Site-specific antibody-drug conjugates comprising benzoselenophene-based compound and use thereof

Abstract

The present invention relates to: an antibody-drug conjugate (ADC) in which a linker drug is site-specifically conjugated to an antibody through an engineered cysteine at a specific position of the antibody; and a pharmaceutical composition for preventing or treating proliferative diseases, comprising same. The site-specifically conjugated ADC according to the present invention exhibited improved physicochemical, pharmacological, and / or pharmacokinetic properties. The ADC of the present invention has binding properties similar to those of wild-type antibodies, excellent in vivo efficacy, increased therapeutic index, and / or improved stability, and thus can exhibit therapeutic effects on various diseases including cancer superior to those of conventional antibodies or ADCs.

Description

Site-specific antibody-drug conjugate comprising benzoselenophene compound and use thereof

[0001] The present invention relates to an antibody-drug conjugate (ADC) and uses thereof, and more particularly, to an antibody-drug conjugate in which a linker drug is site-specifically conjugated to an antibody via an engineered cysteine ​​at a specific position of the antibody, and a pharmaceutical composition for preventing or treating a proliferative disease comprising the same.

[0002]

[0003] Recently, antibody-based diagnostic and therapeutic approaches are being studied for various diseases. In particular, the target specificity of antibodies has led to the development of various therapeutic approaches using antibodies. Various forms of pharmaceuticals containing antibodies, such as antibody-drug conjugates (ADCs), are also being developed. Accordingly, research is ongoing into methods to increase the in vivo stability of antibodies or antibody-drug conjugates and maximize their therapeutic efficacy.

[0004] In addition to monoclonal antibodies and target selection, drugs or linkers are becoming a focus of antibody-drug conjugate development, and the importance of conjugate homogeneity has recently been recognized. Conventional methods for drug attachment to antibodies produce heterogeneous mixtures, with some individual components of the mixture potentially lacking in vivo performance. Novel methods for site-specific drug attachment produce more homogeneous conjugates and allow for greater control over drug attachment sites. These improvements can significantly impact in vivo efficacy, in vivo safety, and the resulting therapeutic index.

[0005] The first site-specific conjugation approach was developed at Genentech by introducing cysteine ​​residues using site-directed mutagenesis at highly thiol-reactive positions as described in WO 2006 / 034488 A. The engineered cysteine ​​approach proved suitable for the development of the site-specific ADC SGN-CD33A (Seattle Genetics), which recently entered a Phase Ib clinical trial in combination with standard-of-care chemotherapy including cytarabine and daunorubicin, as well as a Phase I dose-escalation clinical study for the treatment of acute myeloid leukemia (AML). This ADC comprises a DNA-crosslinking pyrrolobenzodiazepine (PBD) dimer as a drug conjugated to a cleavable dipeptide linker (i.e., valine-alanine) and heavy chain position S239C in the Fc portion of the IgG1 mAb h2H12 (DAR 1.9; Sutherland et al. Blood 2013; 122(8): 1455-1463).

[0006] WO 2006 / 034488 A (Genentech) obtained engineered cysteine ​​residues with high thiol reactivity by substituting specifically surface-accessible valine, alanine, and serine residues that are not involved in antigen binding interactions and are distant from conventional interchain disulfide bonds, and site-specific conjugates were obtained using linker drugs of the MMAE, MMAF, and DM1 series. WO 2014 / 124316 A (Novartis) and WO 2013 / 068874 A (Pfizer) specifically focused on the identification of surface-accessible regions of the constant domains of antibody heavy and light chains, where substitution of cysteine ​​residues enabled efficient conjugation of the payload, resulting in highly stable conjugates. WO 2015 / 177360 A (Byondis) on the other hand obtained engineered cysteine ​​residues by substituting some of the residues that make up the antibody heavy chain constant and variable domains. A series of inventions suggest that the efficacy of site-specific conjugates utilizing cysteine ​​mutations can be optimized depending on the location of the mutant cysteine ​​and the type of linker drug.

[0007] Although techniques for site-specific conjugation of linker drugs have been able to generate FDA-approved antibody-drug conjugates and are currently used to fabricate a majority of ADCs in preclinical and clinical trials, there is still a need for novel conjugation strategies to improve the physicochemical, pharmacokinetic, pharmacological and / or toxicological properties of ADCs to obtain ADCs with acceptable antigen binding characteristics, in vivo efficacy, therapeutic index and / or stability.

[0008] Against this backdrop, the inventors of the present invention have continuously researched to develop antibody-drug conjugates with improved physicochemical, pharmacological, and / or pharmacodynamic properties, and as a result, through in silico analysis, have confirmed that an antibody-drug conjugate can be produced by introducing a cysteine ​​mutation at a specific position within the heavy chain constant region of an antibody, thereby site-specifically conjugating the drug. In particular, in the case of ADCs based on highly hydrophobic payloads such as benzoselenophene duocarmycin, by performing site-specific conjugation at the specific position derived by the inventors, it was confirmed that not only antigen binding characteristics similar to the parent antibody but also excellent in vivo efficacy, improved stability, and / or increased therapeutic index were exhibited, and the present invention was completed based on this.

[0009]

[0010] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0011]

[0012] Summary of the invention

[0013] An object of the present invention is to provide an antibody-drug conjugate having improved physicochemical, pharmacological and / or pharmacodynamic properties.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a proliferative disease comprising the antibody-drug conjugate.

[0015] Another object of the present invention is to provide a method for preventing or treating a proliferative disease using the antibody-drug conjugate, a use of the antibody-drug conjugate for preventing or treating a proliferative disease, and a use of the antibody-drug conjugate for manufacturing a medicament for preventing or treating a proliferative disease.

[0016]

[0017] To achieve the above object, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof, and a linker drug, wherein the linker drug is conjugated to the antibody or antigen-binding fragment thereof via an engineered cysteine ​​at one or more positions selected from heavy chain positions 151, 152, 153, 154, 157, 158, 160, 163, 164, 167, 168, 169, 170, 171, 172, 173, 174, 180, 182, 201, 203, 205, 206, and 210 (according to EU numbering).

[0018] The present invention also provides a pharmaceutical composition for preventing or treating a proliferative disease comprising the antibody-drug conjugate.

[0019] The present invention also provides a method for preventing or treating a proliferative disease, comprising administering the antibody-drug conjugate, a use of the antibody-drug conjugate for preventing or treating a proliferative disease, and a use of the antibody-drug conjugate for preparing a medicament for preventing or treating a proliferative disease.

[0020]

[0021] Figure 1 is a schematic diagram showing the docking of the selenoduocarmycin linker drug vc-PAB-AMB401 within the Fab cavity of an antibody (superimposition of multiple vc-PAB-AMB401).

[0022] Figures 2a and 2b are graphs showing the docking results of the selenoduocarmycin linker drug vc-PAB-AMB401 within the Fab cavity of five antibodies.

[0023] Figures 3a and 3b are schematic diagrams showing three cysteine ​​manipulation residues and their positions selected through docking results and physicochemical property analysis.

[0024] Figure 4 is a schematic diagram of a selenoduocarmycin linker drug conjugated to a cysteine ​​engineered residue within the Fab cavity of an antibody.

[0025] Figure 5 is a graph showing the results of analysis of conjugates in which mc-vc-PAB-AMB401 is site-specifically bound to anti-FGFR3 antibody cysteine ​​mutants using hydrophobic interaction chromatography (HIC)-HPLC. The linker-drug conjugation ratio of each antibody-drug conjugate was confirmed through HPLC.

[0026] Figure 6 shows an ELISA assay experiment of linker-AMB401 (-■-); linked to trastuzumab (-●-); trastuzumab cysteine ​​mutant (P153C) against HER2 in vitro.

[0027] Figure 7 shows an ELISA assay experiment of linker-AMB401 (-■-); linked to trastuzumab (-●-); trastuzumab cysteine ​​mutant (T169C) against HER2 in vitro.

[0028] Figure 8 shows an ELISA assay experiment of trastuzumab (-●-); linker-AMB401 (-■-); conjugated to trastuzumab cysteine ​​mutant (K205C) against HER2 in vitro.

[0029] Figure 9 shows cell proliferation assays of NCI-N87 cells treated with linker-AMB401 conjugated to trastuzumab-vc-secoDUBA (SYD-985) (-○-); trastuzumab cysteine ​​mutant (P153C) (-●-); trastuzumab cysteine ​​mutant (T169C) (-■-); trastuzumab cysteine ​​mutant (K205C) (-▲-); in vitro.

[0030] Figure 10 shows a cell proliferation assay of SK-BR-3 cells treated with linker-AMB401 conjugated to trastuzumab-vc-secoDUBA (SYD-985) (-○-); trastuzumab cysteine ​​mutant (P153C) (-●-); trastuzumab cysteine ​​mutant (T169C) (-■-); trastuzumab cysteine ​​mutant (K205C) (-▲-); in vitro.

[0031] Figure 11 shows a cell proliferation assay of MCF-7 cells treated with linker-AMB401 conjugated to trastuzumab-vc-secoDUBA (SYD-985) (-○-); trastuzumab cysteine ​​mutant (P153C) (-●-); trastuzumab cysteine ​​mutant (T169C) (-■-); trastuzumab cysteine ​​mutant (K205C) (-▲-); in vitro.

[0032] Figure 12 shows the internalization of antibody-drug conjugates in HER2-overexpressing cell lines NCI-N87 (left) and Calu-3 (right) using flow cytometry. From left, the relative fluorescence measurements after pretreatment with 20 nM of trastuzumab, trastuzumab-Seco-DUBA (SYD-985), and trastuzumab cysteine ​​mutant (T169C)-AM10801, AM11001, and AM11101 are shown compared to the initial values ​​at 37°C.

[0033] Figure 13 shows the cell proliferation assay of Calu-3 cells in vitro for SYD-985 (-○-); and trastuzumab cysteine ​​mutant (T169C) conjugates (-■-) of mc-PEG4-vc-PAB-PEG4-AMB401 (AM10801), mc-PEG4-vc-PAB-PEG24-AMB401 (AM11101), and mal-PEG4-β-glucuronide-DMEDA-AMB401 (AM11001).

[0034] Figure 14 shows the cell proliferation assay of SK-OV-3 cells in vitro for SYD-985 (-○-); and trastuzumab cysteine ​​mutant (T169C) conjugates (-■-) of mc-PEG4-vc-PAB-PEG4-AMB401 (AM10801), mc-PEG4-vc-PAB-PEG24-AMB401 (AM11101), and mal-PEG4-β-glucuronide-DMEDA-AMB401 (AM11001).

[0035] Figure 15 shows the cell proliferation assay of MDA-MB-453 cells in vitro for SYD-985 (-○-); and trastuzumab cysteine ​​mutant (T169C) conjugates (-■-) of mc-PEG4-vc-PAB-PEG4-AMB401 (AM10801), mc-PEG4-vc-PAB-PEG24-AMB401 (AM11101), and mal-PEG4-β-glucuronide-DMEDA-AMB401 (AM11001).

[0036] Figure 16 shows the cell proliferation assay of HER2 low-expressing SW-620 cells in vitro for SYD-985 (-○-); and trastuzumab cysteine ​​mutant (T169C) conjugates (-■-) of mc-PEG4-vc-PAB-PEG4-AMB401 (AM10801), mc-PEG4-vc-PAB-PEG24-AMB401 (AM11101), and mal-PEG4-β-glucuronide-DMEDA-AMB401 (AM11001).

[0037] Figure 17 shows the cell proliferation assay of HER2 low-expressing NCI-H520 cells in vitro for SYD-985 (-○-); and trastuzumab cysteine ​​mutant (T169C) conjugates (-■-) of mc-PEG4-vc-PAB-PEG4-AMB401 (AM10801), mc-PEG4-vc-PAB-PEG24-AMB401 (AM11101), and mal-PEG4-β-glucuronide-DMEDA-AMB401 (AM11001).

[0038] Figure 18 is a graph showing the results of measuring tumor volumes over 32 days after intravenous injection of mc-PEG4-vc-PAB-PEG4-AMB401 (AM10801), mc-PEG4-vc-PAB-PEG24-AMB401 (AM11101), and mal-PEG4-β-glucuronide-DMEDA-AMB401 (AM11001) conjugates (-■-) at a dose of 3 mg / kg once, 100 μL each, in nude mice xenografted with NCI-N87 cell lines on day 0.

[0039] Figure 19 is a graph showing the results of an ELISA assay experiment of trastuzumab (-●-) against HER2 in vitro; linker-AMB401 (-●-) bound to trastuzumab cysteine ​​mutant (T169C); linker-AMB401 (-○-) bound to trastuzumab cysteine ​​mutant (T169C) via hydrolyzed maleimide;

[0040] Figure 20 shows cell proliferation assays of SK-BR-3 cells treated in vitro with trastuzumab-vc-secoDUBA (SYD-985) (-○-); AM11001 bound to trastuzumab cysteine ​​mutant (T169C) (-■-); AM11001 bound to maleimide binding site hydrolyzed trastuzumab cysteine ​​mutant (T169C) (-□-); AM11101 bound to trastuzumab cysteine ​​mutant (T169C) (-■-); AM11101 bound to maleimide binding site hydrolyzed trastuzumab cysteine ​​mutant (T169C) (-□-).

[0041] Figure 21 shows cell proliferation assays for AM11001, AM11101(-■-); conjugated to trastuzumab-vc-secoDUBA(-○-) and trastuzumab cysteine ​​mutant (T169C) with an antibody-drug ratio of 1.8 in vitro. Figure 21a shows treatment in the NCI-N87 gastric cancer cell line, Figure 21b shows treatment in the SK-BR-3 breast cancer cell line, and Figure 21c shows treatment in the MCF-7 estrogen hormone receptor-expressing breast cancer cell line.

[0042] Figure 22 is a graph showing the results of measuring tumor volume over 35 days after intravenous injection of 1 mg / kg of PBS (buffer) (-●-); mal-PEG4-β-glucuronide-DMEDA-AMB401 (AM11001), mc-PEG4-vc-PAB-PEG24-AMB401 (AM11101) conjugate (-■-) and trastuzumab-vc-secoDUBA (DAR 1.8) (-○-); once on day 0 in nude mice xenografted with NCI-N87 cell lines.

[0043] Figures 23a and 23b are graphs showing the results of administering 1 mg / kg or 3 mg / kg of 100 μL of PBS (buffer) (-●-); a control antibody-drug conjugate conjugated with AM11001 (DAR 1.8) and vc-secoDUBA (DAR 2.7) (-○-); and trastuzumab conjugated with AM11001 (DAR 1.8) and vc-secoDUBA (DAR 2.7) (-●-) once via intravenous injection on day 0 in nude mice xenografted with HBCx-10, a cell line derived from a triple-negative breast cancer patient, and measuring tumor volume over the following 31 days.

[0044] Figure 24 is a graph showing the hydrophobic interaction chromatography (HIC-HPLC) results for an antibody-drug conjugate in which the AMB401 linker-drug is site-specifically conjugated to the heavy chain sequence T169C mutant antibody of trastuzumab, an anti-HER2 antibody.

[0045] Figure 25 is a graph showing the results of hydrophobic interaction chromatography (HIC-HPLC) for an antibody-drug conjugate in which the AMB401 linker-drug is site-specifically conjugated to the light chain sequence V205C mutant antibody of trastuzumab, an anti-HER2 antibody.

[0046] Figure 26 shows the results of the stability evaluation of ADC in human plasma for an antibody-drug conjugate conjugated with AM11001 to trastuzumab cysteine ​​mutant (T169C) with DAR 1.8.

[0047] Figure 27 shows the results of pharmacokinetic analysis in CES1c deficient mice between an antibody-drug conjugate comprising AM11001 conjugated to trastuzumab cysteine ​​mutant (T169C) with a DAR of 1.8 and an antibody-drug conjugate comprising vc-seco-DUBA conjugated to trastuzumab as a control substance with a DAR of 2.7.

[0048]

[0049] Detailed description of the invention and preferred embodiments

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0051]

[0052] Early research on site-specific antibody-drug conjugates (ADCs) utilizing engineered cysteines involved generating hundreds of site-specific ADCs by mutating each amino acid in the antibody chain to cysteine, thereby identifying conjugation sites with superior properties and toxicity. Results were described to vary depending on the linker drug conjugated to the antibody, suggesting that the optimal conjugation site may vary depending on the linker drug (and, by extension, the antibody).

[0053] Among the various protein regions that make up antibodies, there are variable regions and constant regions. The variable region is a region whose amino acid sequence changes depending on the type of antibody, and thus its structure is likely to change. The constant region is a region whose amino acid sequence does not change depending on the type of antibody, and thus its structure is unlikely to change.

[0054] Accordingly, the inventors of the present invention focused on the antibody constant region as the target of the search for a conjugation site to develop an excellent site-specific antibody-drug conjugate.

[0055]

[0056] Accordingly, the present invention relates, in one aspect, to an antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof, and a linker drug, wherein the linker drug is conjugated to the antibody or antigen-binding fragment thereof via an engineered cysteine ​​at one or more positions selected from heavy chain positions 151, 152, 153, 154, 157, 158, 160, 163, 164, 167, 168, 169, 170, 171, 172, 173, 174, 180, 182, 201, 203, 205, 206 and 210 (according to EU numbering).

[0057]

[0058] In the present invention, the linker drug may be characterized in that it is site-specifically conjugated to the antibody through an engineered cysteine ​​at one or more positions selected from heavy chain positions 151, 152, 153, 154, 157, 158, 160, 163, 164, 167, 168, 169, 170, 171, 172, 173, 174, 180, 182, 201, 203, 205, 206 and 210 (according to EU numbering) of the antibody or antigen-binding fragment thereof.

[0059] In the present invention, the engineered cysteine ​​may be characterized by being at one or more positions selected from positions 152, 153, 154, 164, 167, 168, 169, 170, 171, 172, 174, 180, 182 and 205 (according to EU numbering) in the heavy chain constant region CH1 domain of the antibody, but is not limited thereto.

[0060] In the present invention, the engineered cysteine ​​is preferably, but not limited to, located at one or more positions selected from positions 153, 169, and 205 (according to EU numbering) in the CH1 domain of the heavy chain constant region of the antibody.

[0061] In one embodiment of the present invention, various conjugation residue positions expected to optimally interact with the linker-drug to be conjugated to the Fab internal cavity structure where the heavy and light chains are paired in the antibody constant region structure were identified through simulation experiments using the YASARA software package [Krieger et al. Proteins 2009; 77 Suppl 9: 114-122], and the residue positions were evaluated through in silico analysis.

[0062] Docking of linker-drugs into the Fabs of various antibodies was simulated using the commercial VINA algorithm (Trott O and Olson AJ. J. Comput. Chem. 2010; 31: 455-461) as implemented in YASARA. The antibody Fab models used were obtained from X-ray structures or by homology modeling using YASARA.

[0063] The duocarmycin-type linker-drug was found to have a strong preference for binding in the cavity present in the five antibody Fab structures (see Fig. 1 for the docking results of the linker-drug, vc-PAB-AMB401, distributed in the antibody Fab structure). The cysteine ​​residue manipulation positions suitable for linker-drug attachment were identified as amino acid residues that are close to the maleimide, a constituent compound forming the linker-drug, among the amino acid residues forming the cavity, but far from the intrinsic cysteine ​​that is essentially present in the wild-type antibody.

[0064] In the present invention, the variable region of an antibody is not subject to cysteine ​​residue manipulation. The constant region of an antibody is subject to cysteine ​​residue manipulation, and the EU numbering used to describe this is used to specify the positions within the heavy and light chain constant regions of the antibody. In most antibodies, especially IgG antibodies, the constant region heavy chain CH1 domain typically has P at position 151, E at position 152, P at position 153, S at position 157, W at position 158, S at position 160, L at position 163, T at position 164, V at position 167, H at position 168, T at position 169, F at position 170, P at position 171, A at position 172, V at position 173, L at position 174, Y at position 180, L at position 182, N at position 201, N at position 203, K at position 205, P at position 206, and K at position 210.

[0065] The term “EU numbering” of the present invention refers to the EU index as in the literature [Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., NIH publication no. 91-3242, pp. 662, 680, 689 (1991)]. The EU index refers to the residue numbering of the human IgG1 EU antibody (Edelman, GM et al., Proc. Natl. Acad. Sci. USA, 63, 78-85 (1969)). “Kabat numbering” is used to designate amino acid positions within the heavy (HC) and light (LC) chain variable regions.

[0066] In the present invention, heavy chain positions 151, 152, 153, 154, 157, 158, 160, 163, 164, 167, 168, 169, 170, 171, 172, 173, 174, 180, 182, 201, 203, 205, 206 and 210 (EU numbering) are amino acids constituting the Fab portion of an antibody and are located in the corresponding structure.

[0067] The term "engineered cysteine" of the present invention refers to the substitution of a non-cysteine ​​amino acid in the heavy or light chain of an antibody with cysteine. As is well known to those skilled in the art, this can be accomplished at the amino acid or DNA level, for example, using site-directed mutagenesis.

[0068] In the present invention, one or more engineered cysteine ​​residues may be engineered into an antibody using conventional molecular cloning techniques, or the heavy or light chain domain(s) of the antibody bearing the cysteine ​​mutation(s) may be synthesized alone using known (peptide or DNA) synthesis apparatus and procedures.

[0069] In one embodiment of the present invention, it was confirmed that the site-specific conjugated ADC according to the present invention exhibits improved physicochemical, pharmacological, and / or pharmacodynamic properties compared to an ADC in which the linker drug is conjugated via a native interchain disulfide bond of an antibody or an engineered cysteine ​​ADC in which the linker drug is conjugated at a known site. The ADC according to the present invention has binding properties similar to wild-type antibodies, superior in vivo efficacy, increased therapeutic index, and / or improved stability.

[0070]

[0071] In the present invention, any linker drug known in the art of ADC technology can be used for site-specific conjugation to an antibody. Preferably, the linker drug is characterized by a chemical group capable of reacting with the thiol group of the engineered cysteine, typically a maleimide group or a haloacetyl group.

[0072] In the present invention, the drug may be characterized as being, but is not limited to, duocarmycin, calicheamicin, a pyrrolobenzodiazepine (PBD) dimer, a maytansinoid, or an auristatin derivative. Suitable examples of maytansinoid drugs include DM1 and DM4, and suitable examples of auristatin drugs include MMAE and MMAF.

[0073] In the present invention, the drug may be characterized as being a benzoselenophene-based duocarmycin derivative, but is not limited thereto.

[0074] Duocarmycin, first isolated from cultures of Streptomyces species, is a member of a family of antitumor antibiotics that also includes duocarmycin A, duocarmycin SA, and CC-1065. These extremely potent agents derive their bioactivity from their ability to sequence-selectively alkylate DNA at the N3 position of adenine within the minor groove, initiating a cascade of events that culminates in an apoptotic mechanism.

[0075] In the present invention, the drug may be characterized as being a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0076]

[0077] In the above chemical formula 1, X is halogen, and R1 is substituted or unsubstituted C containing one or more N. 3-10 A heterocycloalkyl group or a heteroaryl group, and R2 and R3 are each independently hydrogen, fluorine, chlorine, bromine, iodine, or substituted or unsubstituted C 1-5 An alkyl group, and R4 and R5 are each independently hydrogen, or substituted or unsubstituted C 1-5 It is an alkyl group.

[0078] The above heterocycloalkyl group or the above heteroaryl group may or may not additionally include one or more hetero elements selected from O and S.

[0079] In the present invention, KR 1775440 B discloses a series of drugs including a duocarmycin derivative of AMB401, and the structure of AMB401 used in one embodiment of the present invention is a compound represented by the following chemical formula 2:

[0080]

[0081] Suitable linker-duocarmycin derivatives that can be used in the present invention are disclosed on pages 28-30 of KR 1775440 B. The chemical syntheses of many of these linker-drugs are described in Examples 1-3 of KR 1775440 B, which can be applied to antibody-drug conjugates according to the present invention.

[0082] As is well known in the art, DAR and drug load distribution can be determined using, for example, hydrophobic interaction chromatography (HIC) or reverse phase high performance liquid chromatography (RP-HPLC).

[0083] The compounds of formula 1 or formula 2 according to the present invention can be obtained according to methods and procedures well known to those skilled in the art. Suitable methods for site-specific conjugation of linker drugs can be found in examples 7 and 8 of WO 2005 / 084390 A, which describes a fully reducing strategy for (partial) loading of antibodies with the linker drug vc-MMAE, examples 11 and 12 of WO 2006 / 034488 A, which describes site-specific conjugation of linker drugs comprising maytansine (DM1), and in the literature [Doronina et al. Bioconjugate Chem. 17 (2006): 114-124], which describes conjugation with mc-MMAF.

[0084] In the present invention, one or two engineered cysteines may be introduced into the heavy chain and / or light chain of the antibody under optimal reaction conditions that produce an ADC compound having a DAR of 2 or 4, respectively. When one engineered cysteine ​​is introduced, it is located in the Fab portion of the antibody.

[0085] In the present invention, the linker drug of the antibody-drug conjugate may include thiosuccinate derived from hydrolysis of thiosuccinimide located at the antibody binding site of the linker drug, and the hydrolysis reaction may proceed as follows, but is not limited thereto:

[0086]

[0087] In the present invention, the linker may be characterized as being a cleavable linker or a non-cleavable linker.

[0088] The linker can be cleaved by a cleavage agent present in the intracellular environment, for example, in a lysosome or endosome, and can be a peptide linker that can be cleaved by an intracellular peptidase or protease enzyme, for example, a lysosomal or endosomal protease. Typically, the peptide linker has a length of at least two amino acids. The cleavage agent can include cathepsin B, cathepsin D, or plasmin, and hydrolyzes the peptide to release the drug into the target cell. The peptide linker can be cleaved by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues, and for example, a Phe-Leu or Gly-Phe-Leu-Gly linker can be used. In addition, the peptide linker can be cleaved by, for example, an intracellular protease, and can be a Val-Cit linker or a Phe-Lys linker.

[0089] In the present invention, the cleavable linker is pH-sensitive and may be susceptible to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, it may be an acid-labile linker that can be hydrolyzed in lysosomes, such as a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitic amide, an orthoester, an acetal, a ketal, etc.

[0090] The above linker may also be cleaved under reducing conditions, for example, a disulfide linker. Various disulfide bonds can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).

[0091] The above linker may include a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase, which is present in large numbers in lysosomes or is overexpressed in some tumor cells. Unlike peptide linkers, it has a high hydrophilicity, which has the advantage of increasing the solubility of the antibody-drug complex when combined with a highly hydrophobic drug.

[0092] In this regard, the present invention may use a beta-glucuronide linker disclosed in Korean Patent Publication No. 2015-0137015, for example, a beta-glucuronide linker including a self-immolative group.

[0093] Additionally, the linker may be, for example, a non-cleavable linker, which releases the drug through a single step of antibody hydrolysis, producing, for example, an amino acid-linker-drug conjugate. This type of linker may be a thioether group or a maleimidocaproyl group, and may maintain stability in blood.

[0094] In one embodiment of the present invention, the linker may be characterized by being a compound represented by the following chemical formulas 3 to 6, but is not limited thereto:

[0095]

[0096]

[0097]

[0098]

[0099] In the above chemical formulas 3 to 6, n is an integer from 0 to 12, m is an integer from 0 to 32, and P is a drug.

[0100] In the present invention, n may be characterized as being an integer from 0 to 12, preferably an integer from 0 to 10, more preferably an integer from 0 to 6, and m may be characterized as being an integer from 0 to 32, preferably an integer from 0 to 25, more preferably an integer from 0 to 23, but is not limited thereto.

[0101] In this specification, “antibody” refers to a general term for substances produced in the immune system by antigen stimulation, and the type thereof is not particularly limited. The antibody is an immunoglobulin molecule that is immunologically reactive with a specific antigen, and refers to a protein molecule that acts as a receptor that specifically recognizes the antigen. It may include polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. The antibody may be non-naturally produced, for example, recombinantly or synthetically produced. The antibody may be an animal antibody (e.g., mouse antibody, etc.), chimeric antibody, humanized antibody, or human antibody. The antibody may be a monoclonal antibody. In addition, unless otherwise specified, the antibody may be understood to also include an antigen-binding fragment of an antibody that possesses antigen-binding ability.

[0102] As used herein, the term “heavy chain” refers to a full-length heavy chain and fragments thereof, comprising a variable domain VH and three constant domains CH1, CH2 and CH3, each comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. The term “light chain” also refers to a full-length light chain and fragments thereof, comprising a variable domain VL and a constant domain CL, each comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0103] In the present invention, the antibody may be any antibody, particularly any antibody known to have therapeutic activity or any antibody known in the art of ADC technology.

[0104] The entire antibody according to the present invention is a concept including IgA, IgD, IgE, IgG and IgM forms, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4.

[0105] A full-length antibody consists of two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The heavy-chain constant region is of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light-chain constant region is of the kappa (κ) and lambda (λ) types.

[0106] In the present invention, the antibody may be characterized as being an IgG antibody, preferably an IgG1 antibody, and more preferably an IgG1 antibody having a κ light chain.

[0107] In the present invention, “antigen-binding fragment” refers to a fragment that possesses the function of binding to a target antigen, and is used interchangeably with “antibody fragment” in the present specification with the same meaning. Antigen-binding fragments of the antibody of the present invention include Fab, Fab', and F(ab')2, which include a CH1 domain.

[0108] The Fab has a structure with variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond.

[0109] These antibody fragments can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and can be produced through genetic recombination technology (for example, DNA encoding the heavy chain or its variable region and DNA encoding the light chain or its variable region of the antibody are used as templates, and amplified by PCR (Polymerase Chain Reaction) using a pair of primers, and amplifying by combining DNA encoding a peptide linker and a pair of primers so that both ends are linked to the heavy chain or its variable region and the light chain or its variable region, respectively).

[0110] Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies including bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, or epitope-binding fragments of the above antibodies.

[0111] In the present invention, the antibody may be characterized by binding to an antigen target expressed within or on the cell membrane of a tumor cell (e.g., on the cell surface). Preferably, after binding to the (antigen) target, the antibody is internalized by the cell, and the toxin is then released into the cell.

[0112] Antibodies suitable for the antibody-drug conjugate according to the present invention include anti-annexin A1 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD38 antibody, anti-CD44 antibody, anti-CD47 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD74 antibody, anti-CD79 antibody, anti-CD115 antibody, anti-CD123 antibody, anti-CD138 antibody, anti-CD203c antibody, anti-CD303 antibody, anti-CEACAM antibody, anti-CLL-1 antibody, anti-HGFR (or anti-c-MET) antibody, anti-Cripto antibody, anti-DLL3 antibody, anti-EGFR antibody, anti-EPCAM antibody, anti-EphA2 antibody, anti-EphB3 antibody, anti-ETBR antibody, anti-FcRL5 antibody, Examples include, but are not limited to, anti-FGFR3 antibodies, anti-FOLR1 antibodies, anti-GCC antibodies, anti-GPNMB antibodies, anti-Her2 antibodies, anti-HMW-MAA antibodies, anti-integrin antibodies, anti-Lewis A-like carbohydrate antibodies, anti-Lewis Y antibodies, anti-LIV1 antibodies, anti-mesothelin antibodies, anti-MN antibodies, anti-MUC1 antibodies, anti-MUC16 antibodies, anti-NaPi2b antibodies, anti-nectin-4 antibodies, anti-PSMA antibodies, anti-SIRPα antibodies, anti-SLC44A4 antibodies, anti-STEAP-1 antibodies, anti-5T4 (or anti-TPBG, trophoblast glycoprotein) antibodies, anti-Tag72 antibodies, anti-TF (or anti-tissue factor) antibodies, anti-TROP2 antibodies, and anti-VLA antibodies.

[0113] Preferably, it may be an anti-annexin A1 antibody, an anti-CD20 antibody, an anti-CD115 antibody, an anti-CD123 antibody, an anti-CLL-1 antibody, an anti-c-MET antibody, an anti-EGFR antibody, an anti-FGFR3 antibody, an anti-HER2 antibody, an anti-MUC1 antibody, an anti-PSMA antibody, an anti-5T4 antibody or an anti-TF antibody, and more preferably, it may be characterized by being an anti-CD20 antibody, an anti-EGFR antibody or an anti-HER2 antibody, but is not limited thereto.

[0114]

[0115] From another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a proliferative disease comprising the antibody-drug conjugate.

[0116] In another aspect, the present invention relates to a method for preventing or treating a proliferative disease, comprising administering the antibody-drug conjugate to a subject.

[0117] In another aspect, the present invention relates to the use of the antibody-drug conjugate for the prevention or treatment of proliferative diseases.

[0118] In another aspect, the present invention relates to the use of the antibody-drug conjugate for the manufacture of a medicament for the prevention or treatment of a proliferative disease.

[0119] In this specification, the term “prevention” means any act of inhibiting or delaying the progression of a proliferative disease by administering the composition of the present invention, and “treatment” means inhibiting the development of a proliferative disease, alleviating or eliminating symptoms.

[0120] In the present invention, the proliferative disease may be, but is not limited to, a neoplasm, a tumor, cancer, leukemia, psoriasis, a bone disease, a fibroproliferative disorder, or atherosclerosis.

[0121] In the present invention, the cancer may be a solid tumor, blood cancer, colorectal cancer, uterine cancer, uterine fibroids, meningioma, lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, colon cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, brain metastasis cancer, sarcoma, osteosarcoma, Kaposi sarcoma, or melanoma, but is not limited thereto, and the pharmaceutical composition according to the present invention may be applicable to diseases not limited to cancer as a target of prevention or treatment.

[0122] In the present invention, the pharmaceutical composition may be characterized by comprising a therapeutically effective amount of an antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0123] The above “pharmaceutically acceptable carrier” is a substance that can be added to the active ingredient to help formulate or stabilize the preparation, and does not cause significant harmful toxic effects to the patient. Pharmaceutically acceptable carriers are those commonly used in the preparation of preparations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0124] The pharmaceutical composition may further comprise, in addition to the above ingredients, lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0125]

[0126] The term "administration" of the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.

[0127] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat a proliferative disease.

[0128] The pharmaceutical composition according to the present invention can be used in combination with conventional therapeutic agents. This means that the antibody-drug conjugate according to the present invention and the pharmaceutical composition containing it can be administered simultaneously with, or sequentially or in reverse order with, conventional therapeutic agents such as anticancer agents. The combination can be administered in an appropriate effective amount within the scope of those skilled in the art.

[0129]

[0130] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0131]

[0132] Manufacturing Example 1: Manufacturing of a Linker-Payload Intermediate

[0133] Manufacturing Example 1-1: (S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 2-(4-nitrophenyl)acetate

[0134]

[0135] (S)-N-(2-(1-(chloromethyl)-5-hydroxy-8-methoxy-2,3-dihydro-1H-benzo[e]indole-3-carbonyl)benzo[b]selenophene-5-yl)-3-morpholinopropanamide (PNP, 72.8 mg, 239 μmol, 1.5 eq.) was dissolved in DMF (1 mL), and DIEA (20.6 mg, 159 μmol, 27.8 μL, 1 eq.) was added. The mixture was stirred at 25 °C for 2 h. LC-MS analysis showed that the starting material was completely consumed, and the main peak of the desired mass was detected.

[0136] MTBE was added to the reaction mixture to precipitate, followed by filtration and drying to obtain (S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 2-(4-nitrophenyl)acetate (122 mg, 154 μmol, 96.6% yield) as a yellow solid.

[0137] LC-MS analysis results: Rt = 0.47 min, m / z = 793.1 [M+3H] + .

[0138]

[0139] Manufacturing Example 1-2: (17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-acid

[0140]

[0141] (1) Peptide synthesis: Peptides were synthesized using standard Fmoc chemistry.

[0142] 1) Resin preparation: DIEA (6.00 eq) was added to a solution of 2-CTC resin (7.00 mmol, 1.00 eq, substitution degree 0.64 mmol / g) and Fmoc-Cit-OH (1.00 eq) in DCM (30.0 mL). The mixture was stirred at 25°C under N₂ atmosphere for 2.0 h. Then, MeOH (10.9 mL) was added and stirred again at 25°C for 0.5 h. The reaction mixture was filtered to obtain the resin, which was washed with DMF (300 mL × 5).

[0143] 2) Deprotection: 20% piperidine / DMF (300 mL) was added and stirred at 25°C under an N₂ atmosphere for 30 minutes. The resin was washed with DMF (300 mL × 5), filtered, and used in the next step.

[0144] 3) Coupling reaction: HATU (2.85 eq) and Fmoc-Val-OH (3.00 eq) were dissolved in DMF (30.0 mL) and added to the resin. DIEA (6.00 eq) was then added and stirred at 25°C under an N₂ atmosphere for 30 minutes. After the reaction, the resin was washed with DMF (300 mL × 5).

[0145] 4) Repeat additional amino acid combinations: Repeat steps 2 and 3 above to sequentially combine the amino acids below.

[0146] Fmoc-Val-OH (3.00 eq), HATU (2.85 eq), DIEA (6.00 eq)

[0147] 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-acid (2.00 eq), HATU (1.90 eq), DIEA (4.00 eq)

[0148] (2) Peptide cleavage and purification

[0149] 1) The resin was washed three times with MeOH and then dried under vacuum.

[0150] 2) Cutting buffer (20% HFIP / DCM) was added to the resin and stirred three times for 30 minutes.

[0151] 3) DCM and TFA were removed under reduced pressure.

[0152] 4) The crude peptide was dried under high vacuum for 2 hours to obtain (17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-acid (3.02 g) as a yellow solid.

[0153] LC-MS analysis results: Rt = 0.754 min, m / z = 602.3 [M+H] + .

[0154] 5) Purification was performed using Prep-HPLC (eluent A: 0.1% TFA, eluent B: acetonitrile).

[0155]

[0156] Manufacturing Example 1-3: 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-(4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide

[0157]

[0158] (17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-acid (1.45 g, 2.41 mmol, 1 eq.) and 4-aminobenzoic acid (PAB, 594 mg, 4.82 mmol, 2 eq.) were dissolved in DMF (15 mL), and then DIC (608 mg, 4.82 mmol, 746 μL, 2 eq.) and HOBt (651 mg, 4.82 mmol, 2 eq.) were added. The mixture was stirred at 25°C for 1 h.

[0159] LC-MS analysis showed that the starting material was completely consumed, and the main peak with the desired mass was detected. The reaction mixture was purified by prep-HPLC under TFA conditions, and finally 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-(4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide (921 mg, 1.30 mmol, 54.1% yield) was obtained as a white solid.

[0160] LC-MS analysis results: Rt = 0.32–0.33 min, m / z = 707.5 [M+H]+.

[0161]

[0162] Manufacturing Example 1-4: 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl 4-nitrophenyl carbonate

[0163]

[0164] 1-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-((S)-1-(4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide (921 mg, 1.30 mmol, 1 eq.) and 4-pyridinecarbonyl-4-nitrophenyl carbonate (PNP, 793 mg, 2.61 mmol, 2 eq.) were dissolved in DMF (8 mL), and then DIEA (337 mg, 2.61 mmol, 454 μL, 2 eq.) was added. The mixture was stirred at 25 °C for 2 h.

[0165] LC-MS analysis showed that the starting material was completely consumed, and a major peak with the desired mass was detected. The reaction mixture was purified by prep-HPLC under TFA conditions, and 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl 4-nitrophenyl carbonate (787 mg, 903 μmol, 69.3% yield) was finally obtained as a white solid.

[0166] LC-MS analysis results: Rt = 0.43–0.44 min, m / z = 872.4 [M+H] + .

[0167]

[0168] Manufacturing Example 2: Manufacturing of Linker-Payload AM10601

[0169] Manufacturing Example 2-1: tert-Butyl (4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)ethane-1,2-diylbis(methylcarbamate)

[0170]

[0171] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl 4-nitrophenyl carbonate (50 mg, 57.4 μmol, 1 eq.) and 2-aminooxalide (10.8 mg, 57.4 μmol, 1 eq.) were dissolved in DMF (0.5 mL), and then HOBt (7.75 mg, 57.4 μmol, 1 eq.) and DIEA (14.8 mg, 115 μmol, 20.0 μL, 2 eq.) were added. The mixture was stirred at 25°C for 4 hours.

[0172] LC-MS analysis showed that the starting material was completely consumed, and the main peak of the desired mass was detected. The reaction mixture was purified by prep-HPLC under TFA conditions, and tert-butyl (4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)ethane-1,2-diylbis(methylcarbamate) (30 mg, 32.6 μmol, 56.8% yield) was finally obtained as a white oil.

[0173] LC-MS analysis results: Rt = 0.43 min, m / z = 921.8 [M+H] + .

[0174]

[0175] Manufacturing Example 2-2: 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl methyl(2-(methylamino)ethyl)carbamate

[0176]

[0177] To a solution of tert-butyl (4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)ethane-1,2-diylbis(methylcarbamate) (30 mg, 32.6 μmol, 1 eq.) in DCM (0.4 mL) was added TFA (154 mg, 1.35 mmol, 100 μL, 41.3 eq.). The mixture was stirred at 0 °C for 2 h.

[0178] LC-MS analysis showed that the starting material was completely consumed, and the main peak of the desired mass was detected. Isopropyl ether was added to the reaction mixture to precipitate, and the solid was filtered and dried to obtain 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl methyl(2-(methylamino)ethyl)carbamate (16.6 mg, 20.2 μmol, based on crude material) as a white solid.

[0179] LC-MS analysis results: Rt = 0.30 min, m / z = 821.5 [M+H] + .

[0180]

[0181] Manufacturing Example 2-3: (R)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl (4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)ethane-1,2-diylbis(methylcarbamate)

[0182]

[0183] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl methyl(2-(methylamino)ethyl)carbamate (16.6 mg, 20.2 μmol, 1.00 eq.) and (S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 2-(4-nitrophenyl)acetate (16.0 mg, 20.2 μmol, 1.00 eq.) was dissolved in DMF (1.0 mL), and DIEA (5.23 mg, 40.4 μmol, 7.04 μL, 2.00 eq.) was added. The mixture was stirred at 25°C for 1 h.

[0184] LC-MS analysis showed that the starting material was completely consumed and a major peak with the target mass was detected. The reaction mixture was purified by prep-HPLC under TFA condition to finally give (R)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl (4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)ethane-1,2-diylbis(methylcarbamate)(17.0 mg, 11.5 μmol, 57.0% yield) as a white solid. Obtained in solid form.

[0185] LC-MS analysis results: Rt = 0.42–0.43 min, m / z = 1474.7 [M+H] + .

[0186]

[0187] Manufacturing Example 3: Manufacturing of Linker-Payload AM10701

[0188] Manufacturing Example 3-1: N-(2-(2-hydroxyethoxy)ethyl)-N-(tert-butoxycarbonyl)glycylamine

[0189]

[0190] 2-(2-Aminoethoxy)ethanol (2.00 g, 19.0 mmol, 1.91 mL, 1.00 eq.) and N-(tert-butoxycarbonyl)-2-aminoacetaldehyde (3.29 g, 19.0 mmol, 1.00 eq.) were dissolved in MeOH (20.0 mL) and stirred at 25 °C for 4 h. After the reaction mixture was cooled to 0 °C, NaBH₃CN (2.39 g, 38.0 mmol, 2.00 eq.) was added. The mixture was stirred at 0–25 °C for 16 h and then concentrated under reduced pressure to obtain N-(2-(2-hydroxyethoxy)ethyl)-N-(tert-butoxycarbonyl)glycylamine (8.70 g, crude) as a yellow oily product.

[0191] LC-MS analysis results: Rt = 0.21–0.22 min, m / z = 263.2 [M+H] + .

[0192]

[0193] Manufacturing Example 3-2: N-(9-fluorenylmethyloxycarbonyl)-N-(tert-butoxycarbonyl)-2-(2-hydroxyethoxy)ethylglycylamine

[0194]

[0195] N-(2-(2-hydroxyethoxy)ethyl)-N-(tert-butoxycarbonyl)glycylamine (1.00 g, 1.49 mmol, 39% purity, 1.00 eq.) and TEA (301 mg, 2.97 mmol, 414 μL, 2.00 eq.) were dissolved in DCM (8.0 mL), and Fmoc-Cl (385 mg, 1.49 mmol, 1.00 eq.) was added. The mixture was stirred at 25 °C for 1 h. The completion of the reaction was confirmed by LC-MS. The reaction mixture was diluted with aqueous NaHCO₃ solution and extracted with DCM (50 mL × 3). The organic layer was combined, washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue.

[0196] The residue was purified by prep-HPLC (TFA conditions) to give N-(9-fluorenylmethyloxycarbonyl)-N-(tert-butoxycarbonyl)-2-(2-hydroxyethoxy)ethylglycylamine (191 mg, 394 μmol, 26.5% yield) as a colorless oily substance.

[0197] LC-MS analysis results: Rt = 0.50–0.52 min, m / z = 507.3 [M+Na] + .

[0198]

[0199] Manufacturing Example 3-3: N-(9-fluorenylmethyloxycarbonyl)-2-(2-hydroxyethoxy)ethylglycylamine

[0200]

[0201] N-(9-Fluorenylmethyloxycarbonyl)-N-(tert-butoxycarbonyl)-2-(2-hydroxyethoxy)ethylglycylamine (191 mg, 394 μmol, 1.00 eq.) was dissolved in dioxane (1.0 mL), and then HCl / dioxane (2 M, 1.0 mL) was added. The mixture was stirred at 25°C for 2.5 h.

[0202] LC-MS results showed that the starting material was completely consumed, and a major peak with the target mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain N-(9-fluorenylmethyloxycarbonyl)-2-(2-hydroxyethoxy)ethylglycylamine (203 mg, crude product) as a yellow oil.

[0203] LC-MS analysis results: Rt = 0.36 min, m / z = 385.2 [M+H] + .

[0204]

[0205] Manufacturing Example 3-4: N-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl)-maleimido-PEG₄-valine-citrulline-valine-N-(9-fluorenylmethyloxycarbonyl)-2-(2-hydroxyethoxy)ethylglycylamide

[0206]

[0207] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl 4-nitrophenyl carbonate (20.0 mg, 22.9 μmol, 1.00 eq) and N-(9-fluorenylmethyloxycarbonyl)-2-(2-hydroxyethoxy)ethylglycylamine (14.4 mg, 34.4 μmol, 1.50 eq) were dissolved in DMF (0.50 mL), and then HOBt (3.10 mg, 22.9 μmol, 1.00 eq) and DIEA (2.96 mg, 22.9 μmol, 4.00 μL, 1.00 eq) was added. The reaction mixture was stirred at 25°C for 2 hours. The reaction mixture was purified by prep-HPLC under TFA conditions to finally obtain 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-hydroxyethoxy)ethyl)amino)ethyl)(methyl)carbamate (18.5 mg, 16.5 μmol, 72.1% yield) as a yellow oil.

[0208] LC-MS analysis results: Rt = 0.45–0.46 min, m / z = 1139.8 [M+Na] + .

[0209]

[0210] Manufacturing Example 3-5: 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-((2-(2-hydroxyethoxy)ethyl)amino)ethyl)(methyl)carbamate

[0211]

[0212] N-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl)-maleimido-PEG₄-valine-citrulline-valine-N-(9-fluorenylmethyloxycarbonyl)-2-(2-hydroxyethoxy)ethylglycylamide (18.5 mg, 16.6 μmol, 1.00 eq) was dissolved in DMF (400 μL), and TEA (72.7 mg, 718 μmol, 100 μL, 43.3 eq.) was added. The mixture was stirred at 25°C for 5 h. TFA was added to the reaction mixture to adjust the pH to less than 7, and isopropyl ether was added to cause precipitation. The precipitate was dried under reduced pressure to obtain a crude product, and finally 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-((2-(2-hydroxyethoxy)ethyl)amino)ethyl)(methyl)carbamate was obtained as a yellow oil.

[0213] LC-MS analysis results: Rt = 0.31 min, m / z = 895.6 [M+H] + .

[0214]

[0215] Manufacturing Example 3-6: (R)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl (2-((((4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate

[0216]

[0217] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-((2-(2-hydroxyethoxy)ethyl)amino)ethyl)(methyl)carbamate (14.8 mg, 16.5 μmol, 1.00 eq, theoretical) and 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl 4-nitrophenyl carbonate (13.1 mg, 16.5 μmol, 1.00 eq) was dissolved in DMF (0.50 mL), and then HOBt (2.46 mg, 18.2 μmol, 1.10 eq) and DIEA (4.27 mg, 33.1 μmol, 5.76 μL, 2.00 eq) were added. The mixture was stirred at 25 °C for 4 h. The reaction mixture was purified by prep-HPLC under TFA condition, and finally (R)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl (2-((((4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (7.00 mg, 4.52 μmol, 27.3% yield) was obtained as a gray-white solid.

[0218] LC-MS analysis results: Rt = 0.40–0.41 min, m / z = 1570.9 [M+Na] + .

[0219]

[0220] Manufacturing Example 4: Manufacturing of Linker-Payload AM10801

[0221] Manufacturing Example 4-1: N-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-N-(tert-butoxycarbonyl)oxetan-3-ylmethylamine

[0222]

[0223] 2-(2-(2-aminoethoxy)ethoxy)ethanol (2.00 g, 10.35 mmol, 1.00 eq) and N-(tert-butoxycarbonyl)-2-aminooxetane-3-carboxaldehyde (1.79 g, 10.35 mmol, 1.00 eq) were dissolved in MeOH (20.0 mL) and stirred at 25°C for 4 h. NaBH₃CN (1.30 g, 20.70 mmol, 2.00 eq) was added at 0°C. The mixture was stirred at room temperature for 16 h. Afterwards, the reaction mixture was concentrated under reduced pressure to obtain N-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-N-(tert-butoxycarbonyl)oxetan-3-ylmethylamine (5.09 g, crude product) in the form of a yellow oil.

[0224] LC-MS analysis results: Rt = 0.26 min, m / z = 351.2 [M+H] + .

[0225]

[0226] Manufacturing Example 4-2: N-(9-fluorenylmethoxycarbonyl)-N-(tert-butyloxycarbonyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethylmethylamine

[0227]

[0228] N-(tert-butyloxycarbonyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethylmethylamine (2.00 g, 1.02 mmol, 17.8% purity, 1.00 eq) was dissolved in DCM (20.0 mL), and Fmoc-Cl (525 mg, 2.03 mmol, 2.00 eq) and TEA (205 mg, 2.03 mmol, 282 μL, 2.00 eq) were added. The mixture was stirred at 25 °C for 2.5 h. After completion of the reaction, the mixture was diluted with aqueous NaHCO₃ solution and extracted with DCM (100 mL × 3). The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (TFA conditions) to give N-(9-fluorenylmethoxycarbonyl)-N-(tert-butyloxycarbonyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethylmethylamine (292 mg, 510 μmol, 50.1% yield) as a yellow oil.

[0229] LC-MS analysis results: Rt = 0.52 min, m / z = 595.3 [M+Na] + .

[0230]

[0231] Manufacturing Example 4-3: N-(9-fluorenylmethoxycarbonyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethylmethylamine

[0232]

[0233] N-(9-Fluorenylmethoxycarbonyl)-N-(tert-butyloxycarbonyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethylmethylamine (292 mg, 510 μmol, 1.00 eq) was dissolved in dioxane (1.0 mL), and then HCl / dioxane (2.0 mL) was added. The mixture was stirred at 25°C for 1 h. The reaction mixture was filtered and concentrated under reduced pressure to obtain N-(9-Fluorenylmethoxycarbonyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethylmethylamine (289 mg, crude) as a yellow oil.

[0234] LC-MS analysis results: Rt = 0.38 min, m / z = 473.3 [M+H] + .

[0235]

[0236] Manufacturing Example 4-4: 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (3-(((9H-fluoren-9-yl)methoxy)carbonyl)-14-hydroxy-6,9,12-trioxa-3-azatetradecyl)(methyl)carbamate

[0237]

[0238] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl 4-nitrophenyl carbonate (20.0 mg, 22.9 μmol, 1.00 eq) and N-(9-fluorenylmethoxycarbonyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethylmethylamine (18.3 mg, 34.4 μmol, 89% purity, 1.50 eq) were dissolved in DMF (0.50 mL), and HOBt (3.10 mg, 22.9 μmol, 1.00 eq) and DIEA (2.96 mg, 22.9 μmol, 4.00 μL, 1.00 eq) were added. The mixture was stirred at 25°C for 2 h. The reaction mixture was purified by prep-HPLC under TFA conditions to finally obtain 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (3-(((9H-fluoren-9-yl)methoxy)carbonyl)-14-hydroxy-6,9,12-trioxa-3-azatetradecyl)(methyl)carbamate (14.6 mg, 12.1 μmol, 52.8% yield) as a yellow oil.

[0239] LC-MS analysis results: Rt = 0.45–0.46 min, m / z = 603.6 [(M+H) / 2] + .

[0240]

[0241] Manufacturing Example 4-5: 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (14-hydroxy-6,9,12-trioxa-3-azatetradecyl)(methyl)carbamate

[0242]

[0243] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (3-(((9H-fluoren-9-yl)methoxy)carbonyl)-14-hydroxy-6,9,12-trioxa-3-azatetradecyl)(methyl)carbamate (14.6 mg, 12.1 μmol, 1.00 eq) was dissolved in DMF (0.40 mL), and then TEA (72.7 mg, 718 μmol, 100 μL, 59.3 eq) was added. The mixture was stirred at 25°C for 5 hours. TFA was added to the reaction mixture to adjust the pH to less than 7, and isopropyl ether was added to cause precipitation. The precipitate was filtered and dried to obtain 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (14-hydroxy-6,9,12-trioxa-3-azatetradecyl)(methyl)carbamate as a yellow oil.

[0244] LC-MS analysis results: Rt = 0.32 min, m / z = 983.7 [M+H] + .

[0245]

[0246] Manufacturing Example 4-6: (R)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl (2-((((4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate

[0247]

[0248] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (14-hydroxy-6,9,12-trioxa-3-azatetradecyl)(methyl)carbamate (11.9 mg, 12.1 μmol, 1.00 eq, theoretical) and (S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 2-(4-Nitrophenyl)acetate (9.59 mg, 12.1 μmol, 1.00 eq) was dissolved in DMF (0.50 mL), and then HOBt (1.80 mg, 13.3 μmol, 1.10 eq) and DIEA (3.13 mg, 24.2 μmol, 4.22 μL, 2.00 eq) were added. The mixture was stirred at 25°C for 1 h. The reaction mixture was purified by prep-HPLC under TFA condition to finally give (R)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl (2-((((4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (5.00 mg, 3.06 μmol, 25.2% yield) was obtained as a gray-white solid.

[0249] LC-MS analysis results: Rt = 0.41 min, m / z = 1659.4 [M+H₂O] + .

[0250]

[0251] Manufacturing Example 5: Manufacturing of Linker-Payload AM10901

[0252] Manufacturing Example 5-1: 1-(4-hydroxybenzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose

[0253]

[0254] 2,3,4,6-Tetra-O-acetyl-α-D-galactopyranosyl bromide (2.00 g, 16.4 mmol, 1.00 eq) and 4-hydroxybenzaldehyde (5.85 g, 14.7 mmol, 0.90 eq) were dissolved in acetonitrile (20.0 mL), and silver oxide (Ag₂O, 9.49 g, 40.9 mmol, 2.50 eq) was added. The mixture was degassed three times and stirred at 25 °C for 3 h under N₂ atmosphere. LC-MS analysis showed that 4-hydroxybenzaldehyde was completely consumed, and a major peak with the target mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue, which was diluted with water and extracted with EtOAc (10 mL × 2). The organic layer was separated, washed with aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. After filtration and concentration, the residue was purified by silica gel column chromatography (SiO₂, petroleum ether / ethyl acetate) to obtain 1-(4-hydroxybenzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose (3.36 g, 7.66 mmol, 46.8% yield) as a yellow solid.

[0255] LC-MS analysis results: Rt = 0.42 min, m / z = 461.0 [M+Na] + .

[0256]

[0257] Manufacturing Example 5-2: 1-(4-Formylbenzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose

[0258]

[0259] 1-(4-Hydroxybenzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose (3.36 g, 7.66 mmol, 1 eq.) was placed in a round-bottomed flask and dissolved in DCM (20 mL), and the mixture was cooled to 0°C. A solution of NaBH₄ (319 mg, 8.43 mmol, 1.1 eq.) dissolved in MeOH (20 mL) was added to the reaction mixture using a syringe. The reaction mixture was stirred at 0°C for 10 min. TLC results confirmed that 1-(4-hydroxybenzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose was completely consumed and a new spot was formed. The reaction proceeded cleanly according to TLC. The mixture was diluted with saturated ammonium chloride aqueous solution (100 mL), and the aqueous layer was sequentially extracted with DCM (125 mL * 3). The organic layer was separated, washed with NaCl (200 mL), and dried over Na₂SO₄. After filtration, the residue was concentrated under reduced pressure to obtain the residue, which was purified by prep-HPLC (TFA conditions). Finally, 1-(4-formylbenzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose (2.17 g, 4.93 mmol, 64.2% yield) was obtained as a white solid.

[0260] LC-MS analysis results: Rt = 0.39 min, m / z = 463.0 [M+Na] + .

[0261]

[0262] Manufacturing Example 5-3: 1-(4-(hydroxymethyl)benzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose

[0263]

[0264] 1-(4-Formylbenzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose (3.36 g, 7.66 mmol, 1.00 eq) was placed in a round-bottomed flask and dissolved in DCM (20.0 mL), and the mixture was cooled to 0°C. NaBH₄ (319 mg, 8.43 mmol, 1.10 eq) was dissolved in MeOH (20.0 mL) and added to the reaction mixture using a syringe. The mixture was stirred at 0°C for 10 min. TLC confirmed that the starting material was completely consumed and a new spot formed, indicating that the reaction was completed cleanly. The mixture was diluted with saturated aqueous ammonium chloride solution (100 mL), and the aqueous layer was extracted with DCM (125 mL × 3). The organic layer was separated, washed with NaCl (200 mL), and dried over anhydrous Na₂SO₄. The residue obtained after filtration and concentration under reduced pressure was purified by prep-HPLC (TFA conditions) to obtain 1-(4-(hydroxymethyl)benzyl)-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose (2.17 g, 4.93 mmol, 64.2% yield) as a white solid.

[0265] LC-MS analysis results: Rt = 0.39 min, m / z = 463.0 [M+Na] + .

[0266]

[0267] Manufacturing Example 5-4: 1-(4-(2-((4-nitrophenyl)carbonyl)oxy)benzyl)-D-galactopyranoic acid

[0268]

[0269] 1-(4-(Hydroxymethyl)benzyl)-D-galactopyranoic acid (200 mg, 666 μmol, 1.00 eq) and 4-nitrophenyl formic acid (PNP, 304 mg, 999 μmol, 1.50 eq) were dissolved in DMF (2.0 mL), and DIEA (172 mg, 1.33 mmol, 232 μL, 2.00 eq) was added. The mixture was stirred at 25 °C for 2.5 h. LC-MS analysis showed that the starting material was completely consumed and the main peak of the target mass was detected. Isopropyl ether was added to the reaction mixture to precipitate, and the residue was purified by prep-HPLC (TFA conditions) to obtain 1-(4-(2-((4-nitrophenyl)carbonyl)oxy)benzyl)-D-galactopyranoic acid (49.5 mg, 106 μmol, 15.9% yield) as a white solid.

[0270] LC-MS analysis results: Rt = 0.36 min, m / z = 488.0 [M+Na] + .

[0271]

[0272] Manufacturing Example 5-5: Tert-butyl (17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15-tetraoxa-3-azaheptadecyl)(methyl)carbamate

[0273]

[0274] 1-(14-Amino-3,6,9,12-tetraoxatetradecyl)-1H-pyrrole-2,5-dione (500 mg, 1.58 mmol, 1 eq.) and tert-butyl methyl(2-oxoethyl)carbamate (274 mg, 1.58 mmol, 1 eq.) were dissolved in MeOH (5 mL) and stirred at 25 °C for 4 h. NaBH₃CN (149 mg, 2.37 mmol, 1.5 eq.) was added at 0 °C. The mixture was stirred at room temperature for 6 h. LC-MS analysis showed that 1-(14-amino-3,6,9,12-tetraoxatetradecyl)-1H-pyrrole-2,5-dione was completely consumed, and a major peak with the desired mass was detected. The reaction mixture was extracted with NH₄Cl and DCM, and the organic layer was concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (TFA conditions) to finally obtain tert-butyl (17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15-tetraoxa-3-azaheptadecyl)(methyl)carbamate (108 mg, 228 μmol, 14.4% yield) as a yellow oil.

[0275] LC-MS analysis results: Rt = 0.31 min, m / z = 474.2 [M+H] + .

[0276]

[0277] Manufacturing Example 5-6: (9H-fluoren-9-yl)methyl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxatetradecyl)carbamate

[0278]

[0279] Tert-butyl (17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15-tetraoxa-3-azaheptadecyl)(methyl)carbamate (108 mg, 228 μmol, 1 eq.) was dissolved in DCM (2 mL), and then TEA (46.2 mg, 456 μmol, 63.5 μL, 2 eq.) and Fmoc-Cl (88.5 mg, 342 μmol, 1.5 eq.) were added. The mixture was stirred at 25 °C for 2 h. LC-MS analysis showed that tert-butyl (17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15-tetraoxa-3-azaheptadecyl)(methyl)carbamate was completely consumed, and a major peak with the desired mass was detected. The reaction mixture was diluted with DCM and extracted with aqueous NaHCO₃ solution. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (TFA conditions) to finally obtain (9H-fluoren-9-yl)methyl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxatetradecyl)carbamate (58.1 mg, 83.5 μmol, 36.6% yield) as a white oil.

[0280] LC-MS analysis results: Rt = 0.54 min, m / z = 696.3 [M+H] + .

[0281]

[0282] Manufacturing Example 5-7: (9H-fluoren-9-yl)methyl (14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxatetradecyl)(2-(methylamino)ethyl)carbamate

[0283]

[0284] (9H-Fluoren-9-yl)methyl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxatetradecyl)carbamate (43 mg, 61.8 μmol, 1 eq.) was dissolved in DCM (400 μL), and then TFA (614 mg, 5.38 mmol, 400 μL, 87.1 eq.) was added. The mixture was stirred at 25°C for 1 h. LC-MS analysis results showed that (9H-fluoren-9-yl)methyl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxatetradecyl)carbamate was completely consumed, and the main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to obtain the residue, which was used as is without purification to obtain crude (9H-fluoren-9-yl)methyl (14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxatetradecyl)(2-(methylamino)ethyl)carbamate as a white oil.

[0285] LC-MS analysis results: Rt = 0.39 min, m / z = 596.1 [M+H] + .

[0286]

[0287] Manufacturing Example 5-8: (2S,3S,4S,5R,6S)-6-(4-(7-(((9H-fluoren-9-yl)methoxy)carbonyl)-21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0288]

[0289] (9H-Fluoren-9-yl)methyl (14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxatetradecyl)(2-(methylamino)ethyl)carbamate (28 mg, 60.2 μmol, 1 eq.) and 1-(4-(2-((4-nitrophenyl)carbonyl)oxy)benzyl)-D-galactopyranoic acid (35.8 mg, 60.2 μmol, theoretical, 1 eq.) were dissolved in DMF (1 mL), and then HOBt (8.13 mg, 60.2 μmol, 1 eq.) and DIEA (7.78 mg, 60.2 μmol, 10.5 μL, 1 eq.) were added. The mixture was stirred at 25 °C for 2 h. LC-MS analysis results showed that 1-(4-(2-((4-nitrophenyl)carbonyl)oxy)benzyl)-D-galactopyranoic acid was completely consumed, and a major peak with the desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA conditions) to finally obtain (2S,3S,4S,5R,6S)-6-(4-(7-(((9H-fluoren-9-yl)methoxy)carbonyl)-21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (36.4 mg, 39.5 μmol, 65.6% yield) as a white solid.

[0290] LC-MS analysis results: Rt = 0.44 min, m / z = 944.1 [M+Na] + .

[0291]

[0292] Manufacturing Example 5-9: (2S,3S,4S,5R,6S)-6-(4-(21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0293]

[0294] (2S,3S,4S,5R,6S)-6-(4-(7-(((9H-fluoren-9-yl)methoxy)carbonyl)-21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (36.4 mg, 39.5 μmol, 1 eq.) was dissolved in DMF (0.6 mL), and then TEA (145 mg, 1.44 mmol, 200 μL, 36.4 eq.) was added. The mixture was stirred at 25 °C for 4 h. LC-MS analysis showed that (2S,3S,4S,5R,6S)-6-(4-(7-(((9H-fluoren-9-yl)methoxy)carbonyl)-21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid was completely consumed, and the main peak with the desired mass was detected. Subsequently, TFA was added until the pH of the reaction mixture became 7 or lower, and then the solid was precipitated using isopropyl ether. Finally, crude (2S,3S,4S,5R,6S)-6-(4-(21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid was obtained as a white solid.

[0295] LC-MS analysis results: Rt = 0.25 min, m / z = 700.0 [M+H] + .

[0296]

[0297] Manufacturing Example 5-10: (2S,3S,4S,5R,6S)-6-(4-(7-((((S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophen-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)carbonyl)-21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0298]

[0299] (2S,3S,4S,5R,6S)-6-(4-(21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (27.6 mg, 39.4 μmol, 1 eq.) and (S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 2-(4-nitrophenyl)acetate (31.2 mg, After dissolving HOBt (39.4 μmol, 1 eq.) in DMF (0.5 mL), HOBt (5.86 mg, 43.4 μmol, 1.1 eq.) and DIEA (30.6 mg, 237 μmol, 41.2 μL, 6 eq.) were added. The mixture was stirred at 25°C for 1 h. After completion of the reaction, the reaction mixture was purified by prep-HPLC (FA condition) to finally give (2S,3S,4S,5R,6S)-6-(4-(7-((((S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophen-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)carbonyl)-21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-3-oxo-2,10,13,16,19-pentaoxa-4,7-diazahenicosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (7.5 mg, 5.54 μmol, 14.1% yield) was obtained as a white solid.

[0300] LC-MS analysis results: Rt = 0.40 min, m / z = 1353.0 [M+H] + .

[0301]

[0302] Manufacturing Example 6: Manufacturing of Linker-Payload AM11001

[0303] Manufacturing Example 6-1: (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethylsilyl triacetate

[0304]

[0305] (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethylsiloxane triacetate (2.0 g, 4.12 mmol, 1 eq.) and PNP-Cl (1.66 g, 8.24 mmol, 2 eq.) were dissolved in DCM (20 mL), and pyridine (978 mg, 12.36 mmol, 998 μL, 3 eq.) was added at 0 °C. The mixture was stirred at 25 °C for 2 h. LC-MS analysis detected a major peak with the desired mass. The reaction mixture was filtered to obtain the compound, and finally (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethyl triacetate (2.68 g, crude) was obtained as a white solid.

[0306] LC-MS analysis results: Rt = 0.48 min, m / z = 673.2 [M+Na] + .

[0307]

[0308] Manufacturing Example 6-2: (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethylsilyl triacetate

[0309]

[0310] (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethylsiloxane)triacetate (2.58 g, 3.97 mmol, 1 eq.), tert-butyl methyl(2-(methylamino)ethyl)carbamate (747 mg, 3.97 mmol, 1 eq.), and HOBt (536 mg, 3.97 mmol, 1 eq.) were dissolved in DMF (20 mL), and then DIEA (513 mg, 3.97 mmol, 691 μL, 1 eq.) was added. The mixture was stirred at 25 °C for 1.5 h. After concentrating the reaction mixture under reduced pressure, the residue was purified by prep-HPLC (TFA conditions) to finally obtain (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-diosa-4,7-diazaundecyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethyl triacetate (2.39 g, 3.41 mmol, 86.1% yield) as a white solid.

[0311] LC-MS analysis results: Rt = 0.47 min, m / z = 722.3 [M+Na] + .

[0312]

[0313] Manufacturing Example 6-3: (2S,3S,4S,5R,6S)-3,4,5-Trihydroxy-6-(2-nitro-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecyl)phenoxy)tetrahydro-2H-pyran-2-carboxylic acid

[0314]

[0315] (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-nitro-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-diosa-4,7-diazaundecyl)phenoxy)tetrahydro-2H-pyran-3,4,5-trimethyl triacetate (700 mg, 1.00 mmol, 1 eq.) was dissolved in THF (4 mL), and then LiOH·H₂O (1 M, 10 mL, 10 eq.) was added. The mixture was stirred at 0°C for 5 minutes. After concentrating the reaction mixture under reduced pressure, the residue was purified by prep-HPLC (TFA conditions) to finally obtain (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(2-nitro-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-diosa-4,7-diazaoundecyl)phenoxy)tetrahydro-2H-pyran-2-carboxylic acid (396 mg, 708 μmol, 70.7% yield) as a yellow solid.

[0316] LC-MS analysis results: Rt = 0.34 min, m / z = 582.2 [M+Na] + .

[0317]

[0318] Manufacturing Example 6-4: (2S,3S,4S,5R,6S)-6-(2-amino-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0319]

[0320] A 100 mL round-bottom flask was purged with argon three times, and Pd / C (dry, 35 mg) was carefully added. Then, MeOH (1 mL) was added to completely wet the Pd / C, and a solution of (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(2-nitro-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecyl)phenoxy)tetrahydro-2H-pyran-2-carboxylic acid (366 mg, 655 μmol, 1 eq.) in MeOH (3 mL) was slowly added under an argon atmosphere. The resulting mixture was degassed and purged with hydrogen three times. The reaction mixture was stirred at 25 °C for 1.5 h under a hydrogen atmosphere. The reaction progress was monitored by TLC (DCM:MeOH = 1:1, Rf = 0.5). The reaction mixture was carefully filtered under N₂ atmosphere, and the organic layer was concentrated under reduced pressure to obtain a crude product. Finally, (2S,3S,4S,5R,6S)-6-(2-amino-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaoundecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (270 mg, 510 μmol, 78.0% yield) was obtained as a yellow solid.

[0321] LC-MS analysis results: Rt = 0.28 min, m / z = 530.2 [M+H] + .

[0322]

[0323] Manufacturing Example 6-5: (2S,3S,4S,5R,6S)-6-(2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido)-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0324]

[0325] 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-acid (72.4 mg, 210 μmol, 1 eq.) was dissolved in DMF (1 mL), and HATU (87.7 mg, 231 μmol, 1.1 eq.) and DIEA (73.0 μL, 630 μmol, 3 eq.) were added and stirred at 25°C for 5 minutes. To this was added (2S,3S,4S,5R,6S)-6-(2-amino-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-diosa-4,7-diazaundecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (74 mg, 210 μmol, 1 eq.) and stirred at 25°C for an additional hour. After concentrating the reaction mixture under reduced pressure, the residue was purified by prep-HPLC (TFA condition) to finally obtain (2S,3S,4S,5R,6S)-6-(2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido)-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaoundecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (23.2 mg, 27.1 μmol, 19.5% yield) as a colorless oil.

[0326] LC-MS analysis results: Rt = 0.37 min, m / z = 879.3 [M+Na] + .

[0327]

[0328] Manufacturing Example 6-6: (2S,3S,4S,5R,6S)-6-(2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido)-4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0329]

[0330] (2S,3S,4S,5R,6S)-6-(2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido)-4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (31.7 mg, 37.0 μmol, 1 eq.) was dissolved in DCM (0.4 mL), and then TFA (100 μL) was added. The mixture was stirred at 0 °C for 25 min. After completion of the reaction, the reaction mixture was added dropwise to isopropyl ether to precipitate the crude product. The residue was purified by prep-HPLC (TFA conditions) to finally obtain (2S,3S,4S,5R,6S)-6-(2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido)-4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (19 mg, 25.1 μmol, 67.9% yield) as an oil.

[0331] LC-MS analysis results: Rt = 0.25 min, m / z = 757.3 [M+H] + .

[0332]

[0333] Manufacturing Example 6-7: (2S,3S,4S,5R,6S)-6-(4-((((2-((((S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophen-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0334]

[0335] (2S,3S,4S,5R,6S)-6-(2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido)-4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (4 mg, 5.29 μmol, 1 eq.) and (S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 2-(4-nitrophenyl)acetate (5.19 mg, 6.55 μmol, 1.24 eq.) was dissolved in DMF (0.3 mL), and DIEA (1.37 mg, 10.6 μmol, 1.84 μL, 2 eq.) was added. The mixture was stirred at 25°C for 2.5 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by prep-HPLC (TFA conditions), and finally (2S,3S,4S,5R,6S)-6-(4-((((2-((((S)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophen-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl)-2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (5.0 mg, 3.27 μmol, 61.9% yield, TFA salt) was obtained in the form of a white solid.

[0336] LC-MS analysis results: Rt = 0.40 min, m / z = 1410.5 [M+H] + .

[0337]

[0338] Manufacturing Example 7: Manufacturing of linker-payload AM11101

[0339] Manufacturing Example 7-1: 2-(Trimethylsilyl)ethyl (2-((tert-butoxycarbonyl)amino)ethyl)(methyl)carbamate

[0340]

[0341] 2,5-Dioxopyrrolidin-1-yl (2-(trimethylsilyl)ethyl)carbonate (1.79 g, 6.89 mmol, 1.2 eq.), tert-butyl (2-(methylamino)ethyl)carbamate (1 g, 5.74 mmol, 1 eq.), and K₂CO₃ (793 mg, 5.74 mmol, 1 eq.) were added to DCM (10 mL) and H₂O (10 mL), and the mixture was degassed three times with N₂ and stirred at 25 °C for 3 h under N₂ atmosphere. The reaction was monitored by LC-MS (Rt = 0.447 min). The mixture was washed with 1 N HCl, and the organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to obtain the crude product. Finally, 2-(trimethylsilyl)ethyl (2-((tert-butoxycarbonyl)amino)ethyl)(methyl)carbamate (1.98 g, crude) was obtained as a colorless oil.

[0342] 1H NMR (400 MHz, CDCl₃): δ ppm: 4.09 - 4.18 (m, 2 H), 3.18 - 3.38 (m, 4 H), 2.88 (s, 3 H), 1.40 (s, 9 H), 0.93 - 1.01 (m, 2 H), -0.03 - 0.02 (m, 9 H).

[0343] LC-MS analysis results: Rt = 0.447 min, m / z = 341.1 [M+Na] + .

[0344]

[0345] Manufacturing Example 7-2: 2-(Trimethylsilyl)ethyl (2-((tert-butoxycarbonyl)amino)ethyl)(methyl)carbamate

[0346]

[0347] NaH (352 mg, 8.79 mmol, 60% purity, 2 eq.) was dissolved in DMF (11 mL), and 2-(trimethylsilyl)ethyl (2-((tert-butoxycarbonyl)amino)ethyl)(methyl)carbamate (1.4 g, 4.40 mmol, 1 eq.) was dissolved in DMF (3 mL), and the mixture was stirred at 5 °C under N₂ atmosphere. After 0.5 h, 3-bromoprop-1-yne (1.31 g, 11.0 mmol, 947 μL, 2.5 eq.) was added. The mixture was heated to 25 °C and stirred for 4 h. The reaction was monitored by TLC (PE: EtOAc = 5:1, Rf = 0.4). After the reaction mixture was cooled to 0-5℃, it was slowly added to a saturated NH₄Cl solution, which was stirred while maintaining the temperature below 15℃ under a N₂ atmosphere. The aqueous solution was then extracted with EA. The organic layers were combined, dried over Na₂SO₄, and concentrated to obtain a residue. The residue was purified by silica gel column chromatography (SiO₂, petroleum ether: ethyl acetate = 100:1 to 80:1) to obtain 2-(trimethylsilyl)ethyl (2-((tert-butoxycarbonyl)(prop-2-yn-1-yl)amino)ethyl)(methyl)carbamate (1.36 g, 3.81 mmol, 86.8% yield) as a yellow oil.

[0348] 1H NMR (400 MHz, CDCl₃): δ ppm: 4.00 - 4.27 (m, 4 H), 3.43 (br s, 4 H), 2.84 - 2.97 (m, 3 H), 2.14 - 2.26 (m, 1 H), 1.45 (s, 9 H), 0.92 - 1.04 (m, 2 H), -0.03 - 0.12 (m, 9 H).

[0349]

[0350] Manufacturing Example 7-3: Tert-butyl (2-(methylamino)ethyl)(prop-2-yn-1-yl)carbamate

[0351]

[0352] 2-(Trimethylsilyl)ethyl (2-((tert-butoxycarbonyl)(prop-2-yn-1-yl)amino)ethyl)(methyl)carbamate (1.36 g, 3.81 mmol, 1 eq.) was dissolved in THF (13 mL), and TBAF (1 M, 11.83 mL, 3.1 eq.) was added. The mixture was degassed three times and then stirred under N₂ atmosphere at 25 °C for 3 h. TLC (PE: EA = 5:1, PMA) showed that 2-(trimethylsilyl)ethyl (2-((tert-butoxycarbonyl)(prop-2-yn-1-yl)amino)ethyl)(methyl)carbamate was completely consumed and a new spot was formed. The reaction mixture was concentrated under reduced pressure, and the residue was purified by prep-HPLC (TFA conditions, ELSD) to give tert-butyl (2-(methylamino)ethyl)(prop-2-yn-1-yl)carbamate (800 mg, 3.77 mmol, 98.8% yield) as a yellow oil.

[0353] 1H NMR (400 MHz, CDCl₃): δ 4.05 (br s, 2H), 3.66 (br s, 2H), 3.15-3.29 (m, 2H), 2.74 (br s, 3H), 2.27 (s, 1H), 1.47 (s, 9H).

[0354] LC-MS analysis results: Rt = 0.216 min, m / z = 213.1 [M+H] +

[0355]

[0356] Manufacturing Example 7-4: Tert-butyl (2-(methylamino)ethyl)(prop-2-yn-1-yl)carbamate

[0357]

[0358] Tert-butyl (2-(methylamino)ethyl)(prop-2-yn-1-yl)carbamate (1.36 g, 3.81 mmol, 1 eq.) was dissolved in THF (13 mL), and TBAF (1 M, 11.83 mL, 3.1 eq.) was added. The reaction mixture was degassed, purged with N₂ three times, and stirred at 25 °C for 3 h under a N₂ atmosphere. TLC (PE:EA = 5:1, PMA) confirmed that tert-butyl (2-(methylamino)ethyl)(prop-2-yn-1-yl)carbamate was completely consumed and a new reaction site was formed. After concentrating the reaction mixture under reduced pressure, the residue was purified by prep-HPLC (TFA conditions, ELSD detection) to finally obtain tert-butyl (2-(methylamino)ethyl)(prop-2-yn-1-yl)carbamate (800 mg, 3.77 mmol, 98.8% yield) as a yellow oil.

[0359] 1H NMR (400 MHz, CDCl₃): δ 4.05 (br s, 2H), 3.66 (br s, 2H), 3.15-3.29 (m, 2H), 2.74 (br s, 3H), 2.27 (s, 1H), 1.47 (s, 9H).

[0360] LC-MS analysis results: Rt = 0.22 min, m / z = 213.1 [M+H] + .

[0361]

[0362] Manufacturing Example 7-5: 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-((tert-butoxycarbonyl)(prop-2-yn-1-yl)amino)ethyl)(methyl)carbamate

[0363]

[0364] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl 4-nitrophenyl carbonate (50 mg, 57.4 μmol, 1 eq.) was dissolved in DMF (0.5 mL), and then tert-butyl (2-(methylamino)ethyl)(prop-2-yn-1-yl)carbamate (18.3 mg, 86.0 μmol, 1.5 eq.), HOBt (7.75 mg, 57.4 μmol, 1 eq.), TEA (14.5 mg, 143 μmol, 20.0 μL, 2.5 eq.) was added dropwise to a DMF solution containing 4-nitrophenyl carbonate. The mixture was stirred at 25°C for 1 h. LC-MS analysis showed that 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl 4-nitrophenyl carbonate was completely consumed, and the main peak with the desired mass was detected. The reaction mixture was added dropwise into cooled isopropyl ether to precipitate the residue, and finally 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-((tert-butoxycarbonyl)(prop-2-yn-1-yl)amino)ethyl)(methyl)carbamate (54 mg, crude) was obtained as a yellow oil.

[0365] LC-MS analysis results: Rt = 0.42 min, m / z = 945.4 [M+H] + .

[0366]

[0367] Manufacturing Example 7-6: 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl methyl(2-(prop-2-yn-1-ylamino)ethyl)carbamate

[0368]

[0369] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-((tert-butoxycarbonyl)(prop-2-yn-1-yl)amino)ethyl)(methyl)carbamate (54 mg, 57.1 μmol, 1 eq.) was dissolved in DCM (0.4 mL), and 20% TFA was added. The mixture was stirred at 0 °C for 4 h. After completion of the reaction, the residue was precipitated by adding dropwise into cooled isopropyl ether, and finally 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl methyl(2-(prop-2-yn-1-ylamino)ethyl)carbamate (42 mg, 49.7 μmol, 87.0% yield) was obtained as a yellow oil.

[0370] LC-MS analysis results: Rt = 0.30 min, m / z = 867.3 [M+Na] + .

[0371]

[0372] Manufacturing Example 7-7: 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-(((1-(tetracosaoxatricheptacontan-73-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethyl)(methyl)carbamate

[0373]

[0374] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl methyl(2-(prop-2-yn-1-ylamino)ethyl)carbamate (42 mg, 49.7 μmol, 1 eq.), 73-azido-tetracosaoxatriheptacontane (55.4 mg, 49.7 μmol, 1 eq.), VcNa (19.7 mg, 99.4 μmol, 2 eq.), THPTA (21.6 mg, 49.7 μmol, 1 eq.) After dissolving in a mixed solvent of t-BuOH (0.25 mL) / H₂O (0.25 mL), CuSO₄ (0.4 M, 124 μL, 1 eq.) was added. The reaction mixture was degassed and purged three times with N₂, and stirred at 25°C for 1 h under a N₂ atmosphere. After completion of the reaction, the residue was purified by prep-HPLC (TFA conditions) to finally obtain 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-(((1-(tetracosaoxatricheptacontan-73-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethyl)(methyl)carbamate (30 mg, 15.3 μmol, 30.8% yield) in the form of an oil.

[0375] LC-MS analysis results: Rt = 0.37 min, m / z = 980.5 [½M+H] + .

[0376]

[0377] Manufacturing Example 7-8: (R)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl (2-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)((1-(2-(2-methoxyethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate

[0378]

[0379] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazahenicosane-21-amido)benzyl (2-(((1-(tetracosaoxatricheptacontan-73-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)ethyl)(methyl)carbamate (15 mg, 7.66 μmol, 1 eq.) (S)-1-(Chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl 2-(4-nitrophenyl)acetate (6.06 mg, 7.66 μmol, 1 eq.) was dissolved in DMF (0.3 mL), and then HOBt (1.03 mg, 7.66 μmol, 1 eq.) and DIEA (1.98 mg, 15.3 μmol, 2.67 μL, 2 eq.) were added. The mixture was stirred at 25 °C for 1 h. After completion of the reaction, the residue was purified by prep-HPLC (TFA conditions) to finally obtain (R)-1-(chloromethyl)-8-methoxy-3-(5-(3-morpholinopropanamido)benzo[b]selenophene-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl (2-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)((1-(2-(2-methoxyethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate (11.8 mg, 4.18 μmol, 54.6% yield, TFA salt) was obtained as an oil.

[0380] LC-MS analysis results: Rt = 0.43 min, m / z = 1307.2 [½M+H] + .

[0381]

[0382] Example 1: In silico analysis and docking simulation

[0383] Example 1-1: Fab Modeling and Optimization

[0384] To identify cysteine ​​mutation sites optimized for linker-drug conjugation of benzoselenophene-based drugs, in silico docking simulations were performed for specific mutant cysteines against three-dimensional antibody models. To perform the docking simulations, three-dimensional Fab structure data for various antibodies were obtained from the PDB webpage (rcsb.org), and the three-dimensional Fab structure data for the anti-FGFR3 antibody were acquired using a homology modeling technique (Table 1).

[0385]

[0386] The three-dimensional Fab structure of the antibody obtained through this process was subjected to a structural optimization process to make it suitable for performing docking simulations, facilitating subsequent analysis. In addition, the homology modeling for inferring the Fab structure of the anti-FGFR3 antibody adopted a method of suggesting a specific template, and the criteria for selecting the template for modeling were as follows: [1] It must be a human IgG1 Fab structure with a lambda light chain, [2] There must be no impurities in the three-dimensional data, [3] There must be no manipulation of the constituent residues, and [4] It must be a human-derived protein.

[0387] Additionally, Fab structures of other antibodies that satisfy all the same conditions as those for homology modeling were found from the PDB webpage, and the results are shown in Table 2 below. Using the structural data, homology modeling of the antibody was performed, and structural optimization was preliminarily completed to make it suitable for running docking simulations.

[0388]

[0389] Example 1-2: Docking Simulation

[0390] Six different optimized Fab structures were used as receptor structures in docking simulations. The molecule used as the ligand structure was vc-PAB-AMB401. For the docking simulations, the shape of the ligand structure was sampled 100 times for each receptor structure. During sampling, cubic boundaries were set with a spacing of 3-10 angstroms for the receptor structure to ensure that the linker-drug shape could be sampled intensively within the Fab structure.

[0391]

[0392] Example 2: In silico analysis and residue screening

[0393] Example 2-1: Docking simulation results

[0394] The results of selecting residues that are effectively close to the linker-drug from the results of all docking simulations and the characteristics of those residues are shown in Table 3 below.

[0395]

[0396]

[0397] Among the characteristics, solvent accessibility refers to the degree to which a residue can be accessed by a solvent, and CA-CA refers to the distance from the alpha carbon of the residue to the alpha carbon of the nearest endogenous cysteine. The mutation site was primarily selected through the residue characteristics. Among these, residues with a CA-CA value farther than at least 8 angstroms were selected to prevent unwanted cysteine-cysteine ​​bonds. That is, residues such as P151, E152, P153, V154, L163, T164, V167, H168, T169, F170, P171, A172, V173, L174, L182, N203, K205, P206, and K210 are considered suitable.

[0398]

[0399] Example 2-2: Calculating pKa

[0400] The pKa value was calculated as an indicator to predict the stability when the linker-drug is conjugated to the mutant cysteine, and the results are shown in Table 4 below. Based on the research result that the higher the pKa value of the mutant cysteine, the stronger the bond with the linker-drug conjugated at that position tends to be formed (Breanna S Vollmar, et al., Bioconjug Chem. 2017 Oct 18;28(10):2538-2548), the pKa of the mutant cysteines was analyzed to select the position that can show physicochemical superiority during linker-drug conjugation. Accordingly, linker-drug conjugation was attempted including residues whose mutant cysteines were analyzed to have a pKa of 8 or higher, especially E152C, V154C, H168C, T169C, A172C, L174C, and L182C with a pKa of 10 or higher (Table 4).

[0401]

[0402] Example 3: Preparation of engineered cysteine ​​(mutant) antibodies

[0403] When the results of the in silico analysis were synthesized, the docking simulation confirmed a high angstrom value, which predicted excellent binding ability, and the high pKa allowed the selection of sites where excellent physicochemical properties were predicted. It was predicted that a cysteine ​​mutant antibody containing positions 153, 169, and 205 (according to EU numbering) in the CH1 domain of the heavy chain constant region of an antibody satisfying both conditions would exhibit excellent properties against benzoselenophene-based duocarmycin derivatives such as AMB401.

[0404] Accordingly, mutant antibodies were produced by introducing cysteine ​​into the constant region of antibodies in the form of monoclonal antibodies, anti-FGFR3 antibody AMB009 (KR 2022-0099103 A), anti-HER2 antibody trastuzumab, or immunoglobulin subtype 1. The antibodies containing the engineered cysteine ​​include at least one of the heavy chain sequences (SEQ ID NO: 1 to SEQ ID NO: 23) present in the constant region (Table 5).

[0405]

[0406]

[0407] Example 4: Preparation and analysis of ADCs through site-specific conjugation

[0408] The cysteine-mutated antibody present in a buffer solution of 20 mM histidine-HOAc, 150 mM NaCl, pH 5.5 was adjusted to pH 8.0 with a buffer solution of 1 M Tris, pH 8.5, and then treated with a 200 mM EDTA aqueous solution. A 100 mM DTT aqueous solution was then added to the solution. The solution prepared through the above process was allowed to react at 22°C until the next day. DTT was removed using a G25 desalting column, and the buffer containing the reduced antibody was exchanged. The unbound antibody was then mixed with a 200 mM EDTA aqueous solution and a freshly prepared 100 mM DHAA solution. The antibody solution was allowed to react at 22°C for 1 hour. After reoxidation, the solution was adjusted to pH 5 with a 10% HOAc aqueous solution and then diluted 5-fold with buffer A. The diluted solution was purified on an SP column to remove the coagulant. The antibody aliquot obtained through purification was collected and placed in 15 mL Amicon TMThe product was concentrated through a filter. The concentrated, unbound antibody was sterilized by passing it through a 0.22 μm PVDF disposable filter, and the aggregates, mass, and endotoxin were analyzed, respectively. The product was aliquoted after adding 0.2 M EDTA to make the final EDTA concentration 2 mM, and the aliquots were stored long-term at -80°C.

[0409] The unbound antibody was mixed in a buffer containing 25 mM NaOAC, 150 mM NaCl, pH 5.0, and the pH was adjusted to 7.0 with 50 mM PBS, pH 8.0. DMA and linker-drug (to a final DMA ratio of 10%) were then slowly added. The binding solution was reacted at 22°C for 1 hour, and the reaction was stopped by adding 10 mM NAC aqueous solution. The stopping solution was reacted at 22°C for 15 minutes, and then separated on a 10 mL zeba column (with a critical point of 40 kDa) and 15 mL Amicon TM The antibody-drug conjugate was purified via (50 kDa). The final product was sterilized by passing it through a 0.22 μm PVDF disposable filter and analyzed for concentration, aggregates, MS-DAR, endotoxin, and free drug levels. The final product was stored long-term at -80°C.

[0410] To confirm the physicochemical superiority of site-specific conjugation for key residues predicted in silico, physical property analyses were performed for various residues (Fig. 5). As a result of analysis via HIC-HPLC, anti-FGFR3_HC-P153C-vc-PAB-AMB401, which achieved an excellent score in the in silico analysis prediction and was selected, was well confirmed to have site-specific and homogeneous conjugation with a DAR of 1.80. However, in the case of anti-FGFR3_HC-F170C-vc-PAB-AMB401, which had a low in silico analysis score, numerous non-specific conjugations were confirmed, forming a heterogeneous antibody-drug conjugate with a DAR of 2.39.

[0411] In addition, a physical property analysis was performed on the antibody-drug conjugate including the anti-FGFR3 antibody and anti-HER2 antibody produced in this example, and the antibody-drug conjugate's antibody-drug conjugate's antibody-drug conjugation ratio for key residues predicted in silico was 1.7 or higher, purity was 80% or higher, free linker-drug was less than 1%, and endogenous toxin was less than 1 EU / mg, etc., and the physical property analysis was performed (Tables 6 and 7).

[0412] For anti-FGFR3 antibodies, in the case of antibody-drug conjugates introducing the commonly known conjugation sites LC-V205C and HC-S442C variants, low purities such as 47.23% and 50.82% were observed, and in the antibody-drug conjugates introducing other variants, the non-specific conjugation rate was high, indicating that the linker drug attachment pattern was undesirable, and in the conjugates introducing variants at other sites, the antibody-drug conjugation rate was low. In contrast, in the embodiments conjugated to P153C, T169C, and K205C, the conjugation purities were all over 85%, and the DAR was confirmed to be close to or higher than 1.8. This is a result confirming that the key residues predicted in the present invention (HC-P153C, T169C, K205C) are sites specifically suitable for linker-payload compounds including AMB401 (Table 6).

[0413] To confirm the superiority of the P153C, T169C, and K205C positions, the same pattern was confirmed by applying it to an anti-HER2 antibody. When AMB401 was conjugated to the LC-V205C position, the DAR was 1.51 (Figure 25) and the purity was 59.86%, showing an unstable conjugation efficiency. However, when AMB401 was conjugated to the HC-T169C position, the DAR was 1.73 (Figure 24) and the purity was 99.97%, showing an excellent conjugation efficiency (Table 6). In particular, the antibody-drug conjugate conjugated to the HC-T169C position showed relatively high hydrophilicity, which can be predicted to have superior physical properties. This result confirms that the mutant cysteine ​​residue derived through in silico analysis can show specifically excellent conjugation ability for the linker-drug containing AMB401. In addition, it is well known that stable DAR conjugation and homogeneity and purity of the conjugate play a very important role in the excellent efficacy of antibody-drug conjugates. Therefore, it is suggested that AMB401 will exhibit excellent efficacy specifically at the site verified through in silico analysis and implementation.

[0414]

[0415] Accordingly, various linker-payloads based on AMB401 were conjugated to the key residues predicted in the present invention (HC-P153C, T169C, K205C) for anti-HER2 antibodies. All antibody-drug conjugates for these residues were close to the target DAR of 1.8-2.0 and exhibited excellent conjugation efficiencies with high homogeneity and purity (Table 7). This confirmed that the cysteine ​​mutation sites (P153C, T169C, K205C) proposed in the present invention exhibited specific and excellent conjugation efficiencies for the linker-drug structure containing the payload referred to as AMB401.

[0416]

[0417]

[0418] 1 To achieve a DAR of 1.8, antibody drug conjugates were prepared by adding additional LP equivalents per antibody during the process.

[0419]

[0420] Example 5: Confirmation of antigen binding affinity of antibody-drug conjugates

[0421] The amino acid sequence of the parent antibody, trastuzumab, and the sequence containing the engineered cysteine ​​are as follows:

[0422] All of the parent antibody light chain (Kappa light chain)

[0423] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25)

[0424] All of the constant region of the parent antibody heavy chain (Heavy chain constant region)

[0425] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26)

[0426]

[0427] Based on the sequence, the antigen binding affinity of anti-HER2 ADCs in which linker drugs (AM10601, AM10701, AM10801, AM10901, AM11001, AM11101) including drug AMB401 were site-specifically conjugated to three trastuzumab cysteine ​​mutants (P153C, T169C, K205C) and the control antibody trastuzumab was evaluated through ELISA (Figs. 6 to 8).

[0428] The binding affinity of all site-specific conjugated anti-HER2 ADCs was confirmed to show equivalent binding affinity with a deviation of approximately 30% compared to the control, trastuzumab (EC in the range of 0.5 to 0.9 nM). 50 ; Table 8).

[0429]

[0430] Therefore, it was confirmed that the ADC formed through all site-specific conjugations maintained antigen binding affinity similar to the parent antibody, trastuzumab antibody, which does not contain any separate mutations.

[0431]

[0432] Example 6: In vitro cell proliferation and cytotoxicity

[0433] The efficacy of ADC, which site-specifically conjugated AMB401 to an anti-FGFR3 antibody, was evaluated using a cell proliferation assay on AMB-BT-0050T, a brain tumor patient-derived cell line expressing FGFR3.

[0434] A 40 μL aliquot of cell culture containing approximately 350 AMB-BT-0050T cells in the medium was deposited into each well of a 384-well clear plate. 50 μL of PBS was added to the surrounding wells where no cell lines were aliquoted. After culturing the cells for 1 day, ADC was added to 10 μL of the culture medium at 5-fold concentrations to give final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into the well plates where the cells were cultured. After culturing for 7 days in a 37°C carbon dioxide incubator, 10 μL of Calcein was added to each well, and the staining was stabilized for 30 minutes, and then measured using a high-resolution fluorescence analyzer, or 10 μL of CellTiterGlo was added to each well, and then the signal was measured using a high-resolution EnVision.

[0435] As a result, anti-FGFR3-HC-T155C-vc-PAB-AMB401 and anti-FGFR3-HC-T169C-vc-PAB-AMB401 showed effective drug efficacy compared to PBS, while anti-FGFR3_HC-T169C-vc-PAB-AMB401 (DAR 1.78) had an IC of 18.9 nM compared to anti-FGFR3_HC-T155C-vc-PAB-AMB401 (DAR 2.60). 50 , exhibited superior tumor killing efficacy despite possessing a low DAR. That is, it was confirmed that AMB401 ADC site-specifically conjugated to the cysteine ​​mutation site derived through in silico analysis showed a statistically significant difference in efficacy compared to ADCs conjugated to other cysteine ​​mutation sites (Table 9).

[0436]

[0437] Based on the fact that the present invention shows great advantages by site-specifically conjugating AMB401 to the mutant cysteine ​​site derived through in silico analysis, we aimed to evaluate the efficacy of trastuzumab ADCs site-specifically conjugated with AMB401 containing various linker structures in NCI-N87 (gastric cancer), SK-BR-3 (breast cancer) expressing HER2, which is the target of trastuzumab, and MCF-7 (estrogen receptor-expressing breast cancer) that does not express HER2. AMB401 was conjugated to specific mutant cysteine ​​sites of trastuzumab containing more than six types of linker structures.

[0438] Forty-μL ​​aliquots of cell culture containing approximately 350 (NCI-N87), 250 (SK-BR-3), and 500 (MCF-7) cells each (NCI-N87, SK-BR-3, MCF-7, and various other cell lines) were seeded into each well of a 384-well opaque plate. 50 μL of PBS was added to the surrounding wells where no cell lines were seeded. After culturing the cells for 1 day, ADC was added to 10 μL of culture medium at a 5-fold concentration to give final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into well plates where cells were cultured. After culturing for 6 days in a 37°C carbon dioxide incubator, 10 μL of CellTiterGlo (Promega) was added to each well, and the signal was stabilized for 10 minutes before being measured using a high-resolution luminescence spectrometer.

[0439] As a result, for NCI-N87 cells and SK-BR-3 cell lines expressing HER2, the efficacy of the trastuzumab ADC conjugated to site-specifically AMB401 showed superior efficacy (IC within the range of 0.05 to 0.3 nM) compared to SYD-985, a duocarmycin-conjugated trastuzumab ADC in phase 3 clinical trials (secoDUBA drug conjugated through stochastic linkage to endogenous disulfide), despite having a lower antibody-to-drug ratio in most implementations. 50 ; Table 10, Figs. 9 and 10).

[0440]

[0441]

[0442] In addition, in the present example, the control substance SYD-985 has a higher antibody-drug conjugation ratio (DAR) than the site-specifically conjugated trastuzumab ADC of the present invention. Therefore, trastuzumab-vc-secoDUBA (DAR 1.7), which conjugates the seco-DUBA drug with a low DAR through stochastic linkage to the endogenous disulfide in the same manner as SYD-985, was manufactured, and the efficacy of AMB401, a payload of the same duocarmycin series, was compared with that of the site-specifically conjugated trastuzumab ADC. The efficacy comparison was performed in vitro and compared in HER2-expressing cell lines NCI-N87 and SK-BR-3 (Figs. 21a and 21b). The site-specifically conjugated trastuzumab ADC had an IC of <1 nM and <0.05 nM in NCI-N87 and SK-BR-3 cell lines, respectively. 50 was produced, which shows a very strong efficacy compared to trastuzumab-vc-secoDUBA (DAR 1.7) conjugated with the same DAR through probabilistic binding.

[0443]

[0444] In addition, in this example, the nonspecific cytotoxicity of the site-specifically conjugated trastuzumab ADC was evaluated against the HER2 low-expressing MCF-7 cell line, and the site-specifically conjugated antibody drug conjugate AMB401 showed nonspecific cytotoxicity equivalent to that of SYD-985, which shares the same class of drugs (IC of 500 nM or more). 50 ; Table 11 and Fig. 11).

[0445]

[0446]

[0447] In addition, in this example, trastuzumab-vc-secoDUBA (DAR 1.7), which was conjugated to endogenous disulfides through stochastic linkage in the same manner as SYD-985, was constructed and the non-specific cytotoxicity of the site-specifically conjugated trastuzumab ADC was compared in vitro in the HER2 low-expressing cell line MCF-7 (Fig. 21c). In this example, the site-specifically conjugated trastuzumab ADC showed higher target-specific tumor killing efficacy than trastuzumab-vc-secoDUBA in the HER2-expressing cell line, but did not show non-specific cytotoxicity in any HER2 low-expressing cell line. This suggests that the AMB401-based antibody-drug conjugate constructed through site-specific conjugation can exhibit high target specificity and therapeutic window.

[0448]

[0449] Among the antibody-drug conjugates site-specifically conjugated to the engineered cysteine ​​described above in this example, trastuzumab-T169C-AM10801, trastuzumab-T169C-AM11001, and trastuzumab-T169C-AM11101, which showed particularly excellent in vitro efficacy and low nonspecific toxicity, were evaluated for in vitro efficacy in HER2-overexpressing and -lowexpressing cell lines. In various other cell lines expressing HER2, the trastuzumab ADC site-specifically conjugated to AMB401 showed excellent efficacy according to HER2 expression despite having a lower antibody-to-drug ratio compared to SYD-985 (Table 12 and FIGS. 13 to 17).

[0450]

[0451] Example 7: Evaluation of internalization of antibody-drug conjugates

[0452] Among the trastuzumab cysteine ​​mutant (T169C) ADCs, the intracellular internalization of three ADCs (trastuzumab-T169C-AM10801, -AM11001, -AM11101) that showed excellent efficacy in in vitro cell proliferation assays, as well as the control substance SYD-985 and the anti-HER2 antibody trastuzumab, was evaluated.

[0453] All antibodies and ADCs were bound to HER2-overexpressing NCI-N87 and SK-OV-3 cell lines at 4°C for 30 min, and then Alexa488-conjugated anti-human IgG1 antibodies were conjugated to the cells. The reduction in cell surface antibody-drug conjugates was calculated by dividing the average FITC fluorescence intensity immediately after cell binding by the average fluorescence intensity after acclimation at 37°C for 0, 1, 4, and 24 h, and multiplying by 100.

[0454] Figure 12 is a graph showing the results of an analysis of the internalization rate, calculated by subtracting the surface antibody-drug conjugate reduction amount for each time point from 100 (%). According to the analysis, trastuzumab, the control substance, Trastuzumab-vc-secoDUBA (SYD-985), and three AMB401 site-specific binding ADCs all showed comparable internalization rates at the same time point (Figure 12).

[0455]

[0456] Example 8: Efficacy of antibody-drug conjugates for tumor growth inhibition in xenograft mice harboring HER2 high-expressing cells.

[0457] The in vivo efficacy of three ADCs (trastuzumab-T169C-AM10801, -AM11001, -AM11101) was evaluated in an NCI-N87 gastric cancer xenograft model. Tumors were propagated by subcutaneous administration into athymic T-cell-deficient Balb / c-nu mice. After the xenograft mouse model reached a tumor volume of 100 mm3, the three ADCs conjugated to AMB401, the control substance SYD-985, and the PBS buffer control were each treated once at a dose of 3 mg / kg and observed for 32 days.

[0458] After 32 days, tumor size was smallest for SYD-985 and trastuzumab-T169C-AM11001, which were control compounds with relatively high antibody-to-drug ratios. Trastuzumab-T169C-AM11101 also exhibited tumor suppression of over 90% (Fig. 18). This suggests that the site-specific conjugation mode of AMB401 exhibits excellent tumor suppression efficacy despite its relatively low drug-to-drug ratio.

[0459]

[0460] Example 9: Confirmation of antigen binding affinity of antibody-drug conjugates with hydrolyzed antibody binding sites

[0461] In an anti-HER2 ADC in which a linker-payload (AMB401) was site-specifically conjugated to two types of trastuzumab cysteine ​​mutants, which were confirmed to have excellent tumor suppression efficacy through Example 8, the antibody binding site of the linker-payload was hydrolyzed, and the antigen binding affinity of trastuzumab before and after hydrolysis and as a control was evaluated through ELISA.

[0462] As a result, the antibody-drug conjugate with the antibody binding site hydrolyzed showed no significant difference in antigen binding affinity before and after hydrolysis, and it was confirmed that it exhibited antigen binding affinity similar to that of the parent antibody, trastuzumab (Fig. 19). This means that even if various manipulations are applied after conjugation of the linker-drug including AMB401 with the maleimide conjugation method for site-specific conjugation to the cysteine ​​of the antibody, the site-specific antibody-drug conjugate of AMB401 can maintain excellent conjugation ability.

[0463]

[0464] Example 10: In vitro cell proliferation and cytotoxicity of antibody-drug conjugates hydrolyzed at the antibody binding site

[0465] Using a cell proliferation assay, the in vitro cytotoxicity of trastuzumab ADC site-specifically conjugated to AMB401 and linker-payload was evaluated before and after hydrolysis to modify the maleimide binding site by hydrolyzing the antibody binding site in HER2-expressing SK-BR-3 (breast cancer).

[0466] A 40 μL aliquot of cell culture medium containing approximately 250 SK-BR-3 cells in the medium was deposited into each well of a 384-well opaque plate. 50 μL of PBS was added to the surrounding wells where no cell lines were seeded. After culturing the cells for 1 day, ADC was added to 10 μL of the culture medium at 5-fold concentrations to give final concentrations of 500,000, 166,700, 55,600, 18,500, 6,200, 2,100, 685.9, 228.6, 76.2, 25.4, 8.5, 2.8, 0.9, and 0.3 pM, respectively, and dispensed into the well plates where the cells were cultured. After culturing for 6 days in a 37°C carbon dioxide incubator, 10 μL of CellTiterGlo (Promega) was added to each well, and the signal was stabilized for 10 minutes before being measured using a high-resolution luminescence analyzer.

[0467] As a result, the efficacy of the site-specifically conjugated trastuzumab ADC was comparable before and after hydrolysis against the HER2-expressing SK-BR-3 cell line (Fig. 20). In particular, the site-specifically conjugated trastuzumab ADC showed superior drug efficacy compared to trastuzumab-vc-seco-DUBA conjugated to the same DAR. This demonstrates the superior efficacy of the site-specific conjugate compared to SYD-985, and suggests that the site-specific conjugate of AMB401 can exhibit superior tumoricidal efficacy either when conjugated to a mutant cysteine ​​through a maleimide or when the ring structure of the maleimide is modified through hydrolysis of the conjugate site.

[0468]

[0469] Example 11: Efficacy of antibody-drug conjugates with equivalent antibody-drug ratios for tumor growth inhibition in xenograft mice bearing HER2-high expressing cells.

[0470] The in vivo efficacy of Trastuzumab-T169C-AM11001 (DAR 1.8) with hydrolyzed antibody conjugates and Trastuzumab-T169C-AM11101 (DAR 1.8) with hydrolyzed antibody conjugates was evaluated in an NCI-N87 gastric cancer xenograft model. Tumors were propagated by subcutaneous administration into athymic T cell-deficient Balb / c-nu mice. After the xenograft mouse model reached a tumor volume of 100 mm3, the two AMB401-conjugated ADCs and the control substance Trastuzumab-vc-secoDUBA (SYD-985, DAR 2.7), Trastuzumab-vc-secoDUBA (DAR 1.7) with an equivalent antibody-to-drug ratio, and PBS buffer control were treated once at a dose of 1 mg / kg and observed for 35 days.

[0471] After 35 days, tumor size showed a tumor inhibition rate of about 50% in both SYD-985, a clinical-stage comparator with a relatively high antibody-to-drug ratio, and trastuzumab-T169C-AM11001 and trastuzumab-T169C-AM11101 with a low antibody-to-drug ratio. In contrast, trastuzumab-vc-secoDUBA ADC with an antibody-to-drug ratio of approximately 1.8 showed no significant tumor inhibition effect, showing tumor growth equivalent to that of the PBS control group. This demonstrates that the site-specific antibody-drug conjugate for AMB401 developed through the present invention exhibits relatively superior tumor inhibition efficacy compared to ADCs conjugated to the same class of drugs under equivalent or lower DAR conditions (Fig. 22).

[0472]

[0473] Example 12: Tumor growth inhibition efficacy of antibody-drug conjugates conjugated with AMB401 site-specifically in patient-derived cell line xenograft mice

[0474] Trastuzumab-T169C-AM11001 (DAR 1.8) and a control antibody-T169C-AM11001 (DAR 1.7) with a nonspecific antigen were evaluated in the HBCx-10 xenograft model. Tumors were propagated by subcutaneous administration into athymic T cell-deficient Athymic-nu mice. In addition, trastuzumab-vc-secoDUBA (DAR 1.8), trastuzumab-vc-secoDUBA (DAR 2.7) and a control antibody-vc-secoDUBA (DAR 2.7) with a nonspecific antigen were produced for comparison between experimental groups and their efficacy was evaluated. After the tumor volume reached 150 mm3, the xenograft mouse model was treated once with each antibody-drug conjugate and PBS buffer control at a dose of 1 mg / kg or 3 mg / kg and observed for 31 days.

[0475] After 31 days, the tumor size showed a tumor inhibition rate of more than 100% at 3 mg / kg for both trastuzumab-vc-secoDUBA (DAR 2.7), a control substance with a relatively high antibody-drug ratio, and trastuzumab-T169C-AM11001 (DAR 1.8), which has a low antibody-drug ratio. In particular, the antibody-drug conjugate in which AM11001 was conjugated to a nonspecific control antibody with a DAR of 1.8 showed the same effect as the PBS control group (Fig. 23a), whereas the antibody-drug conjugate in which vc-secoDUBA was conjugated to the control antibody with a DAR of 2.7 showed a tumor inhibition effect of about 50% in the model despite the lack of tumor specificity due to the nature of the control antibody (Fig. 23b). In addition, while the control substance, trastuzumab-vc-secoDUBA (DAR 2.7), showed a tumor inhibition efficiency of approximately 60% at a dose of 1 mg / kg, trastuzumab-T169C-AM11001 (DAR 1.8) showed tumor inhibition of more than 100% at a dose of only 1 mg / kg.

[0476] Through this, the AMB401 site-specific conjugated drug of the present invention shows superior tumor inhibition efficacy compared to the secoDUBA conjugated antibody-drug conjugate that has proven highly effective in existing clinical studies, and also did not show non-specific cytotoxicity that occurs in stochastic duocarmycin binding when site-specifically conjugated to the control antibody. This confirms that the antibody-drug conjugate site-specifically conjugated to AM401 is an excellent drug that exhibits selective tumor killing ability with high target specificity and stability (Fig. 23).

[0477]

[0478] Example 13: In vitro plasma stability evaluation of antibody-drug conjugates site-specifically conjugated to AMB401

[0479] Trastuzumab-T169C-AM11001 (DAR 1.8) antibody-drug conjugate was subjected to a 7-day stability evaluation in human plasma. To evaluate the antibody-drug conjugate, 20 μL of plasma adapted within 7 days was diluted in PBS / 0.1% Tween 20 solution and 20 μL of Protein A magnetic beads were added. After thorough mixing at room temperature for 2 h, the magnetic beads were washed with PBS / 0.1% Tween 20 solution. Subsequently, 30 μL of cleavage solution (50 mM TRIS, 21.05% ACN containing 10 mM CaCl2) and 10 μL of trypsin were added. Afterwards, 50 μL of beta-glucuronidase (0.2 mg / mL) was added to the sample and incubated for 2 h at 37°C. After adding 10 μL of 2N HCl and mixing well, 50 μL of 100 ng / mL MMAE dissolved in ACN containing 0.1% FA was added for standardization. The analysis sample was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was analyzed by HPLC and LC-MS / MS.

[0480] Additionally, for free toxin drug analysis, 20 μL plasma samples were mixed with 100 μL of IS solution containing 0.1% formic acid (ACN solution containing 100 ng / mL Enalapril). The sample was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was extracted and vacuum-dried. The dried analyte was dissolved in 70 μL of a solution containing 0.1% formic acid in 30% CAN, centrifuged at 13,000 rpm for 3 minutes, and the supernatant was extracted and subjected to LC-MS / MS analysis.

[0481] Our analysis results confirmed that, for the site-specifically conjugated trastuzumab cysteine ​​mutant (T169C)-AM11001 drug, AMB401 maintained stable acDrug and antibody concentrations in human plasma for 7 days. Furthermore, no free payload release of AMB401 was detected in plasma, demonstrating that it can stably exist in the form of a linker-drug conjugate or linker-drug in plasma (Figure 26).

[0482]

[0483] Example 14: Pharmacokinetic evaluation of antibody-drug conjugates conjugated to site-specific AMB401 in CES1c-deficient mice

[0484] The antibody-drug conjugate trastuzumab-T169C-AM11001 (DAR 1.8) was administered to carboxylesterase (CES1c) deficient mutant mice at a concentration of 3 mg / kg and the pharmacokinetics were evaluated for 28 days. The antibody-drug conjugate trastuzumab-vc-secoDUBA (DAR 2.7) was administered as a reference at the same concentration and evaluated. The concentrations of antibody-conjugated drug (acDrug), total antibody, and free drug were analyzed using the same method as described in Example 13.

[0485] Pharmacokinetic analysis revealed that trastuzumab-T169C-AM11001 exhibited an acDrug half-life of approximately 5.3 days, while the reference drug, trastuzumab-vc-secoDUBA, exhibited an acDrug half-life of approximately 2.9 days. Furthermore, both agents exhibited comparable levels of total antibody changes, and no significant free payload was observed in plasma within the measurable range.

[0486] This confirms that site-specifically conjugated trastuzumab-T169C-AM11001 exhibits superior blood stability comparable to or superior to that of the competing duocarmycin drug (Figure 27). This suggests that site-specific conjugation can demonstrate superior in vivo conjugation stability compared to non-specific linker-drug conjugation without a separate change in conjugation method, even when utilizing the same conjugation method, which can be referred to as thio-maleimide.

[0487]

[0488] Compared to ADCs in which linker drugs are conjugated via the native interchain disulfide bond of an antibody or to known engineered cysteine ​​ADCs at other known positions, the site-specifically conjugated ADCs of the present invention exhibit improved physicochemical, pharmacological, and / or pharmacodynamic properties. The ADCs of the present invention have binding properties similar to wild-type antibodies, superior in vivo efficacy, increased therapeutic index, and / or improved stability, and thus may exhibit higher therapeutic efficacy for various diseases, including cancer, compared to existing antibodies or ADCs.

[0489]

[0490] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0491]

[0492] Electronic file attached.

Claims

An antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof, and a linker drug, An antibody-drug conjugate wherein the linker drug is conjugated to the antibody or antigen-binding fragment thereof via an engineered cysteine ​​at one or more positions selected from heavy chain positions 151, 152, 153, 154, 157, 158, 160, 163, 164, 167, 168, 169, 170, 171, 172, 173, 174, 180, 182, 201, 203, 205, 206 and 210 (according to EU numbering) of the antibody or antigen-binding fragment thereof. An antibody-drug conjugate according to claim 1, wherein the engineered cysteine ​​is at one or more positions selected from positions 152, 153, 154, 164, 167, 168, 169, 170, 171, 172, 174, 180, 182, and 205 (according to EU numbering) in the heavy chain constant region CH1 domain of the antibody. An antibody-drug conjugate, characterized in that in claim 1, the engineered cysteine ​​is at one or more positions selected from positions 153, 169, and 205 (according to EU numbering) in the CH1 domain of the heavy chain constant region of the antibody. An antibody-drug conjugate according to claim 1, wherein the drug is duocarmycin, calicheamicin, pyrrolobenzodiazepine (PBD) dimer, maytansinoid, or auristatin derivative. An antibody-drug conjugate according to claim 4, characterized in that the drug is a benzoselenophene-based duocarmycin derivative. In claim 5, the drug is an antibody-drug conjugate characterized in that it is a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: In the above chemical formula 1, X is a halogen, R1 is substituted or unsubstituted C containing one or more N 3-10 A heterocycloalkyl group or a heteroaryl group, R2 and R3 are each independently hydrogen, fluorine, chlorine, bromine, iodine, or substituted or unsubstituted C 1-5 It is an alkyl group, R4 and R5 are each independently hydrogen, or a substituted or unsubstituted C1-5 alkyl group. In claim 6, the drug is an antibody-drug conjugate characterized in that it is a compound represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof: An antibody-drug conjugate according to claim 1, characterized in that the linker is a cleavable linker or a non-cleavable linker. An antibody-drug conjugate according to claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, and a human antibody. An antibody-drug conjugate according to claim 1, characterized in that the antibody is selected from IgA, IgD, IgE, IgG and IgM. An antibody-drug conjugate according to claim 1, characterized in that the antibody binds to an antigen target expressed within or on the cell membrane of a tumor cell. In claim 1, the antibody is an anti-annexin A1 antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD38 antibody, an anti-CD44 antibody, an anti-CD47 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD74 antibody, an anti-CD79 antibody, an anti-CD115 antibody, an anti-CD123 antibody, an anti-CD138 antibody, an anti-CD203c antibody, an anti-CD303 antibody, an anti-CEACAM antibody, an anti-CLL-1 antibody, an anti-HGFR antibody, an anti-Cripto antibody, an anti-DLL3 antibody, an anti-EGFR antibody, an anti-EPCAM antibody, an anti-EphA2 antibody, an anti-EphB3 antibody, an anti-ETBR antibody, an anti-FcRL5 antibody, an anti-FGFR3 antibody, an anti-FOLR1 antibody, An antibody-drug conjugate characterized in that the antibody is selected from the group consisting of anti-GCC antibody, anti-GPNMB antibody, anti-Her2 antibody, anti-HMW-MAA antibody, anti-integrin antibody, anti-Lewis A-like carbohydrate antibody, anti-Lewis Y antibody, anti-LIV1 antibody, anti-mesothelin antibody, anti-MN antibody, anti-MUC1 antibody, anti-MUC16 antibody, anti-NaPi2b antibody, anti-nectin-4 antibody, anti-PSMA antibody, anti-SIRPα antibody, anti-SLC44A4 antibody, anti-STEAP-1 antibody, anti-5T4 antibody, anti-Tag72 antibody, anti-TF antibody, anti-TROP2 antibody, and anti-VLA antibody. A pharmaceutical composition for preventing or treating a proliferative disease, comprising an antibody-drug conjugate according to any one of claims 1 to 12. A pharmaceutical composition according to claim 13, wherein the proliferative disease is a neoplasm, a tumor, cancer, leukemia, psoriasis, a bone disease, a fibroproliferative disorder or atherosclerosis. A pharmaceutical composition according to claim 14, wherein the cancer is a solid tumor, blood cancer, colorectal cancer, uterine cancer, uterine fibroids, meningioma, lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, colon cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, brain metastasis cancer, sarcoma, osteosarcoma, Kaposi sarcoma, or melanoma.

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