Antibody-drug conjugates

Abstract

An antigen-binding molecule that binds to a target antigen, comprising (i) a target antigen-binding moiety and (ii) at least one linker-payload moiety, wherein the antigen-binding molecule comprises (a) 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, more specifically to antibody technology. It also relates to methods for medical treatment and prevention, as well as branched segments and their use in antibody-drug conjugates. [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] While ADCs have shown success in both solid tumors and hematological malignancies, resistance and tumor heterogeneity are major causes of clinical failure (Yamazaki et al., Nat Commun (2021) 12(1):3528). Tumor heterogeneity is known to lead to recurrence, metastasis, and acquired resistance to ADCs and other treatment strategies. Heterogeneous tumors with drug sensitivity differences result in invasive tumor growth, high recurrence rates, and poor survival rates.

[0004] To address these challenges, most chemotherapy regimens consist of drug combinations. Co-delivery of small molecules can overcome resistance, produce additive or synergistic effects, and enhance therapeutic efficacy.

[0005] The emergence of tumors resistant to current therapies has provided a driving force for evaluating novel ADC formats. This challenge has led to the search for dual-drug ADCs capable of simultaneously delivering two mechanistically distinct payloads. Strategies for constructing dual-drug ADCs require either binding both drugs to a single linker or using two different conjugation sites on an antibody, and existing methods are outlined in Nervig et al., J.ADC. (2023) (DOI:10.14229 / jadc.2023.01.05.001). [Overview of the project]

[0006] In a first aspect, the Disclosure provides an antigen-binding molecule that binds to a target antigen, comprising (i) a target antigen-binding moiety and (ii) at least one linker-payload moiety, wherein the antigen-binding molecule comprises (a) a DNA damage response (DDR) inhibitor moiety and (b) a DNA topoisomerase I (TOP1) inhibitor moiety.

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

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[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), 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.

[0014] 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.

[0015] In some embodiments, the antigen-binding molecule includes a linker-payload portion comprising (a) a DDR inhibitor portion and (b) a TOP1 inhibitor portion.

[0016] 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.

[0017] In a second aspect, this disclosure is: (a) an amino group for conjugation to the antigen-binding site, (b) at least one first click base for linking the portion containing the first payload, (c) at least one second click base for linking the portion containing the second payload, (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 the location where the at least one first click group is connected to the branching group. The present invention provides a trifunctional linker portion including a branching group, where c indicates a location in which at least one second click group is linked to the branching group.

[0018] In a third aspect, the disclosure includes a portion derived from the compound of the second aspect. (a) at least one first payload and at least one second payload, (b) Provides a linker between the antigen-binding portion and the linker.

[0019] Therefore, the linker, (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 1~5Selected 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.

[0020] In the fourth aspect, this disclosure is: (a) at least one first payload and at least one second payload, (b) Provides a conjugate including an antigen-binding moiety, Here, the linker between the payload and the antibody includes a portion derived from the compound of the second embodiment.

[0021] Therefore, the conjugate is (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. c includes a branching group, where c indicates a location to which at least one second click group is attached.

[0022] In a fifth aspect, the present disclosure provides a linker-payload molecule for conjugation to an antigen-binding moiety, comprising at least one first payload and at least one second payload, wherein the linker for conjugation to an antibody comprises a moiety derived from the compound of the second aspect.

[0023] Accordingly, the linker-payload molecule is (a) an amino group for conjugation to an antigen-binding moiety, (b) a moiety comprising at least one first payload clicked to a first click group, (c) a moiety comprising at least one second payload clicked to a second click group, (d) a branching group: [Chemical Formula] wherein in the formula, R N is selected from H and -(C 1~5 alkylene)-C(O)OH, wherein one CH2 unit may optionally be replaced with -O-, a indicates the position where the amino group is linked to the branching group, b indicates the position where the at least one first click group is linked to the branching group, c indicates the position where the at least one second click group is linked to the branching group, the branching group.

[0024] In a sixth aspect, the present disclosure provides a modified antigen-binding moiety comprising a moiety derived from the compound of the second aspect.

[0025] Accordingly, the modified antigen-binding moiety is (a) an amino group conjugated to the antigen-binding moiety, (b) at least one first click group for linking a moiety comprising a first payload, (c) at least one second click group for linking a moiety comprising a second payload, (d) a branching 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 the location where the at least one first click group is connected to the branching group. c includes a branching group, where c indicates the location to which at least one second click group is connected.

[0026] This disclosure also provides compositions comprising antigen-binding molecules and pharmaceutically acceptable carriers, diluents, excipients, or auxiliaries.

[0027] 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.

[0028] This disclosure also provides antigen-binding molecules or compositions according to this disclosure for use in the treatment or prevention of cancer.

[0029] This disclosure also provides the use of antigen-binding molecules or compositions according to this disclosure in the manufacture of agents for treating or preventing cancer.

[0030] 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.

[0031] In some embodiments, cancer is a cancer comprising cells that express / overexpress a target antigen. In some embodiments, cancer is a hematological cancer. In some embodiments, cancer is selected from: myeloid hematological cancers, lymphomas, Hodgkin lymphomas, non-Hodgkin lymphomas, AIDS-associated lymphomas, cutaneous T-cell lymphomas, mycosis fungoides, primary central nervous system lymphomas, Césarley syndrome, Waldenström macroglobulinemia, leukemias, T-cell leukemias, B-cell leukemias, acute myeloid leukemias, chronic myeloid leukemias, chronic myelomonocytic leukemias, acute promyelocytic leukemias, chronic promyelocytic leukemias, acute lymphoblastic leukemias, chronic lymphoblastic leukemias, pilocytic cell leukemias, myelomas, multiple myelomas, myelodysplastic syndromes, and myeloproliferative disorders.

[0032] In some embodiments, the cancer is resistant to or recurrent with treatment with DDR inhibitors, and / or hereby resistant to or recurrent with treatment with TOP1 inhibitors.

[0033] 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 a target antigen.

[0034] This disclosure also provides optionally isolated in vitro complexes containing the antigen-binding molecule of this disclosure bound to a target antigen.

[0035] 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]

[0036] [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 shows the percentage of cell death in cancer cell lines after 7 days of in vitro treatment with trastuzumab conjugated with both exatecan and belzocertib (T-(Exa+Ber)), trastuzumab deruxtecan (T-DXd), or an isotype-matched control antibody conjugated with exatecan (isotype control). [Figure 3A] This shows 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 3B] 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 4A] This shows the tumor volume over time in mice possessing a small cell lung cancer xenograft model (cell line SHP77) derived from a human small cell lung cancer cell line, treated with antibodies (TAA(Exa+Ber)) that target tumor-associated antigens expressed by cells of the human small cell lung cancer cell line, either conjugated to PBS (vehicle) or both exatecan and bezocertib. [Figure 4B] This shows the gram (g) body weight over time of mice that possess a small cell lung cancer xenograft model derived from a human small cell lung cancer cell line (cell line SHP77) and were treated with antibodies (TAA(Exa+Ber)) that target tumor-associated antigens expressed by cells of the human small cell lung cancer cell line, either conjugated to PBS (vehicle) or both exatecan and bezocertib. [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-1-B, 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-1-B, or HCT116 live cells, trastuzumab (T-(Exa+Ber)) conjugated to both exatecan and berzocertib, 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-1-B 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 8A] This shows the binding of anti-TAA antibodies (TAA(unworn)), anti-TAA antibodies conjugated to both exatecan and bezocertib (TAA(Exa+Ber)), anti-TAA antibodies conjugated only to exatecan (TAA(Exa)), isotype-matched control antibodies (isotype(unworn)), or isotype-matched control antibodies conjugated to exatecan (isotype(Exa)) to SHP-77 living cells, as measured by flow cytometry. [Figure 8B] This shows the mean fluorescence intensity (MFI) of anti-TAA antibodies (TAA(unworn)) bound to SHP-77 live cells, anti-TAA antibodies conjugated to both exatecan and bezocertib (TAA(Exa+Ber)), anti-TAA antibodies conjugated only to exatecan (TAA(Exa)), isotype-matched control antibodies (isotype(unworn)), or isotype-matched control antibodies conjugated to exatecan (isotype(Exa)), as measured by flow cytometry. [Figure 8C] This shows the binding of anti-TAA antibodies (TAA(unworn)), anti-TAA antibodies conjugated to both exatecan and bezocertib (TAA(Exa+Ber)), anti-TAA antibodies conjugated only to exatecan (TAA(Exa)), isotype-matched control antibodies (isotype(unworn)), or isotype-matched control antibodies conjugated to exatecan (isotype(Exa)) to NCI-H82 living cells, as measured by flow cytometry. [Figure 8D]This shows the mean fluorescence intensity (MFI) of anti-TAA antibodies (TAA(unworn)) conjugated to NCI-H82 live cells, anti-TAA antibodies conjugated to both exatecan and bezocertib (TAA(Exa+Ber)), anti-TAA antibodies conjugated only to exatecan (TAA(Exa)), isotype-matched control antibodies (isotype(unworn)), or isotype-matched control antibodies conjugated to exatecan (isotype(Exa)), as measured by flow cytometry. [Figure 9A] This shows the intracellular localization of anti-TAA antibodies conjugated to both exatecan and bezocertib (TAA(Exa+Ber)) or anti-TAA antibodies conjugated only to exatecan (TAA(Exa)) in NCI-H82 cells after 0, 0.5, or 2 hours of incubation, as measured by immunofluorescence microscopy. [Figure 9B] This shows the intracellular localization of anti-TAA antibodies conjugated to both exatecan and bezocertib (TAA(Exa+Ber)) or anti-TAA antibodies conjugated only to exatecan (TAA(Exa)) in TAA-overexpressing HEK293 cells after 0, 0.5, or 2-hour incubation, as measured by immunofluorescence microscopy. [Figure 10] The graph shows the percentage change in body weight over time after administration to mice at doses of 3 mg / kg body weight, 10 mg / kg body weight, 30 mg / kg body weight, or 60 mg / kg body weight of an anti-TAA antibody conjugated to both exatecan and bezocertib (TAA(Exa+Ber)) or an anti-TAA antibody conjugated only to exatecan (TAA(Exa)), or after administration of the vehicle alone (vehicle). [Figure 11A]This shows the levels of erythrocyte index and anemia parameters in the blood of mice after two doses of anti-TAA antibodies conjugated to both exatecan and berzocertib at 3 mg / kg body weight, 10 mg / kg body weight, 30 mg / kg body weight, or 60 mg / kg body weight (TAA(Exa+Ber)), anti-TAA antibodies conjugated to exatecan only (TAA(Exa)), or vehicle only (vehicle). [Figure 11B] This shows the leukocyte index and platelet levels in the blood of mice after two doses of anti-TAA antibodies conjugated to both exatecan and berzocertib at 3 mg / kg body weight, 10 mg / kg body weight, 30 mg / kg body weight, or 60 mg / kg body weight (TAA(Exa+Ber)), or anti-TAA antibodies conjugated to exatecan only (TAA(Exa)), or vehicle only (vehicle). [Figure 12A] The levels of liver, kidney, and pancreatic indices shown in the blood of mice after two doses of anti-TAA antibodies conjugated to both exatecan and berzocertib at 3 mg / kg body weight, 10 mg / kg body weight, 30 mg / kg body weight, or 60 mg / kg body weight (TAA(Exa+Ber)), anti-TAA antibodies conjugated to exatecan only (TAA(Exa)), or vehicle only (vehicle). [Figure 12B] The levels of electrolytes shown in the blood of mice after two doses of anti-TAA antibodies conjugated to both exatecan and berzocertib at 3 mg / kg body weight, 10 mg / kg body weight, 30 mg / kg body weight, or 60 mg / kg body weight (TAA(Exa+Ber)), anti-TAA antibodies conjugated to exatecan only (TAA(Exa)), or vehicle only (vehicle). [Figure 13A] The Loewe synergy scores of exatecan and belzocertib in HEC1-B cells (upper panel) and the percentage inhibition by exatecan and belzocertib alone and in combination (lower panel) are shown. [Figure 13B]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 14A] The levels of pATR, pCHK1, pH2AX, and CHK1 are shown 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 14B] The levels of pATR, pCHK1, pH2AX, and CHK1 are shown 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 15A] This shows the survival rates 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 15B] This shows the body weight changes 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). [Figure 16A] 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 16B]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 17] 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 18] The image shows antibody concentrations after administration of pertuzumab conjugated to both exatecan and velzocertib at 10 mg / kg ("conjugated Ab (dual-payload ADC)") or pertuzumab at 10 mg / kg ("unconjugated Ab (naked)") in immunocompetent BALB / c mice (upper panel) and immunodeficient NSG mice (lower panel). [Figure 19] 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 20] 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 21] This represents the percentage increase in hydrophobic interaction chromatography retention time (RT) in single-payload ADC and T-(Exa+Ber). [Figure 22] This describes the plasma stability of ADC5 and ADC6 over a 7-day period. [Figure 23]This describes the efficacy of dual-payload ADCs against cells from the NCI-N87 gastric cancer cell line. [Figure 24] The efficacy of dual and single-payload ADCs in cell killing of the NCI-N87 gastric cancer cell line. [Figure 25] The efficacy of dual and single-payload ADCs in cell killing of the NCI-N87 gastric cancer cell line. [Figure 26] This study demonstrates the in vivo antitumor efficacy of dual and single-payload ADCs in xenograft mouse models. [Figure 27] This study describes the in vivo efficacy of dual-payload ADCs containing belzocertib (ATRi) or plexacertib (CHK1i) in mouse models. [Figure 28] This demonstrates the in vivo effectiveness of dual-payload ADCs with different target DAR ratios. [Figure 29A] The results of weight changes after treatment with dual-payload ADC in primate studies are shown. [Figure 29B] The results of biochemical and hematological tests after treatment with dual-payload ADC in primate studies are shown. [Figure 30] The efficacy of dual-payload ADCs with TMTHSI or DBCO moieties against the NCI-N87 gastric cancer cell line. [Figure 31] This is the clearance of dual-payload ADCs with TMTHSI or DBCO moieties compared to non-conjugated control antibodies in immunocompetent mice. [Figure 32] This demonstrates the effectiveness of a dual-payload ADC with different conjugations. Detailed description of the invention

[0037] The present disclosure provides an antigen-binding molecule with unexpected and advantageous properties compared to known antibody-drug conjugates. In particular, DNA damage repair (DDR) is a key mechanism of resistance to topoisomerase I (TOP1) DNA damaging agents, and it is expected that combining both payloads into a single antibody-drug conjugate will likely exhibit improved synergistic effects. Dual-payload ADCs are also expected to have advantageous properties compared to combinations of DDR inhibitors and TOP1 inhibitor ADCs in reducing the overlapping toxicities of DDR inhibitors and TOP1 inhibitor ADCs of chemotherapy agents, such as neutropenia and thrombocytopenia. The dual approach with DDR inhibitors and TOP1 inhibitors is also expected to mitigate resistance to TOP1, which is sometimes observed in treatment with ADCs containing a TOP1 inhibitor payload. Such resistance is described in Mosele, et al., Nat Med (2023) 29(8) 2110-2120 (PMID37488289), Zhang, et al., Br J Cancer (2021) 125(10) 1333-1340 (PMID34294893), and Muai, et al., Mol Cell (2018) 69(3) 371-384 (PMID29395061).

[0038] The combination of DDR inhibitors and TOP1 inhibitors has also been shown preclinically and clinically to resensitize tumors to TOP1 inhibitors, thereby restoring their responsiveness to validated TOP1 inhibitor therapy. Josse et al., Cancer Res (2014) 74(23):6968-6979 describes that belzocertib (ATR inhibitor) enhances the action of irinotecan in the CRC CDX model (COLO205), Coussy et al., Sci Trans Med (2020) 12 (531):eaax2625 describes that belzocertib (ATR inhibitor) increases sensitivity to irinotecan in xenograft tumors derived from SLFN11-negative TNMC patients exhibiting BRCAness, Thomas et al., Cancer Cell (2021) 39(4):566-579.e7 describes that belzocertib (ATR inhibitor) enhances the efficacy of topotecan in chemotherapy-resistant SCLC patients, and Slotkin, et al. (ASCO 2022 Abstract) 11503) describes how prexacertib (a CHK1 inhibitor) resensitizes recurrent or treatment-resistant fibrionic round cell tumors to irinotecan. Further explanation of the combination of DDR inhibitors and TOP1 inhibitors is given below.

[0039] Linker-payload section This disclosure relates to an antigen-binding molecule comprising a linker-payload portion. As used herein, a linker-payload portion refers to a portion comprising one or more payload portions and a linker portion for linking the payload portion(s) to the antigen-binding region of the antigen-binding molecule.

[0040] 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.

[0041] 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."

[0042] 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).

[0043] 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.

[0044] 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).

[0045] 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 (azenocertib), 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).

[0046] In some embodiments, the DDR inhibitor portion is or includes ceraracertib.

[0047] In some embodiments, the DDR inhibitor portion is belzocertib: [ka] It is either or includes it.

[0048] In some embodiments, the DDR inhibitor portion is plexacertib: [ka] It is either or includes it.

[0049] 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.

[0050] 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.

[0051] In some embodiments, the DDR inhibitor portion is nedisertib: [ka] It is either or includes it.

[0052] In some embodiments, the DDR inhibitor portion is not veriparib or does not include it. In some embodiments, where the DDR inhibitor portion is a PARP inhibitor or includes it, the PARP inhibitor is not veriparib. In some embodiments, the DDR inhibitor portion is not a PARP inhibitor or does not include it.

[0053] 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. DNA topoisomerase I inhibitors and their use in the treatment of cancer are 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).

[0054] 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.

[0055] In some embodiments, the TOP1 inhibitory portion is not PBX-7016 as described in WO2023 / 249473A1, or does not contain it (its structure is shown in Example 3 of WO2023 / 249473A1). In some embodiments where the TOP1 inhibitory portion is a camptothecin derivative, or contains it, the camptothecin derivative is not PBX-7016 as described in WO2023 / 249473A1. In some embodiments, the TOP1 inhibitory portion is not a camptothecin derivative as described in WO2023 / 249473A1, or does not contain it.

[0056] In some embodiments, the TOP1 inhibitory portion is exatecan: [ka] It is or includes the TOP1 inhibitor portion. 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] The linker portion of the present disclosure may be any portion suitable for linking the payload portion to the antigen-binding region of the antigen-binding molecule of the present 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.

[0057] In some embodiments, the antigen-binding molecules of this disclosure do not contain veliparib and do not contain PBX-7016 as described in WO2023 / 249473A1.

[0058] 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.

[0059] Part including the payload The portion of this disclosure including a payload may include the payload and a linker portion between the payload and a click group, the click group being selected from the click group pairs described below. The linker portion may be a cleavable linker portion or a non-cleavable portion.

[0060] The linker portion provided in this disclosure may be a detachable linker portion or a non-detachable portion.

[0061] 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).

[0062] In some embodiments, the linker portion according to the Disclosure is a chemically cleaved linker. In some embodiments, the linker portion according to the Disclosure is an enzymatically cleaved linker. In some embodiments, the linker portion is an acid-cleavable linker comprising, for example, a hydrazone group (e.g., 6-maleimidocaproylhydrazone linker or (4-(4-acetylphenoxy)butanoic acid)hydrazone linker), a carbonate group, or a silyl ether group. In some embodiments, the linker portion is a GSH-cleavable linker comprising, for example, a disulfide group. In some embodiments, the linker portion is an Fe(II)-cleavable linker comprising, for example, a 1,2,4-trioxolane group. In some embodiments, the linker portion is a cathepsin-cleavable linker comprising, for example, a dipeptide (e.g., valine-citrulline linker, phenylalanine-lysine linker, or valine-alanine linker), a triglycyl peptide (CX), or a cBu-Cit group. In some embodiments, the linker portion is GGFG (glycine-glycine-phenylalanine-glycine). In some embodiments, the linker portion is a glucuronidase-cleavable linker containing, for example, a β-glucuronide group. In some embodiments, the linker portion is a glycosidase-cleavable linker containing, for example, a β-galactoside group. In some embodiments, the linker portion is a phosphatase-cleavable linker containing, for example, a pyrophosphate group. In some embodiments, the linker portion is a sulfatase-cleavable linker containing, for example, an arylsulfate group. In some embodiments, the linker portion is a photoresponsive linker containing, for example, a heptamethyn cyanine fluorophore group, an O-nitrobenzyl group, or a PC4AP group. In some embodiments, the linker portion is a bioorthogonal-cleavable linker containing, for example, a dsProc group.

[0063] Non-cleavable linkers remain inactive in the general chemical and enzymatic environment within the body, and the payload portion is released after processing of the ADC by cellular lysosomal proteases. Examples of non-cleavable linkers include those containing thioether or maleimidocaproyl groups.

[0064] In some embodiments, the linker portion is a thioether linker. In some embodiments, the linker portion is a maleimidocaproyl linker, for example, comprising a 2-(maleimidomethyl)-1,3-dioxane (MD) group or a Mal-PAB group. In some embodiments, the linker portion comprises a polyethylene glycol (PEG) group and an alkyne, triazole, or piperazine group.

[0065] In some embodiments, the linker-payload portion according to this disclosure has an amino(-NH2) group for linking to the antigen-binding portion, for example, by enzymatic conjugation. In some of these embodiments, enzymatic conjugation using microbial transglutaminase may be used to conjugate the linker-payload portion to the antigen-binding portion.

[0066] 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.

[0067] The payload portion of this disclosure may include or consist of cytotoxic agents. Such payload portions are 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).

[0068] The payload portion of this disclosure may include, or may consist of, a non-cytotoxic agent. Such payload portions are found, for example, in Liu et al., Expert Opinion on Biological Therapy. 2016 16(5), 591-593 (doi:10.1517 / 14712598.2016.1161753), Yu et al., (2018). Next Horizons: ADCs Beyond Oncology. Damelin, M. (eds) Innovations for Next-Generation Antibody-Drug Conjugates. Cancer Drug Discovery and Development (doi:10.1007 / 978-3-319-78154-9_14), McPherson & Hobson, Methods in Molecular Biology. 2019, 2078, 23-36 (doi:10.1007 / 978-1-4939-9929-3_2), and Hobson, Progress in These are described in Medicinal Chemistry. 2023 Volume 62, 1-59 (doi: 10.1016 / bs.pmch.2023.10.001) (all of which are incorporated herein by reference in their entirety).

[0069] In some embodiments, the payload portion of the present disclosure may include, or may consist of, immunotherapies, e.g., nonsteroidal agents, e.g., PDE4 inhibitors, LXR agonists, tyrosine kinase inhibitors, bisphosphonates; steroidal agents, e.g., glucocorticoid receptor modulators (GRMs); and immunostimulants, e.g., TLR7 / 8 agonists, TLR4 agonists, TLR9 agonists, STING agonists, kinesin spindle kinase (KSP) inhibitors. Other immunotherapies include kinase inhibitors, growth factor inhibitors (e.g., EGFR, PDGF, VEGF inhibitors), calcineurin inhibitors, CRAC inhibitors, PARP1 antagonists, PPARy agonists, Kv1.3 antagonists, PP2A agonists, MYD88 inhibitors, BCL-2 inhibitors, adenosine A2A receptor (A2ar) agonists, calcium-activated potassium channel (Kca3.1) agonists, TGF-R1 inhibitors, TGF-R2 inhibitors, GLi1 inhibitors, tankyrase (TNKS) antagonists, Traf2 and Nck interaction kinase (TNIK) antagonists, imides, and vitamin D receptor (VDR) agonists.

[0070] In some embodiments, the payload portion of the present disclosure may include, or may consist of, an HDAC inhibitor, a NAMPT inhibitor, or a DHFR agent.

[0071] In some embodiments, the payload portion according to this disclosure may include, or may consist of, an antibiotic, amantine, or a matrix metalloproteinase (MMP).

[0072] In some embodiments, the payload portion according to this disclosure may include or consist of an amyloid-beta plaque degrading agent. In some embodiments, the payload portion may include or consist of a tau protein aggregate degrading agent.

[0073] In some embodiments, the payload portion according to the present disclosure may include, or may consist of, an atherosclerotic plaque stabilizer.

[0074] In some embodiments, the payload portion according to this disclosure may include or consist of an adipose tissue degrading agent. In some embodiments, the payload portion may include or consist of an insulin resistance modifier or a metabolic pathway activator.

[0075] In some embodiments, the payload portion according to this disclosure may include or consist of coagulation factors. In some embodiments, the payload portion may include or consist of erythropoiesis-stimulating agents.

[0076] In some embodiments, the payload portion of the present disclosure may include or consist of an extracellular matrix degrading agent.

[0077] In some embodiments, the payload portion according to this disclosure may include, or may consist of, microtubule targeting agents, DNA targeting agents, RNA targeting agents, immune system activators, apoptosis promoters, metabolic inhibitors, and proteasome inhibitors.

[0078] In some embodiments, the payload portion of this disclosure comprises or comprises a microtubule-targeting agent. Microtubules play a crucial role in maintaining proper cell morphology, signaling, organelle transport, cell motility, and cell division. Microtubules are formed from tubulin, and agents that disrupt tubulin polymerization dynamics lead to cell cycle arrest and apoptosis. Tubulin inhibitors are more toxic to rapidly dividing cancer cells than to more slowly proliferating non-cancerous cells. Examples of microtubule-targeting agents include tubulin polymerization promoters (e.g., auristatin, taxanes) and tubulin polymerization inhibitors (e.g., mytansinoids, colchicine). In some embodiments, the payload portion of this disclosure comprises or comprises a mytansinoid, e.g., mytansin or its derivatives, e.g., mytansin, DM1 (meltansin), or DM4 (labtansin). In some embodiments, the payload portion comprises or comprises colchicine or its derivatives. In some embodiments, the payload portion includes or comprises auristatin, for example, a dorastatin 10 derivative, for example, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin PE, auristatin PYE, PF-06380101, auristatin F-hydroxypropylamide (AF-HPA), or azastatin. In some embodiments, the payload portion includes or comprises a halichondrin B derivative, for example, eribulin. In some embodiments, the payload portion includes or comprises tubulicin or its derivatives, for example, tubulicin A, D, H, U, or V. In some embodiments, the payload portion includes or comprises cryptophycin or its derivatives, for example, cryptophycin-1, cryptophycin-52, cryptophycin-55, or cryptophycin-55gly. In some embodiments, the payload portion includes or consists of an EG5 (i.e., kinesin / KSP / KIF11) inhibitor, such as ispinesib (SB715992) or a derivative thereof, or filanesib (ARRY-520) or a derivative thereof.In some embodiments, the payload portion includes or comprises a taxane, for example, paclitaxel, docetaxel, or cabazitaxel. In some embodiments, the payload portion includes or comprises a vinca alkaloid, for example, vinblastine, vincristine, vindesine, vinorelbine, or vinflunin. In some embodiments, the payload portion includes or comprises hemiasterlin or a derivative thereof, for example, hemiasterlin, hemiasterlin A, or HTI-286.

[0079] In some embodiments, the payload portion of the present disclosure includes or comprises a DNA targeting agent. Examples of DNA targeting agents include agents that directly or indirectly disrupt DNA by introducing / promoting the formation of single-strand and / or double-strand breaks (e.g., engine, topoisomerase inhibitors), DNA alkylating agents (e.g., pyrrolo[2,1-c][1,4]benzodiazepine, indolinobenzodiazepine, duocalmycin), and DNA crosslinking agents (e.g., mitomycin C). In some embodiments, the payload portion of the present disclosure includes an engine, e.g., calichemycin-like engine, or an anthraquinone fusion engine, e.g., calichemycin γ. I 1. Calcare Sewing Machine θ I1, or comprising or consisting of uncialamicin. In some embodiments, the payload portion comprises or consists of a topoisomerase inhibitor, e.g., a TOP1 or TOP2 inhibitor, e.g., camptothecin or its derivatives, e.g., SN-38, exatecan, exatecan mesylate (DX-8951f), N-glycyl-exatecan, or deruxtecan (Dxd); e.g., anthracyclines, e.g., doxorubicin, daunorubicin, epirubicin, PNU-159682, or idarubicin. In some embodiments, the payload portion comprises or consists of a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer or its derivatives, e.g., PDB, KMR-28-39, SJG-136 SGD-1882, or SG3199 dimer. In some embodiments, the payload portion includes or consists of indolinobenzodiazepine (IGN; monoimine) or a derivative thereof. In some embodiments, the payload portion includes or consists of pyridinobenzodiazepine (PDD) dimer or a derivative thereof, for example, PDD or FGX5-67 dimer. In some embodiments, the payload portion includes or consists of duocalmycin or a derivative thereof, for example, duocalmycin A, CC1065, duocalmycin SA, DUBA, seco-DIBA, or seco-CBI. In some embodiments, the payload portion includes or consists of mitomycin C.

[0080] In some embodiments, the payload portion according to this disclosure includes or comprises an RNA targeting agent. RNA-targeting small molecule inhibitors can kill both proliferating and dormant tumor cells. Examples of RNA targeting agents include RNA splicing inhibitors (e.g., tylanstatin and its derivatives) and RNA polymerase II inhibitors (e.g., amatoxin, RNA polymerase II-IN-2). In some embodiments, the payload portion according to this disclosure includes or comprises tylanstatin or its derivatives, e.g., tylanstatin A, tylanstatin B, tylanstatin C, or FR901464. In some embodiments, the payload portion includes or comprises amatoxin, e.g., α-amanitin or β-amanitin. In some embodiments, the payload portion includes or comprises RNA polymerase II-IN-2.

[0081] In some embodiments, the payload portion of this disclosure comprises or comprises an immune system activator. Immunostimulatory antibody conjugates (ISACs) utilize the action of the innate and / or adaptive immune systems based on small molecules. Various immunomodulatory payloads are under development, including Toll-like receptor (TLR) agonists, interferon gene-stimulating factor (STING) agonists, and glucocorticoid receptor modulators (GRMs). In some embodiments, the payload portion of this disclosure comprises or comprises a TLR agonist, e.g., TLR7, TLR8, or TLR9 agonist. In some embodiments, the payload portion comprises or comprises a STING agonist, e.g., a cyclic dinucleotide (CDN; e.g., 2,3-cGAMP; 3,3-cGAMP; c-di-GMP, or c-di-AMP) or benzimidazole. In some embodiments, the payload portion comprises or comprises a glucocorticoid receptor modulator, e.g., dexamethasone or a derivative thereof.

[0082] In some embodiments, the payload portion of the present disclosure includes or comprises an apoptosis-promoting agent. Anti-apoptotic proteins such as Bcl-xL may play an important role in tumorigenesis, metastasis, and drug resistance. In some embodiments, the payload portion of the present disclosure includes or comprises a Bcl-xL inhibitor. In some embodiments, the payload portion includes or comprises ABT-737.

[0083] In some embodiments, the payload portion of the present disclosure includes or comprises a metabolic inhibitor. Metabolic inhibitors, such as niacinamide phosphate-ribose transferase (NAMPT) inhibitors, regulate the concentration of NAD+ in cells, thereby inducing energy crises and cell death, and inhibit dihydrofolate reductase (DHFR), thereby inhibiting DNA synthesis. Antifolate antagonists, such as methotrexate. In some embodiments, the payload portion of the present disclosure includes or comprises a NAMPT inhibitor, such as FK-866 or A-1293201. In some embodiments, the payload portion of the present disclosure includes or comprises a DHFR inhibitor, such as methotrexate or a derivative thereof.

[0084] In some embodiments, the payload portion according to the present disclosure includes or comprises a proteasome inhibitor. Examples of proteasome inhibitors include carmaficin. In some embodiments, the payload portion according to the present disclosure includes or comprises carmaficin or a derivative thereof, for example, carmaficin A or carmaficin B.

[0085] In some embodiments, the antigen-binding molecule according to the present disclosure comprises a linker-payload moiety, which further comprises both a DDR inhibitor moiety and a TOP1 inhibitor moiety. That is, in some embodiments, the antigen-binding molecule comprises a linker-payload moiety comprising (a) a DDR inhibitor moiety and (b) a TOP1 inhibitor moiety. In some embodiments, the DDR inhibitor moiety and the TOP1 inhibitor moiety are provided in the same linker-payload moiety. In some embodiments, the DDR inhibitor moiety and the TOP1 inhibitor moiety are linked to the antigen-binding portion of the antigen-binding molecule via the same linker moiety.

[0086] Techniques for conjugating multiple payload moieties to a single linker moiety 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.

[0087] In some embodiments, the DDR inhibitor and TOP1 inhibitor payload moieties are provided in the same linker-payload moiety. In some embodiments, the DDR inhibitor and TOP1 inhibitor payload moieties are linked to the antigen-binding portion of the antigen-binding molecule by the same linker moiety.

[0088] In some embodiments, the DDR inhibitor and TOP1 inhibitor payload moieties are linked to the antigen-binding portion of the antigen-binding molecule by a branched linker moiety. The linker may comprise multiple branches to allow for DAR flexibility.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] The branched linker portion described by Kumar et al., Bioorg Med Chem Lett (2018) 28(23-24):3617-3621 has the following structure. [ka]

[0094] 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.

[0095] 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.

[0096] 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 incorporating the payload portion by strain-enhancing azide-dibenzocyclooctin (DBCO) cycloaddition, and (ii) A branched linker moiety is described which includes a methyltetrazine group for incorporating the payload moiety by trans-cyclooctene (TCO) cycloaddition, and two orthogonal functional groups for linking the payload moiety.

[0097] The branched linker portion described in Yamazaki, et al., Nat Commun. (2021) 12(1):3528 has the following structure. [ka]

[0098] 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 lysine-based group), (ii) one or two portions derived from an azide group suitable for incorporating a first payload portion by DBCO cyclization, and (iii) a portion derived from a methyltetrazine group for incorporating a second payload portion by TCO cyclization. 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.

[0099] In some embodiments, the antigen-binding molecule according to the present disclosure comprises a linker-payload moiety that includes (i) a first payload moiety conjugated to the linker moiety via a DBCO cycloaddition reaction between a DBCO group and an azide group, and (ii) a second payload moiety conjugated to the linker moiety via a TCO cycloaddition reaction between a TCO group and a methyltetrazine group. 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.

[0100] In some embodiments, the DDR inhibitor moiety and the TOP1 inhibitor moiety are linked to the linker moiety of the linker-payload moiety via a cysteine group. Such a preparation method for a linker-payload moiety comprising two different payloads is described, for example, in Levengood, et al., Angew Chem Int Ed Engl (2017) 56(3):733-737.

[0101] Levengood, et al., Angew Chem Int Ed Engl (2017) 56(3):733-737 describes a linker-payload moiety comprising two different payload moieties, constructed by sequential deprotection of orthogonally protected cysteines. Each payload is linked to a maleimide group (e.g., via 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. [Chemical Formula]

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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).

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] This disclosure is, (a) an amino group for conjugation to the antigen-binding site, (b) at least one first click base for linking portions including the first payload, (c) at least one second click base for linking the portion containing the second payload, (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 the location where the at least one first click group is connected to the branching group. The present invention provides a trifunctional linker portion including a branching group, where c indicates a location in which at least one second click group is linked to the branching group.

[0117] In some embodiments, R N H is H.

[0118] In some embodiments, R N is, -(C 1~5 The molecule is alkylene)-C(O)OH, where one CH2 unit may be replaced by -O-.

[0119] In some embodiments, R N is, -(C 1~5 It is alkylene-C(O)OH.

[0120] In some embodiments, R N This is -CH2CH2OCH2CH2C(O)OH.

[0121] In some embodiments, R N It is CH2C(O)OH.

[0122] 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

[0123] A1 is given by the following formula: -(CH2) xa -(C2H4O) xb -(CH2) xc - 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.

[0124] In some embodiments, xb is 0, and xa + xc is 1 to 7, for example, 5 (i.e., A1 is -(CH2)5-).

[0125] In other embodiments, xb is 1 to 12, xa is 0, and xc is 0 or 1.

[0126] 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.

[0127] 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.

[0128] 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)-.

[0129] The first and second click groups may be selected from any of the following click group pairs. [Table 1]

[0130] Examples of cyclooctin, cyclooctin derivatives, and cyclooctin analogs used in this disclosure include the following: [Table 2-1] [Table 2-2] These groups can alternatively be referred to as cyclic alkynes.

[0131] Examples of tetrazine and tetrazine derivatives used in this disclosure include the following: [Table 3-1] [Table 3-2]

[0132] The strain alkenes used in this disclosure may have the following structures. [Table 4]

[0133] In some embodiments, the click group may be a dibenzoazacyclooctin (DIBAC) group or a 1-ethylhepta-6-enoxy group. [Table 5]

[0134] In some embodiments, the click group may be tetramethylthiocycloheptine sulfoximine (TMTHSI). [Table 6]

[0135] In some embodiments of the trifunctional linking group, the first and second click groups are the same.

[0136] In other embodiments of the trifunctional linking group, the first and second click groups are selected from an orthogonal click group pair.

[0137] In some embodiments of the trifunctional linking group, the first and / or second click group is an azide.

[0138] In some embodiments of the trifunctional linking group, the first and / or second click group is tetrazine or a tetrazine derivative.

[0139] In some embodiments of the trifunctional linking group, the first and / or second click group is an alkyne (-CCH).

[0140] In some embodiments of the trifunctional linking group, the first and / or second click group is cyclooctin or a cyclooctin derivative.

[0141] In some embodiments of the trifunctional linking group, the first and / or second click group is norbornene or a norbornene derivative.

[0142] In some embodiments of the trifunctional linking group, the first and / or second click group is methylcyclopropene (1-MCP).

[0143] 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.

[0144] In some embodiments of the trifunctional linking group, the first click group is an azide, and the second click group is a cyclooctene.

[0145] 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.

[0146] In some embodiments of the trifunctional linking group, the first click group is an azide, and the second click group is methylcyclopropene (1-MCP).

[0147] 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.

[0148] In some embodiments of the trifunctional linking group, the first click group is an alkyne (-CCH) and the second click group is a cyclooctene.

[0149] 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.

[0150] 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).

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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).

[0155] In some embodiments of the trifunctional linking group, the second click group is phenyltetrazine.

[0156] In some embodiments of the trifunctional linking group, the second click group is selected from the following group: [ka]

[0157] In some embodiments of the trifunctional linking group, the second click group is [ka] That is the case.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] In some embodiments, xl4 is 0. In some embodiments, xl4 is 1.

[0163] 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.

[0164] 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.

[0165] In some of these embodiments of the trifunctional linking group, the second spacer group (B1) is of formula (B1-2): [ka] It is, In the formula, 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.

[0166] 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.

[0167] 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.

[0168] In some embodiments, xe1 is 0. In some embodiments, xe1 is 1.

[0169] 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.

[0170] In some embodiments, xg1 is 0. In some embodiments, xg1 is 1.

[0171] In some embodiments, xh1 is 0. In some embodiments, xh1 is 1.

[0172] In some embodiments, xe2 is 0. In some embodiments, xe2 is 1.

[0173] 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.

[0174] In some embodiments, xg2 is 0. In some embodiments, xg2 is 1.

[0175] In some embodiments, xe1 is 1, xf1 is 2, xg1 is 0, and xh1 is 0.

[0176] In some embodiments, xe1 is 0, xf1 is 0, xg1 is 0, and xh1 is 1.

[0177] In some embodiments, xe1 is 1, xf1 is 2, xg1 is 1, and xh1 is 1.

[0178] In some embodiments, xe2 is 1, xf2 is 2, xg2 is 0, and xh2 is 0.

[0179] In some embodiments, xe2 is 0, xf2 is 0, xg2 is 0, and xh2 is 1.

[0180] In some embodiments, xe2 is 1, xf2 is 2, xg2 is 1, and xh2 is 1.

[0181] In some embodiments of the trifunctional linking group, the second spacer group (B1) is selected from the group including: [ka]

[0182] In some embodiments of the trifunctional linking group, the second spacer group (B1) includes the following: [ka] Selected from: In some embodiments of the trifunctional linking group, the second spacer group (B2) is of 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.

[0183] 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.

[0184] In some embodiments, xl6 is 0. In some embodiments, xl6 is 1.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] In some embodiments, R NB4 is H. In some embodiments, R NB4 is -(C2H4O) xj2 -(CH2) xk2 -(NH) xl2 -(C(=O)CH2) xm2 -. In other embodiments, R NB4 is the same as R NB3 .

[0189] In some embodiments, xi is 0 to 2. In some embodiments, xi is 0 to 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.

[0190] In some embodiments, xj1 is 0. In some embodiments, xj1 is 1.

[0191] In some embodiments, xk1 is 0 to 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.

[0192] In some embodiments, xl1 is 0. In some embodiments, xl1 is 1.

[0193] In some embodiments, xm1 is 0. In some embodiments, xm1 is 1.

[0194] In some embodiments, xj2 is 0. In some embodiments, xj2 is 1.

[0195] 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.

[0196] In some embodiments, xl2 is 0. In some embodiments, xl2 is 1.

[0197] In some embodiments, xj1 is 1, xk1 is 2, xl1 is 0, and xm1 is 0.

[0198] In some embodiments, xj1 is 0, xk1 is 0, xl1 is 0, and xm1 is 1.

[0199] In some embodiments, xj1 is 1, xk1 is 2, xl1 is 1, and xm1 is 1.

[0200] In several embodiments, xj2 is 1, xk2 is 2, xl2 is 0, and xm2 is 0.

[0201] In some embodiments, xj2 is 0, xk2 is 0, xl2 is 0, and xm2 is 1.

[0202] In several premises, xj2 is 1, xk2 is 2, xl2 is 1, and xm2 is 1.

[0203] In some embodiments of the trifunctional linking group, the third spacer group (B2) is selected from the group including: [ka]

[0204] In some embodiments of the trifunctional linking group, the third spacer group (B2) is selected from the group including: [ka]

[0205] 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).

[0206] In some embodiments of the trifunctional linking group, the second spacer group (B1) and the third spacer group (B2), together with the nitrogen atom to which they are bonded, form one of the following groups: [ka]

[0207] In some embodiments of the trifunctional linking group, the second spacer group (B1) and the third spacer group (B2), together with the nitrogen atom to which they are bonded, form one of the following groups: [ka] [ka]

[0208] This disclosure is, (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 NHowever, 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.

[0209] Examples of click groups are shown below. [Table 7-1] [Table 7-2] 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.

[0210] The amino group may be linked to the branched group by the first spacer group (A1) defined above.

[0211] At least one first click group may be linked to a branching group by a second spacer group (B1) as defined above.

[0212] At least one second click group may be linked to the branching group by a third spacer group (B2) as defined above.

[0213] In some embodiments, the linker includes one of the following groups: [ka] [ka] [ka] [ka]

[0214] 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.

[0215] 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.

[0216] 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 X This is a case where Q is an amino acid residue, a dipeptide residue, or a tripeptide residue. X is [ka] And, 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.

[0217] 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.

[0218] c can be 0 or 1. In some of these embodiments, c is 1.

[0219] 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.

[0220] 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.

[0221] In one embodiment, Q is an amino acid residue. The amino acid may be a natural amino acid or a non-natural amino acid.

[0222] In one embodiment, Q is selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, Ser, and Trp, where Cit is citrulline. Alternatively, Q may be a serine derivative (see WO2018 / 234636A1).

[0223] In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of native and non-native amino acids. In some embodiments, the dipeptide comprises native amino acids. When the linker is a cathepsin-instability linker, the dipeptide is the site of action for cathepsin-mediated cleavage. At the same time, the dipeptide is the recognition site for cathepsin.

[0224] In one embodiment, Q is CO -Phe-Lys- NH、 CO -Val-Ala- NH、 <000101>-Val-Lys- NH、 <000103>-Ala-Lys-<000104> <000105>-Val-Cit-<000106> <000107>-Phe-Cit-<000108> <000109>-Leu-Cit-<000110> <000111>-Ile-Cit-<000112> <000113>-Phe-Arg-<000114> <000115>-Gly-Cit-<000116> <000117>-Gly-Ala-<000118>, and <000119>-Trp-Cit-<000120>are selected from Here, Cit is citrulline.

[0225] In some of these embodiments, Q is <000121>-Phe-Lys-<000122> <000123>-Val-Ala-<000124> <000125>-Val-Lys-<000126> <000127>-Ala-Lys-<000128> <000129>-Val-Cit-<000130>is selected from.

[0226] In a further embodiment, Q is<000131>-Phe-Lys-<000132>,CO -Val-Cit- NH , and CO -Val-Ala- NH Selected from.

[0227] 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.

[0228] 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.

[0229] In some embodiments, the TOP1 inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0230] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0231] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0232] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0233] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0234] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0235] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0236] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0237] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0238] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0239] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0240] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0241] In some embodiments, the DDR inhibitor moiety linked to the click group has the following structure: [ka] It has.

[0242] In some embodiments, the antigen-binding molecules of this disclosure do not include, or are not of, the structure of "trastuzumab-veliparib(4)-25-6(4)" as described in WO2023 / 249473A1, for example, as described in Example 7 of WO2023 / 249473A1.

[0243] Antigen-binding molecule and antigen-binding portion An "antigen-binding molecule" refers to a molecule that binds to a given target antigen. An antigen-binding molecule comprises one or more antigen-binding moieties, thereby binding to its target antigen(s). For example, aspects and embodiments of this disclosure relate to antigen-binding molecules comprising target antigen-binding moieties.

[0244] 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.

[0245] 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).

[0246] Those skilled in the art can easily produce antigen-binding molecules that bind to a given target antigen, taking into account the techniques well known in the art. For example, Park and Smolen, Advances in Protein Chemistry (2001) 56:369-421, describe methods for producing monoclonal antibodies suitable for human use, including the production of heterologous antibodies and their subsequent humanization, identification by human antibody gene-phage display, and antibody production in transgenic mice possessing human antibody genes. In summary, in the human antibody gene-phage display technique, genes encoding VH and VL chains are generated from "naive" human lymphocytes by PCR amplification and cloning, assembled into a library, and these genes can be expressed from them 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 a filamentous bacteriophage, and then Fab or scFv capable of binding to a target of interest can be identified by screening the library with an antigen. Molecular evolution or affinity maturation methods may be used to enhance the affinity of Fab / scFv fragments. In transgenic mouse technology, mice in which the endogenous mouse Ig gene locus has been replaced with its human homolog by homologous recombination are immunized with an antigen to obtain fully human monoclonal antibodies, and the monoclonal antibodies are prepared using conventional hybridoma technology.

[0247] 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.

[0248] 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. 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.

[0249] The aspects and embodiments of this disclosure relate to antigen-binding molecules that bind to a given target (i.e., bind to a given target by an antigen-binding moiety).

[0250] The target antigen of the target antigen-binding moiety according to this disclosure may be any molecule. In some embodiments, the target antigen may be a peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or a fragment thereof. The target antigen may be expressed on the cell surface of a cell expressing the target antigen.

[0251] In some embodiments, the target antigen is a disease-related antigen. A “disease-related antigen” refers to an antigen whose presence indicates the presence of a given disease / condition, or an antigen whose elevated level is positively correlated with the given disease / condition. A disease-related antigen may be an antigen whose expression is associated with the onset, progression, or severity of symptoms of a given disease. A disease-related antigen may be associated with the cause or pathology of a disease, or may be abnormally expressed as a result of a disease. A disease-related antigen may be an antigen of an infectious agent or pathogen, a cancer-related antigen, an autoimmune disease-related antigen, a neurological disease-related antigen, a cardiovascular disease-related antigen, a metabolic disease-related antigen, a hematological disease-related antigen, or a fibrous disease-related antigen.

[0252] In some embodiments, the disease-associated antigen is the antigen of a pathogen. The pathogen may be a prokaryote (bacteria), a eukaryote (e.g., protists, helminths, fungi), a virus, or a prion. In some embodiments, the pathogen is an intracellular pathogen. In some embodiments, the pathogen is a virus, such as those described above. In some embodiments, the pathogen is a bacterium.

[0253] In some embodiments, the target antigen is a cancer-associated antigen. A cancer-associated antigen is an antigen whose expression or overexpression is associated with cancer. In some embodiments, the cancer-associated antigen is a receptor molecule, e.g., a cell surface receptor. In some embodiments, the cancer-associated antigen is a cell signaling molecule, e.g., a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the cancer-associated antigen is a growth factor or hormone. In some embodiments, the cancer-associated antigen is a viral antigen. Cancer cell antigens may be abnormally expressed by cancer cells (e.g., cancer cell antigens may be expressed with abnormal localization) or with abnormal structures. Cancer cell antigens may also induce an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen bound by the antigen-binding molecule described herein is presented on the external surface of the cancer cell (i.e., it is extracellular). Cancer cell antigens can be cancer-associated antigens. In some embodiments, cancer cell antigens are antigens whose expression is associated with the onset, progression, or severity of cancer. Cancer-associated antigens may be associated with the cause or pathology of cancer, or may be abnormally expressed as a result of cancer. In some embodiments, cancer cell antigens are antigens whose expression (e.g., at the RNA and / or protein level) is upregulated by cancer cells compared to, for example, the level of expression by comparable non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type). In some embodiments, cancer-associated antigens may be preferentially expressed by cancer cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type). In some embodiments, cancer-associated antigens may be products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer-associated antigens may be products of overexpressed cellular proteins, cancer antigens produced by oncogenic viruses, carcinoembryonic antigens, or cell surface glycolipids or glycoproteins.

[0254] Cancer-related antigens are Zarour HM, DeLeo A, Finn OJ, et al.Categories of Tumor Antigens. Kufe DW, Pollock RE, Weichselbaum RR, et al., editors.Holland-Frei Cancer Medicine.6 th This is outlined in edition. Hamilton(ON):BC Decker;2003. Cancer-related antigens include carcinoembryonic antigens: CEA, immature laminin receptor, TAG-72; and oncogenic viral antigens, such as HPV. E6 and E7; Overexpressed proteins: fibroblast-activating protein (FAP), B-cell maturation antigen (BCMA), CD19, HER2 / neu, BING-4, calcium-activated chloride ion channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1, Survivin; Cancer testicular antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; Series restriction antigens: MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate-specific antigen; Mutant antigens: β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; Post-translational altered antigen: MUC1; Idiotype antigens: Ig, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose regulatory protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placenta-like 2 (ALPPL-2), siglec-5, stress-induced phosphorylated protein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophyllin B. In some embodiments, the cancer cell antigen is one described in Zhao and Cao, Front Immunol. (2019) 10:2250 (which is incorporated herein by reference in its entirety).

[0255] In some embodiments, the target antigen is an immune cell surface molecule. An immune cell surface molecule is any molecule expressed on an immune cell or on the cell membrane of an immune cell. In some embodiments, the portion of the immune cell surface molecule bound by the antigen-binding moiety is located on the external surface of the immune cell (i.e., extracellular). Immune cell surface molecules can be expressed on the cell surface of any immune cell. In some embodiments, the immune cell may be a hematopoietic cell, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. Lymphocytes may be, for example, T cells, B cells, natural killer (NK) cells, NKT cells, or innate lymphocytes (ILCs), or their precursors (e.g., thymocytes or pre-B cells). The immune cell may express CD3 polypeptide (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), TCR polypeptide (TCRα or TCRβ), CD27, CD28, CD4, or CD8. In some embodiments, the immune cells are T cells, e.g., CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are helper T cells (TH cells). In some embodiments, the T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)). In some embodiments, the immune cells are T cells or NK cells.

[0256] In some embodiments, the immune cell surface molecule may be a CD3-TCR complex polypeptide, e.g., TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, or CD3η. In some embodiments, the immune cell surface molecule may be CD3, CD8, CD4, or CD28. In some embodiments, the immune cell surface molecule may be a checkpoint molecule (e.g., PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, or BTLA) or a ligand for a checkpoint molecule (e.g., PD-L1, PD-L2, CD80, CD86, MHC class I, MHC class II, galectin 9, VSIG3, VSIG8, LRIG1, PSGL1, CD155, or HVEM). In some embodiments, the immune cell surface molecule is a co-stimulatory molecule (e.g., CD28, OX40, 4-1BB, ICOS, or CD27) or a ligand for a co-stimulatory molecule (e.g., CD86, CD80, OX40L, 4-1BBL, ICOSL, or CD70).

[0257] 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).

[0258] 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).

[0259] 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.

[0260] 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.

[0261] 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.

[0262] Several different rules for defining antibody CDR and FR, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th VBASE2 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.

[0263] In some embodiments, the antigen-binding molecule according to this disclosure includes or comprises an Fv region that binds to a suitable target antigen. 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).

[0264] 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).

[0265] In some embodiments, the antigen-binding molecules described herein include or consist of a full-length antibody that binds to a suitable target antigen. 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).

[0266] 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).

[0267] In some embodiments, the antigen-binding molecule comprises or consists of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that bind to a suitable target antigen.

[0268] 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 derived from an IgG heavy chain constant region sequence (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, e.g., 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 derived from a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0269] 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: 2 or 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% 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: 3, 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: 4 or 19, 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: 5 or 8, 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 NOs. 9, 10, 20, or 21, 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: 1, 6, 17, or 18, 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.

[0270] 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.

[0271] 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. 11, 12, 13, 14, 15, or 16, 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.

[0272] 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.

[0273] 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 8]

[0274] 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.

[0275] 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 9]

[0276] 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.

[0277] 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.

[0278] The aspects and embodiments of this disclosure are intended to be multispecific antigen-binding molecules. "Multispecific" means that the antigen-binding molecule binds to two or more target antigens (for example, one, two, three, four, five, six, or more target antigens).

[0279] In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding moieties. In some embodiments, the antigen-binding molecule comprises at least two antigen-binding moieties, where each antigen-binding moiety binds to a different target antigen.

[0280] In some embodiments, the antigen-binding molecule comprises (i) an antigen-binding polypeptide (e.g., scFv, scFab, polypeptide aptamer, or VhH) or antigen-binding polypeptide complex (e.g., Fv, Fab, or full-length antibody) that binds to a first antigen, and (ii) an antigen-binding polypeptide (e.g., scFv, scFab, polypeptide aptamer, or VhH) or antigen-binding polypeptide complex (e.g., Fv, Fab, or full-length antibody) that binds to an antigen other than the first antigen. In some embodiments, the antigen-binding molecule comprises (i) an antigen-binding moiety comprising VH and VL of an antibody that binds to a first antigen, and (ii) an antigen-binding moiety comprising VH and VL of an antibody that binds to an antigen other than the first antigen.

[0281] The multispecific antigen-binding molecules provided herein may be provided in any preferred format, e.g., the format described in Brinkmann and Kontermann, MAbs(2017) 9(2):182-212 (which is incorporated herein by reference in its entirety). Examples of multispecific antigen-binding molecule formats include those shown in Figure 2 of Brinkmann and Kontermann, MAbs(2017) 9(2):182-212: antibody conjugates, e.g., IgG2, F(ab')2, or CovX-Body; IgG or IgG-like molecules, e.g., IgG, chimeric IgG, κλ-body common HC; CH1 / CL fusion proteins, e.g., scFv2-CH1 / CL, VHH2-CH1 / CL; and bispecific antigen-binding molecules of "variable domain only," e.g., tandem scFv(taFV). Triple body, diabody (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple head, tandem dAb / VHH, tetravalent dAb.VHH; non-Ig fusion protein, e.g., scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, mini-antibody, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2, ImmTAC(TCR-scFv); modified Fc and CH3 fusion proteins, e.g., scFv-Fc(kih), scFv-Fc(CH3 charge pair), scFv-Fc(EW-RVT), scFv-fc(HA-TF), scFv-Fc(SEEDbody), taFv-Fc(kih), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT) Fab-scFv-Fc(SEEDbody), DART-Fc, scFv-CH3(kih), TriFabs; Fc fusions, e.g., Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions, e.g., Dia-diabody, scDb-CH3;IgE / IgM CH2 fusions, e.g., scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, e.g., Fab-scFv(bibody), Fab-scFv2(tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, e.g., DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g., IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-IgG(kih), Fab-scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pair + CH1 / CL charge pair, hinge / CH3 charge pair, SEEDbody, Duobody, four-in-one CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc*; added and Fc-modified IgG, e.g., IgG(kih)-Fv, IgG HA-TF-Fv, IgG(kih)-scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, Half DVD-Ig, DVI-Ig (Four-in-One), CrossMab-Fab; Modified Fc and CH3 fusion proteins, e.g., Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEED body, TriFab; Added IgG-HC fusions, e.g., IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-Orthogonal Fab, IgG-(CαCβ)Fab, scFv-HC-IgG, Tandem Fab-IgG(Orthogonal Fab), Fab-IgG(CαCβ) Fab), Fab-IgG(CR3), Fab-Hinge-IgG(CR3); IgG-LC fusions, e.g., IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; IgG-HC and LC fusions, e.g., DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, Zybody; Fc fusions, e.g., Fab-scFv-Fc, scFv4-Ig;F(ab')2 fusions, e.g., F(ab')2-scFv2; CH1 / CL fusion proteins, e.g., scFv2-CH1-hinge / CL; modified IgGs, e.g., DAF (two-in-one IgG), DutaMab, Mab; 2 ; and non-Ig fusions, e.g., DNL-Fab4-IgG.

[0282] Those skilled in the art can design and prepare multispecific antigen-binding polypeptide / polypeptide complexes containing at least two antigen-binding moieties. Methods for producing bispecific antigen-binding polypeptide / polypeptide complexes include, for example, chemically crosslinking an antigen-binding molecule or antibody fragment using, for example, a reducible disulfide bond or an unreducible thioether bond, as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16 (which is incorporated herein by reference in its entirety). For example, N-succinimidyl 3-(2-pyridyldithio)propionic acid (SPDP) can be used, for example, to chemically crosslink a Fab fragment via the SH- group of the hinge region to produce a disulfide-bonded bispecific F(ab)2 heterodimer.

[0283] Other methods for producing bispecific antigen-binding polypeptide / polypeptide complexes include, for example, fused antibody-producing hybridomas with polyethylene glycol to produce quadroma cells capable of secreting bispecific antibodies, as described in DMand Bast, BJ2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16. Antigen-binding polypeptide / polypeptide complexes according to this disclosure may also be prepared by chemical synthesis, e.g., liquid-phase or solid-phase synthesis. For example, peptides / polypeptides may be synthesized using, for example, the methods described in Chandrudu et al., Molecules (2013), 18:4373-4388 (which is incorporated herein by reference in its entirety).

[0284] For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 thMolecular biology techniques suitable for the recombinant production of antigen-binding polypeptide / polypeptide complexes are well known in the art, such as those described in (Cold Spring Harbor Press, 2012) and Nat Methods (2008); 5(2):135-146 (both of which are incorporated herein by reference in their entirety). Methods for the recombinant production of antigen-binding polypeptide / polypeptide complexes 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). The multispecific (e.g., bispecific) antigen-binding polypeptide / polypeptide complexes described herein may be recombinantly produced, for example, by expression from nucleic acid constructs encoding polypeptides for antigen-binding polypeptide / polypeptide complexes, as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz), or French, How to make bispecific antibodies, Methods Mol. Med. 2000; 40: 333-339 (the entire contents of both of these are incorporated herein by reference).

[0285] For example, a DNA construct containing sequences encoding light and heavy chain variable domains of two antigen-binding moieties and a suitable linker or dimerization domain between antigen-binding fragments can be prepared by molecular cloning techniques. The recombinant bispecific antigen-binding polypeptide / polypeptide complex can then be produced by expression of the construct (e.g., in vitro) in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific polypeptide / polypeptide complex can then be optionally purified.

[0286] Fc area 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.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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: 9 or 10, 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: 9 or 10, 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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.

[0295] 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: 20 or 21, 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: 20 or 21, 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.

[0296] 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: The antigen-binding molecule binds to cells expressing the target antigen; The antigen-binding molecule inhibits the proliferation of cells expressing the target antigen; It enhances the killing of cells expressing the target antigen of antigen-binding molecules; The antigen-binding molecule inhibits the proliferation of cells near cells expressing the target antigen (e.g., cells that do not express the target antigen) and / or enhances its killing effect; To inhibit tumor growth and / or reduce tumor size / volume (e.g., inhibiting tumor growth and / or reducing tumor size / volume of cancers expressing the target antigen of an antigen-binding molecule); To extend the survival time of subjects with cancer (for example, cancer that expresses the target antigen of an antigen-binding molecule).

[0297] 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.

[0298] 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).

[0299] Analysis of the results of such assays 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, and this may also be referred to as "EC50". Depending on the characteristics, EC50 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.

[0300] In some embodiments, the antigen-binding molecule of this disclosure binds to its target antigen in a region accessible to the antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when the relevant antigen is expressed on the cell surface (i.e., within or on the cell membrane). In some embodiments, the antigen-binding molecule binds to its target antigen when it is expressed on the cell surface. In some embodiments, the antigen-binding molecule binds to a cell expressing its target antigen.

[0301] 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 cells expressing a given target antigen can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0302] In some embodiments, antigen-binding molecules inhibit the proliferation of cells expressing the target antigen of the antigen-binding molecule. 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 3 H-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).

[0303] In some embodiments, the antigen-binding molecules of the present invention can inhibit the proliferation of cells expressing the target antigen of the antigen-binding molecule in a given assay to less than 1 times the proliferation level of the same 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 cells expressing the appropriate target antigen), for example, 0.99 times or less, 0.95 times or less, 0.9 times or less, 0.85 times or less, 0.8 times or less, 0.75 times or less, 0.7 times or less, 0.65 times or less, 0.6 times or less, 0.55 times or less, 0.5 times or less, 0.45 times or less, 0.4 times or less, 0.35 times or less, 0.3 times or less, 0.25 times or less, 0.2 times or less, 0.15 times or less, 0.1 times or less, 0.05 times or less, or 0.01 times or less.

[0304] In some embodiments, the antigen-binding molecules described herein inhibit the proliferation of cells expressing the target antigen of the antigen-binding molecule 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.

[0305] In some embodiments, the antigen-binding molecule inhibits the proliferation of cells near cells expressing the target antigen of the antigen-binding molecule (e.g., cells that do not express the target antigen).

[0306] As used herein, "near" a cell expressing the target antigen of an antigen-binding molecule refers to a region / range within 100 μm of the cell expressing the target antigen. Cells near a cell expressing the target antigen of an antigen-binding molecule may also be referred to as surrounding cells or bystander cells. Such cells may or may not express the target antigen of the antigen-binding molecule itself. In some embodiments, cells near a cell expressing the target antigen of an antigen-binding molecule may be located within 100 μm of the cell expressing the target antigen (e.g., within 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm).

[0307] In some embodiments, the antigen-binding molecule of the present invention can inhibit the proliferation of cells near cells expressing the target antigen of the antigen-binding molecule (e.g., cells that do not express the target antigen) to less than 1x the level of proliferation of the same 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 cells near cells expressing the target antigen of the antigen-binding molecule), 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.

[0308] In some embodiments, the antigen-binding molecule does not inhibit the proliferation of cells near the cells expressing the target antigen of the antigen-binding molecule (e.g., cells that do not express the target antigen).

[0309] In some embodiments, the antigen-binding molecules according to this disclosure enhance (i.e., upregulate, promote) cytotoxicity in cells containing / expressing the target antigen of the antigen-binding molecule.

[0310] In some embodiments, the antigen-binding molecules according to this disclosure may inhibit the growth of cancer or reduce its metastasis, including cells containing / expressing the target antigen of the antigen-binding molecule. In some embodiments, the antigen-binding molecules may enhance (i.e., upregulate, promote) cytotoxicity of cells containing / expressing the target antigen of the antigen-binding molecule. In some embodiments, the antigen-binding molecules may enhance (i.e., upregulate, promote) cytotoxicity of cells near cells containing / expressing the target antigen of the antigen-binding molecule (e.g., cells that do not contain / express the target antigen). In some embodiments, the antigen-binding molecules do not enhance (i.e., upregulate, promote) cytotoxicity of cells near cells containing / expressing the target antigen of the antigen-binding molecule (e.g., cells that do not contain / express the target antigen). In some embodiments, the antigen-binding molecules may inhibit the proliferation of cancer cells containing / expressing the target antigen of the antigen-binding molecule, or inhibit the growth of tumors containing such cells. In some embodiments, the antigen-binding molecules may inhibit the metastasis of cancer / tumors containing cells containing / expressing the target antigen of the antigen-binding molecule.

[0311] In some embodiments, the antigen-binding molecules according to this disclosure may inhibit the growth of cancer or reduce its metastasis, including (i) cells containing / expressing the target antigen of the antigen-binding molecule, and (ii) cells that do not contain / do not express the target antigen of the antigen-binding molecule. In some embodiments, the antigen-binding molecules may inhibit the proliferation of cancer cells including (i) cells containing / expressing the target antigen of the antigen-binding molecule, and (ii) cells that do not contain / do not express the target antigen of the antigen-binding molecule, or may inhibit the growth of tumors containing such cells. In some embodiments, the antigen-binding molecules may inhibit the metastasis of cancer / tumors including (i) cells containing / expressing the target antigen of the antigen-binding molecule, and (ii) cells that do not contain / do not express the target antigen of the antigen-binding molecule.

[0312] 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.

[0313] In some embodiments, the antigen-binding molecules of this disclosure can reduce the number / ratio of cells expressing the target antigen of the antigen-binding molecule. In some embodiments, the antigen-binding molecules of this disclosure can deplete / enhance such cells.

[0314] 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 one of the cancers described herein, for example, a cancer that expresses / overexpresses the target antigen of the antigen-binding molecule.

[0315] 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 expressing the target antigen of the antigen-binding molecule. Cancer may include cells expressing the target antigen of the antigen-binding molecule and cells that do not express the target antigen of the antigen-binding molecule.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] In some embodiments, the antigen-binding molecules of the present disclosure can reduce tumor size / volume (e.g., the mean tumor size / volume of a treatment group in an in vivo model of cancer expressing the target antigen of the antigen-binding molecule) 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 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. 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.

[0320] 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.

[0321] In some embodiments, the antigen-binding molecules of the present disclosure can extend the median survival of subjects with cancer in a given assay (e.g., in an in vivo model of cancer expressing the target antigen of the antigen-binding molecule) 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 subjects in the appropriate treatment group may be expressed in days from the start of the experiment. It should be understood that cancers expressing the target antigen of an antigen-binding molecule may include cells that contain / express the target antigen, and may further include cells that do not contain / express the target antigen.

[0322] In some embodiments, the antigen-binding molecules according to the present disclosure have one or more novel, equivalent, or improved functional properties compared to (i) antigen-binding molecules comprising the same target antigen-binding moiety but comprising only the TOP1 inhibitor moiety (i.e., not further comprising the DDR inhibitor moiety), and / or (ii) antigen-binding molecules comprising the same target antigen-binding moiety but comprising only the DDR inhibitor moiety (i.e., not further comprising the TOP1 inhibitor moiety).

[0323] In some embodiments, the antigen-binding molecules according to this disclosure have one or more novel, comparable, or improved functional properties compared to (i) an antigen-binding molecule that includes the same target antigen-binding moiety as the antigen-binding molecule and includes only the same TOP1 inhibitor moiety (i.e., does not include the DDR inhibitor moiety of the antigen-binding molecule), and / or (ii) an antigen-binding molecule that includes the same target antigen-binding moiety as the antigen-binding molecule and includes only the same DDR inhibitor moiety (i.e., does not include the TOP1 inhibitor moiety of the antigen-binding molecule). For brevity, in the following paragraphs, “an antigen-binding molecule that includes the same target antigen-binding moiety as the antigen-binding molecule and includes only the same TOP1 inhibitor moiety (i.e., does not include the DDR inhibitor moiety of the antigen-binding molecule)” will be simply referred to as “the corresponding antigen-binding molecule that includes only the TOP1 inhibitor moiety,” and “an antigen-binding molecule that includes the same target antigen-binding moiety as the antigen-binding molecule and includes only the same DDR inhibitor moiety (i.e., does not include the TOP1 inhibitor moiety of the antigen-binding molecule)” will be simply referred to as “the corresponding antigen-binding molecule that includes only the DDR inhibitor moiety.”

[0324] In some embodiments, the antigen-binding molecule according to this disclosure may represent one or more of the following: Equivalent binding of antigen-binding molecules to cells expressing appropriate target antigens, compared to corresponding antigen-binding molecules containing only the TOP1 inhibitor moiety and / or corresponding antigen-binding molecules containing only the DDR inhibitor moiety; Enhanced killing of cells expressing an appropriate target antigen, compared to killing of cells expressed by an antigen-binding molecule represented by a corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or a corresponding antigen-binding molecule containing only the DDR inhibitor portion; Equal killing of cells near cells expressing the target antigen (e.g., cells that do not express the target antigen) by the antigen-binding molecule; Enhanced inhibition of tumor growth and / or greater reduction of tumor size / volume (e.g., in cancers expressing the appropriate target antigen of the antigen-binding molecule) compared to the reduction of tumor growth inhibition / tumor size / volume shown by the corresponding antigen-binding molecule containing only the TOP1 inhibitor moiety and / or the corresponding antigen-binding molecule containing only the DDR inhibitor moiety; The degree of internal migration of antigen-binding molecules into cells expressing appropriate target antigens is comparable to that shown by corresponding antigen-binding molecules containing only the TOP1 inhibitor moiety and / or corresponding antigen-binding molecules containing only the DDR inhibitor moiety; The toxicity is comparable to that of the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion.

[0325] According to the previous paragraph, a given characteristic / result level "of the same magnitude" as the reference level can be one of the following: 0.5 times or more and 2 times or less of the reference level, e.g., 0.55 times or more and 1.9 times or less, 0.6 times or more and 1.8 times or less, 0.65 times or more and 1.7 times or less, 0.7 times or more and 1.6 times or less, 0.75 times or more and 1.5 times or less, 0.8 times or more and 1.4 times or less, 0.85 times or more and 1.3 times or less, 0.9 times or more and 1.2 times or less, or 0.95 times or more and 1.1 times or less. In some embodiments, a given level of characteristic / outcome that is "enhanced" compared to the reference level may be more than 1x the reference level, for example, one of 1.01x or greater, 1.02x or greater, 1.03x or greater, 1.04x or greater, 1.05x or greater, 1.1x or greater, 1.2x or greater, 1.3x or greater, 1.4x or greater, 1.5x or greater, 1.6x or greater, 1.7x or greater, 1.8x or greater, 1.9x or greater, 2x or greater, 3x or greater, 4x or greater, or 5x or greater. In some embodiments, the level of a given characteristic / result that is "reduced" compared to the reference level may be less than 1x the reference level, 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.

[0326] For such evaluation, it will be understood that a comparison can be made between an equivalent amount of antigen-binding molecule and a control antigen-binding molecule containing only the TOP1 / DDR inhibitor portion. In some embodiments, different amounts of antigen-binding molecules are used to provide equivalent amounts of each payload.

[0327] In some embodiments, the antigen-binding molecules of the present disclosure are such that, in a given assay, the corresponding antigen-binding molecules containing only the TOP1 inhibitor moiety and / or the corresponding antigen-binding molecules containing only the DDR inhibitor moiety bind to appropriate cells. 50 E-commerce of a similar level 50 The antigen-binding molecule then binds to cells expressing the appropriate target antigen. In some embodiments, the EC of the binding of the antigen-binding molecule to cells expressing the appropriate target antigen is determined by the same assay. 50 This refers to the EC of the binding of the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion to the same type of cell. 50 It is one of the following: 0.5 times or more and 2 times or less, for example, 0.55 times or more and 1.9 times or less, 0.6 times or more and 1.8 times or less, 0.65 times or more and 1.7 times or less, 0.7 times or more and 1.6 times or less, 0.75 times or more and 1.5 times or less, 0.8 times or more and 1.4 times or less, 0.85 times or more and 1.3 times or less, 0.9 times or more and 1.2 times or less, or 0.95 times or more and 1.1 times or less.

[0328] In some embodiments, the antigen-binding molecules of the present disclosure enhance the killing of cells expressing a suitable target antigen of the antigen-binding molecule in a given assay to a level exceeding the killing level of the same cells indicated by the corresponding antigen-binding molecule containing only the TOP1 inhibitor moiety and / or the corresponding antigen-binding molecule containing only the DDR inhibitor moiety. In some embodiments, the antigen-binding molecule enhances the killing of cells expressing the appropriate target antigen of the antigen-binding molecule (e.g., cancer cells expressing the appropriate target antigen of the antigen-binding molecule) to a level greater than 1x the level of cell killing of the appropriate cells indicated by the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, for example, 1.01x, 1.02x or greater, 1.03x or greater, 1.04x or greater, 1.05x or greater, 1.1x or greater, 1.2x or greater, 1.3x or greater, 1.4x or greater, 1.5x or greater, 1.6x or greater, 1.7x or greater, 1.8x or greater, 1.9x or greater, 2x or greater, 3x or greater, 4x or greater, 5x or greater, 6x or greater, 7x or greater, 8x or greater, 9x or greater, or 10x or greater, as determined in the same assay.

[0329] In some embodiments, the antigen-binding molecules of the present disclosure, in a given assay, kill cells expressing the appropriate target antigen of the antigen-binding molecule, while the corresponding antigen-binding molecule containing only the TOP1 inhibitor moiety and / or the corresponding antigen-binding molecule containing only the DDR inhibitor moiety kill the appropriate cells. 50 Smaller e-commerce 50 In some embodiments, the antigen-binding molecule of this disclosure kills cells expressing the appropriate target antigen, as determined by the same assay. 50 This involves the killing of the same type of cells in the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion. 50It is less than 1x, 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.

[0330] In some embodiments, the antigen-binding molecules of the present disclosure, in a given assay, kill cells near cells expressing the target antigen of the antigen-binding molecule (e.g., cells not expressing the target antigen) by the corresponding antigen-binding molecule containing only the TOP1 inhibitor moiety and / or the corresponding antigen-binding molecule containing only the DDR inhibitor moiety, thereby killing the appropriate cells. 50 E-commerce of a similar level 50 In some embodiments, the antigen-binding molecule of this disclosure kills cells near cells expressing the target antigen (e.g., cells that do not express the target antigen) when determined by the same assay. 50 This involves the killing of the same type of cells in the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion. 50 It is one of the following: 0.5 times or more and 2 times or less, for example, 0.55 times or more and 1.9 times or less, 0.6 times or more and 1.8 times or less, 0.65 times or more and 1.7 times or less, 0.7 times or more and 1.6 times or less, 0.75 times or more and 1.5 times or less, 0.8 times or more and 1.4 times or less, 0.85 times or more and 1.3 times or less, 0.9 times or more and 1.2 times or less, or 0.95 times or more and 1.1 times or less.

[0331] In some embodiments, the antigen-binding molecules of the present disclosure reduce tumor size / volume to a level exceeding the level of tumor size / volume reduction observed after treatment with a corresponding antigen-binding molecule containing only a TOP1 inhibitor moiety and / or a corresponding antigen-binding molecule containing only a DDR inhibitor moiety in a given assay. In some embodiments, the antigen-binding molecule, when determined in the same assay, can reduce tumor size / volume (e.g., the mean tumor size / volume of the treatment group in an in vivo model of cancer expressing the appropriate target antigen of the antigen-binding molecule) to less than 1x of the tumor size / volume observed at the same time point after treatment with the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, e.g., ≤0.99x, ≤0.9x, ≤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 administration of the initial dose of the appropriate 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.

[0332] In some embodiments, the antigen-binding molecules of the present disclosure achieve a level of tumor growth inhibition in a given assay that exceeds the level of tumor growth inhibition observed after treatment with a corresponding antigen-binding molecule containing only a TOP1 inhibitor moiety and / or a corresponding antigen-binding molecule containing only a DDR inhibitor moiety. In some embodiments, the antigen-binding molecule achieves a level of tumor growth inhibition (expressed, for example, as % tumor growth inhibition calculated in comparison to tumor growth observed with treatment with an appropriate control antigen-binding molecule) that is greater than 1x the level of tumor growth inhibition observed at the same time point after treatment with the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, when determined in the same assay. For example, 1.01x, 1.02x or greater, 1.03x or greater, 1.04x or greater, 1.05x or greater, 1.1x or greater, 1.2x or greater, 1.3x or greater, 1.4x or greater, 1.5x or greater, 1.6x or greater, 1.7x or greater, 1.8x or greater, 1.9x or greater, 2x or greater, 3x or greater, 4x or greater, 5x or greater, 6x or greater, 7x or greater, 8x or greater, 9x or greater, or 10x or greater. In some embodiments, the assessment of tumor size / volume for such comparison is performed in the appropriate model more than 5 days after administration of the initial dose of the appropriate 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.

[0333] In some embodiments, the antigen-binding molecules of the present disclosure achieve a synergistic level of cell killing of cells expressing the appropriate target antigen of the antigen-binding molecule, compared to the level of cell killing achieved individually by the corresponding antigen-binding molecule containing only the TOP1 inhibitor or DDR inhibitor moiety. That is, in some embodiments, the antigen-binding molecules of the present disclosure achieve a level of cell killing that is synergistic (i.e., super-additive) compared to that observed when the corresponding antigen-binding molecule containing only the TOP1 inhibitor or DDR inhibitor moiety is used alone.

[0334] In some embodiments, the antigen-binding molecules of the present disclosure achieve a synergistic level of tumor growth inhibition and / or synergistic reduction of tumor size / volume compared to the level achieved individually by the corresponding antigen-binding molecules containing only the TOP1 inhibitor or DDR inhibitor moiety. That is, in some embodiments, the antigen-binding molecules of the present disclosure achieve a level of tumor growth inhibition and / or reduction of tumor size / volume that is synergistic (i.e., super-additive) compared to that observed when the corresponding antigen-binding molecule containing only the TOP1 inhibitor or DDR inhibitor moiety is used alone.

[0335] As used herein, the “synergistic” or “hyperadditive” level of relevant effects of a given antigen-binding molecule (e.g., cell killing, inhibition of tumor growth, reduction of tumor size / volume) refers to a level of effect that exceeds the sum of the effects observed with the individual comparative molecules when used alone (i.e., the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and the corresponding antigen-binding molecule containing only the DDR inhibitor portion).

[0336] Quantitative methods for evaluating synergistic effects are described, for example, in Tallarida, Genes Cancer. (2011) 2(11):1003-1008 and Chou, Cancer Res (2010) 70:440-446 (both of which are incorporated herein by reference in their entirety). Additive, synergistic, and antagonistic effects can be evaluated in experiments in which various different doses of the antigen-binding molecules of this disclosure and the corresponding antigen-binding molecules containing only the TOP1 or DDR inhibitor portion are evaluated for the relevant effects. Dose-response curves may be plotted and evaluated to determine whether the antigen-binding molecules of this disclosure achieve a synergistic level of relevant effect compared to the corresponding antigen-binding molecules containing only the TOP1 or DDR inhibitor portion. In some embodiments, synergistic effects can be evaluated using combination / composite index (CI) values ​​calculated using the Chou-Talaray method described in Chou, Cancer Res (2010) 70:440-446. According to the Chow-Talalay method, for a given antigen-binding molecule of this disclosure, CI=1 shows an additive effect, CI<1 shows a synergistic effect, and CI>1 shows an antagonistic effect.

[0337] In some embodiments, the antigen-binding molecules of this disclosure exhibit a similar degree of internal transfer to cells of the same type, compared to the internal transfer of antigen-binding molecules to cells expressing appropriate target antigens, as indicated by the corresponding antigen-binding molecules containing only the TOP1 inhibitor moiety and / or the corresponding antigen-binding molecules containing only the DDR inhibitor moiety. In some embodiments, the antigen-binding molecule is internally transferred to cells expressing the appropriate target antigen of the antigen-binding molecule to a level that is 0.5 to 2 times the level of internal transfer to appropriate cells of the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, as determined in the same assay. For example, 0.55 to 1.9 times, 0.6 to 1.8 times, 0.65 to 1.7 times, 0.7 to 1.6 times, 0.75 to 1.5 times, 0.8 to 1.4 times, 0.85 to 1.3 times, 0.9 to 1.2 times, or 0.95 to 1.1 times. In some embodiments, the evaluation of internal migration for such comparison is performed after incubating the appropriate antigen-binding molecule with the appropriate cells for more than 5 minutes, for example, more than 30 minutes, more than 1 hour, more than 1.5 hours, more than 2 hours, or more than 2.5 hours.

[0338] In some embodiments, the antigen-binding molecules of this disclosure exhibit comparable toxicity to subjects administered with the antigen-binding molecule compared to subjects administered with the corresponding antigen-binding molecule containing only the TOP1 inhibitor moiety and / or the corresponding antigen-binding molecule containing only the DDR inhibitor moiety. In some embodiments, the antigen-binding molecules have a similar toxicological profile compared to the corresponding antigen-binding molecule containing only the TOP1 inhibitor moiety and / or the corresponding antigen-binding molecule containing only the DDR inhibitor moiety.

[0339] In some embodiments, when determined by the same assay, the number / ratio of erythrocytes / leukocytes / lymphocytes / monocytes / neutrophils / platelets in the peripheral blood of a subject after administration of the antigen-binding molecule according to the present disclosure is at least 0.5 times and no more than 2 times the number / ratio of such cells observed after treatment with the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, for example, at least 0.55 times and no more than 1.9 times, at least 0.6 times and no more than 1.8 times, at least 0.65 times and no more than 1.7 times, at least 0.7 times and no more than 1.6 times, at least 0.75 times and no more than 1.5 times, at least 0.8 times and no more than 1.4 times, at least 0.85 times and no more than 1.3 times, at least 0.9 times and no more than 1.2 times, or at least 0.95 times and no more than 1.1 times.

[0340] In some embodiments, when determined by the same assay, the hemoglobin concentration / hematocrit percentage / mean corpuscular volume, mean corpuscular hemoglobin amount / mean corpuscular hemoglobin concentration in the peripheral blood of a subject after administration of the antigen-binding molecule according to the present disclosure is at least 0.5 times and at least 2 times the level observed after treatment with the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, for example, at least 0.55 times and at least 1.9 times, at least 0.6 times and at least 1.8 times, at least 0.65 times and at least 1.7 times, at least 0.7 times and at least 1.6 times, at least 0.75 times and at least 1.5 times, at least 0.8 times and at least 1.4 times, at least 0.85 times and at least 1.3 times, at least 0.9 times and at least 1.2 times, or at least 0.95 times and at least 1.1 times.

[0341] In some embodiments, when determined by the same assay, the levels of liver function correlates (e.g., alkaline phosphatase, alanine aminotransferase, albumin, total protein) in the peripheral blood of a subject after administration of the antigen-binding molecule according to the present disclosure are at least 0.5 times and no more than 2 times the levels observed in the peripheral blood of a subject after treatment with the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, for example, at least 0.55 times and no more than 1.9 times, at least 0.6 times and no more than 1.8 times, at least 0.65 times and no more than 1.7 times, at least 0.7 times and no more than 1.6 times, at least 0.75 times and no more than 1.5 times, at least 0.8 times and no more than 1.4 times, at least 0.85 times and no more than 1.3 times, at least 0.9 times and no more than 1.2 times, or at least 0.95 times and no more than 1.1 times.

[0342] In some embodiments, when determined by the same assay, the levels of renal function correlates (e.g., blood urea nitrogen and / or creatinine) in the subject's peripheral blood after administration of the antigen-binding molecule according to the present disclosure are at least 0.5 times and at least 2 times the levels observed in the subject's peripheral blood after treatment with the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, for example, at least 0.55 times and at least 1.9 times, at least 0.6 times and at least 1.8 times, at least 0.65 times and at least 1.7 times, at least 0.7 times and at least 1.6 times, at least 0.75 times and at least 1.5 times, at least 0.8 times and at least 1.4 times, at least 0.85 times and at least 1.3 times, at least 0.9 times and at least 1.2 times, or at least 0.95 times and at least 1.1 times.

[0343] In some embodiments, when determined by the same assay, the levels of pancreatic function correlates (e.g., glucose and / or amylase) in the peripheral blood of a subject after administration of the antigen-binding molecule according to the present disclosure are at least 0.5 times and at least 2 times the levels observed in the peripheral blood of a subject after treatment with the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, for example, at least 0.55 times and at least 1.9 times, at least 0.6 times and at least 1.8 times, at least 0.65 times and at least 1.7 times, at least 0.7 times and at least 1.6 times, at least 0.75 times and at least 1.5 times, at least 0.8 times and at least 1.4 times, at least 0.85 times and at least 1.3 times, at least 0.9 times and at least 1.2 times, or at least 0.95 times and at least 1.1 times.

[0344] In some embodiments, when determined by the same assay, the sodium / potassium / phosphate / calcium levels in the subject's peripheral blood after administration of the antigen-binding molecule according to this disclosure are at least 0.5 times and no more than 2 times the levels observed in the subject's peripheral blood after treatment with the corresponding antigen-binding molecule containing only the TOP1 inhibitor portion and / or the corresponding antigen-binding molecule containing only the DDR inhibitor portion, for example, at least 0.55 times and no more than 1.9 times, at least 0.6 times and no more than 1.8 times, at least 0.65 times and no more than 1.7 times, at least 0.7 times and no more than 1.6 times, at least 0.75 times and no more than 1.5 times, at least 0.8 times and no more than 1.4 times, at least 0.85 times and no more than 1.3 times, at least 0.9 times and no more than 1.2 times, or at least 0.95 times and no more than 1.1 times.

[0345] additional array The antigen-binding molecules and their constituent polypeptides of this disclosure may additionally include further amino acids or amino acid sequences.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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).

[0350] 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.

[0351] 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).

[0352] Signs and conjugates In some embodiments, the antigen-binding molecules of this disclosure and their constituent polypeptides include detectable portions.

[0353] 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.

[0354] 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 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 , Tellurium 121m , Tellurium 122m , Tellurium 125m ,thulium 165 ,thulium 167 ,thulium168 ,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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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).

[0359] 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).

[0360] 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.

[0361] 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.

[0362] 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).

[0363] 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) thAs 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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).

[0370] 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).

[0371] 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.

[0372] 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.

[0373] 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.

[0374] 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.

[0375] 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).

[0376] 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.

[0377] 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.

[0378] 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.

[0379] 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.

[0380] 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.

[0381] 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.

[0382] 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 a glycine-functionalized payload to the newly generated C-terminus. A Strep II tag, used to assist in the removal of the saltase-recognized motif and unreacted antibody, is 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.

[0383] 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). The N297Q mutation prior to this conjugation results in two additional reaction sites (resulting in a 4-linker-payload conjugation). 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.

[0384] DAR depends on the number of payloads per linker-payload portion being conjugated.

[0385] 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.

[0386] 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.

[0387] In some embodiments, the linker-payload is terminated with an amino group, which is conjugated to an antigen-binding molecule using transglutaminase.

[0388] 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.

[0389] This disclosure also provides antigen-binding molecules that can be obtained or obtained by the methods of this disclosure.

[0390] composition This disclosure provides compositions comprising antigen-binding molecules as described herein.

[0391] 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.

[0392] 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).

[0393] 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.

[0394] 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.

[0395] 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.

[0396] In some embodiments, the pharmaceutical composition / drug is formulated for injection or infusion into, for example, blood vessels, tissues / organs of interest, or tumors.

[0397] 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.

[0398] 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.

[0399] Therapeutic and preventive applications The antigen-binding molecules and compositions described herein are useful in the treatment and prevention of diseases, such as cancer.

[0400] It will be understood that the antigen-binding molecules and compositions of this disclosure may be used for therapeutic or preventive benefits by reducing the level of expression or activity of the target antigen of the antigen-binding molecule, or for the treatment / prevention of any disease / condition that would be subject to a reduction in the number or activity of cells containing / expressing the target antigen of the antigen-binding molecule.

[0401] For example, a disease / condition may be a disease / condition in which cells expressing / overexpressing the target antigen of an antigen-binding molecule or the target antigen of an antigen-binding molecule are pathologically involved, such as an increase in the level / activity of the target antigen of an antigen-binding molecule, or an increase in the number / ratio of cells containing / expressing the target antigen of an antigen-binding molecule, 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 the target antigen of an antigen-binding molecule, or an increase in the number / ratio of cells containing / expressing the target antigen of an antigen-binding molecule, may be a risk factor for the onset, progression, or advancement of the disease / condition.

[0402] This disclosure provides antigen-binding molecules or compositions described herein for use in methods of medical treatment or prevention.

[0403] In some embodiments, the disease / condition is cancer. 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.

[0404] 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).

[0405] 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.

[0406] 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, endometrial cancer, pancreatic cancer, thymic cancer, NSCLC, hematological cancer, and sarcoma.

[0407] In some embodiments, the cancer is breast cancer, lung cancer (e.g., small cell lung cancer), gastric cancer, or endometrial cancer.

[0408] In some embodiments, cancer is a hematological cancer. In some embodiments, cancer is a myeloid hematological cancer, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, AIDS-associated lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, primary central nervous system lymphoma, Césarley syndrome, Waldenström macroglobulinemia, leukemia, T-cell leukemia, B-cell leukemia, acute myeloid leukemia, chronic myelomonocytic leukemia, acute promyelocytic leukemia, chronic promyelocytic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, pilocytic cell leukemia, myeloma, multiple myeloma, myelodysplastic syndrome, or myeloproliferative disorder.

[0409] In some embodiments, the cancer to be treated / prevented includes cells expressing the target antigen of the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented is a cancer that is positive for the target antigen of the antigen-binding molecule. In some embodiments, the cancer includes cells that overexpress the target antigen of the antigen-binding molecule. Overexpression can be determined by detecting an expression level higher than the expression level in the corresponding non-cancerous cells / non-tumor tissue.

[0410] In some embodiments, the cancer to be treated / prevented is a cancer that is heterogeneous in the expression of the target antigen of the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented comprises a population of cells in which the expression of the target antigen of the antigen-binding molecule is heterogeneous.

[0411] In some embodiments, the cancer to be treated / prevented includes cells that do not express the target antigen of the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented includes cells that are negative for the target antigen of the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented includes cells that express the target antigen of the antigen-binding molecule and cells that do not express the target antigen of the antigen-binding molecule. In some embodiments, the cancer to be treated / prevented includes cells that are positive for the target antigen of the antigen-binding molecule and cells that are negative for the target antigen of the antigen-binding molecule.

[0412] 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 a target antigen of an antigen-binding molecule, 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.

[0413] In some embodiments, cancer is a cancer in which the target antigen of the antigen-binding molecule is pathologically involved. That is, in some embodiments, cancer is a cancer caused or exacerbated by the expression of the target antigen of the antigen-binding molecule, a cancer in which the expression of the target antigen of the antigen-binding molecule is a risk factor, and / or a cancer in which the expression of the target antigen of the antigen-binding molecule is positively associated with the onset, progression, progression, severity, or metastasis of the cancer. Cancer may be characterized by the expression of the target antigen of the antigen-binding molecule, for example, cancer may include cells that express the target antigen of the antigen-binding molecule (e.g., cells of tumor tissue). Such cancer may be referred to as positive for the target antigen of the antigen-binding molecule. Cancer that is "positive" for the target antigen of the antigen-binding molecule may include cells that express the target antigen of the antigen-binding molecule (e.g., on the cell surface). Cancer that is "positive" for the target antigen of the antigen-binding molecule may overexpress the target antigen of the antigen-binding molecule.

[0414] In some embodiments, the cancer being treated / prevented includes cells having a gene variant (e.g., a mutation) that causes enhanced expression and / or activity of the target antigen (gene and / or protein) of an antigen-binding molecule 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.

[0415] Mutations that “result” in enhanced expression of the target antigen of an antigen-binding molecule may be known, predicted, or associated with enhanced gene / protein expression of the target antigen of the antigen-binding molecule. Mutations that result in enhanced expression and / or activity of the target antigen of an antigen-binding molecule may be referred to as “activating” mutations.

[0416] Mutations that cause enhanced expression of the target antigen of an antigen-binding molecule may result in the gene or protein expression of the target antigen of the antigen-binding molecule that is not expressed by equivalent cells without the mutation and / or encoded by its genomic nucleic acid. In other words, the expression of the target antigen of an antigen-binding molecule may be a result of a mutation, and therefore, "enhanced expression" may be an enhancement of expression from non-expression.

[0417] Mutations that cause enhanced expression of the target antigen of an antigen-binding molecule may result in enhanced gene or protein expression of the target antigen of the antigen-binding molecule expressed and / or encoded by its genomic nucleic acid in equivalent cells that do not contain the mutation. For example, a cell may contain such mutation(s) that results in an increased transcription level of the nucleic acid encoding the target antigen of the antigen-binding molecule compared to the transcription level of the relevant nucleic acid(s) in equivalent cells that do not contain the mutation(s).

[0418] In some embodiments, a mutation that causes enhanced expression of the target antigen of an antigen-binding molecule may cause enhanced gene expression of the gene encoding the relevant antigen compared to equivalent cells that do not contain the mutation. In some embodiments, a mutation that causes enhanced expression of the target antigen of an antigen-binding molecule may cause enhanced expression of the relevant protein compared to equivalent cells that do not contain the mutation.

[0419] In some embodiments, a mutation that causes enhanced expression of the target antigen of an antigen-binding molecule may result in an increase in the level of the target antigen of the antigen-binding molecule on the cell surface of cells containing the mutation, or on the cell surface of the antigen-binding molecule, compared to equivalent cells that do not contain the mutation.

[0420] Cells in which the expression of the antigen-binding molecule's target antigen is enhanced (e.g., as a result of a mutation) compared to the expression level of the antigen-binding molecule's target antigen in reference cells may be described as “overexpressing” or “upregulated expression” of the antigen-binding molecule's target antigen. For example, a cancer containing cells with a mutation that results in enhanced expression of the antigen-binding molecule's target antigen compared to equivalent cells lacking the mutation may be described as a cancer containing cells exhibiting overexpression / upregulated expression of the antigen-binding molecule's target antigen. 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).

[0421] Mutations that cause an enhancement of the target antigen activity of an antigen-binding molecule may result in an enhancement of the target antigen-mediated activity of the antigen-binding molecule compared to the level of target antigen-mediated activity of an equivalent cell that does not contain the mutation.

[0422] In some embodiments, cancer treated / prevented in accordance with this disclosure may be characterized, for example, by enhanced expression and / or activity (i.e., gene and / or protein expression) of the target antigen of the antigen-binding molecule 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, for example, by enhanced expression and / or activity of the target antigen of the antigen-binding molecule compared to the levels of expression and / or activity observed in equivalent non-cancerous cells / non-tumor tissues.

[0423] 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] DDR inhibitors and their use in the treatment of cancer 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). 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-1002, RBN-2397, fluzoparib, NMS-03305293, AZD9574), ATM inhibitors (e.g., CP-4 66722, KU-55933, KU-60019, KU-59403, AZ31, AZ32, AZD0156, AZD1390), ATR inhibitors (e.g., M6620 (belzocertib), M4344 (VX-803), AZD6738 (ceraracertib), BAY1895344 (elimsertib), RP3500 (camoncertib), ATRN119, ART380, IMP9064, HRS2398, M1774, IMP9064, SC0245, LF0397, NU6027), WEE1 inhibitors (e.g., adavocertib, Debio0123, PD0166285, PD0407824, AZD1775, ZN-c3 (azenocertib), IMP7068, SY4835, SCO191, IMP7068), CHK1 / 2 inhibitors (e.g., CBP-501, prexacertib, MK-8776, GDC-0575, SRA-737, PF-00477736, AZD7762, LY2603618) (e.g., nedisertib, ly2880070, XL884, BEBT260, CC-115, MU380, NU7441, KU-5778), DNA-PK inhibitors (e.g., CC-115, LY-3023414, AsiDNA, M3814 (nedisertib, peposertib), M9831 (VX-984), BR-101801, XRD-0394, SL901, XZP- 6877, IMP-11, ZL-2201, BR-2006, AZD7648, NU7441), PLK1 inhibitors (e.g., BI-6727 (vorasertib), PCM-075 (onvancertib), CYC140 (progocertib)), Polθ inhibitors (e.g., ART4215, ART6043, novobiosin, RP-6685, RP-3467), RAD51 inhibitors Harmful agents (e.g., CYT0851), USP inhibitors (e.g., inhibitors of USP11, USP7, USP4, USP37, USP39, USP45, USP24, and / or USP1; e.g., KSQ-4279), PKMYT1 inhibitors (e.g., RP6306), or Aurora A inhibitors (e.g., aricertib, WJ05129 (JS112), JAB-2485).

[0428] 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.

[0429] Example 4 of this specification demonstrates that the antigen-binding molecule comprising the DDRi inhibitor and TOP1 inhibitor portions according to this disclosure is useful for killing cancer cells that are resistant / insensitive to treatment with the same antibody conjugated only to the TOP1 inhibitor portion. Example 11 of this specification demonstrates that the antigen-binding molecule comprising the DDRi inhibitor and TOP1 inhibitor portions according to this disclosure is useful for inhibiting tumor growth in a mouse model derived from a cancer cell line that is resistant / insensitive to treatment with the same antibody conjugated only to the TOP1 inhibitor portion.

[0430] Example 5 in this specification demonstrates that treatment with an antigen-binding molecule comprising the DDRi inhibitor and TOP1 inhibitor portions according to this disclosure achieves a far more potent inhibition of tumor growth in a cell line-derived ductal carcinoma mouse model compared to treatment with the same antibody conjugated only to the TOP1 inhibitor portion.

[0431] DNA topoisomerase I inhibitors and their use in the treatment of cancer are 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 inhibitor according to this disclosure is 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, calenitecan, silatecan, and namithecan. N, Eromotecan, DRF-1042, Delimotecan, NSC606985, Kimitecan, ZBH-1205, Genz-644282, Non-CPT1, Indotecan (LMP-400), Injimitecan (LMP-776), LMP744, AZ14170132, SHR9265, Ed-04, KL610023, A1.9, ZD06519, P1003, P1021, VIP126, and ZBH-01.

[0432] 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.

[0433] 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.

[0434] 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.

[0435] 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).

[0436] The antigen-binding molecules described herein may be used to treat / prevent diseases / conditions other than cancer. Antibody-drug conjugates are useful in treating / preventing diseases / conditions including infectious diseases, autoimmune diseases, neurological diseases, cardiovascular diseases, metabolic diseases, hematological diseases, and fibrotic diseases (see, for example, Mike Ward, “Exploring the potential of ADCs beyond oncology”, Drug Target Review, published August 6, 2024).

[0437] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is an infectious disease / condition, e.g., a disease / condition caused by a pathogen infection (e.g., a disease / condition caused by a bacterial, viral, fungal, or parasitic infection). According to such embodiments and forms, the target antigen of the target antigen-binding moiety may be an antigen of a pathogen as described herein. In embodiments and forms where the disease / condition treated / prevented is a disease / condition caused by a pathogen infection (e.g., a disease / condition caused by a bacterial, viral, fungal, or parasitic infection), it will be understood that the target antigen of the target antigen-binding moiety of the antigen-binding molecule of this disclosure may be an antigen of the relevant pathogen (i.e., an antigen of the relevant bacteria, virus, fungus, or parasite).

[0438] For example, an antigen-binding molecule according to this disclosure, including a payload portion having cytotoxic activity, may be used to kill / enhance the killing of a relevant pathogen / a cell infected with the relevant pathogen.

[0439] In some embodiments, the infectious disease / condition described herein is a disease / condition caused by / associated with a bacterial infection, such as a Bacillus species, Bordetella pertussis, Clostridium species, Corynebacterium species, Vibrio chloerae, Staphylococcus species, Streptococcus species, Escherichia, Klebsiella, Proteus, Yersinia, Erwina, Salmonella, Listeria species, Helicobacter pylori, mycobacteria (e.g., Mycobacterium tuberculosis), or Pseudomonas aeruginosa. For example, the bacterial infection could be sepsis or tuberculosis. In some embodiments, the infectious disease is a disease caused by / associated with a virus, such as influenza virus, SARS-CoV, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus, or human papillomavirus (HPV). In some embodiments, the infectious disease is a disease caused by / associated with a fungal infection, such as an infection with species of the genera Alternaria, Aspergillus, Candida, and Histoplasma. The fungal infection may be fungal sepsis or histoplasmosis. In some embodiments, the infectious disease is a disease caused by / associated with a parasitic infection, such as an infection with a Plasmodium species (e.g., Plasmodium falciparum, Plasmodium yoeli, Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi). In some embodiments, the disease / condition may be malaria, leishmaniasis, or toxoplasmosis. In some embodiments, the disease / condition may be a disease caused by / associated with a bacterial infection or a disease caused by / associated with a viral infection.

[0440] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is an autoimmune disease / condition. In such embodiments, the target antigen of the target antigen-binding moiety may be an autoimmune disease-related antigen. In embodiments where the disease / condition treated / prevented is an autoimmune disease, it will be understood that the target antigen of the target antigen-binding moiety of the antigen-binding molecule of this disclosure may be an antigen related to the relevant autoimmune disease / condition. For example, the target antigen may be an antigen expressed by autoreactive immune cells (e.g., autoreactive T cells or autoreactive B cells), an antigen of an inflammatory mediator associated with the pathology of the relevant autoimmune disease / condition, or another antigen otherwise associated with the pathology of the autoimmune disease / condition.

[0441] For example, antigen-binding molecules according to this disclosure, including a payload portion having cytotoxic activity, may be used to kill / enhance the killing of autoreactive immune cells and / or other inflammatory mediators associated with the pathology of the associated autoimmune disease / condition.

[0442] In some embodiments, the autoimmune disease / condition is selected from: type 1 diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, arthritis (e.g., rheumatoid arthritis), and systemic lupus erythematosus. In some embodiments, the autoimmune disease / condition is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or type 1 diabetes.

[0443] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a neurological disease / neurological condition. In such embodiments, the target antigen of the target antigen-binding moiety may be a neurological disease-related antigen. In embodiments where the disease / condition treated / prevented is a neurological disease / neurological condition, it will be understood that the target antigen of the target antigen-binding moiety of the antigen-binding molecule of this disclosure may be an antigen related to the relevant neurological disease / neurological condition. For example, the target antigen may be an antigen expressed by a pathological effector of the relevant neurological disease / neurological condition, or an antigen otherwise related to the pathology of the neurological disease / neurological condition.

[0444] For example, antigen-binding molecules according to this disclosure, including a payload portion having cytotoxic activity, may be used to kill or enhance the killing of pathological effectors (e.g., immune cells) associated with related neurological diseases / conditions. For example, antigen-binding molecules according to this disclosure, including a suitable payload portion, may be used to enhance the degradation of plaques (e.g., amyloid-beta plaques) or protein aggregates (e.g., tau protein aggregates) associated with the pathology of a disease / condition.

[0445] In some embodiments, the neurological disease / neurological condition is selected from: Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and hippocampal atrophy. In some embodiments, the neurological disease / neurological condition is Alzheimer's disease or multiple sclerosis.

[0446] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a cardiovascular disease / condition. In such embodiments, the target antigen of the target antigen-binding moiety may be a cardiovascular disease-related antigen. In embodiments where the disease / condition treated / prevented is a cardiovascular disease / condition, it will be understood that the target antigen of the target antigen-binding moiety of the antigen-binding molecule of this disclosure may be an antigen related to the relevant cardiovascular disease / condition. For example, the target antigen may be an antigen expressed by a pathological effector of the relevant cardiovascular disease / condition, or an antigen otherwise related to the pathology of the cardiovascular disease / condition.

[0447] For example, antigen-binding molecules according to this disclosure, including a payload portion having cytotoxic activity, may be used to kill or enhance pathological effectors of cardiovascular remodeling / fibrosis. For example, antigen-binding molecules according to this disclosure, including a suitable payload portion, may be used to stabilize atherosclerotic plaques.

[0448] In some embodiments, the cardiovascular disease / condition is selected from: atherosclerosis, heart failure (e.g., heart failure with preserved ejection fraction (HFpEF) or heart failure with reduced ejection fraction (HFrEF)), dilated tortuous veins, cerebral infarction, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, interstitial fibrosis, replacement fibrosis, Brugada syndrome, myocarditis, endocardial fibrosis, myocardial infarction, fibrous vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), arteriosclerosis, chronic pulmonary hypertension, and AIDS-associated pulmonary hypertension. In some embodiments, the cardiovascular disease / condition is atherosclerosis or heart failure.

[0449] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a metabolic disease / condition. In such embodiments, the target antigen of the target antigen-binding moiety may be a metabolic disease-related antigen. In embodiments where the disease / condition treated / prevented is a metabolic disease / condition, it will be understood that the target antigen of the target antigen-binding moiety of the antigen-binding molecule of this disclosure may be an antigen related to the relevant metabolic disease / condition. For example, the target antigen may be an antigen expressed by a pathological effector of the relevant metabolic disease / condition, or an antigen otherwise related to the pathology of the metabolic disease / condition.

[0450] For example, antigen-binding molecules according to this disclosure, including a payload portion having cytotoxic activity, may be used to kill / enhance the killing of pathological effectors of metabolic diseases (e.g., inflammatory immune cells), or to enhance the degradation of adipocytes / tissues. For example, antigen-binding molecules according to this disclosure, including a suitable payload portion, may be used to modulate insulin resistance and / or activate metabolic pathways.

[0451] In some embodiments, the metabolic disorder / condition is selected from: diabetes mellitus, obesity, prediabetes, metabolic syndrome, pregnancy-related hyperglycemia (i.e., gestational diabetes mellitus), hyperglycemia, amyloidosis, hypertension, and hypercholesterolemia. In some embodiments, the metabolic disorder / condition is diabetes mellitus or obesity.

[0452] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a hematological disorder / condition. In such embodiments, the target antigen of the target antigen-binding moiety may be a hematological disorder-related antigen. In embodiments where the disease / condition treated / prevented is a hematological disorder / condition, it will be understood that the target antigen of the target antigen-binding moiety of the antigen-binding molecule of this disclosure may be an antigen related to the relevant hematological disorder / condition. For example, the target antigen may be an antigen expressed by a pathological effector of the relevant hematological disorder / condition, or an antigen otherwise related to the pathology of the hematological disorder / condition.

[0453] For example, an antigen-binding molecule according to this disclosure, including an appropriate payload portion, may be used to deliver coagulation factors (i.e., for the treatment of hemorrhagic disorders) or to deliver erythropoiesis-stimulating agents (i.e., for the treatment of anemia).

[0454] In some embodiments, the blood disorder / condition is selected from: hemorrhagic disorders (e.g., hemophilia or von Willebrand disease) or anemia (e.g., nutritional anemia, hemolytic anemia, or aplastic anemia). In some embodiments, the blood disorder / condition is hemophilia or anemia.

[0455] In some embodiments, the disease / condition treated / prevented in accordance with this disclosure is a disease / condition characterized by fibrosis (i.e., a fibrous disease / condition). In such embodiments and aspects, the target antigen of the target antigen-binding moiety may be a fibrous disease-associated antigen. In embodiments and aspects where the disease / condition treated / prevented is a fibrous disease / condition, it will be understood that the target antigen of the target antigen-binding moiety of the antigen-binding molecule of this disclosure may be an antigen associated with the relevant fibrous disease / condition. For example, the target antigen may be an antigen expressed by a pathological effector of the relevant fibrous disease / condition, or an antigen otherwise related to the pathology of the fibrous disease / condition.

[0456] For example, an antigen-binding molecule according to this disclosure, including a payload portion having cytotoxic activity, may be used to kill / enhance the killing of pathological effectors of fibrosis (e.g., myofibroblasts). For example, an antigen-binding molecule according to this disclosure, including a suitable payload portion, may be used to enhance the degradation of the extracellular matrix.

[0457] In some embodiments, diseases / conditions characterized by fibrosis are selected from: pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), asthma, cystic fibrosis, chronic obstructive pulmonary disease (COPD), hepatic fibrosis, chronic liver disease, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, primary biliary cirrhosis (PBC), progressive nodular fibrosis, cardiovascular fibrosis, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), atrial fibrosis, atrial fibrillation, ventricular fibrosis, ventricular fibrillation, myocardial fibrosis, systemic sclerosis, scleroderma, renal fibrosis, chronic kidney disease (CKD), Alport syndrome, diabetic nephropathy, chronic glomerulonephritis, ocular fibrosis, Graves' ophthalmopathy, epiretinal fibrosis, retinal fibrosis, subretinal fibrosis, diabetic retinopathy, glaucoma, and arthritis. In some embodiments, the disease / condition characterized by fibrosis is pulmonary fibrosis or hepatic fibrosis.

[0458] 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.

[0459] 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.

[0460] 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 of antigen-binding molecules to cells expressing the target antigen; Inhibition of the proliferation of cells expressing the target antigen of antigen-binding molecules; Killing cells expressing the target antigen of an antigen-binding molecule; Inhibition of proliferation and / or enhancement of killing of cells near cells expressing the target antigen of an antigen-binding molecule (e.g., cells that do not express the target antigen); Inhibition of tumor growth and / or reduction of tumor size / volume, e.g., inhibition of tumor growth and / or reduction of tumor size / volume of cancers expressing the target antigen of an antigen-binding molecule; Furthermore / or extension of survival time for subjects with cancer, for example, cancer expressing the target antigen of an antigen-binding molecule.

[0461] 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.

[0462] 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 the proliferation of cells expressing the target antigen of antigen-binding molecules; Killing cells expressing the target antigen of an antigen-binding molecule; Inhibition of proliferation and / or enhancement of killing of cells near cells expressing the target antigen of the antigen-binding molecule (e.g., cells that do not express the target antigen); Inhibition of tumor growth and / or reduction of tumor size / volume, e.g., inhibition of tumor growth and / or reduction of tumor size / volume of cancer expressing the target antigen of an antigen-binding molecule, and / or Extending the survival period of subjects with cancer, for example, cancer that expresses a target antigen of an antigen-binding molecule.

[0463] 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.

[0464] 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.

[0465] 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.

[0466] In some embodiments and aspects, the articles of the Disclosure may be administered to a tissue / organ of interest (e.g., a tissue / organ suffering from a disease / condition affected by a condition (e.g., a tissue / organ showing symptoms of the disease / condition)). 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.

[0467] 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.

[0468] 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).

[0469] 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.

[0470] 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.

[0471] In some embodiments, a patient may be selected for the treatment described herein based on the detection of cancer expressing / overexpressing the target antigen of the antigen-binding molecule in a sample taken from the subject (e.g., tumor biopsy material).

[0472] 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.

[0473] The kit may have at least one container containing a predetermined amount of an antigen-binding molecule or composition as described herein.

[0474] 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.

[0475] 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).

[0476] 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.

[0477] 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.

[0478] Linker-payload molecule This disclosure provides a linker-payload molecule for conjugating an antigen-binding moiety, comprising at least one first payload and at least one second payload, wherein the linker for conjugation to an antibody comprises a moiety derived from a compound of the second embodiment.

[0479] Therefore, the linker-payload molecule is (a) an amino group for conjugation 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. c includes a branching group, where c indicates a location to which at least one second click group is attached.

[0480] In some embodiments, a linker-payload molecule containing one of the following groups: [ka]

[0481] Modified antigen-binding molecules This disclosure also provides modified antigen-binding molecules that include a moiety derived from the compound of the second embodiment.

[0482] Therefore, the modified antigen-binding portion is (a) an amino group conjugated to the antigen-binding site, (b) at least one first click base for linking the portion containing the first payload, (c) at least one second click base for linking the portion containing the second payload, (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 the location where the at least one first click group is connected to the branching group. c includes a branching group, where c indicates the location to which at least one second click group is connected.

[0483] In some embodiments, the modified antigen-binding molecule includes one of the following groups: [ka]

[0484] 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.

[0485] array [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5]

[0486] This disclosure includes the described embodiments and preferred feature combinations unless such combinations are clearly impossible or expressly avoided.

[0487] The headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subjects described.

[0488] 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.

[0489] 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 an explicitly stated integer or step or group of integers or steps, but not to mean excluding any other integer or step or group of integers or steps.

[0490] 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.

[0491] Where nucleic acid sequences are disclosed or referred to herein, their reverse complements are also expressly intended.

[0492] 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.

[0493] 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]

[0494] 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. 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

[0495] 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)

[0496] 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.

[0497] 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 11]

[0498] 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 12]

[0499] C: Exatecan and seracertib (ATR inhibitor) in HEC-1-B 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.

[0500] IC with Ceraraceltib alone 50 The concentration was 2.118 μM. [Table 13]

[0501] 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 50The value was 4.466 nM. [Table 14]

[0502] 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. cell line HEC-1-B (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)

[0503] 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.

[0504] 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).

[0505] The background was subtracted using the luminescence values ​​of wells containing only culture medium (no cells).

[0506] 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.

[0507] The results are shown in Figures 13A and 13B. The synergistic effect was observed across various exatecan and bezocertib concentrations.

[0508] 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.

[0509] 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... 1The 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.

[0510] 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.

[0511] 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).

[0512] 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.

[0513] 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).

[0514] 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).

[0515] 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.

[0516] 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.

[0517] 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.

[0518] 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.

[0519] 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.

[0520] 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).

[0521] 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(I8) [ka] Compound I5 (150 mg, 168 μmol, 1.0 equivalent) and belzocertib (86 mg, 185 μmol, 1.1 equivalent) were dissolved in DMF (1.5 mL), to which 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 mass of the target product 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.

[0522] 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.

[0523] 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).

[0524] 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.

[0525] 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.

[0526] 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.

[0527] 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.

[0528] 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.

[0529] 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).

[0530] 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] Triethylamine (6 mL) was added to a solution of compound I15 (2.0 g) in DMF (14 mL). The mixture was stirred at 25°C for 12 hours. After 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, but R=H].

[0531] 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%.

[0532] 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.

[0533] 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.

[0534] 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.

[0535] 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.

[0536] 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.

[0537] 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.

[0538] 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.

[0539] 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.

[0540] 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.

[0541] 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.

[0542] 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.

[0543] 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.

[0544] 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.

[0545] 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 percent yield). MS (ESI): MS calculated value: 873.41, MS measured value: [M+H] + = 874.0.

[0546] 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.

[0547] 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 percent yield). MS (ESI): MS calculated value: 926.5, MS measured value: [M+H] + = 927.5.

[0548] 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.

[0549] 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.

[0550] 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.

[0551] 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%. 11H NMR (400 MHz, DMSO-d6) 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 measured value: [M+H] + = 1229.4. Purity (HPLC, 254 nm) = 95.5%.

[0552] xxi) DBCO-PEG3-VA-PABC-SN38 (LP-5)

Chemical Formula

[0553] 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(...

Claims

1. An antigen-binding molecule that binds to a target antigen, comprising (i) a target antigen-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 is a DDR inhibitor, or contains one, and the DDR inhibitor is (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) The antigen-binding molecule according to claim 1 or 2, which is a DNA-PK inhibitor, wherein the DDR inhibitor portion is or comprises nedisertib.

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 the location where at least one first click group is connected to the branching group, The antigen-binding molecule according to claim 6, comprising the branching group, wherein c indicates a location where at least one second click group is linked to the branching group.

8. A composition comprising an antigen-binding molecule according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier, diluent, excipient, or auxiliary agent.

9. An antigen-binding molecule according to any one of claims 1 to 7, or a composition according to claim 8, for use in a medical treatment or preventive method, or a diagnostic or prognostic method.

10. An antigen-binding molecule according to any one of claims 1 to 7, or a composition according to claim 8, for use in the treatment or prevention of cancer.

11. Use of an antigen-binding molecule according to any one of claims 1 to 7, or the composition according to claim 8, in the manufacture of a drug for treating or preventing cancer.

12. A method for treating or preventing cancer, comprising administering to a target an antigen-binding molecule according to any one of claims 1 to 7, or the composition according to claim 8, in an amount effective for treatment or prevention.

13. The antigen-binding molecule or composition for use according to claim 10, the use according to claim 11, or the method according to claim 12, wherein the cancer is selected from cancers comprising cells expressing / overexpressing the target antigen.

14. An antigen-binding molecule or composition for use according to claim 10 or 13, the use according to claim 11 or 13, or the method according to claim 12 or 13, wherein the cancer is resistant to or recurrent in response to treatment with a DDR inhibitor, and / or the cancer is resistant to or recurrent in response to treatment with a TOP1 inhibitor.

15. Use of an antigen-binding molecule according to any one of claims 1 to 7, or the composition according to claim 8, for the purpose of removing or enhancing the killing of cells expressing a target antigen.

16. An optionally isolated in vitro complex comprising an antigen-binding molecule according to any one of claims 1 to 7, bound to the target antigen.

17. (a) an amino group for conjugation to the antigen-binding site, (b) at least one first click base for linking the portion containing the first payload, (c) at least one second click base for linking the portion containing the second payload, (d) Branched group: 【Chemistry 2】 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 the location where at least one first click group is connected to the branching group, A trifunctional linker portion including the branching group, wherein c indicates a location where at least one second click group is linked to the branching group.

18. R Ｎ However, H or CH ２ The trifunctional linker portion according to claim 17, wherein it is C(O)OH.

19. The amino group is linked to the branched group by a first spacer group (A1), which is given by the following formula: -(CH ２ ) ｘａ -(C ２ H ４ O) ｘｂ -(CH ２ ) ｘｃ - The trifunctional linker portion according to claim 17 or claim 18, wherein xa is 0 or 1, xb is 0 to 12, and xc is 0 to 6, where at least one of xa and xb is 1.

20. A1 is - (CH ２ ) ５ -, -(C ２ H ４ )-O-(C ２ H ４ ) -, or - (C ２ H ４ O) ３ - (C ２ H ４ ) - The trifunctional linker portion according to claim 19.

21. The trifunctional linker moiety according to any one of claims 17 to 20, wherein the first and second click groups are selected from an orthogonal click group pair.

22. The first click group is an azide, and the second click group is as follows: 【Transformation 3】 The trifunctional linker portion according to claim 21.

23. The at least one first click group is of formula (B1-1): 【Chemistry 4】 It is connected to the branching group by a second spacer group (B1), R Ｌ１ However, -(C ２ H ４ O) 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 However, -(C ２ H ４ O) ｘｅ１ - (CH2) ｘｆ１ - (NH) ｘｇ１ - (C(=O)CH ２ ) 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 However, H or -(C ２ H ４ O) ｘｅ２ - (CH ２ ) ｘｆ２ - (NH) ｘｇ２ - (C(=O)CH ２ ) 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. The at least one second click group is defined by formula (B2-1): 【Transformation 5】 It is connected to the branching group by a third spacer group (B2), R Ｌ２ However, -(C ２ H ４ O) ｘｌ５ - (CH2) ｘｉ - (C (=O)) ｘｌ６ - and Here, xl6 is between 0 and 4, xi is between 0 and 3, and xl6 is 0 or 1. R ＮＢ３ However, -(C ２ H ４ O) ｘｊ１ - (CH2) ｘｋ１ - (NH) ｘｌ１ - (C(=O)CH ２ ) 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 However, H or -(C ２ H ４ O) ｘｊ２ - (CH ２ ) ｘｋ２ - (NH) ｘｌ２ - (C(=O)CH ２ ) ｘｍ２ - and The trifunctional linker portion according to any one of claims 17 to 22, wherein xj2 is 0 to 4, xk2 is 0 to 2, xl2 is 0 or 1, and xm2 is 0 or 1.

24. The second spacer group (B1) and the third spacer group (B2), together with the nitrogen atom to which they are bonded, 【Chemistry 6-1】 【Chemistry 6-2】 The trifunctional linker moiety according to claim 23, which forms a group selected from the above. 【Request Item 25】 【Chemistry 7】 A trifunctional linker portion according to claim 17, selected from the above.

26. (a) at least one first payload and at least one second payload, (b) A conjugate comprising an antigen-binding moiety, The conjugate comprises a linker between the payload and the antibody, the linker being derived from a compound according to any one of claims 17 to 25.

27. A linker-payload molecule for conjugation to an antigen-binding moiety, comprising at least one first payload and at least one second payload, wherein the linker for conjugation to an antibody comprises a portion derived from a compound according to any one of claims 17 to 25.

28. A modified antigen-binding moiety comprising a portion derived from the compound according to any one of claims 17 to 25.

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