ADC with thiol multiplex linkers

Abstract

To provide thiol multiplexed antibody drug conjugates (TM-ADCs), and multiplex linking assemblies (MLAs) that comprise multiple drug moieties in a single linking assembly.SOLUTION: The invention provides: an MLA compound represented by formula (I) in the figure; and a TM-ADC comprising an antibody and 1-10 covalently attached MLA units. In the formula, A1 is a first linking group; each A2 is independently a bond or a second linking group; each of A1 and A2 comprises 0 or 1 partitioning group (Y) covalently attached to the first or second linking group, or a divalent linking component of the first or second linking group; M is a multiplexing group or a first thiol multiplexing group (TMC1); each TMC2 is a second thiol multiplexing group; m is 0 or 1; where if m is 0 then M is TMC1 in a disulfide form or is TMC1 attached to two drug moieties (DMs), and if m is 1 then each TMC2 is in a disulfide form or is attached to two DMs.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Patent Application No. 62 / 783,707, filed December 21, 2018, and U.S. Patent Application No. 62 / 783,582, filed December 21, 2018, each of which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention Antibody-drug conjugates (ADCs) combine the tumor-targeting specificity of monoclonal antibodies with the potent cell-killing activity of cytotoxic warheads. In part due to recent clinical successes of ADCs, including the approval of brentuximab vedotin (ADCETRIS®) in recurrent Hodgkin's lymphoma and anaplastic large cell lymphoma, and the approval of ado-trastuzumab mertansine (KADCYLA®) in HER2-positive metastatic breast cancer, interest has grown in the design of new ADC formats. Most of these new methodologies have focused on addressing some of the shortcomings of existing clinical molecules, such as heterogeneous drug loading, limited drug-linker stability, and warheads with limited activity in a subset of cancer types. Many notable advances have been made in this field to enable improvements in ADCs. These include site-specific drug-linker conjugation strategies that allow homogeneous loading, drug-linker attachment modes with improved stability, potent new payloads, and linker strategies with alternative release mechanisms.

[0003] Here, we describe a usable multiplex drug conjugation technology for native unengineered IgG as well as engineered IgG, showing the first use of payload attachment where high drug loading is achieved via a single linking chemistry. Depending on the steric parameters of the payload attachment and the utilization of interchain disulfide conjugation as well as conjugation with engineered cysteine ​​residues, ADCs with enhanced in vitro and in vivo activity compared to regular ADCs are then prepared. Summary of the Invention

[0004] Provided herein are multiplexer-linked assembly (MLA) compounds, thiol-multiplexed antibody-drug conjugates (TM-ADCs) comprising an antibody and 1-10 covalently linked multiplexer-linked assembly (MLA) units, where each of the 1-10 covalently linked multiplexer-linked assembly units is covalently bound to a sulfur atom from a cysteine ​​thiol provided by a reduced interchain disulfide bond of the antibody and / or to a sulfur atom from an engineered cysteine ​​residue introduced into the antibody. Each covalently linked multiplexer-linked assembly unit has 2-4 drug moieties (D M ) and an optional splitting group (Y). A particular embodiment of a multiplexer linker assembly (MLA) compound, which is the precursor of the MLA unit in a TM-ADC, has formula (Ia) shown below: Provided by TIFF2024178320000001.tif41128, During the ceremony, A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each contains zero or one dividing group (Y) covalently bonded to the first linking group or the second linking group, or a divalent linking component of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (TMC1 ) and; Each T MC2 are independently thiol multiplexing groups; The subscript m is 0 or 1; where When the subscript m is 0, M represents the first thiol multiplexing group (T MC1 ) or two drug moieties (D M ) attached to a first thiol multiplexing group (T MC1 ) and If the subscript m is 1, then each T MC2 is in disulfide form, dithiol form with appropriate protection of the sulfur atom, or is a dithiol having two drug moieties (D M )

[0005] Also provided herein are thiol multimeric antibody drug conjugates (TM-ADCs), generally prepared using MLA compounds of formula Ia.

[0006] In another aspect, provided herein is a compound having formula (i) and (ii): or a salt thereof, wherein R is H or an amino protecting group, and R 1 teeth, TIFF2024178320000003.tif98128.

[0007] In yet other aspects, provided herein are pharmaceutical compositions and methods of treating diseases using the described TM-ADCs. [The present invention 1001] Formula (I): Multiplexor Ligation Assembly (MLA) compounds with TIFF2024178320000004.tif33128: During the ceremony, A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each contains zero or one dividing group (Y) covalently bonded to the first linking group or the second linking group, or a divalent linking component of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and; Each T MC2 is a second thiol multiplexing group; The subscript m is 0 or 1; where When the subscript m is 0, M represents the first thiol multiplexing group (T MC1 ) or two drug moieties (D M ) attached to a first thiol multiplexing group (T MC1 ) and If the subscript m is 1, then each T MC2 is in disulfide form or has two drug moieties (D M ) [The present invention 1002] A 1 but, TIFF2024178320000005.tif35148, wherein R is selected from the group consisting of H and an amine protecting group; Y is a splitting group; and the wavy line indicates a covalent bond to M. [The present invention 1003] M, T MC1 and The MLA compound of the present invention 1002 is selected from the group consisting of TIFF2024178320000006.tif17128. [The present invention 1004] M, T MC1 and TIFF2024178320000007.tif24150, and the wavy line adjacent to the sulfur is -A 2 -T MC2 1002 MLA compounds of the present invention showing the covalent attachment site to the group. [The present invention 1005] Each TMC2 but, TIFF2024178320000008.tif24150, wherein each T MC2 The wavy lines adjacent to each sulfur represent the drug moiety (D M 1004 MLA compounds of the present invention showing the binding site to [The present invention 1006] Each T MC2 but, The MLA compound of the present invention 1004 is selected from the group consisting of TIFF2024178320000009.tif17128. [The present invention 1007] A 1 but, 1001. An MLA compound of the present invention having a formula selected from the group consisting of: TIFF2024178320000010.tif33138, wherein the wavy line indicates the bond to M. [The present invention 1008] M, T MC1 and The MLA compound of the present invention 1007 is selected from the group consisting of TIFF2024178320000011.tif17128. [The present invention 1009] The subscript m is 1 and M is T MC1 and TIFF2024178320000012.tif24150; and MC2 but, The MLA compound of the present invention 1001 or 1007 selected from the group consisting of TIFF2024178320000013.tif17128. [The present invention 1010] (A 2 -T MC2 ) are respectively, TIFF2024178320000014.tif39137, wherein the succinimide ring is in hydrolyzed form and each R is independently selected from the group consisting of H and an amine protecting group; Y is a splitting group; the wavy line to the left of the succinimide ring is T MC11001 or 1007 MLA compounds of the present invention showing a thioether bond to [The present invention 1011] The subscript m is 0 and M is T MC1 and The MLA compound of the present invention 1001 or 1007 selected from the group consisting of TIFF2024178320000015.tif97128. [The present invention 1012] The subscript m is 0 and M is T MC1 and The MLA compound of the present invention 1001 or 1007 selected from the group consisting of TIFF2024178320000016.tif127140. [The present invention 1013] I-1, I-2, I-1a, and I-2a: having a formula selected from the group consisting of: TIFF2024178320000017.tif182128; During the ceremony, The subscript w is an integer from 0 to 8; each W is independently a natural or unnatural amino acid; each R is independently H or an amine protecting group; and Y is a splitting group. 1001 MLA compounds of the present invention. [The present invention 1014] The MLA compound of the present invention, wherein the dividing group Y comprises a polyethylene glycol group. [The present invention 1015] The MLA compound of the present invention 1001 has a formula selected from TIFF2024178320000018.tif75128. [The present invention 1015] A 1 The MLA compound of the present invention 1001 or 1002, wherein is selected from the group consisting of a maleimide group and a halomethylcarbonyl group. [The present invention 1016] The MLA compound of the present invention 1001 further comprising a group Y which is a splitting group. [The present invention 1017] The MLA compound of the present invention, wherein the dividing group Y comprises a polyethylene glycol group. [The present invention 1019] A 1 but, TIFF2024178320000019.tif34128, wherein R is selected from the group consisting of H and an amine protecting group; and Y is a splitting group. [The present invention 1020] The splitting group is PEG3, PEG6, PEG 12 , and PEG 24 wherein the subscript indicates the number of repeating polyethylene glycol subunits linked in a linear chain. [The present invention 1021] A 1 The MLA compound of the present invention 1001, wherein the MLA compound comprises an azide or alkyne functional group that can participate in click chemistry to form a 1,2,3-triazole moiety. [The present invention 1022] m is 1 and M has the structure: TIFF2024178320000020.tif34128, and each T MC2 but, The MLA compound of the present invention is selected from the group consisting of TIFF2024178320000021.tif17128. [The present invention 1018] A 1 -M- is a group of the formula: MLA compound of the present invention 1001 having TIFF2024178320000022.tif43128. [The present invention 1019] Each A 2 is a bond and A 1 -M-(T MC2 )2 is expressed by the formula: MLA compound of the present invention 1001 having TIFF2024178320000023.tif45128. [The present invention 1020] R is H or an amino protecting group, Y is a splitting group, and R 1 but, TIFF2024178320000024.tif89128, A compound having TIFF2024178320000025.tif40128 or a salt thereof. [The present invention 1021] R 1 but, The compound of the present invention 1025 selected from the group consisting of TIFF2024178320000026.tif17128. [The present invention 1022] formula: Compound 1025 of the present invention having TIFF2024178320000027.tif25128. [The present invention 1023] A thiol-multiplexed antibody drug conjugate (TM-ADC) comprising an antibody and 1 to 10 covalently linked multiplexer-linked assembly (MLA) units, the thiol-multiplexer-linked assembly units each being linked to cysteine ​​thiols of reduced interchain disulfide bonds in an antibody and / or to engineered cysteine ​​residues, or to modified functional groups introduced into an antibody, or heterocyclos derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions, and each of the covalently linked multiplexer-linked assembly units having 2 to 4 drug moieties attached thereto, and an optional resolving group (Y). [The present invention 1024] The TM-ADC of the present invention, wherein each of the MLAs has two attached drug moieties. [The present invention 1030] The TM-ADC of the present invention, wherein the drug moiety is derived from a cytotoxic agent. [The present invention 1031] Formula (I Ab ): TIFF2024178320000028.tif36128, During the ceremony, Ab is from antibody; S* is selected from the group consisting of a sulfur atom of a cysteine ​​residue of a reduced interchain disulfide bond, a sulfur atom from an engineered cysteine ​​residue of the antibody, or a modified functional group introduced into the antibody, or is a heterocyclo derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions; A 1 is a first linking group; 1 optionally includes a splitting group (Y) covalently bonded to the first linking group or is a divalent linking moiety of the first linking group; a bond or an independently selected second linking group T MC1 is the first thiol multiplexing group; Here, T MC1 consists of two drug moieties (D M ) and The subscript p is an integer from 1 to 10. TM-ADC of the present invention 1028. [The present invention 1025] Formula (II Ab ): TIFF2024178320000029.tif38128, During the ceremony, Ab is from antibody; S* is selected from the group consisting of a sulfur atom of a cysteine ​​residue of a reduced interchain disulfide bond, a sulfur atom from an engineered cysteine ​​residue of the antibody, or a modified functional group introduced into the antibody, or is a heterocyclo derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions; A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2each optionally includes a splitting group (Y) covalently bonded to the first linking group or the second linking group or is a divalent linking moiety of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and; Each T MC2 is a second thiol multiplexing group in reduced form; Each D M is the reduced form of T MC2 a drug moiety attached to the sulfur atom of the group; and The subscript p is an integer from 1 to 10. TM-ADC of the present invention 1028. [The present invention 1026] Formula (I Ab A): TIFF2024178320000030.tif35128, During the ceremony, Ab is from antibody; S* is selected from the group consisting of a sulfur atom of a cysteine ​​residue of a reduced interchain disulfide bond, a sulfur atom from an engineered cysteine ​​residue of the antibody, or a modified functional group introduced into the antibody, or is a heterocyclo derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions; A 1 is a first linking group that optionally includes a splitting group (Y) covalently bonded to the first linking group or the second linking group, or is a divalent linking moiety of the first linking group or the second linking group; T MC1 is a thiol multiplexing group; Each S is T MC1 a sulfur atom selected from the group consisting of: Each D M is a drug moiety; and The subscript p is an integer from 1 to 10. TM-ADC of the present invention 1028. [The present invention 1027] Formula (I Ab b): has TIFF2024178320000031.tif47128, During the ceremony, Ab is from antibody; S* is selected from the group consisting of a sulfur atom of a cysteine ​​residue of a reduced interchain disulfide bond, a sulfur atom from an engineered cysteine ​​residue of the antibody, or a modified functional group introduced into the antibody, or is a heterocyclo derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions; A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each independently contains zero or one splitting group (Y) covalently bonded to the first linking group or the second linking group, or a divalent linking component of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and; Each T MC2 is independently a second thiol multiplexing group; Each S is a second thiol multiplexing group (T MC2 ) sulfur atoms; and Each D M is the drug moiety, TM-ADC of the present invention 1028. [The present invention 1028] A 1 but, TIFF2024178320000032.tif35158, wherein the succinimide ring is optionally in hydrolyzed form; S* is selected from the group consisting of a sulfur atom of a reduced interchain disulfide bond or a sulfur atom from an engineered cysteine ​​unit of said antibody; R is selected from the group consisting of H and an amine protecting group; Y is a splitting group; the wavy line indicates the bond to M; and the dashed line indicates the bond to the antibody. [The present invention 1029] A 1 but, TIFF2024178320000033.tif171128, wherein the succinimide ring is optionally in hydrolyzed form; S* is selected from the group consisting of a sulfur atom of a reduced interchain disulfide linkage or a sulfur atom from an engineered cysteine ​​unit of said antibody; R is selected from the group consisting of H and an amine protecting group; Y is a splitting group; the wavy line indicates a covalent bond to M; and the dashed line indicates a covalent bond to the antibody. [The present invention 1030] M is T MC1 And each T MC1 and T MC2 became independent, TIFF2024178320000034.tif24150, and each T MC2 But it also contains two drug moieties (D M ) is bound to the TM-ADC of the present invention. [The present invention 1031] D M are selected from the group consisting of mc-VC-PAB-D, mc-D, mc-VC-D, MDPr-D, and MDPr-Lys(PEG)-D; and wherein each D is a Drug unit which is derived from a chemotherapeutic nucleoside, antimetabolite, or a hydrophilic or mildly hydrophobic chemotherapeutic agent characterized by a ClogP of 2.5 or less and / or a polar surface area of ​​80 square Angstroms or more. [The present invention 1032] formula: TIFF2024178320000035.tif29128, During the ceremony Ab is from antibody; each S* is a sulfur atom from the antibody; each W is independently a natural or unnatural amino acid; wherein each W optionally comprises a splitting group (Y) covalently attached to W; The subscript w is 0, 1, 2, or 3; D M1 is a first drug moiety; D M2 is a second drug moiety; and The subscript p is an integer from 1 to 10. Any of TM-ADCs of the present invention 1028 to 1033. [The present invention 1040] A TM-ADC of the present invention 1039, wherein the subscript w is 0. [The present invention 1041] 1039. A TM-ADC of the present invention, wherein the subscript w is 1 and the separating group (Y) is a PEG unit composed of 8 to 72, 8 to 36, or 8 to 24, or 12 to 24 consecutive polyethylene glycol subunits. [The present invention 1033] W w is a dipeptide residue or a tripeptide residue. [The present invention 1034] W w 1042. The TM-ADC of the invention, wherein each amino acid present in is independently an amino acid residue selected from the group consisting of glycine, alanine, β-alanine, and lysine. [The present invention 1035] D M1 and D. M2 each represents a first drug linker (D L1 ) and a second drug linker (D L2 ), where each D L1 and each D L2 are independently selected from maleimido-caproyl (mc), maleimido-caproyl-valine-citrulline (mc-vc), maleimido-caproyl-valine-citrulline-paraaminobenzyloxycarbonyl (mc-vc-PABC), and maleimidodiaminopropionyl-valine-citrulline (MDPr-vc), where either the mc or MDpr moiety has been converted to its corresponding succinimide moiety, optionally in hydrolyzed form. [The present invention 1036] Each D M1 and each D M2 independently incorporates the structure of a free drug, said free drug being selected from a chemotherapeutic nucleoside, antimetabolite, or a hydrophilic or mildly hydrophobic chemotherapeutic agent characterized by a ClogP of 2.5 or less and / or a polar surface area of ​​80 square Angstroms or more. [Brief description of the drawings]

[0008] [Figure 1] An exemplary reaction scheme for preparing thiol-multiplexed antibody drug conjugates (TM-ADCs) is shown. In the first reaction (left), an antibody thiol from a cysteine ​​residue reacts with a linking group (A1) covalently attached to a thiol-multiplexed (TMC) group (the "A1-TMC" component) to form an intermediate in which the disulfide functionality of the thiol-multiplexed (TMC) group is intact. For the second reaction arrow, a reducing agent such as TCEP cleaves the disulfide bond in the thiol-multiplexed (TMC) group, and the newly generated thiol functionality reacts with N-ethylmaleimide (NEM) to form a TM-ADC with a "dummy" drug moiety. In this case, the final product is a thiol-multiplexed antibody conjugate capped with N-ethylmaleimide (NEM), in which the maleimide moiety has been converted to a thio-substituted succinimide moiety. To prepare thiol-multiplexed antibody drug conjugates (TM-ADCs), substitute NEM with the desired drug moiety (DM) in the reaction scheme. NOTE: The diagram of the antibody on the right shows the succinimide ring of the A1 group in either open or hydrolyzed form. The diagram of the antibody in the middle may be in the open ring form, as the resolution of the mass spectrometer cannot distinguish between the Na+ adduct of the product with the closed ring form and the product with the open ring form. [Figure 2A]Provided is PLRP chromatography data for 16-loaded auristatin TM-ADC derived from fully reduced cAC10 antibody, in which the cysteine ​​residues from disulfide bond reduction have each been alkylated with a multiplexer linker assembly (MLA) compound of formula A1-TMC, where A1 is composed of a maleimide moiety, and the hydrophobicity of the auristatin drug unit has been reduced by PEGylation of the linker components of each drug moiety. [Figure 2B] FIG. 1 provides mass spectrometry data for 16-loaded auristatin TM-ADC derived from fully reduced cAC10 antibody, in which the cysteine ​​residues from disulfide bond reduction have each been alkylated with a multiplexer linker assembly (MLA) compound of formula A1-TMC, where A1 is composed of a maleimide moiety, and the hydrophobicity of the auristatin drug unit has been reduced by PEGylation of the linker component of each drug moiety. [Figure 2C] FIG. 1 provides mass spectrometry data for 16-loaded auristatin TM-ADC derived from fully reduced cAC10 antibody, in which the cysteine ​​residues from disulfide bond reduction have each been alkylated with a multiplexer linker assembly (MLA) compound of formula A1-TMC, where A1 is composed of a maleimide moiety, and the hydrophobicity of the auristatin drug unit has been reduced by PEGylation of the linker component of each drug moiety. [Figure 2D] FIG. 1 provides mass spectrometry data for 16-loaded auristatin TM-ADC derived from fully reduced cAC10 antibody, in which the cysteine ​​residues from disulfide bond reduction have each been alkylated with a multiplexer linker assembly (MLA) compound of formula A1-TMC, where A1 is composed of a maleimide moiety, and the hydrophobicity of the auristatin drug unit has been reduced by PEGylation of the linker component of each drug moiety. [Figure 3A]We provide PLRP chromatography data for 16-loaded auristatin TM-ADC derived from fully reduced cAC10 antibody, in which the cysteine ​​residues from disulfide bond reduction have each been alkylated with a multiplexer linker assembly compound of formula A1-TMC, where A1 is composed of a maleimide moiety and the hydrophobicity of each drug moiety is reduced by a hydrophilic linker component. [Figure 3B] 1 provides mass spectrometry data for 16-loaded auristatin TM-ADC derived from fully reduced cAC10 antibody, in which the cysteine ​​residues from disulfide bond reduction have each been alkylated with a multiplexer linker assembly compound of formula A1-TMC, where A1 is composed of a maleimide moiety and the hydrophobicity of each drug moiety is reduced by a hydrophilic linker component. [Figure 3C] 1 provides mass spectrometry data for 16-loaded auristatin TM-ADC derived from fully reduced cAC10 antibody, in which the cysteine ​​residues from disulfide bond reduction have each been alkylated with a multiplexer linker assembly compound of formula A1-TMC, where A1 is composed of a maleimide moiety and the hydrophobicity of each drug moiety is reduced by a hydrophilic linker component. [Figure 4A] An exemplary addition of A1-TMC to an antibody thiol from a cysteine ​​residue and subsequent reduction of the disulfide bond at the thiol multiplexer (TMC) group is shown. In this embodiment of the description, the representation of the antibody on the right is a simplified representation of the chemical structure shown for the antibody in the middle. [Figure 4B]An exemplary reaction scheme for preparing a thiol multiplexed antibody drug conjugate (TM-ADC) with four drug moieties (DMs) is shown. Numbered steps 1 and 2 are similar to those shown in FIG. 4A, except that in this case, the reaction is carried out simultaneously at 10 different cysteine ​​residues of the antibody. After steps 1 and 2, there are two thiol functional groups on each of the growing multiplexer-linked assembly units. Step 3 illustrates the addition of an "A2-TMC2" group to each thiol functional group, followed in step 4 by reducing the disulfide bond in each TMC2 group to provide an antibody in which each multiplexer-linked assembly unit (10 in total) displays four thiol functional groups (40 in total). In step 5, a drug moiety (DM) is attached to each thiol functional group to form a TM-ADC with 40 drug moieties (DMs). An exemplary chemical structure of "A2-TMC2" used in step 3 is shown below the reaction scheme. [Diagram 5] A preparative route for the conjugation of eight MLA units followed by disulfide bond reduction and drug moiety conjugation is illustrated, yielding an ADC with 16 attached drug moieties. [Figure 6] A preparative route for the conjugation of two MLA units is illustrated using an introduced modified functional group that can participate in a click-coupling reaction. In this example, an engineered cysteine ​​is covalently modified with an MPr-PEG-azide compound, which can then react with the appropriate MLA group of a drug moiety precursor bearing an alkyne group. [Figure 7] A variety of MLA compounds suitable for covalent attachment of two or four drug moieties are illustrated. [Figure 8] 1 illustrates the retention time of hydrophilic dendritic gemcitabine ADCs with various drug-to-antibody ratios. [Figure 9] 1 illustrates the in vitro cytotoxicity of hydrophilic dendritic gemcitabine ADCs with various drug-to-antibody ratios against the Hodgkin lymphoma line L540cy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description of the Invention Summary Provided herein are thiol multiplexed antibody drug conjugates (TM-ADCs) and multiplexer-linked assembly (MLA) units of formula (Ia), (Ib) and (Ic), (IIa), (IIIa) and related sub-formulas thereof. The TM-ADCs and MLA units described herein provide higher drug loading in a single linker assembly compared to conventional ADCs due to the multiplexer-linked assembly units. The higher drug loading provided by a single multiplexer-linked assembly is due to the branched nature of the multiplexer-linked assembly units described herein. For example, in some aspects, a single multiplexer-linked assembly unit contains a drug unit (D U ) each consisting of 2 to 32 or more drug moieties (D M ) covalently attached to the antibody. Thus, the present disclosure provides multiplexer-linked assembly units with high drug loading that require only a single attachment chemistry to the antibody. When multiple multiplexer-linked assembly units are attached to a single antibody, delivery of significantly larger doses of free drug is achievable (e.g., 10 multiplexer-linked assembly (MLA) units, each with 4 drug moieties attached thereto, provides a total of 40 drug units).

[0010] A key component of the TM-ADC and multiplexer-linked assembly (MLA) units described herein is the thiol multiplexer (TM MC As described in more detail below, the thiol multiplexer group can be further MC A drug moiety containing a group (D M ) or a component of a multiplexer-linked assembly unit that provides two covalent attachment points (thiol groups) to a linking group (A). Thus, the thiol multiplexer (T MC ) group allows multiple drug moieties (D M ) is covalently linked to the antibody.

[0011] FIG. 1 and FIGS. 2A and 2B provide diagrams showing how to prepare exemplary thiol-multiplexed antibody drug conjugates (TM-ADCs) that contain multiplexer-linked assembly (MLA) units.

[0012] definition Unless otherwise indicated, the following words and phrases as used herein are intended to have the following meanings: When trademarks are used herein, unless the context indicates otherwise, the trademarks include formulations of the product, generic drugs, and the active pharmaceutical ingredients of the trademarked product.

[0013] The term "antibody" as used herein is used in the broadest sense and specifically refers to intact monoclonal, polyclonal, monospecific, and multispecific antibodies (e.g., bispecific antibodies), including intact and antigen-binding antibody fragments that exhibit the desired biological activity, provided that the antigen-binding antibody fragments have the required number of binding sites for the desired number of drug linker moieties attached. The natural form of an antibody is a tetramer, consisting of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) together are primarily responsible for binding to the antigen. The light and heavy chain variable domains consist of a framework region interspersed with three hypervariable regions, also called "complementarity determining regions" or "CDRs". The constant region can be recognized and interacted with by the immune system. (See, e.g., Janeway et al., 2001, Immuno. Biology, 5th Ed., Garland Publishing, New York). Antibodies include any isotype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) thereof. Antibodies can be derived from any suitable species. In some aspects, the antibody is of human or murine origin, and in some aspects the antibody is a human, humanized, or chimeric antibody.

[0014] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates that the properties of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0015] An "intact antibody" is a set of antibodies that, depending on the antibody class, contains an antigen-binding variable region as well as a light chain constant domain (C L ) and the heavy chain constant domain, C H 1. C H 2. C H 3, and C H 4. The constant domains are either native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0016] "Antibody fragment" includes a portion of an intact antibody, including its antigen-binding or variable region. Antibody fragments of the present disclosure contain at least one cysteine ​​residue (natural or engineered) that provides a site for attachment of a multiplexer-linked assembly. In some embodiments, antibody fragments include Fab, Fab', F(ab')2.

[0017] An "antigen" is an entity to which an antibody specifically binds.

[0018] The terms "specific binding" and "specifically bind" mean that an antibody or antibody fragment thereof binds to its corresponding target antigen in a selective manner and does not bind to a number of other antigens. Typically, antibodies and antibody derivatives bind to at least about 1×10 -7 M, more typically 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M, or 10 -12It binds to a given antigen with an affinity of M that is at least twice its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0019] The terms "inhibit" or "inhibition" mean to reduce by a measurable amount or to prevent completely (eg, 100% inhibition).

[0020] The term "therapeutically effective amount" refers to an amount of a TM-ADC described herein that is effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a conjugate provides one or more of the following biological effects: reduction in the number of cancer cells; reduction in tumor size; inhibition (i.e., slowing to some extent and preferably stopping) of cancer cell invasion into peripheral organs; inhibition (i.e., slowing to some extent and preferably stopping) of tumor metastasis; inhibition to some extent of tumor growth; and / or relief to some extent of one or more of the symptoms associated with cancer. To the extent that the free drug can be released from the TM-ADC to inhibit the growth of or kill existing cancer cells, the free drug is cytostatic and / or cytotoxic. For cancer therapy, in some aspects, efficacy is measured by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0021] Unless otherwise stated or implied by context, the terms "substantial" or "substantially" refer to the majority, i.e., >50%, of a population, mixture, or sample, typically greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the population.

[0022] The terms "intracellularly cleaved" and "intracellular cleavage" refer to an intracellular metabolic process or reaction on a thiol multi-antibody drug conjugate (TM-ADC) in which the cellular machinery acts on the TM-ADC, or a fragment thereof, to release free drug, or other degradation products, from the TM-ADC intracellularly. The moieties resulting from that metabolic process or reaction are thus intracellular metabolites.

[0023] The term "cytotoxic activity" refers to the cell-killing effect of a drug or a thiol-multi-antibody drug conjugate (TM-ADC) or an intracellular metabolite of a TM-ADC. Cytotoxic activity is typically measured using a cytotoxicity assay (IC 50 It is expressed as a value, which is the concentration (molar or mass) per unit volume at which half of the cells will survive exposure to a cytotoxic agent.

[0024] The term "cytostatic activity" refers to a cytostatic substance or drug thereof (D M ) or its intracellular metabolites that are cytostatics, other than cytocidal.

[0025] The term "cytotoxic agent" as used herein refers to an agent that has cytotoxic activity and causes destruction of cells. The term is intended to include chemotherapeutic agents, including their synthetic analogs and derivatives, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin.

[0026] The term "cytostatic substance" as used herein refers to a substance that has cytostatic activity, e.g., inhibits a cellular function that is responsible for or contributes to cell growth or proliferation. Cytostatic substances include inhibitors such as protein inhibitors, e.g., enzyme inhibitors.

[0027] The terms "cancer" and "cancerous" refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" contains multiple cancerous cells.

[0028] As used herein, an "autoimmune disease" is a disease or disorder that arises from and is directed against an individual's own tissues or proteins.

[0029] "Patient" as used herein refers to the subject to which TM-ADC is administered.Examples of "patient" include, but are not limited to, mammals such as humans, rats, mice, guinea pigs, non-human primates, pigs, goats, cows, horses, dogs, cats, birds and poultry.Typically, the patient is a rat, mouse, dog, non-human primate or human.In some aspects, the patient is a human being who needs an effective amount of TM-ADC.

[0030] The term "treat" or "treatment", unless otherwise stated or implied by the context, refers to therapeutic treatment and prophylactic measures to prevent recurrence, where the purpose is to inhibit or slow down (reduce) undesired physiological changes or disorders, such as the development or spread of cancer. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or palliation of disease state, and remission (whether partial or total). In some aspects, "treatment" means prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, and in some aspects further include those prone to having the condition or disorder.

[0031] In the context of cancer, the term "treating" includes any or all of the following: inhibiting the growth of tumor cells, cancer cells, or tumors, inhibiting the replication of tumor cells or cancer cells, reducing the overall tumor burden or reducing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.

[0032] In the context of autoimmune disease, the term "treating" includes any or all of the following: inhibiting the replication of cells associated with the autoimmune disease state, including, but not limited to, cells that produce autoimmune antibodies, reducing the amount of autoimmune antibodies, and ameliorating one or more symptoms of the autoimmune disease.

[0033] As used herein, the term "salt" refers to a compound (e.g., a drug moiety (D M), multiplexer linked assembly (MLA) unit, or TM-ADC). In some aspects, the compound contains at least one amino group and is thus capable of forming an acid addition salt with an amino group. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Salts may also include the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. Counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, salt has one or more charged atoms in its structure. When there are multiple charged atoms as part of the salt, there can be multiple counterions. Thus, salt can have one or more charged atoms and / or one or more counterions. "Pharmaceutically acceptable salt" is suitable for administration to the subject described herein, and in some aspects includes the salt described in PH Stahl and CG Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich:Wiley-VCH / VHCA, 2002, which list is specifically incorporated herein by reference.

[0034] Unless otherwise stated or implied by context, the term "alkyl," by itself or as part of another term, refers to an unsubstituted straight or branched, saturated hydrocarbon having the indicated number of carbon atoms (e.g., "-C1-C8 alkyl" or "-C1-C 10 ("Alkyl" refers to alkyl groups having 1 to 8 or 1 to 10 carbon atoms, respectively). If the number of carbon atoms is not indicated, the alkyl group has 1 to 8 carbon atoms. Representative straight chain "-C1-C8 alkyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl; while branched -C1-C8 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; the term "alkenyl" by itself or as part of another term refers to unsubstituted -C2-C8 alkyl, and includes, but is not limited to, -vinyl, -allyl, -1-butenyl, -2 ... Examples of alkynyl include, but are not limited to, -1-pentenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexylenyl, 2-hexylenyl, and -3-hexylenyl; the term "alkynyl" by itself or as part of another term refers to an unsubstituted -C2-C8 alkyl having one or more triple bonds, such as -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1 butynyl.

[0035] Unless otherwise stated or implied by the context, "alkylene" by itself or as part of another term refers to a substituted or unsubstituted saturated or unsaturated branched or straight chain or cyclic hydrocarbon diradical having the stated number of carbon atoms, typically 1 to 10 carbon atoms, and having two monovalent radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,2-ethylene (-CH2CH2-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and the like. In some aspects, the alkylene is a branched or straight chain hydrocarbon (i.e., not a cyclic hydrocarbon). In other aspects, the alkylene is a saturated alkylene, which is typically not a cyclic hydrocarbon.

[0036] Unless otherwise stated or implied by the context, "aryl" by itself or as part of another term means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of 6 to 20 carbon atoms (preferably 6 to 14 carbons) derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as "Ar". Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is a phenyl group.

[0037] Unless otherwise stated or implied by context, “arylene,” by itself or as part of another term, refers to an aryl group in which one of the hydrogen atoms is replaced with a bond (i.e., is divalent) and has the following structure: An aryl group as defined above that can be ortho, meta, or para oriented, with phenyl being an exemplary group, as shown in TIFF2024178320000036.tif22128.

[0038] Unless otherwise stated or implied by the context, "C3-C8 heterocycle" by itself or as part of another term refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also referred to as ring members) and 1 to 4 heteroatom ring members independently selected from N, O, P, or S, and derived by removing one hydrogen atom from a ring atom of a parent ring system. In some aspects, one or more N, C, or S atoms in the heterocycle are oxidized. A ring containing a heteroatom is aromatic in some aspects and non-aromatic in other aspects. Unless otherwise stated, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Representative examples of C3-C8 heterocycles include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, and isoxazolyl.

[0039] Unless otherwise stated or implied by the context, "C3-C8 heterocyclo" by itself or as part of another term refers to a C3-C8 heterocyclo group as defined above in which one of the hydrogen atoms of the heterocyclo group has been replaced with a bond (i.e., it is divalent). In certain aspects, for example when part of a multiplexer linkage assembly includes a heterocyclo, the heterocyclo is a heterocyclo group as defined above in which one or two of the hydrogen atoms of the heterocyclo group have been replaced with a bond (i.e., the heterocyclo is divalent or trivalent).

[0040] Unless otherwise stated or implied by the context, a "C3-C8 carbocycle" by itself or as part of another term is a 3-, 4-, 5-, 6-, 7-, or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbocycle derived by removing one hydrogen atom from a ring atom of a parent ring system. Representative -C3-C8 carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.

[0041] Unless otherwise stated or implied by the context, "C3-C8 carbocyclo" by itself or as part of another term refers to a C3-C8 carbocyclic group as defined above in which one of the carbocyclic group's hydrogen atoms has been replaced by a bond (i.e., it is divalent). In certain aspects, for example when part of a multiplexer linkage assembly includes a carbocyclo, the carbocyclo is a carbocyclic group as defined above in which one or two of the carbocyclic group's hydrogen atoms have been replaced by a bond (i.e., the carbocyclo is divalent or trivalent).

[0042] Unless otherwise stated, the term "heteroalkyl", by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, including fully saturated or 1 to 3 degrees of unsaturation, or combinations thereof, consisting of the stated number of carbon atoms and 1 to 10, preferably 1 to 3, heteroatoms selected from the group consisting of O, N, Si, and S, including aspects where the nitrogen and / or sulfur atoms are oxidized and where the nitrogen heteroatom is quaternized. The heteroatoms O, N, and S are located at any position within the heteroalkyl group and / or at the position at which the alkyl group is attached to the remainder of the molecule. The heteroatom Si is located at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Examples include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(O)-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. In preferred aspects, the C1-C4 heteroalkyl or heteroalkylene has 1-4 carbon atoms and 1 or 2 heteroatoms, and the C1-C3 heteroalkyl or heteroalkylene has 1-3 carbon atoms and 1 or 2 heteroatoms. In other preferred aspects, the heteroalkyl or heteroalkylene is saturated.

[0043] Unless otherwise stated or implied by the context, the term "heteroalkylene" by itself or as part of another substituent refers to a divalent group derived from a heteroalkyl (as defined above), as exemplified by -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. In the case of heteroalkylene groups, in some aspects, heteroatoms occupy one or both of the chain termini. Furthermore, in the case of alkylene and heteroalkylene linking groups, no orientation of the linking group is implied. In certain aspects, for example, when the linking or tethering group comprises a heteroalkylene, the heteroalkylene is a heteroalkyl group as defined above in which one or two of the hydrogen atoms of the heteroalkyl group are replaced with a bond (i.e., the heteroalkylene is divalent or trivalent).

[0044] "Substituted alkyl" and "substituted aryl" refer to alkyl and aryl, respectively, in which one or more hydrogen atoms are each independently replaced with a substituent. Exemplary substituents include -X, -O, -C, -D, -E, -F ... - , -OR, -SR, -S - , -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R, -C(=O)R, -C(=O)NR2, -SO3 - , -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO - 3, -PO3H2, -AsO2H2, -C(=O)X, -C(=S)R, -CO2R, -CO2 - , -C(=S)OR, C(=O)SR, C(=S)SR, C(=S)NR2, or C(=NR)NR2, where each X is independently a halogen: -F, -Cl, -Br, or -I; and each R is independently -H, -C1-C 20 Alkyl, -C6~C 20 Aryl, -C3~C 14Heterocycle, protecting group or prodrug moiety. Exemplary substituents also include (=O). The aforementioned alkylene, carbocycle, carbocyclo, arylene, heteroalkyl, heteroalkylene, heterocycle, and heterocyclo groups are unsubstituted or similarly substituted. In some aspects, "alkyl" and "alkylene" substituents include -X, -O. - , -OR, -SR, S-, NR2, CX3, CN, OCN, SCN, -NRC(=O)R, -C(=O)R, -C(=O)NR2, -SO3 - In some embodiments, the substituents of "aryl", "carbocyclic", "carbocyclo", "arylene", "heteroalkyl", "heteroalkylene", "heterocycle" and "heterocyclo" include -X, -O, -O- ... - , -C1~C 20 Alkyl, -OR, -SR, -S - , NR2, CX3, CN, OCN, -SCN, -NRC(=O)R, -C(=O)R, -C(=O)NR2, -SO3 - , -SO3H, or -CO2R, where each X is independently -F or -Cl, and R is independently -H or -C1-C8 alkyl.

[0045] As used herein, the term "free drug" refers to a bioactive species that is not covalently attached, directly or indirectly, as a drug moiety to any other portion of the TM-ADC or to a degradation product of the TM-ADC. Thus, a free drug is a drug that is not covalently attached, directly or indirectly, to the drug linker (D L ), or following subsequent intracellular transformation or metabolism. In some aspects, the free drug is referred to as the drug moiety present immediately after cleavage from the multiplexer-linked assembly unit via a release mechanism, such as may be provided by HD U or may be present as a charged moiety. The free drug is the pharmacologically active species capable of exerting the desired biological effect. In some aspects, the pharmacologically active species is the parent drug, while in other aspects, it includes components or traces of the multiplexer-linked assembly units that have not undergone subsequent intracellular metabolism.

[0046] As used herein, the term "splitting group" refers to a specific drug unit (D U ) or multiplexor linked assembly (MLA) units. In some aspects, the splitting groups increase the hydrophilicity of the MLA units. In other aspects, the splitting groups improve the pharmacokinetic properties of the TM-ADC to which they are attached.

[0047] The term "self-stabilizing linker assembly" as used herein refers to a substituted succinimide having a basic functional group adjacent to the succinimide, which can catalyze the hydrolysis of the carbonyl-nitrogen bond of the substituted succinimide. The hydrolysis of the substituted succinimide by the basic functional group forms a self-stabilizing linker. Further details of the self-stabilizing linker assembly are described in WO 2013 / 173337, which is specifically incorporated herein by reference. In some aspects, the self-stabilizing linker assembly is an MDPr, which has the structure disclosed herein.

[0048] The term "engineered cysteine ​​residue" or "eCys residue" as used herein refers to a cysteine ​​amino acid or derivative thereof incorporated into an antibody. One or more eCys residues can be incorporated into an antibody, and typically, the eCys residues are incorporated into either the heavy or light chain of the antibody. Generally, the incorporation of an eCys residue into an antibody is performed by mutagenizing the nucleic acid sequence of the parent antibody to encode one or more amino acid residues having a cysteine ​​or a derivative thereof. Suitable mutations include replacing a desired residue in the light or heavy chain of the antibody with a cysteine ​​or a derivative thereof, incorporating an additional cysteine ​​or a derivative thereof at a desired position in the light or heavy chain of the antibody, and adding an additional cysteine ​​or a derivative thereof to the N- and / or C-terminus of a desired heavy or light chain of amino acids. Derivatives of cysteine ​​(Cys) include, but are not limited to, β-2-Cys, β-3-Cys, homocysteine, and N-methylcysteine.

[0049] Aspects and embodiments Provided herein are thiol-multiplexed antibody drug conjugates (TM-ADCs, described below), as well as multiply linked assembly (MLA) compounds, the structure of which comprises a TM-ADC and an attached drug moiety (D M ) and the drug moiety is incorporated into the MLA unit having a drug linker (DL) when it does not contain a drug unit (D U ) and the attached drug unit (D U ) having a drug linker (D L ) containing the TM-ADC, MLA unit, or drug moiety (D M ) is optionally attached to the MLA unit of the thiol multiplexer (T MC ) group or a splitting group (Y) attached to a portion of the linking group component of TM-ADC, or as part of that linking group component.

[0050] The thiol multiplexing technology described herein readily provides antibody drug conjugates (ADCs) with multiple drug moieties attached. Advantageously, antibodies with higher drug loading can provide therapeutically effective doses of one or more free drugs, reducing the total amount of antibody administered compared to conventional ADCs. This is an advantage when the copy number of the target antigen is low. Furthermore, the multiplexer-linked assembly compounds described herein in preparing TM-ADCs use well-understood thiol / maleimide chemistry, as well as thiol / haloacetyl chemistry. In each case, the covalent coupling conditions are mild and well tolerated by other functional groups on the antibody or on other parts of the linker assembly itself.

[0051] Multiplexer Link Assembly A multiplexer linker assembly (MLA) unit is characterized by the following features: (1) at least one linking group (A 1) which (i) provides for the covalent attachment of MLAs to an antibody or antigen-binding fragment of an antibody, or to an antibody or antigen-binding fragment of an antibody that already has a suitable binding site available (e.g., an antibody with an azide or alkylene moiety attached for participation in click chemistry conjugation or Diels-Alder reaction), and (ii) a thiol multiplexer (T MC ) group or multiplexing group (M), 1 ); and (2) at least one thiol multiplexer (T MC ) groups. As described in more detail below, thiol multiplexers (T MC The aryl group may be in the form of a cyclic disulfide or in the form of a dithiol in which the sulfur atom is appropriately protected or in which two thiols are bonded to a drug moiety (D M ) or a branched (non-cyclic) form that forms a thioether linkage to a further linking group (A). The linkage to a further linking group (A) can be via another thiol multiplexer (T MC ) groups, which in turn provide the opportunity for additional drug loading in a single multiplexer linked assembly (MLA) unit. In some embodiments, the MLA unit further comprises one or more partitioning groups (Y) attached thereto. The subscript m indicates the number of a particular group in the compounds described herein, for example, in the MLA compound of formula (I), when the subscript m is 1, two (A 2 -T MC2 ) units are present. Each subscript m in a particular compound has the same value. For example, the MLA compound of formula (I) has two cases of subscript m. Each of these cases is the same, where if one subscript m is 0, then the other subscript m is also 0; and if one subscript m is 1, then the other subscript m is also 1.

[0052] In one principle embodiment, provided herein is a compound of formula (I): TIFF2024178320000037.tif33128, a multiplexer ligated assembly (MLA) compound having During the ceremony, A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each contains zero or one dividing group (Y) covalently bonded to the first linking group or the second linking group, or a divalent linking moiety of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and; Each T MC2 is independently a second thiol multiplexing group; The subscript m is 0 or 1; where When the subscript m is 0, M is the disulfide form of T MC1 or in a suitably protected dithiol form, or in a form containing two drug moieties (D M ) attached to a first thiol multiplexing group (T MC1 ) and If the subscript m is 1, then each T MC2 is in disulfide form or has two drug moieties (D M )

[0053] In one group of embodiments, provided herein are compounds of formula (Ia): TIFF2024178320000038.tif32128, During the ceremony, A 1 is a linking group further comprising zero or one dividing group (Y) covalently bonded to the first linking group or a divalent linking component of the linking group; T MC1 is a thiol multiplexing group; Each S is a thiol multiplex (T MC1 ) group; and Each D M is the drug moiety.

[0054] In another group of embodiments, provided herein are compounds of formula (Ib): TIFF2024178320000039.tif52128, During the ceremony, A 1 is a first linking group; Each A 2 are independently a bond or an independently selected second linking group, which may be the same or different; A 1 and A 2 each contains zero or one dividing group (Y) covalently bonded to the first linking group or the second linking group, or a divalent linking component of the linking group; M is a multiplexing group or the first thiol multiplexing (T MC1 ) group; Each T MC2 is independently a second thiol multiplexing group; Each S is a second thiol multiplex (T MC2 ) group; and Each D M is the drug moiety.

[0055] Thiol Multiplexer (T MC1 and T MC2 ) group The conceptual approach to the thiol multiplexer groups described herein is illustrated below with respect to four examples. In each transformation shown, a group covalently attached at a single site (depicted as "a") is ring-closed or ring-opened to form two thiols (b) that serve as two sites for further attachment (as "c"). In the compounds and conjugates described herein, the label T MC1 and T MC2 is used to refer to the closed (or disulfide) form, the appropriately protected dithiol form, or the trivalent form "c", depending on the MLA compound or TM-ADC. MCThe group may be in either the closed ring (or disulfide) form "a", the open ring (or dithiol) form b', or in a suitably protected dithiol form as exemplified by b', or may be coupled to a Drug unit (D U ) or drug unit / drug linker (D U / D L ) combination of M ) (configuration "c"). Any T that is not at a terminal position of the multiplexer linkage assembly is MC The group is of the form "c". TIFF2024178320000040.tif79138

[0056] As shown in the figure above, the thiol multiplexer (T MC The ) group is part of a multiplexer linked assembly unit present in form "a": a cyclic moiety in the form of a disulfide bond, form b or b': an acyclic moiety, or form "c": a branched linear moiety with two thioether linkages. In general, the disulfide bond in cyclic form or in a suitably protected acyclic form is a precursor to a branched (acyclic) moiety with two thioether linkages. Conversion between form a and form b, or from form b' to form a, is accomplished by cyclizing the disulfide bond, thereby opening the ring to provide two thiol functional groups, or by reductively removing the acetamide protecting group to provide these groups. Each thiol functional group is connected to a drug moiety (D M ) or further (AT MC ) portion (as described above) of a linking group (e.g., A 2 ) to provide a covalent attachment site.

[0057] In addition to the two thiol functional groups, a thiol multiplexer (T MC ) group is a linking group (e.g., A 1 ) and a third functional group that provides a covalent bond to the thiol multiplexer (T MC The third functional group of the group is a linking group (A 1 , A 2 etc.) in the thiol multiplexer (T MC) group. In the diagram above, the thiol multiplexer (T MC The third functional group of the thiol multiplexer (T) is a cyclic or exocyclic amine (as per the above form "a"). One of skill in the art will appreciate that there are several electron donor / electron acceptor pairs that can provide the described covalent bond. For example, in some embodiments, a thiol multiplexer (T MC The third functional group of the linking group (A 1 , A 2 In some embodiments, the group providing the covalent bond in the thiol multiplexer (T MC The third functional group of the linking group (A 1 , A 2 The groups that provide the covalent bond in are amines.

[0058] The thiol multiplexer linked assembly units used in the multiplexer linked assembly compounds and thiol multiplexer ADCs may be based on commercially available components that typically have 5-, 6-, 7-, or 8-membered carbon rings with two adjacent ring vertices replaced with sulfur to form 1,2-dithiolanes, 1,2-dithianes, 1,2-dithiepanes, and 1,2-dithiocanes. Given the general nature of the "multiplexed" synthesis (see above), the 5- and 6-membered rings generally have exocyclic functional groups suitable for the synthetic chemistry described herein. In some embodiments, the larger 7- and 8-membered rings have exocyclic functional groups suitable for the synthetic chemistry described herein, and in other embodiments, another ring vertex is replaced with, for example, a nitrogen (amine) that may serve as a functional group in the linking chemistry provided.

[0059] Examples of suitable thiol multiplexer groups (in disulfide form) are: Provided as TIFF2024178320000041.tif17128.

[0060] Still other suitable thiol multiplexer groups include the following commercially available amines and carboxylic acids: Based on TIFF2024178320000042.tif124135TIFF2024178320000043.tif139136.

[0061] Linking group (A 1 and A 2 ) Linking group (A 1 and A 2 ) is the multiplexer (M) group in the MLA or the thiol multiplexer (T MC The reactive component (A) introduced onto the group 1 In some embodiments, the first linking group A 1 contains a functional group that provides a covalent bond to an antibody cysteine ​​thiol. In other embodiments, the terminus of the first linking group is a moiety that is used in click chemistry for conjugation to a modified antibody with a compatible click moiety. For example, in one embodiment, the first linking group A 1 The terminus of is a moiety bearing a sufficiently strained alkyne functionality that is reactive to a modified antibody bearing a suitable azide functionality. Dipolar cycloaddition between the two functional groups in click chemistry then produces a triazole heterocyclo. In another embodiment, Diels-Alder type chemistry (4+2 cycloaddition, inverse electron demand) is used to covalently attach a MLA bearing a terminal 1,2,4,5-tetrazine to a modified antibody bearing a suitable trans-cyclooctene functionality. As an example, general diagrams of click and Diels-Alder (4+2 cycloaddition) reactions are shown in a) and b), respectively. Those skilled in the art will appreciate that a variety of modifications are possible, including but not limited to varying the substitution pattern of the reactive moieties, switching the moiety (Ab or MLA) to which each reactive moiety is attached (in click chemistry, the alkyne reactive moiety can be present, for example, on the antibody moiety prior to reaction with the azide-bearing MLA). TIFF2024178320000044.tif60140

[0062] Linking group A2 is M (or T MC1 ) to a reactive moiety, which is often a thiol multiplexer (T MC In some embodiments, the A 2 is a bond, and T MC1 is T MC2 is directly linked to the

[0063] The linking group also provides structural separation and may be any alkylene group, amino acid group (e.g., W w (wherein the subscript w is an integer from 0 to 12, and each W is a natural or unnatural amino acid), a splitting group (Y), or other structural moiety as described below.

[0064] Several functional groups suitable as thiol linking groups have been described in the literature and are well known for binding to thiol moieties. These functional groups include maleimide moieties (e.g., self-stabilizing moieties such as maleimidocaproyl and mDPR, see WO 2013 / 173337, which is incorporated herein by reference).

[0065] Exemplary linking groups within the scope of the present disclosure, prior to covalent attachment to a thiol-containing moiety, include those represented by formulas (V) and (VI): TIFF2024178320000045.tif48128 groups, where LG is a leaving group and the wavy line on the right is a thiol multiplexer (T MC ) group or the point of attachment to the remainder of the linking group (A), and R a is as defined below. One of skill in the art will appreciate that the maleimide of formula (V) can react with an antibody cysteine ​​thiol to form a thiol-substituted succinimide moiety, optionally in hydrolyzed form, and with reference to formula VI, the antibody cysteine ​​thiol becomes covalently attached to the carbon bearing LG via nucleophilic attack, replacing the leaving group (LG). Suitable leaving groups are well known to those of skill in the art and include halogens, tosylates, and mesylates.

[0066] In some embodiments, R a is C1~C 10 Alkylene, C1-C 10 Heteroalkylene-, C3-C8 carbocyclo-, -O-(C1-C8 alkyl)-, -arylene-, C1-C 10 Alkylene-arylene-, -arylene-C1~C 10 Alkylene, C1-C 10 Alkylene-(C3-C8 carbocyclo)-, (C3-C8 carbocyclo)-C1-C 10 Alkylene-, C3-C8 heterocyclo-, C1-C 10 Alkylene-(C3-C8 heterocyclo), (C3-C8 heterocyclo)-C1-C 10 Alkylene, C1-C 10 Alkylene-C(=O)-, C1~C 10 Heteroalkylene-C(=O)-, C3-C8 carbocyclo-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O), C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1~C 10 Alkylene-C(=O)-, C1-C8 alkylene-(C3-C8 carbocyclo)-C(=O)-, (C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, C3-C8 heterocyclo-C(=O)-, C1-C 10 Alkylene-(C3-C8 heterocyclo)-C(=O)-, (C3-C8 heterocyclo)-C1-C 10 Alkylene-C(=O)-, C1~C 10 Alkylene-NH-, C1~C 10 Heteroalkylene-NH-, C3-C8 carbocyclo-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH, C1-C 10 Alkylene-arylene-NH, -arylene-C1~C 10 Alkylene-NH-, C1~C 10 Alkylene-(C3-C8 carbocyclo)-NH-, (C3-C8 carbocyclo)-C1-C 10 Alkylene-NH-, C3-C8 heterocyclo-NH-, C1-C 10Alkylene-(C3-C8 heterocyclo)-NH-, (C3-C8 heterocyclo)-C1-C 10 Alkylene-NH-, C1~C 10 Alkylene-S-, C1~C 10 Heteroalkylene-S-, C3C8 carbocyclo-S-, -O-(C1-C8 alkyl)-S-, -arylene-S-, C1-C 10 Alkylene-arylene-S-, -arylene-C1~C 10 Alkylene-S-, C1~C 10 Alkylene-(C3-C8 carbocyclo)-S-, (C3-C8 carbocyclo)-C1-C8 alkylene-S-, C3-C8 heterocyclo-S-, C1-C 10 Alkylene-(C3-C8 heterocyclo)-S- or (C3-C8 heterocyclo)-C1-C 10 Alkylene-S-. Any R 19 The substituents may be substituted or unsubstituted. In some embodiments, R 19 The substituent is unsubstituted.

[0067] In some embodiments, R a is an amino acid or a peptide comprising 2 to 12 natural or unnatural amino acids. a is a combination of one or more of the foregoing components with one or more (up to 12 amino acids). a is a dipeptide or a tripeptide. In some embodiments, R a are independently selected from the group consisting of valine, alanine, β-alanine, glycine, lysine, leucine, and citrulline.

[0068] In some embodiments, the linking group (A), prior to covalent attachment to the thiol-containing moiety, has formula (VII): TIFF2024178320000046.tif28128, where the wavy line on the right represents the thiol multiplexer (T MC ) group or the point of attachment to the remainder of the linking group (A), and R cis hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, or a protecting group, and R b Ha-NH-C 1~5 In some embodiments, R is an alkylene-C(=O)-, or a mono-, di-, tri-, tetra-, or pentapeptide. b is -NH-CH-C(=O)-. In some embodiments, R b is a dipeptide or a tripeptide. In some embodiments, R b are independently selected from the group consisting of valine, alanine, β-alanine, glycine, lysine, leucine, and citrulline.

[0069] In some embodiments, R of formula (V) and (VI) a The substituent may be substituted. In some embodiments, R of formula (V) a The substituents may be unsubstituted or basic units, e.g., -(CH2) x NHR c or -(CH2) x NR c 2, where x is an integer from 1 to 4, and each R c are independently selected from the group consisting of H, C1-C6 alkyl, and C1-C6 haloalkyl, or two R c The groups combine with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl, or piperidinyl group.

[0070] In some embodiments, the linking group (A 1 Or A 2 )teeth, TIFF2024178320000047.tif43128, where the wavy line on the right represents the thiol multiplexer (T MC ) group or a linking group (A 1 Or A 2 ) is the point of attachment to the remainder of the chain, for example an amino acid or a di- or tripeptide.

[0071] In some embodiments, the linking group (A 1 Or A 2 ) can be composed of one or more amino acids, one or more polyethylene glycol segments (e.g., PEG 24 , PEG 12 , PEG, PEG3), or a combination of structural features such as propanamide units (e.g., -NH-C(O)-CH2-CH2-).

[0072] The linking group (A 1 Or A 2 ) does not always include only linear portions. For example, in some embodiments, the linking group (A 1 ) is the following part: TIFF2024178320000048.tif25128, where the wavy lines on the left and right of the moiety represent linking groups (A 1 ) to the remainder of the molecule.

[0073] The triazole cyclic group in the above paragraph is an azide group and It is understood that these are formed via azide-alkyne polar cycloaddition reactions ("click chemistry"), including those described herein.

[0074] In some embodiments, one or more linking groups (A 2 ) is a bond.

[0075] In some embodiments, one or more linking groups (A 1 Or A 2 ) comprises a lysine group. In some embodiments, the amine group on the side chain of lysine is covalently attached to a splitting group (Y). In some embodiments, the splitting group (Y) covalently attached to the amine group on the side chain of lysine is a terminally carboxylated polyethylene glycol group.

[0076] In some embodiments, one or more linking groups (A 1 Or A 2) comprises a dipeptide, where each amino acid is independently selected from the group consisting of glycine, alanine, β-alanine, valine, leucine, phenylalanine, and proline.

[0077] In some embodiments, one or more linking groups (A 1 Or A 2 ) comprises a tripeptide, where each amino acid is independently selected from the group consisting of glycine, alanine, β-alanine, valine, leucine, phenylalanine, and proline.

[0078] In some embodiments, one or more linking groups (A 1 Or A 2 ) comprises a monopeptide, dipeptide, or tripeptide, where at least one amino acid is selected from the group consisting of aspartic acid, glutamate, lysine, and arginine.

[0079] Upon review of this application and the examples provided therein, one of skill in the art will understand that the operability of the multiplexer-linked assembly compounds and TM-ADCs described herein does not depend on the exact structure of any one linking group (A), and that additional structural features not expressly described herein can be incorporated into one or more linking groups (A) without departing from the scope of the present disclosure.

[0080] In addition, one of skill in the art will also appreciate that, for example, the specific conjugation chemistry to the antibody may alter the synthetic steps leading to the product. In particular, when conjugation to a thiol group on the antibody is performed with a thiol-reactive "A" group, the conjugation to the antibody may be performed with a cyclic thiol multiplexing moiety (T MC ) before reducing it.

[0081] Thiol Multiplexer (T MCTaking the above discussion of linking group (A) into context, reference is made to the following scheme: 1 -T MC1 " groups are attached to antibody thiol groups (typically thiols generated by reduction of interchain disulfide groups or through introduced (engineered) cysteine ​​moieties). 1 -T MC1 The multiplicity of the " " moiety is indicated by brackets and subscript p, but is not shown in the remainder of the scheme for simplicity. MC1 " group is reduced to T MC1 In step (3), the free thiol generated in (1) is reacted with A 2 By covalently linking the maleimide moiety of A 2 -T MC2 In step (4), a T MC2 The group is reduced as in step (1) to generate two free thiol groups. In step (5), a drug moiety (D M ) to T MC2 is covalently bonded to each thiol unit of TIFF2024178320000050.tif199103

[0082] Further illustrations of the preparation of MLAs of the present disclosure are found in Figure 1 and Figures 2A and 2B. One of skill in the art would be able to carry out the construction of MLA units with protecting groups and deprotect the D prior to conjugation to the antibody. M It will be appreciated that the compound may be completed with a bond. For example, in the following scheme, four drug moieties (D M During the construction of the MLA compound to which the A is attached, a protecting group (PG) is used. 1 Addition of a "(e.g., a thiol-reactive maleimide bearing group) is performed to complete the MLA assembly. TIFF2024178320000051.tif114128

[0083] Drug portion (D M ) In some embodiments, the drug moiety (D MDescribe the MLA compounds to which each drug moiety (D M ) derived from a thiol functional group. MC ) group to form a thioether linkage.

[0084] The drug moieties described herein (D M ) is linked to a thiol multiplexer (T MC ) covalently attached to a drug unit (D U ), or drug unit / drug linker (D U / D L ) combination, wherein the Drug Unit (D U ) is the drug linker (D L ) and is covalently attached to a drug linker (D L ) is linked to the thiol multiplexer (T MC ) group.

[0085] Drug Linker (D L ) Drug Linker (D L ) may in some embodiments be a Drug Unit (D U ) thiol multiplexer (T MC ) or to introduce a cleavable linking group.

[0086] Several drug linkers are known for attaching a drug unit to a functional group present in an antibody or a site on an assembly unit, and are referred to herein as drug units (D L ) to the thiol multiplexer (T MC ) is useful for binding to

[0087] In some embodiments, the drug linker (D L ) contains a terminal maleimide and a thiol multiplexer (T MCThe terminal maleimide functionality allows reliable covalent attachment to each of the thiol functionalities, particularly the thiol multiplexer (T MC It will be appreciated that the above moieties are most useful for covalent attachment to the aryl aryl group.

[0088] In some embodiments, the drug linker (D L ) is a drug unit (D U In some of these embodiments, the PABC group is substituted with a sugar, such as glucose, or a derivative thereof, to form a glucuronide unit (as described in more detail in WO 2007 / 011968, which is incorporated herein by reference).

[0089] In some embodiments, the drug linker (D L ) is represented by formula (VIII) or (IX): TIFF2024178320000052.tif28128, where R c is hydrogen or a protecting group, the subscript p is an integer from 1 to 5, and R b Ha-NH-C 1~5 Alkylene-C(=O)-, -NH-C 1~5 alkylene-C(=O)-NH-phenylene-CH2-OC(=O)-, -(dipeptide)-NH-phenylene-CH2-OC(=O)-, or mono-, di-, tri-, tetra-, or pentapeptide. In some embodiments, the phenylene in the aforementioned groups is replaced with a sugar, such as glucose, or a derivative thereof, to form a glucuronide unit. In some embodiments, R b The amine group of R will contain methyl (CH) instead of H. In some embodiments, R b is a dipeptide or a tripeptide. In some embodiments, R b is -NH-CH-C(=O)-. In some embodiments, R bThe amino acids of the peptide units are independently selected from the group consisting of valine, alanine, β-alanine, glycine, lysine, leucine, and citrulline. MC It is understood that the "wavy line" represents the Drug unit (D U ) are shown. U ) and drug linker (D L ) depending on the linking chemistry used between b The terminal portion of the group, in some aspects, also includes a nucleophilic group, such as an amine or hydroxyl group, attached to the terminal carbonyl functionality.

[0090] In some embodiments, the drug linker (D L ) is represented by formula (VIIIa) or (IXa): TIFF2024178320000053.tif28128, where R c is hydrogen or a protecting group, the subscript p is an integer from 1 to 5, and R b Ha-NH-C 1~5 Alkylene-C(=O)-NH-phenylene-CH2-OC(=O)-heterosilyl-C 1~4 Alkylene-b 1 -heterocyclyl-b 2 -;-(Dipeptide)-NH-phenylene-CH2-OC(=O)-heterosilyl-C 1~4 Alkylene-b 1 -heterocyclyl-b 2 - where b 1 and b 2 are independently a bond or a heteroatom selected from NH or O, where each heterocyclyl group is a 5- or 6-membered ring having 1 to 3 heteroatom ring members selected from N, O, and S; and each heterocyclyl group is a C 1~4 Alkyl, hydroxyl, alkoxy, carboxyl, and -C(=O)-C 1~4 In some embodiments, b is substituted with 1 to 3 groups selected from alkyl. 1 and b 2R is a heteroatom or heteroatom moiety selected from the group consisting of NH and O. b The amine group of R may contain methyl (CH) instead of H. In some embodiments, R b is a dipeptide or a tripeptide. In some embodiments, R b is -NH-CH-C(=O)-. In some embodiments, R b The amino acids of the peptide units are independently selected from the group consisting of valine, alanine, glycine, leucine, and citrulline. MC It is understood that the "wavy line" represents the Drug unit (D U ) is shown.

[0091] In some embodiments, the drug linker (D L ) is a releasable drug linker (D RL In some other aspects, the drug linker (D L ) is not a releasable drug linker. In embodiments without a releasable drug linker, the drug unit (D U ) is released via the entire proteolytic pathway (i.e., the non-cleavable pathway).

[0092] Drug Linker (D L ) is a releasable drug linker (D RL For those embodiments in which the group is a releasable drug linker (D), the group allows for efficient release of free drug sufficient to exert, for example, an anti-proliferative effect in targeted cells. RL) is designed for efficient release of free drug after the TM-ADC is internalized into the target cell, but may also be designed to release free drug in the vicinity of the targeted cell. A suitable recognition site for cleavage is one that allows for efficient release of the drug unit of the TM-ADC. Preferably, the recognition site is a peptide cleavage site (such as in the case of a peptide-based releasable linker assembly), a sugar cleavage site (such as in the case of a sugar-based releasable linker assembly that is or consists of a glucuronide unit as described in WO 2007 / 011968), or a disulfide cleavage site (such as in the case of a disulfide-based releasable linker assembly). Examples of peptide cleavage sites include those recognized by intracellular proteases, such as those present in lysosomes. Examples of sugar cleavage sites include those recognized by glycosidases, including glucuronidases, such as beta-glucuronidase.

[0093] In some embodiments, each releasable drug linker (D RL ) is a dipeptide. In some embodiments, the dipeptide is -Val-Cit-, -Phe-Lys-, or -Val-Ala-.

[0094] In some embodiments, each releasable drug linker (D RL ) is independently selected from the group consisting of maleimido-caproyl (mc), maleimido-caproyl-valine-citrulline (mc-vc), maleimido-caproyl-valine-citrulline-paraaminobenzyloxycarbonyl (mc-vc-PABC) and MDPr-vc. RL is understood to be further substituted with a basic moiety, such as an aminoalkyl, which is an exemplary basic unit, to form a self-stabilizing succinimide linker, as described above and in more detail in WO 2013 / 173337.

[0095] In some selected embodiments, the drug linker (D L) is, for example, a maleimide-containing linker that is cleavable by a protease. Thus, exemplary protease-cleavable D L Based on the following: TIFF2024178320000054.tif161128, where S is the thiol multiplexer (T MC ) group, and the wavy line on the right is the drug unit (D U ), and the wavy line on the left is the thiol multiplexer (T MC ).

[0096] Drug Linker (D L ) to the drug unit (D U General methods for covalent attachment of the drug unit (D) are known in the art, and linkers known in the art for traditional ADCs may be used with the TM-ADCs of this disclosure. For example, auristatin and maytansine ADCs are currently in clinical development for the treatment of cancer. Monomethyl auristatin E is conjugated to an antibody through a protease-cleavable peptide linker, monomethyl auristatin F is conjugated directly to an antibody through a maleimidocaproic acid residue, maytansine DM1 is conjugated through a disulfide or through a direct heterobifunctional SMCC linker, and maytansine DM4 is conjugated through a disulfide linker. In preferred embodiments, these linker systems are used with the TM-ADCs described herein, providing for release of the free drug by an enzymatically or non-enzymatically cleavable system, depending on the linker system used. Disulfide, thioether, peptide, hydrazine, ester, or carbamate linkages can all be used to link the drug unit (D) to an antibody. U ) to a drug linker (D L ) is an example of a bond that is useful for connecting

[0097] The optional splitting group (Y) is connected to a drug linker (D L The linkage can be through any suitable atom of the group (a) and (b). Methods for making such linkages are known in the art.

[0098] Drug Unit (D U ) As mentioned above, the drug unit (D U ) to the MLA unit, and a thiol multiplexer (T MC ) group or to the MLA units via a drug linker (D L The drug unit (D U ) before binding to the MLA unit, or to the drug unit (D U ) before attachment of the drug linker (D L It is understood that the following may be combined:

[0099] In some embodiments, the Drug Unit (D U ) is a drug with a cytotoxic activity in the range of 1-100 nM. There are several different assays that can be used to determine if a TM-ADC exerts a cytostatic or cytotoxic effect on a cell line. In one example to determine if a TM-ADC exerts a cytostatic or cytotoxic effect on a cell line, a thymidine incorporation assay is used. For example, cells at a density of 5,000 cells / well on a 96-well plate are cultured for 72 hours and 0.5 μCi of TM-ADC is administered for the last 8 hours of the 72 hour period. 3 Exposure to H-thymidine 3 H-thymidine incorporation into cultured cells is measured in the presence and absence of TM-ADC. The cultured cells show a significant difference in their activity compared to cells of the same cell line cultured under the same conditions but not in contact with TM-ADC. 3 If H-thymidine incorporation is reduced, the TM-ADC has a cytostatic or cytotoxic effect.

[0100] In another example, to determine whether a TM-ADC exerts a cytostatic or cytotoxic effect on a cell line, cell viability is measured by determining the uptake of a dye such as neutral red, trypan blue, or ALAMAR™ blue in the cells (see, e.g., Page et al., 1993, Intl. J. of Oncology 3:473-476). In such an assay, cells are incubated in medium containing the dye, the cells are washed, and the remaining dye, which reflects the cellular uptake of the dye, is measured spectrophotometrically. Cytotoxicity can also be measured using the protein-binding dye sulforhodamine B (SRB) (Skehan et al., 1990, J. Nat'l Cancer Inst. 82:1107-12). Preferred TM-ADCs have IC 50 These include values ​​(defined as the mAB concentration exhibiting 50% cell killing) of less than 1000 ng / ml, preferably less than 500 ng / ml, more preferably less than 100 ng / ml, and even most preferably less than 50 or even less than 10 ng / ml.

[0101] In some embodiments, the Drug Unit is one that has a cellular potency that would not be expected to provide an active ADC in vitro when conjugated with 8 or less drugs / mAb. In some embodiments, the Drug Unit incorporates a drug that is not hydrophobic or has a cLogP of ≦2.5. In some embodiments, the Drug has a cLogP of about 0 to about 2.5, about 0 to 2, about 0 to about 1.5, about 0 to about 1, or about 0 to about 0.5. In some embodiments, the Drug Unit incorporates a drug that is more hydrophilic, e.g., a Drug Unit with a cLogP of ≦1.0. In some embodiments, the Drug has a cLogP of about 0 to about 1, e.g., about 0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.

[0102] In some embodiments, the drug unit is a drug having a charged residue (acid, amine, phosphate), sugar, or polyhydroxylated group. In some embodiments, the drug unit is selected from nucleoside analogs; HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs; SN-38 glucuronide, etoposide phosphate; low molecular weight drugs (e.g., drugs having a molecular weight of less than about 600); nitrogen mustard (melphalan); and proteosome inhibitors (lenalidomide).

[0103] In some embodiments, the Drug Unit is selected from a pyrimidine antagonist and a purine antagonist. In some embodiments, the Drug Unit is selected from an antimetabolite, such as an antifolate; methotrexate, pemetrexed, L-leucovorin, GW1843, Raltitrexid, ZD9331, pralatrexate, and lometrexol.

[0104] Drug Unit (D U ) typically corresponds structurally to a cytotoxic, cytostatic, or immunosuppressant drug, also referred to herein as a cytotoxic, cytostatic, or immunosuppressant. In some embodiments, the Drug unit (D U The free drug incorporated into the thiol multiplexer (T MC In another embodiment, the Drug Unit (D U The free drug incorporated into the thiol multiplexer (T MC ) having a carboxylic acid or ester capable of forming a bond with

[0105] In some embodiments, the Drug Unit (D U ) is the drug linker (D L In some embodiments, the Drug Unit (D U ) is the drug linker (D L In some embodiments, the Drug Unit (D U ) is the drug linker (DL In another embodiment, the Drug unit (D U ) is the drug linker (D L In yet another embodiment, the Drug Unit (D U ) have heteroatoms from the hydroxyl groups of the free drug, or from alcohol-containing free drugs, or from carbonyl functional groups from the free drug, and they are L ) and forms a bond.

[0106] Drug Unit (D U ) preferably incorporates hydrophilic or moderately hydrophobic drugs to accommodate the higher loadings achievable by the present invention. If the drug is too hydrophobic, an undesirable amount of aggregation may occur in the resulting TM-ADC, but in certain cases, incorporation of a splitting group in the multiplexer linkage assembly and / or hydrophilic linker group (A) and / or hydrophilic drug linker (D L), particularly those that exist in a substantially ionized form at physiological pH, may improve to an acceptable extent. An exemplary hydrophilic linker group (A) is composed of a self-stabilizing moiety that includes a succinimide moiety in hydrolyzed form and a basic unit (BU). The self-stabilizing moiety is hydrophilic due to the BU having the amine in the protonated form and the hydrolyzed succinimide exhibiting a carboxylate anion. An exemplary hydrophilic drug linker is composed of a glucuronide unit in which the sugar is glucuronic acid. Moderately hydrophobic to hydrophilic drugs have a ClogP of 2.5 or less and / or a polar surface area of ​​80 square Angstroms or more. In some embodiments, the drugs used in the present invention will have a ClogP value of 2.5 or less, 2.0 or less, 1.5 or less, 1.0 or less, 0.5 or less, 0 or less, -0.5 or less, or -1.0 or less. In other embodiments, the free drug used herein will have a polar surface area of ​​about 80 angstroms or more, about 90 angstroms or more, about 100 angstroms or more, 110 angstroms or more, or 120 angstroms or more. In some embodiments, the drug used herein will have a polar surface area of ​​about 80 angstroms to about 140 angstroms, or any value therebetween. For example, about 80 angstroms, about 90 angstroms, about 100 angstroms, about 80 angstroms, about 110 angstroms, about 120 angstroms, about 130 angstroms, about 140 angstroms, or any value therebetween.

[0107] The general procedure for linking drug to linker is known in the art.See, for example, U.S. Patent No. 8,163,888, U.S. Patent No. 7,659,241, U.S. Patent No. 7,498,298, U.S. Patent Application Publication No. US20110256157 and International Patent Application No. WO2011023883 and WO2005112919, each of which is incorporated herein by reference, particularly with respect to the general procedure described above.

[0108] Splitting group (Y) In certain embodiments, the MLAs and TM-ADCs described herein include an attached splitting group (Y). Splitting groups are useful, for example, to mask the hydrophobicity of certain drug units or multiplexer-linked assemblies. Thus, some splitting groups act to increase the hydrophilicity of the TM-ADCs to which they are attached, resulting in reduced aggregation of the ADC, which may occur at the highest drug loadings for moderately hydrophilic drugs having a ClogP of about 2.5 to about 1, or a polar surface area of ​​about 80 to about 100 Angstroms squared.

[0109] Representative splitting groups include polyethylene glycol (PEG) units, cyclodextrin units, polyamides, hydrophilic peptides, polysaccharides and dendrimers. In some embodiments, the splitting group Y comprises a polyethylene glycol group.

[0110] The splitting group is group A or D M , M., T. MC When included in one or more of the split groups, the group may comprise a lysine residue that provides for simple functional conjugation to the multiplexer linked assembly of the split group.

[0111] Polyethylene glycol unit (PEG) Polydisperse PEG, monodisperse PEG and discrete PEG can be used to make the compounds of the present invention.Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from a heterogeneous mixture, thus providing a single chain length and molecular weight.Preferred PEG units are discrete PEGs, i.e. compounds that are synthesized in a stepwise manner, without going through a polymerization process.Discrete PEG provides a single molecule of a defined and specified chain length.

[0112] The PEG units provided herein include one or more polyethylene glycol chains. The polyethylene glycol chains can be linked together, for example, in a linear, branched or star-shaped configuration. Typically, at least one of the polyethylene glycol chains of the PEG unit is linked to a component of a multiplexer-linked assembly unit (e.g., A, M, T, MC , or D M ) is derivatized at one end for covalent attachment to an appropriate site on the multiplexer-linked assembly unit. Exemplary attachments to the multiplexer-linked assembly unit are by a non-conditionally cleavable linkage or via a conditionally cleavable linkage. Exemplary attachments are via an amide linkage, an ether linkage, an ester linkage, a hydrazone linkage, an oxime linkage, a disulfide linkage, a peptide linkage, or a triazole linkage. In some aspects, attachment to the multiplexer-linked assembly unit is by a non-conditionally cleavable linkage. In some aspects, attachment to the multiplexer-linked assembly unit is not via an ester linkage, a hydrazone linkage, an oxime linkage, or a disulfide linkage. In some aspects, attachment to the multiplexer-linked assembly unit is not via a hydrazone linkage.

[0113] A conditionally cleavable linkage refers to a linkage that is not substantially susceptible to cleavage while circulating in plasma, but is susceptible to cleavage in the intracellular or intratumoral environment. A non-conditionally cleavable linkage is one that is not substantially susceptible to cleavage in any biological environment of the subject to which the TM-ADC is administered. Chemical hydrolysis of hydrazones, reduction of disulfides, and enzymatic cleavage of peptide bonds or glycosidic linkages are examples of conditionally cleavable linkages.

[0114] The PEG unit will be directly attached to the TM-ADC (or intermediate thereof) with a multiplexer-linked assembly unit. The other end (or ends) of the PEG unit is free, untethered (i.e., not covalently attached) and may take the form of a methoxy, carboxylic acid, alcohol or other suitable functional group. The methoxy, carboxylic acid, alcohol or other suitable functional group acts as a cap for the terminal polyethylene glycol subunit of the PEG unit. By untethered, it is meant that the PEG unit is not covalently attached to a drug moiety, antibody, or linking moiety to the drug unit and / or antibody at its untethered site. Such an arrangement allows for the hydrophobic drug moiety (D M ) or drug unit (D U) will allow the PEG unit to be long enough to assume a parallel orientation with respect to the PEG-linked assembly unit. For embodiments in which the PEG unit comprises multiple polyethylene glycol chains, the multiple polyethylene glycol chains may be independently selected to be, for example, the same or different chemical moieties (e.g., polyethylene glycol chains of different molecular weights or subunit numbers). The PEG unit having multiple polyethylene glycol chains is attached to the multiplexer-linked assembly unit at a single attachment site. Those skilled in the art will appreciate that in addition to comprising repeating polyethylene glycol subunits, the PEG unit may also comprise non-PEG material (e.g., to facilitate coupling of the multiple polyethylene glycol chains to each other or to the multiplexer-linked assembly unit). Non-PEG material refers to atoms in the PEG unit that are not part of the repeating -CH2CHO- subunit. In some embodiments provided herein, the PEG unit comprises two monomeric PE polyethylene glycol G chains attached to each other via a non-PEG element. In other embodiments provided herein, the PEG unit comprises two linear polyethylene glycol chains attached to a central core that is attached to the multiplexer-linked assembly unit (i.e., the PEG unit itself is branched).

[0115] In some embodiments, the PEG unit is a divalent linking moiety, i.e., both ends are attached to a component of the multiplexer-linked assembly unit. In some embodiments, the attachment points of the PEG unit are to the same component of the multiplexer-linked assembly (e.g., the linking group (A)). In some embodiments, the attachment points of the PEG unit are to two different components of the multiplexer-linked assembly unit (e.g., the linking group (A) and the multiplexer group (M)). In some embodiments, the PEG unit is a divalent linking moiety of the linking group.

[0116] There are several PEG conjugation methods available to one of skill in the art [e.g., Goodson, et al. (1990) Bio / Technology 8:343 (PEGylation of interleukin-2 at its glycosylation sites after site-directed mutagenesis); EP 0 401 384 (coupling of PEG to G-CSF); Malik, et al., (1992) Exp. Hematol. 20:1028-1035 (PEGylation of GM-CSF with tresyl chloride); ACT Publication No. WO 2006 / 023661 (PEGylation of GM-CSF with tresyl chloride); 90 / 12874 (PEGylation of erythropoietin containing recombinantly introduced cysteine ​​residues using cysteine-specific mPEG derivatives); U.S. Pat. No. 5,757,078 (PEGylation of EPO peptides); U.S. Pat. No. 5,672,662 (Propionic or Butanoic Acid Monosubstituted Poly(ethylene glycol) and Related Polymers and Functional Derivatives Thereof for Biotechnological Applications); U.S. Pat. No. 6,077,939 (PEGylation at the N-terminal α-carbon of peptides); Veronese et al., (1985) Appl. Biochem. Bioechnol 11:141-142 (PEGylation at the N-terminal α-carbon of peptides with PEG-nitrophenyl carbonate ("PEG-NPC") or PEG-trichlorophenyl carbonate); and Veronese (2001) Biomaterials 22:405-417 (review of peptide and protein PEGylation)).

[0117] For example, PEG units may be covalently linked to amino acid residues via a polyethylene glycol-containing compound and a reactive group on the amino acid residue. The reactive groups on the amino acid residue include those (e.g., free amino or carboxyl groups) that are reactive to activated PEG molecules. For example, the N-terminal amino acid residue and the lysine (K) residue have a free amino group; and the C-terminal amino acid residue has a free carboxyl group. Thiol groups (e.g., found on cysteine ​​residues) are also useful as reactive groups for forming covalent bonds to PEG units. In addition, enzyme-assisted methods have been described for specifically introducing activated groups (e.g., hydrazide, aldehyde, and aromatic amino groups) to the C-terminus of polypeptides (see Schwarz, et al. (1990) Methods Enzymol. 184:160; Rose, et al. (1991) Bioconjugate Chem. 2:154; and Gaertner, et al. (1994) J. Biol. Chem. 269:7224).

[0118] In some embodiments, the polyethylene glycol-containing compound forms a covalent bond to the amino group using methoxylated PEG ("mPEG") with different reactive moieties. Non-limiting examples of such reactive moieties include succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imidate, paranitrophenyl carbonate (NPC), succinimidyl propionate (SPA), and cyanuric chloride. Non-limiting examples of such mPEGs include mPEG-succinimidyl succinate (mPEG-SS), mPEG2-succinimidyl succinate (mPEG2-SS); mPEG-succinimidyl carbonate (mPEG-SC), mPEG2-succinimidyl carbonate (mPEG2-SC); mPEG-imidate, mPEG-paranitrophenyl carbonate (mPEG-NPC), mPEG-imidate; mPEG2-paranitrophenyl carbonate. mPEG-N-hydroxysuccinimide (mPEG-NHS); mPEG2-N-hydroxysuccinimide (mPEG2-NHS); mPEG-cyanuric chloride; mPEG2-cyanuric chloride; mPEG2-lysinol-NPC, and mPEG2-Lys-NHS.

[0119] Generally, at least one of the polyethylene glycol chains that constitute the PEG unit is functionalized to provide a covalent bond to the multiplexer-linked assembly unit. Functionalization of the precursor polyethylene glycol-containing compound to the PEG unit includes, for example, via amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or other functional groups. In some embodiments, the PEG unit further comprises a non-PEG material (i.e., a material that is not composed of -CH2CHO-) that provides coupling to the multiplexer-linked assembly unit or facilitates coupling of multiple polyethylene glycol chains when constructing the polyethylene glycol-containing compound or PEG unit.

[0120] The presence of PEG units in the multiplexer-linked assembly unit can have two potential effects on the pharmacokinetics of the resulting TM-ADC. The desired effect is a decrease in clearance (and consequently an increase in exposure), resulting from a decrease in non-specific interactions induced by the exposed hydrophobic elements of the drug unit. The second effect is undesirable, and can result from an increase in the molecular weight of the TM-ADC, resulting in a decrease in volume and distribution rate. Increasing the number of polyethylene glycol subunits increases the hydrodynamic radius of the conjugate, typically resulting in a decrease in diffusivity. The decrease in diffusivity then typically reduces the ability of the TM-ADC to penetrate into tumors (Schmidt and Wittrup, Mol Cancer Ther 2009; 8:2861-2871). Because of these two competing pharmacokinetic effects, it is desirable to use a PEG that is large enough to reduce TM-ADC clearance and thus increase plasma exposure, but not so large that it significantly reduces the diffusivity of the TM-ADC to such an extent that it interferes with its ability to reach the intended target cell population (see, e.g., Examples 1, 18, and 21 of US 2016 / 0310612, which are incorporated herein by reference, particularly for methods for selecting the optimal PEG size of the drug-linker moiety).

[0121] In one group of embodiments, the PEG unit comprises one or more linear polyethylene glycol chains, each having at least 2 subunits, at least 3 subunits, at least 4 subunits, at least 5 subunits, at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, at least 21 subunits, at least 22 subunits, at least 23 subunits, or at least 24 subunits. In preferred embodiments, the PEG unit comprises a total of at least 6 subunits, at least 8 subunits, at least 10 subunits, or at least 12 subunits. In some such embodiments, the PEG unit comprises a total of about 72 or less subunits, preferably a total of about 36 or less subunits. In some embodiments, the PEG unit comprises from about 2 to about 12 subunits.

[0122] In another group of embodiments, the PEG units may be a total of 4-72, 4-60, 4-48, 4-36, or 4-24 subunits, 5-72, 5-60, 5-48, 5-36, or 5-24 subunits, 6-72, 6-60, 6-48, 6-36, or 6-24 subunits, 7-72, 7-60, 7-48, 7-36, or 7-24 subunits, 8-72, 8-60, 8-48, 8-36, or 8-24 subunits, 9-72, 9-60, 9-48, 9-36, or 9-24 subunits. 8, 9-36, or 9-24 subunits; 10-72, 10-60, 10-48, 10-36, or 10-24 subunits; 11-72, 11-60, 11-48, 11-36, or 11-24 subunits; 12-72, 12-60, 12-48, 12-36, or 12-24 subunits; 13-72, 13-60, 13-48, 13-36, or 13-24 subunits; 14-72, 14-60, 14-48, 14-36, or 14-24 subunits, 15-72, 15-60, 15-48, 15-36, or 15-24 subunits, 16-72, 16-60, 16-48, 16-36, or 16-24 subunits, 17-72, 17-60, 17-48, 17-36, or 17-24 subunits, 18-72, 18-60, 18-48, 18-36, or 18-24 subunits, 19-72, 19-60, 19-48, 19-36, or 19-24 subunits. 20-72, 20-60, 20-48, 20-36, or 20-24 subunits; 21-72, 21-60, 21-48, 21-36, or 21-24 subunits; 22-72, 22-60, 22-48, 22-36, or 22-24 subunits; 23-72, 23-60, 23-48, 23-36, or 23-24 subunits; or 24-72, 24-60, 24-48, 24-36, or 24 subunits.

[0123] Exemplary linear PEG units that may be used in any of the embodiments provided herein are as follows: TIFF2024178320000055.tif45128, where the wavy lines indicate binding sites to a multiplexer linked assembly, and each n is independently selected from 4 to 72, 6 to 72, 8 to 72, 10 to 72, 12 to 72, 6 to 24, or 8 to 24. In some embodiments, the subscript b is about 8, about 12, or about 24.

[0124] As described herein, the PEG units are selected to improve the clearance of the resulting TM-ADC, but not significantly affect the ability of the conjugate to penetrate into the tumor. In embodiments in which the drug moieties and multiplexer-linked assembly units of the TM-ADC have hydrophobicity comparable to the maleimide-derived glucuronide MMAE drug moieties, the PEG units selected for use will preferably have between 8 subunits and about 24 subunits, more preferably about 12 subunits. In embodiments in which the drug moieties and multiplexer-linked assembly units of the TM-ADC have hydrophobicity greater than the maleimide-derived glucuronide MMAE drug moieties, PEG units with more subunits may be required.

[0125] In preferred embodiments of the present disclosure, the PEG unit is from about 300 daltons to about 5 kilodaltons; from about 300 daltons to about 4 kilodaltons; from about 300 daltons to about 3 kilodaltons; from about 300 daltons to about 2 kilodaltons; from about 300 daltons to about 1 kilodalton; or any value therebetween. In some such aspects, the PEG unit has at least 8, 10, or 12 subunits. In some such aspects, the PEG unit has at least 8, 10, or 12 subunits, but no more than 72 subunits, preferably no more than 36 subunits.

[0126] In preferred embodiments of the disclosure, besides the PEG units, there are no other PEG subunits in the multiplexer-linked assembly (i.e., there are no PEG subunits in any of the other components of the conjugates and linkers provided herein). In other aspects of the invention, besides the PEG units, there are no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 other polyethylene glycol subunits in the multiplexer-linked assembly (i.e., no more than 8, 7, 6, 5, 4, 3, 2, or 1 other polyethylene glycol subunits in the other components of the TM-ADCs provided herein).

[0127] When referring to polyethylene glycol subunits of a PEG unit, for example, when referring to a population of TM-ADC or its intermediate compounds and / or the use of polydisperse PEG, it will be understood that the number of subunits may represent an average number, depending on the context.

[0128] Further Multiplexer Linkage Assembly Aspects In addition to the multiplexer linked assembly (MLA) embodiments described above (Formulas I, Ib, and Ic), the present disclosure provides further MLA embodiments as described below. MC1 , T MC2 ), linking group (A 1 and A 2 ), a splitting group (Y), and a drug moiety (D M ) have the same meaning as set forth in the preceding paragraph.

[0129] Multiplexer (M) group The multiplexer (M) groups in the MLA compounds and TM-ADCs described herein serve as branching moieties (or trifunctional linking groups). The first multiplexer (M) group is a linking group (A 1 ) and two (A 2 -T MC2 ) group. 1 ) and two (A2 -T MC2 Covalent attachment to the multiplexer (M) group is accomplished with one to three functional groups. For example, in some embodiments, the multiplexer (M) group is coupled to a linking group (A 1 ) and two (A 2 -T MC2 Alternatively, in some embodiments, the multiplexer (M) group is comprised of a single functional group, such as a single tertiary amine, that provides a covalent bond to the linking group (A 1 ) and two (A 2 -T MC2 For example, in some embodiments, a thiol, hydroxyl, amine, or other nucleophilic group is linked to the linking group (A 1 ), while providing a covalent bond to (A 2 -T MC2 Covalent bonds to either or both of the C) groups are provided by thiol, hydroxy, amine, or another nucleophilic group. In embodiments where the multiplexer (M) group is comprised of two or more functional groups, the two or more functional groups may be branched or unbranched C 1~8 The linkage is by any suitable group, such as an alkylene moiety.

[0130] In some embodiments, the multiplexer (M) group has the formula: TIFF2024178320000056.tif13128, where the wavy line on the right represents (A 2 -T MC2 ) part, and the wavy line on the left is A 1 It is based on

[0131] In some embodiments, the multiplexer (M) group has the formula: TIFF2024178320000057.tif14128, where the wavy line on the right represents (A 2 -T MC2 ) part, and the wavy line on the left is A 1 It is based on

[0132] In some embodiments, the multiplexer (M) group has the formula: TIFF2024178320000058.tif25128, where the wavy line on the right represents (A 2 -T MC2 ) part, and the wavy line on the left is A 1 It is based on

[0133] In some embodiments, the multiplexer (M) group has the formula: TIFF2024178320000059.tif24128, where the wavy line on the right represents (A 2 -T MC2 ) part, and the wavy line on the left is A 1 It is based on

[0134] In some embodiments, the multiplexer (M) group has the formula: TIFF2024178320000060.tif24128, where the wavy line on the right represents (A 2 -T MC2 ) part, and the wavy line on the left is A 1 It is based on

[0135] From the above description, the thiol multiplexer (T MC It is also clear that the ) group is a type of multiplexer (M).

[0136] Although all of the functional groups of the multiplexer (M) group described above are nucleophilic; 1 or (A 2 -T MC2 It will be understood that the selection of nucleophilic or electrophilic groups for covalent attachment to A may vary without departing from the scope of the present disclosure. 1 or (A 2 -T MC2 It is clear that the nature of the bond between the aryl group and the multiplexer (M) group will depend on the chemical identity of the functional group that provides the covalent bond in the aryl group to the multiplexer (M) group.

[0137] The above description is for the linking group (A1 ) and two (A 2 -T MC2 Although the focus is on the multiplexer (M) group providing the covalent bond to the aryl) group, the multiplexer (M) group can be at any suitable branch position.

[0138] Antibody (Ab) The antibody useful in the TM-ADC described herein is essentially any antibody or fragment thereof that targets an antigen associated with a clinically relevant disease state.This includes antibody fragments as well as antibodies that have four available interchain disulfide bonds, or eight thiols that are generated by reducing these interchain disulfide bonds.The antibody of the present disclosure can be an unengineered antibody, i.e., an antibody that has not been modified to introduce additional amino acids or peptides, or an engineered antibody, i.e., an antibody that has one or more engineered cysteine ​​residues incorporated into the antibody or fragment thereof.

[0139] In some embodiments, the antibody of the present disclosure comprises one or more engineered cysteine ​​(eCys) residues. An eCys residue is a cysteine ​​amino acid or derivative thereof that is incorporated into the heavy or light chain of an antibody, typically one or more eCys residues are incorporated into the antibody by mutagenizing a parent antibody. Further information can be found in U.S. Patent No. 9,000,130, the contents of which are incorporated herein by reference for all purposes. In some embodiments, derivatives of cysteine ​​(Cys) include, but are not limited to, β-2-Cys, β-3-Cys, homocysteine, and N-methylcysteine.

[0140] In some embodiments, the antibody (Ab) is an intact antibody or a fully reduced antibody. The term "fully reduced" refers to an antibody in which all four interchain disulfide linkages have been reduced to linking group A 1 The term refers to an antibody that provides eight thiols to which a carboxyl group can be attached.

[0141] In some embodiments, the antibody (Ab) is an intact antibody or a fully reduced antibody, or an antibody with an engineered cysteine ​​group that has been modified with a functional group that can participate in, e.g., click chemistry or other cycloaddition reactions, for attachment of the MLA components described herein.

[0142] In one group of embodiments, the thiol multiple antibody drug conjugate (TM-ADC) compound has the formula I Ab : Represented by TIFF2024178320000061.tif44128, During the ceremony, Ab is from antibody; S* is a sulfur atom from a reduced interchain disulfide bond or an antibody sulfur atom from an engineered cysteine ​​unit, or S* represents a modified functional group introduced onto the antibody or onto a moiety covalently attached to the antibody resulting from click chemistry, Diels-Alder chemistry, or other cycloaddition reaction; A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each optionally includes a splitting group (Y) covalently bonded to the first linking group or the second linking group or is a divalent linking moiety of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and; Each D M is a drug moiety; and The subscript p is an integer from 1 to 10.

[0143] In some embodiments provided herein, the thiol multimeric antibody drug conjugate compound has formula I Ab A: Represented by TIFF2024178320000062.tif33128, During the ceremony, Ab is from antibody; S* is a sulfur atom from a reduced interchain disulfide bond or from an engineered cysteine ​​unit of the antibody, or S* represents a modifying functional group introduced onto the antibody or onto a moiety covalently attached to the antibody resulting from click chemistry, Diels-Alder chemistry, or other cycloaddition reaction; A 1 is a first linking group optionally including a splitting group (Y) covalently bonded to the first linking group or is a divalent linking moiety of the first linking group; T MC1 is a thiol multiplexing group; Each S is a thiol multiplex group (T MC1 ) is a sulfur atom from; Each D M is a drug moiety; and The subscript p is an integer from 1 to 10.

[0144] In one group of embodiments, the thiol multiple antibody drug conjugate (TM-ADC) compound has the formula II: Ab : Represented by TIFF2024178320000063.tif46128, During the ceremony, Ab is from antibody; S* is a sulfur atom from a reduced interchain disulfide bond or from an engineered cysteine ​​unit of the antibody, or S* represents a modifying functional group introduced onto the antibody or onto a moiety covalently attached to the antibody resulting from click chemistry, Diels-Alder chemistry, or other cycloaddition reaction; A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each optionally includes a splitting group (Y) covalently bonded to the first linking group or the second linking group or is a divalent linking moiety of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and; Each T MC2 is a second thiol multiplexing group in reduced form; Each D M is the reduced form of T MC2 a drug moiety attached to a sulfur atom from the The subscript p is an integer from 1 to 10.

[0145] In one group of embodiments, the thiol multi-antibody drug conjugate (TM-ADC) has the formula I Ab b: Represented by TIFF2024178320000064.tif53128, During the ceremony, Ab is from antibody; S* is a sulfur atom from a reduced interchain disulfide bond or from an engineered cysteine ​​unit of the antibody, or S* represents a modifying functional group introduced onto the antibody or onto a moiety covalently attached to the antibody resulting from click chemistry, Diels-Alder chemistry, or other cycloaddition reaction; A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each contains zero or one dividing group (Y) covalently bonded to the first linking group or the second linking group, or a divalent linking component of the linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and; Each T MC2 is independently a second thiol multiplexing group; Each S is a second thiol multiplexing group (T MC2 ) sulfur atom; and Each D M is the drug moiety.

[0146] In one group of embodiments, the antibody for any TM-ADC described herein is directed to a cancer cell antigen. In another group of embodiments, the antibody is directed to a bacterial associated antigen. In yet another group of embodiments, the antibody is directed to an autoimmune cell antigen. It will be understood that the antibody component in a TM-ADC is an antibody in the residual form such that the Ab in the TM-ADC structure described herein incorporates the structure of an antibody.

[0147] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies against a particular antigenic determinant (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against an antigen of interest can be prepared using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture.

[0148] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. Antibodies include full-length antibodies and antigen-binding fragments thereof. Human monoclonal antibodies may be produced by any of a number of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982, Meth. Enzymol. 92:3-16).

[0149] In some aspects, the term "antibody" further includes functionally active fragments, derivatives, or analogs of antibodies that immunospecifically bind to target cells (e.g., cancer cell antigens, viral antigens, or microbial antigens), or other antibodies that bind to tumor cells or matrices. In this context, "functionally active" means that the fragment, derivative, or analog can immunospecifically bind to a target cell. To determine which CDR sequences bind to an antigen, synthetic peptides containing the CDR sequences are typically used with the antigen in a binding assay by any binding assay method known in the art (e.g., BIAcore assay) (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md; Kabat E et al., 1980, J. Immunology 125(3):961-969).

[0150] In addition, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which typically contain both human and non-human portions, obtained using standard recombinant DNA techniques, are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those with a variable region derived from a mouse monoclonal and a human immunoglobulin constant region. (See, e.g., U.S. Pat. Nos. 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules derived from non-human species, which have one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be prepared by recombinant DNA techniques known in the art, e.g., International Publication No. WO 87 / 02671; European Patent Publication No. 0 184 187; European Patent Publication No. 0 171 496; European Patent Publication No. 0 173 494; International Publication No. WO 86 / 01533; U.S. Pat. No. 4,816,567; European Patent Publication No. 012 023; Berter 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. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559;Morrison, 1985, Science 229:1202-1207;Oi et al., 1986, BioTechniques 4:214;US Patent No. 5,225,539;Jones et al., 1986, Nature 321:552-525;Verhoeyan et al., 1988, Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060, each of which is incorporated herein by reference in its entirety.

[0151] Fully human antibodies are particularly desirable in some embodiments, and are typically produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes.

[0152] Antibodies immunospecific for cancer cell antigens are commercially available or produced by any method known to those of skill in the art, such as, for example, chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens are available, for example, from the GenBank database or a similar database, literature publications, or by routine cloning and sequencing.

[0153] In certain embodiments, known antibodies for cancer treatment are used.Antibodies that are immunospecific for cancer cell antigens are commercially available or produced by any method known to those skilled in the art, such as recombinant expression technology.Antibodies that are immunospecific for cancer cell antigens can be obtained from, for example, GenBank database or similar databases, literature publications, or by routine cloning and sequencing.

[0154] In another specific embodiment, antibodies for the treatment of autoimmune diseases are used in accordance with the compositions and methods of the present invention. If antibodies immunospecific for antigens of cells responsible for the production of autoimmune antibodies are not commercially available or otherwise available, they can be obtained by any method known to those of skill in the art, such as, for example, chemical synthesis or recombinant expression techniques.

[0155] In certain embodiments, useful antibodies are directed against receptors or receptor complexes expressed on activated lymphocytes. The receptors or receptor complexes may include immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins, or complement control proteins.

[0156] An exemplary linkage to an antibody is via a thioether linkage.

[0157] Cancer Cell Antigens Examples of antibodies that can be used for cancer treatment and intracellular antibodies that bind to tumor-associated antigens are reviewed in Franke, AE, Sievers, EL, and Scheinberg, DA, "Cell surface receptor-targeted therapy of acute myeloid leukemia: a review" Cancer Biother Radiopharm. 2000,15, 459-76; Murray, JL, "Monoclonal antibody treatment of solid tumors: a coming of age" Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998.

[0158] Selected aspects of the linked assembly unit and TM-ADC In some embodiments, the A of the MLA represented by formula I, II, III 1 contains a maleimide group.

[0159] MLA is represented by formulas I and II, 1 is the self-stabilizing part.

[0160] In some selected embodiments, the MLA compounds of formula (I) and (II) are 1 but, TIFF2024178320000065.tif34128, wherein R is selected from the group consisting of H and an amine protecting group; Y is a splitting group; and the wavy line represents T MC1 The bond to is shown.

[0161] In some selected embodiments, the MLA compound of formula (I) is MC1 is in disulfide form, and TIFF2024178320000066.tif17128.

[0162] In some selected embodiments, the MLA compound of formula (I) is a compound in which the subscript m is 0 and M is T MC1 and T MC1 but, TIFF2024178320000067.tif97128.

[0163] In some selected embodiments, the MLA compound of formula (I) is a compound in which the subscript m is 0 and T MC1 but, TIFF2024178320000068.tif127149.

[0164] In some selected embodiments, the MLA compound of formula (II) is a compound in which the subscript m is 1 and T MC1 but, TIFF2024178320000069.tif22137; and MC2 is in disulfide form, and TIFF2024178320000070.tif16128.

[0165] In some selected embodiments, the MLA compound of formula (II) is MC1 and T MC2 Each of them is independent. TIFF2024178320000071.tif23142; and each T MC2 is also a compound that is linked to two drug moieties.

[0166] In some selected embodiments, the MLA compound of formula (II) is 2 -T MC2 ) are respectively, TIFF2024178320000072.tif54128, wherein the wavy line to the left of the succinimide ring is T MC1 The wavy lines to the right of the sulfur atom represent the thioether bond to the drug moiety (D M ) indicates a covalent bond to

[0167] In some embodiments, the MLA compound of formula (I) or (II) has a drug moiety attached thereto, each of which is mc-VC-PAB-D U , mc-D U , mc-VC-D U ,MDpr-D U , MDpr-Lys(PEG)-D U , and D. U wherein mc is a maleimide-derived succinimide moiety, optionally in hydrolyzed form, and each D U is a drug unit and incorporates the structure of a free drug selected from the group consisting of nucleoside chemotherapeutic agents; HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs (e.g., SN-38 glucuronide, etoposide phosphate); low molecular weight drugs (e.g., drugs having a molecular weight less than about 600); nitrogen mustards (e.g., melphalan); and proteosome inhibitors (lenolidomide). In some embodiments, D U incorporates the structure of a free drug selected from the group consisting of nucleoside chemotherapeutic agents and antimetabolites such as antifolates (e.g., methotrexate, pemetrexed, L-leucovorin, GW1843, Raltitrexed, ZD9331, pralatrexate, lometrexol).

[0168] In some embodiments, the MLA compound of formula (I) is wherein each R is H or an amine protecting group; each D Mis a drug moiety; and Y is a splitting group. In some embodiments, the splitting group (Y) is a PEG unit.

[0169] In some selected embodiments, the MLA compound of formula (I-1a) or (I-2a) is D M But mc-VC-PAB-D U , mc-D U , mc-VC-D U ,MDpr-D U , MDpr-Lys(PEG)-D U , and D. U where mc is a maleimide-derived succinimide moiety, optionally in hydrolyzed form, and each D U is a drug unit and incorporates the structure of a free drug selected from the group consisting of nucleoside chemotherapeutic agents; HDAC inhibitors; anthracyclines; NAMPT inhibitors; hydrophilic prodrugs (e.g., SN-38 glucuronide, etoposide phosphate); low molecular weight drugs (e.g., drugs having a molecular weight less than about 600); nitrogen mustards (e.g., melphalan); and proteosome inhibitors (e.g., lenolidomide). In some embodiments, D U incorporates the structure of a free drug selected from the group consisting of nucleoside chemotherapeutic agents and antimetabolites such as antifolates (e.g., methotrexate, pemetrexed, L-leucovorin, GW1843, Raltitrexid, ZD9331, pralatrexate, lometrexol).

[0170] In some selected embodiments, each drug linker (D L ) is the MDPr-vc linker.

[0171] In some selected embodiments, in the MLA compounds represented by formulas I, II, and III, each drug linker (D L) are independently selected from the group consisting of maleimido-caproyl (mc), maleimido-caproyl-valine-citrulline (mc-vc), and maleimido-caproyl-valine-citrulline-paraaminobenzyloxycarbonyl (mc-vc-PABC).

[0172] For ease of reference to the compounds and assemblies described herein, the component mc-vc-PAB-D, where mc is a maleimide-derived succinimide moiety, U The structure: has TIFF2024178320000074.tif41128, Component mc-VA-PAB-D, where mc is a maleimide-derived succinimide moiety U The structure: has TIFF2024178320000075.tif22128, Component mc-VA-D, where mc is a maleimide-derived succinimide moiety U The structure: I have TIFF2024178320000076.tif19128, and MDpr is a maleimide-derived succinimide moiety, U The structure: has TIFF2024178320000077.tif54128, mc-VC-PAB-D, where mc and MDPr are maleimide-derived succinimide moieties. U , mc-VA-PAB-D U , mc-VA-D U , and MDpr-PAB(gluc)-D U An exemplary -D coupled to a multiplexer link assembly unit M The wavy line represents the thiol multiplexer (T MC 3 shows the covalent attachment of the succinimide ring of mc or MDpr to the thiol present on the aryl) group.

[0173] In any one of said maleimide-derived succinimide moieties, the succinimide ring is optionally in hydrolyzed form, and for the MDpr-derived succinimide moiety, the succinimide ring is preferably in hydrolyzed form.

[0174] In some embodiments, the MLA compound of formula (I) or (II) is 1 -T MC1 but It is a compound containing TIFF2024178320000078.tif72165.

[0175] In some embodiments, the MLA compound of formula (I) or (II) is 1 -T MC1 but It is a compound containing TIFF2024178320000079.tif31128.

[0176] In some embodiments, the MLA compound of formula (I) or (II) is 1 -T MC1 but It is a compound containing TIFF2024178320000080.tif47150.

[0177] In some embodiments, the MLA compound of formula (I) or (II) is 1 -T MC1 but It is a compound containing TIFF2024178320000081.tif47150.

[0178] In some embodiments, the MLA compound of formula (I) or (II) is 1 -T MC1 but It is a compound containing TIFF2024178320000082.tif21128.

[0179] In some embodiments, the MLA compound of formula (I) or (II) is 1 -T MC1 but It is a compound containing TIFF2024178320000083.tif54128.

[0180] In some embodiments, the MLA compound of formula (I) or (II) is 1 -T MC1 but It is a compound containing TIFF2024178320000084.tif46128.

[0181] Usage Cancer Treatment TM-ADCs are useful for inhibiting tumor or cancer cell proliferation, inducing apoptosis in tumor or cancer cells, or treating cancer in patients. Thus, TM-ADCs can be used in a variety of settings for the treatment of cancer. TM-ADCs can be used to deliver drugs to tumor or cancer cells. Without being bound by theory, in one embodiment, the antibody of the TM-ADC binds to or associates with a cancer cell or tumor cell-associated antigen, and the TM-ADC can be taken up (internalized) into the tumor or cancer cell through receptor-mediated endocytosis or other internalization mechanisms. The antigen can be an extracellular matrix protein that can bind to the tumor or cancer cell or associate with the tumor or cancer cell. Once inside the cell, the drug is released within the cell via a cleavable mechanism. In another embodiment, the drug or drug unit is cleaved from the TM-ADC outside the tumor or cancer cell, and the drug or drug unit subsequently penetrates the cell.

[0182] In one embodiment, the antibody binds to a tumor cell or cancer cell.

[0183] In another embodiment, the antibody binds to a tumor cell or cancer cell antigen that is on the surface of the tumor cell or cancer cell.

[0184] In another embodiment, the antibody binds to a tumor cell or cancer cell antigen that is an extracellular matrix protein associated with tumor cells or cancer cells.

[0185] The specificity of antibodies to specific tumor cells or cancer cells can be important in determining which tumor or cancer is most effectively treated.For example, TM-ADCs that target cancer cell antigens present on hematopoietic cancer cells treat blood cancers in some embodiments.In other embodiments, TM-ADCs that target cancer cell antigens present on abnormal cells of solid tumors treat such solid tumors.

[0186] In other embodiments, the TM-ADCs are targeted to abnormal cells of hematopoietic cancers, such as lymphomas (Hodgkin's and non-Hodgkin's lymphomas) and leukemias, as well as solid tumors.

[0187] Multidisciplinary cancer therapy Cancer, including but not limited to, tumors, metastatic cancers, or other diseases or disorders characterized by abnormal cells characterized by uncontrolled cell growth, is treated or inhibited in some embodiments by administration of a TM-ADC.

[0188] In other embodiments, methods are provided for treating cancer, comprising administering to a patient in need thereof an effective amount of a TM-ADC and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is one with which the cancer has not proven refractory to treatment. In another embodiment, the chemotherapeutic agent is one with which the cancer has proven refractory to treatment. In some of these embodiments, the TM-ADC is administered to a patient who has also undergone surgery as a treatment for the cancer.

[0189] In some embodiments, the patient also receives additional treatment, such as radiation therapy. In certain embodiments, the TM-ADC is administered simultaneously with the chemotherapeutic agent or radiation therapy. In another particular embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of the TM-ADC.

[0190] In other embodiments, the chemotherapy agents are administered over a series of sessions. Any one or combination of chemotherapy agents, such as standard of care chemotherapy agents, can be administered.

[0191] In addition, methods of treating cancer using TM-ADCs as an alternative to chemotherapy or radiation therapy are provided where chemotherapy or radiation therapy has proven or may prove too toxic, e.g., produces unacceptable or intolerable side effects for the subject being treated. In some embodiments, patients being treated with TM-ADCs are also treated with another cancer treatment, such as surgery, radiation therapy, or chemotherapy, depending on which treatment proves to be acceptable or tolerable.

[0192] Treatment of Autoimmune Disease TM-ADC is useful for killing cells that cause autoimmune disease or inhibiting the replication of cells that cause autoimmune disease, or for treating autoimmune disease. Thus, in some embodiments, TM-ADC is used in various situations to treat autoimmune disease in patients. In other embodiments, TM-ADC is used to deliver drugs to target cells. Without being bound by theory, in one of these embodiments, TM-ADC compounds associate with antigens on the surface of target cells, and then TM-ADC compounds are taken up into target cells through receptor-mediated endocytosis. Once inside the cells, the linker unit is cleaved, causing the release of the drug or drug unit. The released drug is then free to move in the cytosol and induce cytotoxic or cytostatic activity. In another embodiment, the drug is cleaved from TM-ADC outside the target cells, and the drug or drug unit subsequently penetrates the cells.

[0193] In one embodiment, the antibody binds to an autoimmune antigen. In one embodiment, the antigen is on the surface of a cell involved in an autoimmune condition.

[0194] In another embodiment, the antibody binds to an autoimmune antigen on the surface of a cell.

[0195] In one embodiment, the antibody binds to activated lymphocytes associated with an autoimmune disease state.

[0196] In further embodiments, the TM-ADC kills or inhibits the proliferation of cells that produce autoimmune antibodies associated with certain autoimmune diseases.

[0197] Specific types of autoimmune diseases that may be treated with TM-ADC include, but are not limited to, Th2 lymphocyte-associated disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft-versus-host disease); Th1 lymphocyte-associated disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis); and activated B lymphocyte-associated disorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes).

[0198] Multidrug therapy for autoimmune diseases Also disclosed are methods for treating an autoimmune disease comprising administering to a patient in need thereof an effective amount of a TM-ADC and another therapeutic agent known for treating the autoimmune disease.

[0199] Compositions and Methods of Administration The present invention provides a pharmaceutical composition comprising the TM-ADC described herein and a pharma- ceutically acceptable carrier. The TM-ADC is in any form that allows it to be administered to a patient for the treatment of a disorder associated with the expression of the antigen to which the antibody binds. For example, the TM-ADC is in liquid or solid form. The preferred route of administration is parenteral. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. In one embodiment, the composition is administered parenterally. In one of these embodiments, the conjugate is administered intravenously. Administration is typically by any convenient route, for example, by infusion or bolus injection.

[0200] The TM-ADC pharmaceutical compositions are formulated to be bioavailable following administration of the composition to a patient, and are in the form of one or more unit dosage forms for injection.

[0201] The materials used in the preparation of pharmaceutical compositions can be non-toxic in the amount used.The optimal dose of active ingredient in pharmaceutical compositions will depend on various factors, which will be clear to those skilled in the art.Relevant factors include, but are not limited to, the type of animal (e.g., human), the specific form of the compound, the mode of administration, and the composition used.

[0202] TM-ADC composition is typically in the form of liquid, suspension, or lyophilized solid.Liquid composition or suspension is useful for delivery by injection, and lyophilized solid is suitable for reconstitution as liquid or suspension with suitable diluent for injection.In the composition administered by injection, one or more surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents are typically included.

[0203] In some embodiments, liquid compositions, whether they are in solution, suspension, or other similar form, may also contain one or more of the following: water for injection, saline, preferably saline, Ringer's solution, sterile diluent such as isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that can act as solvents or suspending media, polyethylene glycol, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, acetates, citrates, or phosphates; surfactants such as non-ionic surfactants, polyols; and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral compositions are typically enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other materials. Saline is an exemplary auxiliary. Injectable compositions are preferably sterile liquid compositions.

[0204] The amount of TM-ADC that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition, and is usually determined by standard clinical techniques.In addition, in vitro or in vivo assays can be used to help identify optimal dosage ranges.The exact dosage used in the composition also depends on the parenteral administration route and the severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances.

[0205] The compositions contain an effective amount of TM-ADC such that a suitable dosage will be obtained, typically at least about 0.01% TM-ADC by weight of the composition.

[0206] In general, the dose of TM-ADC administered to a patient is typically about 0.01 mg to about 100 mg, about 1 to about 100 mg, or about 0.1 to about 25 mg per kg of the subject's body weight. In some embodiments, the dose administered to a patient is about 0.01 mg to about 15 mg per kg of the subject's body weight. In some embodiments, the dose administered to a patient is about 0.1 mg to about 15 mg per kg of the subject's body weight. In some embodiments, the dose administered to a patient is about 0.1 mg to about 20 mg per kg of the subject's body weight. In some embodiments, the dose administered is about 0.1 mg to about 5 mg or about 0.1 mg to about 10 mg per kg of the subject's body weight. In some embodiments, the dose administered is about 1 mg to about 15 mg per kg of the subject's body weight. In some embodiments, the dose administered is about 1 mg to about 10 mg per kg of the subject's body weight. In some embodiments, the dosage administered is preferably about 0.1-4 mg / kg, even more preferably 0.1-3.2 mg / kg, or even more preferably 0.1-2.7 mg / kg of subject body weight during a treatment cycle.

[0207] The term "carrier" refers to a diluent, adjuvant or excipient with which the compound is administered. Such pharmaceutical carriers are liquid. Water is an exemplary carrier when the compound is administered intravenously. Saline and aqueous dextrose and glycerol solutions are also useful as liquid carriers for injections. Suitable pharmaceutical carriers also include glycerol, propylene, glycol or ethanol. The composition will also contain minor amounts of wetting or emulsifying agents, and / or pH buffering agents, if necessary, in some embodiments.

[0208] In some embodiments, the conjugate is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly humans. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer solution. If necessary, the composition in some embodiments also includes a dividing group. The composition for intravenous administration may also include a local anesthetic, such as lignocaine, to ease pain at the site of injection. Generally, the ingredients are supplied separately or mixed in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the TM-ADC is administered by infusion, it may be dispensed, for example, using an infusion bottle containing sterile pharmaceutical grade water or saline. When the conjugate is administered by injection, an ampoule of sterile water for injection or saline is typically provided so that the ingredients can be mixed prior to administration.

[0209] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration. EXAMPLES

[0210] Unless otherwise noted, all solvents and reagents were purchased from commercial sources at the highest purity possible and were not further purified prior to use. Anhydrous solvents, including dimethylformamide (DMF) and CH2Cl2, were purchased from Aldrich.

[0211] Products were purified by flash column chromatography using a Biotage Isolera One flash purification system (Charlotte, NC). Preparative HPLC was performed on a Waters 2454 Binary Gradient Module solvent delivery system configured with a Waters 2998 PDA detector. Products were purified on a Phenomenex Max-RP 4 μm Synergi 80 Å 250 mm reversed-phase column of appropriate diameter, eluting with 0.05% trifluoroacetic acid in water and 0.05% trifluoroacetic acid in acetonitrile unless otherwise noted. UPLC-MS was performed on a Waters single quadrupole detector mass spectrometer interfaced with a Waters Acquity UPLC system using one of the following methods:

[0212] BEH C18 General method: Acquity UPLC BEH C18 2.1 x 50 mm, 1.7 μm reversed phase column; Solvent A - 0.1% formic acid; Solvent B - acetonitrile with 0.1% formic acid. TIFF2024178320000085.tif56166

[0213] BEH C18 Hydrophobic method: Acquity UPLC BEH C18 2.1 x 50 mm, 1.7 μm reversed phase column; Solvent A - 0.1% formic acid; Solvent B - acetonitrile with 0.1% formic acid TIFF2024178320000086.tif56166

[0214] CORTECS C18 General Law : Waters CORTECS C18 1.6 μm, 2.1 × 50 mm, reversed phase column; Solvent A - 0.1% formic acid in water, Solvent B - acetonitrile containing 0.1% formic acid TIFF2024178320000087.tif55166

[0215] CORTECS C18 Hydrophobic Method: Waters CORTECS C18 1.6 μm, 2.1 × 50 mm, reversed-phase column; Solvent A - 0.1% formic acid in water; Solvent B - acetonitrile with 0.1% formic acid TIFF2024178320000088.tif48166

[0216] CORTECS C18 Hydrophilic Method: Waters CORTECS C18 1.6 μm, 2.1 × 50 mm, reversed-phase column; Solvent A - 0.1% formic acid in water; Solvent B - acetonitrile with 0.1% formic acid TIFF2024178320000089.tif48166

[0217] Example 1 TIFF2024178320000090.tif40128 tert-Butyl ((S)-3-(((S)-1,2-dithian-4-yl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxopropyl)carbamate: Iodine (10 mg / mL) was added to a methanolic solution of dithiobutylamine HCl salt (2, 25 mg, 0.144 mmol) until the purple color of the solution persisted, indicating complete oxidation of the disulfide. After several hours, the mixture was concentrated. The residue was dissolved in DMF (0.5 mL), Boc-DPR-OSu (3, 55 mg, 0.144 mmol) was added, and the mixture was treated with DIPEA (100 μL). The reaction mixture was stirred for 1 h and concentrated. Ethyl acetate was added and the insoluble material was removed by filtration. The resulting solution was chromatographed on a 1 mm radial chromatography plate eluting with 5-10% methanol / dichloromethane. The product-containing fractions were further purified on a 1 mm radial chromatography plate eluting with 50% ethyl acetate / hexane followed by 100% ethyl acetate to give 12 mg (21%) of the title compound (4). Analytical UPLC-MS (BEH C18 general method): r =1.85 minutes. MS (ESI+):402.

[0218] TIFF2024178320000091.tif37128(S)-3-Amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1,2-dithian-4-yl)propanamide, trifluoroacetate salt: To a solution of the Boc-protected amine (4, 12 mg) in DCM (4.5 mL) at 0 °C was added trifluoroacetic acid (0.5 mL). The mixture was stirred for 2 h and UPLC-MS analysis showed conversion to the desired amine. The reaction mixture was concentrated under reduced pressure. Anhydrous DCM was added and the mixture was concentrated again to a residue. The material was then dissolved in anhydrous dichloromethane and concentrated under a stream of N2 (2x) followed by high vacuum to give 16.5 mg of a white solid as the trifluoroacetate salt of the title compound (MLA-1 TFA). MLA-1 is an exemplary multiplexer linker assembly compound of formula Ia. Analytical UPLC-MS (BEH C18 general method): r =0.92 minutes. MS (ESI+):302.

[0219] Example 2 TIFF2024178320000092.tif29154((2S)-1-((1,2-dithian-4-yl)amino)-6-amino-1-oxohexan-2-yl)carbamate tert-butyl: 2,5-dioxopyrrolidin-1-yl N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2-(tert-Butoxycarbonyl)-L-lysinate (5, 1.03 g, 1.82 mmol), 1,2-dithiane-4-amine (6, 235 mg, 1.74 mmol), prepared according to the procedure in Lyon, RP et al. Nature Biotechnol. (2014), 32(10) 1059-1065, and DIPEA (0.61 mL, 3.48 mmol) were mixed in anhydrous DMF (4 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. LCMS showed complete conversion of the disulfide after 2 h. The solvent was removed under reduced pressure and the crude product was purified by silica gel chromatography (5% MeOH in DCM) to give the disulfide intermediate ((5S)-6-((1,2-dithian-4-yl)amino)-6-oxohexane-1,5-diyl)dicarbamate (9H-fluoren-9-yl)methyl tert-butyl (7, 652 mg, 1.11 mmol, 64.1%). LCMS:t r = 2.32 min; m / z = 608.05 [M+Na] + ). The disulfide intermediate was then redissolved in 30% diethylamine / DCM (4 mL) and stirred at room temperature for an additional 2 h. After 2 h, the solvent was removed under reduced pressure and the crude product was redissolved in DMSO / water and purified by preparative HPLC to give the title compound as a white solid (8, 462 mg, 1.00 mmol, 89.9%). Analytical UPLC-MS (BEH C18 general method): r = 1.39 min; m / z = 386.07 [M+Na] + .

[0220] Example 3 TIFF2024178320000093.tif67137N-((5R)-6-((1,2-dithian-4-yl)amino)-5-amino-6-oxohexyl)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatetra Heptacontane-74-amide 2,2,2-trifluoroacetate: ((2S)-1-((1,2-dithian-4-yl)amino)-6-amino-1-oxohexan-2-yl) tert-butyl carbamate (8, 102.2 mg, 0.281 mmol) was dissolved in anhydrous DMF (3 mL) containing DIPEA (98 uL, 0.562 mmol). 24 -OSu (9, 375.5 mg, 0.309 mmol) was then added as a white solid. The reaction mixture was stirred at room temperature for 3 h. After 3 h, the solvent was removed and the crude product was purified by silica gel chromatography (5% MeOH in DCM) to give the disulfide intermediate compound (10, 295.2 mg, 0.202 mmol, 71.8%). Analytical UPLC-MS (BEH C18 general method): r =1.81 min;m / z=1463.42 [M+H] + Compound 10 (108 mg, 0.074 mmol) was then redissolved in 10% TFA / DCM and stirred at room temperature for 30 min. After 30 min, the solvent was removed and the title compound (11) as crude product was used directly in the next step without purification. Analytical UPLC-MS (BEH C18 general method): r =1.33min;m / z=1363.28 [M+H] + .

[0221] Example 4 TIFF2024178320000094.tif86145N-((5S)-6-((1,2-dithian-4-yl)amino)-5-((S)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6-oxohexyl)-2,5,8,11,14,17,20,23, 26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-Tetracosaoxatetraheptacontane-74-amide: Amine TFA salt (11, 109 mg, 0.074 mmol) was dissolved in anhydrous DMF (1 mL) followed by the addition of DIPEA (38.6 μL, 0.221 mmol). Then 2,5-dioxopyrrolidin-1-yl (S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (12, 25.3 mg, 0.066 mmol) in anhydrous DMF (0.1 mL) was added. The reaction mixture was stirred at room temperature for 2 h. After 2 h, the reaction mixture was acidified with HOAc (40 μL), diluted with DMSO / water, and purified by preparative HPLC to give the maleimide intermediate (13, 101.1 mg, 0.062 mmol, 84.0%). Analytical UPLC-MS (general method): r =1.73min;m / z=1628.95 [M+H] + Compound 13 (101.1 mg, 0.062 mmol) was redissolved in 10% TFA in DCM and stirred at room temperature for 90 min. After 90 min, the solvent was removed and the crude product was dissolved in DMSO / water and purified by preparative HPLC to give the title compound as a colorless liquid (14, 79.1 mg, 0.048 mmol, 77.6%). Analytical UPLC-MS (BEH C18 general method): r =1.36min;m / z=1529.40 [M+H] + .

[0222] Example 5 TIFF2024178320000095.tif68138 tert-Butyl bis(2-(1,2,5-dithiazepane-5-carboxamido)ethyl)carbamate: tert-Butyl bis(2-aminoethyl)carbamate (15, 220 mg, 1.08 mmol) and di(1H-1,2,4-triazol-1-yl)methanone (16, 1.07 g, 6.49 mmol) were dissolved in DCM (10 mL) followed by the addition of triethylamine (0.30 mL, 2.16 mmol). The reaction mixture was stirred at room temperature for 30 min. After 30 min, LCMS showed complete conversion of the diamine. The solvent was removed and the crude product was purified by silica gel chromatography (0-5% MeOH in DCM) to give tert-butyl bis(2-(1H-1,2,4-triazole-1-carboxamido)ethyl)carbamate (17, 379.0 mg, 0.963 mmol, 89.0%) as a pale yellow solid. Analytical UPLC-MS (BEH C18 general method): r =1.31 min;m / z=394.22 [M+H] + Compound 17 (201 mg, 0.511 mmol) was redissolved in DMF (3 mL) followed by the addition of trimethylamine (0.21 mL, 1.53 mmol) and 1,2,5-dithiazepane (141.6 mg, 1.05 mmol). The reaction mixture was heated at 45° C. for 5 h. After 5 h, the reaction mixture was cooled to room temperature. The solvent was removed under reduced pressure and the crude product was purified by silica gel chromatography (0-7% MeOH in DCM) to give the title bis-disulfide compound as a pale yellow solid (19, 233.3 mg, 0.444 mmol, 86.8%). Analytical UPLC-MS (BEH C18 general method): r =1.91 min; m / z=525.97 [M+H] + .

[0223] Example 6 TIFF2024178320000096.tif20141N,N'-(azanediylbis(ethane-2,1-diyl))bis(1,2,5-dithiazepane-5-carboxamide) 2,2,2-trifluoroacetate: tert-Butyl bis(2-(1,2,5-dithiazepane-5-carboxamide)ethyl)carbamate (19, 135.2 mg, 0.257 mmol) was dissolved in 20% TFA / DCM (3 mL) and the reaction mixture was stirred at room temperature for 1 h. After 1 h, the solvent was removed under reduced pressure and the title compound (20) was used directly as crude in the next step. Analytical UPLC-MS (BEH C18 general method):t r =1.17min;m / z=426.04 [M+H] + .

[0224] Example 7 TIFF2024178320000097.tif68164DBCO-PEG5-acid (21, 20.8 mg, 0.035 mmol, Broadpharm) was dissolved in anhydrous DMF (200 μL) followed by the addition of HATU (13.9 mg, 0.036 mmol) and DIPEA (0.018 mL, 0.1 mmol). After stirring for 5 min at room temperature, the disulfide amine salt (20, 17.9 mg, 0.033 mmol) in 100 μL DMF was then added. The reaction mixture was stirred at room temperature for 30 min. After 30 min, the crude product was diluted with DMSO / water and purified by preparative HPLC to give the DBCO bis-disulfide compound as a white solid (22, 22.7 mg, 0.023 mmol, 68.1%). Analytical UPLC-MS (BEH C8 General Method):t r =2.00min;m / z=1004.18 [M+H] + .

[0225] Example 8 TIFF2024178320000098.tif85158TIFF2024178320000099.tif102140DBCO-bis-disulfide compound (22, 50 mM in DMA, 20 μL, 1.0 μmol) was dissolved in 20 μL of 1:1 DMA / water, followed by the addition of DTPA (500 mM, 1.5 μL), pH 8.0 Tris buffer (1 M, 6 μL) and TCEP (23, 105 mM, 21 μL). The reaction mixture was warmed at 37° C. for 2 hours. After 2 hours, LCMS showed that all disulfide bonds in compound 22 had been reduced. The multiplexer linker assembly compound of Example 1 (MLA-1, 200 mM in DMA, 35 μL, 7 μmol) was then added and the reaction temperature was maintained at 37° C. for an additional 30 minutes. After 30 min, LCMS showed complete conversion of the starting material. The crude reaction mixture was diluted with DMSO and purified by preparative HPLC to give the DABCO-tetrakis disulfide compound as a white solid (24, 1.18 mg, 0.442 umol, 44.2%). Analytical UPLC-MS (BEH C18 hydrophobic method): r =1.00min;m / z=1107.51 [1 / 2M+H] + Compound 24 is an exemplary MLA compound of formula II.

[0226] Example 9 TIFF2024178320000100.tif48128 Azido-amine (25, 2.26 mg, 10.3 uM, Broadpharm) was dissolved in DMA (0.25 mL) and added to another vial containing maleimide-OSu (26, 2.5 mg, 9.39 uM). The reaction mixture was stirred at room temperature for 30 min. After 30 min, LCMS indicated that compound 26 had been consumed and the desired azidomaleimide compound (27) had been produced. The solution (37.6 mM based on maleimide) was used directly without further purification for reaction with cysteine ​​thiols of antibodies or antigen-binding fragments of antibodies as described in Example 11. Analytical UPLC-MS (BEH C18 General Method): r =1.26min;m / z=370.16 [M+H] + .

[0227] The azide functional group presented by the antibody can be reacted with the strained alkyne of the bisdisulfide compound (22) of Example 8 or the tetrakisdisulfide compound (24) of Example 9 through click chemistry (see, for example, Chio, TI and Bane, SL in Antibody Dr Conjugates: Methods and Protocols; Method in Molecular Biology, Tumey, LN (ed.), 2002, vol. 2078, Chap. 6, Springer Nature). When using a fully reduced antibody and the MLA compound of Example 8, a total of 8 x 2 cyclic disulfides are presented by the antibody, which can provide a total of 32 thiol functional groups for drug moiety attachment. When using the MLA compound of Example 9, 8 x 4 cyclic disulfides are presented by the antibody, providing a total of 64 thiol functional groups for drug moiety attachment.

[0228] TIFF2024178320000101.tif43164 Example 10 (S)-N,N'-(((2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)butane-1,4-diyl)bis(sulfanediyl))bis(methylene))diacetamide. To a vial was added (S)-2-aminobutane-1,4-dithiol hydrochloride (28, 200 mg, 1.15 mmol) and N-(hydroxymethyl)acetamide (29, 308 mg, 3.45 mmol) and suspended in water (0.6 mL). The suspension was cooled in an ice / water bath and hydrochloric acid (11.7 M, 0.2 mL, 2.34 mmol) was added dropwise. The reaction mixture was allowed to warm slowly to room temperature. After stirring overnight, the reaction mixture was concentrated at 45° C. to give the intermediate (S)-N,N′-(((2-aminobutane-1,4-diyl)bis(sulfanediyl))bis-(methylene))-diacetamide hydrochloride (30) as a clear semi-solid, which was used without further purification. Analytical UPLC-MS (BEH C18 general method): r = 0.57 min, m / z (ES+) calculated 280.1 (M+H) +, found 280.0. Compound 30 (232 mg, 0.73 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (26, 391 mg, 1.47 mmol) were mixed in a vial and dissolved in DMF (2.5 mL), and DIPEA (0.51 mL, 2.94 mmol) was added dropwise. After stirring at room temperature for 2 h, the reaction was quenched with acetic acid (0.25 mL), diluted with methanol, purified by preparative HPLC, and lyophilized to give the title compound (31, 42 mg, 13.3%). Analytical UPLC-MS (general method): t r =0.89 min, m / z (ES+) calculated 431.1 (M+H) + Found 431.1; Calculated 453.1 (M+Na) + , actual value 453.0.

[0229] Example 11 General preparation and analytical characterization of antibody multiplexer drug conjugates Preparation: The following is a compound of formula I prepared from the multiplexer linker assembly compound (MLA-1) of Example 1: Ab The present invention provides a method for preparing an antibody-multiplexer drug conjugate of the present invention.

[0230] Following the procedure of US 2003 / 00883263, humanized unconjugated control IGg1 antibody was treated with an excess of reducing agent to completely reduce its interchain disulfide bonds, which results in eight available cysteine ​​residues for Michael addition to the maleimide moieties of MLA-1. Briefly, unconjugated antibody (5-10 mg / mL) in phosphate-buffered saline containing 1 mM ethylenediaminetetraacetic acid (EDTA) was treated with 10 equivalents of tris(2-carboxyethyl)phosphine (TCEP) neutralized to pH 7.4 with dipotassium hydrogen phosphate and incubated at 37 °C for 45 min. Separation of low molecular weight components was achieved by size-exclusion chromatography on a Sephadex G25 column.

[0231] The fully reduced antibody was then treated with excess MLA-1, similar to the procedure in US 2005 / 0238649. Briefly, MLA-1 in DMSO was added to the fully reduced unbound antibody in PBS, along with EDTA and excess DMSO, resulting in a total reaction co-solvent of 15% v / v. After 30 minutes at ambient temperature, excess n-acetylcysteine ​​was added to the mixture to quench any unreacted maleimide groups. The reaction mixture was purified by desalting into PBS buffer using Sephadex G25 resin. By fully reducing the interchain disulfides of a human IgG1 antibody and reacting the maleimides to obtain cysteines, each light chain of the antibody has a single maleimide modification and each heavy chain contains three maleimide modifications, where the maleimide moieties of MLA-1 have been converted to thio-substituted succinimide moieties. As more generally described by WO 2013 / 173337, the presence of -CHNH substitution in MLA-1 results in a succinimide ring having the formula I Ab In the conjugate, spontaneous hydrolysis occurs before and / or after reduction of the disulfides of the MLA units to provide the ring-open form.

[0232] Analytical characterization: For simplicity, Figure 1 shows the modification of one cysteine ​​residue of the light chain of an antibody with MLA-1. The m / z of the light chain before reaction with MLA-1 is 23152 AMU, which is expected to increase to 23476 AMU immediately after reaction with MLA-1 (see panel A). The cyclic disulfide bonds of each of the eight MLA units in the Ab-MLA intermediate are then reduced with TCEP to present a total of 16 reactive thiol functional groups. The thiols are then reacted with N-ethylmaleimide (NEM), which serves as a dummy drug moiety precursor, in the same manner as previously described for the reaction of antibody cysteine ​​thiols with MLA-1. During that time, the succinimide ring will undergo spontaneous hydrolysis. However, the Na thiol shown in Figure 1 +It is possible that hydrolysis occurred prior to the NEM reaction, as the adduct was indistinguishable from the ring-open form of the initially generated succinimide Ab-MLA adduct due to insufficient mass spectrometer resolution. The resulting light chain conjugate, in which two dummy NEM drug moieties are covalently attached to a single cysteine ​​thiol through the intermediate of one multiplexer-linked assembly unit, is expected to exhibit m / z=23723 AMU (see panel B). The structure characterized by panel B corresponds to Formula I Ab It is similar to TM-ADC.

[0233] Example 12 Preparation and analytical characterization of auristatin thiol multiplexer antibody drug conjugates preparation : Auristatin thiol multiplex antibody drug conjugate (cAC10-MLA1-D M1 ) replaced the non-binding control antibody with the CD30-binding chimeric antibody cAC10 and the NEM “dummy” drug moiety precursor with PEGylated auristatin drug moiety (D M1 ) precursor. The PEGylated auristatin drug moiety precursor was prepared according to the procedures in WO 2015 / 057699 and WO 2016 / 149535.

[0234] Another auristatin thiol multiplex antibody-drug conjugate (cAC10-MLA1-D M2 ) was prepared according to the generalized procedure of Example 11, using cAC10 and The hydrophilic auristatin drug moiety precursor (D) was used to prepare the compound having the structure TIFF2024178320000103.tif43132. M2 ) was prepared according to the procedure of WO 2015 / 123679.

[0235] Characterization: Size exclusion chromatography (SEC) for the MDL-1-based 16-loaded auristatin ADC of Example 11 shows a low percentage of high molecular weight species (cAC10-MLA1-D M1 and cAC10-MLA1-D M2 2%).

[0236] PLRP chromatography (Figure 2, panel A): L-MLA1-D M1 (2) t r =1.29 minutes;H-MLA1-D M1 (6) t r= 1.97 min. Each light and heavy chain contained 1 and 3 PEGylated MLA1-D M1 Thus, each light chain binds a total of two auristatin drug units and each heavy chain is conjugated to a total of six PEGylated auristatin drug units. The mass spectrometry analysis of the cAC10 light chain conjugated to one molecule of MLA-1 is shown in Figure 2, panel B. PEGylated MLA1-D M1 The mass spectrometry analysis of the cAC10 light chain, bound to one molecule of PEGylated MLA1-D and thus having conjugation to two auristatin drug units, is shown in Figure 2, panel C. M1 Mass spectrometry of the cAC10 heavy chain conjugated to three molecules of , and thus having conjugation to six auristatin drug units, is shown in Figure 2, panel D. The multiple peaks in panel D are due to the G0, G1, and G2 oligosaccharide forms of the heavy chain.

[0237] PLRP chromatography (Figure 3, panel A): L-MLA1-D M2 (2) t r =0.33 minutes;H-MLA1-D M2 (6) t r =1.00. Hydrophilic MLA1-D M2 The mass spectrometry analysis of the cAC10 light chain, bound to one molecule of MLA1-D and thus having conjugation to two auristatin drug units, is shown in Figure 2, panel B. M2Mass spectrometry of the cAC10 heavy chain conjugated to three molecules of , and thus having conjugation to six auristatin drug units, is shown in Figure 2, panel C. The multiple peaks in panel C are due to the G0, G1, and G2 oligosaccharide forms of the heavy chain.

[0238] Example 13 Preparation of gemcitabine drug moiety TIFF2024178320000104.tif32128 (9H-fluoren-9-yl)methyl (1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate: Gemcitabine (32, 782.6 mg, 2.973 mmol) was dissolved in anhydrous pyridine (10 mL). TMSCl (1.89 mL, 14.9 mmol) was added to the vigorously stirred reaction mixture over 5 min. The reaction mixture was stirred for 15 min and a white precipitate formed. Fmoc-Cl (961.5 mg, 3.717 mmol) was added in one portion to the reaction mixture. The reaction mixture turned yellow and then colorless over 30 min and the white precipitate remained over the course of the reaction. H2O (2 mL) was added and the reaction mixture was stirred for 2 h to hydrolyze the TMS group and excess chloroformate. The reaction mixture was diluted with EtOAc (100 mL), washed with 1M HCl (3 x 100 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The crude title compound was purified by flash chromatography 50-100% EtOAc in 100G KP-Sil Hex. R f (33) = 0.15 (1:2 Hex:EtOAc). Fractions containing the desired product were concentrated under reduced pressure to give the title compound as a white solid (33, 1.169 g, 2.407 mmol, 80.9%). r =1.71 min (CORTECS C18 general method); MS (m / z) [M + H] + C 24 H 22 F2N3O6 calculated value 486.45, measured value 486.12.

[0239] TIFF2024178320000105.tif66168 (2R,3S,4R,5R,6R)-2-(2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-acetamido)-4-((((((2R,3R,5R)-5-(4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)(2-(methylsulfonyl)ethyl)-carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate. The MAC linker intermediate compound (34, 185 mg, 0.206 mmol), prepared similarly to the procedures in WO 2015 / 095755 and Kolakowski, RV et al. Angew. Chem. Int'l Ed. (2016) 55: 7948-7951, was dissolved in DCM (2 mL). Paraformaldehyde (185 mg, 6.18 mmol) was added followed by TMSCl (1 mL). The reaction mixture was stirred for 10 min at which point complete conversion was observed by diluting a 2 uL aliquot into 98 uL MeOH and observing the MeOH adduct by UPLC-MS. The reaction mixture was filtered through a syringe filter, washed with DCM (1 mL) and, after concentration, toluene (2 mL) was added to azeotrope the final mixture. The eluent was concentrated under reduced pressure to give a colorless solid. Prior to use, Fmoc-gemcitabine (33) was azeotroped with toluene and dried under high vacuum. Compound 33 (100 mg, 0.206 mmol) was suspended in anhydrous DCM (2 mL) and DIPEA (71.8 μL, 0.412 mmol) was added. The activated linker was dissolved in anhydrous DCM (2 mL) and added dropwise to the stirring reaction mixture at a rate of 10 mL / h. The reaction mixture was stirred for 45 min, at which point complete conversion was observed. The reaction was quenched with MeOH (0.1 mL), filtered, and the eluent was concentrated under reduced pressure to give the title compound as a colorless solid, which was used in the next step without purification (35, 182 mg, 0.130 mmol, crude, 63%). r =1.56 min (CORTECS C18 hydrophobic method); MS (m / z) [M + H]+ C 67 H 69 F2N6O 23 S calculated value 1395.41, actual value 1395.40.

[0240] TIFF2024178320000106.tif36170 (2R,3R,4R,5S,6R)-6-(4-((((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)-oxy)methyl)-2-(2-(methylamino)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid: Compound 35 (182 mg, 0.130 mmol) was dissolved in THF:MeOH 1:1 (2 mL). The reaction solution was cooled in an ice / water bath and then LiOH (31.2 mg, 1.30 mmol) was added. The resulting reaction mixture was stirred for 30 min, after which HO (1 mL) was added. The reaction mixture was then stirred for 60 min. Complete conversion to the deprotected compound was determined by UPLC-MS. The reaction mixture was quenched with AcOH (30 μL), concentrated under reduced pressure, and purified by preparative HPLC using a 21.2×250 mm Max-RP column eluted with a gradient of 5-35-95% MeCN in HO 0.05% TFA. Fractions containing the desired deprotected compound were concentrated under reduced pressure to give the title compound as a colorless solid (36, 65.1 mg, 0.0803 mmol, 62%). r =0.82 min (CORTECS C18 hydrophilic method); MS (m / z) [M + H] + C 30 H 41 F2N6O 16 S calculated value 811.23, actual value 811.04.

[0241] TIFF2024178320000107.tif57152 (2R,3R,4R,5S,6R)-6-(4-((((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)(2-(methylsulfonyl)ethyl)carbamoyl)oxy)-methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)-phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid: Compound 36 (65.1 mg, 0.0803 mmol) was dissolved in anhydrous DMF (0.5 mL). DIPEA (26.5 μL, 0.160 mmol) was added to the reaction mixture followed by N-succinimidyl 3-maleimidopropionate (26, 23.5 mg, 0.0883 mmol, purchased from TCI America, product number S0427). The reaction mixture was stirred for 15 min, at which point complete conversion was observed by UPLC-MS. The reaction was quenched with AcOH (0.020 mL) and purified by preparative HPLC on a 21.2×250 mm Max-RP, eluting with 5-35-95% MeCN in HO 0.05% TFA. Fractions containing the desired product were lyophilized to give the title compound as a colorless powder (37, 41.2 mg, 0.0428 mmol, 53.3%). r =1.29 min (CORTECS C18 hydrophilic method); MS (m / z) [M + H] + C 37 H 46 F2N7O 19 S calculated value 962.25, actual value 962.06.

[0242] The Ab-MLA intermediate of Example 11, in which MAL compound 37 and the humanized non-binding control IGg1 antibody are replaced with chimeric monoclonal cAC10, are processed in the manner described for the preparation of auristatin TM-ADC to yield the analogous gemcitabine TM-ADC.

[0243] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, those skilled in the art will understand that certain changes and modifications may be practiced which are within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

1. Formula (I): A multiplexer ligated assembly (MLA) compound having: During the ceremony, A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each contains zero or one dividing group (Y) covalently bonded to the first linking group or the second linking group, or a divalent linking moiety of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and Each T MC2 is a second thiol multiplexing group; The subscript m is 0 or 1; When the subscript m is 0, M represents the first thiol multiplexing group in disulfide form (T MC1 ) or two drug moieties (D M ) coupled to a first thiol multiplexing group (T MC1 ) and If the subscript m is 1, then each T MC2 is in disulfide form or has two drug moieties (D M )

2. A 1 but, 2. The MLA compound of claim 1, having a formula selected from the group consisting of: wherein R is selected from the group consisting of H and an amine protecting group; Y is a splitting group; and the wavy line indicates a covalent bond to M.

3. M, T MC1 and 3. The MLA compound of claim 2, selected from the group consisting of:

4. M, T MC1 and and the wavy line adjacent to the sulfur is -A 2 -T MC2 3. The MLA compound of claim 2, exhibiting a covalent attachment site to a group.

5. Each T MC2 but, wherein each T MC2 The wavy lines adjacent to each sulfur in M 5. The MLA compound of claim 4, which exhibits a binding site for

6. Each T MC2 but, 5. The MLA compound of claim 4, selected from the group consisting of:

7. A 1 but, 2. The MLA compound of claim 1, having a formula selected from the group consisting of:

8. M, T MC1 and 8. The MLA compound of claim 7, selected from the group consisting of:

9. The subscript m is 1 and M is T MC1 and and T MC2 but, 8. The MLA compound of claim 1 or 7, selected from the group consisting of:

10. (A 2 -T MC2 ) are respectively, wherein the succinimide ring is in hydrolyzed form and each R is independently selected from the group consisting of H and an amine protecting group; Y is a splitting group; the wavy line to the left of the succinimide ring is T MC1 8. The MLA compound of claim 1 or 7, exhibiting a thioether bond to:

11. The subscript m is 0 and M is T MC1 and 8. The MLA compound of claim 1 or 7, selected from the group consisting of:

12. The subscript m is 0 and M is T MC1 and 8. The MLA compound of claim 1 or 7, selected from the group consisting of:

13. I-1, I-2, I-1a, and I-2a: having a formula selected from the group consisting of: During the ceremony, The subscript w is an integer from 0 to 8; each W is independently a natural or unnatural amino acid; each R is independently H or an amine protecting group; and Y is a splitting group.

2. The MLA compound of claim 1.

14. 14. The MLA compound of claim 13, wherein said resolving group Y comprises a polyethylene glycol group.

15. The MLA compound of claim 1 having a formula selected from:

15. A 1 3. The MLA compound of claim 1 or 2, wherein is selected from the group consisting of a maleimide group and a halomethylcarbonyl group.

16. 2. The MLA compound of claim 1, further comprising a group Y of the splitting group.

17. 18. The MLA compound of claim 17, wherein said resolving group Y comprises a polyethylene glycol group.

19. A 1 but, 3. The MLA compound of claim 1 or 2, having a formula selected from the group consisting of: wherein R is selected from the group consisting of H and an amine protecting group; and Y is a splitting group.

20. The splittable group is PEG 3 , PEG 6 , PEG 12 , and PEG 24 18. The MLA compound of claim 17, wherein the PEG unit is selected from the group consisting of:

21. A 1 2. The MLA compound of claim 1, wherein said MLA compound comprises an azide or alkyne functional group capable of participating in click chemistry to form a 1,2,3-triazole moiety.

22. m is 1 and M has the structure: and each T MC2 but, 2. The MLA compound of claim 1, selected from the group consisting of:

18. A 1 -M- is a group of the formula:

2. The MLA compound of claim 1, having the formula:

19. Each A 2 is a bond and A 1 -M-(T MC2 ) 2 But the formula:

2. The MLA compound of claim 1, having the formula:

20. R is H or an amino protecting group, Y is a splitting group, and R 1 but, A compound represented by the formula (i) or (ii): or a salt thereof.

21. R 1 but, 26. The compound of claim 25, selected from the group consisting of:

22. formula:

26. The compound of claim 25, having the formula:

23. A thiol-multiplexed antibody drug conjugate (TM-ADC) comprising an antibody and 1-10 covalently attached multiplexer-linked assembly (MLA) units, the thiol-multiplexer-linked assembly units each being attached to cysteine ​​thiols of reduced interchain disulfide bonds in an antibody and / or to engineered cysteine ​​residues, or attached to modified functional groups introduced into an antibody, or heterocyclos derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions, and each of the covalently linked multiplexer-linked assembly units has 2 to 4 drug moieties attached thereto, and an optional resolving group (Y).

24. 29. The TM-ADC of claim 28, wherein each of said MLAs has two attached drug moieties.

30. 30. The TM-ADC of claim 29, wherein the drug moiety is derived from a cytotoxic agent.

31. Formula (I Ab ): having During the ceremony, Ab is from antibody; S* is selected from the group consisting of a sulfur atom of a cysteine ​​residue of a reduced interchain disulfide bond, a sulfur atom from an engineered cysteine ​​residue of the antibody, or a modified functional group introduced into the antibody, or is a heterocyclo derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions; A 1 is a first linking group; 1 optionally includes a splitting group (Y) covalently bonded to the first linking group or is a divalent linking moiety of the first linking group; a bond or an independently selected second linking group T MC1 is the first thiol multiplexing group; Here, T MC1 consists of two drug moieties (D M ) and The subscript p is an integer from 1 to 10; The TM-ADC of claim 28.

25. Formula (II Ab ): having During the ceremony, Ab is from antibody; S* is selected from the group consisting of a sulfur atom of a cysteine ​​residue of a reduced interchain disulfide bond, a sulfur atom from an engineered cysteine ​​residue of the antibody, or a modified functional group introduced into the antibody, or is a heterocyclo derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions; A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each optionally includes a splitting group (Y) covalently bonded to the first linking group or the second linking group or is a divalent linking moiety of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and Each T MC2 is a second thiol multiplexing group in reduced form; Each D M is the reduced form of T MC2 a drug moiety attached to the sulfur atom of the group; and The subscript p is an integer from 1 to 10; The TM-ADC of claim 28.

26. Formula (I Ab a): having During the ceremony, Ab is from antibody; S* is selected from the group consisting of a sulfur atom of a cysteine ​​residue of a reduced interchain disulfide bond, a sulfur atom from an engineered cysteine ​​residue of the antibody, or a modified functional group introduced into the antibody, or is a heterocyclo derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions; A 1 is a first linking group that optionally includes a splitting group (Y) covalently bonded to the first linking group or the second linking group, or is a divalent linking moiety of the first linking group or the second linking group; T MC1 is a thiol multiplexing group; Each S is T MC1 a sulfur atom selected from the group consisting of: Each D M is a drug moiety; and The subscript p is an integer from 1 to 10; The TM-ADC of claim 28.

27. Formula (I Ab b): having During the ceremony, Ab is from antibody; S* is selected from the group consisting of a sulfur atom of a cysteine ​​residue of a reduced interchain disulfide bond, a sulfur atom from an engineered cysteine ​​residue of the antibody, or a modified functional group introduced into the antibody, or is a heterocyclo derived from click chemistry of Diels-Alder chemistry or other cycloaddition reactions; A 1 is a first linking group; Each A 2 is independently a bond or an independently selected second linking group; A 1 and A 2 each independently contains zero or one dividing group (Y) covalently bonded to the first linking group or the second linking group, or a divalent linking component of the first linking group or the second linking group; M is a multiplexing group or a first thiol multiplexing group (T MC1 ) and Each T MC2 is independently a second thiol multiplexing group; Each S is a second thiol multiplexing group (T MC2 ) sulfur atoms selected from the group consisting of: Each D M is the drug moiety, The TM-ADC of claim 28.

28. A 1 but, wherein the succinimide ring is optionally in hydrolyzed form; S* is selected from the group consisting of a sulfur atom of a reduced interchain disulfide bond or a sulfur atom from an engineered cysteine ​​unit of the antibody; R is selected from the group consisting of H and an amine protecting group; Y is a splitting group; the wavy line indicates the bond to M; and the dashed line indicates the bond to the antibody.

29. A 1 but, wherein the succinimide ring is optionally in hydrolyzed form; S* is selected from the group consisting of a sulfur atom of a reduced interchain disulfide linkage or a sulfur atom from an engineered cysteine ​​unit of the antibody; R is selected from the group consisting of H and an amine protecting group; Y is a splitting group; the wavy line indicates a covalent bond to M; and the dashed line indicates a covalent bond to the antibody.

30. M is T MC1 And each T MC1 and T MC2 became independent, and each T MC2 Also, two drug moieties (D M 33. The TM-ADC of claim 32, wherein the TM-ADC is linked to

31. D M are selected from the group consisting of mc-VC-PAB-D, mc-D, mc-VC-D, MDPr-D, and MDPr-Lys(PEG)-D; wherein each D is a Drug unit that is derived from a chemotherapeutic nucleoside, antimetabolite, or a hydrophilic or mildly hydrophobic chemotherapeutic agent characterized by a ClogP of 2.5 or less and / or a polar surface area of ​​80 square Angstroms or greater.

32. formula: having During the ceremony Ab is from antibody; each S* is a sulfur atom from the antibody; each W is independently a natural or unnatural amino acid; wherein each W optionally includes a splitting group (Y) covalently attached to W; The subscript w is 0, 1, 2, or 3; D M1 is the first drug moiety; D M2 is a second drug moiety; and The subscript p is an integer from 1 to 10; The TM-ADC of any one of claims 28-33.

40. The TM-ADC of claim 39, wherein the subscript w is 0.

41. The TM-ADC of claim 39, wherein subscript w is 1 and said dividing group (Y) is a PEG unit composed of 8 to 72, 8 to 36, or 8 to 24, or 12 to 24 consecutive polyethylene glycol subunits.

33. W w is a dipeptide residue or a tripeptide residue.

34. W w 43. The TM-ADC of claim 42, wherein each amino acid present in is independently an amino acid residue selected from the group consisting of glycine, alanine, β-alanine, and lysine.

35. D M1 and D. M2 each represents a first drug linker (D L1 ) and a second drug linker (D L2 ), where each D L1 and each D L2 are independently selected from maleimido-caproyl (mc), maleimido-caproyl-valine-citrulline (mc-vc), maleimido-caproyl-valine-citrulline-paraaminobenzyloxycarbonyl (mc-vc-PABC), and maleimidodiaminopropionyl-valine-citrulline (MDPr-vc), wherein either the mc or MDpr moiety has been converted to its corresponding succinimide moiety, optionally in hydrolyzed form.

36. Each D M1 and each D M2 independently incorporates the structure of a free drug, wherein the free drug is selected from a chemotherapeutic nucleoside, antimetabolite, or a hydrophilic or mildly hydrophobic chemotherapeutic agent characterized by a ClogP of 2.5 or less and / or a polar surface area of ​​80 square Angstroms or more.

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