Anti-BCMA antibody drug conjugates and uses thereof

Abstract

The present disclosure relates to an antibody-drug conjugate (ADC) targeting B-cell maturation antigen (BCMA) and uses thereof, and more particularly, to an ADC including an antibody or antigen-binding fragment thereof that binds to BCMA, preferably human BCMA, and an active agent; and uses of ADCs for the treatment and / or prevention of diseases, more particularly, hyperproliferative and / or angiogenic diseases, such as cancers.

Description

[0001] Attorney Docket No.: LCH-03725

[0002] ANTI-BCMA ANTIBODY DRUG CONJUGATES AND USES THEREOF Related Applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 63 / 740,625, filed December 31, 2024, U.S. Provisional Application Serial No. 63 / 802,493, filed May 8, 2025, and U.S. Provisional Application Serial No. 63 / 802,463, filed May 8, 2025; the contents of each of which are hereby incorporated by reference in their entirety.

[0004] Background

[0005] Many therapeutic agents administered parenterally may induce unwanted side effects and even serious toxicity at therapeutic doses, as a result of systemic administration. Antibodydrug conjugates (ADCs) have been a subject of research for developing treatments to take advantage of such therapeutic agents while reducing side effects and toxicity. There remains a demand for development of antibody drug conjugates for effective therapeutic methods.

[0006] Summary

[0007] The present disclosure provides, among other things, antibody-drug conjugates (ADCs) targeting B-cell maturation antigen (BCMA), active metabolites of such ADCs, methods for preparation of such ADCs, and compositions and uses of such ADCs for preventing or treating a condition associated with BCMA expression (e.g., a cancer). More particularly, the present invention provides ADCs comprising an antibody specifically binding to BCMA, and a pharmaceutical composition comprising the same. In certain embodiments, the ADCs disclosed herein have improved stability, enhanced efficacy, and / or lower toxicity as compared to ADCs known in the art.

[0008] In some aspects, the present disclosure provides conjugates having a structure represented by Formula A or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Formula A

[0011] wherein

[0012] Ab is an anti-B Cell Maturation Antigen (BCMA) antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, whereinAttorney Docket No.: LCH-03725

[0013] the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

[0014] the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13;

[0015] each B is independently an active agent;

[0016] each L is independently a linker comprising an alkylene or a heteroalkylene, wherein L comprises one or more of:

[0017] (i) one or more unsaturated bonds;

[0018] (ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain); (iii) at least one C1-20 alkyl substituent;

[0019] (iv) at least one isoprenyl group having a structure represented by General Formula II:

[0020] [General Formula II]

[0021]

[0022] (v) a structure represented by Formula B:

[0023]

[0024] Formula B

[0025] wherein:

[0026] X1and X2are each independently N(R3), O, or S;

[0027] R1is H, alkyl, or cycloalkyl;

[0028] R2is alkyl or cycloalkyl; or R1and R2combine to form a cycloalkyl;

[0029] R3is H, alkyl, aryl, or aralkyl;

[0030] Ya1is a connection to Ta;

[0031] Ya2is a connection to Ab;

[0032] each Tais independently a cleavage group, preferably a self-immolative group, more preferably a structure represented by Formula C:Attorney Docket No.: LCH-03725

[0033]

[0034] Formula C

[0035] wherein:

[0036] ""

[0037] G is independently a glucuronic acid moiety

[0038]

[0039] RAand RBare each independently hydrogen, Ci-s alkyl, or C3-8 cycloalkyl;

[0040] Rcis hydrogen or a carboxyl -protecting group;

[0041] each RDis independently hydrogen or a hydroxyl-protecting group;

[0042] W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR', -SONR'-, or -PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of Formula C and the NR' is bonded to Ya3;

[0043] R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, Ci-8 alkylthio, mono- or di-Ci-8 alkylamino, C3-20 heteroaryl, or Ce-2oaryl;

[0044] each Z is independently C1-8 alkyl, halogen, cyano, or nitro;

[0045] n is 0, 1, 2, or 3;

[0046] Ya3is a connection to L;

[0047] Ya4is a connection to B; and

[0048] nA and nB are each independently an integer from 1 to 20.

[0049] In some aspects, the present disclosure provides conjugates having a structure represented by General Formula I or a pharmaceutically acceptable salt thereof:

[0050] [General Formula I]Attorney Docket No.: LCH-03725

[0051]

[0052] wherein

[0053] Ab is an anti-B Cell Maturation Antigen (BCMA) antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein

[0054] the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

[0055] the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13;

[0056] each B’ is an active agent;

[0057] each G is independently a glucuronic acid moiety o

[0058]

[0059] Rcis hydrogen or a carboxyl -protecting group;

[0060] each RDis independently hydrogen or a hydroxyl -protecting group;

[0061] RAand RBare each independently hydrogen, Ci-s alkyl, or C3-8 cycloalkyl;

[0062] Wis -C(0)-, -C(0)NR’-, -C(0)0-, -SO2NR'-, -P(0)R"NR', -SONR'-, or -PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of General Formula I and the NR' is bonded to L;

[0063] R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, C3-20 heteroaryl, or Ce-2oaryl;Attorney Docket No.: LCH-03725

[0064] each Z is independently Ci-s alkyl, halogen, cyano, or nitro;

[0065] n is 0, 1, 2, or 3;

[0066] L comprises: a C1-50 alkylene or C1-50 heteroalkylene wherein the C1-50 alkylene or C1-50 heteroalkylene comprises one or more of:

[0067] (i) one or more unsaturated bonds;

[0068] (ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain);

[0069] (iii) at least one C 1-20 alkyl substituent; and

[0070] (iv) at least one isoprenyl group having a structure represented by General Formula II:

[0071] [General Formula II]

[0072]

[0073] 1 and m are each independently 1 to 20.

[0074] Brief Description of the Drawings

[0075] FIGs. 1A-1B show that ADC4 compared to ADC9 (Blenrep) exhibited superior tumor growth inhibition (FIG. 1A) and improved animal survival (FIG. IB) in the OPM2-Luc xenograft model.

[0076] FIGs. 2A-2B show that ADC1, ADC3, and ADC7 compared to ADC9 exhibited significant tumor growth inhibition (FIG.2A) and improved animal survival rate (FIG.2B) in the NCI-H929 xenograft model.

[0077] FIGs. 3A-3B show that ADC3 and ADC7 compared to ADC9 exhibited superior and sustained tumor growth inhibition (FIG. 3A) and improved animal survival rate (FIG. 3B) in the MM IS xenograft model.

[0078] FIG.4 shows that ADC 10 exhibited significant tumor growth inhibition in the OPM2-Luc xenograft model.

[0079] FIG. 5 shows that ADC5 and ADC11 exhibited significant tumor growth inhibition in the OPM2-Luc xenograft model.

[0080] FIG.6 shows that ADC 12 exhibited significant tumor growth inhibition in the OPM2-Luc xenograft model.

[0081] FIG. 7 shows animal survival rate in the OPM2-Luc xenograft model after administration of ADC5, ADC10, ADC11, and ADC12.Attorney Docket No.: LCH-03725

[0082] FIG. 8 shows the synthesis and structure of exemplified ADC1.

[0083] FIG. 9 shows the synthesis and structure of exemplified ADC2.

[0084] FIG. 10 shows the synthesis and structure of exemplified ADC5.

[0085] FIG. 11 shows the synthesis and structure of exemplified ADC7.

[0086] FIG. 12 shows the synthesis and structure of exemplified ADC8.

[0087] FIG. 13 shows the synthesis and structure of exemplified ADC 10.

[0088] FIG. 14 shows the synthesis and structure of exemplified ADC11.

[0089] FIG. 15 shows the synthesis and structure of exemplified ADC 12.

[0090] Detailed Description

[0091] An antibody-drug conjugate (ADC) is a targeted technology for conjugating a therapeutic agent (e.g., a toxin or a drug) to an antibody that binds to an antigen, by which the agent is released in a cell to cause a desired effect (e.g., cell death of the targeted cell) and the like (e.g., a cancer cell). The ADC enables a drug to be accurately delivered to target cells (e.g., target cancer cells) while minimally affecting healthy cells, and to be released only under specific conditions, and thus has excellent efficacy compared to antibody therapeutic agents themselves and can remarkably reduce the risk of side effects compared to existing agents (e.g., an anticancer agent such as a toxin).

[0092] The basic structure of representative antibody-drug conjugates is “antibody-linker-small molecule drug (toxin)”. In this structure, the linker plays a functional role in linking the antibody and the drug, but in some cases also ensures that the drug is released from the antibody at the appropriate time, for example after reaching target cells. That is, the stability of the linker can play a very important role in the efficacy and safety such as systemic toxicity of an antibody-drug conjugate (Discovery Medicine 2010, 10(53): 329-39).

[0093] The use of monoclonal antibodies for treatment of diseases (e.g., cancer) has had substantial success. For example, monoclonal antibodies are suitable for target-directed addressing of tumor tissue and tumor cells. Antibody-drug conjugates have become a novel and powerful option for the treatment of lymphomas and solid cancers, and immunomodulatory antibodies also have recently had considerable success in clinical trials. The development of therapeutic antibodies is based on deep understanding of cancer serology, protein engineering technology and the action thereof, mechanisms of resistance, and interactions between immune systems and cancer cells.

[0094] Antigens which are expressed on the surface of human cancer cells are defined as a broad range of targets which are over-expressed compared to normal tissues, mutated andAttorney Docket No.: LCH-03725

[0095] selectively expressed. The key challenge is to identify antigens suitable for antibody-based therapies. These therapeutic agents mediate changes in antigen or receptor function (i.e., function as a stimulant or an antagonist), regulate the immune system through Fc and T cell activation, and exhibit efficacy through the delivery of specific drugs that bind to antibodies targeting specific antigens. Molecular techniques that can alter antibody pharmacokinetics, function, size and immune stimulation are emerging as key factors in the development of novel antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients highlights the importance of approaches for selecting optimized antibodies, including affinity and binding of target antigens and antibodies, selection of an antibody structure, and therapeutic approaches (signaling blockade or immune function).

[0096] B-cell maturation antigen (BCMA) is a member of the tumor necrosis family receptor super family (TNFRSF). BCMA’s expression is typically restricted to the B-cell lineage and is reported to increase in terminal B-cell differentiation. BCMA is expressed by human plasma blasts, plasma cells from tonsils, spleen and bone marrow, and also by tonsillar memory B cells and by germinal center B cells (Darce et al, 2007, J. Immunol 179: 7276-7286). BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The expression of BCMA has been recently linked to a number of cancers, autoimmune disorders, and infectious diseases. Cancers with increased expression of BCMA include without limitation hematological cancers, such as multiple myeloma (MM), Hodgkin’s and non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma (DLBCL), various leukemias (e.g., chronic lymphocytic leukemia (CLL)), and glioblastoma.

[0097] Without being bound by any particular scientific theory, the present disclosure includes the recognition that compositions containing antibodies (e.g., an anti-BCMA antibody), such as antibody-drug conjugates (ADCs), are useful for the treatment of diseases (e.g., cancers). In certain aspects, the present disclosure provides antibody-drug conjugates comprising an anti-BCMA antibody. In certain embodiments, the antibody disclosed herein binds to BCMA (e.g., human BCMA) expressed in a tumor and may be used to deliver a drug to the tumor. In certain embodiments, the antibody-drug conjugates disclosed herein have improved stability, enhanced efficacy, and / or lower toxicity as compared to antibody-drug conjugates known in the art. Without being bound by any particular scientific theory, the antibody-drug conjugates disclosed herein enable a drug and / or toxin to be easily, specifically, and efficiently released in a target cell (e.g., a cancer cell) to maximize efficacy, and enable a drug and / or toxin to stably reach the target cell.Attorney Docket No.: LCH-03725

[0098] In various aspects, the present disclosure provides linear or branched linkers, and conjugates comprising those linkers, as well as methods for their synthesis and for their use in therapeutic applications. In particular, the disclosure describes conjugates comprising a structure represented by Formula A and General Formula I. That structure incorporates multiple attachment points for coupling to active agents, allowing for the delivery of multiple payloads or the combination of different functionalities within a single conjugate. The branched structure may enhance the therapeutic efficacy, improve pharmacokinetic properties, or allow for the co-delivery of synergistic agents. The conjugates described herein may be particularly useful in the context of antibody-drug conjugates, but the technology is broadly applicable to various targeting moieties and payloads.

[0099] In various aspects, the present disclosure provides linkers, and conjugates comprising those linkers, as well as methods for their synthesis and for their use in therapeutic applications. In particular, the disclosure describes conjugates comprising a structure represented by Formula A and General Formula I. That structure incorporates multiple attachment points for coupling to active agents, allowing for the delivery of multiple payloads or the combination of different functionalities within a single conjugate. The branched structure may enhance the therapeutic efficacy, improve pharmacokinetic properties, or allow for the co-delivery of synergistic agents. The conjugates described herein may be particularly useful in the context of antibody-drug conjugates, but the technology is broadly applicable to various targeting moieties and payloads. Indeed, the linkers disclosed herein may be used with existing linker technology in multiple ways, as it sits within a well-developed field with numerous available techniques for assembly of conjugates:

[0100] 1. Maleimide conjugation: This method utilizes the reaction between mal eimide groups and thiol groups. For example, antibodies can be partially reduced to expose cysteine residues, which then react with maleimide-functionalized linkers or payloads.

[0101] 2. Cysteine Re-bridging: This technology creates homogeneous and stable ADCs by re-bridging reduced interchain disulfide bonds with bifunctional linkers, without the need for antibody engineering.

[0102] 3. N-Hydroxy succinimide (NHS) ester conjugation: NHS esters react with primary amines, such as lysine residues on proteins. This method is commonly used for attaching linkers or payloads to antibodies or other targeting moieties.Attorney Docket No.: LCH-03725

[0103] Click chemistry: Copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) are widely used for bioorthogonal conjugation. These reactions allow for site-specific attachment of payloads to modified targeting moieties. Click chemistry offers high specificity and can be performed under mild conditions, making it suitable for a wide range of biomolecules. Recently, metal-free SPAAC cycloaddition reactions have been investigated, including dibenzocyclooctyne (DBCO)-derived molecules covalently linked to the azide group of a functionalized phenylalanine such as para-azidophenlyalanine (pAzF) or para-azidomethylphenylalanine (pAMF). Metal-free SPAAC reactions have been shown as promising conjugation reactions for homogeneous, highly stable conjugates with desirable pharmacokinetics and controlled delivery of the potent payload. See e.g., Xenobiotica 2024, Vol. 54, No.

[0104] 8, 469^91.

[0105] Enzymatic conjugation: Methods such as sortase-mediated ligation or transglutaminase-catalyzed conjugation enable site-specific attachment of payloads to targeting moieties under mild conditions. Sortase A (SrtA) catalyzes transpeptidation to insert recognition peptides at the C-terminus of antibody chains, followed by payload attachment. Bacterial transglutaminases (BTGs) form amide bonds between lysine and glutamine residues, enabling stable payload attachment. Additional examples include (i) conjugation via formyl glycine-generating enzyme, which inserts a specific sequence, converting cysteine to formyl glycine, that can then be conjugated to aldehyde-containing payloads; and (ii) prenyltransferase-mediated conjugation, wherein farnesyl transferase attaches a ketone-functionalized isoprenoid to a C-terminal CaaX motif, which is then linked to a hydroxylamine-bearing payload via oxime ligation. See e.g., Biomedicine & Pharmacotherapy 161 (2023) 114408.

[0106] Hydrazone and oxime ligation: These methods involve the reaction between aldehydes or ketones with hydrazides or aminooxy groups, respectively. Exemplary methods include using amino-terminal engineered serine at the N-terminus, which is oxidized to an aldehyde for oxime ligation.

[0107] Thiol-ene and thiol-yne reactions: These photoinitiated reactions allow for the conjugation of thiol-containing molecules to alkene or alkyne-functionalizedAttorney Docket No.: LCH-03725

[0108] components. These methods offer rapid reaction kinetics and can be performed under mild conditions, making them suitable for sensitive biomolecules.

[0109] 8. Diels-Alder cycloaddition: This reaction between a diene and a dienophile can be used for bioconjugation, particularly when using cyclic dienes like transcyclooctene. The reaction proceeds rapidly and specifically under physiological conditions.

[0110] 9. Staudinger ligation: This method involves the reaction between an azide and a phosphine to form an amide bond; the method offers high chemoselectivity and can be performed in aqueous media, making it suitable for bioconjugation.

[0111] 10. Photoclick chemistry: Light-induced cycloadditions, such as the reaction between tetrazoles and alkenes, provide spatiotemporal control over the conjugation process. This can be particularly useful for in situ labeling or activation of conjugates. 11. Ligation to Fab nucleotide-binding sites: A method which uses indole-based linkers to target lysine residues in the Fab region’s nucleotide-binding pocket for sitespecific conjugation. See e.g., Biomedicine & Pharmacotherapy 161 (2023) 114408.

[0112] Each conjugation method offers unique advantages and presents specific challenges, and the choice of conjugation method may depend on factors, as readily appreciated by the skilled artisan, such as the nature of the targeting moiety, the payload, and the desired properties of the final conjugate. In many cases, the targeting moiety or payload may be modified to introduce specific functional groups that facilitate conjugation. For example, antibodies may be engineered to incorporate non-natural amino acids with bioorthogonal reactive groups, allowing for conjugation.

[0113] The skilled artisan can modulate reaction conditions, such as pH, temperature, and reagent concentrations, to achieve desired conjugation efficiencies and product homogeneity. The use of organic co-solvents, catalysts, or additives may be employed to enhance reaction rates or selectivity. In some cases, multiple conjugation steps may be used to assemble complex conjugates with multiple payloads or functionalities.

[0114] In certain aspects, the present disclosure provides conjugates having a structure represented by Formula A or a pharmaceutically acceptable salt thereof:Attorney Docket No.: LCH-03725

[0115]

[0116] Formula A

[0117] wherein

[0118] Ab is an anti-B Cell Maturation Antigen (BCMA) antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein

[0119] the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

[0120] the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13;

[0121] each B is independently an active agent;

[0122] each L is independently a linker comprising an alkylene or a heteroalkylene, wherein L comprises one or more of:

[0123] (i) one or more unsaturated bonds;

[0124] (ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain); (iii) at least one C1-20 alkyl substituent;

[0125] (iv) at least one isoprenyl group having a structure represented by General Formula II:

[0126] [General Formula II]

[0127]

[0128] (v) a structure represented by formula B:

[0129]

[0130] Formula B

[0131] wherein:

[0132] X1and X2are each independently N(R3), O, or S;Attorney Docket No.: LCH-03725

[0133] R1is H, alkyl, or cycloalkyl;

[0134] R2is alkyl or cycloalkyl; or R1and R2combine to form a cycloalkyl;

[0135] R3is H, alkyl, aryl, or aralkyl;

[0136] Ya1is a connection to Ta;

[0137] Ya2is a connection to Ab;

[0138] each Tais independently a cleavage group, preferably a self-immolative group, more preferably a structure represented by Formula C:

[0139]

[0140] Formula C

[0141] wherein:

[0142] G is independently a glucuronic acid moiety

[0143]

[0144] RAand RBare each independently hydrogen, Ci-s alkyl, or C3-8 cycloalkyl;

[0145] Rcis hydrogen or a carboxyl -protecting group;

[0146] each RDis independently hydrogen or a hydroxyl-protecting group;

[0147] W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR', -SONR'-, or -PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of Formula C and the NR' is bonded to Ya3;

[0148] R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, Ci-8 alkylthio, mono- or di-Ci-8 alkylamino, C3-20 heteroaryl, or Ce-2oaryl;

[0149] each Z is independently C1-8 alkyl, halogen, cyano, or nitro;

[0150] n is 0, 1, 2, or 3;

[0151] Ya3is a connection to L;

[0152] Ya4is a connection to B; and

[0153] nA and nB are each independently an integer from 1 to 20.Attorney Docket No.: LCH-03725

[0154] In certain embodiments, when n is 0, the ring does not comprise a Z substituent, and the remaining positions of the ring, other than those bonded to G and W, are occupied by hydrogen.

[0155] In certain embodiments, R1is alkyl, preferably methyl. In other embodiments, R1is H. In certain embodiments, R2is alkyl, preferably methyl. In certain embodiments, R1and R2combine to form a cycloalkyl, preferably cyclobutyl. In certain embodiments, X1is N(R3). In certain embodiments, R3is H. In certain embodiments, X2is O.

[0156] In certain preferred embodiments, L comprises a structure represented by Formula B:

[0157]

[0158] Formula B

[0159] wherein

[0160] X1and X2are each independently N(R3), O, or S;

[0161] R1is H, alkyl, or cycloalkyl;

[0162] R2is alkyl or cycloalkyl; or R1and R2combine to form a cycloalkyl;

[0163] R3is H, alkyl, aryl, or aralkyl;

[0164] Ya1is a connection to Ta; and

[0165] Ya2is a connection to Ab.

[0166] In certain preferred embodiments, Tarepresents a structure represented by Formula C:

[0167]

[0168] Formula C

[0169] wherein

[0170] "

[0171] G is independently a glucuronic acid moiety

[0172]

[0173] Attorney Docket No.: LCH-03725

[0174] RAand RBare each independently hydrogen, Ci-s alkyl, or C3-8 cycloalkyl;

[0175] Rcis hydrogen or a carboxyl -protecting group;

[0176] each RDis independently hydrogen or a hydroxyl -protecting group;

[0177] Wis -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR', -SONR'-, or -PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of Formula C and the NR' is bonded to Ya3;

[0178] R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, C3-20 heteroaryl, or Ce-2oaryl;

[0179] each Z is independently C1-8 alkyl, halogen, cyano, or nitro;

[0180] n is 0, 1, 2, or 3;

[0181] Ya3is a connection to L; and

[0182] Ya4is a connection to B.

[0183] In certain embodiments, the conjugates disclosed herein have a structure represented by General Formula I or a pharmaceutically acceptable salt thereof:

[0184] [General Formula I]

[0185]

[0186] wherein

[0187] Ab is the anti-B Cell Matmation Antigen (BCMA) antibody or antigen-binding fragment thereof;

[0188] each B’ is an active agent;

[0189] "

[0190] each G is independently a glucuronic acid moiety o

[0191]

[0192] Rcis hydrogen or a carboxyl -protecting group;

[0193] each RDis independently hydrogen or a hydroxyl -protecting group;

[0194] RAand RBare each independently hydrogen, C1-8 alkyl, or C3-8 cycloalkyl;Attorney Docket No.: LCH-03725

[0195] Wis -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR', -SONR'-, or -PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of General Formula I and the NR' is bonded to L;

[0196] R' and R" are each independently hydrogen, Ci-s alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, C3-20 heteroaryl, or Ce-2oaryl;

[0197] each Z is independently C1-8 alkyl, halogen, cyano, or nitro;

[0198] n is 0, 1, 2, or 3;

[0199] each L is independently a linker comprising a C1-50 alkylene or C1-50 heteroalkylene, and L comprises one or more of:

[0200] (i) one or more unsaturated bonds;

[0201] (ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain);

[0202] (iii) at least one C 1-20 alkyl substituent; and

[0203] (iv) at least one isoprenyl group having a structure represented by General Formula II:

[0204] [General Formula II]

[0205]

[0206] 1 is nA and m is nB.

[0207] In certain embodiments, L comprises: a C1-50 alkylene or C 1-50 heteroalkylene wherein the Ci-50 alkylene or C1-50 heteroalkylene comprises one or more of:

[0208] (i) one or more unsaturated bonds;

[0209] (ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain); (iii) at least one C1-20 alkyl substituent; and

[0210] (iv) at least one isoprenyl group having a structure represented by General Formula II:

[0211] [General Formula II]

[0212]

[0213] 1 and m are each independently 1 to 20.

[0214] In certain embodiments, when n is 0, the ring does not comprise a Z substituent, and the remaining positions of the ring, other than those bonded to G and W, are occupied by hydrogen.Attorney Docket No.: LCH-03725

[0215] In certain embodiments, each W is independently selected from -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR', -SONR'-, and -PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of General Formula I and the NR' is bonded to L. In certain embodiments, each L independently comprises a C1-50 alkylene or C 1-50 heteroalkylene wherein the Ci-50 alkylene or C1-50 heteroalkylene comprises one or more of:

[0216] (i) one or more unsaturated bonds;

[0217] (ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain); (iii) at least one C1-20 alkyl substituent; and

[0218] (iv) at least one isoprenyl group having a structure represented by General Formula II:

[0219] [General Formula II]

[0220]

[0221] 1 and m are each independently 1 to 20.

[0222] In certain embodiments, each B is independently an active agent.

[0223] The conjugate utilizing the disclosed linkers demonstrates superior efficacy compared to Blenrep, which utilizes a non-cleavable linker. This enhanced performance is attributed to reduced toxicity, improved plasma stability, and optimized pharmacokinetics when the disclosed linkers are combined with the antibodies and cytotoxic agents disclosed herein. The linkers minimize off-target effects, leading to a better safety profile, enhance the stability of the drug in the bloodstream, ensuring sustained activity over time, and exhibit favorable absorption, distribution, metabolism, and excretion (ADME) characteristics, resulting in improved therapeutic outcomes.

[0224] Analytical Methods for Measuring Advantages:

[0225] To quantify and confirm these advantages, the following analytical methods are employed: cytotoxicity assays (e.g., MTT or LDH assays) and in vivo toxicity studies for toxicity assessment, LC-MS / MS or HPLC analysis to monitor degradation and stability profiles in plasma for plasma stability, and LC-MS / MS or bioanalytical methods for determining drug concentration in plasma over time, supported by compartmental PK modeling for pharmacokinetics. These analyses collectively validate the superior efficacy profile of the disclosed linker-based conjugates compared to Blenrep.

[0226] In some embodiments, Ab comprises a heavy chain variable region comprising: the amino acid sequence of SEQ ID NO: 15; a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15 while maintaining the heavy chain CDR1Attorney Docket No.: LCH-03725

[0227] comprising the amino acid sequence of SEQ ID NO: 2, the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 while maintaining the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0228] In some embodiments, Ab comprises a light chain variable region comprising: the amino acid sequence of SEQ ID NO: 16; a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 while maintaining the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16 while maintaining the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, Ab comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

[0229] In some embodiments, Ab is a humanized antibody or a human antibody. In some embodiments, Ab is selected from a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody. In some embodiments, Ab comprises an IgA, IgG, IgM, IgE, or IgD constant domain or is an IgA, IgG, IgM, IgE, or IgD antibody. In some embodiments, Ab comprises an IgG constant domain or is an IgG antibody. In some embodiments, Ab comprises an IgGl, IgG2, IgG3, or IgG4 constant domain or is an IgGl, IgG2, IgG3, or IgG4 antibody.

[0230] In some embodiments, Ab comprises LALA mutations in a heavy chain constant region. In some embodiments, the LALA mutations comprise L234A and L235A according to EU numbering convention. In some embodiments, the LALA mutations are in place of amino acids corresponding to amino acids 238-239 in SEQ ID NO: 17. In some preferredAttorney Docket No.: LCH-03725

[0231] embodiments, Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20.

[0232] In some embodiments, Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20 or 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and alight chain comprising the amino acid sequence of SEQ ID NO: 18. In some preferred embodiments, Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 20 and alight chain comprising the amino acid sequence of SEQ ID NO: 18.

[0233] In some embodiments, each

[0234]

[0235] some embodiments, RAand RBare each hydrogen. In some embodiments, Rcis hydrogen. In some embodiments, n is 0. In some embodiments, each W is -C(O)NR'-. In some embodiments, each RDis independently hydrogen. In some embodiments, RAand RBare each hydrogen; n is 0; and each W is -C(O)NR’-.

[0236] In some embodiments, L comprises a C1-50 heteroalkylene, wherein the C1-50 heteroalkylene comprises an isoprenyl group having a structure represented by General Formula Ila:

[0237] [General Formula Ila]

[0238]

[0239] wherein n4is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, L comprises a peptide comprising at least one hydrophilic amino acid. In some embodiments, the peptide comprises an amino acid having a side chain having a moiety that bears a charge at neutral pH in aqueous solution (e.g., an amine, guanidine, or carboxyl moiety). In some embodiments, the peptide comprises an amino acid selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, histidine, arginine and threonine.Attorney Docket No.: LCH-03725

[0240] In some embodiments, L comprises an oxime. In some embodiments, the oxygen atom of the oxime is on the side of L linked to W and the carbon atom of the oxime is on the side of L linked to Ab. In some embodiments, the carbon atom of the oxime is on the side of L linked to W and the oxygen atom of the oxime is on the side of L linked to Ab. In some embodiments, L is a Ci-50 heteroalkylene comprising an oxime. In some embodiments, the oxygen atom of the oxime is on the side of L linked to W. In some embodiments, the oxygen atom of the oxime is on the side of L linked to Ab. In some embodiments, L comprises at least one isoprenyl group. In some embodiments, L comprises at least one isoprenyl group and the at least one isoprenyl group covalently bonds the oxime to Ab (e.g., the at least one isoprenyl group directly or indirectly bonds the oxime to Ab).

[0241] In some embodiments, L comprises a connecting unit represented by General Formula Illa or General Formula Illb:

[0242] [General Formula Illa]

[0243] -(CH2)r(V(CH2)p)q- [General Formula Illb]

[0244] -(CH2CH2X)W- V is a single bond, -O-, -S-, - NR21-, -C(O)NR22-, -NR23C(O)-, -NR24SO2-, or -SO2NR25-; X is -O-, Ci-8 alkylene, or -NR21-;

[0245] R21to R25are each independently hydrogen, Ci-6 alkyl, Ci-6 alkyl Ce-2o aryl, or Ci-6 alkyl-C3-2o heteroaryl;

[0246] r is 0 to 10; p is 0 to 10; q is 1 to 20; and w is 1 to 20.

[0247] In some embodiments, q is 1 to 10. In some embodiments, r is 1 or 2. In some embodiments, p is 1 or 2. In some embodiments, V is -O-. In some embodiments, q is 1 to 10; r and p are each 1 or 2; and V is -O-. In some embodiments, X is -O-. In some embodiments,

[0248] L comprises at least one polyethylene glycol unit represented by

[0249]

[0250]

[0251] In some embodiments, L comprises an oxime and the at least one polyethylene glycol unit covalently bonds the oxime to W.

[0252] In some embodiments, L further comprises a binding unit formed by a reaction between an alkyne and an azide or between an aldehyde or ketone group and hydrazine orAttorney Docket No.: LCH-03725

[0253] hydroxylamine. In some embodiments, L further comprises a binding unit represented by General Formula IVa, IVb, IVc, IVd, or IVe:

[0254] [General Formula IVa]

[0255] ]

[0256]

[0257] [General Formula IVc]

[0258]

[0259] [General Formula IVd]

[0260]

[0261] wherein

[0262] L1is each independently a single bond or C 1-30 alkylene; and

[0263] R11 is hydrogen or C1-10 alkyl.

[0264] In some embodiments, L is covalently bonded to Ab by a thioether bond and the thioether bond comprises a sulfur atom of a cysteine of Ab. In some embodiments, Ab at the C-terminus comprises an amino acid motif that is recognized by an isoprenoid transferase. In some embodiments, the isoprenoid transferase is farnesyl protein transferase (FTase) orAttorney Docket No.: LCH-03725

[0265] geranylgeranyl transferase (GGTase). In some embodiments, L is covalently bonded to Ab by a thioether bond and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif. In some embodiments, the amino acid motif comprises a CYYX sequence, wherein: C is cysteine; each Y is an aliphatic amino acid; and X is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine. In some embodiments, each Y is independently selected from alanine, isoleucine, leucine, methionine, and valine. In some embodiments, the amino acid motif comprises a CVIM (SEQ ID NO: 22) or CVLL (SEQ ID NO: 23). In some embodiments, at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine. In some embodiments, the amino acid motif has the sequence GGGGGGGCVIM (SEQ ID NO: 21).

[0266] In some embodiments, the conjugate comprises

[0267]

[0268]

[0269] Attorney Docket No.: LCH-03725

[0270] embodiments, the conjugate comprises

[0271]

[0272] . in some embodiments, the conjugate

[0273]

[0274] In some embodiments, L is covalently bonded to Ab by an amide. In some embodiments, the amide is formed from the nitrogen of a side chain of a lysine residue on the Ab. In some embodiments, the amide is part of the side chain of a lysine residue.

[0275] In some embodiments, L comprises

[0276]

[0277] . in some

[0278]

[0279] embodiments, L comprises 0 In some embodiments, L comprises

[0280]

[0281] H H

[0282]

[0283] some embodiments, L comprises S . In some embodiments, L comprises

[0284]

[0285] embodiments, L comprises O o . In some embodiments, L comprises

[0286]

[0287] . , p

[0288] In some embodiments, L is covalently bonded to Ab by a disulfide. In some embodiments, the disulfide is formed from the side chain of a serine residue. In some embodiments, the disulfide is part of the side chain of a serine residue. In some embodiments,Attorney Docket No.: LCH-03725

[0289]

[0290] In some embodiments, L is branched and comprises: i) a branching unit covalently coupled to Ab by a primary linker; ii) a first branch which couples a first B’ to the branching unit; and iiia) a second branch which couples a second B’ to the branching unit; or iiib) a second branch, in which an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) is covalently coupled to the branching unit. In some embodiments, L comprises a second branch which couples a second B’, via a cleavage group, to the branching unit. In some embodiments, L comprises a second branch, in which an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) is covalently coupled to the branching unit.

[0291] In some embodiments, the branching unit has a structure represented by

[0292]

[0293] [General Formula Va]

[0294]

[0295] [General Formula Vb]Attorney Docket No.: LCH-03725

[0296]

[0297] [General Formula Vc]

[0298]

[0299] [General Formula Ve]

[0300] wherein

[0301] G1, G2, and G3each independently represents a bond,

[0302]

[0303] ,

[0304]

[0305] R60is hydrogen or C1-30 alkyl;

[0306] R70is hydrogen or L5-COOR80;

[0307] R80is hydrogen or C1-30 alkyl

[0308] L2, L3, L4’, and L5are each independently a bond or -Cn’FFn-; and

[0309] n' is 1 to 10.

[0310] In some embodiments, at least one branched linker is covalently coupled to Ab; and at least two B’ are covalently coupled to the branched linker. In some embodiments, the conjugateAttorney Docket No.: LCH-03725

[0311] comprises 1, 2, 3, or 4 branched linkers and each branched linker comprises two B’. In some embodiments, the branching unit comprises a lysine residue.

[0312] In some embodiments, the conjugate comprises a structure represented by:

[0313]

[0314] or a pharmaceutically acceptable salt thereof; wherein

[0315] B' and B" are each an active agent;

[0316] nl’ to n3’ are each independently 0 to 30;

[0317] AA is an amino acid group; and

[0318] the wavy bond represents a connection to Ab.

[0319] In certain preferred embodiments, L and (Ta-B)nA together have a structure represented by Formula D or a pharmaceutically acceptable salt thereof:Attorney Docket No.: LCH-03725

[0320]

[0321] Formula D

[0322] wherein

[0323] L1Acomprises a C1-C100 alkylene;

[0324] X3is -N(R4)C(O)-, -C(O)N(R4)-, -C(O)N(R4)(CH2)Z-, -(CH2)ZC(O)N(R4)-, -C(O)-, -C(O)O-, -OC(O)-, -S(O2)N(R4)-, -N(R4)S(O2)-, -P(O)(R4)N(R5)-, -N(R5)P(O)(R4)-, - P(O)2N(R5)-,

[0325] or -N(R5)P(O)2-;

[0326] z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0327] Y1, Y2, and Y3are each independently O, N(R6), or S;

[0328] V1, V2, and V3are each independently a bond, N, NH, C, CH, or CH2;

[0329] R4, R5, and R6are each independently H, alkyl, aryl, or aralkyl;

[0330] L3, L4, and L5each independently comprise an alkylene;

[0331] T1, T2, and T3are each independently a bond, -O-, or a cleavage group;

[0332] B1, B2, and B3are each independently an active agent (e.g., a drug or a toxin), H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, alkylamino, guanidinyl, amido, cyano, azido, alkylthio, cycloalkyl, alkylsulfonyl, sulfonamide, aryl, heteroaryl, or heterocyclyl; or

[0333] T1and B1, T2and B2, or T3and B3together represent a branched moiety (e.g., a branched moiety substituted with one or more active agents); and

[0334] ml, m2, and m3 are each independently 1, 2, 3, or 4.

[0335] In certain embodiments, the conjugate has a structure represented by Formula V:

[0336]

[0337] Attorney Docket No.: LCH-03725

[0338] wherein

[0339] R1is H, alkyl or cycloalkyl;

[0340] R2is alkyl or cycloalkyl; or R1and R2combine to form a cycloalkyl;

[0341] X1and X2are each independently N(R3), O, or S;

[0342] R3is H, alkyl, aryl, or aralkyl;

[0343] each B is an active agent (e.g., a drug or a toxin);

[0344] T is a cleavage group;

[0345] L1is a covalent linker;

[0346] L2is a covalent linker;

[0347] Lg is Ab; and

[0348] nl is l, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0349] In certain embodiments, R1is alkyl, preferably methyl. In other embodiments, R1is H. In certain embodiments, R2is alkyl, preferably methyl. In certain embodiments, R1and R2combine to form a cycloalkyl, preferably cyclobutyl. In certain embodiments, X1is N(R3). In certain embodiments, R3is H. In certain embodiments, X2is O.

[0350] In certain embodiments, T comprises a structure represented by Formula VI:

[0351]

[0352] Formula VI

[0353] wherein

[0354] each Rb1, Rb2, Rb3, and Rb4is independently H, alkyl, aralkyl, or an oxygen protecting group;

[0355] each Rb5is alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, cycloalkyl, alkylsulfonyl, or sulfonamide;

[0356] Wb1is independently - -*C(O)O-, -*S(

[0357]

[0358] Attorney Docket No.: LCH-03725

[0359] wherein the C(0), N, CH2, S, or P marked with an * is bonded to the phenyl ring of Formula VI,

[0360] Rb7and Rb8are each independently hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, alkylamino, heteroaryl, or aryl;

[0361] each Jb1and Jb2are independently H or alkyl; or

[0362] Jb1and Jb2combine to form an oxo;

[0363] tai is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0364] nal is 0, 1, 2, or 3;

[0365] Ab1represents a linkage to B; and

[0366] Ab2represents a linkage to L1.

[0367] In certain embodiments, each Rb1is H. In certain embodiments, each Rb2is H. In certain embodiments, each Rb3is H. In certain embodiments, each Rb5is H. In certain embodiments, each nal is 0. When nal is 0, the ring does not comprise a Rb5substituent, and the remaining positions of the ring are occupied by hydrogen. In certain embodiments, each Wb1is -*C(O)N(Rb7)-. In certain embodiments, each Rb7is H. In certain embodiments, Jb1is H. In certain embodiments, Jb2is H. In other embodiments, Jb1and Jb2combine to form oxo. In certain embodiments, L1is linear. In certain embodiments, each nl is 1. In other embodiments, each nl is 2. In other embodiments, each nl is 3. In other embodiments, each nl is 4. In yet other embodiments, each nl is 5. In certain embodiments, L1comprises a C1-C100 alkylene. In certain embodiments, L1comprises a C1-C50 alkylene. In certain embodiments, L1further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, or 5 ethylene glycol moieties. In certain preferred embodiments, L1further comprises 5 oligoethylene glycol moieties. In other embodiments, the oligoethylene glycol comprises 6 ethylene glycol moieties. In certain embodiments, L1further comprises an amido moiety. In certain embodiments, L1comprises 1, 2, 3, 4, 5, or 6 amido moieties. In certain preferred embodiments, L1comprises 1 or 2 amido moieties. In certain preferred embodiments, the amido moieties have a structure represented by O O

[0368]

[0369] , wherein the wavy lines represent connections to the remainder of L1. In certain embodiments, L1further comprises a heteroaryl (e.g., pyrazolyl, triazolyl). In certain embodiments, L1comprises a structure represented by Formula Vila or Vllb:Attorney Docket No.: LCH-03725

[0370]

[0371] Vila Vllb

[0372] wherein

[0373] each Ad1represents a linkage to T or B; and

[0374] each A2represents a linkage to X1.

[0375] In certain embodiments, L1comprises a structure represented by Formula Villa, Vlllb, or VIIIc:

[0376]

[0377] Villa Vlllb VIIIc wherein

[0378] each A1represents a linkage to B; and

[0379] each A2represents a linkage to X1.

[0380] In certain embodiments, L1comprises a C1-C100 alkylene. In certain embodiments, L1comprises a C1-C50 alkylene. In certain embodiments, L1further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, 4, 5, or 6 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol comprises 5 ethylene glycol moieties. In certain embodiments, L1further comprises an amido moiety. In certain embodiments, L1comprises 1, 2, 3, 4, 5, or 6 amido moieties. In certain preferred embodiments, L1comprises 1 or 2 amido moieties. In certain preferred embodiments, the amido moieties have 0 0

[0381] a structure represented by

[0382]

[0383] , wherein the wavy lines represent connections to the remainder of L1. In certain embodiments, L1further comprises at least one amino acid. In certain embodiments, L1further comprises a heteroaryl (e.g., pyrazolyl, triazolyl).

[0384] In certain embodiments, L1comprises a structure represented by Formula IXa or IXb:Attorney Docket No.: LCH-03725

[0385]

[0386] IXa IXb

[0387] wherein

[0388] each Ae1represents a linkage to T or B; and

[0389] each A4represents a linkage to X1.

[0390] In certain embodiments, L1comprises a structure represented by Formula Xa, Xb, or Xc:

[0391]

[0392] wherein

[0393] each A3represents a linkage to B; and

[0394] each A4represents a linkage to X1.

[0395] In certain embodiments, L1is substituted with an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, or 4 ethylene glycol moieties. In certain embodiments, the oligoethylene glycol is substituted with carboxyalkyl (e.g., carboxyethyl). In certain embodiments, the oligoethylene glycol is substituted with amino (e.g., dimethylamino).

[0396] In certain embodiments, L1further comprises at least one amino acid. In certain embodiments, L2comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, L1comprises 1 amino acid. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is a hydrophilic amino acid. In certain embodiments, the amino acid is arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In certain embodiments, the amino acid is arginine, aspartate, glutamate, histidine, lysine, or ornithine. In certain embodiments, L1further comprises an amino acid (e.g., a hydrophilic amino acid). In certain embodiments, L1furtherAttorney Docket No.: LCH-03725

[0397] comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., hydrophilic amino acids). In certain embodiments, the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine. In certain embodiments, the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine. In certain embodiments, the amino acid(s) are selected from arginine, aspartate, glutamate, histidine, lysine, and ornithine. In certain embodiments, the amino acid(s) are selected from aspartate and glutamate. In certain preferred embodiments, the amino acid(s) are sarcosine.

[0398] In certain embodiments, wherein L1is branched. In some embodiments, the branched linker of the conjugate comprises an amino acid having a side chain with a moiety that bears a charge at neutral pH in aqueous solution, preferably an arginine, aspartate, glutamate, lysine, or ornithine. This amino acid may be present anywhere in the branched linker. For example, it may covalently link an oxime of the branched linker to a polyethylene glycol unit of the branched linker. Alternatively or additionally, such an amino acid may by present in a secondary linker, optionally in each secondary linker.

[0399] The branching unit of the branched linker may comprise an amine or an amino acid having a side chain with a group capable of participating in an amide or ester (preferably amide) bond.

[0400] In some embodiments, the branching unit is a lysine unit. The lysine unit may comprise modifications, such as methylation of the s-amino group, giving methyl-, dimethyl-, and trimethyllysine and even acetylation, sumoylation, and / or ubiquitination. The branching unit may comprise many other amino acids in various embodiments of the invention. For example, an amino acid of the branching unit may be selected from lysine, 5 -hydroxylysine, 4-oxalysine, 4-thialysine, 4-selenalysine, 4-thiahomolysine, 5,5-dimethyllysine, 5,5-difluorolysine, trans-4-dehydrolysine, 2,6-diamino-4-hexynoic acid, cA-4-dehydrolysine, 6-A-methyl lysine, diaminopimelic acid, ornithine, 3-methylornithine, a-methylornithine, citrulline, and homocitrulline. The branching unit may comprise a L-amino acid or a D-amino acid. The branching unit may comprise an a-amino acid or a -amino acid. The branching unit may comprise a naturally-occurring amino acid or a non-naturally-occurring amino acid. The branching unit of the conjugate may comprise other amino acids instead of or in addition to lysine.

[0401] In some embodiments, the branched linker comprises a hydrophilic amino acid, e.g., to increase the water solubility of the conjugate, linker, and / or precursors of the conjugate. TheAttorney Docket No.: LCH-03725

[0402] hydrophilic amino acid may be located proximal to the active agent, proximal to the antibody or antigen-binding fragment thereof, or interposed anywhere along the branched linker, e.g., in the primary linker and / or a secondary linker, preferably each secondary linker. In some embodiments, the hydrophilic amino acid may be, for example, arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In some preferred embodiments, the hydrophilic amino acid is arginine, histidine, lysine, aspartate, or glutamate.

[0403] In certain embodiments, L1further comprises at least one amino acid. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is a hydrophilic amino acid. In certain embodiments, the amino acid is arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In certain embodiments, the amino acid is arginine, aspartate, glutamate, histidine, lysine, or ornithine. In certain embodiments, the amino acid is aspartate or glutamate. In certain embodiments, L1comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, L1comprises 1 amino acid.

[0404] In certain embodiments, a) L1and (B)nior b) L1and (B-T)nicomprise:

[0405] i) a branching unit covalently coupled to X1by a primary linker;

[0406] ii) a first branch, which couples a first active agent, via a first cleavage group, to the branching unit; and

[0407] iiia) a second branch, which couples a second active agent, via a second cleavage group, to the branching unit; or

[0408] iiib) a second branch which couples an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) to the branching unit.

[0409] In certain embodiments, a) L1and (B)nior b) L1and (B-T)ni comprise a second branch which couples a second active agent, via a second cleavage group, to the branching unit. In certain embodiments, a) L1and (B)nior b) L’and (B-T)ni comprise a second branch which couples an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) to the branching unit. In certain embodiments, the branching unit is an amino acid. In certain embodiments, the branching unit has a structure represented by Formula Xia, Xlb, XIc, Xld, or Xie:Attorney Docket No.: LCH-03725

[0410]

[0411] R30is H or alkyl;

[0412] R40is H, alkyl or LD-CO2R50;

[0413] R50is H or alkyl; and

[0414] LA, LB, LC, and LDare each independently a bond or alkylene.

[0415] In certain embodiments, the branching unit has a structure represented by Formula Xlla:

[0416]

[0417] Formula Xlla

[0418] wherein R30is H or alkyl.

[0419] In certain embodiments, the branching unit has a structure represented by Formula Xllb:

[0420]

[0421] Attorney Docket No.: LCH-03725

[0422] Formula Xllb

[0423] wherein R30is H or alkyl.

[0424] In certain embodiments, each cleavage group has a structure represented by Formula XIII or a pharmaceutically acceptable salt thereof:

[0425]

[0426] Formula XIII

[0427] wherein

[0428] Bw is an active agent;

[0429] each Rw2, Rw3, Rw4, and Rw5is independently H, alkyl, aralkyl, or an oxygen protecting group;

[0430] each Rw6is alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, cycloalkyl, alkylsulfonyl, or sulfonamide;

[0431] Ww1is independently -*C(O)-, -*C(O)N(Rw7)-, -*N(Rw7)C(O)-, -*(CH2)twiN(Rw7)C(O)-, - *C(O)O-, -*S(O2)N(RW7)-, -*P(O)( RW8)N(RW7)-, -*S(O)N(RW7)-, or -*P(O2)N(Rw7)-, wherein the C(O), N, CH2, S, or P marked with an * is bonded to the phenyl ring of Formula XIII;

[0432] Rw7and Rw8are each independently hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, alkylamino, heteroaryl, or aryl;

[0433] twl is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0434] nwl is 0, 1, 2, or 3;

[0435] Jw1is H or alkyl;

[0436] Jw2is H or alkyl; orAttorney Docket No.: LCH-03725

[0437] Jw1and Jw2combine to form an oxo; and

[0438] Aw2represents a linkage to the secondary linker or the branching unit.

[0439] In certain embodiments, when nwl is 0, the ring does not comprise a Rw6substituent, and the remaining positions of the ring are occupied by hydrogen.

[0440] In certain embodiments, a) L1and (B)nior b) L1and (B-T)nitogether have a structure represented by Formula XIV or a pharmaceutically acceptable salt thereof:

[0441]

[0442] Formula XIV

[0443] wherein

[0444] L1Acomprises a C1-C100 alkylene;

[0445] X3is -N(R4)C(O)-, -C(O)N(R4)-, -C(O)N(R4)(CH2)Z-, -(CH2)ZC(O)N(R4) -, -C(O)-, -C(O)O-, -OC(O)-, -S(O2)N(R4)-, -N(R4)S(O2)-, -P(O)(R4)N(R5)-, -N(R5)P(O)(R4)-, - P(O)2N(R5)-,

[0446] or -N(R5)P(O)2-;

[0447] z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0448] Y1, Y2, and Y3are each independently O, N(R6), or S;

[0449] V1, V2, and V3are each independently a bond, N, NH, C, CH, or CH2;

[0450] R4, R5, and R6are each independently H, alkyl, aryl, or aralkyl;

[0451] L3, L4, and L5each independently comprise an alkylene;

[0452] T1, T2, and T3are each independently a bond, -O-, or a cleavage group;

[0453] B1, B2, and B3are each independently an active agent (e.g., a drug or a toxin), H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, alkylamino, guanidinyl amido, cyano, azido, alkylthio, cycloalkyl, alkylsulfonyl, sulfonamide, aryl, heteroaryl, or heterocyclyl; or

[0454] T1and B1, T2and B2, or T3and B3together represent a branched moiety (e.g., a branched moiety substituted with an active agent; and

[0455] ml, m2, and m3 are each independently 1, 2, 3, or 4.

[0456] Those of skill in the art will recognize that variables V1and ml, V2and m2, and V3and m3 are related. For example, those of skill in the art will understand that if ml is 3, then V3Attorney Docket No.: LCH-03725

[0457] will have 3 open valencies. Likewise, if ml is 1, then V1will have at least one open valency. Simply put, the skilled artisan will understand that V1and ml, V2and m2, and V3and m3 will be combined in such a way that stable configurations are obtained based on the valencies required.

[0458] In certain embodiments, L1Acomprises a C1-C50 alkylene.

[0459] In certain embodiments, Y1is O.

[0460] In certain embodiments, Y2is O.

[0461] In certain embodiments, Y3is O.

[0462] In certain embodiments, X3is N-C(O)N(R4)-. In certain embodiments, R4is H.

[0463] In certain embodiments, L3comprises a C1-C100 alkylene. In certain embodiments, L3comprises a C1-C50 alkylene. In certain embodiments, L3further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, 4, 5, or 6 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol comprises 5 ethylene glycol moieties. In certain embodiments, L3further comprises an amido moiety. In certain embodiments, L3comprises 1, 2, 3, 4, 5, or 6 amido moieties. In certain preferred embodiments, L3comprises 1 or 2 amido moieties. In certain preferred embodiments, the amido moieties have

[0464] a structure represented by

[0465]

[0466] , wherein the wavy lines represent connections to the remainder of L3. In certain embodiments, L3further comprises at least one amino acid In certain embodiments, L3further comprises a heteroaryl (e.g., pyrazolyl, triazolyl). In certain embodiments, L3comprises a structure represented by Formula XVa or XVb:

[0467]

[0468] XVa XVb

[0469] wherein

[0470] each Ax1represents a linkage to V1; and

[0471] each Ax2represents a linkage to Y1.

[0472] In certain embodiments, L3is substituted with an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, or 4 ethylene glycol moieties. InAttorney Docket No.: LCH-03725

[0473] certain embodiments, the oligoethylene glycol is substituted with carboxyalkyl (e.g., carboxyethyl). In certain embodiments, the oligoethylene glycol is substituted with amino (e.g., dimethylamino).

[0474] In certain embodiments, L3further comprises at least one amino acid. In certain embodiments, L3comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, L3comprises 1 amino acid. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is a hydrophilic amino acid. In certain embodiments, the amino acid is arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In certain embodiments, the amino acid is arginine, aspartate, glutamate, histidine, lysine, or ornithine. In certain embodiments, L3further comprises an amino acid (e.g., a hydrophilic amino acid). In certain embodiments, L3further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., hydrophilic amino acids). In certain embodiments, the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine. In certain embodiments, the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine. In certain embodiments, the amino acid(s) are selected from arginine, aspartate, glutamate, histidine, lysine, and ornithine. In certain embodiments, the amino acid(s) are selected from aspartate and glutamate. In certain preferred embodiments, the amino acid(s) are sarcosine.

[0475] In certain embodiments, V1is a bond. In certain embodiments, V1is a bond and ml is 1.

[0476] In certain embodiments, ml is 1. In other embodiments, ml is 2.

[0477] In certain embodiments, V1is N and V1, T1, and B1together represent a branched moiety having a structure represented by Formula XVIa or a pharmaceutically acceptable salt thereof:

[0478]

[0479] Formula XVIa

[0480] wherein

[0481] Ax3represents a linkage to L3;

[0482] Lx1and Lx2each independently comprise an alkylene;Attorney Docket No.: LCH-03725

[0483] Tx1and Tx2are each independently a bond or a cleavage group; and

[0484] Bx1and Bx2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0485] In certain embodiments, V1is CH and V1, T1, and B1together represent a branched moiety having a structure represented by Formula XVIb or a pharmaceutically acceptable salt thereof::

[0486]

[0487] Formula XVIb

[0488] wherein

[0489] Ax3represents a linkage to L3;

[0490] Lx1and Lx2each independently comprise an alkylene;

[0491] Tx1and Tx2are each independently a bond or a cleavage group; and

[0492] Bx1and Bx2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0493] In certain embodiments, V1is CH and V1, T1, and B1together represent a branched moiety having a structure represented by Formula XVIc or a pharmaceutically acceptable salt thereof:

[0494]

[0495] Formula XVIc

[0496] wherein

[0497] Ax3represents a linkage to L3; and

[0498] Lx1and Lx2each independently comprise an alkylene;

[0499] O O

[0500] Gx1, Gx2, and Gx3are each independently an alkylene,

[0501]

[0502] each Rx1is independently H, alkyl, or aralkyl;

[0503] Tx1and Tx2are each independently a bond or a cleavage group; andAttorney Docket No.: LCH-03725

[0504] Bx1and Bx2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0505] In certain embodiments, V1is C and V1, T1and B1together represent a branched moiety having a structure represented by Formula XVId or a pharmaceutically acceptable salt thereof:

[0506]

[0507] Formula XVId

[0508] wherein:

[0509] Ax3represents a linkage to L3;

[0510] Lx1, Lx2, and Lx3each independently comprise an alkylene;

[0511] Tx1, Tx2, and Tx3are each independently a bond or a self-immolative moiety; and

[0512] Bx1, Bx2, and Bx3are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0513] In certain embodiments, the heteroaryl of Bx1, Bx2, or Bx3is further substituted with alkylene or amido. In certain embodiments, the amido of Bx1, Bx2, or Bx3is further substituted with methyl or penta-l,2,3,4,5-ol.

[0514] In certain embodiments, Lx1comprises a C1-C100 alkylene. In certain embodiments, Lx1comprises a C1-C50 alkylene. In certain embodiments, Lx1further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol comprises 1, 2, 3, or 4 ethylene glycol moieties. In certain preferred embodiments, the oligoethylene glycol further comprises 4 ethylene glycol moieties.

[0515] In certain embodiments, Lx2comprises a C1-C100 alkylene. In certain embodiments, Lx2comprises a C1-C50 alkylene. In certain embodiments, Lx2further comprises an oligoethylene glycol. In certain preferred embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol further comprises 1, 2, 3, or 4 ethylene glycol moieties.Attorney Docket No.: LCH-03725

[0516] In certain embodiments, T1, Tx1, Tx2, or Tx3have a structure represented by Formula XVII or a pharmaceutically acceptable salt thereof:

[0517]

[0518] Formula XVII

[0519] wherein

[0520] each Rx2, Rx3, Rx4, and Rx5is independently H, alkyl, aralkyl, or an oxygen protecting group; each Rx6is alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, cycloalkyl, alkylsulfonyl, or sulfonamide;

[0521] Wx1-*C(O)O-,

[0522]

[0523] wherein the C(O), N, CH2, S, or P marked with an * is bonded to the phenyl ring of Formula XVII; Rx7and Rx8are each independently hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, alkylamino, heteroaryl, or aryl;

[0524] Ja1is H or alkyl;

[0525] Ja2is H or alkyl; or

[0526] Ja1and Ja2combine to form an oxo;

[0527] tx1is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0528] nx1is 0, 1, 2, or 3;

[0529] Ax4represents a linkage to B1, Bx1, Bx2, or Bx3; and

[0530] Ax5represents a linkage to V1, Lx1, Lx2, or Lx3.

[0531] In certain embodiments, Rx2is H.

[0532] In certain embodiments, Rx3is H.

[0533] In certain embodiments, Rx4is H.

[0534] In certain embodiments, Rx5is H.Attorney Docket No.: LCH-03725

[0535] In certain embodiments, nxl is 0. When nxl is 0, the ring does not comprise a Rx6substituent, and the remaining positions of the ring are occupied by hydrogen.

[0536] In certain embodiments, Wx1is -*C(O)N(Rx7)-.

[0537] In certain embodiments, Rx7is H.

[0538] In certain embodiments, Ja1is H. In certain embodiments, Ja2is H. In other embodiments, Ja1and Ja2combine to form oxo.

[0539] In certain embodiments, B1is an active agent.

[0540] In certain embodiments, each T1is a bond.

[0541] In certain embodiments, each B1is H, hydroxyl, amino, (e.g., dimethyl amino), phosphoryl, sulfonamide, carboxyl, amido (e.g., piperazineamide), alkoxy, or heterocyclyl (e.g., a glucuronidyl or morpholinyl).

[0542] In certain embodiments, L4comprises a C1-C100 alkylene. In certain embodiments, L4comprises a C1-C50 alkylene. In certain embodiments, L4further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, 4, 5, or 6 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol comprises 5 ethylene glycol moieties. In certain embodiments, the oligoethylene glycol comprises at least 6 ethylene glycol moieties. In certain embodiments, L4further comprises an amido moiety. In certain embodiments, L4comprises 1, 2, 3, 4, 5, or 6 amido moieties. In certain preferred embodiments, L4comprises 1 or 2 optionally substituted amido moieties e.g., heterocyclylamido, such as piperazinylamido or carboxylamido). In certain preferred embodiments, the amido moieties

[0543]

[0544] In certain embodiments, L4is substituted with an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, or 4 ethylene glycol moieties. In certain embodiments, the oligoethylene glycol is substituted with carboxyalkyl (e.g., carboxyethyl). In certain embodiments, the oligoethylene glycol is substituted with amino (e.g., dimethylamino).

[0545] In certain embodiments, L4further comprises at least one amino acid. In certain embodiments, L4comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments,Attorney Docket No.: LCH-03725

[0546] L4comprises 1 amino acid. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is a hydrophilic amino acid. In certain embodiments, the amino acid is arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In certain embodiments, the amino acid is arginine, aspartate, glutamate, histidine, lysine, or ornithine. In certain embodiments, L4further comprises an amino acid (e.g., a hydrophilic amino acid). In certain embodiments, L4further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., hydrophilic amino acids). In certain embodiments, the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine. In certain embodiments, the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine. In certain embodiments, the amino acid(s) are selected from arginine, aspartate, glutamate, histidine, lysine, and ornithine. In certain embodiments, the amino acid(s) are selected from aspartate and glutamate. In certain preferred embodiments, the amino acid(s) are sarcosine.

[0547] In certain embodiments, L4further comprises a heteroaryl (e.g., pyrazolyl, triazolyl). In certain embodiments, L4further comprises a structure represented by Formula XVIIIa or XVIIIb:

[0548]

[0549] XVIIIa XVIIIb

[0550] wherein

[0551] each Ay1represents a linkage to V2; and

[0552] each Ay2represents a linkage to Y2.

[0553] In certain embodiments, m2 is 1. In other embodiments, m2 is 2.

[0554] In certain embodiments, V2is a bond.

[0555] In certain embodiments, V2is a bond and m2 is 1.

[0556] In certain embodiments, V2is N and V2, T2, and B2together represent a branched moiety having a structure represented by Formula XIXa or a pharmaceutically acceptable salt thereof:

[0557]

[0558] Attorney Docket No.: LCH-03725

[0559] Formula XIXa

[0560] wherein

[0561] Ay3represents a linkage to L4;

[0562] Ly1and Ly2each independently comprise an alkylene;

[0563] Ty1and Ty2are each independently a bond, or a cleavage group; and

[0564] Xy1and Xy2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0565] In certain embodiments, V2is CH and V2, T2, and B2together represent a branched moiety having a structure represented by Formula XlXb or a pharmaceutically acceptable salt thereof:

[0566]

[0567] Formula XlXb

[0568] wherein

[0569] Ay3represents a linkage to L4;

[0570] Ly1and Ly2each independently comprise an alkylene;

[0571] Ty1and Ty2are each independently a bond or a cleavage group; and

[0572] Xy1and Xy2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0573] In certain embodiments, V2is CH and V2, T2, and B2together represent a branched moiety having a structure represented by Formula XIXc or a pharmaceutically acceptable salt thereof:

[0574]

[0575] Formula XIXc

[0576] wherein

[0577] Ay3represents a linkage to L4;

[0578] Ly1and Ly2each independently comprise an alkylene;Attorney Docket No.: LCH-03725

[0579] 0 o Y y

[0580] Gy1, Gy2, and Gy3are each independently an alkylene,Ry, orRy;

[0581] each Ry1is independently H, alkyl, or aralkyl;

[0582] Ty1and Ty2are each independently a bond or a cleavage group; and

[0583] By1and By2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0584] In certain embodiments, m2 is 3.

[0585] In certain embodiments, V2is C and V2, T2and B2together represent a branched moiety having a structure represented by Formula XlXd or a pharmaceutically acceptable salt thereof:

[0586] ""

[0587] """

[0588]

[0589] Formula XlXd

[0590] wherein

[0591] Ay3represents a linkage to L4;

[0592] Ly1, Ly2, and Ly3each independently comprise an alkylene;

[0593] Ty1, Ty2, and Ty3are each independently a bond or a cleavage group; and

[0594] By1, By2, and By3are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0595] In certain embodiments, the heteroaryl of By1, By2, or By3is further substituted with alkylene or amido. In certain embodiments, the amido of By1, By2, or By3is further substituted with methyl or penta-l,2,3,4,5-ol.

[0596] In certain embodiments, Ly1comprises a Ci-Cioo alkylene. In certain embodiments, Ly1comprises a C1-C50 alkylene. In certain embodiments, Ly1further comprises an oligoethylene glycol. In certain embodiments, oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol comprises 1, 2, 3, or 4 ethylene glycol moieties.Attorney Docket No.: LCH-03725

[0597] In certain embodiments, Ly2comprises a C1-C100 alkylene. In certain embodiments, Ly2comprises a C1-C50 alkylene. In certain embodiments, Ly2further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol comprises 1, 2, 3, or 4 ethylene glycol moieties.

[0598] In certain embodiments, T2, Ty1, Ty2, or Ty3has a structure represented by Formula XX or a pharmaceutically acceptable salt thereof:

[0599]

[0600] wherein

[0601] each Ry2, Ry3, Ry4, and Ry5is independently H, alkyl, aralkyl, or an oxygen protecting group;

[0602] each Ry6is alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, cycloalkyl, alkylsulfonyl, or sulfonamide;

[0603] Wy1is independently - -*C(O)O-, -*S(

[0604]

[0605] wherein the C(O), N, CH2, S, or P marked with an * is bonded to the phenyl ring of Formula XX,

[0606] Ry7and Ry8are each independently hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, alkylamino, heteroaryl, or aryl;

[0607] Jb1is H or alkyl;

[0608] Jb2is H or alkyl; orAttorney Docket No.: LCH-03725

[0609] Jb1and Jb2combine to form an oxo;

[0610] tyl is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0611] nyl is 0, 1, 2, or 3;

[0612] Ay4represents a linkage to B2, By1, By2, or By3; and

[0613] Ay5represents a linkage to V2, Ly1, Ly2, or Ly3.

[0614] In certain embodiments, Ry2is H.

[0615] In certain embodiments, Ry3is H.

[0616] In certain embodiments, Ry4is H.

[0617] In certain embodiments, Ry5is H.

[0618] In certain embodiments, each nyl is 0.

[0619] In certain embodiments, Wy1is -*C(O)N(Ry7)-.

[0620] In certain embodiments, Ry7is H.

[0621] In certain embodiments, Jc1is H. In certain embodiments, Jc2is H. In certain embodiments, Jc1and Jc2combine to form oxo.

[0622] In certain embodiments, B2is an active agent.

[0623] In certain embodiments, m2 is 1. In other embodiments, m2 is 2. In yet other embodiments, m2 is 3.

[0624] In certain embodiments, each T2is a bond.

[0625] In certain embodiments, each B2is H, hydroxyl, amino, (e.g., dimethyl amino), phosphoryl, sulfonamido, carboxyl, amido (e.g., piperazineamide), or heterocyclyl (e.g., a glucuronidyl or morpholinyl).

[0626] In certain embodiments, L5comprises a Ci-Cioo alkylene. In certain embodiments, L5comprises a C1-C50 alkylene. In certain embodiments, L5further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, 4, 5, or 6 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol comprises 6 ethylene glycol moieties. In certain embodiments, the oligoethylene glycol further comprises at least one amido moiety. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, or 6 amido moieties. In certain preferred embodiments, the oligoethylene glycol comprises 1 or 2 amido moieties. In certain preferred embodiments, the amido moieties have a structure represented by 0 0

[0627]

[0628] Attorney Docket No.: LCH-03725

[0629] In certain embodiments, L5is substituted with an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, or 4 ethylene glycol moieties. In certain embodiments, the oligoethylene glycol is substituted with carboxyalkyl (e.g., carboxyethyl). In certain embodiments, the oligoethylene glycol is substituted with amino (e.g., dimethylamino).

[0630] In certain embodiments, L5further comprises at least one amino acid. In certain embodiments, L5comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, L5comprises 1 amino acid. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is a hydrophilic amino acid. In certain embodiments, the amino acid is arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In certain embodiments, the amino acid is arginine, aspartate, glutamate, histidine, lysine, or ornithine. In certain embodiments, L5further comprises an amino acid (e.g., a hydrophilic amino acid). In certain embodiments, L5further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., hydrophilic amino acids). In certain embodiments, the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine. In certain embodiments, the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine. In certain embodiments, the amino acid(s) are selected from arginine, aspartate, glutamate, histidine, lysine, and ornithine. In certain embodiments, the amino acid(s) are selected from aspartate and glutamate. In certain preferred embodiments, the amino acid(s) are sarcosine.

[0631] In certain embodiments, L5further comprises a heteroaryl (e.g., pyrazolyl, triazolyl). In certain embodiments, L5comprises a structure represented by Formula XXIa or XXIb:

[0632]

[0633] XXIa XXIb

[0634] wherein:

[0635] each Az1represents a linkage to V3; and

[0636] each Az2represents a linkage to Y3.

[0637] In certain embodiments, wherein m3 is 1. In other embodiments, wherein m3 is 2.Attorney Docket No.: LCH-03725

[0638] In certain embodiments, V3is a bond. In certain embodiments, V3is a bond and m3 is 1.

[0639] In certain embodiments, V3is N and V3, T3, and B3together represent a branched moiety having a structure represented by Formula XXIIa or a pharmaceutically acceptable salt thereof:

[0640]

[0641] " ""

[0642] Formula XXIIa

[0643] wherein:

[0644] Az3represents a linkage to L5;

[0645] Lz1and Lz2each independently comprise an alkylene;

[0646] Tz1and Tz2are each independently a bond or a cleavage group; and

[0647] Bz1and Bz2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0648] In certain embodiments, V3is N and V3, T3, and B3together represent a branched moiety having a structure represented by Formula XXIIb or a pharmaceutically acceptable salt thereof:

[0649]

[0650] " ""

[0651] Formula XXIIb

[0652] wherein:

[0653] Az3represents a linkage to L5;

[0654] Lz1and Lz2each independently comprise an alkylene;

[0655] Tz1and Tz2are each independently a bond or a cleavage group; and

[0656] Bz1and Bz2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0657] In certain embodiments, V3is CH and V3, T3, and B3together represent a branched moiety having a structure represented by Formula XXIIc or a pharmaceutically acceptable salt thereof:Attorney Docket No.: LCH-03725

[0658]

[0659] "" " """

[0660] Formula XXIIc

[0661] wherein:

[0662] Az3represents a linkage to L5; and

[0663] Lz1and Lz2each independently comprise an alkylene;

[0664] O O

[0665] Gz1, Gz2, and Gz3are each independently an alkylene,

[0666]

[0667] ;

[0668] each Rz1is independently H, alkyl, or aralkyl;

[0669] Lz1and Lz2each independently comprise an alkylene;

[0670] Tz1and Tz2are each independently a bond or a cleavage group; and

[0671] Bz1and Bz2are each independently an active agent (e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.

[0672] In certain embodiments, V3is C and V3, T3, and B3together represent a branched moiety having a structure represented by Formula XXIId or a pharmaceutically acceptable salt thereof:

[0673]

[0674] Formula XXIId

[0675] wherein:

[0676] Az3represents a linkage to L5;

[0677] Lz1, Lz2, and Lz3each independently comprise an alkylene;

[0678] Tz1, Tz2, and Tz3are each independently a bond or a self-immolative moiety; and

[0679] Bz1, Bz2, and Bz3are each independently an active agent ( e.g., a drug or a toxin), amido, carboxyl, ester, urea, or heteroaryl.Attorney Docket No.: LCH-03725

[0680] In certain embodiments, Bz1, Bz2, or Bz3is further substituted with alkylene or amido. In certain embodiments, the amido of Bz1, Bz2, or Bz3is further substituted with methyl or penta-1, 2, 3,4,5-oL

[0681] In certain embodiments, wherein Lz1comprises a C1-C100 alkylene. In certain embodiments, Lz1comprises a C1-C50 alkylene. In certain embodiments, Lz1further comprises an oligoethylene glycol. In certain embodiments, Lz1comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain preferred embodiments, Lz1comprises 1, 2, 3, or 4 ethylene glycol moieties.

[0682] In certain embodiments, Lz2comprises a C1-C100 alkylene. In certain embodiments, Lz2comprises a C1-C50 alkylene. In certain embodiments, Lz2further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties (e.g., 1, 2, 3, or 4 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol further comprises 1, 2, 3, or 4 ethylene glycol moieties.

[0683] In certain embodiments, T3, Tz1, Tz2, or Tz3have a structure represented by Formula XXIII or a pharmaceutically acceptable salt thereof:

[0684]

[0685] Formula XXIII

[0686] wherein,

[0687] each Rz2, Rz3, Rz4, and Rz5is independently H, alkyl, aralkyl, or an oxygen protecting group; each Rz6is alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, acetyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, cycloalkyl, alkylsulfonyl, or sulfonamide;Attorney Docket No.: LCH-03725

[0688] 2)tziN(Rz7)C(O)-, -*C(0)0-,

[0689]

[0690] (RZ7)-, or -*P(O2)N(Rz7)-, wherein the C(O), N, CH2, S, or P marked with an * is bonded to the phenyl ring of Formula XXIII,

[0691] Rz7and Rz8are each independently hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, alkylamino, heteroaryl, or aryl;

[0692] Jc1is H or alkyl;

[0693] Jc2is H or alkyl; or

[0694] Jc1and Jc2combine to form an oxo;

[0695] tzl is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0696] nzl is 0, 1, 2, or 3;

[0697] Az4represents a linkage to B3, Bz1, Bz2, or Bz3; and

[0698] Az5represents a linkage to V3, Lz1, Lz2; Lz3.

[0699] In certain embodiments, Rz2is H.

[0700] In certain embodiments, Rz3is H.

[0701] In certain embodiments, Rz4is H.

[0702] In certain embodiments, Rz5is H.

[0703] In certain embodiments, nzl is 0.

[0704] In certain embodiments, Wz1is -*C(O)N(Rz7)-.

[0705] In certain embodiments, Rz7is H.

[0706] In certain embodiments, Jc1is H. In certain embodiments, Jc2is H. In other embodiments, Jc1and Jc2combine to form oxo.

[0707] In certain embodiments, B3is an active agent.

[0708] In certain embodiments, T3is a bond.

[0709] In certain embodiments, each B3is H, hydroxyl, amino, (e.g., dimethyl amino), phosphoryl, sulfonamide, carboxyl, amido (e.g., piperazineamide), alkoxy, or heterocyclyl (e.g., a glucuronidyl or morpholinyl).

[0710] In certain embodiments, L2is a covalent linker comprising an alkylene or a heteroalkylene, wherein L comprises one or more of: (i) one or more unsaturated bonds; (ii) a heterocylene or a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain); and / or (iii) at least one C1-20 alkyl substituent. In certain embodiments, L2comprises a Ci-Cioo alkylene. In certain embodiments, L2comprises a C1-C50 alkylene. In certain embodiments, L5further comprises an oligoethylene glycol. In certain embodiments, the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties e.g., 1, 2,Attorney Docket No.: LCH-03725

[0711] 3, 4, 5, or 6 ethylene glycol moieties). In certain preferred embodiments, the oligoethylene glycol comprises 1, 2, 3, or 4 ethylene glycol moieties. In certain embodiments, L2is substituted with alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino (e.g., hydroxylamino), amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, alkylsulfonyl, or sulfonamido. In certain embodiments, L2further comprises an amido moiety (e.g., 1, 2, 3, 4, 5, or 6 amido moieties, preferably 1 or 2 amido moieties). In certain preferred embodiments, the amido moieties have a structure represented 0 0

[0712] by

[0713]

[0714] , wherein the wavy lines represent connections to the remainder of L2. In certain embodiments, L2further comprises at least one amino acid. In certain embodiments, the amino acid is a naturally occurring amino acid. In certain embodiments, the amino acid is a hydrophilic amino acid. In certain embodiments, the amino acid is arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine. In certain embodiments, the amino acid is arginine, aspartate, glutamate, histidine, lysine, or ornithine. In certain preferred embodiments, the amino acid is aspartate or glutamate. In certain embodiments, L2comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, L2comprises 1 amino acid. In certain embodiments, L2further comprises a heteroaryl (e.g., pyrazolyl, triazolyl). In certain embodiments, L2 comprises a structure represented by Formula XXIVa or XXIVb:

[0715]

[0716] wherein

[0717] each At1represents a linkage to X1; and

[0718] each A6represents a linkage to Lg.

[0719] In certain embodiments, L2comprises a structure represented by Formula XXVa, XXVb, or XXVc:

[0720]

[0721] Attorney Docket No.: LCH-03725

[0722] XXVa XXVb XXVc

[0723] wherein

[0724] each A5represents a linkage to X1; and

[0725] each A6represents a linkage to Lg.

[0726] In certain embodiments, L2comprises a structure represented by Formula XXVI:

[0727]

[0728] wherein pl is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;

[0729] each A7represents a linkage towards X1; and

[0730] each A8represents a linkage to Lg.

[0731] In certain embodiments, pl is 1, 2, 3, 4, or 5.

[0732] In certain embodiments, each active agent is independently a chemotherapeutic agent or a toxin, or a combination thereof. In certain embodiments, each active agent is a chemotherapeutic compound, a cytotoxic compound, an immunomodulatory compound, an anticancer agent, an antiviral agent, antibacterial agent, an antifungal agent, an antiparasitic agent, and a combination thereof. In certain embodiments, each active agent is a cytotoxic compound selected from a mitotic inhibitor, a DNA alkylating agent, a RNA targeting agent and a topoisomerase inhibitor, or a combination thereof. In certain embodiments, the cytotoxic compound is a thailanstatin or an amatoxin. In certain embodiment, the cytotoxic compound is selected from an auristatin, a maytansinoid, a tubulisin, a calicheamicin, a duocarmycin, a pyrrolobenzodiazepine, and a camptothecin, or a combination thereof.

[0733] In other embodiments, the active agent is selected from:

[0734] (a) erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5 -fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacinone, camptothecin, topotecan, deruxtecan, SN-38, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophy cin 1, cryptophy cin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin,Attorney Docket No.: LCH-03725

[0735] spongistatin, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotoxin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1 , dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, antimycin, azaserine, bleomycins, carubicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, 6-diazo-5- oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubucin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidamol, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2’,2”-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, and anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, arabinoside, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, vincristine, vinorelbine, novantrone, teniposide, daunomycin, aminopterin, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or pharmaceutically acceptable salts, solvates or acids of any of the foregoing;

[0736] (b) monokine, a lymphokine, a traditional polypeptide hormone, parathyroid hormone, thyroxine, relaxin, prorelaxin, a glycoprotein hormone, follicle stimulating hormone, thyroid stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-a, tumor necrosisAttorney Docket No.: LCH-03725

[0737] factor-p, Mullerian inhibiting substance, mouse gonadotropin associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, an osteoinductive factor, an interferon, interferon-oc, interferon-P, interferon-y, a colony stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, an interleukin (IL), IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, a tumor necrosis factor, polypeptide factor, LIF, kit ligand, or a combination of any of the foregoing;

[0738] (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin a- amanitin, pyrrolobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, an auristatin, tubulysin, geldanamycin, maytansinoid, SG2285, SG2057, cryptophycin, an enediyne antibiotic, epothilone, taltobulin, azonafide, a toxoid, or a combination of any of the foregoing;

[0739] (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, a stimulating agent, a neurotransmitter, a radioisotope, or a combination of any of the foregoing;

[0740] (e) a radioactive label,32P,35S, a fluorescent dye, an electron dense reagent, an enzyme, biotin, streptavidin, digoxigenin, a hapten, an immunogenic protein, a nucleic acid molecule with a sequence complementary to a target, or a combination of any of the foregoing; (f) an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, and an anti-parasitic agent, or a combination of any of the foregoing;

[0741] (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, LY117018, onapristone, or toremifene;

[0742] (h) 4(5)-imidazoles, megestrol acetate, exemestane, or anastrozole;

[0743] (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, ortroxacitabine;

[0744] (j) an aromatase inhibitor;

[0745] (k) a protein kinase inhibitor;

[0746] (l) a lipid kinase inhibitor;

[0747] (m) an antisense oligonucleotide;

[0748] (n) a ribozyme;

[0749] (o) a vaccine; and

[0750] (p) an anti-angiogenic agent.

[0751] In certain embodiments, each active agent is selected from exatecan, monomethyl auristatin (e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), PBDAttorney Docket No.: LCH-03725

[0752] dimers, FL118, SN-38, Dxd, Dxd2, or a combination thereof. In certain embodiments, each active agent is Dxd2. In certain embodiments, each active agent is monomethyl auristatin such as monomethyl auristatin E (MMAE). In certain embodiments, each active agent is monomethyl auristatin F (MMAF). In certain embodiments, each active agent is a PBD dimer.

[0753] In some embodiments, the active agent is a chemotherapeutic agent or a toxin. In some embodiments, the active agent is selected from:

[0754] (a) a microtubule targeting agent (e.g., a maytansinoid, an auristatin, an eribulin, a tubulysin, a cryptophy cin, or an EG5 inhibitor);

[0755] (b) a DNA damaging agent (e.g., an enediyne, a topoisomerase I inhibitor, a pyrrolo[2,l- c] [ 1 ,4] benzodiazepine (PBD), or a duocarmycin); and

[0756] (c) an RNA targeting agent (e.g., a thailanstatin or an amatoxin).

[0757] In some embodiments, the active agent is selected from MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), PBD dimers and camptothecin and derivatives thereof.

[0758] In some embodiments, the active agent is a pyrrolobenzodiazepine dimer; position N10 of the pyrrolobenzodiazepine dimer is substituted with X or position N’10 is substituted with X’, wherein X or X' links the pyrrolobenzodiazepine dimer to the linker;

[0759] X and X' are each independently -C(O)O-* or -C(O)-*; and

[0760] * refers to a binding site between the pyrrolobenzodiazepine dimer and the linker.

[0761] In some embodiments, the active agent is a pyrrolobenzodiazepine dimer and the pyrrolobenzodiazepine dimer has a structure represented:

[0762]

[0763] wherein

[0764] the wavy line indicates a connection point to the linker;

[0765] a dotted line represents an optional double bond;Attorney Docket No.: LCH-03725

[0766] Ri and Ri are each independently selected from H, OH, =0, =CH2, CN, Rm, OR111, =CH-Rm=C(Rm)2, O-SO2-R111, CO2R111, CORm, halo, and dihalo;

[0767] Rmis selected from Rm, CO2Rm, CORm, CHO, CO2H, and halo;

[0768] Rmis selected from substituted or unsubstituted C1-12 alkyl, substituted or unsubstituted C2-12 alkenyl, substituted or unsubstituted C2-12 alkynyl, substituted or unsubstituted C5-20 aryl, substituted or unsubstituted C3-6 heteroaryl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocyclyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, and substituted or unsubstituted 5- to 7-membered heteroaryl, wherein when the C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7- membered heterocycloalkyl, or 5- to 7-membered heteroaryl is substituted, the respective hydrogen atoms in the C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl may each be independently replaced with methoxy, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl;

[0769] R2 , R3 , R5 , R2 , R3 , and R5 are each independently selected from H, Rm, OH, OR111, SH, SRm, NH2, NHRm, NRmRm, N02, Me3Sn, and halo;

[0770] R4 and R4 are each independently selected from H, Rm, OH, OR111, SH, SRm, NH2, NHRm, NRmRm, NO2, Me tSn, halo, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted C5-12 aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -CN, -NCO, -OR11, - 0C(0)Rn, -0C(0)NRnRn, -OS(O)Rn, -OS(O)2Rn, -SRn, -S(O)Rn, -S(O)2Rn, - S(0)NRnRn, -S(0)2NRnRn, -0S(0)NRnRn’, -0S(0)2NRnRn’, -NRnRn, -NRnC(0)R°, - NRnC(0)0R°, -NRnC(0)NR°R° , -NRnS(0)R°, -NRnS(0)2R°, -NRnS(0)NR°R°’, - NRnS(0)2NR°R° , -C(0)Rn, -C(0)0Rn, and -C(0)NRnRn, wherein the hydrogen atoms in the C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7- membered heterocycloalkyl, C5-12 aryl, and 5- to 7-membered heteroaryl may each be independently replaced with C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C5-12 aryl, 5- to 7-membered heteroaryl, -ORP, -0C(0)Rp, -0C(0)NRpRp’, -OS(O)RP, -OS(O)2RP, -SRP, -S(O)RP, -Attorney Docket No.: LCH-03725

[0771] S(O)2RP, -S(O)NRPRP, -S(O)2NRPRP, -OS(O)NRPRP, -OS(O)2NRPRP, -NRPRP, - NRpC(O)Rq, -NRpC(O)ORq, -NRpC(O)NRqH, -NRpS(O)Rq, -NRpS(O)2Rq, - NRpS(O)NRqH, -NRpS(O)2NRqH, -C(O)RP, -C(O)ORP, or -C(O)NRPRPwhen the Ci-6alkyl, Ci-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7-membered heterocycloalkyl, Cs-n aryl, and 5- to 7-membered heteroaryl;

[0772] Rn, Rn, R°, R° Rp, Rp, and Rqare each independently selected from H, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, C3-13 cycloalkyl, 3- to 7-membered heterocycloalkyl, Ce-io aryl, and 5- to 7-membered heteroaryl;

[0773] X’ is selected from -C(O)O-, -S(O)O-, -C(O)-, -C(O)NR-, -S(O)2NR-, -P(O)R'NR-, - S(O)NR-, and -PO2NR-;

[0774] Xa is a bond or substituted or unsubstituted C1-6 alkylene, wherein C1-6 alkylene is substituted with C1-8 alkyl, or C3-8 cycloalkyl when substituted;

[0775] R and R' each independently denote H, OH, NH2, ONH2, NHNH2, substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C1-8 alkoxy, substituted or unsubstituted C1-8 alkylthio, substituted or unsubstituted Ck2o heteroaryl, substituted or unsubstituted C5-M aryl, or mono- or di- C1-8 alkylamino, wherein the C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-2o heteroaryl, and C5-2o aryl are substituted with a substituent selected from OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halo, C1-6 alkyl, C1-6 alkoxy, and C6-12 aryl when substituted;

[0776] Y’ and Y” are each independently selected from O, S, and N(H);

[0777] Re is a substituted or unsubstituted saturated or unsaturated C3-12 hydrocarbon chain, wherein the chain may be interrupted by one or more heteroatoms, NMe, or a substituted or unsubstituted aromatic ring, the chain or aromatic ring may be substituted with -NH, -NRm, -NHC(O)Rm, -NHC(O)CH2-[OCH2CH2]nC-R, or-[CH2CH2O]nc-R at any one or more positions of hydrogen atoms on the chain or aromatic ring or unsubstituted, wherein Rmand R are each as defined for Rmand R above, and nC is 1 to 12; and R7 and R7 are each independently H, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 3- to 7-membered heterocycloalkyl, substituted or unsubstituted Ce-io aryl, substituted or unsubstituted 5- to 7-membered heteroaryl, -ORr, -OC(O)Rr, -OC(O)NRrRr, -OS(O)Rr, -OS(O)2Rr, - SRr, -S(O)Rr, -S(O)2Rr, -S(O)NRrRr, -S(O)2NRrRr’, -OS(O)NRrRr’, -OS(O)2NRrRr’, - NR'R1, -NRrC(O)Rs, -NRrC(O)ORs, -NRrC(O)NRsRs, -NRrS(O)Rs, -NRrS(O)2Rs, -Attorney Docket No.: LCH-03725

[0778] NRrS(O)NRsRs, -NRrS(O)2NRsRs, -C(O)Rr, -C(O)ORS, or -C(O)NRrRr, wherein the hydrogen atoms in the Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7- membered heterocycloalkyl, Ce-io aryl, and 5- to 7-membered heteroaryl may each be independently replaced with C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7-membered heterocycloalkyl, Ce-io aryl, 5- to 7-membered heteroaryl, -OR1, - " "

[0779]

[0780] " CtOjR1, -CtOjOR1, or -C(O)NRtRtwhen the C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7-membered heterocycloalkyl, Ce-io aryl, and 5- to 7-membered heteroaryl;

[0781] Rr, Rr, Rs, Rs, R‘, R1, Ru, and Ru' are each independently selected from H, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, C3-13 cycloalkyl, 3- to 7-membered heterocycloalkyl, C5-10 aryl, and 5- to 7-membered heteroaryl;

[0782] G is a glucuronide group or a galactoside group;

[0783] O

[0784] each Z is selected from H, C1-8 alkyl, halo, NO2, CN,

[0785]

[0786] ;

[0787] R9, Rio, and Rie are each independently selected from H, C1-8 alkyl, C2-6 alkenyl, C1-6 alkoxy, and alkyloxyalkyl; and

[0788] n30 is 0 to 3.

[0789] In some embodiments, Y’ is O. In some embodiments, is Y” is O. In some embodiments, a dotted line represents presence of a double bond between the carbons bearing Ri and R7 or Ri and R7 In some embodiments, Ri is selected from substituted or unsubstituted Cw alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C5-7 aryl, and substituted or unsubstituted C3-6 heteroaryl. In some embodiments, R2 , R3 , and R5 are each independently H or OH. In some embodiments, R4 is C1-6 alkoxy. In some embodiments, R4 is methoxy, ethoxy, or butoxy. In some embodiments, X’ is selected from -C(O)O-, -C(O)-, and -C(O)NR-. In some embodiments, X’ is -C(O)NR-. In some embodiments, Re is a substituted or unsubstituted saturated or unsaturated C3-8 hydrocarbon chain, wherein one or more of the carbon atoms of the hydrocarbon chain is replaced by a heteroatom or a substituted or unsubstituted aromatic ring, wherein the heteroatom is O, S, or N(H) and the aromatic ring is benzene, pyridine, imidazole, or pyrazole, and the chain or aromatic ring may be substituted with -NHC(O)CH2-[OCH2CH2]nc-R or -[CH2CH2O]nc-R atAttorney Docket No.: LCH-03725

[0790] any one or more positions of hydrogen atoms on the chain or aromatic ring; and nC is 1 to 6. In some embodiments, nC is 1 to 6. In some embodiments, Xa is a bond or C1-3 alkylene.

[0791] 0

[0792] In some embodiments, Z is H,

[0793]

[0794] wherein R9, Rio, and Rie are each independently selected from H, C1-3 alkyl, C1-3 alkoxy, and alkyloxyalkyl. In some embodiments, R9 is methyloxyalkyl. In some embodiments, Rio is methyloxyalkyl. In some embodiments, Rie is methyloxyalkyl. In some embodiments, R9, Rio, or Rie is -(CH2CH2O)m-(CH2)m4CH3, further wherein m is 1-6 and m4 is 0-2. In some embodiments, m is 1. In some embodiments, m2 is 0. In some embodiments, R2 is H. In some embodiments, R3 is H. In some embodiments, R7 is H. In some embodiments, R4 is C1-6 alkoxy (e.g., methoxy). In some embodiments, R5 is OH. In some embodiments, Ri is =CH2, CH3, or phenyl, optionally substituted with methoxy. In some embodiments, Y’ is O.

[0795] In some embodiments, R2” is H. In some embodiments, R3” is H. In some embodiments, R7’ is H. In some embodiments, R4” is C1-6 alkoxy (e.g., methoxy). In some embodiments, R5” is OH. In some embodiments, Ri” is =CH2, CH3, or phenyl, optionally substituted with methoxy. In some embodiments, Y” is O. In some embodiments, X’ is -C(O)O-. In some embodiments, Xa is CH2. In some embodiments, G is a glucuronide group.

[0796] In some embodiments,

[0797]

[0798] some embodiments, n30 is 1.

[0799] O

[0800] In some embodiments, Z is

[0801]

[0802] . In some embodiments, R9 is H. In some embodiments, Rie is alkyloxyalkyl (e.g., methoxyethyl). In some embodiments, Z V ) 'RK10

[0803] is jnsome embodiments, Riois alkyl (e.g., methyl). In some embodiments, Re is C3-12 alkyl (e.g., pentyl).

[0804] In some embodiments, the pyrrolobenzodiazepine dimer is selected from:

[0805]

[0806] Attorney Docket No.: LCH-03725

[0807]

[0808] OMe or -NH2.

[0809] In certain embodiments, the conjugate comprises:

[0810]

[0811] Attorney Docket No.: LCH-03725

[0812]

[0813] Attorney Docket No.: LCH-03725

[0814]

[0815] Attorney Docket No.: LCH-03725

[0816]

[0817] wherein

[0818] MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F; and

[0819] the dotted line represents a connection to Ab.

[0820] In certain embodiments, a conjugate disclosed herein comprises a structure represented in FIG. 8. In certain embodiments, a conjugate disclosed herein comprises a structure represented by:Attorney Docket No.: LCH-03725

[0821]

[0822] In certain embodiments, a conjugate disclosed herein comprises a structure represented in FIG. 9. In certain embodiments, a conjugate disclosed herein comprises a structure represented by:

[0823]

[0824] In certain embodiments, a conjugate disclosed herein comprises a structure represented in FIG. 10. In certain embodiments, a conjugate disclosed herein comprises a structure represented by:

[0825]

[0826] In certain embodiments, a conjugate disclosed herein comprises a structure represented in FIG. 11. In certain embodiments, a conjugate disclosed herein comprises a structure represented by:Attorney Docket No.: LCH-03725

[0827] >

[0828]

[0829] wherein

[0830]

[0831] the linker-payload compound may be conjugated by reacting with one or more sulfur-containing functional groups present in Ab, for example, sulfur atoms of cysteine residues, at any of a plurality of available positions, such that the conjugation sites on Ab are not specifically defined.

[0832] In certain embodiments, a conjugate disclosed herein comprises a structure represented in FIG. 12. In certain embodiments, a conjugate disclosed herein comprises a structure represented by:

[0833]

[0834] wherein

[0835]

[0836] the linker-payload compound may be conjugated by reacting with one or more sulfur-containing functional groups present in Ab, for example, sulfur atoms of cysteine residues, at any of a plurality of available positions, such that the conjugation sites on Ab are not specifically defined.

[0837] In certain embodiments, a conjugate disclosed herein comprises a structure represented in FIG. 13. In certain embodiments, a conjugate disclosed herein comprises a structure represented by:Attorney Docket No.: LCH-03725

[0838]

[0839] In certain embodiments, a conjugate disclosed herein comprises a structure represented in FIG. 14. In certain embodiments, a conjugate disclosed herein comprises a structure represented by:

[0840]

[0841] In certain embodiments, a conjugate disclosed herein comprises a structure represented in FIG. 15. In certain embodiments, a conjugate disclosed herein comprises a structure represented by:

[0842]

[0843] In certain aspects, the present disclosure provides pharmaceutical compositions comprising a conjugate disclosed herein and a pharmaceutically acceptable excipient.Attorney Docket No.: LCH-03725

[0844] In certain aspects, the present disclosure provides methods of treating or preventing a condition associated with BCMA expression in a subject in need thereof comprising administering a conjugate disclosed herein or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the condition associated with BCMA expression is a cancer.

[0845] In some embodiments, the cancer is lung cancer, small-cell lung cancer, non-small-cell lung cancer, gastrointestinal cancer, colon cancer, intestinal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, myeloma, sarcoma, osteosarcoma, Kaposi’s sarcoma or melanoma. In some embodiments, the cancer is a hematological cancer.

[0846] In some embodiments, the cancer is Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), T cell lymphoma, B cell lymphoma, natural killer cell lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, lymphoblastic lymphoma, enteropathy-type intestinal lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-cell acute lymphoid leukemia (“B-ALL”), T-cell acute lymphoid leukemia (“T-ALL”), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, Blastic plasmacytoid dendritic cell neoplasm (BPDCN), Burkitt’s lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, Waldenstrom macroglobulinemia, and combinations thereof. In some embodiments, the cancer is Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, glioblastoma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), or multiple myeloma. In some embodiments, the cancer is multiple myeloma.

[0847] Antibody or Antigen-Binding Fragment thereof

[0848] The present disclosure provides an antibody that binds to B-cell maturation antigen (BCMA). As described herein, the antibody according to the present disclosure is a polypeptide comprising one or more complementarity-determining areas or regions (CDRs).Attorney Docket No.: LCH-03725

[0849] In some embodiments, the CDR is included in a "framework" region, and the framework orients the CDR(s) so that the CDR(s) can have appropriate antigen-binding properties.

[0850] In certain aspects, the present disclosure provides antibody-drug conjugates comprising an anti-BCMA antibody that binds to BCMA. In certain embodiments, the antibody disclosed herein binds to BCMA expressed in a target cell (e.g., a tumor or cancer cell) and may be used to deliver a drug to the target cell (e.g., a tumor or cancer cell). In certain embodiments, the antibody drug conjugates disclosed herein have improved stability, enhanced efficacy, and / or lower toxicity as compared to antibody drug conjugates known in the art.

[0851] In certain embodiments, the antibody comprises, but is not limited to, a monoclonal antibody, a bispecific antibody, a diabody, a multispecific antibody, a polyantibody, a minibody, a domain antibody, an antibody mimetic (or synthetic antibody), a chimeric antibody, a humanized antibody, a human antibody or an antibody fusion (or antibody conjugate), and a fragment thereof, and includes various forms of antibodies disclosed herein.

[0852] In certain embodiments, an antibody fragment of the antibody according to the present disclosure includes Fab, Fab', F(ab')2, scFab, Fv, dsFv, scFV, scFV-Fc, a minibody, a diabody, sc Ab, or dAb.

[0853] In certain embodiments, the antibody according to the present disclosure may consist of a polypeptide of only light chains and / or only heavy chains including the variable regions shown in Table 1.

[0854] CDR sequences that may be included in the heavy and light chain variable regions of the antibody or antigen-binding fragment thereof according to an embodiment of the present disclosure are shown in Table 1.

[0855] An antibody according to the present disclosure shares certain regions or sequences with other antibodies disclosed herein. In certain embodiments, the constant region of the antibody or antigen-binding fragment thereof may be shared. In certain embodiments, Fc regions may be shared. In certain embodiments, the frame of a variable region may be shared.

[0856] The heavy chain variable region and the light chain variable region according to the present disclosure may be linked to at least a part of a human constant region. The selection of a constant region may be determined partially by whether or not antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complementdependent cytotoxicity is required. For example, human isotypes IgGl and IgG3 have complement-dependent cytotoxicity, and human isotypes IgG2 and IgG4 do not have such cytotoxicity. In addition, human IgGl and IgG3 induce a cell-mediated effector functionAttorney Docket No.: LCH-03725

[0857] stronger than that of human IgG2 and IgG4. The light chain constant region may be lambda or kappa.

[0858] A variable region of an immunoglobulin chain generally has the same overall structure and includes a comparatively conserved framework region (FR) linked by three hypervariable regions called “complementarity determining areas or regions or domains” or complementarity determining regions (CDRs). The CDRs of a variable region derived from each chain including a heavy chain / light chain pair are typically aligned by a framework region to form a structure specifically binding to a specific epitope of a target protein. These factors of naturally occurring light chain and heavy chain variable regions are typically included from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The position of amino acid sequences corresponding to each variable region may be determined by Kabat (Kabat et al., (1983) U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest”), Chothia (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)) or in a manner related to the OPAL library (Hye Young Yang et. al., 2009 Mol. Cells 27: 225). The CDRs determined by each definition, when compared to each other, may be subsets which overlap or where one includes another. Those of ordinary skill in the art will be readily able to easily select CDR sequences according to the definitions above, given a variable region sequence of an antibody.

[0859] In certain embodiments, amino acid sequences of CDRs are defined according to Kabat definition. However, it is well known to those skilled in the art that CDRs of an antibody can be defined in the art according to a variety of methods, such as Chothia definition based on the location of a structural loop region (Al-Lazikani, B et al., J Mol Biol 273: 927-48 (1997)), and Kabat definition based on sequence variability (Kabat et al., “Sequences of proteins of immunological interest”, fifth edition, National Institutes of Health, Bethesda, MD. (1991)).

[0860] In certain embodiments, amino acid residues in variable region sequences may also be determined using a Combined definition that incorporates both Kabat definition and Chothia definition. The Combined definition refers to the combination of the ranges of Kabat definition and Chothia definition. It should be understood by those skilled in the art that unless otherwise specified, the terms “CDR” and “complementarity determining region” of a given antibody or region thereof (e.g., a variable region) should be understood to encompass the complementarity determining region as defined according to any of the embodiments described in the present disclosure.

[0861] Although the scope of protection claimed in the claims of the present disclosure is based on the sequences defined according to Kabat definition, amino acid sequences definedAttorney Docket No.: LCH-03725

[0862] according to other CDRs definitions should also fall within the scope of protection of the present disclosure.

[0863] In certain embodiments, the antibody according to the present disclosure is a humanized antibody. A humanized antibody refers to any antibody in which the constant region of a nonhuman antibody is completely substituted with a human form of the constant region, and at least a portion of the variable region of a non-human antibody, except for the three loops of an amino acid sequence outside each variable region that binds to a target structure, is completely or partially substituted with the corresponding portion of a human antibody. In certain embodiments, the antibody according to the present disclosure is a human antibody.

[0864] Certain mutations may be introduced to the framework region to enhance the stability of antibodies while maintaining their antigen binding activity. Stabilization of therapeutic antibodies can result in improved serum half-life, lower dosage requirements, reduced sideeffects, improved shelf-life and reduced shipping and storage costs.

[0865] In certain embodiments, the present disclosure discloses one or more amino acid sequences having substantial sequence identity to one or more amino acid sequences disclosed herein. Substantial identity means that the effects disclosed herein are maintained in the presence of sequence variations. In certain embodiments, the amino acid sequence has about 90% identity, about 95% identity, or about 99% identity to the heavy chain variable regions shown in Table 1. In another embodiment, the amino acid sequence has about 90% identity, about 95% identity, or about 99% identity to the light chain variable regions shown in Table 1. For example, in the case of variants exhibiting 90% identity, 95% identity, or 99% identity to the sequence of the antibody or antigen-binding fragment thereof according to the present disclosure, any mutation occurs in the framework of the variable region rather than the CDRs.

[0866] In certain embodiments, a nucleic acid encoding the antibody or fragment thereof according to the present disclosure is a nucleic acid encoding a full-length antibody including the CDRs disclosed herein, the variable region including the CDRs, and the variable region, and the constant region. Once the amino acid sequence is determined, the nucleic acid sequence may be easily determined in consideration of a known reverse transcription program, codon usage, and the like.

[0867] Antigen Specificity and Affinity for Antibody

[0868] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure has specificity to a BCMA antigen, preferably a human BCMA antigen, and affinity suitable for use as an antibody therapeutic / diagnostic agent. InAttorney Docket No.: LCH-03725

[0869] certain embodiments, the affinity for aggregates may be KD < 1,000 nM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM, and may be, for example, 10’6M to 10’12M.

[0870] Production of Antibody

[0871] The antibody or antigen-binding fragment thereof of the present disclosure may be produced by any means known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination, including recombinant expression, chemical synthesis, and enzymatic digestion of a full-length monoclonal antibody. The recombinant expression may take place in any suitable host cell known in the art, including without limitation to, mammalian host cells, bacterial host cells, yeast host cells, and insect host cells, or in a cell-free system (e.g., Sutro’s Xpress CF platform, World Wide Web at sutrobio.com / technology / ) .

[0872] Knowing the amino acid sequence of the desired sequence, one skilled in the art may readily produce said antibodies or polypeptides, by standard techniques for production of polypeptides. For instance, they may be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, Calif.) and following the manufacturer's instructions. Alternatively, antibodies and other polypeptides may be synthesized by recombinant DNA techniques as is well-known in the art. For example, these fragments may be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they may be later isolated using well-known techniques.

[0873] In particular, described herein are methods of producing an antibody or a polypeptide, which method comprises the steps consisting of: (i) culturing a transformed host cell under conditions suitable to allow expression of said antibody or polypeptide; and (ii) recovering the expressed antibody or polypeptide.

[0874] Antibodies and other polypeptides are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) may also be employed for purification. See,Attorney Docket No.: ECH-03725

[0875] e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.

[0876] The present disclosure also provides an expression vector and a host cell for producing the antibody or antigen-binding fragment thereof described herein. Various expression vectors may be used to express a polynucleotide encoding the antibody or antigen-binding fragment thereof. Both viral-based and nonviral expression vectors may be used to produce antibodies in mammalian host cells. Nonviral vectors and systems include plasmids, episomal vectors (typically, containing an expression cassette for protein or RNA expression), and human artificial chromosomes (e.g., see [Harrington et al., Nat Genet. 15:345, 1997]). Useful viral vectors include without limitation to, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus-based vector, SV40 vectors, papilloma virus vectors, HBP Epstein-Barr virus vectors, vaccinia virus vectors, and Semliki Forest virus (SFV)-based vectors.

[0877] The selection of the expression vector varies based on an intended host cell in which the vector is to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancer) operably linked to a polynucleotide encoding the antibody or antibody fragment (e.g., antigen-binding fragment). In some embodiments, an inducible promoter is used to prevent expression of inserted sequences except under inducing conditions. The inducible promoter includes, for example, arabinose, lacZ, metallothionein promoter, or heat shock promoter. A transformed organism culture may be proliferated under non-inducing conditions without biasing a population for coding sequences of which expression products are better tolerated by the host cell.

[0878] The expression vector may also provide a secretion signal sequence location to form a fusion protein with a polypeptide encoded by the inserted antibody or antibody fragment (e.g., antigen-binding fragment) sequence. More often, the inserted antibody or antibody fragment (e.g., antigen-binding fragment) sequence is included in the vector after being linked to a signal sequence. A vector used to contain a sequence encoding the light and heavy chain variable domains of the antibody or antibody fragment (e.g., antigen-binding fragment) sometimes also encodes a constant region or a portion thereof. Such a vector allows the variable region to be expressed as a fusion protein with the constant region to induce production of a complete antibody or fragment thereof.

[0879] In some embodiments, mammalian host cells are used to express and produce the antibody or antibody fragment (e.g., antigen-binding fragment) polypeptide of the present disclosure. For example, these mammalian host cells may be hybridoma cell lines expressingAttorney Docket No.: LCH-03725

[0880] endogenous immunoglobulin genes (e.g., myeloma hybridoma clones as described in Examples), or mammalian cell lines harboring exogenous expression vectors (e.g., SP2 / 0 myeloma cells to be exemplified below). The mammalian host cells include any normal apoptotic or normal or abnormal immortalized animal or human cells. For example, a plurality of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. Expression vectors for mammalian host cells may include expression control sequences, such as an origin of replication, a promoter, and an enhancer, and necessary processing information sites, such as a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription terminator sequence. These expression vectors generally contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type-specific, step-specific, and / or controllable or regulatable promoters. Useful promoters include a metallothionein promoter, a constitutive adenovirus major late promoter, a dexamethasone-inducible MMTV promoter, a SV40 promoter, a MRP polIII promoter, a constitutive MPSV promoter, a tetracycline-inducible CMV promoter (e.g., a human immediate early CMV promoter), a constitutive CMV promoter, and promoter-enhancer combinations known in the art, but are not limited thereto.

[0881] A method of introducing an expression vector including a polynucleotide sequence of interest varies depending on a type of host cell. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other host cells.

[0882] In the present disclosure, a non-human antibody may be derived from, for example, any antibody-producing animal, for example, a mouse, a rat, a rabbit, a goat, a donkey, or non-human primates (e.g., monkeys such as cynomolgus or rhesus monkey) or apes (e.g., chimpanzees). A non-human antibody may be produced by immunizing an animal by using a method known in the art.

[0883] Chimeric antibodies (e.g., mouse-human chimeras) may be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. The CH domain of a human chimeric antibody may be any region which belongs to human immunoglobulin, such as the IgG class or a subclass thereof, such as IgGl, IgG2, IgG3 and IgG4. Similarly, the CL of a human chimeric antibody may be any region which belongs toAttorney Docket No.: LCH-03725

[0884] Ig, such as the kappa class or lambda class, chimeric and humanized monoclonal antibodies, comprising both human and non-human portions may be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies may be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Patent Publication PCT / US86 / 02269; Akira et al. European Patent Application 184,187; Taniguchi, M. European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT Application WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Patent 5,225,539; Jones etal. (1986) Nature 321:552-525; Verhoeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0885] In addition, humanized antibodies may be made according to standard protocols such as those disclosed in U.S. Patent 5,565,332. In another embodiment, antibody chains or specific binding pair members may be produced by recombination between vectors comprising nucleic acid molecules encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicable generic display package and vectors containing nucleic acid molecules encoding a second polypeptide chain of a single binding pair member using techniques known in the art, e.g., as described in U.S. Patents 5,565,332, 5,871,907, or 5,733,743. Humanized antibodies may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell.

[0886] The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type).

[0887] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e.g. , Riechmann L. et al. 1988; Neuberger M S. et al. 1985). Antibodies may be humanized using a variety of techniquesAttorney Docket No.: LCH-03725

[0888] known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka G M et al. (1994); Roguska M A. et al. (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0889] A fully human antibody may be produced by administering an antigen to a transformed animal including a human immunoglobulin gene locus, or by treating a phage display library expressing a human antibody repertory with an antigen, and then selecting the target antibody. The antibody may be polyclonal or monoclonal, or may be synthesized within a cell host through the expression of recombinant DNA. A monoclonal antibody (mAb) may be produced using a conventional monoclonal antibody method, for example, a standard somatic hybridization technique in the literature (Kohler and Milstein, 1975, Nature 256:495).

[0890] Bispecific or multispecific antibodies described herein may be made according to standard procedures. For example, triomas and hybrid hybridomas are two examples of cell lines that may secrete bispecific or multispecific antibodies. Examples of bispecific and multispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Such antibodies may also be constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan (1986) Immunol. Today 7:241). Alternatively, such antibodiesmay also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling the desired antibodies. They may also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences.

[0891] Method for Expressing Antibody

[0892] The antibody disclosed herein may be expressed in a hybridoma cell line or an expression cell line other than a hybridoma. An expression construct encoding the antibody may be used to transform a mammalian, an insect or a microbial host cell. A construct such as a plasmid may be produced, as described in the foregoing description, using any of various known methods for introducing a polynucleotide into a host cell. The specific method may vary according to the type of host cell. Methods for introducing a heterogeneous polynucleotide into a mammalian cell are widely known in the art, and include, but are not limited to, for example,Attorney Docket No.: LCH-03725

[0893] dextran-mediated transfer, calcium phosphate precipitation, polybrene-mediated transfer, protoplast fusion, electrophoresis, capsulation of a transferred polynucleotide using liposomes, mixing of a nucleic acid and a positively charged lipid, and direct microinjection of DNA into the nucleus.

[0894] Use of Anti-BCMA Antibody Drug-Conjugates for Therapeutic Purposes

[0895] The expression of BCMA is associated with a number of conditions (e.g., cancers such as hematological cancers and B-cell malignancies, autoimmune disorders, and infectious diseases). The expression of BCMA in cancer is associated with unfavorable prognosis of certain cancers (e.g., hematological cancers and B-cell malignancies) and is known to also affect cancer metastasis. For anticancer antibody treatment, for example, the anti-BCMA antibody or antigen binding fragment thereof may, as described herein, be used in a form linked to various active agents via a linker to remove BCMA overexpressing cancer cells. Accordingly, an antibody or antigen binding fragment thereof binding to BCMA may be used in a form bonded to an active agent, and thus can be used as a targeted therapeutic agent for directing to BCMA expressing cells.

[0896] In some aspects, the present disclosure provides a pharmaceutical composition comprising a conjugate disclosed herein and a pharmaceutically acceptable excipient.

[0897] In some aspects, the present disclosure provides a pharmaceutical composition for use in preventing or treating a disease associated with BCMA expression, the composition comprising a conjugate disclosed herein. In some embodiments, the disease associated with BCMA expression is a proliferative disease. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is selected from liver cancer, thyroid cancer, ovarian cancer, brain cancer, multiple myeloma, colon cancer, head and neck cancer, lymphoma, leukemia, bladder cancer, kidney cancer, stomach cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, lung cancer, sarcoma, neuroendocrine tumor, melanoma, and combinations thereof. In some embodiments, the cancer is selected from Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), T cell lymphoma, B cell lymphoma, natural killer cell lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, lymphoblastic lymphoma, enteropathy-type intestinal lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-cell acute lymphoid leukemia (“B-ALL”), T-cell acuteAttorney Docket No.: LCH-03725

[0898] lymphoid leukemia (“T-ALL”), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, Blastic plasmacytoid dendritic cell neoplasm (BPDCN), Burkitt’s lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, Waldenstrom macroglobulinemia, and combinations thereof. In some preferred embodiments, the cancer is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), or multiple myeloma. In some preferred embodiments, the cancer is multiple myeloma.

[0899] Treatment Method: Pharmaceutical Formulation and Administration Route

[0900] In some embodiments, the present disclosure also provides a treatment method using a conjugate disclosed herein or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the conjugate or pharmaceutically acceptable salt or solvate thereof is provided to a patient. The conjugate or pharmaceutically acceptable salt or solvate thereof inhibits cancer cell progression by binding to BCMA expressed on the surface of cancer cells. In some embodiments, the antibody binds to BCMA expressed on the surface of cancer cells in a form bonded to an active agent disclosed herein, thereby specifically delivering the active agent bonded to the antibody to cancer cells, to induce the death of the cancer cells. In some embodiments, the antibody binds to BCMA expressed on the surface of cancer cells in the form of an antibody specific to the same target or another target, thereby increasing specificity of multiple antibodies for cancer cells or inducing connections between cancer cells and other types of cells such as immune cells, to induce the death of the cancer cells.

[0901] In some aspects, the present disclosure provides a method of treating or preventing a disease associated with BCMA expression in a subject in need thereof comprising administering a conjugate disclosed herein or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the disease associated with BCMA expression is a proliferative disease. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is selected from liver cancer, thyroid cancer, ovarian cancer, brain cancer, multiple myeloma, colon cancer, head and neck cancer, lymphoma, leukemia, bladder cancer, kidney cancer, stomach cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, lung cancer, sarcoma, neuroendocrine tumor, melanoma, and combinations thereof. In some embodiments, the cancerAttorney Docket No.: LCH-03725

[0902] is selected from Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), T cell lymphoma, B cell lymphoma, natural killer cell lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, lymphoblastic lymphoma, enteropathy-type intestinal lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-cell acute lymphoid leukemia (“B-ALL”), T-cell acute lymphoid leukemia (“T-ALL”), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, Blastic plasmacytoid dendritic cell neoplasm (BPDCN), Burkitt’s lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, Waldenstrom macroglobulinemia, and combinations thereof. In some preferred embodiments, the cancer is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), or multiple myeloma. In some preferred embodiments, the cancer is multiple myeloma.

[0903] Pharmaceutical compositions

[0904] Provided is also a pharmaceutical composition including a therapeutically effective amount of a conjugate disclosed herein or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, a carrier, a solubilizer, an emulsifier, a preservative, and / or an adjuvant. Also, for example, a method of treating a patient (e.g., a cancer patient) by administering such a pharmaceutical composition is provided. The term “patient” includes human patients.

[0905] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used as a meaning including an excipient, a diluent, or an adjuvant. The carrier may be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, saline, buffer such as PBS, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof.Attorney Docket No.: LCH-03725

[0906] The pharmaceutical composition may be prepared as any formulation according to general methods. The composition may be formulated into formulations for oral administration (e.g., powders, tablets, capsules, syrups, pills or granules) or for parenteral administration (for example, injections). In addition, the composition may be prepared as a systemic or local formulation.

[0907] The pharmaceutical composition may include an effective amount of the antibody or antigen-binding fragment thereof, an anticancer agent, or a combination thereof. The term “effective amount” refers to an amount sufficient to exhibit preventive or therapeutic effects when administered to an individual requiring prevention or treatment. The effective amount may be appropriately selected depending on a cell or individual that is selected by those or ordinary skill in the art. The effective amount may be determined according to factors including the severity of the disease, the age, body weight, health and gender of a patient, sensitivity of a patient to the drug, administration time, administration routes, excretion rate, treatment period, and drugs used in combination or simultaneously with the used composition, and other factors well known in the medical field.

[0908] The dosage of the pharmaceutical composition may range, for example, from 10 pg / kg to about 30 mg / kg, optionally from 0.1 mg / kg to about 30 mg / kg, or alternatively from 0.3 mg / kg to about 20 mg / kg per adult. The pharmaceutical composition may be administered once a day, multiple times a day, once every 1 to 4 weeks, or once to 12 times a year.

[0909] Hereinafter, the present disclosure will be described in more detail with reference to examples and experimental examples.

[0910] The following examples are intended to aid in understanding of the present disclosure and are not intended to limit the scope of the present disclosure.

[0911] Definitions

[0912] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

[0913] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout thisAttorney Docket No.: LCH-03725

[0914] specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0915] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0916] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0917] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

[0918] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

[0919] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0920] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus,Attorney Docket No.: LCH-03725

[0921] prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0922] “Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0923] Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0924] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0925] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus,Attorney Docket No.: LCH-03725

[0926] a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0927] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0928] It is understood that substituent and substitution patterns on the compounds of the present disclosure can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0929] As used herein, the term “optionally substituted” refers to the optional replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2 or -CH2-OP(O)(O-alkyl)2. Preferably, “optionally substituted” refers to the optional replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are optionally replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

[0930] The term “conjugates” as used herein refers to cell binding agents that are covalently bonded to one or more molecules of a cytotoxic compound. In this regard, "cell binding agent" is a molecule having affinity for a biological target, and may be, for example, an antibody, particularly a monoclonal antibody, or an antibody fragment, and the binding agent functions to direct a biologically active compound to a biological target. In certain embodiments, the conjugate may be designed to target tumor cells through cell surface antigens. The antigen may be a cell surface antigen that is overexpressed or expressed in an abnormal cell type. Specifically, the target antigen may be expressed only on proliferative cells (e.g., tumor cells). The target antigen may be selected on the basis of different expression, usually betweenAttorney Docket No.: LCH-03725

[0931] proliferative tissues and normal tissues. In the present disclosure, the antibody is bonded to the linker.

[0932] In the present disclosure, a "variant" of a polypeptide, for example, an antigen-binding fragment, a protein, or an antibody, is a polypeptide in which insertion, deletion, addition, and / or substitution have occurred at one or more amino acid residues compared to other polypeptide sequences, and includes fusion polypeptides. Protein variants also include those modified by protein enzymatic cleavage, phosphorylation or other post-translational modifications, but retaining the biological activity of the antibody disclosed herein, such as binding and specificity to CLDN18.2. Variants may have about 99% identity, about 98% identity, about 97% identity, about 96% identity, about 95% identity, about 94% identity, about 93% identity, about 92% identity, about 91% identity, about 90% identity, about 89% identity, about 88% identity, about 87% identity, about 86% identity, about 85% identity, about 84% identity, about 83% identity, about 82% identity, about 81% identity, or about 80% identity to the sequence of the antibody or antigen-binding fragment thereof according to the present disclosure. Percent identity (%) or homology may be calculated by methods known in the art.

[0933] In certain embodiments, the percent homology or identity can be calculated by 100X[(same position) / min(TGA, TGB)], wherein TGA and TGB are the sum of the number of residues and internal gap positions in sequences A and B to be compared (Russell et al., J. Mol Biol., 244: 332-350 (1994).

[0934] The term "derivative" of a polypeptide as used herein refers to a polypeptide that has chemical modification at one or more residues through conjugation with other chemical moieties, different from insertion, deletion, addition or substitution variants.

[0935] The term "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence. The percentage of sequence identity is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying theAttorney Docket No.: LCH-03725

[0936] result by 100 to yield the percentage of sequence identity. The comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using readily available software programs. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (at world wide web at blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at world wide web at ebi.ac.uk / Tools / psa.

[0937] As used herein, “homology” with respect to a peptide, polypeptide or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.

[0938] The term “affinity” or “avidity” refers to the strength of interaction between an antibody or its antigen-binding fragment and an antigen, determined by characteristics of the antigen such as size, shape, and / or charge, and the CDR sequences of the antibody or antigen-binding fragment. Methods for determining such affinities are known in the art and may also be referenced herein.

[0939] Antibodies or their antigen-binding fragments used in the present invention are said to “specifically bind” to the target, such as the antigen, when the dissociation constant (KD) is <10-6M. Antibodies bind “with high affinity” to the target when KD is <lx 10’8M.

[0940] As used in the present disclosure, the “antigen-binding fragment” of a chain (heavy chain or light chain) of an antibody or immunoglobulin includes a part of an antibody which lacks some amino acids compared to a full-length chain, but can specifically bind to an antigen.Attorney Docket No.: LCH-03725

[0941] This fragment can be considered as having biological activity, in that the fragment can specifically bind to a target antigen, or can compete with other antibodies or antigen binding fragments thereof to bind to a specific epitope. In certain embodiments, such a fragment includes at least one CDR present in a full-length light chain or heavy chain, and in some embodiments, includes a short-chain heavy chain and / or light chain, or part thereof. This biological active fragment may be produced by a recombinant DNA technique or may be produced, for example, by cleaving an intact antibody enzymatically or chemically. An immunologically functional immunoglobulin fragment includes, but is not limited to, Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFV, scFV-Fc, diabody, minibody, scAb, and dAb, and may be derived from any mammal, including, but not being limited to, a human, a mouse, a rat, a camelid, or a rabbit. The functional parts of antibodies such as the one or more CDRs disclosed in the present disclosure may be linked with a secondary protein or a small compound by a covalent bond, and thereby used as a targeted therapeutic agent for a specific target.

[0942] In the present disclosure, the “Fc” region includes two heavy chain fragments including CH2 and CH3 domains of an antibody. These two heavy chain fragments are linked to each other by hydrophobic interaction of two or more of disulfide bonds and a CH3 domain.

[0943] In the present disclosure, the “Fab fragment” consists of one light chain and one heavy chain including a variable region and CHI only. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. In an scFab, two molecules of Fab are linked by a flexible linker.

[0944] In the present disclosure, the “Fab' fragment” includes a Fab fragment and additionally a region between CHI and CH2 domains of a heavy chain. A disulfide bond may form between two heavy chains of Fab' fragments of two molecules, forming aF(ab')2 molecule.

[0945] In the present disclosure, as described above, the “F(ab')2 fragment” includes two light chains and two heavy chains including a variable region CHI and part of a constant region between the CHI and CH2 domains, with an inter-chain disulfide bond formed between the two heavy chains. Accordingly, a F(ab')2 fragment consists of two Fab' fragments, and the two Fab' fragments are joined to each other by the disulfide bond therebetween.

[0946] In the present disclosure, the “Fv region” is a fragment of an antibody which includes each variable region of a heavy chain and a light chain, but does not include constant regions. In an sdFV, a heavy chain and a light chain are linked by a disulfide bond. In an scFc, the Fv is linked by a flexible linker. In a scFv-Fc, a Fc is linked to a scFV. In a minibody, CH3 is linked to a scFV. A diabody includes the scFVs of two molecules.Attorney Docket No.: LCH-03725

[0947] In the present disclosure, the “single chain Fv” or “scFv” antibody fragment includes the VH and VL domains of an antibody, and these domains are present within a single polypeptide chain. An Fv polypeptide may additionally include a polypeptide linker between a Vh domain which enables the scFv to form the target structure for antigen binding, and a VL domain.

[0948] In the present disclosure, the “single-chain antibody scAb)” is a single polypeptide chain including one constant region of a heavy chain or a light chain constant region in which heavy chain and light chain variable regions are linked by a flexible linker. For a single-chain antibody, U.S. Pat. No. 5,260,203 may be referred to, and short-chain antibody is disclosed herein by reference.

[0949] In the present disclosure, the “domain antibody (dAb)” is an immunologically functional immunoglobulin fragment including only a variable region of a heavy chain or a variable region of a light chain. In certain embodiments, two or more VH regions are linked by a covalent bond via a peptide linker, to form a bivalent domain antibody. Two VH regions of this bivalent domain antibody may target the same or different antigens.

[0950] In the present disclosure, “complementarity determining region” (CDR; that is, CDR1, CDR2, and CDR3) denotes amino acid residues of the variable region of an antibody, which are necessary for binding to antigen. Each variable region typically has three CDR domains, identified as CDR1, CDR2, and CDR3.

[0951] In the present disclosure, the “framework region” (FR) is a variable region residue other than the CDR residues. Each variable region typically has four FRs, identified as FR1, FR2, FR3, and FR4.

[0952] In the present disclosure, the “bivalent antigen-binding protein” or “bivalent antibody” includes two antigen-binding sites. The two antigen-binding sites included in a bivalent antibody may have the same antigen specificity, or may be a bispecific antibody where the antigen-biding sites bind to different antigens.

[0953] In the present disclosure, the “multispecific antigen-binding protein” or “multispecific antibody” targets two or more antigens or epitopes.

[0954] As used herein, a “chimeric antibody” refers to an antibody (immunoglobulin) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit theAttorney Docket No.: UCH-03725

[0955] desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat’l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.

[0956] “Humanized” forms of non-human (e.g., murine) antibodies, are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an CDR of the recipient are replaced by residues from an CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, and the like. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the U chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0957] A “human antibody” is one that possesses an amino-acid sequence corresponding to that of an antibody, produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phagedisplay libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies areAttorney Docket No.: LCH-03725

[0958] methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.

[0959] 77 (1985); Boerner et al., J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Nat’l Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0960] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0961] An “affinity-matured” antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s). In one embodiment, an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by VH- and VL-domain shuffling. Random mutagenesis of CDR and / or framework residues is described by, for example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147- 155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0962] In the present disclosure, “linker” refers to a compound which covalently bonds a cytotoxic compound to an antibody.

[0963] In the present disclosure, "unsubstituted or substituted" is used to refer to a parent group which may be unsubstituted or substituted, "substituted" refers to a parent group having at least one substituent, and a substituent refers to a chemical moiety covalently bonded to or fused with a parent group.Attorney Docket No.: LCH-03725

[0964] In the present disclosure, “halo” refers to fluorine, chlorine, bromine, iodine, and the like.

[0965] As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C10 straight-chain alkyl groups or C1-C10 branched-chain alkyl groups. Preferably, the “alkyl” group refers to Ci-Ce straight-chain alkyl groups or Ci-Ce branched-chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl, 1 -heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1 -octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted.

[0966] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0967] The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0968] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0969] The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

[0970] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0971] The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., Ci-30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer.

[0972] Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

[0973] The term “Cx y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. Coalkyl indicates a hydrogen where the group is in a terminal position,Attorney Docket No.: LCH-03725

[0974] a bond if internal. A Ci-ealkyl group, for example, contains from one to six carbon atoms in the chain.

[0975] The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group.

[0976] The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0977] The term “amido”, as used herein, refers to a group

[0978]

[0979] wherein R9and R10each independently represent a hydrogen or hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0980] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by

[0981]

[0982] wherein R9, R10, and R10’ each independently represent a hydrogen or a hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0983] The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

[0984] The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

[0985] The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0986] The term “carbamate” is art-recognized and refers to a groupAttorney Docket No.: LCH-03725

[0987]

[0988] wherein R9and R10independently represent hydrogen or a hydrocarbyl group.

[0989] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0990] The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5 -cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-lH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

[0991] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0992] The term “carbonate” is art-recognized and refers to a group -OCO2-.

[0993] The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

[0994] The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R100) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole,Attorney Docket No.: LCH-03725

[0995] oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, tetrahydroquinoline, and the like.

[0996] The term “ester”, as used herein, refers to a group -C(O)OR9wherein R9represents a hydrocarbyl group.

[0997] The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.

[0998] The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0999] The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

[1000] The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[1001] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[1002] The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

[1003] The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,Attorney Docket No.: LCH-03725

[1004] cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[1005] The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbonhydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[1006] The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

[1007] The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[1008] The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

[1009] The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[1010] The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae

[1011]

[1012] wherein R9and R10independently represents hydrogen or hydrocarbyl.Attorney Docket No.: LCH-03725

[1013] The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

[1014] The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[1015] The term “sulfone” is art-recognized and refers to the group -S(O)2-.

[1016] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxy carbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamide, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[1017] The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

[1018] The term “thioester”, as used herein, refers to a group -C(O)SR9or -SC(O)R9wherein R9represents a hydrocarbyl.

[1019] The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

[1020] The term “urea” is art-recognized and may be represented by the general formula 0

[1021] ANRV

[1022] RxRxAttorney Docket No.: LCH-03725

[1023] wherein Rxand RYindependently represent hydrogen or a hydrocarbyl.

[1024] The term “DBCO” used herein refers to an optionally substituted dibenzocyclooctyne moiety, e.g., the following structure:

[1025]

[1026] The term “sugar moiety” used herein refers to a naturally occurring sugar' or a modified sugar which is part of a larger molecule and is connected to the remainder of the larger molecule through, for example, one of the hydroxyl groups present on the sugar. For example, in some embodiments the sugar is connected to the remainder of the larger molecule via the hydroxyl group on the anomeric carbon. Examples of sugar moieties include, but are not limited to, glucuronosyl and galactosyl.

[1027] A “glycosidase” is an enzyme that breaks down glycosidic bonds in carbohydrates, glycoproteins, and glycolipids. Examples of glycosidases are 0-glucuronidase and 0-galactosidase. P-glucuronidase is a type of glucuronidase that catalyzes hydrolysis of 0-D-glucuronic acid or P-glucuronosyl residues. 0-Galactosidase is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing P-D-galactose or P-galactosyl residues in 0-D-galactosides.

[1028] The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

[1029] The term “cleavage group” refers to a chemical moiety which dissociates when subjected to a stimulus, such as acidic conditions, basic conditions, reducing conditions, oxidizing conditions, light, or heat, or an enzyme, such as an esterase.

[1030] Examples

[1031] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

[1032] Preparation Example 1: Production of Exemplified BCMA Specific Antibodies BCMA (B-cell maturation antigen) is widely expressed in several hematologic malignancies. An exemplified anti-BCMA antibody Clonel (Belantamab), that specifically binds to BCMA, was produced by the methods described in U.S. Patent No. 11,419,945, theAttorney Docket No.: LCH-03725

[1033] entirety of which is incorporated herein by reference. Belantamab is an afucosylated antibody. The afucosylated antibody was produced by CH0-Fut8K0. The amino acid sequences of Clonel are shown in Table 1 below. The amino acid sequences of the CDRs below are defined according to the Kabat definition.

[1034] Table 1. Amino Acid Sequences of Exemplified BCMA Antibody Clonel (Belantamab)

[1035]

[1036] Attorney Docket No.: LCH-03725

[1037]

[1038] For ADC synthesis, an exemplified antibody Clone2 was constructed by introducing a CaaX peptide moiety (GGGGGGGCVIM, SEQ ID NO:21) to the C-terminus of the light chain of SEQ ID NO: 18 in Table 1 with the method disclosed in International Patent Application No. PCT / IB2012 / 001065, the entirety of which is incorporated herein by reference. The amino acid sequences of Clone2 are shown in Table 2 below.

[1039] Table 2. Amino Acid Sequences of Exemplified BCMA Antibody Clone2

[1040]

[1041] Attorney Docket No.: LCH-03725

[1042]

[1043] Exemplified antibody Clone3 was produced using CH0-K1 cells and by introducing LALA mutations (L238A / L239A of SEQ ID NO: 17 in Table 1) into the heavy chain constant region of the Clonel . Clone3 is a fucosylated version of Belantamab. The amino acid sequences of Clone3 are shown in Table 3 below. The amino acid sequences of the CDRs below are defined according to the Kabat definition.

[1044] Table 3. Amino Acid Sequences of Exemplified BCMA Antibody Clone3

[1045]

[1046] Attorney Docket No.: LCH-03725

[1047]

[1048] For ADC synthesis, an exemplified antibody Clone4 was constructed by introducing a CaaX peptide moiety (GGGGGGGCVIM, SEQ ID NO: 21) to the C-terminus of the light chain of SEQ ID NO: 18 in Table 3 with the method disclosed in International Patent Application No. PCT / IB2012 / 001065, the entirety of which is incorporated herein by reference. The amino acid sequences of Clone4 are shown in Table 4 below.

[1049] Table 4. Amino Acid Sequences of Exemplified BCMA Antibody Clone4

[1050]

[1051] Attorney Docket No.: LCH-03725

[1052] Preparation Example 2: Synthesis of Exemplified Compounds 2-1: Preparation of Compound 1

[1053]

[1054] Compound 1 was produced with the method disclosed in U.S. Patent No. 11,173,214, the entirety of which is incorporated herein by reference.

[1055] EI-MS m / z : [M+H]+1248.9.

[1056] 2-2: Preparation of Compound 2

[1057]

[1058] Compound 2 was produced with the method disclosed in U.S. Patent No. 11,173,214, the entirety of which is incorporated herein by reference.

[1059] EI-MS m / z: [M / 2+H]+1623.6, [M / 3+H]+1082.8.

[1060] The structure of MMAE (Monomethyl auristatin E) in Compound 2 is as follows:

[1061]

[1062] Attorney Docket No.: LCH-03725

[1063]

[1064] Compound 3 was produced with the method disclosed in U.S. Patent No. 11,654,197, the entirety of which is incorporated herein by reference.

[1065] EI-MS m / z: [M+H]+1698.2, [M / 2+H]+849.6.

[1066] 2-4: Preparation of Compound 4

[1067]

[1068] Compound 4 was produced with the method disclosed in International Publication No. WO 2024 / 189428 Al, the entirety of which is incorporated herein by reference.

[1069] EI-MS m / z : [M / 2+H]+1340.5, [M / 3+H]+894.1.

[1070] 2-5: Preparation of Compound 21Attorney Docket No.: LCH-03725

[1071]

[1072] Preparation of Compound 5

[1073] To a solution of 3-(6-methyl-2-pyridyl)propanoic acid (2.4 g, 14.53 mmol) in methanol (80 mL) was added thionyl chloride (4 mL, 54.80 mmol) at 0 °C under N2. After 3 hours, the reaction mixture was concentrated under the reduced pressure to afford Compound 5 (2.46 g, 94%) as an ivory solid, which was used for the next step without further purification.

[1074] 1H-NMR (400 MHz, CDCh) 58.12 (t, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 3.65 (s, 3H), 3.57 (t, J = 6.8 Hz, 2H), 3.11 (t, J = 6.8 Hz, 2H), 3.01 (s, 3H).

[1075] Preparation of Compound 6

[1076] To a solution of Compound 5 (3.98 g, 22.21 mmol) in dichloromethane (150 mL) was added 3-chloroperoxybenzoic acid (6.57 g, 26.65 mmol, purity 50-55%). After being stirred at room temperature for 16 hours, the reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated aq. sodium bicarbonate solution (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 6 (2.46 g, 56%) as an ivory solid.

[1077] ‘H-NMR (400 MHz, CDCh) 5 7.21-7.14 (m, 2H), 7.10 (t, J = 7.6 Hz, 1H), 3.66 (s, 3H), 3.23 (t, J = 6.8 Hz, 2H), 2.87 (t, J = 6.8 Hz, 2H), 2.53 (s, 3H).

[1078] Preparation of Compound 7

[1079] To a solution of Compound 6 (1.4 g, 7.17 mmol) in ethyl acetate (15 mL) was added HC1 (4 M in 1,4-dioxane, 0.25 mL,7.17 mmol). After being stirred at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure and dissolved in phosphorus(V) oxychloride (10 mL). Then the reaction mixture was heated at 90 °C. After 16 hours at 90 °C, the reaction mixture was concentrated under reduced pressure and then the residue was adjusted to pH 10 with aq. potassium carbonate solution. The resulting solutionAttorney Docket No.: LCH-03725

[1080] was diluted with dichloromethane (100 mL) and extracted with dichloromethane (70 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 7 (1.2 g, 78%) as a brown oil.

[1081] 1H-NMR (400 MHz, CDCh) 57.01 (d, J = 2.8 Hz, 2H), 3.68 (s, 3H), 3.05 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 7.63 Hz, 2H), 2.49 (s, 3H).

[1082] Preparation of Compound 8

[1083] To a solution of Compound 7 (884 mg, 4.14 mmol) in 1,4-dioxane (18 mL) were added vinylboronic acid pinacol ester (1.06 mL, 6.21 mmol) and 3 -chloroperoxybenzoic acid (6.57 g, 26.65 mmol, purity 50-55%). After being stirred at room temperature for 16 hours, the reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated aq. sodium bicarbonate solution (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 8 (2.46 g, 56%) as an ivory solid.

[1084] ‘H-NMR (400 MHz, CDCh) 5 6.97 (s, 2H), 6.64-6.57 (m, 1H), 5.92 (d, J = 17.6 Hz, 1H), 5.43 (d, J = 10.8 Hz, 1H), 3.67 (s, 3H), 3.07 (t, J = 7.6 Hz, 2H), 2.78 (t, J = 7.6 Hz, 2H), 2.51 (s, 3H). ELMS m / z : [M+H]+206.30.

[1085] Preparation of Compound 9

[1086] To a solution of Compound 8 (180 mg, 0.88 mmol) in tetrahydrofuran (5 mL) was added lithium hydroxide monohydrate (59 mg, 1.40 mmol) in water (5 mL). After being stirred at room temperature for 5 hours, the reaction mixture was adjusted to pH -3.0 with 1 M aq. HC1 solution and concentrated under reduced pressure to afford Compound 9 (260 mg, quant.) as an ivory solid, which was used for the next step without further purification.

[1087] ‘H-NMR (400 MHz, DMSO-d6) 57.84 (d, J= 11.2 Hz, 2H), 6.91-6.84 (m, 1H), 4.49 (d, J = 17.6 Hz, 1H), 5.90 (d, J = 10.8 Hz, 1H), 3.21 (t, J = 7.6 Hz, 2H), 2.86 (t, J = 7.6 Hz, 2H), 2.72 (s, 3H). ELMS m / z : [M+H]+192.32.Attorney Docket No.: LCH-03725

[1088]

[1089] Preparation of Compound 10

[1090] To a solution of pentaethylene glycol (10 g, 41.97 mmol) in dichloromethane (45 mL) were added p-toluenesulfonyl chloride (16 g, 83.94 mmol) and potassium hydroxide (18.84 g, 335.74 mmol) at 0 °C. The reaction mixture was warmed to room temperature under N2. After 16 hours, the reaction mixture was diluted with dichloromethane (80 mL) and washed with distilled water (80 mL). The organic layer was dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure afforded Compound 10 (24 g, quant.) as a clear oil, which was used for the next step without further purification.

[1091] ‘H-NMR (400 MHz, CDCI3) 57.79 (d, J= 6.4 Hz, 4H), 7.34 (d, J= 8.0 Hz, 4H), 4.15 (t, J = 4.8 Hz, 4H), 3.68 (t, J = 4.8 Hz, 4H), 3.60 (s, 4H), 3.58 (s, 8H), 2.45 (s, 6H).

[1092] Preparation of Compound 11

[1093] Compound 10 (28 g, 51.22 mmol) and sodium azide (13.32 g, 204.88 mmol) were dissolved in A i methyl formamide (100 mL). And then the reaction mixture was heated to 80 °C. After being stirred for 16 hours, the reaction mixture was diluted with ethyl acetate (150 mL) and washed with saturated aq. ammonium chloride solution (85 mL) and distilled water (80 mL x 3). The organic layer was dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure afforded Compound 11 (14.3 g, 96.8%) as a clear oil, which was used for the next step without further purification.

[1094] 1H-NMR (400 MHz, CDC13) 5 3.72-3.64 (m, 16H), 3.39 (t, J = 4.8 Hz, 4H). ELMS m / z : [M+H]+311.41.

[1095] Preparation of Compound 12

[1096] To a solution of Compound 11 (14.3 g, 49.60 mmol) in ethyl acetate (180 mL) were added triphenylphosphine (13 g, 49.60 mmol) and 1 N aq. HC1 solution (91.76 mL, 91.76Attorney Docket No.: LCH-03725

[1097] mmol) at 0 °C under N2. After being stirred at room temperature for 16 hours, the reaction mixture was concentrated under reduced pressure and then the residue was adjusted to pH 14 with 1 M aq. sodium hydroxide solution. The mixture was extracted with dichloromethane (100 mL x 2). The combined organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 12 (11 g, 84%) as a clear oil, which was used for the next step without further purification.

[1098] EI-MS m / z : [M+H]+263.43.

[1099] Preparation of Compound 13

[1100] To a solution of Compound 12 (11 g, 41.94 mmol) in dichloromethane (150 mL) was added di - / -butyl dicarbonate (9.83 mL, 42.77 mmol) under N2. After being stirred at room temperature for 5 hours, the reaction mixture was concentrated under reduced pressure. The resulting Compound 13 (15.19 g, quant.) was used for the next step without further purification.

[1101] Preparation of Compound 14

[1102] To a solution of Compound 13 (15.19 g, 41.91 mmol) in tetrahydrofuran (200 mL) were added triphenylphosphine (16.49 g, 62.87 mmol) and distilled water (7.6 mL, 419 mmol) at 0 °C under N2. The reaction mixture was heated to 85 °C and stirred for 16 hours. After the completion of reaction, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 14 (5.82 g, 41%).

[1103] 1H-NMR (400 MHz, CDCh) 55.25 (bs, 1H), 3.70-3.62 (m, 12H), 3.58-3.50 (m, 4H), 3.33-3.28 (m, 2H), 2.87 (t, J= 5.2 Hz, 2H), 1.44 (s, 9H). EI-MS m / z : [M+H]+337.51.

[1104] Preparation of Compound 16

[1105] To a solution of Compound 14 (1.7 g, 3.51 mmol) in N,N-di methylformamide (30 mL) were added Compound 15 (1.3 g, 3.86 mmol, Compound 15 was prepared by the method described in U.S. Patent No. 11,173,214), A,A,A’,A’-tetramethyl-(9-(lH-benzotriazol-l-yljuronium hexafluorophosphate (HBTU, 2.0 g, 5.26 mmol) and A i isopropyl ethyl amine (1.83 mL, 10.53 mmol) under N2 at 0 °C. After being stirred at room temperature for 6 hours, the reaction mixture was diluted with ethyl acetate (70 mL) and washed with saturated aq. ammonium chloride solution (85 mL) and distilled water (80 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reducedAttorney Docket No.: LCH-03725

[1106] pressure. The residue was purified by column chromatography to afford Compound 16 (2.3 g, 81.6%).

[1107] 1H-NMR (400 MHz, CDCh) 57.97 (bs, 1H), 7.46 (dd, h = 8.4 Hz, h = 2.0 Hz, 1H), 7.38 (bs, 1H), 7.04 (d, J = 8.8 Hz, 1H), 5.42-5.28 (m, 3 H), 5.25 (d, J= 7.2 Hz, 1H), 5.18-5.11 (m, 1H), 4.67 (d, J = 5.6 Hz, 2H), 4.21 ( d, J = 5.6 Hz, 1H), 3.79-3.72 (m, 4H), 3.71-3.62 (m, 8H), 3.62-3.56 (m, 6H), 3.27 (t, 7= 5.2 Hz, 3H), 3.29-3.24 (m, 2H), 2.06 (s, 3H), 2.06 (s, 6H), 1.44 (s, 9H). EI-MS m / z : [M+Na]+825.50.

[1108] Preparation of Compound 17

[1109] To a solution of Compound 16 (1 g, 1.25 mmol) in dichloromethane (10 mL) were added bis(pentafluorophenyl)carbonate (589 mg, 1.49 mmol) and A,A-diisopropylethylamine (0.33 mL, 1.87 mmol) at 0 °C under N2. After being stirred at room temperature for 16 hours, the reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated aq. sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 17 (1.23 g, 97%) as a pale yellow foam solid.

[1110] EI-MS m / z : [M+Na]+1013.42.

[1111]

[1112] Attorney Docket No.: LCH-03725

[1113] To a solution of Compound 17 (430 mg, 0.42 mmol) in A,A-dimethylformamide (5 mL) were added MMAF-OMe (348 mg, 0.47 mmol), A,A’-diisopropylethylamine (0.3 mL, 2.12 mmol). After being stirred at room temperature for 12 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (50 mL) and distilled water (30 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 18 (640 mg, 96%) as a white solid.

[1114] ELMS m / z : [M / 2+H+] 788.62.

[1115] Preparation of Compound 19

[1116] To a solution of Compound 18 (200 mg, 0.13 mmol) in dichloromethane (2 mL) at 0 °C was added trifluoroacetic acid (0.6 mL). After being stirred at room temperature for 2 hours under N2, the reaction mixture was concentrated under reduced pressure to afford Compound 19 (202 mg, quant.), which was used without further purification.

[1117] ELMS m / z : [M / 2+H]+738.57.

[1118] Preparation of Compound 20

[1119] To a solution of Compound 19 (132 mg, 0.08 mmol) in A,A-dimethylformamide (3 mL) were added Compound 9 (37 mg, 0.12 mmol), A,A,A’,A’-tetramethyl-(9-(lH-benzotriazoLLyl)uronium hexafluorophosphate (HBTU, 47 mg, 0.12 mmol) and N,N-diisopropylethylamine (0.1 mL, 0.58 mmol) at 0 °C under N2. After being stirred at room temperature for 2 hours, the reaction mixture was diluted with ethyl acetate (60 mL) and washed with distilled water (40 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 20 (136 mg, 99%).

[1120] ELMS m / z : [M / 2+H]+825.16.

[1121] Preparation of Compound 21

[1122] To a solution of Compound 20 (136 mg, 0.08 mmol) in tetrahydrofuran / methanol (1 mL / 1 mL) was added lithium hydroxide monohydrate (17 mg, 0.41 mmol) in distilled water (1 mL) at -50 °C under N2. The reaction mixture was warmed up to 0 °C and kept for 2 hours at this temperature. Then the reaction mixture was neutralized using acetic acid andAttorney Docket No.: LCH-03725

[1123] concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 21 (75 mg, 61%).

[1124] EI-MS m / z : [M / 2+H]+748.10, [M+H]+1494.58.

[1125] 2-6: Preparation of Compound 24

[1126]

[1127] Preparation of Compound 22

[1128] To a solution of Compound 18 (275 mg, 0.18 mmol) in tetrahydrofuran / methanol (1 mL / 1 mL) was added lithium hydroxide monohydrate (37 mg, 0.87 mmol) in distilled water (1 mL) at -50 °C under N2. The reaction mixture was warmed up to 0 °C and kept for 2 hours at this temperature. Then the reaction mixture was neutralized using acetic acid. The reaction mixture was concentrated under reduced pressure to afford Compound 22 (248 mg, quant).

[1129] EI-MS m / z : [M / 2+H]+711.54, [M+H]+1421.47.

[1130] Preparation of Compound 23

[1131] To a solution of Compound 22 (248 mg, 0.17 mmol) in dichloromethane (2 mL) at 0 °C was added trifluoroacetic acid (0.4 mL). After being stirred at room temperature for 2 hours under N2, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 23 (154 mg, 61%).

[1132] EI-MS m / z : [M / 2+H]+738.57.

[1133] Preparation of Compound 24

[1134] To a solution of Compound 23 (154 mg, 0.11 mmol) in A, A-di methyl formamide (2 mL) were added 6-maleimidohexanoic acid A-hydroxysuccin imide ester (40 mg, 0.13 mmol), MA'-di isopropylethylamine (0.06 mL, 0.32 mmol). After being stirred at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 24 (92 mg, 57%).Attorney Docket No.: LCH-03725

[1135] EI-MS m / z : [M / 2+H]+758.06, [M+H]+1514.43.

[1136] 2-7: Preparation of Compound 28

[1137]

[1138] Preparation of Compound 25

[1139] To a solution of Compound 17 (150 mg, 0.15 mmol) in A,A-dimethylformamide (5 mL) were added exatecan mesylate (86.59 mg, 0.16 mmol) and A,A’-diisopropylethylamine (0.1 mL, 0.74 mmol). After being stirred at room temperature for 4 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (50 mL) and distilled water (30 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 25 (187 mg, quant.) as a yellow solid.

[1140] EI-MS m / z : [M+H]+1265.12.

[1141] Preparation of Compound 26

[1142] To a solution of Compound 25 (187 mg, 0.15 mmol) in dichloromethane (3 mL) at 0 °C was added trifluoroacetic acid (0.5 mL). After being stirred at room temperature for 2 hoursAttorney Docket No.: LCH-03725

[1143] under N2, the reaction mixture was concentrated under reduced pressure to afford Compound 26 (189 mg, quant.), which was used without further purification.

[1144] EI-MS m / z : [M / 2+H]+582.86, [M+Na]+1185.99.

[1145] Preparation of Compound 27

[1146] To a solution of Compound 26 (189 mg, 0.16 mmol) in A iniethylforniamide (5 mL) were added Compound 9 (45 mg, 0.24 mmol), A( ACA / '-tetraniethyl-O-(l / / -benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 89 mg, 0.24 mmol) and N,N-diisopropylethylamine (0.11 mL, 0.79 mmol) at 0 °C under N2. After being stirred at room temperature for 4 hours, the reaction mixture was diluted with ethyl acetate (60 mL) and washed with distilled water (40 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 27 (142 mg, 67%).

[1147] EI-MS m / z : [M / 2+H]+669.40, [M+H]+1338.10.

[1148] Preparation of Compound 28

[1149] To a solution of Compound 27 (142 mg, 0.11 mmol) in tetrahydrofuran / methanol (2 mL / 2 mL) was added lithium hydroxide monohydrate (17.8 mg, 0.42 mmol) in distilled water (3 mL) at -50 °C under N2. The reaction mixture was warmed up to 0 °C and kept for 2 hours at this temperature. Then the reaction mixture was neutralized using acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 28 (51 mg, 40%).

[1150] EI-MS m / z : [M / 2+H]+599.41, [M+H]+1197.03Attorney Docket No.: LCH-03725

[1151] 2-8 : Preparation of Compound 33

[1152]

[1153] Preparation of Compound 30

[1154] To a solution of Compound 29 (200 mg, 0.16 mmol, Compound 29 was prepared by the method described in U.S. Patent No. 11,173,214) in N,N-dimethylformamide (5 mL) were added Compound 14 (82 mg, 0.24 mmol), N,N,N’,N’-tetramethyl-(9-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 93 mg, 0.24 mmol) and UV-diisopropylethylamine (0.09 mL, 0.49 mmol) at 0 °C under N2. After being stirred at room temperature for 16 hours, the reaction mixture was diluted with ethyl acetate (60 mL) and washed with saturated aq. ammonium chloride solution (50 mL) and distilled water (40 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 30 (250 mg, 99%).

[1155] ELMS m / z : [M / 2+H]+6774.69, [M+H]+1548.00.

[1156] Preparation of Compound 31

[1157] To a solution of Compound 30 (120 mg, 0.08 mmol) in dichloromethane (5 mL) at 0 °C was added HC1 (4 M in 1,4-dioxane, 0.13 mL). After being stirred at room temperature for 24 hours under N2, the reaction mixture was concentrated under reduced pressure to afford Compound 31 (115 mg, quant.), which was used without further purification.

[1158] ELMS m / z : [M / 2+H]+724.36, [M+H]+1446.99.Attorney Docket No.: LCH-03725

[1159] Preparation of Compound 32

[1160] To a solution of Compound 31 (115 mg, 0.08 mmol) in A,A-dimethylformamide (5 mL) were added Compound 9 (30 mg, 0.10 mmol), A( ACA / '-tetraniethyl-O-(l / / -benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 44 mg, 0.12 mmol) and N,N-diisopropylethylamine (0.09 mL, 0.54 mmol) at 0 °C under N2. After being stirred at room temperature for 3 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (40 mL) and distilled water (40 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 32 (111 mg, 88%) as a pale yellow foam solid.

[1161] ELMS m / z : [M / 2+H]+810.97, [M+H]+1621.21.

[1162] Preparation of Compound 33

[1163] To a solution of Compound 32 (111 mg, 0.07 mmol) in tetrahydrofuran / methanol (2 mL / 1 mL) was added lithium hydroxide monohydrate (14.38 mg, 0.34 mmol) in distilled water (3 mL) at -50 °C under N2. The reaction mixture was warmed up to 0 °C and kept for 2 hours at this temperature. Then the reaction mixture was neutralized using acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 33 (66 mg, 60%).

[1164] ELMS m / z : [M / 2+H]+740.98, [M+H]+1480.09.Attorney Docket No.: LCH-03725

[1165] 2-9 : Preparation of Compound 43

[1166]

[1167] Preparation of Compound 35

[1168] To a solution of Compound 34 (1.2 g, 2.31 mmol, Compound 34 was prepared by the method described in the International Publication No. WO2021 / 137646 Al, the entirety of which is incorporated herein by reference) in tetrahydrofuran (40 mL) were added triphosgene (274 mg, 0.93 mmol) and triethylamine (1.3 mL, 9.25 mmol) at 0 °C under N2. After being stirred at 0 °C for 10 min, a solution of Compound 16 (2.2 g, 2.78 mmol) and triethylamine (0.3 mL, 2.31 mmol) in tetrahydrofuran (20 mL) were added to the reaction mixture at 0 °C under N2. After being stirred at room temperature for 17 hours, the reaction mixture was diluted with ethyl acetate (200 mL) and washed with saturated aq. sodium bicarbonate solution (100 mL) and distilled water (100 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 35 (2.0 g, 64%).

[1169] ‘H-NMR (400 MHz, DMSO-d6) 59.12 (s, 1H), 7.93 (s, 1H), 7.66 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.21-7.13 (m, 2H), 6.73 (s, 1H), 6.05-5.91 (m, 1H), 5.79-5.72 (m, 1H), 5.49 (t, JAttorney Docket No.: LCH-03725

[1170] = 9.6 Hz, 1H), 5.31-5.27 (m, 1H), 5.25-5.16 (m, 2H), 5.12-5.03 (m, 3H), 4.96 (s, 1H), 4.79-4.70 (m, 1H), 4.56 (d, J = 5.5 Hz, 2H), 3.97 (t, J = 6.3 Hz, 3H), 3.75 (s, 3H), 3.63 (s, 3H), 3.58-3.45 (m, 16H), 3.05 (q, J = 6.0 Hz, 3H), 2.63 (s, 1H), 2.01 (s, 11H), 1.36 (s, 9H), 0.80 (d, J = 41.8 Hz, 9H), 0.21 (s, 4H), -0.17 (s, 2H). EI-MS m / z : [M+H]+1348.82.

[1171] Preparation of Compound 36

[1172] To a solution of Compound 35 (2.0 g, 1.48 mmol) in tetrahydrofuran (5 mL) and water (5 mL) was added acetic acid (5 mL) at 0 °C under N2. After being stirred at room temperature for 20 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. sodium bicarbonate solution (50 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 36 (1.3 g, 69%).

[1173] EI-MS m / z : [M+H]+1234.65.

[1174] Preparation of Compound 37

[1175] To a solution of Compound 36 (1.3 g, 1.02 mmol) in dichloromethane (30 mL) was added Dess-Martin periodinane (563 mg, 1.33 mmol) at 0 °C under N2. After being stirred at room temperature for 5 hours, the reaction mixture was diluted with dichloromethane (50 mL) and washed with saturated aq. sodium bicarbonate solution (50 mL) and distilled water (50 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 37 (991 mg, 79%).

[1176] EI-MS m / z : [M+H]+1232.63.

[1177] Preparation of Compound 38

[1178] To a solution of Compound 37 (500 mg, 0.41 mmol) in A,A-dimethylformamide (4 mL) were added tetrakis(triphenylphosphine)palladium(0) (188 mg, 0.16 mmol), pyrrolidine (0.04 mL, 0.49 mmol) under N2. After being stirred at room temperature for 2 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (40 mL) and distilled water (40 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 38 (434 mg, 90%) as a pale yellow foam solid.Attorney Docket No.: LCH-03725

[1179] EI-MS m / z : [M+H]+1192.59.

[1180] Preparation of Compound 40

[1181] To a solution of Compound 38 (434 mg, 0.36 mmol) in A,A-dimethylformamide (3 mL) were added Compound 39 (175 mg, 0.55 mmol, Compound 39 was prepared by the method described in the International Publication No. WO2021 / 137646 Al, the entirety of which is incorporated herein by reference, A,A,A’,A’-tetramethyl-(9-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 180 mg, 0.47 mmol) and UV-diisopropylethylamine (0.13 mL, 0.73 mmol) at 0 °C under N2. After being stirred at room temperature for 2 hours, the reaction mixture was diluted with ethyl acetate (60 mL) and washed with distilled water (40 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 40 (430 mg, 79%).

[1182] EI-MS m / z : [M / 2+H]+747.70, [M+H]+1493.99.

[1183] Preparation of Compound 41

[1184] To a solution of Compound 40 (197 mg, 0.13 mmol) in dichloromethane (2 mL) at 0 °C was added trifluoroacetic acid (0.3 mL). After being stirred at room temperature for 2 hours under N2, the reaction mixture was concentrated under reduced pressure to afford Compound 41 (198 mg, quant.), which was used without further purification.

[1185] EI-MS m / z : [M / 2+H]+697.81, [M+H]+1393.86.

[1186] Preparation of Compound 42

[1187] To a solution of Compound 41 (198 mg, 0.13 mmol) in A,A-dimethylformamide (5 mL) were added Compound 9 (51 mg, 0.17 mmol), A,A,A’,A’-tetramethyl-(9-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 75 mg, 0.20 mmol) and NJV-diisopropylethylamine (0.11 mL, 0.66 mmol) at 0 °C under N2. After being stirred at room temperature for 17 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (40 mL) and distilled water (40 mL x 3). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 42 (173 mg, 84%) as a pale yellow foam solid.

[1188] EI-MS m / z : [M / 2+H]+784.93, [M+H]+1567.16.Attorney Docket No.: LCH-03725

[1189] Preparation of Compound 43

[1190] To a solution of Compound 42 (173 mg, 0.11 mmol) in tetrahydrofuran / methanol (1 mL / 1 mL) was added lithium hydroxide monohydrate (19 mg, 0.44 mmol) in distilled water (1 mL) at -50 °C under N2. The reaction mixture was warmed up to 0 °C and kept for 2 hours at this temperature. Then the reaction mixture was neutralized using acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 43 (110 mg, 70%).

[1191] EI-MS m / z : [M / 2+H]+714.45, [M+H]+1426.91.

[1192] 2-10 : Preparation of Compound 57

[1193]

[1194] 44 45

[1195] Preparation of Compound 44

[1196] To a solution of A-boc-hydroxylamine (5 g, 37.55 mmol) in A iniethylforniamide (20 mL) were added potassium carbonate (8.8 g, 63.84 mmol) and ethyl-2-bromoisobutyrate (15.38 g, 78.86 mmol). After being stirred at room temperature for 16 hours, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with saturated aq. ammonium chloride solution (50 mL), distilled water (50 mL), and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 44 (10.5 g, 100%).

[1197] ‘H-NMR (400 MHz, CDCh) 54.21 (p, J= 6.8 Hz, 2H), 1.49 (s, 6H), 1.47 (s, 9H), 1.31 (q, J = 6.9 Hz, 3H).

[1198] Preparation of Compound 45

[1199] To a solution of Compound 44 (47 g, 190.1 mmol) in methanol (400 mL) was added lithium hydroxide monohydrate (16 g, 380 mmol) in water (100 mL). After being stirred at room temperature for 3 hours, the reaction mixture was adjusted to pH 3 with aq. 1 M HC1 solution and concentrated. Then the reaction mixture was extracted with ethyl acetate (200 mL x 3). The combined organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and triturated with ether. The resulting solid was filtered to afford Compound 45 (38 g, 91%) as a white solid.Attorney Docket No.: LCH-03725

[1200] 1H-NMR (400 MHz, CDCI3) 57.32 (s, 1H), 1.53 (s, 6H), 1.49 (s, 9H).

[1201]

[1202] Preparation of Compound 46

[1203] To a solution of pentaethylene glycol (10 g, 41.97 mmol) in dichloromethane (45 mL) were added p-toluenesulfonyl chloride (16 g, 83.94 mmol) and potassium hydroxide (18.84 g, 335.74 mmol) at 0 °C. The reaction mixture was warmed to room temperature under N2. After 16 hours, the reaction mixture was diluted with dichloromethane (80 mL) and washed with distilled water (80 mL). The organic layer was dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure afforded Compound 46 (24 g, quant.) as a clear oil, which was used for the next step without further purification.

[1204] ‘H-NMR (400 MHz, CDCI3) 57.79 (d, J= 6.4 Hz, 4H), 7.34 (d, J= 8.0 Hz, 4H), 4.15 (t, J = 4.8 Hz, 4H), 3.68 (t, J = 4.8 Hz, 4H), 3.60 (s, 4H), 3.58 (s, 8H), 2.45 (s, 6H).

[1205] Preparation of Compound 47

[1206] Compound 46 (28 g, 51.22 mmol) and sodium azide (13.32 g, 204.88 mmol) were dissolved in .A i methyl formamide (100 mL). And then the reaction mixture was heated to 80 °C. After being stirred for 16 hours, the reaction mixture was diluted with ethyl acetate (150 mL) and washed with saturated aq. ammonium chloride solution (85 mL) and distilled water (80 mL x 3). The organic layer was dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure afforded Compound 47 (14.3 g, 96.8%) as a clear oil, which was used for the next step without further purification.

[1207] ‘H-NMR (400 MHz, CDCI3) 5 3.72-3.64 (m, 16H), 3.39 (t, J = 4.8 Hz, 4H). ELMS m / z : [M+H]+311.41.

[1208] Preparation of Compound 48Attorney Docket No.: LCH-03725

[1209] To a solution of Compound 47 (14.3 g, 49.60 mmol) in ethyl acetate (180 mL) were added triphenylphosphine (13 g, 49.60 mmol) and 1 N aq. HC1 solution (91.76 mL, 91.76 mmol) at 0 °C under N2. After being stirred at room temperature for 16 hours, the reaction mixture was concentrated under reduced pressure and then the residue was adjusted to pH 14 with 1 M aq. sodium hydroxide solution. The mixture was extracted with dichloromethane (100 mL x 2). The combined organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 48 (11 g, 84%) as a clear oil, which was used for the next step without further purification.

[1210] ELMS m / z : [M+H]+263.43.

[1211] Preparation of Compound 49

[1212] To a solution of Compound 48 (1.04 g, 3.96 mmol) in A,A-dimethylformamide (15 mL) were added Compound 45 (955 mg, 4.35 mmol), A,A,A’,A’-tetramethyl-(9-(lH-benzotriazoLLyljuronium hexafluorophosphate (HBTU, 2.25 g, 5.94 mmol), and A,A-diisopropylethylamine (1.37 mL, 7.91 mmol) under N2. After being stirred at room temperature for 16 hours, the reaction mixture was diluted with ethyl acetate (60 mL) and washed with saturated aq. ammonium chloride solution (60 mL), saturated aq. sodium bicarbonate solution (60 mL) and brine (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 49 (923 mg, 50%).

[1213] ELMS m / z : [M+H]+464.42.

[1214] Preparation of Compound 50

[1215] To a solution of Compound 49 (923 mg, 1.99 mmol) in tetrahydrofuran (20 mL) were added triphenylphosphine (1.56 g, 5.97 mmol) and distilled water (0.35 mL, 19.9 mmol) at 0 °C under N2. The reaction mixture was heated to 85 °C and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 50 (703 mg, 80%).

[1216] ELMS m / z : [M+H]+438.22.

[1217] Preparation of Compound 51

[1218] To a solution of Compound 50 (703 mg, 1.61 mmol) in A i methyl formamide (8 mL) were added Compound 15 (702 mg, 1.45 mmol), A,A,A’,A’-tetramethyl-(9-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 916 mg, 2.41 mmol) and N,N-Attorney Docket No.: LCH-03725

[1219] diisopropylethylamine (0.56 mL, 3.22 mmol) under N2 at 0 °C. After being stirred at room temperature for 6 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (50 mL) and distilled water (30 mL x 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 51 (780 mg, 53%).

[1220] ELMS m / z : [M+H]+904.28.

[1221]

[1222] Preparation of Compound 53

[1223] To a solution of Compound 52 (500 mg, 0.35 mmol, Compound 52 was prepared by the method described in the International Publication No. WO2021 / 137646 Al) in dichloromethane (20 mL) were added triphosgene (41 mg, 0.14 mmol), N,N- diisopropylethylamine (0.03 mL, 1.05 mmol) and dibutyltin dilaurate (0.02mL, 0.04 mmol) at 0 °C under N2. After being stirred at 0 °C for 10 min, a solution of Compound 51 (411 mg, 0.45 mmol) and A N-di isopropylethyl amine (0.03 mL, 1.05 mmol) in dichloromethane (20 mL) were added to the reaction mixture at 0 °C under N2. After being stirred at room temperature for 17 hours, the reaction mixture was diluted with ethyl acetate (30 mL) and washed with saturated aq. sodium bicarbonate solution (20 mL) and distilled water (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated underAttorney Docket No.: LCH-03725

[1224] reduced pressure. The resulting residue was purified by column chromatography to afford Compound 53 (403 mg, 48%).

[1225] EI-MS m / z : [M / 2+H]+1177.32.

[1226] Preparation of Compound 54

[1227] To a solution of Compound 53 (403 mg, 0.17 mmol) in tetrahydrofuran (5 mL) and water (5 mL) was added acetic acid (5 mL) at 0 °C under N2. After being stirred at room temperature for 20 hours, the reaction mixture was diluted with ethyl acetate (30 mL) and washed with saturated aq. sodium bicarbonate solution (50 mLx 2). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 54 (360 mg, 98%).

[1228] EI-MS m / z : [M+H]+2121.94

[1229] Preparation of Compound 55

[1230] To a solution of Compound 54 (360 mg, 0.16 mmol) in dichloromethane (30 mL) was added Dess-Martin periodinane (86 mg, 0.20 mmol) at 0 °C under N2. After being stirred at room temperature for 5 hours, the reaction mixture was diluted with dichloromethane (30 mL) and washed with saturated aq. sodium bicarbonate solution (30 mL) and distilled water (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 55 (326 mg, 95%).

[1231] EI-MS m / z : [M+H]+2117.86.

[1232] Preparation of Compound 56

[1233] To a solution of Compound 55 (326 mg, 0.15 mmol) in tetrahydrofuran / methanol (12 mL / 12 mL) was added lithium hydroxide monohydrate (116 mg, 2.77 mmol) in distilled water (12 mL) at -50 °C under N2. The reaction mixture was warmed up to 0 °C and kept for 2 hours at this temperature. The reaction mixture was adjusted to pH 4~5 with acetic acid and concentrated under reduced pressure to afford Compound 56 (310 mg, crude), which was used without further purification.

[1234] EI-MS m / z : [M+H]+1837.75.

[1235] Preparation of Compound 57Attorney Docket No.: LCH-03725

[1236] To a solution of compound 56 (160 mg, 0.08 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.6 mL) at 0 °C. The reaction mixture was stirred at room temperature for 5 hours and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 57 (81 mg, 52%) as a white solid.

[1237] EI-MS m / z : [M+H]+1738.41, [M / 2+H]+869.77.

[1238] 2-11 : Preparation of Compound 81

[1239]

[1240] Preparation of Compound 58

[1241] To a mixture of hexaethylene glycol (30 g, 106 mmol), silver (I) oxide (29.55 g, 128 mmol) and potassium iodide (1.76 g, 10.6 mmol) in dichloromethane (2 mL) was added p-toluenesulfonyl chloride (20.26 g, 106 mmol) at 0 °C. The reaction mixture was warmed to room temperature under N2. After 16 hours, the reaction mixture was filtered through a celite pad, concentrated under reduced pressure and purified by column chromatography to yield Compound 58 (28.5 g, 61%) as a colorless oil.

[1242] ‘H-NMR (400 MHz, CDCh) 57.80 (d, J= 8.4 Hz, 2H), 7.34 (d, J= 8.4 Hz, 2H), 4.16 (m, 2H), 3.72-3.58 (m, 22H), 2.97 (br, 1H), 2.45 (s, 3H). EI-MS m / z : [M+H]+437.79.

[1243] Preparation of Compound 59

[1244] Compound 58 (28 g, 64.1 mmol) and sodium azide (6.25 g, 96.2 mmol) were dissolved in A(A i m ethyl formamide (70 mL). Then the reaction mixture was heated to 100 °C. After being stirred for 3 hours, the reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography to yield Compound 59 (11.3 g, 57%) as a colorless oil.

[1245] 1H-NMR (400 MHz, CDCh) 53.77-3.69 (m, 2H), 3.71-3.63 (m, 18H), 3.67-3.58 (m, 2H), 3.40 (t, 7 = 5.1 Hz, 2H). EI-MS m / z : [M+H]+308.65.

[1246] Preparation of Compound 60Attorney Docket No.: LCH-03725

[1247] To a solution of Compound 59 (10 g, 32.5 mmol) in tetrahydrofuran (60 mL) were added triphenylphosphine (9.39 g, 35.8 mmol) and distilled water (5.9 mL, 325 mmol) at 0 °C under N2. The reaction mixture was heated to 85 °C and stirred for 4 hours. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by column chromatography to yield Compound 60 (7.7 g, 84%) as a colorless oil.

[1248] 1H-NMR (400 MHz, CDCh) 53.75-3.57 (m, 22H), 3.56-3.49 (m, 2H), 2.92-2.83 (m, 2H). EI-MS m / z : [M+H]+282.34.

[1249] Preparation of Compound 61

[1250] To a solution of Compound 60 (7.7 g, 27.4 mmol) in acetonitrile (50 mL) were added benzyl chloroformate (4.7 mL, 32.8 mmol) and a solution of sodium bicarbonate (6.9 g, 82.1 mmol) in distilled water (60 mL). After being stirred at room temperature for 16 hours, the reaction mixture was diluted with dichloromethane (100 mL) and washed with distilled water (30 mL x 3). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to yield Compound 61 (9.27 g, 82%).

[1251] ‘H-NMR (400 MHz, CDCh) 57.35 (d, J = 4.7 Hz, 5H), 5.10 (d, J = 3.8 Hz, 2H), 3.77-3.50 (m, 22H), 3.40 (t, J = 5.6 Hz, 2H), 3.01 (d, J= 6.5 Hz, 1H). EI-MS m / z : [M+H]+416.34.

[1252] Preparation of Compound 62

[1253] To a solution of Compound 61 (2.2 g, 5.30 mmol) in dichloromethane (50 mL) were added triethylamine (2.23 mL, 15.89 mmol) and p-toluenesulfonyl chloride (1.31 g, 6.88 mmol) at 0 °C. After being stirred at room temperature for 4 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with aq. 10% citric acid solution (30 mL x 3) and brine (30 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to yield Compound 62 (1.9 g, 63%) as a colorless oil.

[1254] EI-MS m / z : [M+H]+570.07.

[1255]

[1256] Attorney Docket No.: LCH-03725

[1257] To a solution of hexaethylene glycol (65.37 g, 232 mmol) in tetrahydrofuran (100 mL) was added sodium hydride (60 % dispersion in mineral oil, 151 mg, 6.29 mmol) at 0 °C. After being stirred at 0 °C for 30 minutes, the reaction mixture was added tert-butyl acrylate (10 mL, 68.7 mmol). After being stirred at room temperature for 16 hours, the reaction mixture was quenched with acetic acid (1 mL) and concentrated under reduced pressure. Then, the resulting residue was diluted with distilled water (500 mL) and extracted with ethyl acetate (500 mL x 4). The combined organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 63 (28 g, 100 %) as a colorless oil, which was used without further purification.

[1258] ELMS m / z : [M+Na]+433.18.

[1259] Preparation of Compound 64

[1260] To a solution of Compound 63 (900 mg, 2.19 mmol) in dichloromethane (20 mL) were added triethylamine (0.92 mL, 6.58 mmol) and p-toluenesulfonyl chloride (627 mg, 3.29 mmol) at 0 °C. After being stirred at room temperature for 25 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (30 mL x 3) and brine (30 mL). The organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to yield Compound 64 (900 mg, 73%) as a pale yellow oil.

[1261] ’H-NMR (400 MHz, CDCh) 5 7.83-7.76 (m, 2H), 7.37-7.31 (m, 2H), 4.19-4.12 (m, 2H), 3.75-3.66 (m, 4H), 3.66-3.43 (m, 22H), 2.50 (t, J = 6.6 Hz, 2H), 2.45 (s, 4H), 1.45 (s, 9H). ELMS m / z : [M+Na]+587.33.

[1262]

[1263] Preparation of Compound 65

[1264] To a solution of bis(2-hydroxyethyl)ether (20 g, 188.5 mmol) in dichloromethane (400 mL) were added p-toluenesulfonyl chloride (79 g, 414.6 mmol) and potassium hydroxide (42 g, 753.9 mmol) at 0 °C under N2. After being stirred at room temperature for 18 hours, theAttorney Docket No.: LCH-03725

[1265] reaction mixture was washed with saturated aq. ammonium chloride solution (100 mL x 2) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 65 (78 g, 99%) as a white solid.

[1266] ’H-NMR (400 MHz, CDC13) 5 7.79-7.77 (m, 4H), 7.36-7.34 (m, 4H), 4.10-4.08 (m, 4H), 3.62-3.60 (m, 4H), 2.45 (s, 6H).

[1267] Preparation of Compound 66

[1268] To a solution of Compound 65 (78 g, 188.2 mmol) in A,A-dimethylformamide (470 mL) was added sodium azide (61 g, 940.9 mmol) under N2. After being stirred at 80 °C for 18 hours, the reaction mixture was diluted with ethyl acetate (I L) and washed with saturated aq. ammonium chloride solution (500 mL x 2) and brine (500 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 66 (24 g, 83%) as a pale yellow oil.

[1269] 1H-NMR (400 MHz, CDCh) 53.70-3.67 (m, 4H), 3.43-3.40 (m, 4H).

[1270] Preparation of Compound 67

[1271] To a solution of Compound 66 (24 g, 153.7 mmol) in ethyl acetate (500 mL) were added triphenylphosphine (40 g, 153.7 mmol) and aq. 1 M HC1 solution (284 mL) at 0 °C under N2. After being stirred at room temperature for 17 hours, the reaction mixture was concentrated under reduced pressure and then the residue was adjusted to pH 10 with 1 M sodium hydroxide solution. The residue was extracted with dichloromethane (100 mL x 2). The combined organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 67 (9.6 g, 48%) as a colorless oil.

[1272] ‘H-NMR (400 MHz, CDCh) 53.68 (t, J = 4.8 Hz, 2H), 3.54 (t, J = 4.8 Hz, 2H), 3.40 (t, J = 4.8 Hz, 2H), 3.90 (t, . / = 4.8 Hz, 2H), 1.37 (bs, 2H).

[1273] Preparation of Compound 68

[1274] To a solution of Compound 67 (8.9 g, 68.4 mmol) in dichloromethane (150 mL) were added Compound 45 (15 g, 68.4 mmol), A,A,A’,A’-tetramethyl-(9-(lH-benzotriazol-l-yljuronium hexafluorophosphate (HBTU, 33.8 g, 88.95 mmol), and A,A-diisopropylethylamine (23.8 mL, 137 mmol) under N2. After being stirred at room temperatureAttorney Docket No.: LCH-03725

[1275] for 14 hours, the reaction mixture was washed with saturated aq. ammonium chloride solution (20 mL), saturated aq. sodium bicarbonate solution (20 mL), and brine (40 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 68 (20 g, 88%) as a yellow oil.

[1276] 1H-NMR (400 MHz, CDCh) 57.04 (s, 1H), 3.66 (t, J = 5.0 Hz, 2H), 3.58 (s, 2H), 3.47 (t, J = 5.6 Hz, 2H), 3.41 (t, J = 5.0 Hz, 2H), 1.48 (s, 9H), 1.46 (s, 6H). EI-MS m / z : [M+H]+332.249.

[1277] Preparation of Compound 69

[1278] To a solution of Compound 68 (21 g, 63.3 mmol) in tetrahydrofuran (150 mL) were added triphenylphosphine (18 g, 69.7 mmol) and distilled water (22 mL) at 0 °C under N2. After being stirred at 80 °C for 20 hours, the reaction mixture was concentrated and the resulting residue was purified by column chromatography to afford Compound 69 (15 g, 77%) as a yellow oil.

[1279] ‘H-NMR (400 MHz, CDCh) 53.56 (t, J = 5.4 Hz, 2H), 3.53-3.48 (m, 4H), 3.46 (q, J = 5.5 Hz, 2H), 2.90 (t, J = 5.0 Hz, 2H), 1.48 (s, 9H), 1.46 (s, 6H). ELMS m / z : [M+H]+306.28.Attorney Docket No.: LCH-03725

[1280]

[1281] Preparation of Compound 70

[1282] To a solution of methyl 3,4,5-trihydroxybenzoate (methyl gallate, 70 g, 380.1 mmol) in .A i methyl formamide (600 mL) were added 4-methoxybenzyl chloride (51.8 mL, 380.1 mmol), potassium bicarbonate (114.17 g, 1140.4 mmol), and potassium iodide (0.38 g, 0.006 mmol). The reaction mixture was heated to 60 °C under N2. After 16 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was diluted with water (300 mL) and extracted with dichloromethane (300 mL x 2). The combined organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by column chromatography to afford Compound 70 (58.17 g, 50%) as a white solid.

[1283] 1H-NMR (400 MHz, CDCh) 57.30 (d, J = 8.0 Hz, 2H), 7.19 (s, 2H), 6.90 (d, J = 8.0 Hz, 2H), 5.40 (s, 2H), 5.05 (s, 2H), 3.88 (s, 3H), 3.82 (s, 3H). ELMS m / z : [M+H]+305.31.Attorney Docket No.: LCH-03725

[1284] Preparation of Compound 71

[1285] To a solution of Compound 70 (1.5 g, 4.93 mmol) in N,N-di methylformamide (10 mL) were added potassium bicarbonate (3.41 g, 24.6 mmol) and Compound 62 (6.46 g, 11 -34 mmol) under N2. After being stirred at 80 °C for 17 hours, the reaction mixture was concentrated under reduced pressure and the resulting residue was diluted with ethyl acetate (30 mL). The organic layer was washed with saturated aq. ammonium chloride solution (30 mL), saturated aq. sodium bicarbonate solution (30 mL), and brine (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by column chromatography to afford Compound 71 (5.6 g, 100%) as a yellow oil.

[1286] ELMS m / z : [M+H]+1100.46.

[1287] Preparation of Compound 72

[1288] To a solution of Compound 71 (3.6 g, 3.28 mmol) in dichloromethane (30 mL) were added anisole (5.3 mL, 49.1 mmol) and trifluoroacetic acid (1.3 mL) at 0 °C under N2. After being stirred at room temperature for 40 minutes, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 72 (3.6 g, 100%) as a pale yellow oil.

[1289] ELMS m / z : [M+H]+979.45.

[1290] Preparation of Compound 73

[1291] To a solution of Compound 72 (2 g, 2.04 mmol) in N, A-di methyl formamide (10 mL) were added potassium bicarbonate (1.02 g, 10.21 mmol) and Compound 64 (1.6 g, 2.86 mmol) under N2. The reaction mixture was stirred at 65 °C for 12 hours. The reaction mixture was concentrated under reduced pressure and the resulting residue was diluted with ethyl acetate (30 mL). The organic layer was washed with saturated aq. ammonium chloride solution (10 mL x 3), saturated aq. sodium bicarbonate solution (10 mL x 3), and brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to afford Compound 73 (1.9 g, 68%) as a pale yellow oil.

[1292] ELMS m / z : [M+H]+1371.69.

[1293] Preparation of Compound 74

[1294] To a solution of Compound 73 (1.8 g, 1.31 mmol) in methanol (12 mL) was added aq.

[1295] 1 N sodium hydroxide solution (6.56 mL, 6.56 mmol) at 0 °C under N2. After 7 hours, the reaction mixture was titrated with aq. 1 N HC1 solution to pH 3~4 at 0 °C, and the mixture wasAttorney Docket No.: LCH-03725

[1296] extracted with dichloromethane (30 mL x 3). The combined organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to afford Compound 74 (914 mg, 51%) as a yellow oil.

[1297] EI-MS m / z : [M+Na]+1379.60.

[1298] Preparation of Compound 75

[1299] To a solution of Compound 74 (190 mg, 0.14 mmol) in A,A-dimethylformamide (5 mL) were added Compound 69 (45 mg, 0.15 mmol), ACA / '-tetraniethyl-O-(I H-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU, 69 mg, 0.18 mmol), and N,N-diisopropylethylamine (0.06 mL, 0.42 mmol) under N2. After being stirred at room temperature for 16 hours, the reaction mixture was diluted with ethyl acetate (30 mL) and washed with saturated aq. ammonium chloride solution (30 mL), saturated aq. sodium bicarbonate solution (30 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 75 (140 mg, 61%).

[1300] EI-MS m / z [M+H]+1645.04, [M / 2+H]+823.39.

[1301] Preparation of Compound 76

[1302] To a solution of Compound 75 (140 mg, 0.09 mmol) in methanol (5 mL) was added palladium / charcoal (10 wt. %, 15 mg) under hydrogen atmosphere. After being stirred at room temperature for 2 hours, the reaction mixture was filtered through a celite pad and concentrated under reduced pressure to afford Compound 76 (90 mg, 77%) as a yellow oil.

[1303] EI-MS m / z : [M+H]+1376.80, [M / 2+H]+689.21.Attorney Docket No.: LCH-03725

[1304]

[1305] Preparation of Compound 77

[1306] To a solution of compound 76 (1.90 g, 1.38 mmol) in dichloromethane (30 mL) were added compound 15 (1.47 g, 4.62 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1, 609 mg, 6.30 mmol), 1 -hydroxybenzotriazole (HOBt, 448 mg, 3.31 mmol) and N-methylmorpholine (0.91 mL, 8.28 mmol) under N2. After being stirred at room temperature for 12 hours, the reaction mixture was washed with saturated aq. sodium bicarbonate solution (30 mL) and saturated aq. ammonium chloride solution (30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to afford Compound 77 (1.3 g, 41%) as a colorless oil.

[1307] ELMS m / z : [M+Na]+2330.22.

[1308] Preparation of Compound 78

[1309] To a solution of Compound 77 (670 mg, 0.29 mmol) in dichloromethane (10 mL) were added bis(pentafluorophenyl)carbonate (286 mg, 0.725 mmol) and pyridine (0.093 mL, 1.16 mmol) at 0 °C under N2. After being stirred at room temperature for 18 hours, the reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated aq. sodium bicarbonate solution (50 mL), aq. 0.5 N HC1 solution (50 mL), and aq. 2% NaOH solution (50Attorney Docket No.: LCH-03725

[1310] mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 78 (995 mg, crude), which was used without further purification.

[1311] EI-MS m / z : [M / 2+H]+1364.73.Attorney Docket No.: LCH-03725

[1312]

[1313] Attorney Docket No.: LCH-03725

[1314] Preparation of Compound 79

[1315] To a solution of Compound 78 (200 mg, 0.07 mmol) and MMAF-OMe (109 mg, 0.15 mmol) in A A I methyl formamide (3 mL) were added l-hydroxy-7-azabenzotriazole (HOAt, 3 mg, 0.02 mmol) and A A-di isopropylethylamine (0.1 mL, 0.59 mmol) at 0 °C under N2. The reaction mixture was warmed to room temperature and stirred for 12 hours. The reaction mixture was diluted with ethyl acetate (30 mL) and washed with saturated aq. ammonium chloride solution (20 mL), distilled water (20 mL) and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford Compound 79 (220 mg, 78%) as a white solid.

[1316] ELMS m / z : [M / 3+H]+1285.75.

[1317] Preparation of Compound 80

[1318] To a solution of Compound 79 (220 mg, 0.06 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (24 mg, 0.57 mmol) in water (1 mL) at -78 °C. After being stirred at -5 °C for 6 hours, the reaction mixture was adjusted to pH 4~5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 80 (140 mg, 69%) as a white solid.

[1319] ELMS m / z : [M / 3+H]+1183.02.

[1320] Preparation of Compound 81

[1321] To a solution of Compound 80 (140 mg, 0.04 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.3 mL) at 0 °C under N2. After being stirred at room temperature for 4 hours, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 81 (65 mg, 49%) as a white solid.

[1322] ELMS m / z : [M / 3+H]+1130.82.Attorney Docket No.: LCH-03725

[1323] 2-12 : Preparation of Compound 85

[1324]

[1325] Preparation of Compound 83

[1326] Compound 82 (Dxd2) was produced with the method disclosed in U.S. Patent No.

[1327] 10,195,288, the entirety of which is incorporated herein by reference. To a solution of Compound 78 (204 mg, 0.07 mmol) in A(A i methyl formamide (3 mL) were added Compound 82 (99 mg, 0.15 mmol) and A(A i isopropylethylamine (0.05 mL, 0.29 mmol) under N2. After being stirred at room temperature for 4 hours, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aq. ammonium chloride solution (10 mL), distilled water (10 mL), and brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford Compound 83 (254 mg, crude), which was used without further purification.

[1328] ELMS m / z : [M / 2+H]+1702.09. [M / 3+H]+1135.05.

[1329] Preparation of Compound 84

[1330] To a solution of Compound 83 (254 mg, 0.07 mmol) in tetrahydrofuran (1 mL) and methanol (1 mL) was added lithium hydroxide monohydrate (25 mg, 0.59 mmol) in water (1 mL) at -78 °C. The reaction mixture was stirred at 5 °C for 4 hours. The reaction mixture was adjusted to pH 4~5 with acetic acid and concentrated under reduced pressure. The resulting residue was purified by HPLC to afford Compound 84 (80 mg, 34% in 2 steps) as a yellow solid.

[1331] ELMS m / z : [M / 2+H]+1561.83. [M / 3+H]+1041.60.Attorney Docket No.: LCH-03725

[1332] Preparation of Compound 85

[1333] To a solution of Compound 84 (80 mg, 0.03 mmol) in acetonitrile (2 mL) was added phosphoric acid (85%, 0.4 mL) at 0 °C. After being stirred at room temperature for 5 hours, the reaction mixture was purified by HPLC to afford Compound 85 (53 mg, 70%) as a yellow solid.

[1334] EI-MS m / z : [M / 2+H]+1483.60, [M / 3+H]+989.53.

[1335] Preparation Example 3: Synthesis of Exemplified ADCs

[1336] Table 5. Exemplified ADCs

[1337]

[1338] *Blenrep indicated Blenrep biosimilar made via maleimide conjugation of belantamab and mcMMAF

[1339] 3-1 : Preparation of Exemplified ADCs by Site-Specific Conjugation Exemplified anti-BCMA antibody-drug conjugates (ADC1, ADC2, ADC3, ADC4, ADC5, ADC6, ADC10, ADC11, ADC12) were produced with the below two steps. LCB14-0606 was produced with the method disclosed in U.S. Patent No. 9,669,107, the entirety of which is incorporated herein by reference. The structural formula of LCB 14-0606 is as follows:

[1340]

[1341] Step 1: Production of Prenylated Antibody with LCB 14-0606Attorney Docket No.: LCH-03725

[1342] An exemplified anti-BCMA antibody Clone2 and Clone4 were prepared according to Preparation Example 1. A mixture comprising exemplified anti-BCMA antibody Clone2 or Clone4 for the prenylation reaction was prepared and reacted at 25 °C for 16 hours. The reaction mixture contained a total of 54.5 pM antibody clone, 0.6 pM FTase (Sigma-Aldrich #344145), and 436 pM LCB 14-0606 in a buffer solution (50 mM Tris-HCl, pH 7.4, 5 mM MgCh, 10 pM ZnCh, 0.25 mM DTT). In order to assembly of reduced antibody, the mixture was performed oxidation process by addition 10 molar of DHAA (Dehydroascorbic Acid) at 25 °C with stirring at 80 rpm for 4 hours. After the reaction, the prenylated antibody was desalted using Amicon® Ultra centrifugal filter (Merck Millipore, 10 MWKO) equilibrated with PBS buffer.

[1343] Step 2: Drug-Conjugation Method

[1344] Oxime Bond Reaction of ADCs (conjugation by oxime bond formation) The oxime bond formation reaction between the prenylated antibody and the linkerdrug compound was carried out by mixing 100 mM sodium acetate buffer, pH 4.5 or 5.2, 10% DMSO, 10 mg / ml of prenylated antibody, and 4 equivalents (40 mg / ml or 60 mg / ml) of the linker-payload (Compound 1, 2, 3, 4, 57, 81 or 85 of Preparation Example 2) at 25 °C with stirring at 80 rpm for 3 hours. After the reaction, excess low molecular weight compounds were removed by Amicon® Ultra centrifugal filter (Merck Millipore, 30MWKD), and the desired protein fraction was collected and concentrated.

[1345] The drug-antibody ratio (DAR) purity of ADCs was analyzed using an HIC-HPLC or LC-MS, according to standard protocols.

[1346] 3-2 : Preparation of Exemplified ADCs by Random Conjugation (Maleimide) Exemplified anti-BCMA antibody-drug conjugates (ADC8, ADC9) were produced by random conjugation. The exemplified anti-BCMA antibody Clonel or Clone4 of Preparation Example 1 was conjugated with Compound 24 of Preparation Example 2 or mcMMAF were purchased from MCE (#HY-15578). The structural formula of mcMMAF is as follows:

[1347] &

[1348]

[1349] Attorney Docket No.: LCH-03725

[1350] mcMMAF

[1351] The anti-BCMA antibody Clonel or Clone4 was incubated with tris(2-carboxyethyl)-phosphine (TCEP) at 22 °C for 18 hours. The amount of TCEP is 1-3 times the molar equivalent of the antibody. The linker-payload (Compound 24 or mcMMAF) for maleimide conjugation was added to the mixed solution of antibody clone and TCEP. The amount of linker-payload is 5~10 times the molar equivalent of the antibody and was incubated for 1 hour at 22 °C.

[1352] After conjugation, the mixture was desalted by Amicon® Ultra centrifugal filter (Merck Millipore, 30 MWCO) to remove excess small molecules. The drug-antibody ratio (DAR) was determined by hydrophobic interaction chromatography, according to standard protocols.

[1353] 3-3 : Preparation of Exemplified ADCs by Random Conjugation (Permalink) An exemplified anti-BCMA antibody-drug conjugate (ADC7) was produced by random conjugation. The exemplified anti-BCMA antibody Clone4 of Preparation Example 1 was conjugated with Compound 21 of Preparation Example 2.

[1354] The anti-BCMA antibody Clone4 was reduced with 1-2.5 equivalents of TCEP and conjugated to 4-8 equivalents of PermaLink-linker payload (Compound 21) using the conditions described in paper [Leukemia Research 108 (2021) 106626], The conjugate (>4.0 mg / ml, >27 pM) was isolated from the excess linker-payload and organic solvent into PBS using desalting columns following manufacturer’s instructions after the mixture was incubated at 22 °C for 22 hours. The ADCs were analyzed using reduced LC-MS to determine the DAR, according to standard protocols.

[1355] Experimental Example 1: In vitro Cell Toxicity Evaluation of Exemplified BCMA ADCs

[1356] The cytotoxic activity of exemplified BCMA ADCs was measured by cell proliferation assay using the Cell titer gio assay following the manufacturer's protocol (Promega, CellTiter-Glo® Luminescent Cell Viability Assay Protocol). Commercially available human multiple myeloma cell lines (H929, MM1.S, RPML8226) were used.

[1357] Briefly, 80 pl of RPMI-1640 cell culture medium containing the optimized cell number (1,000 cells / well) for each cell line was dispensed into each well of a 96-well plate. After overnight incubation, 20 pl aliquots of ADC, were added to the experimental wells andAttorney Docket No.: LCH-03725

[1358] incubated for 120 h. Then, 50 pl of CellTiter-Glo reagent was added to each well and the luminescence signal was read with Envision.

[1359] The efficacy of exemplified BCMA ADCs was tested in comparison with Blenrep™. Both ADC2 and ADC4 showed 2.7- to 11.7-fold higher cell killing activity than Blenrep in H929, MMES and RPMI-8226 cell lines. The IC50 values of anti-BCMA ADCs in each cell line are shown in Table 6.

[1360] Table 6. In vitro Cytotoxicity of Exemplified BCMA ADCs in Multiple Myeloma Cell Line

[1361]

[1362] Experimental Example 2: In vivo Efficacy Evaluation of Exemplified BCMA ADCs

[1363] General Methods

[1364] Commercially available immunodeficient mice and commercially available human multiple myeloma cell lines were used.

[1365] Tumor volumes were measured twice weekly using calipers and calculated according to the following formula:

[1366] Tumor Volume (mm3) = (length x width2) / 2.

[1367] Tumor growth inhibition (TGI) was calculated at each timepoint using the following formula:

[1368] TGI (%) = [1 - (Tt - To) / (Ct - Co)] x 100,

[1369] wherein Tt and Ct represent the mean tumor volumes of the treated group and the vehicle group, respectively, at a given timepoint, and To and Co represent the mean tumor volumes at the initiation of treatment.

[1370] Survival was assessed based on animal health status and tumor burden, and animals reaching predefined ethical endpoint criteria were humanely euthanized in accordance with institutional animal care and use guidelines.Attorney Docket No.: LCH-03725

[1371] OPM2-Luc Xenograft Model_l

[1372] Female CB17 / SCID mice were subcutaneously inoculated with 0PM2-Luc cells, a human multiple myeloma cell line. When the mean tumor volume reached approximately 150-200 mm3, the mice were randomized into treatment groups (n = 6 per group) and administered a single intravenous dose of vehicle, ADC4 (0.3 mg / kg), ADC4 (0.6 mg / kg) or ADC9 (0.75 mg / kg). Tumor volumes were measured twice weekly, and tumor growth inhibition (TGI) was calculated relative to the vehicle-treated control group at each timepoint, as described in the General Methods.

[1373] The results are summarized in Table 7 and FIG. 1A. In control group or treatment groups exhibiting weak or no antitumor efficacy, excessive tumor burden led to the animals reaching predefined ethical endpoint criteria, and the affected animals were humanely euthanized. As a result, the number of evaluable animals was reduced, and TGI values beyond Day 18 could not be determined.

[1374] Table 7. Tumor Growth Inhibition (%) in OPM2-Luc Xenograft Model

[1375]

[1376] As shown in FIG. 1A, ADC4 demonstrated superior antitumor efficacy relative to ADC9, despite being administered at both a lower drug-to-antibody ratio (DAR 2) and a lower dose than ADC9 (DAR 4). ADC4 further exhibited a clear dose-dependent inhibition of tumor growth, with the 0.6 mg / kg dose showing sustained tumor growth suppression.

[1377] Survival was monitored up to Day 32 post-treatment. The results are represented in Table 9 and FIG. IB. ADC4 administered at 0.6 mg / kg resulted in a survival rate of 100% at Day 32, whereas ADC9-treated animals exhibited a survival rate of 17%.

[1378] Table 9. Survival Rate (%) in OPM2-Luc Xenograft Model

[1379]

[1380] Attorney Docket No.: LCH-03725

[1381]

[1382] These results demonstrate that ADC4 provides enhanced antitumor efficacy and survival benefit compared to ADC9 in a multiple myeloma xenograft model.

[1383] NCI-H929 Xenograft Model

[1384] Female NOD / SCID mice were subcutaneously inoculated with NCI-H929 cells, a human multiple myeloma cell line. When the mean tumor volume reached approximately 80- 110 mm3, the mice were randomized into treatment groups (n = 8 per group) and administered a single intravenous dose of vehicle, ADC1 (3.0 mg / kg), ADC3 (3.0 mg / kg), ADC7 (3.0 mg / kg) or ADC9 (3.0 mg / kg). Tumor volumes were measured twice weekly, and tumor growth inhibition (TGI) was calculated relative to the vehicle-treated control group at each timepoint, as described in the General Methods.

[1385] The results are summarized in Table 10 and FIG. 2A. In control group or treatment groups exhibiting weak or no antitumor efficacy, excessive tumor burden led to the animals reaching predefined ethical endpoint criteria, and the affected animals were humanely euthanized. As a result, the number of evaluable animals was reduced, and TGI values beyond Day 24 could not be determined.

[1386] Table 10. Tumor Growth Inhibition (%) in NCI-H929 Xenograft Model

[1387]

[1388] As shown in FIG.2A, ADC1, ADC3, and ADC7 resulted in pronounced inhibition of tumor growth and demonstrated antitumor activity that was comparable to or greater than that observed with ADC9 across multiple timepoints. Notably, ADC3 and ADC7 exhibitedAttorney Docket No.: LCH-03725

[1389] consistently high levels of tumor growth inhibition, while ADC1 showed increasing tumor growth inhibition at later timepoints.

[1390] Survival was monitored up to Day 42 post-treatment. The results are represented in Table 11 and FIG.2B. As shown in Table 10, animals treated with ADC1, ADC3, or ADC7 demonstrated improved survival outcomes relative to those treated with ADC9.

[1391] Table 11. Survival Rate (%) in NCI-H929 Xenograft Model

[1392]

[1393] All ADCs evaluated comprised the same cytotoxic payload (MMAF). ADC9 utilized a maleimidocaproyl (MC)-based linker, whereas ADC1, ADC3, and ADC7 each employed a P-glucuronide based linker.

[1394] Taken together, these results demonstrate that BCMA antibody-drug conjugates comprising P-glucuronide based linker exhibit robust and sustained antitumor efficacy in a multiple myeloma xenograft model, and that such efficacy is maintained irrespective of the specific conjugation method employed.

[1395] MM1S Xenograft Model

[1396] Female NOD / SCID mice were subcutaneously inoculated with MM1S cells, a human multiple myeloma cell line. When the mean tumor volume reached approximately 80-100 mm3, the mice were randomized into treatment groups (n = 8 per group) and administered a single intravenous dose of vehicle, ADC3 (3.0 mg / kg), ADC7 (3.0 mg / kg) or ADC9 (3.0 mg / kg). Tumor volumes were measured twice weekly, and tumor growth inhibition (TGI) was calculated relative to the vehicle-treated control group at each timepoint, as described in the General Methods.

[1397] The results are summarized in Table 11 and FIG. 3A. In control group or treatment groups exhibiting weak or no antitumor efficacy, excessive tumor burden led to the animals reaching predefined ethical endpoint criteria, and the affected animals were humanelyAttorney Docket No.: LCH-03725

[1398] euthanized. As a result, the number of evaluable animals was reduced, and TGI values beyond Day 30 could not be determined.

[1399] Table 12. Tumor Growth Inhibition (%) in MM1S Xenograft Model

[1400]

[1401] As shown in Table 12, ADC3 and ADC7 rapidly induced tumor growth inhibition, achieving TGI values exceeding 110% as early as Day 9 post-administration, which were maintained through Day 30. In contrast, ADC9 exhibited only transient tumor growth inhibition, with TGI values decreasing after Day 20, indicating a loss of sustained antitumor activity. Consistent with the TGI results, treatment with ADC3 or ADC7 resulted in sustained tumor regression throughout the observation period, whereas ADC9-treated animals showed evidence of tumor regrowth at later stages, as shown in FIG.3A.

[1402] Survival was monitored up to Day 34 post-treatment. The results are represented in Table 13 and FIG.3B. As shown in Table 12, animals treated with ADC3 or ADC7 exhibited a survival rate of 100%, whereas treatment with ADC9 resulted in a reduced survival rate of 87.5%.

[1403] Table 13. Survival Rate (%) in MM1S Xenograft Model

[1404]

[1405] All ADCs evaluated comprised the same cytotoxic payload (MMAF). ADC9 utilized a maleimidocaproyl (MC)-based linker, whereas ADC3, and ADC7 each employed a 0- glucuronide based linker, with distinct conjugation methods used for ADC3, and ADC7, respectively.Attorney Docket No.: LCH-03725

[1406] Taken together, these results demonstrate that BCMA antibody-drug conjugates comprising a P-glucuronide-based linker provide earlier onset, more durable antitumor activity, and improved survival outcomes compared to an otherwise comparable ADC comprising a maleimidocaproyl-based linker in a multiple myeloma model.

[1407] OPM2-Luc Xenograft Model_2

[1408] Female CB17 / SCID mice were subcutaneously inoculated with 0PM2-Luc cells, a human multiple myeloma cell line. When the mean tumor volume reached approximately 150-200 mm3, the mice were randomized into treatment groups (n = 6 per group) and administered a single intravenous dose of vehicle, ADC10 (0.6 mg / kg), ADC11 (3.0 mg / kg), ADC5 (3.0 mg / kg) or ADC12 (3.0 mg / kg). Tumor volumes were measured at Day 18, and tumor growth inhibition (TGI) was calculated relative to the vehicle-treated control group at each timepoint, as described in the General Methods.

[1409] The results are summarized in Table 14 and FIGs. 4-6. In control group, excessive tumor burden caused multiple animals to reach predefined ethical endpoint criteria, and the affected animals were humanely euthanized. As a result, the number of evaluable animals was reduced, and TGI values beyond Day 18 could not be determined.

[1410] Table 14. Tumor Growth Inhibition (%) in OPM2-Luc Xenograft Model

[1411]

[1412] Survival was monitored up to Day 21 post-treatment. The results are represented in Table 15 and FIG.7.

[1413] Table 15. Survival Rate (%) in OPM2-Luc Xenograft Model

[1414]

[1415] Attorney Docket No.: LCH-03725

[1416]

[1417] As shown in Tables 14 and 15 and FIGs. 4-7, BCMA-targeting ADCs comprising a P-glucuronide-based linker exhibited robust antitumor activity in the multiple myeloma model. Notably, ADCs comprising different cytotoxic payloads, including PBD, MMAF, MMAE, and Dxd2, demonstrated high tumor growth inhibition while maintaining 100% survival.

[1418] These results suggest that incorporation of the P-glucuronide-based linker into a BCMA-targeting ADC contributes to enhanced in vivo efficacy and favorable tolerability.

[1419] Incorporation by Reference

[1420] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[1421] Equivalents

[1422] While specific embodiments of the disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Attorney Docket No.: LCH-03725We claim:

1. A conjugate having a structure represented by Formula A or a pharmaceutically acceptable salt thereof:Formula AwhereinAb is an anti-B Cell Maturation Antigen (BCMA) antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; andthe light chain variable region comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13;each B is independently an active agent;each L is independently a linker comprising an alkylene or a heteroalkylene,wherein L comprises one or more of:(i) one or more unsaturated bonds;(ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain);(iii) at least one C 1-20 alkyl substituent;(iv) at least one isoprenyl group having a structure represented by General Formula II:[General Formula II]Attorney Docket No.: LCH-03725(v) a structure represented by Formula B:Formula Bwherein:X1and X2are each independently N(R3), O, or S;R1is H, alkyl, or cycloalkyl;R2is alkyl or cycloalkyl; or R1and R2combine to form a cycloalkyl; R3is H, alkyl, aryl, or aralkyl;Ya1is a connection to Ta;Ya2is a connection to Ab;each Tais independently a cleavage group, preferably a self-immolative group, more preferably a structure represented by Formula C:Formula Cwherein:G is independently a glucuronic acid moiety oRAand RBare each independently hydrogen, Ci-s alkyl, or C3-8 cycloalkyl;Rcis hydrogen or a carboxyl -protecting group;each RDis independently hydrogen or a hydroxyl-protecting group; W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR', -SONR'-, orAttorney Docket No.: LCH-03725-PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of Formula C and the NR' is bonded to Ya3;R' and R" are each independently hydrogen, Ci-s alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, C3-20 heteroaryl, or Ce-2oaryl;each Z is independently C1-8 alkyl, halogen, cyano, or nitro;n is 0, 1, 2, or 3;Ya3is a connection to L;Ya4is a connection to B; andnA and nB are each independently an integer from 1 to 20.

2. The conjugate of claim 1, wherein Ab comprises a heavy chain variable region comprising:the amino acid sequence of SEQ ID NO: 15;a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:15 while maintaining the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; ora sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:15 while maintaining the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

3. The conjugate of claim 1 or 2, wherein Ab comprises a light chain variable region comprising:the amino acid sequence of SEQ ID NO: 16;a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:16 while maintaining the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; orAttorney Docket No.: LCH-03725a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:16 while maintaining the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

4. The conjugate of claim 1, wherein Ab comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

5. The conjugate of any one of claims 1-4, wherein Ab is a humanized antibody or a human antibody.

6. The conjugate of any one of claims 1-5, wherein Ab is selected from a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

7. The conjugate of any one of claims 1-6, wherein Ab comprises an IgA, IgG, IgM, IgE, or IgD constant domain or is an IgA, IgG, IgM, IgE, or IgD antibody.

8. The conjugate of claim 7, wherein Ab comprises an IgG constant domain or is an IgG antibody.

9. The conjugate of claim 8, wherein Ab comprises an IgGl, IgG2, IgG3, or IgG4 constant domain or is an IgGl, IgG2, IgG3, or IgG4 antibody.

10. The conjugate of any one of claims 7-9, wherein Ab comprises LALA mutations in a heavy chain constant region.

11. The conjugate of claim 10, wherein the LALA mutations comprise L234A and L235A according to EU numbering convention.Attorney Docket No.: ECH-0372512. The conjugate of claim 10, wherein the LALA mutations are in place of amino acids corresponding to amino acids 238-239 in SEQ ID NO: 17.

13. The conjugate of claim 1, wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 or 20 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

14. The conjugate of any one of claims 1-13, wherein E comprises a structure represented by Formula B:Formula BwhereinX1and X2are each independently N(R3), O, or S;R1is H, alkyl, or cycloalkyl;R2is alkyl or cycloalkyl; or R1and R2combine to form a cycloalkyl;R3is H, alkyl, aryl, or aralkyl;Ya1is a connection to Ta; andYa2is a connection to Ab.

15. The conjugate of any one of claims 1-14, wherein Tarepresents a structure represented by FormulaC:Formula CwhereinAttorney Docket No.: LCH-03725G is independently a glucuronic acid moietyRAand RBare each independently hydrogen, Ci-s alkyl, or C3-8 cycloalkyl;Rcis hydrogen or a carboxyl -protecting group;each RDis independently hydrogen or a hydroxyl -protecting group;W is -C(0)-, -C(0)NR’-, -C(0)0-, -SO2NR'-, -P(0)R"NR', -SONR'-, or -PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of Formula C and the NR' is bonded to Ya3;R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, C3-20 heteroaryl, or Ce-2oaryl;each Z is independently C1-8 alkyl, halogen, cyano, or nitro;n is 0, 1, 2, or 3;Ya3is a connection to L; andYa4is a connection to B.

16. The conjugate of any one of claims 1-13, having a structure represented by General Formula I or a pharmaceutically acceptable salt thereof:[General Formula I]whereineach B’ is an active agent;Attorney Docket No.: LCH-03725each G is independently a glucuronic acid moiety oRcis hydrogen or a carboxyl -protecting group;each RDis independently hydrogen or a hydroxyl -protecting group;RAand RBare each independently hydrogen, Ci-s alkyl, or C3-8 cycloalkyl;W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR', -SONR'-, or -PO2NR'-, wherein the C, S, or P is directly bonded to the phenyl ring of General Formula I and the NR' is bonded to L;R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono- or di-Ci-8 alkylamino, C3-20 heteroaryl, or Ce-2oaryl;each Z is independently C1-8 alkyl, halogen, cyano, or nitro;n is 0, 1, 2, or 3;each L is independently a linker comprising a C1-50 alkylene or C 1-50 heteroalkylene wherein the Ci-50 alkylene or C1-50 heteroalkylene, and L comprises one or more of:(i) one or more unsaturated bonds;(ii) a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain);(iii) at least one C 1-20 alkyl substituent; and(iv) at least one isoprenyl group having a structure represented by General Formula II:[General Formula II]1 is nA and m is nB.Attorney Docket No.: LCH-0372517. The conjugate of claim 16, wherein each18. The conjugate of claim 16 or 17, wherein RAand RBare each hydrogen.

19. The conjugate of any one of claims 16-18, wherein Rcis hydrogen.

20. The conjugate of any one of claims 16-19, wherein n is 0.

21. The conjugate of any one of claims 16-20, wherein each W is -C(O)NR'-.

22. The conjugate of any one of claims 16-21, wherein each RDis independently hydrogen.

23. The conjugate of any one of claims 16-17, wherein:RAand RBare each hydrogen;n is 0; andeach W is -C(O)NR’-.

24. The conjugate of any one of claims 16-23, wherein L comprises a C1-50 heteroalkylene, wherein the C 1-50 heteroalkylene comprises an isoprenyl group having a structure represented by General Formula Ila:[General Formula Ila]wherein n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

25. The conjugate of any one of claims 16-24, wherein L comprises a peptide comprising at least one hydrophilic amino acid.Attorney Docket No.: LCH-0372526. The conjugate of claim 25, wherein the peptide comprises an amino acid having a side chain having a moiety that bears a charge at neutral pH in aqueous solution (e.g., an amine, guanidine, or carboxyl moiety).

27. The conjugate of claim 25 or 26, wherein the peptide comprises an amino acid selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine, histidine, arginine and threonine.

28. The conjugate of any one of claims 16-27, wherein L comprises an oxime.

29. The conjugate of claim 28, wherein the oxygen atom of the oxime is on the side of L linked to W and the carbon atom of the oxime is on the side of L linked to Ab.

30. The conjugate of claim 28, wherein the carbon atom of the oxime is on the side of L linked to W and the oxygen atom of the oxime is on the side of L linked to Ab.

31. The conjugate of any one of claims 16-30, wherein L is a C 1-50 heteroalkylene comprising an oxime.

32. The conjugate of claim 31 , wherein the oxygen atom of the oxime is on the side of L linked to W.

33. The conjugate of claim 31 , wherein the oxygen atom of the oxime is on the side of L linked to Ab.

34. The conjugate of any one of claims 16-33, wherein L comprises at least one isoprenyl group.

35. The conjugate of any one of claims 31-34, wherein L comprises at least one isoprenyl group and the at least one isoprenyl group covalently bonds the oxime to Ab (e.g., the at least one isoprenyl group directly or indirectly bonds the oxime to Ab).

36. The conjugate of any one of claims 16-35, wherein L comprises a connecting unit represented by General Formula Illa or General Formula Illb:[General Formula Illa]Attorney Docket No.: LCH-03725-(CH2)r(V(CH2)P)q- [General Formula Illb]-(CH2CH2X)W- V is a single bond, -O-, -S-, - NR21-, -C(O)NR22-, -NR23C(O)-, -NR24SO2-, or -SO2NR25-; X is -O-, Ci-8 alkylene, or -NR21-;R21to R25are each independently hydrogen, Ci-6 alkyl, Ci-6 alkyl Ce-2o aryl, or Ci-6 alkyl-C3-2o heteroaryl;r is 0 to 10;p is 0 to 10;q is 1 to 20; andw is 1 to 20.

37. The conjugate of claim 36, wherein q is 1 to 10.

38. The conjugate of claim 36 or 37, wherein r is 1 or 2.

39. The conjugate of any one of claims 36-38, wherein p is 1 or 2.

40. The conjugate of any one of claims 36-39, wherein V is -O-.

41. The conjugate of claim 40, wherein:q is 1 to 10;r and p are each 1 or 2; andV is -O-.

42. The conjugate of any one of claims 36-41, wherein X is -O-.

43. The conjugate of any one of claims 16-42, wherein L comprises at least onepolyethylene glycol unit represented byorAttorney Docket No.: LCH-0372544. The conjugate of claim 43, wherein L comprises an oxime and the at least one polyethylene glycol unit covalently bonds the oxime to W.

45. The conjugate of any one of claims 16-44, wherein L further comprises a binding unit formed by a reaction between an alkyne and an azide or between an aldehyde or ketone group and hydrazine or hydroxylamine.

46. The conjugate of any one of claims 16-44, wherein L further comprises a binding unit represented by General Formula IVa, IVb, IVc, IVd, or IVe:[General Formula IVa][General Formula IVb][General Formula IVc][General Formula IVd]Attorney Docket No.: LCH-03725whereinL1is each independently a single bond or C 1-30 alkylene; andR11 is hydrogen or C1-10 alkyl.

47. The conjugate of any one of claims 16-46, wherein L is covalently bonded to Ab by a thioether bond and the thioether bond comprises a sulfur atom of a cysteine of Ab.

48. The conjugate of any one of claims 16-47, wherein Ab at the C-terminus comprises an amino acid motif that is recognized by an isoprenoid transferase.

49. The conjugate of claim 48, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

50. The conjugate of claim 48 or 49, wherein L is covalently bonded to Ab by a thioether bond and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

51. The conjugate of any one of claims 48-50, wherein the amino acid motif comprises a CYYX sequence, wherein:C is cysteine;each Y is an aliphatic amino acid; andX is selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine.

52. The conjugate of claim 51 , wherein each Y is independently selected from alanine, isoleucine, leucine, methionine, and valine.

53. The conjugate of any one of claims 48-52, wherein the amino acid motif comprises a CVIM (SEQ ID NO: 22) or CVLL (SEQ ID NO: 23).

54. The conjugate of any one of claims 48-53, wherein at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine.

55. The conjugate of any one of claims 48-54, wherein the amino acid motif has the sequence GGGGGGGCVIM (SEQ ID NO: 21).Attorney Docket No.: LCH-0372556. The conjugate of any one of claims 16-55, wherein the conjugate comprises57. The conjugate of any one of claims 16-55, wherein the conjugate comprises58. The conjugate of any one of claims 16-57, wherein L is branched and comprises: i) a branching unit covalently coupled to Ab by a primary linker;ii) a first branch which couples a first B’ to the branching unit; andiiia) a second branch which couples a second B’ to the branching unit; oriiib) a second branch, in which an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) is covalently coupled to the branching unit.

59. The conjugate of claim 58, wherein L comprises a second branch which couples a second B’, via a cleavage group, to the branching unit.

60. The conjugate of claim 58 or 59, wherein L comprises a second branch, in which an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) is covalently coupled to the branching unit.

61. The conjugate of any one of claims 58-60, wherein the branching unit has a structure represented byAttorney Docket No.: LCH-03725[General Formula Va][General Formula Vb][General Formula Vc][General Formula Ve]whereinAttorney Docket No.: LCH-03725G1, G2, and G3each independently represents a bondR60is hydrogen or C1-30 alkyl;R70is hydrogen or L5-COOR80;R80is hydrogen or C1-30 alkylL2, L3, L4’, and L5are each independently a bond or -CnThi-; andn' is 1 to 10.

62. The conjugate of any one of claims 58-61, wherein:at least one branched linker is covalently coupled to Ab; andat least two B’ are covalently coupled to the branched linker.

63. The conjugate of claim 62, wherein the conjugate comprises 1, 2, 3, or 4 branched linkers and each branched linker comprises two B’.

64. The conjugate of any one of claims 58-63, wherein the branching unit comprises a lysine residue.

65. The conjugate of any one of claims 16-64, wherein the conjugate comprises astructure represented by:Attorney Docket No.: LCH-03725or a pharmaceutically acceptable salt thereof; whereinB' and B" are each an active agent;nl’ to n3’ are each independently 0 to 30;AA is an amino acid group; andthe wavy bond represents a connection to Ab.

66. The conjugate of any one of claims 1-15, wherein L and (Ta-B)nA together have a structure represented by Formula D or a pharmaceutically acceptable salt thereof:Formula DwhereinL1Acomprises a Ci-Cioo alkylene;X3is -N(R4)C(O)-, -C(O)N(R4)-, -C(O)N(R4)(CH2)Z-, -(CH2)ZC(O)N(R4)-, -C(O)-, -C(O)O-, -OC(O)-, -S(O2)N(R4)-, -N(R4)S(O2)-, -P(O)(R4)N(R5)-, -N(R5)P(O)(R4)-, -Attorney Docket No.: LCH-03725P(O)2N(R5)-,or -N(R5)P(O)2-;z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;Y1, Y2, and Y3are each independently O, N(R6), or S;V1, V2, and V3are each independently a bond, N, NH, C, CH, or CH2;R4, R5, and R6are each independently H, alkyl, aryl, or aralkyl;L3, L4, and L5each independently comprise an alkylene;T1, T2, and T3are each independently a bond, -O-, or a cleavage group;B1, B2, and B3are each independently an active agent (e.g., a drug or a toxin), H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, alkylamino, guanidinyl, amido, cyano, azido, alkylthio, cycloalkyl, alkylsulfonyl, sulfonamide, aryl, heteroaryl, or heterocyclyl; orT1and B1, T2and B2, or T3and B3together represent a branched moiety (e.g., a branched moiety substituted with one or more active agents); andml, m2, and m3 are each independently 1, 2, 3, or 4.

67. The conjugate of any one of claims 1-13, wherein the conjugate comprises a structure represented by Formula V, or a pharmaceutically acceptable salt thereof:Formula VwhereinR1is H, alkyl or cycloalkyl;R2is alkyl or cycloalkyl; or R1and R2combine to form a cycloalkyl;X1and X2are each independently N(R3), O, or S;R3is H, alkyl, aryl, or aralkyl;each B is an active agent (e.g., a drug or a toxin);T is a cleavage group;L1is a covalent linker;L2is a covalent linker;Lg is Ab; andAttorney Docket No.: LCH-03725nl is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

68. The conjugate of claim 67, wherein each T has a structure represented by Formula VI:Formula VIwhereineach Rb1, Rb2, Rb3, and Rb4is independently H, alkyl, aralkyl, or an oxygen protecting group;each Rb5is alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, cycloalkyl, alkylsulfonyl, or sulfonamide;Wb1is independently - -*C(O)O-, -*S(wherein the C(O), N, CH2, S, or P marked with an * is bonded to the phenyl ring of Formula VI,Rb7and Rb8are each independently hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, alkylamino, heteroaryl, or aryl;each Jb1and Jb2are independently H or alkyl; orJb1and Jb2combine to form an oxo;tai is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;nal is 0, 1, 2, or 3;Ab1represents a linkage to B; andAb2represents a linkage to L1.

69. The conjugate of claim 67 or 68, wherein each of Rb1, Rb2, Rb3, and Rb4is H.

70. The conjugate of any one of claims 67-69, wherein each nal is 0.Attorney Docket No.: LCH-0372571. The conjugate of any one of claims 67-70, wherein each Wb1is -*C(O)N(Rb7)- and wherein each Rb7is H.

72. The conjugate of claim 67, wherein nl is 1.

73. The conjugate of claim 67, wherein L1is linear.

74. The conjugate of claim 67, wherein L1is branched.

75. The conjugate of claim 73 or 74, wherein L1comprises a Ci-Cioo alkylene, preferably a C1-C50 alkylene.

76. The conjugate of claim 75, wherein L1further comprises an oligoethylene glycol.

77. The conjugate of claim 76, wherein the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties, preferably comprising 1, 2, 3, 4, or 5 ethylene glycol moieties.

78. The conjugate of claim 77, wherein the oligoethylene glycol comprises 5 ethylene glycol moieties or 6 ethylene glycol moieties.

79. The conjugate of any one of claims 67-78, wherein L1further comprises an amido moiety.

80. The conjugate of claim 79, wherein L1comprises 1, 2, 3, 4, 5, or 6 amido moieties, preferably 1 or 2 amido moieties.

81. The conjugate of any one of claims 67-80, wherein L1further comprises a heteroaryl (e.g., pyrazolyl, triazolyl).

82. The conjugate of claim 81, wherein L1comprises a structure represented by Formula Vila or Vllb:Attorney Docket No.: LCH-03725Vila Vllbwhereineach Ad1represents a linkage to T or B; andeach A2represents a linkage to X1.

83. The conjugate of claim 81, wherein L1comprises a structure represented by Formula Villa, Vlllb, or VIIIc:Villa Vlllb VIIIc whereineach A1represents a linkage to B; andeach A2represents a linkage to X1.

84. The conjugate of any one of claims 67-83, wherein L1is substituted with an oligoethylene glycol.

85. The conjugate of claim 84, wherein the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties, preferably 1, 2, 3, or 4 ethylene glycol moieties.

86. The conjugate of claim 84 or 85, wherein the oligoethylene glycol is substituted with carboxyalkyl (e.g., carboxyethyl) or amino (e.g., dimethylamino).

87. The conjugate of any one of claims 67-86, wherein L1further comprises an amino acid (e.g., a hydrophilic amino acid), preferably L1further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., hydrophilic amino acids).

88. The conjugate of claim 87, wherein the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine,Attorney Docket No.: LCH-03725serine, and threonine, preferably from arginine, aspartate, glutamate, histidine, lysine, and ornithine, more preferably the amino acid(s) are selected from aspartate, glutamate, and sarcosine.

89. The conjugate of any one of claims 67-88, wherein L1and (B-T)ni comprise:i) a branching unit covalently coupled to X1by a primary linker;ii) a first branch, which couples a first active agent, via a first cleavage group, to the branching unit; andiiia) a second branch, which couples a second active agent, via a second cleavage group, to the branching unit; oriiib) a second branch which couples an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) to the branching unit.

90. The conjugate of claim 89, wherein L1and (B-T)nicomprise a second branch which couples a second active agent, via a second cleavage group, to the branching unit.

91. The conjugate of claim 89, wherein L1and (B-T)nicomprise a second branch which couples an alkyl or heteroalkyl (e.g., a polyethylene glycol monomer or a polyethylene glycol oligomer) to the branching unit.

92. The conjugate of any one of claims 89-91, wherein the branching unit is an amino acid.

93. The conjugate of any one of claims 89-92, wherein the branching unit has a structure represented by Formula Xia, Xlb, XIc, Xld or Xie:Xia Xlb XIc Xld Xie whereinAttorney Docket No.: LCH-03725R30is H or alkyl;R40is H, alkyl or LD-CO2R50;R50is H or alkyl; andLA, LB, LC, and LDare each independently a bond or alkylene.

94. The conjugate of any one of claims 89-93, wherein each cleavage group has a structure represented by Formula XIII:Formula XIIIwhereinBw is an active agent;each Rw2, Rw3, Rw4, and Rw5is independently H, alkyl, aralkyl, or an oxygen protecting group;each Rw6is alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, cycloalkyl, alkylsulfonyl, or sulfonamide;Attorney Docket No.: LCH-03725-*P(O2)N(RW7)-, wherein the C(0), N, CH2, S, or P marked with an * is bonded to the phenyl ring of Formula XIII;Rw7and Rw8are each independently hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, alkylamino, heteroaryl, or aryl;twl is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;nwl is 0, 1, 2, or 3;Jw1is H or alkyl;Jw2is H or alkyl; orJw1and Jw2combine to form an oxo; andAw2represents a linkage to the branching unit.

95. The conjugate any one of claims 67-88, wherein L1and (B-T)ni together have a structure represented by Formula XIV or a pharmaceutically acceptable salt thereof:Formula XIVwhereinL1Acomprises a C1-C100 alkylene;X3is -N(R4)C(O)-, -C(O)N(R4)-, -C(O)N(R4)(CH2)Z-, -(CH2)ZC(O)N(R4)-, -C(O)-, -C(O)O-, -OC(O)-, -S(O2)N(R4)-, -N(R4)S(O2)-, -P(O)(R4)N(R5)-, -N(R5)P(O)(R4)-, - P(O)2N(R5)-,or -N(R5)P(O)2-;z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;Y1, Y2, and Y3are each independently O, N(R6), or S;V1, V2, and V3are each independently a bond, N, NH, C, CH, or CH2;R4, R5, and R6are each independently H, alkyl, aryl, or aralkyl;L3, L4, and L5each independently comprise an alkylene;T1, T2, and T3are each independently a bond, -O-, or a cleavage group;Attorney Docket No.: LCH-03725B1, B2, and B3are each independently an active agent (e.g., a drug or a toxin), H, alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, alkylamino, guanidinyl, amido, cyano, azido, alkylthio, cycloalkyl, alkylsulfonyl, sulfonamide, aryl, heteroaryl, or heterocyclyl; orT1and B1, T2and B2, or T3and B3together represent a branched moiety (e.g., a branched moiety substituted with one or more active agents); andml, m2, and m3 are each independently 1, 2, 3, or 4.

96. The conjugate of claim 95, wherein L1Acomprises a C1-C50 alkylene.

97. The conjugate of claim 95 or 96, wherein each of Y1, Y2, and Y3is O.

98. The conjugate of any one of claims 95-97, wherein X3is -C(O)N(R4)-, further wherein R4is H.

99. The conjugate of any one of claims 95-98, wherein each of L3, L4, and L5comprises a C1-C100 alkylene, preferably a C1-C50 alkylene.

100. The conjugate of claim 99, wherein each of L3, L4, and L5further comprises an oligoethylene glycol.

101. The conjugate of claim 100, wherein the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties, preferably 1, 2, 3, 4, 5, or 6 ethylene glycol moieties, more preferably the oligoethylene glycol comprises 5 ethylene glycol moieties.

102. The conjugate of any one of claims 95-101, wherein each of L3, L4, and L5further comprises an amido moiety.

103. The conjugate of claim 102, wherein each of L3, L4, and L5comprises 1, 2, 3, 4, 5, or 6 amido moieties, preferably 1 or 2 amido moieties.

104. The conjugate of any one of claims 95-103, wherein each of L3, L4, and L5further comprises a heteroaryl (e.g., pyrazolyl, triazolyl).Attorney Docket No.: LCH-03725105. The conjugate of claim 104, wherein each of L3, L4, and L5comprises a structure represented by Formula XVa or XVb:XVa XVbwhereineach Ax1represents a linkage to V1; andeach Ax2represents a linkage to Y1.

106. The conjugate of any one of claims 95-105, wherein each of L3, L4, and L5is substituted with an oligoethylene glycol.

107. The conjugate of claim 106, wherein the oligoethylene glycol comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ethylene glycol moieties, preferably 1, 2, 3, or 4 ethylene glycol moieties.

108. The conjugate of claim 106 or 107, wherein the oligoethylene glycol is substituted with carboxyalkyl (e.g., carboxy ethyl) or amino (e.g., dimethylamino).

109. The conjugate of any one of claims 95-108, wherein each of L3, L4, and L5further comprises an amino acid (e.g., a hydrophilic amino acid), preferably L3, L4, or L5further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., hydrophilic amino acids).

110. The conjugate of claim 109, wherein the amino acid(s) are selected from arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, sarcosine, serine, and threonine, preferably arginine, aspartate, glutamate, histidine, lysine, and ornithine, more preferably, the amino acid(s) are selected from aspartate, glutamate, and sarcosine.

111. The conjugate of any one of claims 95-110, wherein each of V1, V2, and V3is a bond.

112. The conjugate of any one of claims 95-111, wherein each of ml, m2, and m3 is 1.Attorney Docket No.: LCH-03725113. The conjugate of any one of claims 95-112, wherein T1, T2, and T3each independently has a structure represented by Formula XVII or a pharmaceutically acceptable salt thereof:whereineach Rx2, Rx3, Rx4, and Rx5is independently H, alkyl, aralkyl, or an oxygen protecting group;each Rx6is alkyl, alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino, amido, cyano, nitro, azido, alkylthio, cycloalkyl, alkylsulfonyl, or sulfonamide;O-,wherein the C(O), N, CH2, S, or P marked with an * is bonded to the phenyl ring of Formula XVII, Rx7and Rx8are each independently hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, alkylamino, heteroaryl, or aryl;Ja1is H or alkyl;Ja2is H or alkyl; orJa1and Ja2combine to form an oxo;tx1is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;nx1is 0, 1, 2, or 3;Ax4represents a linkage to B1, B2, or B3; andAx5represents a linkage to V1, V2, or V3.Attorney Docket No.: LCH-03725114. The conjugate of claim 113, wherein each of Rx2, Rx3, and Rx4is H.

115. The conjugate of claim 113 or 114, wherein nxl is 0.

116. The conjugate of any one of claims 113-115, wherein Wx1is -*C(O)N(Rx7)-, further wherein Rx7is H.

117. The conjugate of any one of claims 113-116, wherein Ja1and Ja2combine to form oxo.

118. The conjugate of any one of claims 95-112, wherein T2is a bond.

119. The conjugate of any one of claims 95-118, wherein B2is H, hydroxyl, amino, (e.g., dimethyl amino), phosphoryl, sulfonamide, carboxyl, amido (e.g., piperazineamide), or heterocyclyl (e.g., a glucuronidyl or morpholinyl).

120. The conjugate of any one of claims 95-119, wherein each of B1and B3is independently an active agent, H, hydroxyl, amino, (e.g., dimethyl amino), phosphoryl, sulfonamide, carboxyl, amido (e.g., piperazineamide), or heterocyclyl (e.g., a glucuronidyl or morpholinyl).

121. The conjugate of any one of claims 67-120, wherein L2is a covalent linker comprising an alkylene or a heteroalkylene,wherein L comprises one or more of:(i) one or more unsaturated bonds;(ii) a heterocylene or a heteroarylene (e.g., a heteroarylene in the alkylene or heteroalkylene chain); and / or(iii) at least one C 1-20 alkyl substituent.

122. The conjugate of any one of claims 67-121, wherein L2comprises a structure represented by Formula XXVI:Attorney Docket No.: LCH-03725XXVIwherein pl is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;each A7represents a linkage towards X1; andeach A8represents a linkage to Lg.

123. The conjugate of claim 122, wherein pl is 1, 2, 3, 4, or 5.

124. The conjugate of any one of claims 67-123, wherein L2is substituted with alkenyl, alkynyl, halo, hydroxyl, carboxyl, acyl, ester, thioester, alkoxy, phosphoryl, amino (e.g., hydroxylamino), amido, cyano, nitro, azido, alkylthio, alkenyl, alkynyl, cycloalkyl, alkylsulfonyl, or sulfonamido.

125. The conjugate of any one of claims 1-124, wherein the active agent is a chemotherapeutic agent or a toxin.

126. The conjugate of any one of claims 1-124, wherein each active agent is a cytotoxic compound selected from a mitotic inhibitor, a DNA alkylating agent, a RNA targeting agent and a topoisomerase inhibitor, or a combination thereof.

127. The conjugate of claim 126, wherein the cytotoxic compound is selected from an auristatin, a maytansinoid, a tubulisin, a calicheamicin, a duocarmycin, a pyrrolobenzodiazepine, and a camptothecin, or a combination thereof.

128. The conjugate of any one of claims 1-124, wherein the active agent is selected from: (a) a microtubule targeting agent (e.g., a maytansinoid, an auristatin, an eribulin, a tubulysin, a cryptophy cin, or an EG5 inhibitor);(b) a DNA damaging agent (e.g., an enediyne, a topoisomerase I inhibitor, a pyrrolo[2,l- c] [ 1 ,4] benzodiazepine (PBD), or a duocarmycin); and(c) an RNA targeting agent (e.g., a thailanstatin or an amatoxin).

129. The conjugate of any one of claims 1-124, wherein the active agent is selected from MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), PBD dimers and camptothecin and derivatives thereof.Attorney Docket No.: LCH-03725130. A pharmaceutical composition comprising the conjugate of any one of claims 1-129 and a pharmaceutically acceptable excipient.

131. A method of treating or preventing a condition associated with BCMA expression in a subject in need thereof comprising administering a conjugate of any one of claims 1-129 or a pharmaceutically acceptable salt thereof to the subject.

132. The method of claim 131, wherein the condition associated with BCMA expression is cancer.

133. The method of claim 132, wherein the cancer is lung cancer, small-cell lung cancer, non-small-cell lung cancer, gastrointestinal cancer, colon cancer, intestinal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, myeloma, sarcoma, osteosarcoma, Kaposi’s sarcoma or melanoma.

134. The method of claim 133, wherein the cancer is a hematological cancer.

135. The method of claim 133, wherein the cancer is Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), T cell lymphoma, B cell lymphoma, natural killer cell lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, lymphoblastic lymphoma, enteropathy-type intestinal lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, anaplastic large cell lymphoma, peripheral T cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-cell acute lymphoid leukemia (“B-ALL”), T-cell acute lymphoid leukemia (“T-ALL”), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, Blastic plasmacytoid dendritic cell neoplasm (BPDCN), Burkitt’s lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, nonHodgkin's lymphoma, plasmablastic lymphoma, Waldenstrom macroglobulinemia, and combinations thereof.Attorney Docket No.: LCH-03725136. The method of claim 135, wherein the cancer is Hodgkin’s lymphoma, nonHodgkin’s lymphoma, glioblastoma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), or multiple myeloma.

137. The method of claim 135 or 136, wherein the cancer is multiple myeloma.

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