not identical to the amino acid residue at the corresponding position in the corresponding unsubstituted amino acid sequence. In some embodiments, the substituted amino acid may be a non-natural amino acid residue, i.e., an amino acid residue other than those listed in the Preamble. Examples of non-natural amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs, such as those described in Ellman, et al., Meth. Enzym. 202 (1991) 301-336.

[0067] In some embodiments, substitutions may be biochemically conserved. In some embodiments, if the amino acid to be substituted is shown in one of rows 1-5 of the table below, the substituted amino acid is a different, non-identical amino acid shown in the same row. [Table 1]

[0068] As an example, in some embodiments where the substitution is of a Met residue, the substituted amino acid may be selected from Ala, Val, Leu, Ile, Trp, Tyr, Phe, and norleucine.

[0069] In some embodiments, the substituted amino acid in a substitution may have the same side-chain polarity as the amino acid residue it replaces. In some embodiments, the substituted amino acid in a substitution may have the same side-chain charge (pH 7.4) as the amino acid residue it replaces. [Table 2]

[0070] In other words, in some embodiments, nonpolar amino acids are substituted with other nonpolar amino acids that are not identical. In some embodiments, polar amino acids are substituted with other polar amino acids that are not identical. In some embodiments, acidic polar amino acids are substituted with other acidic polar amino acids that are not identical. In some embodiments, basic polar amino acids are substituted with other basic polar amino acids that are not identical. In some embodiments, neutral amino acids are substituted with other neutral amino acids that are not identical. In some embodiments, positive amino acids are substituted with other positive amino acids that are not identical. In some embodiments, negative amino acids are substituted with other negative amino acids that are not identical.

[0071] In some embodiments, the substitution(s) may be functionally conserved. That is, in some embodiments, the substitution may not affect (or substantially affect) one or more functional properties (e.g., binding to a target) of the antigen-binding molecule containing the substitution compared to the corresponding unsubstituted molecule.

[0072] The antigen-binding molecule of this disclosure includes an antigen-binding moiety that binds to HER2.

[0073] In some embodiments, the antigen-binding moiety includes a CDR of the antigen-binding moiety that can bind to HER2. In some embodiments, the antigen-binding moiety includes a FR of the antigen-binding moiety that can bind to HER2. In some embodiments, the antigen-binding moiety includes a CDR and FR of an antibody that can bind to HER2. That is, in some embodiments, the antigen-binding moiety includes a VH region and a VL region of an antibody that can bind to HER2.

[0074] In some embodiments, the antigen-binding moiety capable of binding to HER2 according to this disclosure may be or be derived from a HER2-binding antibody selected from the following: trastuzumab (DrugBank accession number: DB00072; formed from a polypeptide having the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13), pertuzumab (DrugBank accession number: DB06366), margetuximab (DrugBank accession number: DB14967), thymigutuzumab (e.g., as described in Fiedler et al., ESMO Open. (2018) 3(4):e000381), CT-P6 (e.g., as described in Jeong et al., Expert Opin Biol Ther (2019) 19(10):1085-1095), PF-05280014 (e.g., as described in Paik, BioDrugs (2018) (as described in 32(5):515-518), SB3 (e.g., Lamb, BioDrugs (2018) 32(3):293-296), ABP-980 (e.g., Dhillon, BioDrugs (2018) 32(5):511-514), MYL-1410 (e.g., Rugo et al., JAMA (2017) 317:37-47), BCD-022 (e.g., Alexeev et al., BMC Cancer (2020) 20:783), HD201 (e.g., Pivot et al., Clin Ther. (2018) 40(3):396-405.e4), and HLX22 (e.g., Yang et al. BioDrugs. 2022;36(3):393-409). In some embodiments, the antigen-binding moiety is or is derived from trastuzumab.

[0075] In some embodiments, the antigen-binding moiety can bind to the same DLL3 region as the HER2 region bound by the antigen-binding molecule containing the VH and VL sequences of the HER2-binding antibody described above, or to a region of HER2 that overlaps with it. In some embodiments, the antigen-binding moiety can bind to the same HER2 region as the HER2 region bound by the antigen-binding molecule containing the VH and VL sequences of trastuzumab (i.e., an antigen-binding molecule containing VH having the amino acid sequence of SEQ ID NO: 14 and VL having the amino acid sequence of SEQ ID NO: 22), or to a region of HER2 that overlaps with it.

[0076] In some embodiments, the antigen-binding moiety can be bound to a polypeptide containing or consisting of one of the amino acid sequences of SEQ ID NOs: 1, 7, or 9.

[0077] The ability of the antigen-binding moiety to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (e.g., Western blotting), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), or biolayer interference (see, e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507).

[0078] In embodiments where the antigen-binding moiety can bind to a peptide / polypeptide containing a reference amino acid sequence, the peptide / polypeptide may contain one or more additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments, the peptide / polypeptide contains, 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 (or more) added to one or both ends of the reference sequence (i.e., the N-terminus and C-terminus) correspond to the terminal positions of the reference sequence in the context of the HER2 amino acid sequence.

[0079] In some embodiments, the antigen-binding moiety can be bound to a peptide / polypeptide conjugated by an antibody containing the VH and VL sequences of trastuzumab (i.e., an antigen-binding molecule containing VH having the amino acid sequence of SEQ ID NO: 14 and VL having the amino acid sequence of SEQ ID NO: 22).

[0080] In some embodiments, the antigen-binding portion can be bound to a peptide / polypeptide conjugated by an antibody containing the VH and VL sequences of the HER2-binding antibody described above.

[0081] In some embodiments, the antigen-binding moiety includes the heavy chain CDR and light chain CDR of the HER2-binding antibody described above. In some embodiments, the antigen-binding moiety includes the VH and VL of the HER2-binding antibody described above. In some embodiments, the antigen-binding moiety includes the heavy chain polypeptide (i.e., including the VH, CH1, CH2, and CH3 region sequences) and the light chain polypeptide (i.e., including the VL and CL region sequences) of the HER2-binding antibody described above.

[0082] In some embodiments, the antigen-binding moiety includes the heavy chain CDR and light chain CDR of trastuzumab. In some embodiments, the antigen-binding moiety includes the VH and VL of trastuzumab. In some embodiments, the antigen-binding moiety includes the heavy chain polypeptide (i.e., the VH, CH1, CH2, and CH3 region sequences) and the light chain polypeptide (i.e., the VL and CL region sequences) of trastuzumab.

[0083] In some embodiments, the antigen-binding portion is The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 15, HC-CDR2 having the amino acid sequence of SEQ ID NO: 16, HC-CDR3 having the amino acid sequence of SEQ ID NO: 17, Alternatively, variants in which 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 other amino acids. The VH region incorporating, and The following CDR: 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, Alternatively, variants in which 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 substituted with other amino acids. Includes a VL region incorporating it.

[0084] In some embodiments, the antigen-binding portion is A VH region containing an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 14, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity; and The VL region includes an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 22, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity.

[0085] In some embodiments, the antigen-binding portion is In some embodiments, the antigen-binding moiety comprises (i) one or more (e.g., two) polypeptides having an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 12, more preferably 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity; and (ii) comprising an amino acid sequence having at least 70% sequence identity of SEQ ID NO: 13, more preferably 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity, or comprising one or more (e.g., two) polypeptides consisting of such an amino acid sequence.

[0086] In some embodiments, the antigen-binding molecule of the present disclosure (e.g., its antigen-binding portion) includes an Fc region. As used herein, “Fc region” refers to a polypeptide complex formed by the interaction of two polypeptides, each polypeptide containing a CH2-CH3 region of the constant region sequence of an immunoglobulin (Ig) heavy chain.

[0087] In this specification, "CH2 domain" refers to the amino acid sequence corresponding to the CH2 domain of immunoglobulin (Ig). The CH2 domain is the Ig region formed at positions 231-340 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. "CH3 domain" refers to the amino acid sequence corresponding to the CH3 domain of immunoglobulin (Ig). The CH3 domain is the Ig region formed at positions 341-447 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85. "CH2-CH3 region" refers to the amino acid sequences corresponding to the CH2 and CH3 domains of immunoglobulin (Ig). The CH2-CH3 region is the Ig region formed at positions 231-447 of the immunoglobulin constant domain, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85.

[0088] In some embodiments, the CH2 domain, CH3 domain, and / or CH2-CH3 region according to this disclosure corresponds 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 corresponds to the CH2 domain / CH3 domain / 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 a human IgG1 allotype (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.

[0089] The Fc region induces functional effects through interactions with Fc receptors and other molecules of the immune system. Fc-mediated effector functions are outlined, for example, in Jefferis et al., Immunol Rev 1998 163:59-76 (which is incorporated herein by reference in its entirety), and are induced by Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) via interactions between the Fc region and Fc receptors expressed by such immune cells, recruitment of complement pathway components via binding of the Fc region to complement protein C1q, and resulting activation of the complement cascade. Functions mediated by Fc include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), membrane invasion complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0090] In some embodiments, the antigen-binding molecule according to the present disclosure comprises one or more (e.g., two) polypeptides having an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 38 or 39, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity. In some embodiments, the antigen-binding molecule includes an Fc region comprising one or more (e.g., two) polypeptides containing an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 38 or 39, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity.

[0091] Modifications of antibody Fc regions 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 (which is incorporated herein by reference in its entirety). 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 molecules of this disclosure include an Fc region that includes modifications to enhance or reduce Fc-mediated function compared to an antigen-binding molecule that includes a corresponding unmodified Fc region. Where an Fc region / CH2 / CH3 is described as including a modification "corresponding" to a reference substitution(s), equivalent substitution(s) in homologous Fc / CH2 / CH3 are intended.

[0092] In some embodiments, the antigen-binding molecule of the Disclosure includes an Fc region with modifications. In some embodiments, the antigen-binding molecule of the Disclosure includes an Fc region with modifications to one or more of the CH2 and / or CH3 regions.

[0093] In some embodiments, the Fc region includes modifications to reduce / prevent Fc-mediated functions (e.g., ADCC, ADCP, CDC). In some embodiments, the Fc region includes modifications to reduce / prevent ADCC. In some embodiments, the Fc region includes modifications to reduce / prevent CDC. In some embodiments, the Fc region includes modifications to reduce / prevent binding to the Fc receptor. In some embodiments, the Fc region includes modifications to reduce / prevent binding to the Fcγ receptor. In some embodiments, the Fc region includes modifications to reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0094] In some embodiments, the Fc region includes modifications with the amino acid residue corresponding to N297. In some embodiments, the Fc region includes modifications corresponding to N297A, N297Q, or N297G, as described in Leabman et al., Mabs. (2013) 5:896-903. Substitution of "N297" with "A", "G", or "Q" is known to remove glycosylation, thereby reducing the binding of Fc to the C1q and Fcγ receptors, and thereby reducing CDC and ADCC. In some embodiments, the Fc region includes modifications corresponding to N297A.

[0095] In some embodiments, the antigen-binding molecule according to the present disclosure comprises one or more (e.g., two) polypeptides containing an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 49 or 50, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity. In some embodiments, the antigen-binding molecule includes an Fc region comprising one or more (e.g., two) polypeptides containing an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 49 or 50, for example, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity.

[0096] 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 described below.

[0097] Linker-payload section The aspects and embodiments of this disclosure relate to antigen-binding molecules including a linker-payload portion. As used herein, a linker-payload portion refers to a portion including one or more payload portions and a linker portion for linking the payload portion(s) to the antigen-binding region of an antigen-binding molecule.

[0098] The payload portion of this disclosure includes or consists of cytotoxic agents. The payload portion is described, for example, in Parslow et al., Biomedicines. 2016 Sep;4(3):14, Goundry and Parker, Org. Process Res. Dev. (2022) 26,8,2121-2123, Fu et al., Signal Transduction and Targeted Therapy (2022) 7:93, Wang et al., Acta Pharmaceutica Sinica B (2023) 13(10):4025-4059, and Conilh et al., J. Hematol. & Oncol. (2023) 16:3 (all of which are incorporated herein by reference in their entirety).

[0099] In particular, this disclosure relates to an antigen-binding molecule comprising at least one linker-payload portion, wherein the payload portion comprises (a) a DNA damage response (DDR) inhibitor and (b) a DNA topoisomerase I (TOP1) inhibitor.

[0100] For the sake of brevity, the "DDR inhibitor payload portion" may be simply referred to as the "DDR inhibitor portion," and similarly, the "TOP1 inhibitor payload portion" may be simply referred to as the "TOP1 inhibitor portion."

[0101] The DNA damage response (DDR) is a complex network of mechanisms for detecting and repairing DNA damage to maintain genomic stability. The DDR is outlined, for example, in Groelly et al., Nature Reviews Cancer (2023) 23:78-94 and Molinaro et al., Cancers (Basel). (2021) 13(15):3819 (both of which are incorporated herein by reference in their entirety).

[0102] The detection of DNA damage and the initiation of repair pathways are mediated by proteins such as ATM (ataxia telangiectasia mutation) and ATR (ataxia telangiectasia and Rad3-related gene). ATM is a protein kinase activated by double-strand breaks in DNA, which initiates downstream signaling. ATR is activated by DNA damage and replication stress, and in particular responds to single-strand breaks and stalled DNA replication forks. CHK1 and CHK2 (checkpoint kinases 1 and 2) are downstream effectors of ATM and ATR, phosphorylating various target proteins to halt cell cycle progression and promote DNA repair. PARP (poly-ADP-ribose polymerase) is involved in the repair of single-strand DNA breaks, assisting in the recruitment of repair factors and the formation of repair complexes at the DNA damage site. DNA-PK (DNA-dependent protein kinase) assists in assembling cleaved DNA ends for non-homologous end joining (NHEJ) to repair double-strand breaks. DDR is promoted by cell cycle regulation by WEE1 and PLK1 (polo-like kinase 1). WEE1 is a kinase that phosphorylates and inhibits CDK (cyclin-dependent kinase), thereby delaying cell cycle progression and giving more time for DNA damage repair before cell division. PLK1 regulates cell cycle checkpoints and promotes repair processes by phosphorylating Polθ. RAD51 is an ATPase involved in DNA repair. Ubiquitin-specific proteases (USPs) regulate DDR by influencing the ubiquitination of proteins involved in DDR. Membrane-bound tyrosine / threonine protein kinase 1 (PKMYT1) regulates the cell cycle and is involved in DDR-related signaling. Aurora A may contribute to the G2 DNA damage checkpoint through PLK1 and CDC25B activation and is important in the mitotic DNA damage response.

[0103] Most cancer cells exhibit a higher dependence on DDR compared to non-cancerous cells. DDR inhibitors and their use in cancer treatment are described, for example, in Cheng et al., Eur J Med Chem. (2022) 230:114109, Wang et al., Front Immunol. (2022) 13:854730, and Choi and Lee, Int J Mol Sci. (2022) 23(3):1701 (all of which are incorporated herein by reference in their entirety).

[0104] In some embodiments, the DDR inhibitor portion of this disclosure is or includes a DDR inhibitor selected from the following: (a) PARP inhibitors (e.g., olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, simmiparib, senaparib, SC-10914, 2X-121, AMXI-5001, JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-1002, RBN-2397, fluzoparib, NMS-03305293, AZD9574); (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 (belzocertib), M4344 (VX-803), AZD6738 (ceraracertib), BAY1895344 (elimsertib), RP3500 (camoncertib), ATRN119, ART380, IMP9064, HRS2398, M1774, IMP9064, SC0245, LF0397, NU6027); (d) WEE1 inhibitors (e.g., adavocertib, Debio 0123, PD0166285, PD0407824, AZD1775, ZN-c3, IMP7068, SY4835, SCO191, IMP7068); (e) CHK1 / 2 inhibitors (e.g., CBP-501, prexasertib, MK-8776, GDC-0575, SRA-737, PF-00477736, AZD7762, LY2603618 (labucertib), LY2880070, XL884, BEBT260, MU380, NU7441, KU-5778); (f) DNA-PK inhibitors (e.g., CC-115, LY-3023414, AsiDNA, M3814 (Nedicertib, Pepocertive), 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 (voracertib), PCM-075 (onvancertib), CYC140 (progocertib)); (h) Polθ inhibitors (e.g., ART4215, ART6043, novobiosin, RP-6685, RP-3467); (i) RAD51 inhibitors (e.g., CYT0851); (j) Inhibitors of ubiquitin-specific protease (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., aricertib, WJ05129 (JS112), JAB-2485).

[0105] In some embodiments, the DDR inhibitor portion is or includes ceraracertib.

[0106] In some embodiments, the DDR inhibitor portion is belzocertib: [ka] It is either or includes it.

[0107] In some embodiments, the DDR inhibitor portion is plexacertib: [ka] It is either or includes it.

[0108] In some embodiments, the DDR inhibitor portion of adavocertib may be linked as follows, or similarly via other positions: [ka] It is either or includes it.

[0109] In some embodiments, the DDR inhibitor portion may be connected as follows, or similarly, via other locations AZD0156: [ka] It is either or includes it.

[0110] In some embodiments, the DDR inhibitor portion is nedisertib: [ka] It is either or includes it.

[0111] During DNA replication and transcription, a large torsional strain exists on the DNA helix, which is mitigated by the action of DNA topoisomerases I and II (TOP1 and TOP2), which break the DNA strand and allow it to unwind before the breaks can be rejoined (see, e.g., Delgado et al., Biochem J. (2018) 475(2):373-398). DNA topoisomerase inhibitors inhibit the rejoining step, thereby leading to DNA fragmentation and cell death. The use of DNA topoisomerase I inhibitors and their application in the treatment of cancer is described, for example, 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).

[0112] In some embodiments, the TOP1 inhibitor portion of the present disclosure is or comprises a TOP1 inhibitor selected from the following: camptothecin or its derivatives, exatecan, exatecan mesylate (DX-8951f), N-glycyl-exatecan, SN-38, DXd(1), DXd(2), irinotecan, etirinotecan, FL118, topotecan, gimatecan, berotecan, deruxtecan, berotecan, rubitecan, raltotecan, diflomotecan, calenitecan (K arenitecan), shiratecan, namitecan, eromotecan, DRF-1042, delimotecan, NSC606985, kimitecan, ZBH-1205, Genz-644282, non-CPT1, indotecan (LMP-400), injimitecan (LMP-776), AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021, VIP126, ZBH-01, and LMP-744.

[0113] In some embodiments, the TOP1 inhibitory portion is exatecan: [ka] It is either or includes it.

[0114] In some embodiments, the TOP1 inhibitor portion is berotecan: [ka] It is or includes the above. In some embodiments, the TOP1 inhibitor portion is SN38: [ka] It is or includes the TOP1 inhibitor portion, DXd: [ka] It is either or includes it.

[0115] The linker portion of this disclosure may be any portion suitable for linking the payload portion to the antigen-binding region of the antigen-binding molecule of this disclosure. Accordingly, they generally include a group that enables linkage to the payload portion, a group that links the conjugation to the antigen-binding region of the antigen-binding molecule, and a linker core.

[0116] For example, the linker portion is described 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.

[0117] The linker portion provided in this disclosure may be a detachable linker portion or a non-detachable portion.

[0118] Scleavable linkers typically release the payload in a targeted manner by utilizing the differences between the systemic circulation environment and the cancer cell environment / tumor microenvironment. Examples of cleavable linkers include chemical cleavage linkers (e.g., acid-cleavable linkers, GSH-cleavable linkers, Fe(II)-cleavable linkers) and enzymatic cleavage linkers (e.g., cathepsin-cleavable linkers, glycosidase-cleavable linkers, phosphatase-cleavable linkers, sulfatase-cleavable linkers).

[0119] In some embodiments, the linker moiety according to the present disclosure is a chemically cleavable linker. In some embodiments, the linker moiety according to the present disclosure is an enzymatically cleavable linker. In some embodiments, the linker moiety is an acid-cleavable linker, comprising, for example, a hydrazone group (e.g., a 6-maleimidocaproyl hydrazone linker or a (4-(4-acetylphenoxy)butanoic acid) hydrazone linker), a carbonate group, or a silyl ether group. In some embodiments, the linker moiety is a GSH-cleavable linker, comprising, for example, a disulfide group. In some embodiments, the linker moiety is an Fe(II)-cleavable linker, comprising, for example, a 1,2,4-trioxolane group. In some embodiments, the linker moiety is a cathepsin-cleavable linker, comprising, for example, 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 moiety is GGFG (glycine-glycine-phenylalanine-glycine). In some embodiments, the linker moiety is a glucuronidase-cleavable linker, comprising, for example, a β-glucuronide group. In some embodiments, the linker moiety is a glycosidase-cleavable linker, comprising, for example, a β-galactoside group. In some embodiments, the linker moiety is a phosphatase-cleavable linker, comprising, for example, a pyrophosphate group. In some embodiments, the linker moiety is a sulfatase-cleavable linker, comprising, for example, an aryl sulfate group. In some embodiments, the linker moiety is a photoresponsive linker, comprising, for example, a heptamethine cyanine fluorophore group, an o-nitrobenzyl group, or a PC4AP group. In some embodiments, the linker moiety is a bioorthogonal cleavable linker, comprising, for example, a dsProc group.

[0120] Non-cleavable linkers remain inert in the general chemical and enzymatic environments within the body, and the payload moiety is released after processing of the ADC by lysosomal proteases in the cell. Non-cleavable linkers include linkers containing thioether or maleimidocaproyl groups.

[0121] In some embodiments, the linker moiety is a thioether linker. In some embodiments, the linker moiety is a maleimidocaproyl linker, including, for example, a 2-(maleimidomethyl)-1,3-dioxane (MD) group or a Mal-PAB group. In some embodiments, the linker moiety comprises a polyethylene glycol (PEG) group and an alkyne, triazole, or piperazine group.

[0122] In some embodiments, the linker-payload moiety according to the present disclosure has an amino (-NH2) group for linking to the antigen-binding moiety, for example, via enzymatic conjugation. In some of these embodiments, enzymatic conjugation using a microbial transglutaminase may be used to conjugate the linker-payload moiety to the antigen-binding moiety.

[0123] In some embodiments, the linker portion further includes a spacer portion. The spacer portion is sometimes required due to the bulky nature of the payload portion. Commonly used spacer portions include para-aminobenzyl carbamate (PABC), hemiaminal groups, PEG groups, polar acyl sulfamide groups, polar carbamoyl sulfamide groups, and HydraSpace (e.g., described in Verkade et al., Antibodies (Basel) (2018) 7(1):12 and WO2016 / 053107A1 (both of which are incorporated herein by reference in their entirety)). PABC is commonly used as a spacer portion in cathepsin-cleavable dipeptide linkers, β-glucuronidase-cleavable linkers, β-galactosidase-cleavable linkers, and phosphatase-cleavable linkers. In some embodiments, para-aminobenzyl (PAB) is used as a spacer group.

[0124] In some embodiments, the antigen-binding molecule according to the Disclosure comprises a linker-payload portion, which further comprises both a DDR inhibitor portion and a TOP1 inhibitor portion. That is, in some embodiments, the antigen-binding molecule comprises a linker-payload portion comprising (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 by 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] Techniques for combining multiple payload portions into a single linker portion are described, for example, in 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 by 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 by the same linker portion.

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

[0128] In some embodiments, the DDR inhibitor and TOP1 inhibitor payload portions are linked to the antigen-binding portion of the antigen-binding molecule by an enhanced hydrophilic linker portion.

[0129] In some embodiments, the DDR inhibitor and TOP1 inhibitor payload portions are linked to the antigen-binding portion of the antigen-binding molecule by a branched hydrophilic linker portion.

[0130] In some embodiments, the DDR inhibitor moiety and the TOP1 inhibitor moiety are linked to the linker moiety of the linker-payload moiety via orthogonal functional groups. In some embodiments, the linker-payload moiety includes a trifunctional linker moiety that results in the linkage of the antigen-binding moiety to two different payload moieties. Methods for constructing linker-payload moieties containing two different payloads are described, for example, 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 (a) A group for linking to the antigen-binding site (which is a self-stabilizing N-arylmaleimide), (b) (i) Alkyne groups for the incorporation of the payload portion by copper-mediated azide-alkyne cycloaddition (CuAAC), and (ii) A branched linker moiety is described which includes a ketone group for incorporating the payload moiety by an amino-oxy reaction that brings about an oxime bond, and two orthogonal functional groups for linking the payload moiety.

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

[0133] Accordingly, in some embodiments, the linker-payload portion according to the present disclosure includes a linker portion comprising (i) a portion derived from a group for linking to an antigen-binding portion (e.g., a self-stabilizing N-arylmaleimide group), (ii) a portion derived from an alkyne group suitable for incorporating the first payload portion by CuAAC (i.e., divalent triazole), and (iii) a portion derived from a ketone group suitable for incorporating the second payload portion by an aminooxy reaction resulting in oxime bonding (i.e., an oxime). According to such embodiments, in some examples, the first payload portion is a DDR inhibitor portion as described herein, and the second payload portion is a TOP1 inhibitor portion as described herein.

[0134] In some embodiments, the antigen-binding molecule according to the present disclosure comprises a linker-payload portion including (i) a first payload portion conjugated to the linker portion by a CuAAC reaction between an azide group and an alkyne group, and (ii) a second payload portion conjugated to the linker portion via an oxime bond between an alkoxyamine or hydrazide group and a ketone group. According to such embodiments, in some examples, the first payload portion is a DDR inhibitor portion as described herein, and the second payload portion is a TOP1 inhibitor portion as described herein.

[0135] Yamazaki, et al., Nat Commun. (2021) 12(1):3528, (a) A group for (lysine-based) linkage to the antibody, (b) (i) one or two azide groups for incorporation of payload moieties via strain-promoted azide-dibenzocyclooctyne (DBCO) cycloaddition, and (ii) a methyltetrazine group for incorporation of a payload moiety via trans-cyclooctene (TCO) cycloaddition, describing a branched linker moiety comprising two orthogonal functional groups for conjugating the payload moieties.

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

Chemical Formula

[0137] Accordingly, in some embodiments, the linker-payload moiety according to the present disclosure comprises a linker moiety comprising (i) a moiety derived from a group for conjugation to an antigen-binding moiety (e.g., a lysine-based group), (ii) one or two moieties derived from azide groups suitable for incorporating a first payload moiety via DBCO cycloaddition, and (iii) a moiety derived from a methyltetrazine group for incorporation of a second payload moiety via TCO cycloaddition. According to such embodiments, in some examples, the first payload moiety is a DDR inhibitor moiety as described herein, and the second payload moiety is a TOP1 inhibitor moiety as described herein. In some embodiments, the first payload moiety is a TOP1 inhibitor moiety as described herein, and the second payload moiety is a DDR inhibitor moiety as described herein.

[0138] In some embodiments, the antigen-binding molecule according to the present disclosure comprises a linker-payload portion including (i) a first payload portion conjugated to the linker portion by a DBCO cycloaddition reaction between a DBCO group and an azide group, and (ii) a second payload portion conjugated to the linker portion by a TCO cycloaddition reaction between a TCO group and a methyltetrazine group. According to such embodiments, in some examples, the first payload portion is a DDR inhibitor portion as described herein, and the second payload portion is a TOP1 inhibitor portion as 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. Such a method for fabricating a linker-payload portion containing two different payloads is described, for example, 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 describes a linker-payload moiety containing two distinct payload moieties constructed by sequential deprotection of orthogonally protected cysteine. Each payload is linked to a maleimide group (e.g., by a cleavable linker), and the maleimide 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: [ka]

[0141] Accordingly, in some embodiments, the linker-payload portion according to the present disclosure includes (i) a first payload portion conjugated to the linker-payload portion by reduction of a cysteine ​​residue having a disulfide protecting group (e.g., an S-(tert-butyl) disulfide group or an S-(isopropyl) disulfide group) and subsequent incorporation of the first payload portion by a thiol-maleimide reaction, and (ii) a second payload portion conjugated to the linker-payload portion by reduction of a cysteine ​​residue having an acetamidomethyl protecting group and subsequent incorporation of the first payload portion by a thiol-maleimide reaction. According to such embodiments, in some examples, the first payload portion is a DDR inhibitor portion as described herein, and the second payload portion is a TOP1 inhibitor portion as described herein.

[0142] In some of these embodiments, the linker-payload portion is (a) an amino group for conjugation to the antigen-binding site, (b) A portion comprising at least one first payload clicked to a first click base, wherein the portion comprising the first payload comprises a DDR inhibitor portion, (c) A portion comprising at least one second payload clicked to a second click base, wherein the portion comprising the second payload comprises a TOP1 inhibitor portion, (d) Branched group: [ka] And in the formula, R N However, H and -(C 1~5 Selected from alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates the location where the amino group is linked to the branched group. b indicates a location where at least one first click group is attached to the branching group. c includes a branching group, where c indicates a location to which at least one second click group is attached.

[0143] In some embodiments, the DDR inhibitor portion and the TOP1 inhibitor portion of the antigen-binding molecule of the Disclosure are linked to the antigen-binding portion of the antigen-binding molecule via different linker portions.

[0144] In some embodiments, the antigen-binding molecule according to the present disclosure comprises (a) a linker-payload portion containing a DDR inhibitor portion, and (b) a linker-payload portion containing 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 of which comprises a TOP1 inhibitor portion.

[0145] Techniques for linking multiple payload portions to an antigen-binding portion via multiple linker portions are described, for example, in Swiderska et al., Intl J Mol Sci (2018) 19:2098, Nilchan et al., Antib.Ther. (2019) 2:71-78, and Wang et al., Acta Pharmaceutica Sinica B (2023) 13(10):4025-4059 (all of which are incorporated herein by reference in their entirety).

[0146] Swiderska et al., Intl J Mol Sci (2018) 19:2098 describes a method in which (i) a first linker-payload portion (specifically, maleimide-Val-Cit-PAB-α-amanitin) is conjugated to a cysteine ​​residue of the polypeptide by a thiol-maleimide reaction, and (ii) a second linker-payload portion (specifically, an azide linked to MMAE) is conjugated to the same polypeptide by CuAAC-mediated conjugation to the alkyne group of a manipulated N-propargyl-L-lysine (PrK) residue.

[0147] Accordingly, in some embodiments, the antigen-binding molecule according to the Disclosure comprises (i) a first linker-payload portion conjugated to the antigen-binding portion by a thiol-maleimide reaction between a cysteine ​​residue of the antigen-binding portion and a maleimide group of the linker-payload portion, and (ii) a second linker-payload portion conjugated by a CuAAC reaction between an azide group of the linker-payload portion and an alkyne group of an N-propargyl-L-lysine residue of the antigen-binding portion. According to such embodiments, in some examples, the first linker-payload portion comprises a DDR inhibitor portion as described herein, and the second linker-payload portion comprises a TOP1 inhibitor portion as described herein.

[0148] Nilchan et al., Antib.Ther. (2019) 2:71-78 describes a double conjugation method in which (i) the first linker-payload portion is conjugated to the antigen-binding portion by selenoether conjugation between the selenocysteine ​​residue of the antigen-binding portion and the iodoacetamide group of the linker-payload portion, and (ii) the second linker-payload portion is conjugated to the same antigen-binding portion by a reaction between the cysteine ​​residue of the antigen-binding portion and the methylsulfonephenyloxadiazole (MSODA) group of the linker-payload portion.

[0149] Accordingly, in some embodiments, the antigen-binding molecule according to the present disclosure comprises (i) a first linker-payload portion conjugated to the antigen-binding portion by selenoether conjugation between a selenocysteine ​​residue of the antigen-binding portion and an iodoacetamide group of the linker-payload portion, and (ii) a second linker-payload portion conjugated by a reaction between a cysteine ​​residue of the antigen-binding portion and an MSODA group of the linker-payload portion. According to such embodiments, in some examples, the first linker-payload portion comprises a DDR inhibitor portion as described herein, and the second linker-payload portion comprises a TOP1 inhibitor portion as described herein.

[0150] Further aspects of the present disclosure provide a DDR inhibitor moiety linked to a click group, wherein the click group is suitable for conjugation of a linker moiety to a corresponding click group, and the linker moiety is conjugated to or suitable for conjugation to an antigen-binding moiety.

[0151] Further aspects of the present disclosure provide a TOP1 inhibitor moiety linked to a click group, wherein the click group is suitable for conjugation of a linker moiety to a corresponding click group, and the linker moiety is conjugated to or suitable for conjugation to an antigen-binding moiety.

[0152] According to such embodiments, in some cases the click group is selected from the following: (i) Azide group; (ii) alkyne; (iii) Tetrazine or tetrazine derivative group; (iv) cyclooctin, cyclooctin derivatives, or cyclooctin analog groups; and (v) Distortion Alken.

[0153] The trifunctional linkers of this disclosure are modular in design, allowing for easy modification of the number of linkers and thus payloads bound to the antigen-binding moiety, which has been shown to be important in the development of clinically significant antibody-drug conjugates. Furthermore, the use of click groups to attach payload-containing moieties to branching groups allows for the conjugation of a wide range of payload types. When orthogonal click moieties are used, two different payload moieties, including those with different and possibly complementary modes of action, can be linked.

[0154] The trifunctional linking group of this disclosure has a hydrophilic branching group, which can result in reduced toxicity and improved biophysical properties, stability properties, and pharmacokinetic properties.

[0155] As mentioned above, in some embodiments, the linker-payload portion is (a) an amino group for conjugation to the antigen-binding site, (b) a moiety comprising at least one first payload clicked to a first click group, wherein the moiety comprising the first payload comprises a DDR inhibitor moiety, the moiety comprising the at least one first payload, (c) a moiety comprising at least one second payload clicked to a second click group, wherein the moiety comprising the second payload comprises a TOP1 inhibitor moiety, the moiety comprising the at least one second payload, (d) a branching group:

Chemical Formula

[0156] In some embodiments, R N is H.

[0157] In some embodiments, R N is -(C 1~5 alkylene)-C(O)OH, wherein one CH2 unit may optionally be replaced with -O-.

[0158] In some embodiments, R N is -(C 1~5 alkylene)-C(O)OH.

[0159] In some embodiments, R N is -CH2CH2OCH2CH2C(O)OH.

[0160] In some embodiments, RN It is CH2C(O)OH.

[0161] The amino group may be linked to the branched group by a first spacer group. The first spacer group (A1) is (I C 1~7 Alkylene group; and / or (ii) May contain PEG1-12 groups

[0162] A1 is given by the following formula: -(CH2) xa -(C2H4O) xb -(CH2) x c- The equation can be such that xa is 0 or 1, xb is between 0 and 12, and xc is between 0 and 6, where at least one of xa and xb is 1.

[0163] In some embodiments, xb is 0, and xa + xc is 1 to 7, for example, 5 (i.e., A1 is -(CH2)5-).

[0164] In other embodiments, xb is 1 to 12, xa is 0, and xc is 0 or 1.

[0165] 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.

[0166] In some embodiments, if xb is 1 to 12, then xc is 1 to 6 or 1 to 2. In some of these embodiments, xc is 1. In some of these embodiments, xc is 2.

[0167] In some embodiments, xa is 0, xb is 1 to 6, and xc is 2. In some of these embodiments, A1 is -(C2H4)-O-(C2H4)-. In some of these embodiments, A1 is -(C2H4O)3-(C2H4)-.

[0168] The first and second click groups may be selected from any of the following click group pairs. [Table 3]

[0169] Examples of cyclooctin, cyclooctin derivatives, and cyclooctin analogs used in this disclosure include the following: [ka] [ka]

[0170] These groups can alternatively be referred to as cyclic alkynes.

[0171] Examples of tetrazine and tetrazine derivatives used in this disclosure include the following: [ka] [ka]

[0172] The strain alkenes used in this disclosure may have the following structures. [ka]

[0173] In some embodiments, the click group may be a dibenzoazacyclooctin (DIBAC) group or a 1-ethylhepta-6-enoxy group. [ka]

[0174] In some embodiments, the click group may be tetramethylthiocycloheptine sulfoximine (TMTHSI). [ka]

[0175] The linkage between the payload portion (e.g., the DDR inhibitor portion or the TOP1 inhibitor portion) and the click group according to this disclosure may be, for example, a cleavable linker portion or a non-cleavable portion, as described above.

[0176] In some embodiments of the trifunctional linking group, the first and second click groups are the same.

[0177] In other embodiments of the trifunctional linking group, the first and second click groups are selected from an orthogonal click group pair.

[0178] In some embodiments of the trifunctional linking group, the first and / or second click group is an azide.

[0179] In some embodiments of the trifunctional linking group, the first and / or second click group is tetrazine or a tetrazine derivative.

[0180] In some embodiments of the trifunctional linking group, the first and / or second click group is an alkyne (-CCH).

[0181] In some embodiments of the trifunctional linking group, the first and / or second click group is cyclooctin or a cyclooctin derivative.

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

[0183] In some embodiments of the trifunctional linking group, the first and / or second click group is methylcyclopropene (1-MCP).

[0184] In some embodiments of the trifunctional linking group, the first click group is an azide, and the second click group is a tetrazine or a tetrazine derivative.

[0185] In some embodiments of the trifunctional linking group, the first click group is an azide, and the second click group is a cyclooctene.

[0186] In some embodiments of the trifunctional linking group, the first click group is an azide, and the second click group is norbornene or a norbornene derivative.

[0187] In some embodiments of the trifunctional linking group, the first click group is an azide, and the second click group is methylcyclopropene (1-MCP).

[0188] In some embodiments of the trifunctional linking group, the first click group is an alkyne (-CCH), and the second click group is a tetrazine or a tetrazine derivative.

[0189] In some embodiments of the trifunctional linking group, the first click group is an alkyne (-CCH) and the second click group is a cyclooctene.

[0190] In some embodiments of the trifunctional linking group, the first click group is an alkyne (-CCH), and the second click group is norbornene or a norbornene derivative.

[0191] In some embodiments of the trifunctional linking group, the first click group is an alkyne (-CCH) and the second click group is methylcyclopropene (1-MCP).

[0192] In some embodiments of the trifunctional linking group, the first click group is cyclooctin or a cyclooctin derivative, and the second click group is tetrazine or a tetrazine derivative.

[0193] In some embodiments of the trifunctional linking group, the first click group is cyclooctin or a cyclooctin derivative, and the second click group is cyclooctene.

[0194] In some embodiments of the trifunctional linking group, the first click group is cyclooctyne or a cyclooctyne derivative, and the second click group is norbornene or a norbornene derivative.

[0195] In some embodiments of the trifunctional linking group, the first click group is cyclooctin or a cyclooctin derivative, and the second click group is methylcyclopropene (1-MCP).

[0196] In some embodiments of the trifunctional linking group, the second click group is phenyltetrazine.

[0197] In some embodiments of the trifunctional linking group, the second click group is selected from the following group: [ka]

[0198] In some embodiments of the trifunctional linking group, the second click group is [ka] That is the case.

[0199] At least one first click group may be connected to a branching group by a second spacer group (B1). In some embodiments, the second spacer group is branched such that two first click groups are connected to the branching group. In other embodiments, the second spacer group is not branched such that a single first click group is connected to the branching group.

[0200] At least one second click group may be connected to a branching group by a third spacer group (B2). In some embodiments, the third spacer group is branched such that two second click groups are connected to the branching group. In other embodiments, the third spacer group is not branched such that a single second click group is connected to the branching group.

[0201] In some embodiments of the trifunctional linking group, the second spacer group (B1) is given by formula (B1-1): [ka] It is, R L1 is -(C2H4O) xl3 -(CH2) xd -(C(=O)) xl4 -and, Here, xl3 is between 0 and 4, xd is between 0 and 3, and xl4 is 0 or 1. R NB1 is -(C2H4O) xe1 -(CH2) xf1 -(NH) xg1 -(C(=O)CH2) xh1 -and, Here, xe1 is between 0 and 4, xf1 is between 0 and 2, xg1 is 0 or 1, and xh1 is 0 or 1. R NB2 is H or -(C2H4O) xe2 -(CH2) xf2 -(NH) xg2 -(C(=O)CH2) xh2 -and, Here, xe2 is between 0 and 4, xf2 is between 0 and 2, xg2 is 0 or 1, and xh2 is 0 or 1.

[0202] In some embodiments, xl3 is between 0 and 2. In some embodiments, xl3 is 0. In some embodiments, xl3 is 1. In some embodiments, xl3 is 2.

[0203] In some embodiments, xl4 is 0. In some embodiments, xl4 is 1.

[0204] In some embodiments, xe1 is 2 to 4. In some embodiments, xe1 is 2. In some embodiments, xe1 is 3. In some embodiments, xe1 is 4.

[0205] In some embodiments, xe2 is 2 to 4. In some embodiments, xe2 is 2. In some embodiments, xe2 is 3. In some embodiments, xe2 is 4.

[0206] In some of these embodiments of the trifunctional linking group, the second spacer group (B1) is of formula (B1-2): [ka] In this equation, xd is between 0 and 3. R NB1 is -(C2H4O) xe1 -(CH2) xf1 -(NH) xg1 -(C(=O)CH2) xh1 -and, Here, xe1 is 0 or 1, xf1 is 0 to 2, xg1 is 0 or 1, and xh1 is 0 or 1. R NB2 is H or -(C2H4O) xe2 -(CH2) xf2 -(NH) xg2 -(C(=O)CH2) xh2 -and, Here, xe2 is 0 or 1, xf2 is 0 to 2, xg2 is 0 or 1, and xh2 is 0 or 1.

[0207] In some embodiments, R NB2 H is H. In some embodiments, R NB2 is -(C2H4O) xe2 -(CH2) xf2 -(NH) xg2-(C(=O)CH2) xh2 -In other embodiments, R NB2 R NB1 It is the same as this.

[0208] In some embodiments, xd is between 0 and 2. In some embodiments, xd is between 0 and 1. In some embodiments, xd is 0. In some embodiments, xd is 1. In some embodiments, xd is 2. In some embodiments, xd is 3. In some embodiments, xd is 0 or 2.

[0209] In some embodiments, xe1 is 0. In some embodiments, xe1 is 1.

[0210] In some embodiments, xf1 is between 0 and 1. In some embodiments, xf1 is 0. In some embodiments, xf1 is 1. In some embodiments, xf1 is 2. In some embodiments, xf1 is 0 or 2.

[0211] In some embodiments, xg1 is 0. In some embodiments, xg1 is 1.

[0212] In some embodiments, xh1 is 0. In some embodiments, xh1 is 1.

[0213] In some embodiments, xe2 is 0. In some embodiments, xe2 is 1.

[0214] In some embodiments, xf2 is between 0 and 1. In some embodiments, xf2 is 0. In some embodiments, xf2 is 1. In some embodiments, xf2 is 2. In some embodiments, xf2 is 0 or 2.

[0215] In some embodiments, xg2 is 0. In some embodiments, xg2 is 1.

[0216] In some embodiments, xe1 is 1, xf1 is 2, xg1 is 0, and xh1 is 0.

[0217] In some embodiments, xe1 is 0, xf1 is 0, xg1 is 0, and xh1 is 1.

[0218] In some embodiments, xe1 is 1, xf1 is 2, xg1 is 1, and xh1 is 1.

[0219] In some embodiments, xe2 is 1, xf2 is 2, xg2 is 0, and xh2 is 0.

[0220] In some embodiments, xe2 is 0, xf2 is 0, xg2 is 0, and xh2 is 1.

[0221] In some embodiments, xe2 is 1, xf2 is 2, xg2 is 1, and xh2 is 1.

[0222] In some embodiments of the trifunctional linking group, the second spacer group (B1) is selected from the group including: [ka]

[0223] In some embodiments of the trifunctional linking group, the second spacer group (B1) is selected from the group including: [ka] In some embodiments of the trifunctional linking group, the second spacer group (B2) is given by formula (B2-1): [ka] It is, R L2 is -(C2H4O) xl5 -(CH2)xi -(C(=O)) xl6 -and, Here, xl6 is between 0 and 4, xi is between 0 and 3, and xl6 is either 0 or 1. R NB3 is -(C2H4O) xj1 -(CH2) xk1 -(NH) xl1 -(C(=O)CH2) xm1 -and, Here, xj1 is between 0 and 4, xk1 is between 0 and 2, xl1 is 0 or 1, and xm1 is 0 or 1. R NB4 is H or -(C2H4O) xj2 -(CH2) xk2 -(NH) xl2 -(C(=O)CH2) xm2 -and, Here, xj2 is between 0 and 4, xk2 is between 0 and 2, xl2 is 0 or 1, and xm2 is 0 or 1.

[0224] In some embodiments, xl5 is between 0 and 2. In some embodiments, xl5 is 0. In some embodiments, xl5 is 1. In some embodiments, xl5 is 2.

[0225] In some embodiments, xl6 is 0. In some embodiments, xl6 is 1.

[0226] In some embodiments, xj1 is 2 to 4. In some embodiments, xj1 is 2. In some embodiments, xj1 is 3. In some embodiments, xj1 is 4.

[0227] In some embodiments, xj2 is 2 to 4. In some embodiments, xj2 is 2. In some embodiments, xj2 is 3. In some embodiments, xj2 is 4.

[0228] In some embodiments of the trifunctional linking group, the third spacer group (B2) is given by formula (B2-2): [ka] It is, In the formula, xi is between 0 and 3. R NB3 is -(C2H4O) xj1 -(CH2) xk1 -(NH) xl1 -(C(=O)CH2) xm1 -and, Here, xj1 is 0 or 1, xk1 is 0 to 2, xl1 is 0 or 1, and xm1 is 0 or 1. R NB4 is H or -(C2H4O) xj2 -(CH2) xk2 -(NH) xl2 -(C(=O)CH2) xm2 -and, Here, xj2 is 0 or 1, xk2 is 0 to 2, xl2 is 0 or 1, and xm2 is 0 or 1.

[0229] In some embodiments, R NB4 H is H. In some embodiments, R NB4 is -(C2H4O) xj2 -(CH2) xk2 -(NH) xl2 -(C(=O)CH2) xm2 -In other embodiments, R NB4 R NB3 It is the same as this.

[0230] In some embodiments, xi is between 0 and 2. In some embodiments, xi is between 0 and 1. In some embodiments, xi is 0. In some embodiments, xi is 1. In some embodiments, xi is 2. In some embodiments, xi is 3. In some embodiments, xi is 0 or 2.

[0231] In some embodiments, xj1 is 0. In some embodiments, xj1 is 1.

[0232] In some embodiments, xk1 is between 0 and 1. In some embodiments, xk1 is 0. In some embodiments, xk1 is 1. In some embodiments, xk1 is 2. In some embodiments, xk1 is 0 or 2.

[0233] In some embodiments, xl1 is 0. In some embodiments, xl1 is 1.

[0234] In some embodiments, xm1 is 0. In some embodiments, xm1 is 1.

[0235] In some embodiments, xj2 is 0. In some embodiments, xj2 is 1.

[0236] In some embodiments, xk2 is between 0 and 1. In some embodiments, xk2 is 0. In some embodiments, xk2 is 1. In some embodiments, xk2 is 2. In some embodiments, xk2 is 0 or 2.

[0237] In some embodiments, xl2 is 0. In some embodiments, xl2 is 1.

[0238] In some embodiments, xj1 is 1, xk1 is 2, xl1 is 0, and xm1 is 0.

[0239] In some embodiments, xj1 is 0, xk1 is 0, xl1 is 0, and xm1 is 1.

[0240] In some embodiments, xj1 is 1, xk1 is 2, xl1 is 1, and xm1 is 1.

[0241] In several embodiments, xj2 is 1, xk2 is 2, xl2 is 0, and xm2 is 0.

[0242] In some embodiments, xj2 is 0, xk2 is 0, xl2 is 0, and xm2 is 1.

[0243] In several premises, xj2 is 1, xk2 is 2, xl2 is 1, and xm2 is 1.

[0244] In some embodiments of the trifunctional linking group, the third spacer group (B2) is selected from the group including: [ka]

[0245] In some embodiments of the trifunctional linking group, the third spacer group (B2) is selected from the group including: [ka]

[0246] In some embodiments of the trifunctional linking group, the second spacer group (B1) is the same as the third spacer group (B2). In some embodiments of the trifunctional linking group, the second spacer group (B1) is different from the third spacer group (B2).

[0247] In some embodiments of the trifunctional linking group, the second spacer group (B1) and the third spacer group (B2), along with the nitrogen atom to which they are bonded, are one of the following groups: [ka] It forms a structure.

[0248] In some embodiments of the trifunctional linking group, the second spacer group (B1) and the third spacer group (B2), along with the nitrogen atom to which they are bonded, are one of the following groups: [ka] This disclosure forms (a) an amino group conjugated to the antigen-binding site, (b) A portion comprising at least one first payload clicked onto a first click base, (c) A portion comprising at least one second payload clicked onto a second click base, (d) Branched group: [ka] And in the formula, R N However, H and -(C 1~5 Selected from alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-, a indicates the location where the amino group is linked to the branched group. b indicates a location where at least one first click group is attached to the branching group. The present invention provides a linker comprising a branching group, wherein c indicates a location where at least one second click group is linked to the branching group.

[0249] Examples of click groups are shown below. [ka]

[0250] As shown in the table above, the reaction between the first and second members of a click group pair can result in two isomeric products, i.e., a mixture. If only one is shown, this disclosure encompasses both isomeric forms.

[0251] The amino group may be linked to the branched group by the first spacer group (A1) defined above.

[0252] At least one first click group may be linked to a branching group by a second spacer group (B1) as defined above.

[0253] At least one second click group may be linked to the branching group by a third spacer group (B2) as defined above.

[0254] In some embodiments, the linker includes one of the following groups: [ka] [ka] [ka] [ka]

[0255] The linkage between the payload portion (e.g., the DDR inhibitor portion or the TOP1 inhibitor portion) and the click group according to this disclosure may be, for example, a cleavable linker portion or a non-cleavable portion, as described above.

[0256] In some embodiments, a functional group is present to link the click group. This functional group can be selected from carbonyl (C=O) and oxy (O). If the click group is DBCO or TMTHSI, the functional group may be carbonyl. If the click group is TCO, the functional group may be oxy.

[0257] In some embodiments, the linker between the payload portion and the click portion is a connecting portion, [ka] Includes, Q is, [ka] And in the formula, Q XThis is a case where Q is an amino acid residue, a dipeptide residue, or a tripeptide residue. X is [ka] In the equation, a = 0 to 5, b = 0 to 8, c = 0 or 1, and d = 0 to 5. a can be 0, 1, 2, 3, 4, or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 1.

[0258] 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 be 0, 1, 2, 3, or 4. In further embodiments, b is 3.

[0259] c can be 0 or 1. In some of these embodiments, c is 1.

[0260] 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 further embodiments, d is 2. In other further embodiments, d is 1. In yet another further embodiment, d is 0.

[0261] In some embodiments of X, a is 0, c is 1, d is 2, and b can be 0 to 8. In some of these embodiments, b is 0, 4, or 8.

[0262] In one embodiment, Q is an amino acid residue. The amino acid may be a natural amino acid or a non-natural amino acid.

[0263] In one embodiment, Q is selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, Ser, and Trp, wherein Cit is citrulline. In one aspect, Q is a serine derivative (see WO2018 / 234636A1).

[0264] In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. At the same time, the dipeptide is the recognition site for cathepsin.

[0265] In one embodiment, 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 selected from Here, Cit stands for citrulline.

[0266] In some of these embodiments, Q is CO -Phe-Lys- NH , CO -Val-Ala- NH , CO -Val-Lys- NH , CO -Ala-Lys- NH , CO -Val-Cit- NH Selected from.

[0267] In further embodiments, Q is CO -Phe-Lys- NH , CO -Val-Cit- NH , and CO -Val-Ala- NH Selected from.

[0268] In some embodiments, the linkage between the payload portion (e.g., DDR inhibitor portion or TOP1 inhibitor portion) and the click group comprises a PABC, a cathepsin-cleavable dipeptide, and a PEG2-PEG4 (e.g., PEG3) group. In some of these embodiments, the linkage between the payload portion and the click group is [ka] It includes, or is, that.

[0269] In some embodiments, the linkage between payload portions includes GGFG (glycine-glycine-phenylalanine-glycine). This may be directly linked to the payload or linked via a CH2 group. In some of these embodiments, the linkage between the payload portion and the click group is [ka] It includes, or is, that.

[0270] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0271] In some embodiments, the TOP1 inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0272] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0273] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0274] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0275] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0276] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0277] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0278] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0279] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0280] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0281] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0282] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It holds.

[0283] Functional properties of antigen-binding molecules The antigen-binding molecules described herein may be characterized by reference to certain functional properties. In some embodiments, the antigen-binding molecules described herein may have one or more of the following properties: It binds to cells that express HER2; Inhibits the proliferation of cells that express HER2; Enhances the killing of HER2-expressing cells; To inhibit tumor growth and / or reduce tumor size / volume (e.g., to inhibit tumor growth and / or reduce tumor size / volume in HER2-expressing cancers); To extend the survival time of patients with cancer (for example, cancer that expresses HER2).

[0284] It will be understood that a given antigen-binding molecule may exhibit two or more of the properties listed in the previous paragraph. A given antigen-binding molecule may be evaluated for the properties listed in the previous paragraph using a suitable assay. For example, the assay may be, for example, an in vitro assay, optionally a cell-based assay, or a cell-free assay. In some embodiments, the assay may be, for example, an in vivo assay, i.e., it may be performed on a non-human animal. In some embodiments, the assay may be, for example, an ex vivo assay, i.e., it may be performed using cells / tissues / organs obtained from a subject.

[0285] If the assay is a cell-based assay, it may involve treating cells with an antigen-binding molecule to determine whether the antigen-binding molecule exhibits one or more of the listed properties. The assay may use species labeled with a detectable entity to facilitate detection. The assay may involve evaluating the listed properties after separately treating cells with a given antigen-binding molecule in a range of amounts / concentrations (e.g., a dilution series).

[0286] Analysis of the results of such assay may include determining the concentration at which 50% of the maximum level of the relevant activity is achieved. The concentration of a given drug at which 50% of the maximum level of the relevant activity is achieved may be referred to as the “50% effective concentration” of the drug with respect to the relevant activity, which is the “EC 50 It can also be called "EC". Depending on the characteristics, 50 This refers to "50% inhibitory concentration" or "IC 50 This can also be referred to as the "maximum inhibitory level," which is the concentration of a drug at which 50% of the maximum inhibitory level of a given characteristic is observed.

[0287] In some embodiments, 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) when HER2 is expressed on the cell surface (i.e., within or on the cell membrane). In some embodiments, the antigen-binding molecule binds to HER2 expressed on the cell surface of a HER2-expressing cell. In some embodiments, the antigen-binding molecule binds to a HER2-expressing cell.

[0288] The ability of an antigen-binding molecule to bind to a given cell type can be analyzed by bringing cells into contact with the antigen-binding molecule and, for example, detecting the antigen-binding molecule bound to the cells after a washing step to remove unbound antigen-binding molecules. The ability of an antigen-binding molecule to bind to HER2-expressing cells can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0289] In some embodiments, antigen-binding molecules inhibit the proliferation of HER2-expressing cells (e.g., HER2-expressing cancer cells). The ability of an antigen-binding molecule to inhibit the proliferation of a given cell type can be analyzed by bringing cells into contact with the antigen-binding molecule and then evaluating the proliferation of those cells (i.e., after a sufficient period of time for the effect on cell proliferation to be observed). Cell proliferation can be measured, for example, by detecting changes in cell number over time, or 3H-thymidine uptake can be evaluated by in vitro analysis or by a CFSE dilution assay, for example, as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564 (the whole of which is incorporated herein by reference).

[0290] In some embodiments, the antigen-binding molecule of the present invention can inhibit the proliferation of HER2-expressing cells in a given assay to less than 1x 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, 0.99x or less, 0.95x or less, 0.9x or less, 0.85x or less, 0.8x or less, 0.75x or less, 0.7x or less, 0.65x or less, 0.6x or less, 0.55x or less, 0.5x or less, 0.45x or less, 0.4x or less, 0.35x or less, 0.3x or less, 0.25x or less, 0.2x or less, 0.15x or less, 0.1x or less, 0.05x or less, or 0.01x or less.

[0291] In some embodiments, the antigen-binding molecules described herein inhibit the proliferation of human HER2-expressing cells to one of the following ICs: 100 nM or less, preferably 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, or 500 pM or less. 50 It inhibits it.

[0292] In some embodiments, the antigen-binding molecules according to this disclosure enhance (i.e., upregulate, promote) cellular killing of cells containing / expressing HER2.

[0293] In some embodiments, antigen-binding molecules according to this disclosure may inhibit the growth of cancer containing / expressing HER2 cells or reduce its metastasis. In some embodiments, antigen-binding molecules may enhance (i.e., upregulate, promote) cytotoxicity of HER2-containing / expressing cells. In some embodiments, antigen-binding molecules may inhibit the proliferation of cancer cells containing / expressing HER2 cells or inhibit the growth of tumors containing them. In some embodiments, antigen-binding molecules may inhibit the metastasis of cancer / tumors containing HER2-containing / expressing cells.

[0294] Cell killing can be investigated using any of the methods outlined, for example, in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616 (the entire work is incorporated herein by reference). Examples of in vitro assays for cytotoxicity / cell killing assays include release assays, e.g., 51 Examples include Cr release assays, lactate dehydrogenase (LDH) release assays, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assays, ATP release assays using Cell Titre Glo, and calcein-acetoxymethyl (calcein-AM) release assays. These assays measure cell killing based on the detection of factors released from lysed cells.

[0295] In some embodiments, the antigen-binding molecules of this disclosure can reduce the number / proportion of cells expressing HER2. In some embodiments, the antigen-binding molecules of this disclosure can deplete / enhance such cells.

[0296] In some embodiments, the antigen-binding molecules of this disclosure exhibit anticancer activity. In some embodiments, the antigen-binding molecules enhance the killing of cancer cells. In some embodiments, the antigen-binding molecules cause, for example, a reduction in the number of cancer cells in vivo compared to appropriate control conditions. The cancer may be a cancer such as those described herein, for example, a cancer that expresses / overexpresses HER2.

[0297] In some embodiments, the antigen-binding molecules of this disclosure reduce / inhibit the growth of cancer and / or cancerous tumors. In some embodiments, the antigen-binding molecules reduce tissue invasion by cancer cells. In some embodiments, the antigen-binding molecules reduce cancer metastasis. In some embodiments, the antigen-binding molecules exhibit anticancer activity. In some embodiments, the antigen-binding molecules reduce the growth / proliferation of cancer cells. In some embodiments, the antigen-binding molecules reduce the survival of cancer cells. In some embodiments, the antigen-binding molecules enhance the killing of cancer cells. In some embodiments, the antigen-binding molecules of this disclosure cause a reduction in the number of cancer cells, for example, in vivo. Cancer may include cells that express HER2.

[0298] The antigen-binding molecules of this disclosure can be analyzed for the properties described in the preceding paragraph in a suitable assay. Such assays include, for example, in vivo models.

[0299] In some embodiments, administration of antigen-binding molecules according to the present disclosure may cause one or more of the following: for example, inhibition of cancer progression / progression, delay / prevention of cancer onset, 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 symptoms of cancer, reduction of the number of cancer cells, reduction of cancer burden, reduction of tumor size / volume, and / or extension of survival (e.g., progression-free survival or overall survival) of a subject with cancer.

[0300] It will be understood that the characteristics listed in the previous paragraph should be evaluated after a sufficient period of time for the effects associated with treatment using antigen-binding molecules to be observed. Tumor growth can be monitored by investigating tumor volume over time (e.g., mm). 3 This can be evaluated by measuring the tumor volume.

[0301] In some embodiments, the antigen-binding molecules of the present disclosure can reduce the tumor size / volume (e.g., the mean tumor size / volume of a treatment group in an in vivo model of HER2-expressing cancer) in a given assay to less than 1x of the tumor size / volume observed at the same time point in the absence of treatment with the antigen-binding molecule (or after treatment with a suitable control antigen-binding molecule known not to affect tumor growth), for example, ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x. In some embodiments, the assessment of tumor size / volume for such comparison is performed in the appropriate model more than 5 days after the administration of the initial dose of the antigen-binding molecule, for example, after one of the following periods: 10 days or more, 15 days or more, 20 days or more, 25 days or more, 30 days or more, 35 days or more, 40 days or more, 35 days or more, 50 days or more, 55 days or more, 60 days or more, 65 days or more, 70 days or more, 75 days or more, 80 days or more, 85 days or more, 90 days or more, 95 days or more, or 100 days or more.

[0302] In some embodiments, the antigen-binding molecules of the present disclosure achieve a level of tumor growth inhibition (expressed, for example, as % tumor growth inhibition calculated compared to tumor growth observed with treatment with a suitable control antigen-binding molecule) that is greater than 1x the level of tumor growth inhibition observed at the same time point in a given assay in the absence of treatment with the antigen-binding molecule (or after treatment with a suitable control antigen-binding molecule known not to affect tumor growth), for example, 1.01x, ≥1.02x, ≥1.03x, ≥1.04x, ≥1.05x, ≥1.1x, ≥1.2x, ≥1.3x, ≥1.4x, ≥1.5x, ≥1.6x, ≥1.7x, ≥1.8x, ≥1.9x, ≥2x, ≥3x, ≥4x, ≥5x, ≥6x, ≥7x, ≥8x, ≥9x, or ≥10x (expressed, for example, as % tumor growth inhibition calculated compared to tumor growth observed with treatment with a suitable control antigen-binding molecule). In some embodiments, the evaluation of tumor growth inhibition for such comparison is performed in a suitable model more than 5 days after the administration of the initial dose of the antigen-binding molecule, for example, after one of the following periods: 10 days or more, 15 days or more, 20 days or more, 25 days or more, 30 days or more, 35 days or more, 40 days or more, 35 days or more, 50 days or more, 55 days or more, 60 days or more, 65 days or more, 70 days or more, 75 days or more, 80 days or more, 85 days or more, 90 days or more, 95 days or more, or 100 days or more.

[0303] In some embodiments, the antigen-binding molecules of the present disclosure can extend the median survival of a subject with cancer (e.g., in an in vivo model of HER2-expressing cancer) in a given assay to more than one of the median survival observed in the absence of treatment with the antigen-binding molecule (or after treatment with a suitable control antigen-binding molecule known not to affect the survival of subjects 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. The median survival of a subject in the appropriate treatment group may be expressed in days from the start of the experiment.

[0304] additional array The antigen-binding molecules and their constituent polypeptides of this disclosure may additionally include further amino acids or amino acid sequences.

[0305] The polypeptides of this disclosure may include one or more linker sequences between amino acid sequences. Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369 (which is incorporated herein by reference in its entirety). In some embodiments, the linker sequences may be mobile linker sequences. Mobile linker sequences allow relative movement of the amino acid sequences linked by the linker sequence. Mobile linkers are known to those skilled in the art and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369. Mobile linker sequences often contain a high proportion of glycine and / or serine residues.

[0306] 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 following structure: (GxS)n or (GxS)nGm, where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5, or 6, m = 0, 1, 2, or 3. In some embodiments, the linker sequence comprises one or more copies (e.g., 1, 2, 3, 4, 5, or 6 copies) of the sequence motif G4S (e.g., in tandem). In some embodiments, the linker sequence comprises or consists of (G4S)4 or (G4S)6. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.

[0307] The antigen-binding molecules and constituent polypeptides of this disclosure may include amino acid sequences(s) to facilitate the expression, folding, transport, processing, purification, or detection of the antigen-binding molecules / polypeptides. For example, the antigen-binding molecules and polypeptides of this disclosure may additionally include amino acid sequences that form a detectable moiety, such as those described below.

[0308] The antigen-binding molecules and their constituent polypeptides of this disclosure may additionally include signal peptides (also known as leader sequences or signal sequences). Signal peptides typically consist of a sequence of 5 to 30 hydrophobic amino acids that form a single α-helix. Secretory proteins and proteins expressed on the cell surface often contain signal peptides. Signal peptides for numerous proteins are known and recorded 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).

[0309] Signal peptides may be present at the N-terminus of a polypeptide or in newly synthesized polypeptides. Signal peptides facilitate the efficient transport of polypeptides. In many cases, signal peptides are removed by cleavage and are therefore not present in mature polypeptides.

[0310] Numerous protein signal peptides are publicly known and recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted 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).

[0311] Signs and conjugates In some embodiments, the antigen-binding molecules of this disclosure and their constituent polypeptides include detectable portions.

[0312] In some embodiments, the detectable portion is fluorescently labeled, phosphorescently labeled, luminescently labeled, immunodetectable (e.g., epitope tag), radioactively labeled, chemically labeled, nucleic acid labeled, or enzymatically labeled. Antigen-binding molecules or their constituent polypeptides may be covalently or noncovalently labeled using the detectable portion.

[0313] Examples of fluorescent labels include fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), rare earth chelates such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Examples of radioactive labels include radioisotopes such as hydrogen. 3 ,sulfur 35 ,carbon 14 , Lin 32 iodine 123 iodine 125 iodine 126 iodine 131 iodine 133 ,bromine 77 ,technetium99m ,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 Examples of luminescent labels include, for example, emitroluminescence, chemiluminescence (e.g., acridinium esters, luminol, isoluminol), and bioluminescent labels. Examples of immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands, such as biotin, avidin, streptavidin, or digoxigenin. Examples of nucleic acid labels include aptamers.

[0314] In some embodiments, the antigen-binding molecule or its constituent polypeptide contains a radionuclide. Such a conjugate may be referred to as a radionuclide-drug conjugate (RDC) or radioimmunoconjugate (RIC). In some embodiments, the antigen-binding molecule or its constituent polypeptide contains a chelator group capable of chelating the radionuclide. The radionuclide may be a radioisotope, e.g., those listed above, or others, e.g., lutetium. 177 Actinium 225 , and strontium 90 These are some examples.

[0315] In some embodiments, antigen-binding molecules or their constituent polypeptides optionally include epitope tags, e.g., 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) at the N or C terminus of the antigen-binding molecule / polypeptide.

[0316] In some embodiments, antigen-binding molecules or their constituent polypeptides include a sub-mole having detectable activity, such as an enzymatic moiety. Examples of enzymatic labels include luciferase, glucose oxidase, galactosidase (e.g., β-galactosidase), glucuronidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., wasabi peroxidase), and cholinesterase.

[0317] Nucleic acids and vectors This disclosure provides nucleic acids or a plurality of nucleic acids that encode antigen-binding molecule / antigen-binding polypeptide complexes or their constituent polypeptides. In some embodiments, the nucleic acids include or consist of DNA and / or RNA.

[0318] The antigen-binding molecule / antigen-binding polypeptide complexes or their constituent polypeptides according to this disclosure may be produced intracellularly by translation of RNA encoding the polypeptide(s). The antigen-binding molecule / antigen-binding polypeptide complexes or their constituent polypeptides may be produced intracellularly by transcription from nucleic acids encoding the polypeptide(s), and subsequent translation of the transcribed RNA.

[0319] In some embodiments, nucleic acids(s) may be or may be contained within a vector or multiple vectors. As used herein, “vector” refers to a nucleic acid molecule used as a medium for introducing exogenous nucleic acids into cells.

[0320] Accordingly, this disclosure also provides a vector or a plurality of vectors comprising nucleic acids or plurality of nucleic acids according to this disclosure. The vector may facilitate the delivery of nucleic acids(s) encoding polypeptides according to this disclosure to cells. The vector may be an expression vector comprising elements required to express polypeptides according to this disclosure. The vector may comprise elements that facilitate the integration of nucleic acids(s) into the genomic DNA of cells into which the vector is introduced.

[0321] The nucleic acids and vectors provided herein may be provided in purified or isolated forms, i.e., in forms purified or isolated from other nucleic acids or naturally occurring biological materials.

[0322] A vector can be a vector for the expression of nucleic acids in cells (i.e., an expression vector). Such a vector may include a promoter sequence operably ligated to a nucleotide sequence encoding an antigen-binding molecule or polypeptide according to the Disclosure. The vector may also include a stop codon (i.e., the 3' end of the nucleotide sequence encoding the polypeptide(s) in the vector's nucleotide sequence) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the Art may be used to express a peptide or polypeptide from the vector according to the Disclosure.

[0323] The term “operatably linked” may encompass situations in which the nucleic acid encoding the polypeptide and the regulatory nucleic acid sequence(s) (e.g., promoter and / or enhancer) (e.g., promoter and / or enhancer) are covalently linked so as to place the expression of the polypeptide-encoding nucleic acid under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette). Thus, if the regulatory sequence can influence the nucleic acid sequence, the regulatory sequence is operatably linked to the selected nucleic acid sequence. The resulting transcript(s) can then be translated into the desired polypeptide(s).

[0324] Vectors intended in connection with this disclosure include, for example, DNA vectors, RNA vectors, plasmids (e.g., conjugating plasmids (e.g., F plasmids), non-conjugating plasmids, R plasmids, colisin plasmids, episomes), viral vectors (e.g., retroviral vectors, e.g., gamma retroviral vectors (e.g., mouse leukemia virus (MLV)-derived vectors, e.g., 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), as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan, and Boyerinas, Biomedicines (2016) 4:9 (both of which are incorporated herein by reference in their entirety).

[0325] In some embodiments, the vector may be a eukaryotic vector, for example, a vector containing elements necessary for protein expression from the vector within a eukaryotic cell. In some embodiments, the vector may be a mammalian vector containing, for example, a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0326] The constituent polypeptides of the antigen-binding molecule / antigen-binding polypeptide complex according to this disclosure may be encoded by different nucleic acids from among multiple nucleic acids, or by different vectors from among multiple vectors.

[0327] Production of antigen-binding molecules The antigen-binding molecules according to this disclosure can be prepared according to methods for the preparation of antibody-drug conjugates known to those skilled in the art.

[0328] The antigen-binding moieties according to this disclosure may be prepared by chemical synthesis, for example, liquid-phase synthesis or solid-phase synthesis. For example, peptides / polypeptides may be synthesized using the methods described, for example, Chandrudu et al., Molecules (2013), 18:4373-4388 (which is incorporated herein by reference in its entirety).

[0329] Alternatively, the antigen-binding moiety according to this disclosure can be produced by recombinant expression. For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th Molecular biology techniques suitable for recombinant polypeptide production, such as those described in (Edition), Cold Spring Harbor Press, 2012, and Nat Methods. (2008); 5(2):135-146 (both of which are incorporated herein by reference in their entirety), are well known in the art. Methods for recombinant production of antigen-binding polypeptides are also described in Frenzel et al., Front Immunol. (2013); 4:217, and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (both of which are incorporated herein by reference in their entirety).

[0330] In some examples, the antigen-binding moiety of the present disclosure is composed of two or more polypeptide chains. In such cases, the production of the antigen-binding moiety may involve the transcription and translation of two or more polypeptides, as well as the subsequent association of polypeptide chains to form the antigen-binding moiety.

[0331] In the recombinant production according to this disclosure, any cell suitable for polypeptide expression may be used. The cell may be a prokaryote or a eukaryote. In some embodiments, the cell may be a prokaryotic cell, e.g., an archaeal or bacterial cell. In some embodiments, the bacterium may be a Gram-negative bacterium, e.g., a bacterium of the family Enterobacteriaceae, e.g., Escherichia coli. In some embodiments, the cell may be a eukaryotic cell, e.g., a yeast cell, plant cell, insect cell, or mammalian cell, e.g., the cells mentioned above. In some cases, the cell may not be a prokaryotic cell because some prokaryotic cells do not allow the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotes, and the protein may be easier to purify from eukaryotes using appropriate tags. Certain plasmids that enhance the secretion of the protein into the culture medium may also be utilized.

[0332] In some embodiments, polypeptides may be prepared by cell-free protein synthesis (CFPS) according to the system described, for example, Zemella et al. Chembiochem (2015) 16(17):2420-2431 (which is incorporated herein by reference in its entirety).

[0333] The production of antigen-binding moieties may involve culturing or fermenting eukaryotic cells modified to express the polypeptide(s) of interest. Culturing or fermentation may be carried out in a bioreactor with adequate supply of nutrients, air / oxygen, and / or growth factors. The secreted proteins may be collected by fractionating the culture medium / fermentation broth from the cells, extracting the protein contents, and separating individual proteins to isolate the secreted polypeptide(s). Culturing, fermentation, and isolation techniques are well known to those skilled in the art, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4) th As described in the Edition (incorporated herein by the above reference). A bioreactor comprises one or more vessels in which cells can be cultured. Culturing in a bioreactor may be carried out continuously, involving the continuous inflow of reactants into the reactor and the continuous outflow of cultured cells from the reactor. Alternatively, culturing may be carried out in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, inflow rate into and out of the vessels, and agitation within the vessels, so as to provide optimal conditions for the cells being cultured.

[0334] After culturing cells expressing a polypeptide(s), the polypeptide(s) of interest may be isolated. Any suitable method known in the art for separating proteins from cells may be used. To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated from the culture medium containing the secreted polypeptide(s) of interest by centrifugation. If the polypeptide(s) of interest accumulate within the cells, protein isolation may include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with lysis buffer, and cell disruption by, for example, sonication, rapid freeze-thaw, or osmotic lysis.

[0335] Next, it may be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common method for separating protein components from the supernatant or culture medium is by precipitation. Differently soluble proteins precipitate with different concentrations of precipitating agents, such as ammonium sulfate. For example, low concentrations of precipitating agents extract water-soluble proteins. Thus, differently soluble proteins can be distinguished by adding gradually increasing concentrations of precipitating agents. Dialysis may then be used to remove ammonium sulfate from the separated proteins. Other methods for distinguishing different proteins, such as ion exchange chromatography and size chromatography, are known in the art. These may be used instead of precipitation or performed after precipitation.

[0336] After isolating the polypeptide(s) of interest from the culture, it may be desirable or necessary to concentrate the polypeptide(s). Numerous methods for concentrating proteins, such as ultrafiltration or lyophilization, are known in the art.

[0337] The antigen-binding polypeptide / polypeptide complex according to this disclosure can be conjugated to the linker-payload portion according to this disclosure by any suitable technique that is well known to those skilled in the art and routinely used in the art for the preparation of the antigen-binding molecule according to this disclosure.

[0338] General methods for conjugating antigen-binding polypeptide / polypeptide complexes to linker-payload portions 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). The conjugation of the antigen-binding portion and the linker-payload portion, as well as the purification of the antigen-binding molecule produced by such conjugation, are described, for example, 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, (2016) Wiley.

[0339] General methods for conjugating antigen-binding polypeptide / polypeptide complexes to linker-payload portions 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). The conjugation of the antigen-binding portion and the linker-payload portion, as well as the purification of the antigen-binding molecule produced by such conjugation, are described, for example, 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.

[0340] The antigen-binding portion of this disclosure is well known to those skilled in the art and can be conjugated to the linker-payload portion of this disclosure by any suitable technique commonly 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).

[0341] The conjugation of the antigen-binding portion and the linker-payload portion, and the purification of the antigen-binding molecule produced by such conjugation, may be carried out as described, for example, in Beck et al., (2017) Nat Rev Drug Discov 16:315-337, Peters and Brown Biosci Rep (2015) 35:art:e00225, McCombs and Owen, The AAPS Journal (2015) 17:339-351, Jackson, Org Process Res Dev (2016) 20:852-866, or Olivier and Hurvitz, Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcome to Target Cancer, (2016) Wiley (all of which are incorporated herein by reference in their entirety). Other relevant disclosures regarding conjugations and linkers are as follows: Tsuchikama and An, Protein Cell. (2018) 9(1):33-46, Khongorzul et al., Mol Cancer Res (2020) 18 (1):3-19, and Drago et al., Nature Reviews Clinical Oncology (2021) 18:327-344 (all of which are incorporated herein by reference in their entirety).

[0342] Lysine amide coupling Lysine-based conjugation is one of the most widely used nonspecific conjugation strategies. Such conjugation occurs at the reactive amine side chains of lysine residues due to the good nucleophilicity of those side chains. Immunoglobulin scaffolds contain more than 80 lysine residues, most of which are exposed on the surface of the molecule. Of these surface lysine residues, more than 20 have been shown to have high solvent accessibility and may serve as candidate sites for ADC conjugation. Lysine conjugation follows two main strategies that result in the formation of stable amide or amidine bonds between the protein and the drug-linker complex. Generally speaking, activated esters on the drug-linker complex, often O-succinimide reagents such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS esters, react with antibody lysine residues to achieve amide-bond conjugation. On the other hand, stable amidine bonds can be generated on the antibody by the reaction of imide ester compounds, e.g., Trout's reagents, with antibody lysine residues.

[0343] One-step conjugation of a drug-linker moiety containing an amine-reactive group to an antibody via an amide bond is known, as well as two-step conjugation. In the two-step conjugation, in the first step, a small bifunctional reagent containing both an amine-reactive and a thiol-reactive functional group is reacted with an available lysine ε-amino group to function as a chemical adapter, thereby leaving a free thiol-reactive group on the antibody. In the second step, the payload drug or drug-linker complex is conjugated to the pre-introduced thiol-reactive group to form an ADC. The two-step method is often used as an alternative route when the drug / drug-linker complex contains a thiol-reactive module, or when it is known to be difficult to introduce an amine-reactive module into the drug or drug-linker complex. The following four small adapters are commonly used in two-step conjugation: SPDB disulfide, MCC (maleimidomethylcyclohexane-1-carboxylate), sulfo-SPDB, and hydrazine.

[0344] Cysteine ​​coupling Cysteine ​​modification most commonly results from the addition of a 1,4-conjugate to an N-substituted maleimide. Maleimides are particularly attractive reagents due to their ease of synthesis and rapid reaction rates with cysteine ​​under mild conditions. The resulting thiosuccinimide conjugates are inherently unstable due to their tendency to undergo retro-Michael addition. This instability can be mitigated by forcing hydrolysis of the thiosuccinimide after conjugation, thereby creating a stable chemical bond. Consequently, numerous "autohydrolyzable" maleimides are currently being developed, whose ring-opening is catalyzed by adjacent functional groups, among the most promising being primary amines, polyethylene glycol (PEG), and N-aryl groups. Other reagents include α-halocarbonyls, palladium oxidative addition complexes, ethynylphosphoneamides, vinyl phosphonitrides, and ethynylbenzoiodoxolones.

[0345] Several non-maleimidocysteine ​​conjugations are summarized in Kang, et al., Chem Sci (2021) 12, 13613-13647 (doi:10.1039 / D1SC02973H), and include the following uses: (i) Alkynylcarboxylic acid derivatives; (ii) 5-Methylenepyrrolone (5MP); (iii) 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB); (iv) Phenyloxadiazole sulfone (PODS); (v) Azadibenzocyclooctin (DBCO); (vi) phosphoamidite; (vii)3-arylpropionitrile (APN); (viii) Perfluoroarenes; (ix) Ethinylbenzoiodoxolone (EBX); (x) Bicyclo[1.1.0]butane(BCB) carboxylic acid amide; and (xi) Allenamide.

[0346] Another usable technique in which 2-methylsulfonylpyrimidine is used as a substitute for maleimide is described in Cheng, et al., Front. Oncol. 12:951589 (doi: 10.3389 / fonc.2022.951589).

[0347] Genetic modification of the number of accessible cysteine ​​residues on the antibody surface is a method for achieving site-selective and uniform modification. For example, in THIOMABs, genetically modified cysteine, mutated at heavy-chain alanine 114 (HC-A114), is introduced into anti-MUC16 antibodies. In other methods, antibodies are genetically modified to contain cysteine ​​mutations such as D265C, S239C, E269C, K326C, or A327C, or to insert additional cysteine ​​before or after the positions of HC-S239, HC-A114, and LC-V205.

[0348] Incorporation of non-natural amino acids through genetic engineering Site-specific incorporation of non-standard amino acids (ncAAs) into antibodies provides an efficient method for site-specific modification of antibodies and the resulting homogeneous ADCs.

[0349] NcAAs possessing unique functional groups, such as ketone, azide, cyclopropene, or diene functional groups, have been developed and incorporated into antibodies. Examples of such ncAAs include p-acetylphenylalanine (pAcF) with a ketone side chain capable of participating in oxime ligation reactions; Nε-(1-methylcyclopropa-2-enecarboxamide)-lysine (CpK) with a cyclopropene side chain capable of participating in IEDDA reactions; para-azide methylphenylalanine (pAMF) with an azide side chain capable of causing click reactions; spiro[2.4]hepta-4,6-diene-lysine (SCpHK) with a spiro[2.4]hepta-4,6-diene side chain capable of participating in Diels-Alder reactions; and N6-(2-azideethoxy)-carbonyl-L-lysine (AzK) with an azide side chain capable of causing click reactions.

[0350] Azide-containing ncAAs can undergo rapid CuAAC or SPAAC reactions under physiological conditions. Para-azidophenylalanine (pAzF) can react with, for example, cyclooctin-functionalized linkers and dibenzylcyclooctin (DBCO)-functionalized linkers. The cyclopropene derivative of lysine (Nε-[((2-methylcyclopropane-2-en-1-yl)methoxy)carbonyl]-l-lysine; CypK) can undergo rapid and efficient reverse electron-demanded Diels-Alder (IEDDA) reactions with tetrazine-functionalized linkers.

[0351] Cyclopentadiene-containing ncAAs, spiro[2,4]hepta-4,6-diene-lysine (SCpHK), and cyclopentadiene-lysine (CpHK) can undergo irreversible Diels-Alder cyclization with maleimide-modified drugs.

[0352] Enzyme-mediated conjugation Enzymes can be used to achieve site-selective antibody modification due to their high specificity and mild reaction conditions. Enzymes can either directly conjugate a payload to a specific amino acid sequence, or introduce reactive functional groups into an antibody that can be further functionalized using the desired payload.

[0353] Peptide transfer using saltase Soltase-mediated antibody conjugation (SMAC) technology is an enzymatic ligation method. SMAC technology utilizes S. aureus saltase A, a transpeptidase that cleaves the amide bond between the threonine and glycine residues in the LPXTG (X = any amino acid) pentapeptide motif and subsequently catalyzes the binding of the glycine-functionalized payload to the newly generated C-terminus. A saltase-recognition motif and a Strep II tag, used to assist in the removal of unreacted antibodies, are fused to the light and heavy chain C-terminuses of various antibodies. Soltase-mediated conjugation can then be used to conjugate a series of pentaglysin-tagged payloads.

[0354] Peptide transfer using microbial transglutaminase The use of bacterial transglutaminase is a powerful technique for site-specific incorporation of payloads into antibodies. Transglutaminase derived from Streptomyces mobaraensis catalyzes a peptide transfer in which a primary amine-containing linker is covalently bonded to the primary amide side chain of a specific glutamine (Q295) in a deglycosylated antibody, resulting in an ADC with a predetermined DAR, which is caused by two linker-payload conjugations (one conjugation site per heavy chain). A pre-conjugation N297Q mutation results in two additional reaction sites (resulting in four linker-payload conjugations). An alternative version uses peptide sequence-specific transglutaminase. This enzyme recognizes and utilizes a genetically integrated LLQG motif, thereby resulting in site-specific antibody-drug conjugation. Another advantage of this LLQG-specific bacterial transglutaminase is the flexibility in positioning the conjugation site by inserting this short peptide motif into the antibody structure. Further alternative methods allow for the use of transglutaminase without deglycosylation.

[0355] DAR depends on the number of payloads per linker-payload portion being conjugated.

[0356] N-glycan operation Asn297 (N297) within the Fc domain and the N-glycan on this residue are conserved across all IgG classes, making these components attractive reaction sites for widely applicable ADC conjugations. By incorporating aldehyde groups into the N-glycan termini using β-1,4-galactosyltransferase (GalT) and α-2,6-sialyltransferase (SialT), sialic acid is introduced to each N-glycan termini, which is then converted to an aldehyde group under mild oxidation conditions using NaIO4. The resulting aldehyde group can then be used to conjugate an aminooxy-functionalized payload.

[0357] Another approach involves incorporating a non-natural saccharide with an orthogonal reaction handle into the antibody. A technique based on this strategy is GlycoConnect, in which the glycan chain of Asn297 is removed using the endoglycosidase Endo S2, and then the azide group is introduced using the mutant galactosyltransferase GalT (Y289L) and N-azidoacetylgalactosamine (GalNAz). The azide handle can then be used in a strain-enhanced click reaction with the payload.

[0358] In some embodiments, the linker-payload is terminated with an amino group, which is conjugated to an antigen-binding molecule using transglutaminase.

[0359] In some embodiments, the method further includes purifying / isolating the antigen-binding molecule (i.e., from unreacted precursors and / or by-products). In some embodiments, the antigen-binding molecule may be purified / isolated by chromatography, for example, size exclusion chromatography.

[0360] This disclosure also provides antigen-binding molecules that can be obtained or obtained by the methods of this disclosure.

[0361] composition This disclosure provides compositions comprising antigen-binding molecules as described herein.

[0362] The antigen-binding molecules described herein may be formulated as pharmaceutical compositions or agents for clinical use and may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Accordingly, this disclosure provides pharmaceutical compositions / agents comprising the antigen-binding molecules described herein.

[0363] The pharmaceutical compositions / agents of this disclosure include one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), auxiliaries, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants It may contain agents (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).

[0364] As used herein, the term “pharmaceutically acceptable” means a compound, component, material, composition, dosage form, etc., that is suitable for use in contact with the tissue of the subject in question (e.g., a human subject) without causing excessive toxicity, irritation, allergic response, or other problems or complications, within the bounds of sound medical judgment, and that is commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, auxiliary, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener of a composition disclosed herein must also be “acceptable” in the sense that it is compatible with the other components of the formulation. Suitable carriers, diluents, excipients, auxiliaries, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, or sweeteners are listed in standard pharmaceutical textbooks, e.g., Remington's 'The Science and Practice of Pharmacy' (Ed. A. Adejare), 23 rdThis can be found in Edition (2020), Academic Press.

[0365] The pharmaceutical compositions and agents of this disclosure may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, subarachnoid, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, subarachnoid, oral, or transdermal administration routes. In some embodiments, the pharmaceutical compositions / agents may be formulated for administration by injection or infusion, or by oral ingestion.

[0366] Suitable formulations may include antigen-binding molecules provided in a sterile or isotonic medium. Drugs and pharmaceutical compositions may be formulated in liquid form, including gel form. Liquid formulations may be formulated for administration by injection or infusion (e.g., via catheter) to a selected area of ​​the human or animal body.

[0367] In some embodiments, the pharmaceutical composition / drug is formulated for injection or infusion into, for example, blood vessels, tissues / organs of interest, or tumors.

[0368] This disclosure also provides a method for preparing pharmaceutically useful compositions, which may include one or more steps selected from the following: To produce antigen-binding molecules as described herein, To isolate / purify the antigen-binding molecules described herein, and / or mixing the antigen-binding molecules described herein with a pharmaceutically acceptable carrier, auxiliary agent, excipient, or diluent.

[0369] For example, further aspects of this disclosure relate to a method for formulating or preparing a drug or pharmaceutical composition for use in the treatment of a disease / condition (e.g., a disease / condition described herein), the method comprising formulating the pharmaceutical composition or drug by mixing an antigen-binding molecule described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0370] Therapeutic and preventive applications The antigen-binding molecules and compositions described herein are useful in the treatment and prevention of diseases, such as cancer.

[0371] It will be understood that the antigen-binding molecules and compositions of this disclosure may be used to treat / prevent any disease / condition that would benefit from a reduction in the level of HER2 expression or activity, or a reduction in the number or activity of cells containing / expressing HER2.

[0372] For example, a disease / condition may be a disease / condition in which cells expressing / overexpressing HER2 or HER2 are pathologically involved, such as an increase in the level / activity of HER2, or an increase in the number / ratio of cells containing / expressing HER2, which may be positively associated with the onset, progression, or advancement of the disease / condition, and / or the severity of one or more symptoms of the disease / condition. In some embodiments, an increase in the level / activity of HER2, or an increase in the number / ratio of cells containing / expressing HER2, may be a risk factor for the onset, progression, or advancement of the disease / condition.

[0373] This disclosure provides antigen-binding molecules or compositions described herein for use in methods of medical treatment or prevention. Also provided are antigen-binding molecules or compositions described herein for use in methods of treating or preventing cancer (e.g., cancers described herein). Also provided are the use of antigen-binding molecules or compositions described herein in the manufacture of agents for treating or preventing cancer (e.g., cancers described herein). Also provided are methods of treating or preventing a target cancer (e.g., cancers described herein), comprising administering a therapeutically or prophylactically effective amount of an antigen-binding molecule or composition described herein to a target.

[0374] This method may be effective in reducing the progression or progression of cancer, alleviating cancer symptoms, or reducing cancer pathology. This method may be effective in preventing cancer progression, for example, by preventing cancer worsening or slowing the rate of cancer progression. In some embodiments, this method may result in cancer improvement, for example, a reduction in cancer symptoms or a reduction in several other correlations of cancer severity / activity. In some embodiments, this method may prevent progression to later stages of cancer (e.g., chronic or metastatic).

[0375] As used herein, “cancer” means any unwanted cell proliferation (or any disease resulting from such unwanted cell proliferation), neoplasm, or tumor, or may include such a condition. Cancer may be benign or malignant. Cancer may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be present in any tissue. Cancer may originate from tissues / cells of the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, bile duct, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, uterine muscle, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, or leukocytes.

[0376] The tumors treated may be neurological or non-neurological tumors. Neurological tumors may originate from either the central or peripheral nervous system (e.g., glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwann cell tumor, neurofibrosarcoma, astrocytoma, and oligodendroglioma). 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 syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermal carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, hematological cancer, and sarcoma.

[0377] In some embodiments, the cancer to be treated / prevented includes cells expressing EGFR family members (e.g., HER2, EGFR, HER3, or HER4) and / or ligands of EGFR family members. In some embodiments, the cancer to be treated / prevented includes cells expressing variants or wild-type versions of EGFR family members (e.g., HER2, EGFR, HER3, or HER4). In some embodiments, the cancer to be treated / prevented is a cancer that is positive for EGFR family members. In some embodiments, the cancer includes cells that overexpress EGFR family members and / or ligands of EGFR family members. Overexpression can be determined by detecting expression levels higher than those of corresponding non-cancerous cells / non-tumor tissues.

[0378] Expression can be measured by any suitable means. Expression may be gene expression or protein expression. Gene expression can be measured, for example, by detecting mRNA encoding HER2, for example by quantitative real-time PCR (qRT-PCR). Protein expression can be measured, for example, by antibody-based methods, for example by Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[0379] In some embodiments, cancer is a cancer in which HER2 is pathologically involved. That is, in some embodiments, cancer is a cancer caused or exacerbated by HER2 expression, a cancer in which HER2 expression is a risk factor, and / or a cancer in which HER2 expression is positively associated with the onset, progression, progression, severity, or metastasis of the cancer. Cancer may be characterized by HER2 expression, for example, cancer may include cells that express HER2 (e.g., cells in tumor tissue). Such cancer may be referred to as HER2-positive. Cancer that is "HER2-positive" may include cells that express HER2 (e.g., on the cell surface). Cancer that is "HER2-positive" may overexpress HER2.

[0380] In some embodiments, the cancer being treated / prevented includes cells having a gene variant (e.g., a mutation) that causes enhanced HER2 (gene and / or protein) expression and / or activity compared to equivalent cells having a reference allele (e.g., a non-mutant or "wild-type" allele) that does not contain the gene variant. The gene variant may be, or include, an insertion, deletion, substitution, or a larger translocation / relocation thereof in the nucleotide sequence compared to the reference allele.

[0381] Mutations that “result” in increased HER2 expression may be known, predicted, or associated with increased HER2 gene / protein expression. Mutations that “result” in increased HER2 activity may be known, predicted, or associated with increased HER2-mediated signaling and / or EGFR-mediated signaling. Mutations that result in increased HER2 expression and / or activity may be referred to as “activating” mutations.

[0382] Mutations that cause enhanced HER2 expression may result in the expression of the HER2 gene or protein that is not expressed in equivalent cells that do not carry the mutation, and / or encoded by their genomic nucleic acid. In other words, HER2 expression can be a result of a mutation, and therefore, "enhanced expression" can be an enhancement of expression from non-expression.

[0383] Mutations that cause enhanced HER2 expression may result in enhanced expression of the HER2 gene or protein in equivalent cells that do not contain the mutation, and / or in the HER2 gene encoded by their genomic nucleic acid. For example, a cell may contain the mutation that results in an increased transcription level of the HER2-encoding nucleic acid compared to the transcription level of the HER2-encoding nucleic acid in equivalent cells that do not contain the mutation.

[0384] In some embodiments, a mutation that causes enhanced HER2 expression may result in enhanced HER2 gene expression compared to equivalent cells that do not contain the mutation.

[0385] In some embodiments, a mutation that enhances HER2 expression may result in an increase in the level of HER2 on the cell surface or at the cell surface of cells containing the mutation compared to equivalent cells that do not contain the mutation.

[0386] Cells exhibiting enhanced HER2 expression (e.g., as a result of a mutation) compared to the HER2 expression level of reference cells may be described as “overexpressing” HER2 or “upregulated” HER2 expression. For example, a cancer containing cells with a mutation that results in enhanced HER2 expression compared to equivalent cells lacking the mutation may be described as a cancer containing cells exhibiting HER2 overexpression / upregulated expression. In some embodiments, the reference cells lacking the mutation may be non-cancerous cells (e.g., equivalent cell types) or cancerous cells (e.g., equivalent cancer types).

[0387] Mutations that enhance HER2 activity may result in enhanced HER2-mediated signaling compared to the level of HER2-mediated signaling in equivalent cells that do not contain the mutation.

[0388] In some embodiments, cancer treated / prevented in accordance with this disclosure may be characterized, for example, by enhanced HER2 expression and / or activity (i.e., gene and / or protein expression) in the diseased organ / tissue / subject compared to a normal organ / tissue / subject (i.e., in the absence of the disease / condition). In some embodiments, cancer cells and / or tumors treated / prevented may be characterized by enhanced HER2 expression and / or activity compared to, for example, the levels of expression and / or activity observed in equivalent non-cancerous cells / non-tumor tissues.

[0389] HER2-overexpressing cancers can overexpress HER2 as a result of amplification of the ERBB2 gene.

[0390] In some embodiments, the cancer treated / prevented in accordance with this disclosure is ERBB2-amplified cancer.

[0391] ERBB2 amplification can be identified using techniques well known in the art, for example, by immunohistochemical analysis and in situ hybridization (see, e.g., Wesola and Jelen, Adv Clin Exp Med. (2015) 24(5):899-903). For example, ERBB2 amplification can be evaluated by fluorescence in situ hybridization, as described, e.g., Stocker et al., PLoS One (2016) 11(7):e0159176. When measured by in situ hybridization, ERBB2-to-centromere 17 (CEP17) ratios of 2 or more (e.g., 4 or more, 8 or more) may be present.

[0392] The association and role of HER2 with cancer are outlined, for example, in Oh and Bang, Nat Rev Clin Oncol (2020) 17:33-48, Hudis, NEJM (2007) 357(1):39-51, Arteaga and Engelman, Cancer Cell. (2014) 25(3):282-303, and Yan et al., Cancer Metastasis Rev. (2015) 34(1):157-164 (all of which are incorporated herein by reference in their entirety).

[0393] ERBB2 amplification has been observed in various cancers, including breast cancer, gastric cancer, and esophageal cancer (Koboldt et al., Nature. (2012) 490:61-70). Potential activating mutations in HER2 (i.e., gain-of-function mutations) have also been reported in cancers such as lobular breast cancer, lung cancer, gastric cancer, bladder cancer, and endometrial cancer. A very high percentage of the following cancers express HER2 (i.e., are HER2-positive; see Table 2 in Yan et al., Cancer Metastasis Rev. (2015) 34(1):157-164): bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, esophageal / esophagogastric junction cancer, gallbladder cancer, gastric adenocarcinoma, gastrointestinal stromal tumor, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, colon cancer, kidney cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, melanoma, neuroendocrine tumor, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibrous tumor, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer.

[0394] In some embodiments, cancer is selected from: cancers containing cells expressing / overexpressing EGFR family members, cancers containing cells expressing / overexpressing HER2, cancers containing cells not overexpressing EGFR family members, cancers containing cells not overexpressing HER2, HER2-low-expressing cancers, HR-positive cancers, solid tumors, bladder cancer, breast cancer, HER2-positive breast cancer, metastatic HER2-positive breast cancer, HER2-low-expressing breast cancer, unresectable or metastatic HER2-low-expressing breast cancer, HR-positive breast cancer, triple-negative breast cancer, cervical cancer, gastric cancer, HER2-positive gastric cancer, locally advanced or metastatic HER2-positive gastric cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer / gastric junction cancer, gallbladder cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, HER2-positive gastric cancer / gastric junction adenocarcinoma, locally advanced or metastatic HER2-positive gastric cancer / gastric junction adenocarcinoma, gastrointestinal stromal tumors, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, intestinal cancer, kidney cancer, lung cancer, non-small cell lung cancer, unresectable or metastatic non-small cell lung cancer with ERBB2 activating mutations, small cell lung cancer, melanoma, neuroendocrine tumors, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibrous tumor, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer.

[0395] In some embodiments, the cancers according to this disclosure are cancers for which fam-trastuzumab deruxtecan-nxki is an approved therapeutic agent. In some embodiments, the cancers are 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 with activating ERBB2 mutations; and locally advanced or metastatic HER2-positive gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

[0396] In some embodiments, cancer is selected from: cancers comprising cells that do not overexpress EGFR family members (e.g., HER2, EGFR, HER3, or HER4); cancers comprising 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 comprising cells expressing 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.

[0397] In this specification, “HER2 low-expression” cancer means cancer with an immunohistochemical (IHC) score of 1+ for HER2 expression or cancer with an IHC score of 2+ for HER2 expression, provided that such cancer does not include ERBB2 amplification as determined by in situ hybridization (ISH) analysis. IHC analysis, HER2 expression scoring, and ISH analysis of ERBB2 amplification are described, for example, in Wolff et al., J Clin Oncol. (2018) 36(20):2105-2122 (which is incorporated herein by reference in its entirety).

[0398] In this specification, "ERBB2 activating mutations" may enhance ERBB2 transcription, increase the level of ERBB2-encoded RNA, decrease the degradation of ERBB2-encoded RNA, increase the level of ERBB2-encoded protein, enhance (promote) normal splicing of ERBB2-encoded premRNA, enhance translation of mRNA encoding ERBB2-encoded protein, enhance (promote) normal post-translational processing of ERBB2-encoded protein, enhance (promote) normal transport of ERBB2-encoded protein, decrease the degradation of ERBB2-encoded protein, increase the functional level of ERBB2-encoded protein, and / or confer new properties to ERBB2-encoded protein.

[0399] In some embodiments, cancer is selected from: cancers comprising cells that do not overexpress EGFR family members (e.g., HER2, EGFR, HER3, or HER4); cancers comprising 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 comprising cells expressing 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.

[0400] In some embodiments, the cancer may be recurrent cancer. As used herein, “recurrent” cancer refers to cancer that responded to treatment (e.g., first-line cancer treatment) but subsequently reappeared / progressed, for example, after a period of remission. For example, recurrent cancer may be cancer whose growth / progression was inhibited by treatment (e.g., first-line cancer treatment) and subsequently proliferated / progressed. Cancer that is recurrent to a given treatment may be described as having acquired resistance to such treatment.

[0401] In some embodiments, the cancer may be a treatment-resistant cancer. As used herein, “treatment-resistant” cancer refers to cancer that has not responded to treatment (e.g., first-line cancer treatment). For example, a treatment-resistant cancer may be cancer whose growth / progression was not inhibited by treatment (e.g., first-line cancer treatment). In some embodiments, a treatment-resistant cancer may be cancer in which a subject treated for cancer did not show a partial or complete response to said treatment. A cancer that is treatment-resistant to a given treatment may be described as having intrinsic resistance to such treatment.

[0402] In some embodiments, the cancer is recurrent or refractory to treatment with DNA damage response (DDR) inhibitors. In some embodiments, the cancer is refractory to treatment with DDR inhibitors. In some embodiments, the cancer is recurrent to treatment with DDR inhibitors. In some embodiments, the cancer has intrinsic resistance to treatment with DDR inhibitors. In some embodiments, the cancer has acquired resistance to treatment with DDR inhibitors. According to such embodiments, the DDR inhibitor may be administered in the form of an antigen-binding molecule containing a payload portion comprising the DDR inhibitor, or in a non-conjugate form.

[0403] In this specification, when cancer is described as recurrent / treatment-resistant / resistant to a given intervention, it may simply be expressed as “recurrent / treatment-resistant / resistant” to the intervention in question.

[0404] The use of DDR inhibitors and their application in the treatment of cancer is described, for example, in Cheng et al., Eur J Med Chem. (2022) 230:114109, Wang et al., Front Immunol. (2022) 13:854730, and Choi and Lee, Int J Mol Sci. (2022) 23(3):1701 (all of which are 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, lucaparib, niraparib, talazoparib, veliparib, pamiparib, simmiparib, senaparib, SC-10914, 2X-121, AMXI-5001, JPI-547, AZD5305, IDX-1197, TQB-3823, HWH-340, AsiDNA, STP-100) 2. RBN-2397), ATM inhibitors (e.g., CP-466722, KU-55933, KU-60019, KU-59403, AZ31, AZ32, AZD0156, AZD1390), ATR inhibitors (e.g., M6620 (belzocertib), M4344 (VX-803), AZD6738 (ceraracertib), BAY1895344 (elimsertib)), WEE1 inhibitors (e.g., adavocertib, Debio 0123, PD0166285, PD0407824, AZD1775), CHK1 / 2 inhibitors (e.g., CBP-501, prexasertib, MK-8776, GDC-0575, SRA-737), DNA-PK inhibitors (e.g., CC-115, LY-3023414, AsiDNA, M3814 (Nedisertib), M9831 (VX-984)), and PLK1 inhibitors (e.g., BI-6727 (Voracertib), PCM-075 (Onvancertib)).

[0405] In some embodiments, the cancer is recurrent or refractory to treatment with DNA topoisomerase I (TOP1) inhibitors. In some embodiments, the cancer is refractory to treatment with TOP1 inhibitors. In some embodiments, the cancer is recurrent to treatment with TOP1 inhibitors. In some embodiments, the cancer has intrinsic resistance to treatment with TOP1 inhibitors. In some embodiments, the cancer has acquired resistance to treatment with TOP1 inhibitors. According to such embodiments, the TOP1 inhibitor may be administered in the form of an antigen-binding molecule containing a payload portion comprising the TOP1 inhibitor, or in a non-conjugate form.

[0406] The use of DNA topoisomerase I inhibitors and their application in the treatment of cancer is described, for example, 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). In some embodiments, the TOP1 inhibitors according to this disclosure are selected from: camptothecin, irinotecan, etilinotecan, SN-38, DX-8951f (exatecan mesylate), DXd(1), DXd(2), exatecan, FL118, topotecan, gimatecan, berotecan, deruxtecan, berotecan, rubitecan, raltotecan, diflomotecan, karenitecan, silatecan, namithecan, aeromothecan, DRF-1042, delimotecan, NSC606985, quimitecan, ZBH-1205, Genz-644282, non-CPT1, indothecan (LMP-400), indimitecan (LMP-776), and LMP744.

[0407] In some embodiments, the cancer treated / prevented in accordance with this disclosure is a cancer that is recurrent or resistant to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein) and recurrent or resistant to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). In some embodiments, the cancer is resistant to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein) and resistant to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). In some embodiments, the cancer is recurrent to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein) and resistant to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). In some embodiments, the cancer is resistant to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein) and resistant to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). In some embodiments, the cancer is recurrent to treatment with DDR inhibitors (e.g., DDR inhibitors as described herein) and resistant to treatment with TOP1 inhibitors (e.g., TOP1 inhibitors as described herein). According to such embodiments, the TOP1 inhibitor and / or DDR inhibitor may be in the form of an antigen-binding molecule containing a payload portion comprising a TOP1 inhibitor / DDR inhibitor, or may be administered in a non-conjugated form.

[0408] Treatment of cancer by the methods of the present disclosure achieves one or more of the following treatment effects: reduction of the number of cancer cells in the subject; reduction of the size of cancerous tumors / lesions in the subject; inhibition of cancer cell proliferation (e.g., prevention or slowing) in the subject; inhibition of cancer tumor / lesion growth (e.g., prevention or slowing) in the subject; inhibition of cancer progression (e.g., to late stages or metastasis) (e.g., prevention or slowing); reduction of the severity of cancer symptoms in the subject; extension of survival (e.g., progression-free survival or overall survival) in the subject; reduction of the number or activity of cancer cells in the subject; and / or reduction of cancer burden in the subject.

[0409] The subjects may be evaluated according to the revised therapeutic response criteria: Lugano classification (for example, as described in Cheson et al., J Clin Oncol (2014) 32:3059-3068, incorporated above by reference) to determine the response to the treatment. In some embodiments, treatment of the subjects by the method of this disclosure achieves one of the following: complete response, partial response, or disease stabilization.

[0410] Prevention may refer to the prevention of the onset of cancer and / or the progression of cancer, such as the prevention of progression to later stages of cancer (e.g., metastasis).

[0411] In some embodiments, administration of an antigen-binding molecule / composition according to this disclosure may be associated with one or more of the following: inhibition of cancer onset / progression, delay / prevention of cancer onset, 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 symptoms of cancer, reduction of the number of cancer cells, reduction of cancer burden, reduction of tumor size / volume, and / or extension of survival (e.g., progression-free survival or overall survival) of a subject with cancer.

[0412] Depending on 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 extending the survival time of subjects with cancer.

[0413] Various aspects of this disclosure provide methods for or including (for example, cancer, e.g., in the context of the treatment / prevention of cancer as described herein): Binding to cells expressing HER2; Inhibition of proliferation of HER2-expressing cells; Killing cells that express HER2; Inhibition of tumor growth and / or reduction of tumor size / volume, e.g., inhibition of tumor growth and / or reduction of tumor size / volume in HER2-expressing cancers; and / or Extending the survival time of patients with cancer, such as HER2-expressing cancer.

[0414] The present disclosure provides antigen-binding molecules and compositions for use in such methods, as well as the use of the present disclosure antigen-binding molecules and compositions in the manufacture of compositions (e.g., pharmaceuticals) for use in such methods. It will be understood that the methods typically involve administering the antigen-binding molecules of the present disclosure to a target.

[0415] Similarly, one or more of the following may be observed in subjects after the treatment or preventive intervention provided by this disclosure (compared to, for example, pre-intervention levels / numbers / proportions): Inhibition of proliferation of HER2-expressing cells; Killing cells that express HER2; Inhibition of tumor growth and / or reduction of tumor size / volume, e.g., inhibition of tumor growth and / or reduction of tumor size / volume in HER2-expressing cancers; and / or Extending the survival time of patients with cancer, such as HER2-expressing cancer.

[0416] In some embodiments, the therapeutic / preventive interventions described herein may be expressed as “associated” with one or more of the effects described in the preceding paragraph. Those skilled in the art can readily evaluate such properties using techniques routinely practiced in the art.

[0417] The administration of the antigen-binding molecules and compositions of this disclosure is preferably in a “therapeutably effective” or “preventively effective” dose, which is sufficient to demonstrate a therapeutic or preventive effect on the subject. The actual amount administered, as well as the rate of administration and its course over time, depends on the nature and severity of the disease / condition and the specific item being administered. The prescription of the treatment, e.g., the determination of the dosage, is the responsibility of the general practitioner and other medical doctors, and typically takes into account the disease / disorder being treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols described above can be found in Remington's 'The Science and Practice of Pharmacy' (Ed. A. Adejare), 23 rd This can be found in Edition (2020), Academic Press.

[0418] Administration of the antigen-binding molecules and compositions of this disclosure may, for example, be parenteral, systemic, topical, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, subarachnoid, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral, or transdermal. Administration may be by injection, infusion, or oral ingestion.

[0419] In some embodiments and aspects, the articles of the Disclosure may be administered to a tissue / organ of interest (e.g., a tissue / organ affected by a disease / condition, e.g., a tissue / organ on which symptoms of the disease / condition appear). In some embodiments and aspects, the articles of the Disclosure may be administered into the bloodstream by injection or infusion (e.g., via a cannula) (i.e., intravenous / intra-arterial administration), or subcutaneously or orally. In some embodiments and aspects, the articles of the Disclosure may be administered to a tumor.

[0420] In some embodiments, the therapeutic or preventive interventions described herein may further include administering another agent for the treatment / prevention of the relevant disease / condition. The antigen-binding molecules and compositions described herein may be administered alone or in combination with other treatments, concurrently or sequentially, depending on the condition being treated. Concurrent administration means administering together with another therapeutic agent, for example, as a pharmaceutical composition (combination preparation) containing both agents, 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., into the same tissue, artery, vein, or other blood vessel). Sequential administration means administering one agent followed by another agent separately after a given time interval. The two agents do not need to be administered via the same route, but in some embodiments, they are so. The time interval can be any time interval.

[0421] Repeated doses of antigen-binding molecules and compositions may be provided. Repeated doses may be separated by predetermined time intervals, which may be selected to be 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. As an example, doses may be given once every 7, 14, 21, or 28 days (±3, 2, or 1 day).

[0422] Detection method This disclosure also provides articles of this disclosure for use in methods for detecting, identifying, or imaging HER2 or cells expressing HER2.

[0423] The antigen-binding molecules described herein may be used in methods comprising detecting the binding of the antigen-binding molecule to HER2. Such methods may include detecting the binding complex of the antigen-binding molecule and HER2. It will be understood that HER2 may be HER2 expressed by cells, for example, HER2 expressed inside or on the surface of cells that express HER2.

[0424] Accordingly, a method is provided that includes contacting a sample containing or suspected of containing HER2, and detecting the formation of a complex between an antigen-binding molecule and HER2. A method is also provided that includes contacting a sample containing or suspected of containing cells expressing HER2, and detecting the formation of a complex between an antigen-binding molecule and cells expressing HER2.

[0425] Preferred method formats, including immunoassays such as sandwich assays and ELISAs, are well known in the art. These methods may involve labeling an antigen-binding molecule or target(s) or both with a detectable portion, such as a fluorescent label, phosphorescent label, luminescent label, immunodetectable label, radioactive label, chemical, nucleic acid, or enzymatic label as described herein. Detection techniques are well known to those skilled in the art and may be selected in correspondence with the labeling agent.

[0426] Methods involving the detection of HER2 or cells expressing HER2 include methods for diagnosing / predicting the prognosis of the diseases / conditions described herein.

[0427] This type of method can be performed in vitro on or after processing of a patient sample. Since the patient does not need to be present to perform the in vitro method once the sample has been collected, this method may not be performed on a human or animal body. In some embodiments, the method is performed in vivo.

[0428] Such methods may include, for example, detecting or quantifying HER2 and / or HER2-expressing cells in a patient sample. If the method includes quantifying relevant factors, it may further include comparing the measured amounts to standard or reference values ​​as part of a diagnosis or prognosis assessment. Other diagnostic / prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis prediction, or to confirm the results obtained by using the tests described herein.

[0429] Detection in a sample may be used for diagnosing a disease / condition (e.g., cancer), predisposition to a disease / condition, or to provide prognosis prediction (determine prognosis) for a disease / condition, such as those described herein. The diagnosis or prognosis prediction may be related to an existing (previously diagnosed) disease / condition.

[0430] The sample may be taken from any tissue or bodily fluid. The sample may include, or may be derived from: a certain amount of blood; a certain amount of serum derived from the blood of an individual, which may include the fluid portion of the blood obtained after the removal of fibrin thrombi and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from the individual. In some embodiments, the sample may be taken from, or may be derived from, a tissue or multiple tissues affected by the disease / condition (e.g., a tissue or multiple tissues on which the symptoms of the disease appear or which are involved in the pathogenesis of the disease / condition).

[0431] Subjects may be selected for diagnostic / prognostic evaluation based on the presence of symptoms indicating the diseases / conditions described herein, or based on subjects who are considered to be at risk of developing the diseases / conditions described herein.

[0432] This disclosure also provides methods for selecting / stratifying subjects for treatment with HER2-targeted drugs. In some embodiments, subjects are selected for treatment / prevention by the methods of this disclosure, or identified as subjects who would benefit from such treatment / prevention, based, for example, on the detection / quantification of HER2 or HER2-expressing cells in a sample taken from an individual.

[0433] subject In the embodiments described herein, the subject may be any animal or human. The subject may be a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. The subject may be a patient. The subject may have been diagnosed with a disease or condition requiring treatment (e.g., cancer, e.g., cancer as described herein), may be suspected of having such a disease / condition, or may be at risk of developing / being affected by such a disease / condition.

[0434] In some embodiments, the subjects treated according to the therapeutic or preventive methods of the Disclosure herein are subjects who have cancer, for example, cancer as described herein, or who are at risk of developing it. In embodiments of the Disclosure herein, subjects may be selected for treatment by the Method based on characterization of certain markers for such disease / condition.

[0435] In some embodiments, a patient may be selected for the treatment described herein based, for example, on the detection of HER2-expressing / overexpressing cancer in a sample taken from the subject (e.g., tumor biopsy material).

[0436] kit This disclosure also provides a kit of parts. The kit provided by this disclosure may include components for carrying out the methods described herein, either in whole or in part.

[0437] The kit may have at least one container containing a predetermined amount of an antigen-binding molecule or composition as described herein.

[0438] In some aspects of this disclosure, a kit of parts is provided. In some embodiments, the kit may comprise an antigen-binding molecule or composition described herein, which may be provided in predetermined quantities.

[0439] The kit may provide the antigen-binding molecules or compositions described herein, along with instructions for use to administer them to a patient to treat a specific disease / condition (e.g., one of the diseases / conditions described herein, e.g., cancer).

[0440] The kit may provide an antigen-binding portion and a linker-payload portion according to the disclosure. The kit may further include reagents for conjugating the antigen-binding portion and the linker-payload portion.

[0441] The kit may further include reagents, buffers, and / or reference materials required to carry out the methods according to this disclosure. The kit according to this disclosure may include, for example, instructions for use in the form of an instruction booklet or leaflet. The instructions for use may include protocols for carrying out any one or more of the methods described herein.

[0442] 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 the sequences have been aligned and gaps introduced, if necessary, to achieve the maximum possible sequence identity percentage between the sequences. Pairwise and multiple sequence alignments for determining the percentage of sequence identity between two or more amino acid sequences or nucleic acid sequences can be achieved using various methods known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Soding, 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 such software, it is preferable to use default parameters, such as default parameters for gap penalty and elongation penalty.

[0443] array [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13]

[0444] This disclosure includes the described embodiments and preferred feature combinations unless such combinations are clearly impossible or expressly avoided.

[0445] The headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subjects described.

[0446] Next, aspects and embodiments of this disclosure are illustrated by reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0447] Throughout this Specification, including the subsequent claims, unless the context requires otherwise, the words “comprise” and variations such as “comprises” and “comprising” are understood to mean including any explicitly stated component or step or group of components or steps, but not to mean excluding any other component or step or group of components or steps.

[0448] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural nouns unless otherwise explicitly indicated by the context. In this specification, a range may be expressed as “about” one particular value and / or “about” another particular value. Where such a range is expressed, an alternative embodiment encompasses one particular value and / or the other. Similarly, where a value is expressed as an approximation using the antecedent “about,” it will be understood that the particular value forms an alternative embodiment.

[0449] Where nucleic acid sequences are disclosed or referred to herein, their reverse complements are also expressly intended.

[0450] The methods described herein may preferably be performed in vitro. The term “in vitro” is intended to encompass procedures performed using cells in culture, while the term “in vivo” is intended to encompass procedures using / for intact multicellular organisms.

[0451] In this specification, values ​​may be expressed as "approximately" a specific value. Similarly, in this specification, ranges may be expressed as "approximately" from one specific value and / or "approximately" to another specific value. The term "approximately" in relation to numbers is optional and means, for example, + / - 10%. For example, a reference to "approximately 10%" should be interpreted as 9% to 11%. Wherever "approximately" is used in this specification, the value it precedes is also specifically intended. For example, a reference to "approximately 10%" also specifically intends 10%. [Examples]

[0452] Example 1. In vitro combination of DDR inhibitor and TOP1 inhibitor To further demonstrate the advantages of combining DDR inhibitors and TOP1 inhibitors, certain inhibitors were combined in the following two-dimensional growth assay.

[0453] cell line HCT-116 HEC-1B Test compound Exatecan (MedChemExpress, #HY-13631) Ceraracertib (MedChemExpress, #HY-19323) Plexacertib (MedChemExpress, #HY-18174) contrast Vehicle control (0.25% DMSO) Cells only Detection reagent Cell Titre Glo 2.0, Promega #G9243

[0454] HEC-1B cells were seeded at a density of 7,000 cells per well, and HCT-116 cells at a density of 3,000 cells per well, in wells of a 96-well white plate, with each well containing 150 μl of culture medium. In single-compound experiments, 25 μl of the test compound and 25 μl of culture medium were added to each well at different concentrations. In synergistic experiments, 25 μl of compound 1 and 25 μl of another compound were added to each well at different concentrations. The plates were then incubated at 37°C and 5% CO2 for 3 days. After incubation, 50 μl of detection reagent was added per well and the plates were shaken at 600 rpm for 20 minutes. The resulting luminescence was measured using a Perkin Elmer Victor Nivo, and the percentage inhibition was calculated using the following formula. % inhibition = 100 - ((Lum 処置 / Lum ビヒクル )*100)

[0455] Next, the synergistic effects between the test compounds (Loewe synergy score) were determined using the Synergy Finder tool at synergyfinder.fimm.fi. A Loewe synergy score greater than 10 indicates synergistic effects, scores between 10 and -10 indicate additive effects, and scores below -10 indicate antagonistic effects.

[0456] A: Exatecan and seracertib (ATR inhibitor) in HCT-116 cells Figure 1A shows the percentage inhibition for exatecan and ceraracertib alone and in combination. The Loewe synergy scores are shown in Table 1A below. IC with exatecan alone 50 The molecular weight was 0.626 nM, 0.080 nM when combined with 375 nM ceraracertib, and 0.044 nM when combined with 750 nM ceraracertib. [Table 5]

[0457] B: Exatecan and prexasertib (CHK1 inhibitor) in HCT-116 cells Figure 1B shows the percentage inhibition for exatecan and plexacertib alone and in combination. The Loewe synergy scores are shown in Table 1B below. IC with exatecan alone 50 The ion strength was 0.626 nM, 0.166 nM when combined with 50 nM plexacertib, and 0.158 nM when combined with 100 nM plexacertib. IC 50 The value was 61.14 nM. [Table 6]

[0458] C: Exatecan and seracertib (ATR inhibitor) in HEC-1B cells Figure 1C shows the percentage inhibition for exatecan and ceraracertib alone and in combination. The Loewe synergy scores are shown in Table 1C below. IC with exatecan alone 50 The mM was 113.9 nM, 19.01 nM when combined with 0.37 μM ceraracertib, and 3.719 nM when combined with 1.1 μM ceraracertib.

[0459] IC with Ceraraceltib alone50 The concentration was 2.118 μM. [Table 7]

[0460] D: Exatecan and prexasertib (CHK1 inhibitor) in HEC-1B cells Figure 1D shows the percentage inhibition for exatecan and plexacertib alone and in combination. The Loewe synergy scores are shown in Table 1D below. IC with exatecan alone 50 The initial value was 113.9 nM, and when combined with 1.9 nM plexacertib, it was 8.988 nM, and when combined with 3.8 nM plexacertib, it was 2.013 nM. IC with plexacertib alone 50 The value was 4.466 nM. [Table 8]

[0461] In additional experiments, the additional DDR inhibitor belzocertib (an ATR inhibitor) was combined with the TOP1 inhibitor exatecan in the following two-dimensional proliferation assay.

[0462] cell line HEC-1B (HTB-113) HCT-116 (CCL-247) Test compound Exatecan (MedChemExpress, #HY-13631) Belzocertib (Selleckchem, #S7102) detection CellTiter-Glo® 2.0D Cell Viability Assay (Promega, #G9243)

[0463] Cells were seeded in 150 μl of culture medium in 96-well white opaque plates and incubated at 37°C and 5% CO2 for 24 hours. HEC-1B was seeded at 7000 cells / well, and HCT-116 was seeded at 3000 cells / well.

[0464] The following day, 25 μl of exatecan, verzocertib, or exatecan and verzocertib at different concentrations were added to the cells and incubated at 37°C and 5% CO2 for 3 days. After incubation, 50 μl of CellTiter-Glo reagent was added to the plate and incubated for 25 minutes with gentle shaking at 600 rpm. Cell viability was measured by luminescence using a Victor Nivo (PerkinElmer).

[0465] The background was subtracted using the luminescence values ​​of wells containing only culture medium (no cells).

[0466] Percent inhibition was calculated using the following formula: 100 - {(Lum of cells treated with the drug / Lum of cells treated with the buffer control) * 100} (Lum = luminescence). The Loewe synergy score was calculated using the Synergy Finder tool at synergyfinder.fimm.fi.

[0467] The results are shown in Figures 8 and 9. The synergistic effect was observed across various exatecan and bezocertib concentrations.

[0468] In summary, the in vitro synergistic effects of exatecan in combination with two clinical-stage ATR inhibitors (belzocertib and seracertib) were tested in the TOP1 inhibitor-low sensitivity cell line HEC-1B and the TOP1 inhibitor-high sensitivity cell line HCT-116. Both belzocertib and seracertib showed synergistic effects with exatecan over a wide range of concentrations in the tested cell lines. The effect in HEC-1B demonstrates that the combination of a TOP1 inhibitor and a DDR inhibitor can sensitize cells that are inherently less sensitive to TOP1 inhibitor therapy.

[0469] General conditions All chemicals, raw materials, and solvents were purchased from private suppliers unless otherwise specified. All chemical reactions were carried out under ambient conditions unless otherwise specified. Flash column chromatography was performed using CombiFlash® NEXTGEN 100, with columns purchased from Agela Technologies. Preparative HPLC purification was performed using AUNO LC-2000. The columns used were Phenomenex Luna C18, 250 × 100 mm, 10 μm, 10 nm. 1 The 1H NMR spectrum was recorded using a Bruker spectrometer (400 MHz). From tetramethylsilane onwards, the low-field side. 1 The 1H NMR chemical shifts were expressed in parts per million (δ) (using the CDCl3 peak at 7.26 ppm as the reference). Mass spectrometry data were recorded using SHIMADZU LCMS-2020 (ESI-MS) and Agilent 1260\G6125B (ESI-MS). The columns used were Kinetex® EVO C18 4.6×50mm, 5μm, Kinetex® EVO C18 2.1×30mm, 5μm, Shim-pack Scepter C18-120 3.0×33mm, 3μm, and Poroshell 120 EC C18 2.7μm, 3.0×30mm.

[0470] Example 2. Synthesis of Linker-Payload Molecule-1 (LP-1) [ka] i) (1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azapentadecane-15-oil)-L-valyl-L-alanine (compound I1) [ka] Compound I1 was synthesized using standard solid-phase Fmoc chemistry. a) Resin loading: CH2Cl2 (200 mL) was added to 2-chlorotrityl chloride resin (6.0 mmol, 1.00 equivalent), followed by the addition of Fmoc-Ala-OH (1.0 equivalent) and DIPEA (6.0 equivalents). The mixture was stirred at 25°C for 2 hours under an N2 atmosphere. Then, methanol (9.5 mL) was added to the resin and stirring was continued for 30 minutes. The resin was then filtered and washed with DMF (300 mL x 3). b) Deprotection: 20% piperidine in DMF (200 mL) was added to the resin and stirred at 25°C for 30 minutes under an N2 atmosphere. The resin was washed with DMF (200 mL x 5) and filtered to obtain a resin containing reactive amine groups. c) Coupling: A solution of HBTU (2.85 equivalents) and Fmoc-Val-OH (3.0 equivalents) in DMF (200 mL) was added to the resin, followed by the addition of DIEPA (6.0 equivalents). The mixture was stirred at 25°C for 30 minutes under an N2 atmosphere. The resin was then washed with DMF (200 mL x 3). d) Repeat step b to deprotect the Fmoc 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 under an N2 atmosphere at 25°C for 30 minutes. Wash the resulting resin with DMF (200 mL x 3). e) Peptide cleavage and purification: The resin was washed with methanol (200 mL x 3) and dried under reduced pressure. The dried resin was treated with a cleavage buffer consisting of 20% HFIP in CH2Cl2, stirred for 30 minutes, and filtered. Crude compound I1 (1.71 g) was obtained by concentrating the filtrate under reduced pressure and used in the next step without further purification.

[0471] ii) ((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 (9H-fluoren-9-yl)methyl (compound I3) [ka] Compound I2 (657 mg, 5.34 mmol, 2.0 equivalents) and EEDQ (1.32 g, 5.34 mmol, 2.0 equivalents) 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. LC-MS analysis showed that the starting material I1 was completely consumed and the mass of the target product was detected. The reaction mixture was added to 160 mL of isopropyl ether and then centrifuged to obtain crude compound I3 (2.00 g) as yellow oil. The crude product was used in subsequent reactions without further purification. MS (ESI): [M+Na] + : 727.4.

[0472] iii) ((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 (9H-fluoren-9-yl)methyl (compound I5) [ka] Compound I3 (2.00 g, 2.6 mmol, 1.0 equivalent) and compound I4 (1.58 g, 5.2 mmol, 2.0 equivalent) were dissolved in DMF (20 mL) and DIEPA (671 mg, 5.20 mmol, 905 μL, 2.0 equivalent) was added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed 31% formation of the target product and 2% of the starting material, alcohol I3. The mixture was directly purified by preparative HPLC (TFA conditions), and the solvent was removed to obtain compound I5 (680 mg, 765 μmol, 29.4% yield, 97.9% purity) as a yellow solid. MS (ESI): [M+Na] + : 870.4.

[0473] iv)((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]indolidino[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 (9H-fluoren-9-yl)methyl (compound I6) [ka] Compound I5 (200 mg, 225 μmol, 1.0 equivalent) and exatecan (119 mg, 225 μmol, 1.00 equivalent) were dissolved in DMF (3.40 mL), to which HOBt (33.4 mg, 247 μmol, 1.1 equivalent) and DIPEA (58 mg, 450 μmol, 78.4 μL, 2.0 equivalent) were added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed that compound I5 was completely consumed and the mass of the target product was identified. The mixture was directly purified by preparative HPLC (TFA conditions) to obtain compound I6 (180 mg, 148 μmol, 66% yield, 96.3% purity) as a yellow solid. MS (ESI): [M+H] + : 1167.7.

[0474] v)((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]indolidino[1,2-b]quinoline-1-yl)carbamate 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)benzyl(compound I7) [ka] To a solution of compound I6 (180 mg, 148 μmol, 1.0 equivalent) in DMF (1.26 mL), triethylamine (392 mg, 3.88 mmol, 0.54 mL, 26 equivalents) was added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed that compound I6 was completely consumed and the mass of the target product was detected. Compound I7 (140 mg, crude product) was obtained as a brown liquid and used directly in the next step. MS (ESI): [M+H] + : 945.4.

[0475] vi) Synthesis of LP-1 [ka] To a solution of compound I7 (140 mg, 148 μmol, 1.0 equivalent) in DMF (1.26 mL), triethylamine (392 mg, 3.9 mmol, 0.54 mL, 26.1 equivalents) and DBCO-OSu (59.6 mg, 148 μmol, 1.0 equivalent) were added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound I7 was completely consumed and the target mass was detected. When the reaction mixture was added to 18.0 mL of isopropyl ether, the crude product slowly precipitated. The crude product was obtained by centrifugation, and the supernatant was discarded. The residue was purified by preparative HPLC (TFA conditions) to obtain LP-1 (61.0 mg, 47.5 μmol, 32.5% yield, 97.5% purity) as a yellow solid. MS (ESI): [M+Na] + : 1253.6; 1H NMR (400 MHz, DMSO-d6) δ ppm 9.98 (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, 4 H), 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, 4 H), 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, 3 H), 0.84 - 0.91 (m, 6 H), 0.81 (br d, J =6.75 Hz (3 H).

[0476] Example 3. Synthesis of Linker-Payload Molecule-2 (LP-2) [ka] i) (4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((17S,20S)-1-(9H-fluoren-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo-2,7,10,13-tetraoxa-4,16,19-triazahenicosan-21-amide)benzyl (compound I8) [ka] To a solution of compound I5 (150 mg, 168 μmol, 1.0 equivalent) and belzocertib (86 mg, 185 μmol, 1.1 equivalent) in DMF (1.5 mL), HOBt (25.0 mg, 185 μmol, 1.10 equivalents) and DIPEA (43.6 mg, 337 μmol, 58.8 μL, 2.00 equivalents) were added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed that compound I5 was completely consumed and the target mass was detected. The mixture was directly purified by preparative HPLC (TFA conditions) to obtain compound I8 (150 mg, 121 μmol, 72.2% yield, 97.1% purity) as a yellow solid. MS (ESI): [M+H] + : 1194.6.

[0477] ii) (4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)benzyl (compound I9) [ka] To a solution of compound I8 (150 mg, 121 μmol, 1.0 equivalent) in DMF (1.0 mL), triethylamine (327 mg, 3.23 mmol, 450 μL, 26.5 equivalents) was added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed that compound I8 was completely consumed and the mass of the target product was observed. The reaction mixture was added to 15.0 mL of isopropyl ether and centrifuged to obtain crude compound I9 (200 mg, crude product) as a yellow oil. MS (ESI): [M+H] + : 972.5.

[0478] iii)(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((15S,18S)-1-(((E)-cycloocta-4-en-1-yl)oxy)-15-isopropyl-18-methyl-1,13,16-trioxo-5,8,11-trioxa-2,14,17-triazanonadecane-19-amide)benzyl(LP-2) [ka] To a solution of compound I9 (190 mg, 169 μmol, 1.0 equivalent) and TCO-NHS (45.2 mg, 169 μmol, 1.0 equivalent) in DMF (1.5 mL), NMM (17.1 mg, 169 μmol, 18.6 μL, 1.00 equivalent) was added. The mixture was stirred at 0°C for 3 hours. LC-MS showed that compound I9 was completely consumed. The resulting reaction mixture was directly purified by preparative HPLC (neutral conditions) to obtain compound LP-2 (55.0 mg, 45.0 μmol, 27.6% yield, 95.6% purity) as a pale yellow solid. MS (ESI): [M+Na] + :1146.6; 1H NMR (400 MHz, DMSO-d6): δ ppm 9.99 (s, 1 H), 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, 10 H), 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).

[0479] Example 4. Synthesis of Linker-Payload Molecule-3 (LP-3) i) 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azapentadecane-15-acid 2,5-dioxopyrrolidine-1-yl (compound I11) [ka] Compound I10 (25.5 g, 59.4 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (250 mL), to which 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. The mixture was stirred at 25°C for 12 hours. After stirring was complete, the reaction mixture was filtered and concentrated under reduced pressure to obtain compound I11 as yellow oil (31.7 g, crude product), which was used without further purification. MS (ESI): MS calculated value: 526.53, MS measured value: [M+H] + = 527.1.

[0480] ii) (1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azapentadecane-15-oil)-L-valyl-L-alanine (compound I12) [ka] Compound I11 (31.3 g, 59.4 mmol, 1.0 equivalent) and dipeptide Val-Ala-OH (11.1 g, 59.4 mmol, 1.0 equivalent) were dissolved in DMF (300 mL), to which DIPEA (7.68 g, 59.4 mmol, 10.3 mL, 1.0 equivalent) was added. 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 obtain compound I12 (23.5 g, 37.5 mmol, 63% yield in 2 steps) as a white solid. MS (ESI): MS calculated value: 599.67, MS measured value: [M+H] + = 600.3.

[0481] iii) ((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 (9H-fluoren-9-yl)methyl (compound I13) [ka] Compound I12 (23.5 g, 37.5 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (235 mL), to which 4-aminobenzyl alcohol (9.25 g, 75 mmol, 2.0 equivalents) and EEDQ (18.5 g, 75.0 mmol, 2.0 equivalents) were added. The mixture was stirred under dark conditions at 25°C for 12 hours. After completion, the mixture was concentrated and washed with isopropyl ether (2.35 L) to obtain compound I13 (34 g, crude product) as a yellow gelatinous solid, which was used without further purification. MS (ESI): MS calculated value: 704.8, MS measured value: [M+H] + = 705.3.

[0482] iv)((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-diazaheptadecan-17-yl)carbamate (9H-fluoren-9-yl)methyl (compound I14) [ka] Compound I13 (34 g, 45.4 mmol, 1.0 equivalent) was dissolved in DMF (320 mL) and PNP2O (34.5 g, 113 mmol, 2.5 equivalents) and DIPEA (15 mL, 90.8 mmol, 2.0 equivalents) were added. The mixture was stirred at 25°C for 4 hours. After stirring was complete, the reaction mixture was purified by preparative HPLC to obtain compound I14 (22 g, 24.6 mmol, 63% yield in 2 steps) as a yellow solid. MS (ESI): MS calculated value: 869.35, MS measured value: [M+H] + = 870.4.

[0483] v)(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((17S,20S)-1-(9H-fluoren-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo-2,7,10,13-tetraoxa-4,16,19-triazahenicosan-21-amide)benzyl(compound I15) [ka] Compound I14 (2.1 g, 2.35 mmol, 1.0 equivalent) and belzocertib (1.14 g, 2.47 mmol, 1.05 equivalents) were dissolved in DMF (11 mL), to which HOBt (350 mg, 2.59 mmol, 1.1 equivalents) and DIPEA (778 μL, 4.71 mmol, 2.0 equivalents) were added. The mixture was stirred at 28 °C for 2 hours. LC-MS analysis showed that compound I14 was completely consumed. The reaction product was then concentrated and tritulate with isopropyl ether (3 × 110 mL). The resulting residue was further purified by preparative HPLC to obtain compound I15 (2.4 g, 1.97 mmol, 86% yield) as a yellow solid. MS (ESI): MS calculated value: 1193.49, MS measured value: [M+H]+ = 1194.5.

[0484] vi) (E)-2-(cycloocta-4-en-1-yloxy)acetic acid (compound I16) [ka] To a solution of compound TCO-OH (200 mg, 1.58 mmol, 1.0 equivalent) in anhydrous THF (2.0 mL), NaH (60% dispersion in mineral oil, 190 mg, 4.75 mmol, 3.0 equivalents) was added. The mixture was stirred at 25°C for 1 hour under an N2 atmosphere. Then, 2-bromoacetic acid (264 mg, 1.9 mmol, 1.2 equivalents) and KI (26.3 mg, 158 μmol, 0.10 equivalents) were added. The mixture was stirred at 70°C for 12 hours. After stirring was complete, the reaction mixture was quenched with H2O (10 mL) and the pH was adjusted to 2 using 1N HCl. The product was extracted with CH2Cl2 (6 × 5 mL). The combined organic layers were washed with brine (3 × 5 mL), dried on anhydrous sodium 2SO4, filtered, concentrated, and purified by preparative HPLC to obtain compound I16 (82 mg, 445 μmol, 28% yield) 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).

[0485] vii)(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((2S,5S)-20-(((E)-cycloocta-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4,7,19-trioxo-9,12,15-trioxa-3,6,18-triazycosaneamide)benzyl(LP-3) [ka] To a solution of compound I15 (2.0 g) in DMF (14 mL), triethylamine (6 mL) was added. The mixture was stirred at 25°C for 12 hours. After stirring 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 was used directly in the next step without further purification. [Note: I15* has the same chemical formula as I15, where R=H].

[0486] To a solution of crude product 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 equivalents) and DIPEA (89.7 μL, 542 μmol, 2.0 equivalents) were added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis indicated completion of the reaction. The reaction mixture was purified by preparative HPLC to obtain LP-3 (205 mg, 49% yield) as a pale yellow solid. 1H 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 actual value: [M+H]+ = 1138.0. HPLC purity (254nm) = 96.8%.

[0487] Example 5. Synthesis of linker-payload molecules LP-4, LP-5, LP-6, LP-7, LP-8, and LP-9 [ka] i)((2S,5S)-1-((4-(chloromethyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecan-17-yl)carbamate (9H-fluoren-9-yl)methyl (compound I17) [ka] To a solution of compound I13 (180 mg, 250 μmol, 1.0 equivalent) in CH2Cl2 (2.0 mL), SOCl2 (15.2 μL) was slowly added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis indicated completion of the reaction. The reaction mixture was added dropwise to ice-cold isopropyl ether (20 ml), centrifuged, and the desired product I17 was isolated as a yellow oily residue (160 mg, crude product) and used directly in the next reaction.

[0488] ii) General procedure for mooring the payload (General Procedure 1A): To a solution of compound I14 (140 μmol, 1.2 equivalents) in DMF (approximately 1.0 mL), DIPEA (3.0 equivalents) and payload molecule (1.0 equivalent) were added at 25°C. The mixture was stirred at 25°C for 2 hours. The progress of the reaction was monitored by LC-MS analysis. After stirring was complete, the reaction mixture was concentrated, and the residue was used directly or purified by preparative HPLC.

[0489] iii) General procedure for mooring the payload (General procedure 1B): To a solution of the payload molecule (140 μmol, 1.0 equivalent) in CH2Cl2 (1.0 mL), DMAP (3.0 equivalents) and triphosgene (0.8 equivalents) were added. The mixture was stirred at 25°C for 2 minutes. DIPEA (2.0 equivalents), followed by compound I13 (1.0 equivalent), was added. The mixture was stirred at 25°C for 2 hours. The reaction was monitored by LC-MS analysis. After stirring was complete, the reaction mixture was poured into ice-cooled isopropyl ether. The precipitate was collected and further purified by preparative HPLC.

[0490] iv) General procedure for mooring the payload (General procedure 1C): To a solution of compound I17 (approximately 110 μmol, 1.0 equivalent) in DMF (800 μL), payload molecules (0.7 equivalents) and DIPEA (3.0 equivalents) were added. The mixture was stirred at 25°C for 12 hours. The reaction was monitored by LC-MS analysis. After stirring was complete, the reaction mixture was poured into ice-cooled isopropyl ether. The precipitated product was collected and used directly without further purification.

[0491] v)(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 4-((17S,20S)-1-(9H-fluoren-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo-2,7,10,13-tetraoxa-4,16,19-triazahenicosan-21-amide)benzyl(compound I18a) [ka] Prepared according to general procedure 1A. Payload: Belotecan. I18a was obtained as a white solid (42.0 mg, 76% yield). MS (ESI): MS calculated value: 1164.3, MS measured value: [M+H] + = 1164.0.

[0492] vi)((2S,5S)-1-((4-((((((S)-9-((tert-butyldimethylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[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 (9H-fluoren-9-yl)methyl (compound I18b) [ka] Prepared according to general procedure 1B. Payload: SN38-(OTBS). I18b was obtained as a yellow solid (75 mg, 57%). MS (ESI): MS calculated value: 1236.5, MS measured value: [M+H] + = 1237.5.

[0493] vii)((2S,5S)-1-((4-((((3-(2-(3-((5-cyanopyrazine-2-yl)amino)-1H-pyrazole-5-yl)-3-methoxyphenoxypropyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl)carbamate (9H-fluoren-9-yl)methyl (compound I18c) [ka] Prepared according to general procedure 1A. Payload: Plexasertib. I18c was obtained as a colorless solid (150 mg, crude product) and used directly without further purification. MS (ESI): MS calculated value: 1095.48, MS measured value: [M+H] + = 1096.1.

[0494] viii)1-(4-((17S,20S)-1-(9H-fluoren-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo-2,7,10,13-tetraoxa-4,16,19-triazahenicosan-21-amide)benzyl)-4-(4-((2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridine-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidine-6-yl)amino)phenyl)-1-methylpiperazine-1-ium (compound I18d) [ka] Prepared according to general procedure 1C. Payload: adavocertib. I18d was obtained as yellow oil (70 mg, crude product). MS (ESI): MS calculated value: 1187.6, MS measured value: [M+H] += 1188.9.

[0495] ix)N-(4-((17S,20S)-1-(9H-fluoren-9-yl)-17-isopropyl-20-methyl-3,15,18-trioxo-2,7,10,13-tetraoxa-4,16,19-triazahenicosan-21-amide)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)propane-1-aminium (compound I18e) [ka] Prepared according to general procedure 1C. Payload: AZD0156. I18e was obtained as yellow oil (70 mg, crude product). MS (ESI): MS calculated value: 1148.5, MS measured value: [M+H] + = 1149.5.

[0496] x)((2S,5S)-1-((4-(((((S)-(2-chloro-4-fluoro-5-(7-morpholinoquinazoline-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 (9H-fluoren-9-yl)methyl (compound I18f) [ka] Prepared according to general procedure 1A. Payload: Nedicertib. I18f (50 mg, 40%, white solid). MS (ESI): MS calculated: 1211.45, MS measured: [M+H] + = 1212.0.

[0497] xi) General procedure for deprotecting NHFmoc (General procedure 2): To a solution of Fmoc-protected amine (approximately 40-60 μmol, 1.0 equivalent) in DMF (approximately 0.60 mL), triethylamine (approximately 0.25 mL) was added. The reaction mixture was then stirred at 25°C for 3 hours. After stirring was complete, the solvent was removed, and the mixture was purified by preparative HPLC to obtain the desired product.

[0498] xii)(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)benzyl(compound I19a) [ka] Preparation was carried out according to general procedure 2. I19a was obtained as a white solid (30 mg, 88% yield). MS (ESI): MS calculated value: 942.0, MS observed value: [M] + = 942.0.

[0499] xiii)((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)carbonate 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)benzyl(compound I19b) [ka] Preparation was carried out according to general procedure 2. I19b was obtained as a yellow solid (50 mg, crude product). LC(ESI): MS calculated value: 900.3, MS observed value: [M+H] + =901.5.

[0500] xiv)(3-(2-(3-((5-cyanopyrazine-2-yl)amino)-1H-pyrazole-5-yl)-3-methoxyphenoxy)propyl)carbamate 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)benzyl(compound I19c) [ka] Preparation was carried out according to general procedure 2. I19c was obtained as a white solid (30 mg, 42% yield). MS (ESI): MS calculated value: 873.41, MS measured value: [M+H] + = 874.0.

[0501] xv)4-(4-((2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridine-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidine-6-yl)amino)phenyl)-1-(4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)benzyl)-1-methylpiperazine-1-ium (compound I19d) [ka] Preparation was carried out according to general procedure 2. I19d was obtained as a yellow solid (40 mg, 70% yield). MS (ESI): MS calculated value: 965.5, MS observed value: [M+H] + = 966.5.

[0502] xvi)N-(4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)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)propane-1-aminium (compound I19e) [ka] Preparation was carried out according to general procedure 2. I19e was obtained as a yellow solid (40 mg, 31% yield). MS (ESI): MS calculated value: 926.5, MS measured value: [M+H] + = 927.5.

[0503] xvii)((S)-(2-chloro-4-fluoro-5-(7-morpholinoquinazoline-4-yl)phenyl)(6-methoxypyridazine-3-yl)methyl)carbonate 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)benzyl(compound I19f) [ka] Preparation was carried out according to general procedure 2. I19f was isolated as a yellow solid (40 mg, 98% yield). MS (ESI): MS calculated value: 989.39, MS measured value: [M+H] + = 990.4.

[0504] xviii) General procedure for DBCO coupling (General procedure 3A): To a solution of amine (30 μmol, 1.0 equivalent) in DMF (1.0 mL), DBCO-OSu (2.0 equivalents) and DIPEA (3.0 equivalents) were added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis showed completion of the conversion of the amine to the target product. The reaction mixture was concentrated under reduced pressure and purified directly by preparative HPLC to obtain the target product.

[0505] xix) General procedure for TCO coupling (General procedure 3B): Compound I16 (1.0 equivalent), HATU (2.0 equivalents), and DIPEA (2.0 equivalents) 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 completion of the conversion of the amine to the target product. The reaction mixture was concentrated under reduced pressure, and the residue was directly purified by preparative HPLC to obtain the target product.

[0506] xx)N-[2-[(19S)-19-ethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-10-yl]ethyl]-N-isopropylcarbamate[4-[[(2S)-2-[[(2S)- 2-[[2-[2-[2-[[4-(2-Azatricyclo[10.4.0.04,9]Hexadeca-1(12),4(9),5,7,13,15-Hexaen-10-in-2-yl)-4-Oxo-butanoyl]amino]ethoxy]ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl(LP-4) [ka] LP-4 was synthesized from I19a according to general procedure 3A. Yield: 45%. 1 1H NMR (400 MHz, DMSO-d 6. ) δ ppm 10.13 - 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 value: 1229.3, MS actual value: [M+H] + = 1229.4. Purity (HPLC, 254nm) = 95.5%.

[0507] xxi)DBCO-PEG3-VA-PABC-SN38(LP-5) [ka] LP-5 was synthesized from I19b according to general procedure 3A. Yield: 38%. 11H NMR (400 MHz, DMSO-d6) δ ppm 10.16 - 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, 2 H), 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, 1 H), 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, 2 H), 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, 1 H), 2.31 - 2.36 (m, 1 H), 2.12 - 2.28 (m, 4 H), 2.06 - 2.10 (m, 1 H), 1.96 - 2.04 (m, 3 H), 1.86 - 1.94 (m, 2 H), 1.28 - 1.32 (m, 6 H), 1.23 - 1.26 (m, 3 H), 0.87 - 0.92 (m, 6 H), 0.80 - 0.83 (m, 3 H). MS(ESI): MS calculated value: 1187.4, MS observed value: [M+H] + = 1188.7. Purity (HPLC, 254 nm) = 96.2%.

[0508] xxii)(3-(2-(3-((5-cyanopyrazine-2-yl)amino)-1H-pyrazole-5-yl)-3-methoxyphenoxy)propyl)carbamate 4-((2S,5S)-20-(((E)-cycloocta-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4,7,19-trioxo-9,12,15-trioxa-3,6,18-triazycosaneamide)benzyl(LP-6) [ka] LP-6 was synthesized from I18c according to general procedure 3B. Yield: 46%. 1 H NMR(400 MHz, DMSO-d6) 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 = 5.8 Hz, 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 actual value: [M+H]+ = 1040.5. Purity (HPLC, 254nm) = 96.4%.

[0509] xxiii)4-(4-((2-allyl-1-(6-(2-hydroxypropane-2-yl)pyridine-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidine-6-yl)amino)phenyl)-1-(4-((2S,5S)-20-(((E)-cycloocta-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4,7,19-trioxo-9,12,15-trioxa-3,6,18-triazycosaneamide)benzyl)-1-methylpiperazine-1-ium(LP-7) [ka] LP-7 was synthesized using I19d according to general procedure 3B. Yield: 37%. 11H NMR (400 MHz, DMSO-d6) δ ppm 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%.

[0510] xxiv)N-(4-((2S,5S)-20-(((E)-cycloocta-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4,7,19-trioxo-9,12,15-trioxa-3,6,18-triazycosaneamide)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)propane-1-aminium(LP-8) [ka] LP-8 was synthesized from I19e according to general procedure 3B. Yield: 32% (obtained as a mixture of quaternary salts). 1H NMR (400 MHz, DMSO-d6) 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, 4 H), 6.97 - 7.04 (m, 1 H), 5.47 - 5.57 (m, 1 H), 5.29 - 5.38 (m, 1 H), 5.11 - 5.21 (m, 1 H), 4.50 - 4.54 (m, 2 H), 4.42 - 4.46 (m, 2 H), 4.35 - 4.40 (m, 1 H), 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, 6 H), 2.64 - 2.77 (m, 4 H), 2.30 - 2.38 (m, 4H), 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, 254nm): 99.9% (fourth grade mixture).

[0511] xxv)(4-((2S,5S)-20-(((E)-cycloocta-4-en-1-yl)oxy)-5-isopropyl-2-methyl-4,7,19-trioxo-9,12,15-trioxa-3,6,18-triazycosaneamide)benzyl)carbonate(S)-(2-chloro-4-fluoro-5-(7-morpholinoquinazoline-4-yl)phenyl)(6-methoxypyridazine-3-yl)methyl(LP-9) [ka] LP-9 was synthesized from I19f according to general procedure 3B. Yield: 39%. 1H NMR (400 MHz, DMSO-d6) δ ppm 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, 254nm): 95.2%.

[0512] xxvi)N-[20-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-17,20-dioxo-4,7,10,13-tetraoxa-16-azaicosan-1-oil]glycyl-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]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide (LP-10) [ka] N-[20-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-17,20-dioxo-4,7,10,13-tetraoxa-16-azaicosan-1-oil]glycyl-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]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide is commercially available from suppliers such as Key Organics Ltd., UK (CAS number: 2694856-51-2).

[0513] Example 6. Synthesis of linker-payload molecule LP-11 i)((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]indolidino[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 (9H-fluoren-9-yl)methyl(I2O) [ka] Compound I14 (2.5 g, 2.8 mmol, 1.0 equivalent) and exatecan (1.49 g, 2.8 mmol, 1.0 equivalent) were dissolved in DMF (25 mL), to which HOBt (416 mg, 3.08 mmol, 1.1 equivalent) and DIPEA (725 mg, 5.61 mmol, 927 μL, 2.0 equivalent) were added. The mixture was stirred at 25°C for 2 hours. LC-MS analysis indicated completion of the reaction. Isopropyl ether (25 mL) was then added to the reaction mixture, and the resulting precipitate was collected by centrifugation to obtain compound I20 (4.0 g, crude product) as brown oil, which was used directly in the next step. MS (ESI): MS calculated value: 1165.4, MS measured value: [M+H] + = 1166.1.

[0514] ii) ((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]indolidino[1,2-b]quinoline-1-yl)carbamate 4-((2S,5S)-17-amino-5-isopropyl-2-methyl-4,7-dioxo-9,12,15-trioxa-3,6-diazaheptadecanamide)benzyl(I20*) To a solution of compound I20 (4.0 g, 1.0 equivalent) in DMF (28 mL), triethylamine (12 mL) was added. The mixture was stirred at 25 °C for 12 hours. After stirring 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 using LC-MS analysis and used directly in the next step. MS (ESI): MS calculated value: 943.4, MS measured value: [M+H]+ = 944.2.

[0515] iii) N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2,14 .0 4,13.0 6,11 .0 20,24 ]Tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamate[4-[[(2S)-2-[[(2S)-3-methyl-2-[[2-[2-[2-[2-[(3,3,6,6-tetramethyl-1-oxo-1λ 6 -Tiacyclohepta-4-in-1-ylidene)carbamoylamino]ethoxy]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]propanoyl]amino]phenyl]methyl (LP-11): [ka] 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 equivalent) 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 stirring was complete (observed by LC-MS analysis), the solvent was removed under reduced pressure, and the residue was purified by preparative HPLC to obtain LP-11 (23 mg) as a yellow solid. Purity by HPLC (220 nm): 95.6%, retention time = 10.720 mins. MS (ESI): [(M+H)] + =1169.4, [(M+2H)] 2+ = 585.5. 1H 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, 3 H), 0.88 (d, J=7.13 Hz, 6 H), 0.83 (d, J=6.63 Hz, 3 H).

[0516] Example 7. Synthesis pathway up to linker 1 [ka] a) Compound 2 can be prepared from Compound 1 by coupling tert-butyl 2-bromoacetate with triethanolamine (TEA) in THF, for example.

[0517] [ka] b) Compound 4: Boc protection Compound 4 can be prepared from compound 3 by treating it with (Boc)2O and a base (e.g., TEA) under anhydrous conditions, for example, in acetonitrile.

[0518] c) Compound 5: SN2 conversion from alcohol to azide Compound 5 can be prepared from Compound 4 by treating it with diphenyl phosphoryl azide (DPPA) and 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU) in an anhydrous aprotic solvent (e.g., toluene / DMF) for, for example, 48 hours.

[0519] d) Compound 6: Boc deprotection Compound 6 can be prepared from compound 5 by removing the Boc group, for example, using HCl in ethyl acetate.

[0520] e) Compound 7 Compound 7 can be prepared from compound 6 by, for example, using potassium carbonate in acetonitrile to bond compound 6B at, for example, 60°C.

[0521] f) Compound 8 Compound 8 can be prepared from compound 7 by removing the Boc group, for example, using HCl in ethyl acetate.

[0522] g) Compound 9 Compound 9 can be prepared from compound 8, for example, by using NaBH(OAc)3 in DCE to bond 9H-fluoren-9-ylmethyl N-(2-oxoethyl)carbamate.

[0523] [ka] h) Compound 10 Compound 10 can be prepared from compound 9 by using a urea formation reaction with compound 2.

[0524] i) Compound 11 Compound 11 can be prepared from compound 10 by removing the Fmoc group.

[0525] j) Compound 13 Compound 13 can be prepared from compound 11 by coupling with 2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetic acid.

[0526] k) Linker 1 Linker 1 can be prepared from compound 13 by removing the Boc and t-butoxy groups, for example, using HCl in ethyl acetate.

[0527] Example 8. Synthesis pathway up to linker 2 [ka] a) Compound 14 Compound 14 can be prepared from the compound by, for example, using NaBH(OAc)3 in DCE to bond 9H-fluoren-9-ylmethyl N-(2-oxoethyl)carbamate.

[0528] b) Compound 15 Compound 15 can be prepared from compound 14 by coupling with 2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetic acid.

[0529] c) Compound 16 Compound 16 can be prepared from compound 15 by removing the Fmoc protecting group.

[0530] d) Compound 17 Compound 17 can be prepared from compound 16 by coupling with 2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetic acid.

[0531] e) Linker 2 Linker 2 can be prepared from compound 17 by removing the Boc and t-butoxy groups, for example, using HCl in ethyl acetate.

[0532] Example 9. Synthesis of Linkers 3 and 4 i) Synthesis of common intermediate AA_9: i(a)3-(2-(2-oxoethoxy)ethoxy)methyl propanoate (compound AA_2) [ka] A solution of compound AA_1 (37 g, 192 mmol, 1.0 equivalent) in CH2Cl2 (100 mL) was added to a solution of Dess Martin periodinane (DMP) (122 g, 288 mmol, 1.5 equivalents) in CH2Cl2 (200 mL). The mixture was stirred at 25°C for 4 hours. The reaction contents were then poured into a saturated aqueous solution of NaHCO3 (100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were washed with a saturated aqueous solution of Na2S2O3 (100 mL) and brine (3 × 50 mL), dried on anhydrous Na2SO4, and concentrated to obtain the residue. The residue was tritulated with MeOH (200 mL) for 2 hours, and the filtrate was concentrated to obtain compound AA_2 (over 38 g, crude product) as a yellow oil. The crude product was used directly in the next step without further purification.

[0533] i(b) 4,7,13,16-tetraoxa-10-azanonadecanedioic acid 1-(tert-butyl)19-methyl (compound AA_4) [ka] Compound AA_2 (36.6 g, 194 mmol, 1.0 equivalent) was dissolved in MeOH (500 mL), to which compound AA_3 (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. 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 obtain compound AA_4 (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, 1 H), 3.49 (s, 1 H) 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 actual value: [M+H] + = 408.

[0534] i(c)10-((2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-yl)carbamoyl)-4,7,13,16-tetraoxa-10-azanonadecanediic acid 1-(tert-butyl)19-methyl(compound AA_6) [ka] 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 an ice-cold 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 equivalent) in CH2Cl2 (100 mL) was slowly added over 10 minutes or more at 0°C. The mixture was stirred at 0°C for a further 2 hours. The reaction mixture was poured into a saturated aqueous solution of NaHCO3 (100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were washed with brine (3 × 50 mL), dried on anhydrous Na₂SO₄, concentrated, and purified by preparative HPLC to obtain compound AA_6 (22 g, 54% yield) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 5.94 - 6.06 (m, 1 H), 5.08 (br s, 1 H), 3.75 (t, J = 6.40 Hz, 2 H), 3.70 (s, 3 H), 3.58 - 3.67 (m, 22 H), 3.55 (br d. 1.46 (d, J = 1.51 Hz, 18 H). LCMS: MS calculated value: 725, MS measured value: [M+H] + = 726.

[0535] 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-euic acid (compound AA_7): [ka] To a solution of compound AA_6 (22 g, 30.3 mmol, 1.0 equivalent) in THF / H2O (1:1, 220 mL), LiOH·H2O (1.9 g, 45.4 mmol, 1.5 equivalents) was added. The mixture was stirred at 0°C for 2 hours. After stirring, the reaction product was acidified to pH 3 with 1N HCl and extracted with CH2Cl2 (3 × 500 mL). The combined organic phase was dried on anhydrous Na2SO4 and concentrated under reduced pressure to obtain compound AA_7 (20 g, crude product) as a white solid, which was used without further purification. LCMS: MS calculated value: 711, MS measured value: [M+H] + = 712.

[0536] i(e)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-heptaoxa-12,22-diazahentriacontane-31-ate tert-butyl (compound AA_8) [ka] Compound AA_7 (20 g, 30.9 mol, 1.0 equivalent) was dissolved in CH2Cl2 (200 mL) and compound AA_14 (12.9 g, 30.9 mmol, 1.1 equivalent), HATU (14 g, 42.1 mmol, 1.5 equivalent), and DIPEA (6.96 g, 42.1 mmol, 1.5 equivalent) were added. The mixture was stirred at 25°C for 2 hours. This was then added to H2O (100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined organic phase was washed with brine (50 mL × 3), dried on anhydrous Na2SO4, and concentrated under reduced pressure to obtain compound AA_8 (26 g, crude product) as a yellow solid, which was 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).

[0537] i(f)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-heptaoxa-12,22-diazahentriacontane-31-euic acid (compound AA_9) [ka] A solution of compound AA_8 (26 g, 32.3 mmol, 1.0 equivalent) in 2N HCl (260 mL) in 1,4-dioxane was stirred at 0°C for 2 hours. The reaction mixture was then concentrated under reduced pressure to obtain the intermediate acid (25 g, crude product), which was redissolved in CH2Cl2 (250 mL). Subsequently, (Boc)2O (8.55 g, 39.1 mmol, 1.5 equivalent) and triethylamine (5.51 mL, 39.1 mmol, 1.5 equivalent) were added. 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 obtain compound AA_9 (18.6 g, 17.5 mmol, 58% yield in 2 steps) as a white solid. 1 H NMR (400 MHz, DMSO-d6) 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, MS actual value: [M+H] + = 1058.

[0538] ii) Synthesis of intermediates AA_13 and AA_18 ii(a)(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecane-17-yl)carbamate tert-butyl (compound AA_12) [ka] Compound AA_11 (6.7 g, 29 mmol, 1.0 equivalent) was dissolved in DMF (67 mL) and DIPEA (13.1 mL, 79.4 mmol, 2.73 equivalents), HATU (30.2 g, 79.4 mmol, 2.73 equivalents), and compound AA_10 (13.4 g, 39.68 mmol, 1.36 equivalents) were added. The mixture was stirred at 25°C for 2 hours. After completion, the reaction mixture was concentrated under reduced pressure and purified by preparative HPLC to obtain compound AA_12 (10 g, 63% yield) as a pale pink solid. MS (ESI): Calculated accurate mass: 548.3, MS measured value: [M+H] + = 549.1.

[0539] ii(b)N-(14-amino-3,6,9,12-tetraoxatetradecyl)-2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)acetamide (compound AA_13) [ka] Compound AA_12 (10 g, 18.2 mmol, 1.0 equivalent) was mixed with 2N HCl (100 mL) in 1,4-dioxane. The reaction mixture was stirred at 25°C for 2 hours. The solvent was then concentrated under reduced pressure to obtain compound AA_13 (8.1 g, 84% yield) as a pale pink solid, which was used without further purification. MS (ESI): Calculated accurate mass: 448.3, MS measured value: [M+H] + = 449.

[0540] ii(c) Bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamate tert-butyl (compound AA_15) [ka] Compound AA_14 (12 g, 28.6 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (120 mL), to which (Boc)2O (12.5 g, 57.2 mmol, 2.0 equivalents) and triethylamine (9.9 mL, 71.2 mmol, 2.5 equivalents) 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 × 200 mL). The combined organic layer was washed with brine (3 × 50.0 mL), dried on anhydrous Na2SO4, filtered, concentrated, and purified by flash silica gel chromatography to obtain compound AA_15 (13.6 g, 87% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ ppm 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 actual value: [M+H] + = 520.2.

[0541] ii(d) Bis(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate tert-butyl (compound AA_16) [ka] To a solution of compound AA_15 (13.6 g, 26.2 mmol, 1.0 equivalent) in THF (100 mL), triphenylphosphine (20.6 g, 78.7 mmol, 3.0 equivalents) was added, and the reaction mixture was stirred at 25°C for 3 hours. Then, H2O (100 mL) was added, and the mixture was stirred for a further 12 hours at 80°C. After stirring was complete, the reaction product was diluted with CH2Cl2 (500 mL) and stirred for 5 minutes. The organic layer was discarded, the aqueous layer was concentrated, and freeze-dried to obtain compound AA_16 (12 g, crude product) as a colorless oil, which was used without further purification. 1¹H 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.

[0542] ii(e) Bis(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-oxo-6,9,12-trioxa-3-azatetradecane-14-yl)carbamate tert-butyl (compound AA_17) [ka] Compound AA_11 (2.21 g, 9.63 mmol, 2.5 equivalents), DCC (1.98 g, 9.63 mmol, 2.5 equivalents), HOBt (1.56 g, 11.5 mmol, 3.0 equivalents), and DIPEA (3.82 mL, 23.1 mmol, 6.0 equivalents) were added to a solution of compound AA_16 (1.85 g, 3.85 mmol, 1.0 equivalent) in CH2Cl2 (40 mL). The reaction mixture was stirred at room temperature for 6 hours. After stirring was complete, the contents were diluted with CH2Cl2 (200 mL) and washed with water (400 mL). The aqueous layer was extracted with CH2Cl2 (2 × 200 mL). The combined organic layers were washed with brine (3 × 50 mL), dried on anhydrous Na₂SO₄, filtered, concentrated, and purified by flash silica gel chromatography to obtain compound AA_17 (2.79 g, 81% yield) 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 actual value: [M+Na] + = 914.7.

[0543] ii(f)N,N'-(3,6,9,15,18,21-Hexaoxa-12-Azatricosan-1,23-diyl)bis(2-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)acetamide) (Compound AA_18) [ka] Compound AA_17 (1.41 g, 1.59 mmol, 1.0 equivalent) was added to 1,4-dioxane in 2N HCl (15 mL), and the mixture was stirred at 25°C for 2 hours. After stirring was complete, the reaction mixture was concentrated under reduced pressure to obtain compound AA_18 (1.3 g, crude product) as a purple oil, which was used without further purification.

[0544] iii) Synthesis of linkers 3 and 4 iii(a)(14-(1-azido-12-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-13-oxo-3,6,9,16,19-pentaoxa-12-azahenicosan-21-yl)-41-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-13,23,40-trioxo-3,6,9,17,20,27,30,33,36-nonanoxa-12,14,24,39-tetraazahentetracontyl) tert-butyl carbamate (compound AA_19) [ka] Compound AA_9 (7.7 g, 7.28 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (80 mL) and compound AA_13 (3.92 g, 8.7 mmol, 1.2 equivalents), HATU (4.14 g, 10.9 mmol, 1.5 equivalents), and DIPEA (2.41 mL, 14.5 mmol, 2.0 equivalents) were added. The mixture was stirred at 25°C for 30 minutes. The reaction mixture was then concentrated and passed through a silica plug to obtain 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.

[0545] iii(b)10-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-N1,N1-bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N19-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecane-17-yl)-4,7,13,16-tetraoxa-10-azanonadecanediamide (linker 3) [ka] A solution of compound AA_19 (10 g, 6.7 mmol, 1.0 equivalent) in 2N HCl (90 mL) in 1,4-dioxane was stirred at 0°C for 30 minutes. 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-d6) 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): MS calculated value: 1836, MS actual value: [M+H] + = 1837. HPLC purity (254nm) = 98.6%.

[0546] iii(c)(14-(1-azido-12-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-13-oxo-3,6,9,16,19-pentaoxa-12-azahenicosan-21-yl)-38-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-24-(1-(4-(6-methyl-1, 2,4,5-Tetradin-3-yl)phenyl)-2-oxo-6,9,12-trioxa-3-azatetradecane-14-yl)-13,23,37-trioxo-3,6,9,17,20,27,30,33-octaoxa-12,14,24,36-tetraazaoctatricantyl)carbamate tert-butyl (compound AA_20) [ka] Compound AA_9 (7.7 g, 7.28 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (77 mL), to which compound AA_18 (7.25 g, 8.75 mmol, 1.2 equivalents), HATU (4.16 g, 10.9 mmol, 1.5 equivalents), and DIPEA (2.41 mL, 14.6 mmol, 2.00 equivalents) were added. The mixture was stirred at 25°C for 30 minutes. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was purified by silica plug column to obtain compound AA_20 (14.7 g, crude product) as a purple solid, which was used directly without further purification. 1 H NMR (400 MHz, DMSO-d6) 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.

[0547] iii(e)10-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-N1,N1-bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N19,N19-bis(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-2-oxo-6,9,12-trioxa-3-azatetradecane-14-yl)-4,7,13,16-tetraoxa-10-azanonadecanediamide (linker 4) [ka] A solution of compound AA_20 (14.7 g, 8.02 mmol, 1.0 equivalent) in 2N HCl (150 mL) in 1,4-dioxane was stirred at 0°C for 30 minutes. The reaction was monitored by LC-MS analysis. After stirring was complete, the reaction mixture was concentrated under reduced pressure, purified by preparative HPLC (0.01% TFA), and lyophilized to obtain linker 4 (4.9 g, 39% yield in 2 steps, 96.7% purity) 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 = 6.78Hz, 4H). MS(ESI): MS calculated value: 1729, MS measured value: [(M+2H) / 2] 2+ =866. HPLC purity (254nm)=96.7%

[0548] Example 10. Synthesis of Linker 5 i) Intermediate AA_57 [ka] 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 equivalents) in DCE (75.0 mL) was added and stirred for 5 minutes. Then, NaBH(OAc)3 (8.71 g, 41.1 mmol, 2.0 equivalents) was added to the mixture at 0°C. The mixture was allowed to rise naturally to room temperature and stirred at 25°C for 16 hours. After stirring was complete, the reaction mixture was quenched by adding H2O (10.0 mL), and the reaction product was concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (TFA conditions) to obtain AA_57 (6.74 g, 10.8 mmol, 52.7% yield, TFA salt) as a colorless oil. LCMS: Retention time: 0.88 min, MS calculated value: 508.3, MS measured value: [M+Na] + =531.4. HPLC: ES27056-40-p1cc, retention time: 4.08 minutes, purity: 97.27%. 1 H NMR: 400 MHz, CDCl3δ: 7.76 (d, J = 7.53 Hz, 2 H), 7.62 (d, J = 7.28 Hz, 2 H), 7.37 - 7.45 (m, 2 H), 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 = 4.77Hz, 4H).

[0549] ii) Intermediate AA_58 [ka] 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 obtain AA_58 (2.73 g, 9.53 mmol, 71.9% yield) as a yellow oil.

[0550] LCMS: Retention time: 0.45 minutes, MS calculated value: 286.2, MS measured value: 287.1 [M+H] + . HPLC: ES27056-60-p1c1, retention time: 2.27 minutes, purity: 84.56%. 1 H NMR: 400 MHz, CDCl3δ: 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).

[0551] iii) Intermediate AA_59 [ka] To a solution of AA_58 (1.0 g, 3.49 mmol, 1.0 equivalent) in DCE (30.0 mL), 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 at 0°C. The mixture was slowly allowed to reach room temperature and stirred at 25°C for 16 hours. After stirring was complete, 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 obtain AA_59 (560 mg, 841 μmol, 24% yield, TFA salt) as a yellow oil. LCMS: ES27056-83-p1a, Retention time: 1.07 min, MS calculated value: 551.3, MS measured value: 569.3 [M+H2O] + . 1H NMR: 400 MHz, CDCl3δ: 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).

[0552] iv) Intermediate AA_60 [ka] To a solution of AA_59 (743 mg, 953 μmol, 1.00 equivalent) in dichloromethane (18.5 mL), (Boc)2O (438 μL, 1.91 mmol, 2.00 equivalent) and triethylamine (133 μL, 953 μmol, 1.0 equivalent) were added. The mixture was stirred at 25°C for 2 hours. After stirring was complete, the reaction mixture was diluted with H2O (50.0 mL) and extracted with dichloromethane (25.0 mL x 3). The combined organic layer was 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 obtain AA_60 (609 mg, 934 μmol, 98% yield) as a white solid. LCMS: Retention time: 0.95 minutes, MS calculated value: 651.4, MS measured value: 652.3 [M+H] + . 1H NMR:, 400 MHz, CDCl3δ: 7.77 (d, J = 7.53 Hz, 2 H), 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).

[0553] v) Intermediate AA_61 [ka] 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 reduced pressure to obtain the residue. The residue was further purified by column chromatography (SiO2, dichloromethane / MeOH = 100 / 1 to 15 / 1) to obtain AA_61 (328 mg, 764 μmol, 82% yield) as a milky white solid. LCMS: Retention time: 1.03 min, MS calculated value: 429.3, MS measured value: 430.2 [M+H] + . 1 H NMR: 400 MHz, CDCl3δ: 3.60 - 3.66 (m, 4 H), 3.57 (t, J = 5.90 Hz, 4 H), 3.39 (t, J = 4.89 Hz, 4 H), 3.29 (br s, 4 H), 2.85 (t, J = 6.53 Hz, 2 H), 2.80 (t, J = 5.77 Hz, 4 H), 2.74 (br s, 2 H), 1.47 (s, 9 H).

[0554] vi) Intermediate AA_62 [ka] AA_61 (328 mg, 764 μmol, 1.0 equivalent) and Tz-OSu (375 mg, 1.15 mmol, 1.50 equivalents) were dissolved in CH2Cl2 (5.0 mL), to which DIPEA (200 μL, 1.15 mmol, 1.50 equivalents) was added. The mixture was stirred at 25°C for 3 hours. After stirring was complete, the reaction mixture was diluted with H2O (5.0 mL) and extracted with dichloromethane (5.0 mL x 3). The combined organic layer was washed with brine (approximately 5.0 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was then purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to obtain AA_62 (485 mg, 756 μmol, 99.0% yield) as a purple oil. LCMS: Retention time: 0.87 minutes, MS calculated value: 641.4, MS measured value: 642.5 [M+H] + . 1 H NMR: ES27056-100-p1b1, 400 MHz, CDCl3δ: 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, 2 H), 3.40 (br s, 4 H), 3.36 (t, J = 4.89 Hz, 4 H), 3.23 (br s, 1 H), 3.10 (s, 3 H), 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, 9 H).

[0555] vii) Intermediate compound AA_63 [ka] AA_62 (485 mg, 756 μmol, 1.0 equivalent) was dissolved in 2N HCl / siRNA (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 obtain AA_63 (456 mg, 592 μmol, 78.4% yield, TFA salt) as a purple oil. LCMS: Retention time: 0.76 min, MS calculated value: 541.3, MS measured value: 542.4 [M+H] + . HNMR: 400 MHz, CDCl3δ: 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, 3 H).

[0556] viii) Intermediate compound AA_64 [ka] To a solution of triphosgene (114 mg, 385 μmol, 0.65 equivalents) in dichloromethane (7.5 mL), R26 (225 mg, 770 μmol, 1.30 equivalents) and triethylamine (495 μL, 3.55 mmol, 6.00 equivalents) in dichloromethane (7.50 mL) were added at 0°C. The mixture was stirred at 0°C for 1 hour. Next, the resulting mixture was added dropwise to a solution of AA_63 (456 mg, 592 μmol, 1.0 equivalent) in dichloromethane (10.0 mL) at 0°C. The mixture was stirred at 25°C for 2 hours. After stirring was complete, the reaction mixture was diluted with dichloromethane (approximately 25 mL). This was then 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 obtain AA_64 (573 mg, 666 μmol) as a purple oil. LC-MS: Retention time: 0.88 min, MS calculated value: 859.5, MS measured value: 860.8 [M+H] + . HPLC: Retention time: 3.48 minutes, purity: 93.87%. 1 H NMR: 400 MHz, CDCl3δ: 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).

[0557] ix) Linker 5 [ka] AA_64 (509 mg, 592 μmol, 1.00 equivalent) was dissolved in 2N HCl / siRNA (10.0 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 purified by preparative HPLC (TFA conditions). The eluate 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 reduced pressure to obtain Linker 5 (180 mg, 237 μmol, 40% yield) as a purple oil. LCMS: ES27056-112-p1a1, retention time: 0.78 min, 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, CDCl3δ: 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, 2 H), 2.79 (t, J = 5.52 Hz, 4 H), 2.75 (br d, J = 4.77 Hz, 2H).

[0558] Example 11. Synthesis of Linker 6 i) Intermediate AA_65 [ka] To a solution of AA_58 (1.3 g, 4.54 mmol, 2.0 equivalents) in THF (25 mL), AcOH (780 μL, 13.6 mmol, 6.0 equivalents) was added, followed by the addition of aldehyde 1 (1.24 g, 2.27 mmol, 1.0 equivalent) and NaBH3CN (571 mg, 9.08 mmol, 4.00 equivalents) to the mixture. The mixture was stirred at 45°C for 72 hours. The mixture was then diluted with H2O (approximately 5 mL) and concentrated under reduced pressure to obtain the residue. The residue was further purified by preparative HPLC (TFA conditions) to obtain AA_65 (1.06 g, 1.30 mmol, 57% yield) as a colorless oil. LCMS: ES27056-114-p2a5, Retention time: 0.97 mins, MS calculated value: 816.4, MS measured value: 839.6 [M+Na] + . HPLC: ES27056-114-p1c, retention time: 4.756 minutes, purity: 90%. 1 H NMR: 400 MHz, CDCl3δ: 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, 4 H), 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).

[0559] ii) Intermediate compound AA_66 [ka] To a solution of AA_65 (393 mg, 422 μmol, 1.0 equivalent) in dichloromethane (4.00 mL), (Boc)2O (388 μL, 1.69 mmol, 4.00 equivalent) and triethylamine (88.1 μL, 633 μmol, 1.50 equivalent) 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 obtain AA_66 (354 mg, 386 μmol, 91% yield) as a colorless oil. LCMS: ES27056-123-p1a9, retention time: 1.05 min, MS calculated value: 916.5, MS measured value: 917.8 [M+H] + . HPLC: ES27056-123-p1c, retention time: 5.27 minutes, purity: 92.87%. 1 H NMR: 400 MHz, CDCl3δ: 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).

[0560] ii) Intermediate compound AA_67 [ka] 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 reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, dichloromethane / MeOH = 50 / 1~5 / 1) to obtain AA_67 (242 mg, 512 μmol, 71.5% yield) as a colorless oil. LCMS: Retention time: 1.61 min, MS calculated value: 472.3, MS measured value: 473.3 [M+H]+ .

[0561] HNMR:, 400 MHz, CDCl3δ: 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, 9H).

[0562] iii) Intermediate compound AA_68 [ka] To a solution of Tz-acid (methyltetradic acid (CAS: 1380500-88-8)) (377 mg, 1.64 mmol, 3.2 equivalents) in dichloromethane (2.5 mL), DIPEA (446 μL, 2.56 mmol, 5.0 equivalents) and HATU (623 mg, 1.64 mmol, 3.2 equivalents) were added at 0°C. 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 layer was washed with brine (approximately 5.0 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was further purified by preparative HPLC (TFA conditions) to obtain AA_68 (351 mg, 391 μmol, 76% yield) as a purple oil. LC-MS: Retention time: 0.94 min, MS calculated value: 896.5, MS measured value: 898.5 [M+H] +. HPLC: Retention time: 10.37 minutes, purity: 93.28%. 1H NMR: 400 MHz, CDCl3δ: 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, 1 H), 3.09 (s, 6 H), 2.57 - 2.99 (m, 4 H), 1.44 - 1.51 (m, 9 H).

[0563] iv) Intermediate compounds of AA_69 [ka] AA-68 (91.6 mg, 102 μmol, 1.0 equivalent) was dissolved in 2N HCl / siRNA (500 μL) and stirred at 25°C for 2 hours. LC-MS showed that AA-68 was completely consumed and the target mass peak was identified. The mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (TFA conditions) to obtain AA-69 (81.0 mg, 102 μmol, 99.5% yield) as a purple oil. LC-MS: ES27056-125-p3a1, retention time: 0.85 min, MS calculated value: 796.4, MS measured value: 819.8 [M+Na] + . HPLC: Retention time: 2.87 minutes, purity: approximately 100%.

[0564] v) Intermediate compound AA_70 [ka] To a solution of triphosgene (19.4 mg, 65.3 μmol, 0.65 equivalents) in DCM (1.50 mL), 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) were added at 0°C. The mixture was stirred at 0°C for 1 hour. TLC indicated completion of the reaction. Next, this mixture was added to a solution of AA_69 (80.0 mg, 100 μmol, 1.0 equivalent) in dichloromethane (1.0 mL). The mixture was stirred at 25°C for 2 hours. After this, the reaction mixture was diluted with dichloromethane (2.0 mL), washed with H2O (2.0 mL), and extracted with CH2Cl2 (15 mL × 3). The combined organic layers were washed with brine (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 obtain AA_70 (118 mg, 106 μmol) as a purple oil. LCMS: Retention time: 4.47 min, MS calculated value: 1115.3, MS measured value: 1116.6 [M+H] + . HPLC: Purity: Approximately 100%. 1 H NMR: 400 MHz, CDCl3δ: 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).

[0565] vi) Linker 6 [ka] AA_70 (111 mg, 99.5 μmol, 1.0 equivalent) was dissolved in 2N HCl / siRNA (1.50 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, which was purified by preparative HPLC (TFA conditions). The eluate 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 reduced pressure to obtain Linker 6 (50 mg, 49.3 μmol, 49% yield) 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, CDCl3δ: 8.43 - 8.60 (m, 4 H), 7.35 - 7.68 (m, 5 H), 3.91 (s, 1 H), 3.31 - 3.81 (m, 40 H), 3.18 - 3.30 (m, 2 H), 3.09 (s, 6 H), 2.85 - 3.00 (m, 2 H), 2.71 - 2.82 (m, 6 H).

[0566] Example 12. (4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((2S,5S,31S,34S)-18-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl)-35-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3 Synthesis of ,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[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-hexaoxa-3,6,18,30,33-pentazapentatricontanamid)benzyl (LP-13) i) 2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]tert-butyl acetate (compound I28) [ka] To a solution of oxalyl chloride (5.9 mL, 136 mmol, 4.0 equivalents) in CH2Cl2 (90.0 mL), DMSO (9.6 mL, 68.1 mmol, 2.0 equivalents) was added at -78°C, followed by the addition of compound I27 (9.0 g, 34.0 mmol, 1.0 equivalent). The reaction mixture was stirred at the same temperature for 1 hour. Then, triethylamine (28.4 mL, 204 mmol, 6.0 equivalents) was added at -78°C, and the mixture was stirred for another hour before being allowed to rise naturally to room temperature. After stirring was complete, the reaction mixture was filtered and concentrated under reduced pressure to obtain compound I28 (12.0 g, crude product) as a colorless oil, which was used without further purification. 1 H NMR (400 MHz, DMSO-d6): 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).

[0567] ii) 2-[2-[2-[2-[2-[2-[2-(2-tert-butoxy-2-oxo-ethoxy)ethoxy]ethoxy]ethylamino]ethoxy]ethoxy]ethoxy]tert-butyl acetate (compound I30): [ka] Compound I28 (12.0 g, 1.0 equivalent) and Compound I29 (17.9 g, 681 μmol, 1.5 equivalent) were dissolved in MeOH (120 mL), to which NaBH3CN (2.85 g, 454 mmol, 1.0 equivalent) was added. The mixture was stirred at 20-25°C for 1 hour. After stirring was complete, the reaction mixture was extracted with CH2Cl2 (200 mL) and H2O (100 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain Compound I30 (18.0 g, crude product) as a yellow oil. MS (ESI): [M+H] + :510.6.

[0568] iii) 2-[2-[2-[2-[2-[2-[2-(2-tert-butoxy-2-oxo-ethoxy)ethoxy]ethoxy]ethyl-(9H-fluoren-9-ylmethoxycarbonyl)amino]ethoxy]ethoxy]ethoxy]tert-butyl acetate (compound I31): [ka] To a solution of compound I30 (18.0 g, 1.0 equivalent) and FmocOSu (17.9 g, 681 μmol, 1.5 equivalents) in THF (90 mL) and H2O (90 mL), NaHCO3 (5.9 g, 70.6 mmol, 2.0 equivalents) was added. The mixture was stirred at 20-25°C for 1 hour. After stirring was complete, LC-MS of the reaction product showed that compound I30 had been completely consumed. The reaction product was extracted with DCM (200 mL) and H2O (100 mL). The combined organic components were 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 obtain compound I31 (2.1 g, 2.87 mmol, 98.1% purity, and 2.2 g, 3.01 mmol, 92.4% purity, 16.6% yield) as a white solid. Purity by HPLC (220 nm): 98.1%; MS (ESI): [M+H] + :732.3; 1 H NMR (400 MHz, DMSO-d6): 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 = 5.4 Hz, 1 H), 3.95 - 3.99 (m, 4 H), 3.46 - 3.56 (m, 16 H), 3.38 - 3.44 (m, 4 H), 3.13 (br s, 4 H), 1.40 - 1.42 (m, 18H).

[0569] iv) 2-[2-[2-[2-[2-[2-[2-(2-tert-butoxy-2-oxo-ethoxy)ethoxy]ethoxy]ethyl-(9H-fluoren-9-ylmethoxycarbonyl)amino]ethoxy]ethoxy]ethoxy]tert-butyl acetate (compound I32): [ka] 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. LC-MS 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 directly purified by preparative HPLC (TFA conditions) to obtain compound I32 (700 mg, 1.03 mmol, 35.9% yield) as a colorless oil. Purity by HPLC (220 nm): 98.2%; MS (ESI): [M+H] + :676.3; 1 H NMR (400 MHz, DMSO-d6): 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)

[0570] v)(2S,5S)-18-(((9H-Fluoren-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]indolidino[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-triazanonacosane-29-euic acid (compound I33): [ka] Compound I32 (200 mg, 295 μmol, 1.0 equivalent) and Compound I26 (CAS: 2845164-91-0) (308 mg, 377 μmol, 1.3 equivalents) were dissolved in DMF (2.0 mL), to which DIPEA (58.6 μL, 354 μmol, 1.2 equivalents), HOBt (47.9 mg, 354 μmol, 1.2 equivalents), and EDCI (67.9 mg, 354 μmol, 1.2 equivalents) were added, respectively. The mixture was stirred at 20-25°C for 3 hours. LC-MS analysis showed that Compound I32 was completely consumed. The resulting mixture was directly purified by preparative HPLC (TFA conditions) to obtain Compound I33 (900 mg, 637 μmol, 68.3% yield) as a white solid. Purity by HPLC (220nm): 97.3%; MS (ESI): [M+H] + :1413.5. The reaction was carried out in parallel to obtain a total of 900 mg of I33.

[0571] vi)(2S,5S)-18-(((9H-Fluoren-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]indolidino[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-triazanonacosane-29-euic acid (compound I34) [ka] 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 reaction product was triturated with isopropyl ether (10 mL) at 0–5°C to obtain compound I34 (500 mg, crude product) as a colorless oil. MS (ESI): [M+H] + :1356.8.

[0572] vii)((2S,5S)-1-((4-((((4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamoyl)oxy)methyl)phenyl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo-9,12,15-trioxa-3,6-diazaheptadecane-17-yl)((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]indolidino[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 (9H-fluoren-9-yl)methyl (compound I35) [ka] To a solution of compound I34 (500 mg, 368 μmol, 1.0 equivalent) and compound I24 (392 mg, 479 μmol, 1.3 equivalents) in DMF (5.0 mL), DIPEA (152 μL, 921 μmol, 2.5 equivalents), HOBt (74.7 mg, 552 μmol, 1.5 equivalents), and EDCI (105 mg, 552 μmol, 1.5 equivalents) were sequentially added. The mixture was stirred at 20-25°C for 2 hours. LC-MS analysis showed that compound I35 was consumed and the target mass was identified. The reaction mixture was used directly in the next step without further purification. MS (ESI): [(M+2H) / 2]2 + :1062.5.

[0573] viii)(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 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,1 2H-benzo[de]pyrano[3',4':6,7]indolidino[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-hexaoxa-3,6,18,30,33-pentazapentatricontanamid)benzyl (compound I36): [ka] Triethylamine (1.0 mL) was added to the above solution of compound I35. The reaction mixture was stirred at 20-25°C for 4 hours. LC-MS showed that compound I35 was completely consumed and the target mass was detected. The resulting mixture was directly purified by preparative HPLC (TFA conditions) to obtain compound I36 (200 mg, 105 μmol, 28.5% purity) as a white solid. Purity by HPLC (220 nm): 92.9%; MS (ESI): [(M+2H) / 2] 2+ :950.5.

[0574] ix)(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((2S,5S,31S,34S)-18-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-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]indolidino[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-hexaoxa-3,6,18,30,33-pentazapentatricontanaamide)benzyl(LP-13) [ka] Compound I36 (30 mg, 14.9 μmol, 1.0 equivalent) and compound MC-acid (6.2 mg, 29.3 μmol, 2.0 equivalent) were dissolved in DMF (300 μL), to which DIPEA (6.10 μL, 37.2 μmol, 2.5 equivalents), HOBt (4.0 mg, 29.8 μmol, 2.0 equivalents), and EDCI (5.7 mg, 29.8 μmol, 2.0 equivalents) were added. The mixture was stirred at 20-25°C for 1 hour. LC-MS indicated the completion of compound I36 consumption, and the peak of the target product was detected. The solvent was removed under reduced pressure, and the residue was purified by preparative HPLC (AcOH conditions) to obtain LP-13 (13.1 mg) as a white solid. Purity by HPLC (220 nm): 96.9%; MS (ESI): [(M+2H) / 2] 2+ :1047.1. 1H NMR (400 MHz, DMSO-d6) 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, 4 H), 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)。

[0575] Example 13. N-[[4-[5-[3-amino-6-(4-isopropylsulfonylphenyl)pyrazine-2-yl]isoxazole-3-yl]phenyl]methyl]-N-methyl-carbamate[4-[[(2S)-2-[[(2S)-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-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamoyloxymethyl]anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-2-oxo-ethoxy]ethoxy]ethoxy]ethyl-[2-[2-[2-[2-[(3,3,6,6-tetramethyl-1-oxo-1λ 6 Synthesis of -thiacyclohepta-4-in-1-ylidene)carbamoylamino]ethoxy]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (LP-12) i)(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((2S,5S,31S,34S)-18-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azapentadecane-15-oil)-35-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di Oxo-2,3,9,10,13,15-Hexahydro-1H,12H-Benzo[de]pyrano[3',4':6,7]indolidino[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-hexaoxa-3,6,18,30,33-pentazapentatricontanamid)benzyl (compound I37): [ka] Compound I36 (85 mg, 42.2 μmol, 1.0 equivalent) and compound R1-2 (36.4 μL, 82.4 μmol, 2.0 equivalent) were dissolved in DMF (1.0 mL), to which DIEA (17.4 μL, 105 μmol, 2.5 equivalents), HOBt (11.4 mg, 84.4 μmol, 2.0 equivalents), and EDCI (16.1 mg, 84.4 μmol, 2.0 equivalents) were sequentially added. The mixture was stirred at 20-25°C for 1 hour. LC-MS showed that compound I36 was completely consumed and the target mass was detected. The reaction mixture was used directly in the next step. MS (ESI): [(M+2H) / 2]2 + :1155.9.

[0576] ii)(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazine-2-yl)isoxazole-3-yl)benzyl)(methyl)carbamate 4-((2S,5S,31S,34S)-18-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetyl)-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]indolidino[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-hexaoxa-3,6,18,30,33-pentazapentatricontanamid)benzyl (compound I38): [ka] 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 target mass was detected. The resulting reaction mixture was directly purified by preparative HPLC (TFA conditions) to obtain compound I38 (55 mg, 26.3 μmol, 62% yield) as a white solid. Purity by HPLC (220 nm): 98.5%; MS (ESI): [(M+2H) / 2] 2+ :1045.1.

[0577] iii) N-[[4-[5-[3-amino-6-(4-isopropylsulfonylphenyl)pyrazine-2-yl]isoxazole-3-yl]phenyl]methyl]-N-methyl-carbamic acid[4-[[(2S)-2-[[(2S)-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-Diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16(24),17,19-heptaen-23-yl]carbamoyloxymethyl]anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-2-oxo-ethoxy]ethoxy]ethoxy]ethyl-[2-[2-[2-[2-[(3,3,6,6-tetramethyl-1-oxo-1λ 6 -Tiacyclohepta-4-in-1-ylidene)carbamoylamino]ethoxy]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (LP-12): [ka] To a solution of compound I38 (40 mg, 19.1 μmol, 1.0 equivalent) in DMF, 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, respectively. The mixture was stirred at 20-25°C for 2 hours and monitored by LC-MS analysis. After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by preparative HPLC (HCOOH conditions) to obtain LP-12 (20 mg, 8.64 μmol, 45.1% yield) as a white solid. Purity by HPLC (220 nm): 97.7%; MS (ESI): [(M+2H) / 2] 2+ :1157.8; 1H NMR (400 MHz, DMSO-d6): 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, 1 H), 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).

[0578] Example 14: Synthesis of N,N-bis(2-(2-azidoethoxy)ethyl)-6-(2-(methylsulfonyl)pyrimidine-5-yl)hexa-5-inamide (linker 7): [ka] Compound R1 (40 mg, 149 μmol, 1.0 equivalent) and Compound R2 (36.2 mg, 149 μmol, 1.00 equivalent) were dissolved in DMF (400 μL), to which HOBt (40.2 mg, 298 μmol, 2.0 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. The mixture was stirred at 25°C for 1 hour. LC-MS showed consumption of the starting materials. The reaction mixture wa...

Claims

1. 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.

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

3. The DDR inhibitor portion, (a) An ATR inhibitor, wherein the DDR inhibitor portion is or contains belzocertib, (b) A CHK1 / 2 inhibitor, wherein the DDR inhibitor portion is or contains plexacertib. (c) A WEE1 inhibitor, wherein the DDR inhibitor portion is or contains adavocertib. (d) an ATM inhibitor, wherein optionally the DDR inhibitor portion is AZD0156 or includes it, (e) A DNA-PK inhibitor, wherein optionally the DDR inhibitor portion is nedisertib or a DDR inhibitor comprising the same, or an antigen-binding molecule according to claim 1 or 2.

4. The TOP1 inhibitory portion is camptothecin or its derivatives, exatecan, exatecan mesylate (DX-8951f), N-glycyl-exatecan, SN-38, DXd(1), DXd(2), irinotecan, etirinotecan, FL118, topotecan, gimatecan, berotecan, deruxtecan, berotecan, rubitecan, raltotecan, diflomotecan, karenitecan, silatecan, namithecan, aeromothecan, DRF-1042 An antigen-binding molecule according to any one of claims 1 to 3, which is a TOP1 inhibitor selected from, delimotecan, NSC606985, quimitecan, ZBH-1205, Genz-644282, non-CPT1, indotecan, indimitecan, AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021, VIP126, ZBH-01, and LMP-744, or comprising the same.

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

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

7. The linker-payload portion is (a) an amino group for conjugation to the antigen-binding site, (b) A portion comprising at least one first payload clicked to a first click group, wherein the portion comprising the first payload comprises a DDR inhibitor portion, (c) A portion comprising at least one second payload clicked to a second click group, wherein the portion comprising the second payload comprises a TOP1 inhibitor portion, (d) Branched group: 【Chemistry 1】 And in the formula, R Ｎ However, H and -(C １～５ Selected from alkylene)-C(O)OH, where one CH ２ The unit may be replaced with -O-. a indicates the location where the amino group is connected to the branched group. b indicates a location where at least one first click group is connected to the branching group. The antigen-binding molecule according to claim 6, comprising a branched group, wherein c indicates a location where at least one second click group is linked to the branched group.

8. The HER2 coupling portion, (i) The following CD-Rs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 15, HC-CDR2 having the amino acid sequence of SEQ ID NO: 16, HC-CDR3 having the amino acid sequence of SEQ ID NO: 17 A heavy-chain variable (VH) region incorporating, and (ii) The following CD-Rs: 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 An antigen-binding molecule according to any one of claims 1 to 7, comprising a light chain variable (VL) region incorporating the above.

9. The antigen-binding portion that binds to HER2, A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 14, and A VL region containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

22. An antigen-binding molecule according to any one of claims 1 to 8, comprising:

10. The antigen-binding portion that binds to HER2, 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 thereof, having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:

13. An antigen-binding molecule according to any one of claims 1 to 9, comprising:

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 auxiliary agent.

12. An antigen-binding molecule according to any one of claims 1 to 10, or a composition according to claim 11, for use in a medical treatment or preventive method, or a diagnostic or prognostic method.

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

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

15. A method for treating or preventing cancer, comprising administering to a target an antigen-binding molecule according to any one of claims 1 to 10, or the composition according to claim 11, in an amount effective for treatment or prevention.

16. The aforementioned cancers include cancers containing cells that express / overexpress EGFR family members, cancers containing cells that express / overexpress HER2, cancers containing cells that do not overexpress EGFR family members, cancers containing cells that do not overexpress 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, bile duct cancer, colorectal cancer, esophageal cancer / esophagogastric junction cancer, gallbladder cancer, gastric adenocarcinoma, An antigen-binding molecule or composition for use according to claim 13, selected from gastroesophageal junction adenocarcinoma, HER2-positive gastric cancer / gastroesophageal junction adenocarcinoma, locally advanced or metastatic HER2-positive gastric cancer / gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, glioblastoma multiforme, glioma, head and neck cancer, hepatocellular carcinoma, intestinal 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 tumor, oligodendroglioma, ovarian cancer, pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcoma, solitary fibrous tumor, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer, the use according to claim 14, or the method according to claim 15.

17. The antigen-binding molecule or composition for use according to claim 13 or 16, the use according to claim 14 or 16, or the method according to claim 15 or 16, wherein the cancer is resistant to or recurrent in response to treatment with a DNA damage repair inhibitor, and / or the cancer is resistant to or recurrent in response to treatment with a DNA topoisomerase I inhibitor.

18. Use of an antigen-binding molecule according to any one of claims 1 to 10, or the composition according to claim 11, to remove or enhance the killing of cells expressing HER2.

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

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