Peptide-containing linkers for antibody-drug conjugates

Abstract

To provide a targeting moiety-drug conjugate that is biodegradable and biocompatible and exhibits high drug load as well as strong binding to a target antigen.SOLUTION: Provided is a conjugate comprising a targeting moiety and one or more linker-drug moieties covalently bonded to the targeting moiety, each linker-drug moiety including a multifunctional linker that connects the targeting moiety to one or more drug units through intermediacy of a releasable assembly unit for each drug unit and connects a hydrophilic group to the drug units of each linker-drug moiety, the releasable assembly units being capable of releasing a free drug in proximity to a target site targeted by the targeting moiety, the multifunctional linker comprising a peptide moiety between the targeting moiety and the hydrophilic group, and the peptide moiety includes at least two amino acids.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims priority and benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos. 62 / 425,895, filed November 23, 2016, and 62 / 572,010, filed October 13, 2017, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] background Traditionally, pharmaceuticals have mainly consisted of small molecules administered orally (as solid pills and liquids) or as injections. Over the past 30 years, formulations (i.e., compositions that control the route and / or rate of drug delivery and enable delivery of therapeutic agents at the required site) have become increasingly popular and complex. Nevertheless, many problems and challenges regarding the development of new treatments and the mechanisms for administering them remain unaddressed. For example, many drugs generally exhibit limited or otherwise reduced efficacy and therapeutic effects, either because they are subject to partial degradation before reaching the desired target in the body, or because they accumulate in tissues other than the target, or both.

[0003] Thus, one goal in the field of drug delivery systems is to deliver intact pharmaceuticals to specifically targeted areas of the body through systems that can utilize either physiological or chemical mechanisms, or both, to stabilize the drug and control the in vivo transport of the therapeutic agent.

[0004] Antibody-drug conjugates have been developed as target-specific therapeutic agents. Antibodies against various cancer cell surface antigens have been shown to bind to microtubules (e.g., maytansinoids, auristatins, and taxanes; see, e.g., U.S. Patent Nos. 5,208,020 (Patent Document 1); 5,416,064 (Patent Document 2); 6,333,410 (Patent Document 3); 6,441,163 (Patent Document 4); 6,340,701 (Patent Document 5); 6,372,738 (Patent Document 6); 6,436,931 (Patent Document 7); 6,596,757 (Patent Document 8); and 7,276,497 (Patent Document 9)). No. 5,585,499; No. 5,846,545; No. 6,534,660; No. 6,756,397; and No. 6,630,579). Antibody conjugates with some of these cytotoxic drugs are being actively investigated in hospitals for cancer therapy (see, for example, Ricart, AD, and Tolcher, AW, 2007, Nature Clinical Practice, 4, 245-255 (Non-Patent Document 1); Krop et al., 2010, J. Clin. Oncol., 28, 2698-2704 (Non-Patent Document 2)). However, existing antibody-drug conjugates have shown several limitations. The main limitations are the limited number of antigens they target and the inability to deliver sufficient drug concentrations to the target site due to the relatively mild cytotoxicity of cancer drugs such as methotrexate, daunorubicin, maytansinoids, taxanes, and vincristine. One approach to achieving significant cytotoxicity is to directly or indirectly link large amounts of drug molecules to antibodies.However, such heavily modified antibodies often exhibit impaired binding to target antigens and / or rapid in vivo clearance from the bloodstream. Thus, there is a need to improve the ability to deliver sufficient concentrations of drugs to a target so that maximal cytotoxicity for the drug is achieved. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 5,208,020 [Patent Document 2] U.S. Patent No. 5,416,064 [Patent Document 3] U.S. Patent No. 6,333,410 [Patent Document 4] U.S. Patent No. 6,441,163 [Patent Document 5] U.S. Patent No. 6,340,701 [Patent Document 6] U.S. Patent No. 6,372,738 [Patent Document 7] U.S. Patent No. 6,436,931 [Patent Document 8] U.S. Patent No. 6,596,757 [Patent Document 9] U.S. Patent No. 7,276,497 [Patent Document 10] U.S. Patent No. 5,475,092 [Patent Document 11] U.S. Patent No. 5,585,499 [Patent Document 12] U.S. Patent No. 5,846,545 [Patent Document 13] U.S. Patent No. 6,534,660 [Patent Document 14] U.S. Patent No. 6,756,397 [Patent Document 15] U.S. Patent No. 6,630,579 [Non-patent literature]

[0006] [Non-Patent Document 1] Ricart, AD, and Tolcher, AW, 2007, Nature Clinical Practice, 4, 245-255 [Non-patent document 2] Krop et al., 2010, J. Clin. Oncol., 28, 2698-2704 Summary of the Invention

[0007] overview The present disclosure features targeting moiety-drug conjugates that are biodegradable, biocompatible, and exhibit high drug loading and strong binding to target antigens. For example, the targeting moiety is a protein-based recognition molecule (PBRM). The present disclosure also features peptide-containing scaffolds useful for conjugating with a PBRM, a drug, or both to obtain targeting moiety-drug conjugates.

[0008] In one aspect, the present disclosure provides a conjugate comprising a targeting moiety and one or more linker-drug moieties covalently attached to the targeting moiety, each linker-drug moiety comprises a multifunctional linker that links a targeting moiety to one or more drug units via a releasable assembly unit for each drug unit, and links a hydrophilic group to the drug unit of each linker-drug moiety; The releasable assembly unit is capable of releasing free drug near the target site targeted by the targeting moiety; and The multifunctional linker comprises a peptide moiety between the targeting moiety and the hydrophilic group, the peptide moiety comprising at least two amino acids. Concerning conjugates.

[0009] In another aspect, the present disclosure provides a conjugate comprising a targeting moiety and one or more linker-drug moieties covalently attached to the targeting moiety, each linker-drug moiety comprises a multifunctional linker that links a targeting moiety to one or more drug units via a releasable assembly unit for each drug unit, and links a polyalcohol or a derivative thereof to the drug unit of each linker-drug moiety; The releasable assembly unit is capable of releasing free drug near the target site targeted by the targeting moiety; Concerning conjugates.

[0010] The present disclosure also relates to a conjugate of formula (I): TIFF2025114641000002.tif43128 formula, a1 is an integer of 0 to 1; a2 is an integer of 1 to 3; a3 is an integer from 0 to 1; a4 is an integer from 1 to about 5; a5 is an integer from 1 to 3; d 13 is an integer from 1 to about 14; PBRM stands for protein-based recognition molecule; L P’ PBRM M P and its corresponding monovalent moiety L P is a functional group W that can form a covalent bond with the functional group of the PBRM. P Contains; M P is the stretcher unit; L M is a bond or a trivalent or tetravalent linker, and L M is a bond, a2 is 1, and L M is a trivalent linker, a2 is 2, or L M is a tetravalent linker, a2 is 3; L 3is a carbonyl-containing moiety; M A comprises a peptide portion containing at least two amino acids; T 1 is a hydrophilic group, and T 1 and M A Between TIFF2025114641000003.tif12128 is T 1 and M A means a direct or indirect connection with; each occurrence of D is independently a therapeutic agent having a molecular weight of ≦about 5 kDa; and L D Each occurrence of independently converts D to M A and contains at least one cleavable bond, such that upon bond rupture D is released in an active form for its intended therapeutic effect.

[0011] In yet another aspect, the present disclosure relates to a peptide-containing scaffold having any of formulas (II)-(IX): TIFF2025114641000004.tif171128TIFF2025114641000005.tif94128In the formula, a1 is an integer of 0 to 1; a2 is an integer of 1 to 3; a3 is an integer from 0 to 1; a4 is an integer from 1 to about 5; a5 is an integer from 1 to 3; d 13 is an integer from 1 to about 14; PBRM stands for protein-based recognition molecule; L P’ PBRM M P and its corresponding monovalent moiety L P is a functional group W that can form a covalent bond with the functional group of the PBRM. P Contains; M P is the stretcher unit; LM is a bond or a trivalent or tetravalent linker, and L M is a bond, a2 is 1, and L M is a trivalent linker, a2 is 2, or L M is a tetravalent linker, a2 is 3; L 3 is a carbonyl-containing moiety; M A comprises a peptide portion containing at least two amino acids; T 1 is a hydrophilic group, and T 1 and M A Between TIFF2025114641000006.tif12128 is T 1 and M A means a direct or indirect connection with; W M Each occurrence of independently may be replaced by forming a hydrogen, a protecting group, a leaving group, or a covalent bond to L M M P is a functional group capable of linking to; W D is independently a functional group capable of forming a covalent bond with a functional group of a therapeutic agent (“D”) having a molecular weight of ≦about 5 kDa; and L D Each occurrence of is independently D Or D to M A and L D contains at least one cleavable bond, and upon bond rupture D is released in an active form for its intended therapeutic effect.

[0012] The conjugates and scaffolds of the present disclosure can include one or more of the following features, where applicable.

[0013] For example, the drug unit and the hydrophilic group are each linked to a multifunctional linker in a side-by-side orientation.

[0014] For example, the targeting moiety is a protein-based recognition molecule (PBRM), for example, the PBRM is an antibody or antibody fragment.

[0015] For example, the peptide portion in the multifunctional linker comprises from 3 to about 16 amino acids, such as about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids.

[0016] For example, the peptide portion in the polyfunctional linker comprises from 3 to about 10 amino acids, such as about 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0017] For example, the peptide portion contains from 3 to about 10 amino acids selected from glycine, serine, glutamic acid, aspartic acid, lysine, cysteine, stereoisomers thereof (e.g., isoglutamic acid or isoaspartic acid), and combinations thereof.

[0018] For example, the peptide moiety contains at least four glycines and at least one serine.

[0019] For example, the peptide moiety contains at least four glycines, at least one serine, and at least one glutamic acid or isoglutamic acid.

[0020] For example, the hydrophilic group includes a polyalcohol or a derivative thereof, a polyether or a derivative thereof, or a combination thereof.

[0021] For example, the hydrophilic group comprises an aminopolyalcohol, such as glucamine or bis-glucamine.

[0022] For example, the hydrophilic group is Includes TIFF2025114641000007.tif16128.

[0023] For example, the hydrophilic group is Includes TIFF2025114641000008.tif25128.

[0024] For example, aminopolyalcohols include TIFF2025114641000009.tif6128, where n1 is an integer from 0 to about 6; Each R 58 are independently hydrogen or C 1~8 is alkyl; R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 -, where R 59 is H, alkyl, cycloalkyl, or arylalkyl; R 61 is CH2OR 62 , COOR 62 , -(CH2) n2 COOR 62 or heterocycloalkyl substituted with one or more hydroxyl; R 62 is H or C 1~8 is alkyl; and n2 is an integer of 1 to about 5.

[0025] For example, the hydrophilic group is TIFF2025114641000010.tif16128, where: n4 is an integer from 1 to about 25; Each R 63 are independently hydrogen or C 1~8 is alkyl; R 64 is a bond or C 1~8 is an alkyl linker; R 65 is H, C 1~8 Alkyl or -(CH2) n2 COOR 62 and; R 62 is H or C 1~8 is alkyl; and n2 is an integer of 1 to about 5.

[0026] For example, the hydrophilic group comprises polyethylene glycol, eg, polyethylene glycol having about 6 to about 24 PEG subunits, preferably about 6 to about 12 PEG subunits or about 8 to about 12 PEG subunits.

[0027] For example, L 3 If present, -XC 1~10 alkylene-C(O)-, and X is L M wherein X is CH, O, or NR, and R is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, COOH, or COO-C 1~6 It is alkyl.

[0028] For example, L 3 If present, -NR5-(CH2) v -C(O)- or -CH2-(CH2) v -C(O)-NR5-(CH2) v -C(O)-, where each v is independently an integer of 1 to 10 (e.g., each v is independently an integer of 1 to 6 or 2 to 4, or 2). For example, L 3 is —NH—(CH2)2—C(O)— or —(CH2)2—C(O)—NH—(CH2)2—C(O)—.

[0029] For example, a4 can be 1, 2, or 3.

[0030] For example, d 13 is an integer from 1 to about 10, for example, d 13 is 4 or 5.

[0031] For example, each W P is, if present, independently TIFF2025114641000011.tif148156TIFF2025114641000012.tif197170TIFF2025114641000013.tif40146, where Ring A is cycloalkyl or heterocycloalkyl; Ring B is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 1K is a leaving group; R 1A is a sulfur protecting group; R 1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; R 2J is hydrogen, an aliphatic, aryl, heteroaliphatic, or carbocyclic moiety; R 3J is C 1~6 alkyl, and each of Z1, Z2, Z3, and Z7 is independently a carbon or nitrogen atom; R 4j are hydrogen, halogen, OR, -NO2, -CN, -S(O)2R, C 1~24 Alkyl (e.g., C 1~6 alkyl), or 6- to 24-membered aryl or heteroaryl, where C 1~24 Alkyl (e.g., C 1~6 alkyl), or 6- to 24-membered aryl or heteroaryl, optionally substituted with one or more aryl or heteroaryl; or two R 4j together form a fused cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R is hydrogen, alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl; R 5j is C(R 4j )2, O, S, or NR; and z1 is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0032] For example, R 1Kis halo or RC(O)O—, where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety. For example, R 1A teeth, TIFF2025114641000014.tif23128, where r is 1 or 2, and R s1 , R s2 , and R s3 Each of is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0033] For example, ring A is TIFF2025114641000015.tif33128; where R 6j is hydrogen, halogen, C 1~24 Alkyl (e.g., C 1~6 alkyl), or 6- to 24-membered aryl or heteroaryl, where C 1~24 Alkyl (e.g., C 1~6 alkyl), or 6- to 24-membered aryl or heteroaryl may be substituted with one or more aryls or heteroaryls.

[0034] For example, ring A is It could be TIFF2025114641000016.tif29128.

[0035] For example, M P If present, -(Z4)-[(Z5)-(Z6)] z - and Z4 is L P’ or L P The Z6 is connected to the L M where: z is 1, 2, or 3; The Z4 is TIFF2025114641000017.tif112147, where * is L P’ or L P and ** means connection to Z5 or Z6, if present, or to L if both Z5 and Z6 are absent. Mmeans connection to; b1 is an integer of 0 to 6; e1 is an integer from 0 to 8, R 17 is C 1~10 Alkylene, C 1~10 Heteroalkylene, C 3~8 Cycloalkylene, O-(C 1~8 Alkylene, arylene, -C 1~10 Alkylene-arylene-, -arylene-C 1~10 Alkylene-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-, -(C 3~8 Cycloalkylene-C 1~10 Alkylene-, 4- to 14-membered heterocycloalkylene, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-, -C 1~10 Alkylene-C(=O)-, -C 1~10 Heteroalkylene-C(=O)-, -C 3~8 Cycloalkylene -C(=O)-, -O-(C 1~8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1~10 Alkylene-arylene-C(=O)-, -arylene-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-C(=O)-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-C(=O)-, -4 to 14-membered heterocycloalkylene-C(=O)-, -C 1~10 Alkylene-(4-14 membered heterocycloalkylene)-C(=O)-, -(4-14 membered heterocycloalkylene)-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-NH-, -C 1~10 Heteroalkylene-NH-, -C 3~8 Cycloalkylene-NH-, -O-(C 1~8 alkyl)-NH-, -arylene-NH-, -C 1~10Alkylene-arylene-NH-, -arylene-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-NH-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-NH-, -4 to 14-membered heterocycloalkylene-NH-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-NH-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-S-, -C 1~10 Heteroalkylene -S-, -C 3~8 Cycloalkylene -S-, -OC 1~8 Alkyl)-S-, -arylene-S-, -C 1~10 Alkylene-arylene-S-, -arylene-C 1~10 Alkylene-S-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-S-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-S-, -4 to 14-membered heterocycloalkylene-S-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-S-, or -(4- to 14-membered heterocycloalkylene)-C1-C 10 alkylene-S-; Each Z5 is independently absent, R 57 -R 17 or a polyether unit; Each R 57 are independently bonded, NR 23 , S, or O; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; and Each Z6 may independently be absent, -C 1~10 Alkyl-R3-, -C 1~10 Alkyl-NR5-, -C1~10 Alkyl-C(O)-, -C 1~10 Alkyl-O-, -C 1~10 Alkyl-S-, or -(C 1~10 alkyl-R3) g1 -C 1~10 alkyl-C(O)-; each R3 is independently -C(O)-NR5- or -NR5-C(O)-; Each R5 is independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, COOH, or COO-C 1~6 is alkyl; and g1 is an integer of 1 to 4.

[0036] For example, M P If exists, TIFF2025114641000018.tif143150, where * is L P’ or L P and ** means connection to L M means connection to; R3 is -C(O)-NR5 or -NR5-C(O)-; R4 is a bond or -NR5-(CR 20 R 21 )-C(O)-; R5 is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; R 17 is C 1~10 Alkylene, C 1~10 Heteroalkylene, C 3~8 Cycloalkylene, O-(C 1~8 Alkylene, arylene, -C 1~10 Alkylene-arylene-, -arylene-C 1~10 Alkylene-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-, -(C 3~8Cycloalkylene-C 1~10 Alkylene-, 4- to 14-membered heterocycloalkylene, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-, -C 1~10 Alkylene-C(=O)-, -C 1~10 Heteroalkylene-C(=O)-, -C 3~8 Cycloalkylene -C(=O)-, -O-(C 1~8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1~10 Alkylene-arylene-C(=O)-, -arylene-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-C(=O)-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-C(=O)-, -4 to 14-membered heterocycloalkylene-C(=O)-, -C 1~10 Alkylene-(4-14 membered heterocycloalkylene)-C(=O)-, -(4-14 membered heterocycloalkylene)-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-NH-, -C 1~10 Heteroalkylene-NH-, -C 3~8 Cycloalkylene-NH-, -O-(C 1~8 alkyl)-NH-, -arylene-NH-, -C 1~10 Alkylene-arylene-NH-, -arylene-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-NH-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-NH-, -4 to 14-membered heterocycloalkylene-NH-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-NH-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-S-, -C 1~10Heteroalkylene -S-, -C 3~8 Cycloalkylene -S-, -OC 1~8 Alkyl)-S-, -arylene-S-, -C 1~10 Alkylene-arylene-S-, -arylene-C 1~10 Alkylene-S-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-S-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-S-, -4 to 14-membered heterocycloalkylene-S-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-S-, or -(4- to 14-membered heterocycloalkylene)-C1-C 10 alkylene-S-; Each R 20 and R 21 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; each b1 is independently an integer from 0 to 6; e1 is an integer from 0 to 8, each f1 is independently an integer from 1 to 6; and g2 is an integer of 1 to 4.

[0037] For example, M P If exists, TIFF2025114641000019.tif104141, where * is L P’or L P and ** means connection to L M means connection to

[0038] For example, L M is a bond and a2 is 1.

[0039] For example, a2 is 2 and L M teeth, TIFF2025114641000020.tif39128TIFF2025114641000021.tif189128, where TIFF2025114641000022.tif12128, if it exists, is P Connection to or M P If there is no L P or L P’ means connection to; Y1, if present, is L 3 Connection to or L 3 If there is no M A means connection to; R and R′ are each independently hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, optionally substituted C 3~19 Branched alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10 Aryl, optionally substituted heteroaryl, optionally substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, optionally substituted C 2~6 Alkanoyl, optionally substituted C 2~6 Alkanoyloxy, optionally substituted C 2~6Substituted alkanoyloxy, -COOH, or -COO-C 1~6 is alkyl; each of c1, c2, c3, c4, c5, c7, and c8 is independently an integer ranging from 0 to 10; and Each of d1, d2, d3, d4, d5, and d7 is independently an integer in the range of 0 to 10.

[0040] For example, a2 is 2 and L M teeth The file is TIFF2025114641000023.tif37128.

[0041] For example, a2 is 3 and L M teeth TIFF2025114641000024.tif99134TIFF2025114641000025.tif210140TIFF2025114641000026.tif143137; where: TIFF2025114641000027.tif12128, if it exists, is P Connection to or M P If there is no L P or L P’ means connection to; Y1, if present, is L 3 Connection to or L 3 If there is no M A means connection to; R and R′ are each independently hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, optionally substituted C 3~19 Branched alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10 Aryl, optionally substituted heteroaryl, optionally substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, optionally substituted C 2~6 Alkanoyl, optionally substituted C 2~6 Alkanoyloxy, optionally substituted C 2~6 Substituted alkanoyloxy, -COOH, or -COO-C 1~6 is alkyl; each of c1, c2, c3, c4, c5, c6, c7, and c8 is independently an integer ranging from 0 to 10; each of d1, d2, d3, d4, d5, d6, d7, and d8 is independently an integer ranging from 0 to 10; and Each of e1, e2, e3, e4, e5, e6, e7, and e8 is independently an integer in the range of 0 to 10.

[0042] For example, a2 is 3 and L M teeth, TIFF2025114641000028.tif37128.

[0043] For example, M A comprises a peptide portion containing at least about 5 amino acids. For example, M A includes a peptide portion containing at most about 16 amino acids, e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids. A comprises a peptide portion containing at most about 10 amino acids, for example, about 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0044] For example, M A comprises a peptide moiety containing 3 to about 10 amino acids selected from glycine, serine, glutamic acid, aspartic acid, lysine, cysteine, stereoisomers thereof (e.g., isoglutamic acid or isoaspartic acid), and combinations thereof.

[0045] For example, M A comprises a peptide portion containing at least four glycines and at least one serine.

[0046] For example, M A comprises a peptide portion containing at least four glycines and at least one glutamic acid.

[0047] For example, M A comprises a peptide portion containing at least four glycines, at least one serine, and at least one glutamic acid.

[0048] For example, the ratio of D to PBRM or the ratio of drug unit to targeting moiety can be greater than 1:1 and up to 50:1, e.g., 2:1 to 40:1; 5:1 to 20:1; 10:1 to 50:1, 25:1 to 50:1, or 30:1 to 50:1. Examples of PBRM include, but are not limited to, full-length antibodies such as IgG and IgM, antibody fragments such as Fab, scFv, camelid, and Fab2, small proteins, and peptides.

[0049] For example, the ratio of D to PBRM or the ratio of drug units to targeting moieties is about 50:1, 40:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0050] For example, the ratio of D to PBRM or the ratio of drug units to targeting moieties can be about 25:1, 20:1, 15:1, 10:1, 5:1, or 2:1.

[0051] For example, the conjugates disclosed herein are used for the manufacture of a medicament useful for treating or lessening the severity of such disorders characterized by abnormal cell growth (eg, cancer).

[0052] For example, the drug unit or D is delivered locally to a specific target cell, tissue, or organ.

[0053] The present disclosure also provides compositions comprising the conjugates, methods for their preparation, and methods for their use in the treatment of various disorders, including, but not limited to, cancer.

[0054] In one aspect, the present disclosure further relates to a pharmaceutical composition comprising a scaffold or conjugate described herein and a pharmaceutically acceptable carrier.

[0055] In another aspect, the present disclosure relates to a method of treating a disorder in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate disclosed herein.

[0056] In yet another aspect, the present disclosure relates to a method of diagnosing a disorder in a subject suspected of having the disorder, the method comprising administering to the subject an effective amount of a conjugate described herein, or performing an assay to detect the target antigen / receptor in a sample from the subject, and determining whether the subject expresses the target antigen or receptor.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, the singular also includes the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0058] [The present invention 1001] A conjugate comprising a targeting moiety and one or more linker-drug moieties covalently attached to the targeting moiety, each linker-drug moiety comprises a multifunctional linker that links a targeting moiety to one or more drug units via a releasable assembly unit for each drug unit, and links a hydrophilic group to the drug unit of each linker-drug moiety, the releasable assembly unit being capable of releasing free drug near a target site targeted by the targeting moiety; and The multifunctional linker comprises a peptide moiety between the targeting moiety and the hydrophilic group, the peptide moiety comprising at least two amino acids. Conjugates. [The present invention 1002] 1001. A conjugate of the present invention, wherein the targeting moiety is a protein based recognition molecule (PBRM). [The present invention 1003] Any of the preceding conjugates of the invention, wherein the PBRM is an antibody or antibody fragment. [The present invention 1004] Any of the conjugates of the present invention, wherein the peptide portion comprises 3 to about 10 amino acids. [The present invention 1005] Any of the preceding conjugates of the invention, wherein the peptide moiety comprises at least 4 amino acids or at least 5 amino acids. [The present invention 1006] Any of the aforementioned conjugates of the invention, wherein the hydrophilic group comprises a polyalcohol or a derivative thereof, a polyether or a derivative thereof, or a combination thereof. [The present invention 1007] Any of the aforementioned conjugates of the invention, wherein the hydrophilic group comprises an aminopolyalcohol. [The present invention 1008] The aminopolyalcohol TIFF2025114641000029.tif6128, where n1 is an integer from 0 to about 6; Each R 58 are independently hydrogen or C 1~8 is alkyl; R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 -, where R 59 is H, alkyl, cycloalkyl, or arylalkyl; R 61 is CH2OR 62 , COOR 62 , -(CH2) n2 COOR 62 or heterocycloalkyl substituted with one or more hydroxyl groups; and R 62 is H or C 1~8 is alkyl; and n2 is an integer from 1 to about 5; Any of the conjugates of the present invention. [The present invention 1009] Any of the conjugates of the invention described above, wherein the hydrophilic group comprises glucamine. [The present invention 1010] The hydrophilic group is Any of the conjugates of the present invention, comprising TIFF2025114641000030.tif25128. [The present invention 1011] The hydrophilic group is TIFF2025114641000031.tif16128, where: n4 is an integer from 1 to about 25; Each R 63 are independently hydrogen or C 1~8 is alkyl; R 64 is a bond or C 1~8 is an alkyl linker; R 65 is H, C 1~8 Alkyl, or -(CH2) n2 COOR 62 and; R 62is H or C 1~8 is alkyl; and n2 is an integer from 1 to about 5; Any of the conjugates of the present invention. [The present invention 1012] Any of the aforementioned conjugates of the invention, wherein the hydrophilic group comprises polyethylene glycol. [The present invention 1013] Any of the conjugates of the present invention described above, wherein the hydrophilic group comprises polyethylene glycol having about 6 to about 24 PEG subunits, preferably about 6 to about 12 PEG subunits or about 8 to about 12 PEG subunits. [The present invention 1014] A conjugate comprising a targeting moiety and one or more linker-drug moieties covalently attached to the targeting moiety, Each linker-drug moiety comprises a multifunctional linker that links a targeting moiety to one or more drug units via a releasable assembly unit for each drug unit, and links a polyalcohol or a derivative thereof to the drug unit of each linker-drug moiety, wherein the releasable assembly unit is capable of releasing free drug near a target site targeted by the targeting moiety; Conjugates. [The present invention 1015] The conjugate of claim 1014, wherein the polyalcohol or derivative thereof comprises an aminopolyalcohol. [The present invention 1016] The aminopolyalcohol TIFF2025114641000032.tif6128, where n1 is an integer from 0 to about 6; Each R 58 are independently hydrogen or C 1~8 is alkyl; R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 -, where R 59is H, alkyl, cycloalkyl, or arylalkyl; R 61 is CH2OR 62 , COOR 62 , -(CH2) n2 COOR 62 , -(CH2) 1~5 COOH, or heterocycloalkyl substituted with one or more hydroxyls; R 62 is H or C 1~8 is alkyl; and n2 is an integer from 1 to about 5; A conjugate of the present invention. [The present invention 1017] The conjugate of any one of claims 1014 to 1016, wherein the polyalcohol or derivative thereof comprises glucamine. [The present invention 1018] A conjugate of formula (I): TIFF2025114641000033.tif43128 formula, a1 is an integer of 0 to 1; a2 is an integer of 1 to 3; a3 is an integer from 0 to 1; a4 is an integer from 1 to about 5; a5 is an integer from 1 to 3; d 13 is an integer from 1 to about 14; PBRM stands for protein-based recognition molecule; L P’ PBRM M P and its corresponding monovalent moiety L P is a functional group W that can form a covalent bond with the functional group of the PBRM. P Contains; M P is the stretcher unit; L M is a bond or a trivalent or tetravalent linker, and L M is a bond, a2 is 1, and L Mis a trivalent linker, a2 is 2, or L M is a tetravalent linker, a2 is 3; L 3 is a carbonyl-containing moiety; M A comprises a peptide portion containing at least two amino acids; T 1 is a hydrophilic group, and T 1 and M A Between TIFF2025114641000034.tif12128 is T 1 and M A means a direct or indirect connection with; each occurrence of D is independently a therapeutic agent having a molecular weight of ≦about 5 kDa; and L D Each occurrence of independently converts D to M A and contains at least one cleavable bond, such that upon bond rupture D is released in an active form for its intended therapeutic effect. Any of the conjugates of the present invention. [The present invention 1019] A peptide-containing scaffold having any of formulas (II) to (XIV), TIFF2025114641000035.tif170128TIFF2025114641000036.tif20899In the formula, a1 is an integer of 0 to 1; a2 is an integer of 1 to 3; a3 is an integer from 0 to 1; a4 is an integer from 1 to about 5; a5 is an integer from 1 to 3; d 13 is an integer from 1 to about 14; PBRM stands for protein-based recognition molecule; L P’ PBRM M P and its corresponding monovalent moiety L Pis a functional group W that can form a covalent bond with the functional group of the PBRM. P Contains; M P is the stretcher unit; L M is a bond or a trivalent or tetravalent linker, and L M is a bond, a2 is 1, and L M is a trivalent linker, a2 is 2, or L M is a tetravalent linker, a2 is 3; L 3 is a carbonyl-containing moiety; M A comprises a peptide portion containing at least two amino acids; T 1 is a hydrophilic group, and T 1 and M A Between TIFF2025114641000037.tif12128 is T 1 and M A means a direct or indirect connection with; W M Each occurrence of independently may be replaced by forming a hydrogen, a protecting group, a leaving group, or a covalent bond to L M M P is a functional group capable of linking to; W D is independently a functional group capable of forming a covalent bond with a functional group of a therapeutic agent (“D”) having a molecular weight of ≦about 5 kDa; and L D Each occurrence of is independently D Or D to M A and L D contains at least one cleavable bond, and upon bond rupture D is released in an active form for its intended therapeutic effect; Peptide-containing scaffolds. [The present invention 1020] L 3 If present, -XC 1~10 alkylene-C(O)-, and X is LM wherein X is CH, O, or NR, and R is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, COOH, or COO-C 1~6 Any of the conjugates or scaffolds of the present invention which is alkyl. [The present invention 1021] L 3 If present, -NR5-(CH2) v -C(O)- or -CH2-(CH2) v -C(O)-NR5-(CH2) v Any of the conjugates or scaffolds of the present invention, wherein each v is independently an integer of 1 to 10. [The present invention 1022] L 3 When present, is —NH—(CH)—C(O)— or —(CH)—C(O)—NH—(CH)—C(O)—. [The present invention 1023] Any of the conjugates or scaffolds of the present invention, wherein each v is independently an integer of 1 to 6 or 2 to 4, or 2. [The present invention 1024] Any of the preceding conjugates or scaffolds of the invention, wherein a4 is 1, 2, or 3. [The present invention 1025] d 13 is 4 or 5. [The present invention 1026] Each W P , if present, independently, TIFF2025114641000038.tif107155TIFF2025114641000039.tif226169TIFF2025114641000040.tif43143, where Ring A is cycloalkyl or heterocycloalkyl; Ring B is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 1K is a leaving group; R 1A is a sulfur protecting group; R 1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; R 2J is hydrogen, an aliphatic, aryl, heteroaliphatic, or carbocyclic moiety; R 3J is C 1~6 alkyl, and each of Z1, Z2, Z3, and Z7 is independently a carbon or nitrogen atom; R 4j are hydrogen, halogen, OR, -NO2, -CN, -S(O)2R, C 1~24 alkyl, or 6- to 24-membered aryl or heteroaryl, where C 1~24 The alkyl, or 6- to 24-membered aryl or heteroaryl may be substituted with one or more aryl or heteroaryl; or two R 4j together form a fused cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R is hydrogen, alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl; R 5j is C(R 4j )2, O, S, or NR; and z1 is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Any of the conjugates or scaffolds of the present invention. [The present invention 1027] R 1K is halo or RC(O)O—, where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety. [The present invention 1028] R 1A but, TIFF2025114641000041.tif23128, where r is 1 or 2, and R s1 , R s2 , and R s3 each of which is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; Any of the conjugates or scaffolds of the present invention. [The present invention 1029] M P If exists, -(Z4)-[(Z5)-(Z6)] z - and Z4 is L P’ or L P The Z6 is connected to the L M where: z is 1, 2, or 3; The Z4 is TIFF2025114641000042.tif104147, where * is L P’ or L P and ** means connection to Z5 or Z6, if present, or to L if both Z5 and Z6 are absent. M means connection to; b1 is an integer of 0 to 6; e1 is an integer from 0 to 8, R 17 is C 1~10 Alkylene, C 1~10 Heteroalkylene, C 3~8 Cycloalkylene, O-(C 1~8 Alkylene, arylene, -C 1~10 Alkylene-arylene-, -arylene-C 1~10 Alkylene-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-, -(C 3~8 Cycloalkylene-C 1~10 Alkylene-, 4- to 14-membered heterocycloalkylene, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-, -(4- to 14-membered heterocycloalkylene)-C1~10 Alkylene-, -C 1~10 Alkylene-C(=O)-, -C 1~10 Heteroalkylene-C(=O)-, -C 3~8 Cycloalkylene -C(=O)-, -O-(C 1~8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1~10 Alkylene-arylene-C(=O)-, -arylene-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-C(=O)-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-C(=O)-, -4 to 14-membered heterocycloalkylene-C(=O)-, -C 1~10 Alkylene-(4-14 membered heterocycloalkylene)-C(=O)-, -(4-14 membered heterocycloalkylene)-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-NH-, -C 1~10 Heteroalkylene-NH-, -C 3~8 Cycloalkylene-NH-, -O-(C 1~8 alkyl)-NH-, -arylene-NH-, -C 1~10 Alkylene-arylene-NH-, -arylene-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-NH-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-NH-, -4 to 14-membered heterocycloalkylene-NH-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-NH-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-S-, -C 1~10 Heteroalkylene -S-, -C 3~8 Cycloalkylene -S-, -OC 1~8 Alkyl)-S-, -arylene-S-, -C 1~10 Alkylene-arylene-S-, -arylene-C 1~10Alkylene-S-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-S-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-S-, -4 to 14-membered heterocycloalkylene-S-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-S-, or -(4- to 14-membered heterocycloalkylene)-C1-C 10 alkylene-S-; Each Z5 is independently absent, R 57 -R 17 or a polyether unit; Each R 57 are independently bonded, NR 23 , S, or O; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; and Each Z6 may independently be absent, -C 1~10 Alkyl-R3-, -C 1~10 Alkyl-NR5-, -C 1~10 Alkyl-C(O)-, -C 1~10 Alkyl-O-, -C 1~10 Alkyl-S-, or -(C 1~10 alkyl-R3) g1 -C 1~10 alkyl-C(O)-; each R3 is independently -C(O)-NR5- or -NR5-C(O)-; Each R5 is independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, COOH, or COO-C 1~6 is alkyl; and g1 is an integer from 1 to 4. Any of the conjugates or scaffolds of the present invention. [The present invention 1030] MP But if it exists, TIFF2025114641000043.tif54155TIFF2025114641000044.tif73148, where * is L P’ or L P and ** means connection to L M means connection to; R3 is -C(O)-NR5 or -NR5-C(O)-; R4 is a bond or -NR5-(CR 20 R 21 )-C(O)-; R5 is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; R 17 is C 1~10 Alkylene, C 1~10 Heteroalkylene, C 3~8 Cycloalkylene, O-(C 1~8 Alkylene, arylene, -C 1~10 Alkylene-arylene-, -arylene-C 1~10 Alkylene-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-, -(C 3~8 Cycloalkylene-C 1~10 Alkylene-, 4- to 14-membered heterocycloalkylene, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-, -C 1~10 Alkylene-C(=O)-, -C 1~10 Heteroalkylene-C(=O)-, -C 3~8 Cycloalkylene -C(=O)-, -O-(C 1~8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1~10 Alkylene-arylene-C(=O)-, -arylene-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-(C3~8 Cycloalkylene)-C(=O)-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-C(=O)-, -4 to 14-membered heterocycloalkylene-C(=O)-, -C 1~10 Alkylene-(4-14 membered heterocycloalkylene)-C(=O)-, -(4-14 membered heterocycloalkylene)-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-NH-, -C 1~10 Heteroalkylene-NH-, -C 3~8 Cycloalkylene-NH-, -O-(C 1~8 alkyl)-NH-, -arylene-NH-, -C 1~10 Alkylene-arylene-NH-, -arylene-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-NH-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-NH-, -4 to 14-membered heterocycloalkylene-NH-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-NH-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-S-, -C 1~10 Heteroalkylene -S-, -C 3~8 Cycloalkylene -S-, -OC 1~8 Alkyl)-S-, -arylene-S-, -C 1~10 Alkylene-arylene-S-, -arylene-C 1~10 Alkylene-S-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-S-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-S-, -4 to 14-membered heterocycloalkylene-S-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-S-, or -(4- to 14-membered heterocycloalkylene)-C1-C 10 alkylene-S-; Each R20 and R 21 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; each b1 is independently an integer from 0 to 6; e1 is an integer from 0 to 8, each f1 is independently an integer from 1 to 6; and g2 is an integer from 1 to 4. Any of the conjugates or scaffolds of the present invention. [The present invention 1031] M P But if it exists, TIFF2025114641000045.tif104142, where * is L P’ or L P and ** means connection to L M means connection to Any of the conjugates or scaffolds of the present invention. [The present invention 1032] L M any of the preceding conjugates or scaffolds of the invention, wherein a is a bond and a2 is 1. [The present invention 1033] a2 is 2 and L M but, TIFF2025114641000046.tif238117, where TIFF2025114641000047.tif12128, if it exists, is P Connection to or M P If there is no L P or L P’ means connection to; Y1, if present, is L 3 Connection to or L 3 If there is no M A means connection to; R and R′ are each independently hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, optionally substituted C 3~19 Branched alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10 Aryl, optionally substituted heteroaryl, optionally substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, optionally substituted C 2~6 Alkanoyl, optionally substituted C 2~6 Alkanoyloxy, optionally substituted C 2~6 Substituted alkanoyloxy, -COOH, or -COO-C 1~6 is alkyl; each of c1, c2, c3, c4, c5, c7, and c8 is independently an integer ranging from 0 to 10; and Each of d1, d2, d3, d4, d5, and d7 is independently an integer ranging from 0 to 10. Any of the conjugates or scaffolds of the present invention. [The present invention 1034] a2 is 2 and L M but TIFF2025114641000048.tif37128, Any of the conjugates or scaffolds of the present invention. [This invention 1035] a2 is 3 and L M but TIFF2025114641000049.tif204134TIFF2025114641000050.tif205140TIFF2025114641000051.tif45136, During the ceremony, TIFF2025114641000052.tif12128, if it exists, is P Connection to or M P If there is no L P or L P’ means connection to; Y1, if present, is L 3 Connection to or L 3 If there is no M A means connection to; R and R′ are each independently hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, optionally substituted C 3~19 Branched alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10 Aryl, optionally substituted heteroaryl, optionally substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, optionally substituted C 2~6 Alkanoyl, optionally substituted C 2~6 Alkanoyloxy, optionally substituted C 2~6 Substituted alkanoyloxy, -COOH, or -COO-C1~6 is alkyl; each of c1, c2, c3, c4, c5, c6, c7, and c8 is independently an integer ranging from 0 to 10; each of d1, d2, d3, d4, d5, d6, d7, and d8 is independently an integer ranging from 0 to 10; and each of e1, e2, e3, e4, e5, e6, e7, and e8 is independently an integer ranging from 0 to 10; Any of the conjugates or scaffolds of the present invention. [The present invention 1036] a2 is 3 and L M but TIFF2025114641000053.tif37128, Any of the conjugates or scaffolds of the present invention. [This invention 1037] M A any of the foregoing conjugates or scaffolds of the invention, wherein said conjugate or scaffold comprises a peptide portion containing at least about 5 amino acids. [The present invention 1038] M A any of the foregoing conjugates or scaffolds of the invention, wherein said conjugate or scaffold comprises a peptide portion containing at most about 10 amino acids. [This invention 1039] M A any of the conjugates or scaffolds of the present invention, wherein the conjugate or scaffold comprises a peptide moiety containing 3 to about 10 amino acids selected from glycine, serine, glutamic acid, aspartic acid, lysine, cysteine, and combinations thereof. [The present invention 1040] M A any of the conjugates or scaffolds of the invention, wherein said conjugate or scaffold comprises a peptide moiety containing at least four glycines and at least one serine. [The present invention 1041] M Aany of the conjugates or scaffolds of the invention, wherein said conjugate or scaffold comprises a peptide moiety containing at least four glycines and at least one glutamic acid. [The present invention 1042] M A any of the conjugates or scaffolds of the invention, wherein said conjugate or scaffold comprises a peptide moiety containing at least four glycines, at least one serine, and at least one glutamic acid. [This invention 1043] Any of the conjugates of the present invention selected from those in Table B. [This invention 1044] Any of the scaffolds of the present invention selected from those in Table C. [This invention 1045] A conjugate of formula (XXX): TIFF2025114641000054.tif21153In formula, each R A but, TIFF2025114641000055.tif111132, Any of the conjugates of the present invention. [The present invention 1046] A conjugate of formula (XXX): TIFF2025114641000056.tif20153 In the formula, each R A but, TIFF2025114641000057.tif212155TIFF2025114641000058.tif209164TIFF2025114641000059.tif201159TIFF2025114641000060.tif190158TIFF2025114641000061.tif80145 Any of the conjugates of the present invention. [This invention 1047] Each R A but, TIFF2025114641000062.tif101140, Any of the conjugates of the present invention. [This invention 1048] Each R A but, TIFF2025114641000063.tif92135, Any of the conjugates of the present invention. [This invention 1049] Each R A but, TIFF2025114641000064.tif108148, Any of the conjugates of the present invention. [The present invention 1050] Each R A but, TIFF2025114641000065.tif101131, Any of the conjugates of the present invention. [This invention 1051] Each R A but, TIFF2025114641000066.tif100130, Any of the conjugates of the present invention. [This invention 1052] Each R A but, TIFF2025114641000067.tif80145, Any of the conjugates of the present invention. [This invention 1053] Each R A but, TIFF2025114641000068.tif79145, Any of the conjugates of the present invention. [This invention 1054] Each R A but, TIFF2025114641000069.tif94163, Any of the conjugates of the present invention. [This invention 1055] Each R A but, TIFF2025114641000070.tif82141, Any of the conjugates of the present invention. [This invention 1056] A pharmaceutical composition comprising any of the conjugates of the present invention and a pharmaceutically acceptable carrier. [This invention 1057] A method of treating a disorder in a subject in need thereof, comprising the step of administering to the subject an effective amount of any of the conjugates of the present invention. Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0059] [Figure 1] 1 shows the antitumor efficacy of a trastuzumab-drug conjugate (conjugate 43B, see Example 18) as measured in the N-87 mouse tumor xenograft model. [Figure 2]

[0023] Figure 2 shows the antitumor efficacy of XMT-1535-drug conjugates (Example 24, Conjugate 53D; Example 25, Conjugate 55A; Example 29, Conjugate 66) measured in an OVCAR3 mouse tumor xenograft model. XMT-1535 is an antibody disclosed in co-pending application US 15 / 457,574, filed March 13, 2017. [Figure 3] 2 shows the antitumor efficacy of XMT-1535-drug conjugates (Example 25, Conjugate 55C; Example 26, Conjugate 57; Example 28, Conjugate 61; and Example 27, Conjugate 59) measured in an OVCAR3 mouse tumor xenograft model. [Figure 4] 3 shows the antitumor efficacy of trastuzumab-drug conjugates (Example 30, Conjugate 67; and Example 31, Conjugate 68) as measured in the Calu-3 mouse tumor xenograft model. [Figure 5]1 shows the antitumor efficacy of XMT-1535-drug conjugates (Example 33, Conjugate 76; Example 26, Conjugate 57; Example 25, Conjugate 55C; and Example 27, Conjugate 59) measured in an OVCAR3 mouse tumor xenograft model. [Figure 6] 1 shows the antitumor efficacy of XMT-1535-drug conjugates (Example 26, Conjugate 57; Example 33, Conjugate 76; and Example 34, Conjugate 78) measured in an OVCAR3 mouse tumor xenograft model. [Figure 7A] 1 shows total antibodies measured for PBRM-polymer-drug conjugates (Example 35, Conjugate 79A; Example 35, Conjugate 7B; Example 37, Conjugate 83; and Example 38, Conjugate 85) after administration of the conjugates to mice. [Figure 7B] 3 shows total AF-HPA measured for PBRM-polymer-drug conjugates (Example 35, Conjugate 79A; Example 35, Conjugate 7B; Example 37, Conjugate 83; and Example 38, Conjugate 85) after administration of the conjugates to mice. [Figure 7C] 3 shows the concentration of conjugated AF-HPA for PBRM-polymer-drug conjugates (Example 35, Conjugate 79A; Example 35, Conjugate 7B; Example 37, Conjugate 83; and Example 38, Conjugate 85) measured after administration of the conjugates to mice. DETAILED DESCRIPTION OF THE INVENTION

[0060] Detailed Description The present disclosure provides novel targeting moiety-drug conjugates, scaffolds for making the conjugates, synthetic methods for making the conjugates or scaffolds, pharmaceutical compositions containing them, and various uses of the conjugates.

[0061] Also, definitions of some compounds and certain functional groups of the present disclosure are described in more detail herein. For purposes of this disclosure, chemical elements are defined according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., inside cover, and specific functional groups are generally defined as described herein. Furthermore, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference. Furthermore, those skilled in the art will recognize that synthetic methods such as those described herein utilize a variety of protecting groups.

[0062] The use of the articles "a," "an," and "the" in both the following description and the claims shall be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "is," "includes," and "containing," such as in "comprise," "have," and "is of the formula," should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise specified, and allow for, but do not require, the inclusion of additional elements or steps. For example, a scaffold of a certain formula includes all components shown in the formula and may also include additional components not shown in the formula. Furthermore, whenever "comprising" or another open-ended term is used in an embodiment, it should be understood that the same embodiment may be more narrowly claimed using the intermediate term "consisting essentially of" or the limiting term "consisting of."

[0063] As used herein, the expressions "one or more of A, B, or C," "one or more of A, B, or C," "one or more of A, B, and C," "one or more of A, B, and C," etc. are used interchangeably and all refer to a selection from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof.

[0064] The terms "about," "approximately," or "approximately," when used in reference to a numerical value, are meant to include a set or range of values. For example, "about X" includes a range of values that are ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is the numerical value. In one embodiment, the term "about" refers to a range of values that are 5% greater or less than the specified value. In another embodiment, the term "about" refers to a range of values that are 2% greater or less than the specified value. In another embodiment, the term "about" refers to a range of values that are 1% greater or less than the specified value.

[0065] Unless otherwise specified herein, the description of a range of values is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, and each separate value is incorporated herein as if it were individually described herein. As used herein, a range includes the two limits of the range unless otherwise specified. For example, the expressions "x is an integer between 1 and 6" and "x is an integer from 1 to 6" both mean "x is 1, 2, 3, 4, 5, or 6", i.e., the terms "between X and Y" and "range from X to Y" include X and Y and the integers therebetween.

[0066] "Protecting group": As used herein, the term protecting group means that a specific functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be selectively carried out at another reactive site in a multifunctional compound. In preferred embodiments, the protecting group reacts selectively in high yield to give a stable protected substrate for the intended reaction; the protecting group must be selectively removed in high yield by a readily available, preferably non-toxic, reagent that does not attack other functional groups; the protecting group forms an easily separable derivative (more preferably, without creating a new stereogenic center); and the protecting group minimizes the addition of functionality and avoids the creation of additional reactive sites. As described in detail herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. For example, in some embodiments, certain exemplary oxygen protecting groups may be utilized. These oxygen protecting groups include methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), and PMBM (p-methoxybenzyloxymethyl ether)), substituted ethyl ethers, substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilyl ether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether, and TBDPS (t-butyldiphenylsilyl ether), esters (e.g., formates, acetates, benzoates (Bz), trifluoroacetates, and dichloroacetates), carbonates, cyclic acetals, and ketals. In certain other exemplary embodiments, nitrogen protecting groups are utilized. Nitrogen protecting groups and protection and deprotection methods are known in the art. Nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl, and substituted ethyl carbamates (e.g., Troc)), amides, cyclic imide derivatives, N-alkyl and N-aryl amines, imine derivatives, and enamine derivatives. In yet other embodiments, certain exemplary sulfur protecting groups may be utilized.Sulfur protecting groups include, but are not limited to, the oxygen protecting groups previously described, as well as aliphatic carboxylic acids (e.g., acrylic acid), maleimide, vinylsulfonyl, and optionally substituted maleic acid. While certain other exemplary protecting groups are detailed herein, the present disclosure is not intended to be limited to these protecting groups; rather, it will be recognized that a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the present disclosure. Furthermore, various protecting groups are described in "Protective Groups in Organic Synthesis," Third Ed. Greene, TW and Wuts, PG, Eds., John Wiley & Sons, New York: 1999, the entire contents of which are incorporated herein by reference.

[0067] "Leaving group" refers to the molecular fragment that leaves with the electron pair upon cleavage of an anisotropic bond. Leaving groups can be anions or neutral molecules. Leaving groups include halides, e.g., Cl. - , Br - , and I - , sulfonic acid esters, such as para-toluenesulfonate ("tosylate", TsO - ), and RC(O)O—, where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0068] "Antibody" refers to a full-length antibody or a functional fragment of an antibody, including an immunoglobulin. By "functional fragment" is meant that a sufficient portion of the immunoglobulin or antibody is provided such that the portion effectively binds to or complexes with a cell surface molecule, e.g., human carcinoembryonic antigen, on the target cell population.

[0069] Immunoglobulins can be purified, recombinantly produced, synthetically produced, or a combination thereof, using techniques known to those skilled in the art. Immunoglobulins within or derived from IgG antibodies are particularly well suited for use in the conjugates or scaffolds of the present disclosure, although immunoglobulins from any class or subclass may be selected, e.g., IgG, IgA, IgM, IgD, and IgE. Preferably, the immunoglobulin is of class IgG, including but not limited to IgG subclasses (IgG1, 2, 3, and 4), or class IgM, which are capable of specifically binding to a particular epitope of an antigen. The antibody can be an intact immunoglobulin from a natural or recombinant source, or an immunoreactive portion of an intact immunoglobulin. Antibodies can exist in a variety of forms, including for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, intrabodies ("intrabodies"), recombinant antibodies, anti-idiotypic antibodies, domain antibodies, linear antibodies, multispecific antibodies, antibody fragments such as Fv, Fab, F(ab)2, F(ab)3, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem / bis-scFv, Fc, pFc', scFvFc (or scFv-Fc), disulfide Fv (dsfv), bispecific antibodies (bc-scFv), such as BiTE antibodies; camelid antibodies, resurfaced antibodies antibody), humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as NANOBODY®), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies, e.g., Dual Affinity Retargeting Protein (DART™), divalent (or bivalent) single-chain variable fragments (di-scFv, bi-scFv), including but not limited to minibodies, diabodies, triabodies or tribodies, tetrabodies, and multivalent antibodies. An "antibody fragment" refers to at least a portion of the variable region of an immunoglobulin molecule that binds to its target, i.e., the antigen-binding region.As used herein, the term "antibody" refers to both full length antibodies and antibody fragments, unless otherwise specified.

[0070] "Protein-based recognition molecule" or "PBRM" refers to a molecule that recognizes and binds to a cell surface marker or receptor, such as a transmembrane protein, a surface-anchored protein, or a proteoglycan. Examples of PBRMs include, but are not limited to, antibodies (e.g., trastuzumab, cetuximab, rituximab, bevacizumab, epratuzumab, veltuzumab, labetuzumab, B7-H4, B7-H3, CA125, CD33, CXCR2, EGFR, FGFR1, FGFR2, FGFR3, FGFR4, HER2, NaPi2b, c-Met, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1, c-Kit, MUC1, MUC13, and anti-5T4) or peptides (LHRH receptor targeting peptide, EC-1 peptide), lipocalins, e.g., anticalins, proteins, e.g., interferons, lymphokines, growth factors, colony-stimulating factors, peptides or peptidomimetics, etc. In addition to targeting the conjugate to specific cells, tissues, or locations, the protein-based recognition molecule may also have specific therapeutic effects, such as antiproliferative (cytostatic and / or cytotoxic) activity against the target cell or pathway. The protein-based recognition molecule may contain or be engineered to contain at least one chemically reactive group, such as -COOH, primary amine, secondary amine, -NHR, -SH, or a chemically reactive amino acid moiety or side chain, such as tyrosine, histidine, cysteine, or lysine. In one embodiment, the PBRM may be a ligand (LG) or targeting moiety that specifically binds to or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population. After the specific binding or complex formation of the ligand with its receptor, the cell allows the uptake of the ligand or ligand-drug conjugate, which is then internalized into the cell. As used herein, a ligand that "specifically binds to or complexes with" or "targets" a cell surface molecule preferentially associates with the cell surface molecule via intermolecular forces.For example, the ligand can preferentially associate with cell surface molecules with a Kd of less than about 50 nM, less than about 5 nM, or less than 500 pM.Techniques for measuring the binding affinity of ligands to cell surface molecules are well known; for example, one suitable technique is called surface plasmon resonance (SPR).In one embodiment, the ligand is used for targeting and does not have detectable therapeutic effect when separated from the drug it delivers.In another embodiment, the ligand functions as both a targeting moiety and a therapeutic agent or immunomodulator (for example, to enhance the activity of active drugs or prodrugs).

[0071] As used herein, "biocompatible" is intended to describe a compound that produces minimal disruptive or host-responsive effects during contact with body fluids or living cells or tissues. Thus, a biocompatible group, as used herein, refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl, or heteroaryl moiety that falls within the definition of the term biocompatible as defined above and herein. The term "biocompatible" is also used herein to mean that a compound produces minimal interaction with recognition proteins, such as naturally occurring antibodies, cellular proteins, cells, and other components of biological systems, unless such interaction is specifically desired. Thus, substances and functional groups, such as drugs and prodrugs, that are specifically designed to produce the above-mentioned minimal interaction are considered to be biocompatible. Preferably (excluding compounds intended to be cytotoxic, e.g., anti-neoplastic agents), a compound is "biocompatible" if its addition to normal cells in vitro at concentrations similar to its intended systemic in vivo concentration results in 1% or less cell death over a time period equivalent to the compound's half-life in vivo (e.g., the period required for 50% of the administered compound to be removed / cleared in vivo), and if its in vivo administration induces minimal and medically acceptable inflammation, foreign body reaction, immunotoxicity, chemical toxicity, and / or other such adverse effects. In the above sentence, the term "normal cells" refers to cells that are not intended to be destroyed or otherwise significantly affected by the compound being tested.

[0072] "Biodegradable": As used herein, a "biodegradable" compound or moiety is a compound that, once taken up by a cell, can be broken down by lysosomal or other chemical mechanisms, or by hydrolysis, into components that the cell can reuse or dispose of without significant toxic effects to the cell. The term "biocleavable" as used herein has the same meaning as "biodegradable." The degradation fragments preferably do not induce, or induce little to no organ or cellular overload or pathological processes in vivo that are due to such overload or other adverse effects. Examples of biodegradable processes include enzymatic and non-enzymatic hydrolysis, oxidation, and reduction. Suitable conditions for non-enzymatic hydrolysis of the biodegradable conjugates described herein (or their components, e.g., peptide-containing scaffolds and linkers between the scaffolds and antibodies or drug molecules) include, for example, exposing the biodegradable conjugates to water at the temperature and pH of the intracellular compartment of the lysosomal compartment. Biodegradation of some conjugates (or their components, e.g., peptide-containing scaffolds and linkers between the scaffold and antibody or drug molecules) can also be increased extracellularly, for example, in low pH regions of an animal's body, such as in inflammation, near activated macrophages or other cells that release factors that promote degradation. The integrity of the conjugates or scaffolds disclosed herein can be measured, for example, by size-exclusion HPLC. While more rapid degradation may be preferred in some cases, it is generally more desirable for the conjugates or scaffolds disclosed herein to degrade intracellularly at a rate that does not exceed the rate of metabolization or excretion of fragments by the cell. In preferred embodiments, the biodegradation byproducts of the conjugates or scaffolds disclosed herein are biocompatible.

[0073] "Bioavailability": The term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug or compound administered to a subject. Bioavailability is an absolute term that indicates a measure of both the time (rate) and total amount (extent) of a drug or compound that reaches the systemic circulation from an administered dosage form.

[0074] "Hydrophilic": The term "hydrophilic" essentially refers to a chemical moiety that contains ionic, polar, or polarizable atoms or that can otherwise be solvated by water molecules, consistent with the common meaning of this term in the art. Thus, as used herein, a hydrophilic moiety or group refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl, or heteroaryl moiety that falls within the definition of the term hydrophilic, as defined above. Examples of suitable specific hydrophilic organic moieties include, but are not limited to, aliphatic or heteroaliphatic groups containing chains of atoms ranging from about 1 to 12 atoms, hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic acid ester, thioester, aldehyde, nitrile, isonitrile, nitroso, hydroxylamine, mercaptoalkyl, heterocycle, carbamate, carboxylic acid and its salts, sulfonic acid and its salts, sulfonate ester, phosphoric acid and its salts, phosphate ester, polyglycol ether, polyamine, polycarboxylate, polyester, polythioester, polyalcohol, and derivatives thereof. In some embodiments, the hydrophilic substituent is a carboxyl group (COOH), an aldehyde group (CHO), a ketone group (COC 1~4 alkyl), methylol (CH2OH) or glycol (e.g., CHOH-CH2OH or CH-(CH2OH)2), NH2, F, cyano, SO3H, PO3H, and the like.

[0075] The hydrophilicity of the compounds disclosed herein (including drugs, conjugates, and scaffolds) can be measured directly via determination of hydration energy, or can be determined via testing between two liquid phases, or by chromatography on a solid phase of known hydrophobicity, such as C4 or C18.

[0076] "Physiological conditions": The phrase "physiological conditions," as used herein, relates to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions that would be encountered in the extracellular fluid of living tissues. In most normal tissues, physiological pH is about 7.0 to 7.4. Circulating plasma and normal interstitial fluid represent typical examples of normal physiological conditions.

[0077] "Polysaccharide," "carbohydrate," or "oligosaccharide": The terms "polysaccharide," "carbohydrate," or "oligosaccharide" are known in the art and generally refer to a carbohydrate having the chemical formula (CHO) n and their derivatives, where n>2 generally. Carbohydrates are polyhydroxyaldehydes or polyhydroxyketones, or can be converted to such substances by simple chemical transformation (e.g., hydrolysis, oxidation, or reduction). Typically, carbohydrates exist in the form of cyclic acetals or ketals (e.g., glucose or fructose). These cyclic units (monosaccharides) can be linked to each other to form molecules with a few (oligosaccharides) or many (polysaccharides) monosaccharide units. Often, carbohydrates with a well-defined number, type, and arrangement of monosaccharide units are called oligosaccharides, while carbohydrates consisting of a mixture of molecules with variable numbers and / or arrangements of monosaccharide units are called polysaccharides. The terms "polysaccharide," "carbohydrate," and "oligosaccharide" are used interchangeably herein. Polysaccharides can include natural sugars (eg, glucose, fructose, galactose, mannose, arabinose, ribose, and xylose) and / or derivatives of natural sugars (eg, 2'-fluororibose, 2'-deoxyribose, and hexose).

[0078] "Drug": As used herein, the term "drug" refers to a compound (e.g., an active pharmaceutical ingredient) that is biologically active and provides a desired physiological effect after administration to a subject in need thereof.

[0079] "Prodrug": As used herein, the term "prodrug" refers to a precursor of an active drug, i.e., a compound that can be converted into an active drug. Typically, such a prodrug is subjected to in vivo processing, which converts the drug into a physiologically active form. In some cases, the prodrug itself may have a desired physiological effect. The desired physiological effect may be, for example, a therapeutic effect, a cytotoxic effect, an immunomodulatory effect, etc.

[0080] "Cytotoxic": As used herein, the term "cytotoxic" means toxic to a cell or a selected population of cells (e.g., cancer cells). A toxic effect can result in cell death and / or lysis. In some cases, a toxic effect can be a destructive effect that does not result in cell death, such as slowing or preventing cell growth. To achieve a cytotoxic effect, the drug or prodrug can be selected from the group consisting of a DNA damaging agent, a microtubule disrupting agent, or a cytotoxic protein or polypeptide, among others.

[0081] "Cytostatic": As used herein, the term "cytostatic" refers to a drug or other compound that inhibits or stops cell growth and / or proliferation.

[0082] "Small molecule": As used herein, the term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or artificially created (e.g., by chemical synthesis). Preferred small molecules are biologically active in that they produce a local or systemic effect in an animal, preferably a mammal, and more preferably a human. In certain preferred embodiments, the small molecule is a drug, and the small molecule is referred to as a "drug molecule" or "drug" or "therapeutic agent." In certain embodiments, the drug molecule has a MW of about 5 kDa or less. In other embodiments, the drug molecule has a MW of about 1.5 kDa or less. In embodiments, the drug molecule is selected from vinca alkaloids, auristatins, duocarmycins, kinase inhibitors, MEK inhibitors, KSP inhibitors, PI3 kinase inhibitors, calicheamicins, SN38, camptothecin, topoisomerase inhibitors, non-naturally occurring camptothecins, protein synthesis inhibitors, RNA polymerase inhibitors, pyrrolobenzodiazepines, maytansinoids, DNA binding agents, DNA intercalating agents, and analogs thereof. Preferably, although not necessarily, the drug is one that has already been deemed safe and effective for use by an appropriate government agency or body, such as the FDA. For example, drugs for human use listed by the FDA at 21 CFR §§ 330.5, 331-361, and 440-460, which are incorporated herein by reference; drugs for veterinary use listed by the FDA at 21 CFR §§ 500-589, all of which are deemed suitable for the methods, conjugates, and scaffolds disclosed herein.Classes of drug molecules that can be used in the practice of the present invention include, but are not limited to, anti-cancer agents, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, antivirals, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubricants, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonians, antispasmodics and muscle contractants (including channel blockers, miotics, and anticholinergics), antiglaucoma compounds, antiparasitic and / or antiprotozoan compounds, modulators of cell-extracellular matrix interactions (including cell growth inhibitors and anti-adhesion molecules), vasodilators, inhibitors of DNA, RNA, or protein synthesis, antihypertensives, analgesics, antipyretics, steroidal and nonsteroidal anti-inflammatory agents, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, ophthalmic agents, prostaglandins, antidepressants, antipsychotics, antiemetics, and imaging agents. Many large molecules are also drugs, and such large molecules can be used in the conjugates and other constructs described herein.Examples of suitable large molecules include, for example, amino acid-based molecules.Amino acid-based molecules can include, for example, peptides, polypeptides, enzymes, antibodies, immunoglobulins, or their functional fragments.

[0083] A more complete (though not exhaustive) list of classes and specific drugs suitable for use in the present disclosure can be found in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", Edited by Susan Budavari et al., CRC Press, 1996, both of which are incorporated herein by reference. In preferred embodiments, the drugs used in the present disclosure are therapeutic agents that have antiproliferative (cytostatic and / or cytotoxic) activity against target cells or pathways. Drugs may contain a chemically reactive group, e.g., -COOH, primary amine, secondary amine, -NHR, -OH, -SH, -C(O)H, -C(O)R, -C(O)NHR. 2b , C(S)OH, -S(O)OR 2b , -P(O)2OR 2b , -CN, -NC, or -ONO, where R is an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; 2b is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocyclic moiety.

[0084] As used herein, "active form" refers to the form of a compound that exhibits the intended pharmaceutical efficacy in vivo or in vitro. In particular, when a drug molecule intended to be delivered by a conjugate of the present disclosure is released from the conjugate, the active form may be the drug itself or a derivative thereof that exhibits the intended therapeutic properties. Release of the drug from the conjugate can be achieved by cleavage of the biodegradable bond of the linker that connects the drug to the scaffold or conjugate of the present disclosure. Thus, an active drug derivative may include a portion of the linker.

[0085] "Diagnostic Label": As used herein, the term diagnostic label refers to an atom, group of atoms, moiety or functional group, nanocrystal, or other element of a discrete composition of matter that can be detected in vivo or ex vivo using analytical methods known in the art. When associated with a conjugate of the invention, such a diagnostic label allows for monitoring of the conjugate in vivo. Alternatively or additionally, constructs and compositions comprising a diagnostic label can be used to monitor biological function or structure. Examples of diagnostic labels include, but are not limited to, labels that can be used in medical diagnostic procedures, such as radioisotopes (radionuclides) for gamma-scintigraphy and positron emission tomography (PET), contrast agents for magnetic resonance imaging (MRI) (e.g., paramagnetic atoms and superparamagnetic nanocrystals), contrast agents for computed tomography and other x-ray based imaging methods, agents for ultrasound-based diagnostic methods (sonography), agents for neutron activation (e.g., boron, gadolinium), fluorophores for various optical procedures, and generally, moieties (e.g., gamma rays, x-rays, radio waves, microwaves, light), particles (e.g., alpha particles, electrons, positrons, neutrons, protons), or other forms of radiation, such as ultrasound, that can emit, reflect, absorb, scatter, or otherwise affect electromagnetic fields or waves (e.g., gamma rays, x-rays, radio waves, microwaves, light), particles (e.g., alpha particles, electrons, positrons, neutrons, protons), or other forms of radiation, such as ultrasound.

[0086] "Animal": The term animal, as used herein, refers to human and non-human animals, including at any stage of development, for example, mammals, birds, reptiles, amphibians, fish, worms, and single cells. Cell cultures and live tissue samples are considered to be animals. Preferably, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, primate, or pig). The animal may be a transgenic animal or a human clone. The term "subject" encompasses animals.

[0087] "Effective amount": Generally, when referring to an active agent or drug delivery device, the term "effective amount" refers to the amount necessary to elicit a desired biological response. As will be recognized by those skilled in the art, the effective amount of an agent or device can vary depending on factors such as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like. For example, an effective amount of antigen-containing microparticles to be delivered to immunize an individual is the amount that generates an immune response sufficient to prevent infection with the administered antigen-bearing organism.

[0088] As used herein, "natural amino acid" refers to any one of the common naturally occurring L-amino acids found in naturally occurring proteins, such as glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gln), cysteine (Cys), methionine (Met), or a stereoisomer thereof, such as isoglutamic acid (iGlu) or isoaspartic acid (iAsp). Unless otherwise specified, reference to an amino acid includes the amino acid itself and its stereoisomers. For example, the term "glutamic acid" includes both Glu and iGlu, while the term "aspartic acid" includes both Asp and iAsp.

[0089] "Unnatural amino acid," as used herein, refers to any amino acid that is not a natural amino acid. This includes, for example, amino acids that contain α-, β-, γ-, D-, or L-aminoacyl residues. More generally, unnatural amino acids are those having the general formula TIFF2025114641000071.tif18128, where the side chain R is other than a naturally occurring amino acid side chain. Exemplary unnatural amino acids include, but are not limited to, sarcosine (N-methylglycine), citrulline (cit), homocitrulline, β-ureidoalanine, thiocitrulline, hydroxyproline, allothreonine, pipecolic acid (homoproline), α-aminoisobutyric acid, tert-butylglycine, tert-butylalanine, allo-isoleucine, norleucine, α-methylleucine, cyclohexylglycine, β-cyclohexylalanine, β-cyclopentylalanine, α-methylproline, phenylglycine, α-methylphenylalanine, and homophenylalanine.

[0090] "Alkyl," as used herein by itself or as part of another term, refers to a substituted or unsubstituted, straight or branched chain, saturated or unsaturated hydrocarbon having the indicated number of carbon atoms (e.g., "-C 1~8 alkyl" or "-C 1~10 ("Alkyl" refers to an alkyl group having 1 to 8 or 1 to 10 carbon atoms, respectively. If the number of carbon atoms is not specified, the alkyl group has 1 to 8 carbon atoms. Representative straight chain "-C 1~8 "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 -C 1~8 Alkyl includes, but is not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated -C 2~8Alkyl includes, but is not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl. In some embodiments, the alkyl group is unsubstituted. The alkyl group can be substituted with one or more groups. In other aspects, the alkyl group is saturated.

[0091] "Alkylene," as used herein by itself or as part of another term, refers to a substituted or unsubstituted, saturated or unsaturated, branched, straight-chain, or cyclic hydrocarbon group having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane, and having a specified number of carbon atoms, typically 1 to 10 carbon atoms. Typical alkylene groups include, but are not limited to, methylene (-CH-), 1,2-ethyl (-CHCH-), 1,3-propyl (-CHCHCH-), 1,4-butyl (-CHCHCHCH-), and the like. In some embodiments, the alkylene is a branched or straight-chain hydrocarbon (i.e., it is not a cyclic hydrocarbon). In any of the embodiments provided herein, the alkylene can be a saturated alkylene.

[0092] "Aryl," as used herein by itself or as part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon group of 6 to 20 carbon atoms (preferably 6 to 14 carbon atoms) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented by the exemplary structure as "Ar." Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is the phenyl group.

[0093] "Arylene," as used herein by itself or as part of another term, refers to an aryl group defined above in which one of the aryl atoms has been replaced with a bond (i.e., it is divalent), and can be represented by the following structure, with phenyl as an exemplary group: It can be in the ortho, meta, or para orientation as shown in TIFF2025114641000072.tif30128.

[0094] In some embodiments, for example, when a polyfunctional linker or drug unit comprises an arylene, the arylene is an aryl group defined above in which one or two of the aryl group's hydrogen atoms have been replaced with a bond (i.e., the arylene can be divalent or trivalent).

[0095] "Heterocycle," as used herein by itself or as part of another term, refers to a ring structure having a number of (e.g., 3 to 8 or C 3~8) carbon atoms (also called ring members) and 1 to 4 heteroatom ring members independently selected from N, O, P, or S, and derived by the removal of a single hydrogen atom from a ring atom of the parent ring system. One or more N, C, or S atoms in a heterocycle can be oxidized. A ring containing a heteroatom can be aromatic or non-aromatic. Unless otherwise specified, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Heterocycles (e.g., C 3~8 Representative examples of 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.

[0096] "Heterocyclo" or "heterocyclo-", as used herein, refers to a heterocyclic group defined above in which one or more of the additional hydrogen atoms of the heterocycle has been replaced with a bond (e.g., C 3~8 In some embodiments, when the hydrophilic group, polyfunctional linker, or linker-drug moiety comprises a heterocycle, the heterocycle is a heterocycle group defined above in which one or two of the heterocycle group's hydrogen atoms have been replaced with a bond (i.e., the heterocycle can be divalent or trivalent).

[0097] "Carbocycle," as used herein by itself or as part of another term, refers to a ring system having a number (e.g., 3 to 8 or C) of ring atoms derived by the removal of one hydrogen atom from a ring atom of a parent ring system. 3~8A monovalent substituted or unsubstituted aromatic ("aryl") or saturated or unsaturated non-aromatic ("cycloalkyl") monocyclic, bicyclic, tricyclic, or tetracyclic carbocyclic ring system having 1) carbon atoms (also called ring members). The carbocyclic ring can be 3, 4, 5, 6, 7, or 8 members. Representative C 3~8 Carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, phenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl.

[0098] "Carbocyclo" or "carbocyclo-", as used herein by itself or as part of another term, refers to a C as defined above. 3~8 It refers to a carbocyclic group in which one or more of the carbocyclic group's hydrogen atoms have been replaced with a bond (i.e., it is divalent). In selected embodiments, for example, when a hydrophilic group, a polyfunctional linker, or a linker-drug moiety contains a carbocyclo, the carbocyclo is a carbocyclic group defined above in which one or two of the carbocyclic group's hydrogen atoms have been replaced with a bond (i.e., the carbocyclo can be divalent or trivalent).

[0099] "Heteroalkyl," as used herein, by itself or in combination with another term, means, unless otherwise specified, a stable straight- or branched-chain hydrocarbon, or combination thereof, consisting of the specified number of carbon atoms and 1 to 10, preferably 1 to 3, heteroatoms selected from the group consisting of O, N, Si, and S, and which is fully saturated or contains 1 to 3 degrees of unsaturation, and wherein the nitrogen and sulfur atoms can be optionally oxidized and the nitrogen heteroatom can be quaternized. The heteroatoms O, N, and S can be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. The heteroatom Si can be placed 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 a preferred embodiment, C 1~4 Heteroalkyl or heteroalkylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms, C 1~3 The heteroalkyl or heteroalkylene has 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some aspects, the heteroalkyl or heteroalkylene is saturated.

[0100] "Heteroalkylene," as used herein by itself or as part of another substituent, refers to a divalent group derived from a heteroalkyl (as discussed above), as exemplified by -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can occupy either or both of the chain termini. Furthermore, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied. In selected embodiments, for example, when a hydrophilic group, a polyfunctional linker, or a linker-drug moiety comprises a heteroalkylene, the heteroalkylene is a heteroalkyl group defined above in which one or two of the heteroalkyl group's hydrogen atoms have been replaced with a bond (i.e., the heteroalkylene can be divalent or trivalent).

[0101] "Optionally substituted," as used herein, means that a chemical moiety (e.g., alkyl, heteroalkyl, carbocycle, heterocycle, etc.) is either substituted or unsubstituted. Unless otherwise specified, chemical moieties disclosed herein may be substituted. When a chemical moiety is substituted, one or more hydrogen atoms are each independently replaced with a substituent. Exemplary substituents include -X', -R', -O, -OR', -SR', -S - , -N(R')2, -N(R')3, =NR', -C(X')3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)N(R')2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 - , -PO3H2, -AsO2H2, -C(=O)R', -C(=O)X', -C(=S)R', -CO2R', -CO2 -, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)N(R')2, C(=S)N(R')2, or C(=NR')N(R')2, where each X' is independently a halogen -F, -CI, -Br, or -I; and each R' is independently -H, -C 1~20 Alkyl, -C 6~20 Aryl, -C3~C 14 It may be a heterocycle, a protecting group, or a prodrug moiety. Typical substituents include oxo (=O).

[0102] "Linker-drug moiety," as used herein, refers to the non-targeting portion of a conjugate disclosed herein. The linker component of the linker-drug moiety has a release mechanism, called a releasable assembly unit, interposed between the multifunctional linker and the drug unit.

[0103] As used herein, a "multifunctional linker" refers to a linker that links one or more hydrophilic groups, one or more drug units, and a targeting moiety (e.g., a PBRM) to form a conjugate or scaffold as disclosed herein. The linkage of these components to the multifunctional linker can be in parallel or in series. In some embodiments, the multifunctional linker includes a peptide portion between the targeting moiety and the hydrophilic group, wherein the peptide portion includes at least two amino acids. In other embodiments, when the hydrophilic group is a polyalcohol or a derivative thereof, the multifunctional linker does not need to include a peptide portion of at least two amino acids. In other embodiments, when the hydrophilic group is a glucosylamine, a di-glucosylamine, a tri-glucosylamine, or a derivative thereof, the multifunctional linker does not need to include a peptide portion of at least two amino acids.

[0104] As used herein, the phrases "parallel orientation," "parallel arrangement," "parallel linkage," or similar terms refer to a configuration in which the parallel-arranged or parallel-oriented or parallel-linked components are connected to a polyfunctional linker such that each component has one end constrained to the polyfunctional linker and one free end. The term "parallel" is used herein to indicate that the two components are side-by-side in space or have the same distance between them over some or all of their length. If the parallel-oriented component itself is branched and has multiple ends, it still has only one constrained end. In some embodiments, only the hydrophilic groups required to mask the hydrophobicity of a given linker-drug moiety are in a parallel orientation relative to the drug unit, although this does not necessarily require that all of the drug units and hydrophilic groups linked to the polyfunctional linker be in a parallel orientation relative to each other. In other embodiments, all of the drug units and hydrophilic groups linked to the polyfunctional linker are in a parallel orientation relative to each other.

[0105] The phrase "tandem orientation" or "tandem arrangement" or "tandem linkage" or similar terms refer to a configuration of components in a conjugate or scaffold of the present disclosure in which the tandemly oriented components are connected such that they have two constrained ends, each end being linked to a different component of the conjugate or scaffold of the present disclosure. For example, one or more (OCH2CH2) subunits, such as those characterizing a PEG unit or subunit, are interposed between the drug unit and the targeting moiety.

[0106] As used herein, "free drug" refers to a biologically active form of a drug moiety that is not covalently bound directly or indirectly to a hydrophilic group or degradation product of a ligand unit. The free drug can refer to a drug, as it exists immediately after cleavage from a polyfunctional linker via a release mechanism provided by a releasable assembly unit in the linker-drug moiety, or after subsequent intracellular transformation or metabolism. In some aspects, the free drug may have the form HD or exist as a charged moiety. The free drug is a pharmacologically active species that can exert a desired biological effect. In some aspects, the pharmacologically active species may not be the parent drug, but may include a component of the linker that connects the drug to the targeting moiety without subsequent intracellular metabolism.

[0107] Hydrophobicity can be measured using SlogP, which is defined as the logarithm of the octanol / water partition coefficient (including implicit hydrogen) and can be calculated using the program MOE™ from Chemical Computing Group (SlogP values are calculated using Wildman, SA, Crippen, GM; Prediction of Physiochemical Parameters by Atomic Contributions; J. Chem. Inf. Comput. Sci. 39 No. 5 (1999) 868-873).

[0108] In certain embodiments, the present disclosure provides a targeting moiety-drug conjugate composition comprising a population of targeting moiety-drug conjugates. A targeting moiety-drug conjugate comprises one targeting moiety unit and multiple linker-drug moieties connected thereto. Preferably, there are an average of about 2 to about 14, about 3 to about 10, or about 3 to about 5 linker-drug moieties (e.g., d of Formula (I)) per targeting moiety in the conjugate. 13) Exemplary attachment to the targeting moiety is via a thioether bond. Exemplary conjugation sites for the targeting moiety are thiol groups resulting from reduction of interchain disulfide residues and / or thiol-containing residues introduced into the targeting moiety (e.g., introduced cysteine). Attachment can be, for example, via thiol residues from the interchain disulfide and from 0 to 8 introduced cysteine residues.

[0109] As used herein, the "molecular weight" or "MW" of a polymer refers to the weight average molecular weight, unless otherwise specified.

[0110] The present disclosure is intended to encompass all isotopes of atoms occurring in the present compounds.Isotopes include atoms with the same atomic number but different mass numbers.By way of general example and without limitation, hydrogen isotopes include tritium and deuterium.Carbon isotopes include C-13 and C-14.

[0111] The present disclosure is intended to encompass all isomers of the compounds (e.g., drugs, conjugates, and scaffolds disclosed herein), which refers to and includes optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers, and non-chiral isomers, and optical isomers include isolated optical isomers and mixtures of optical isomers, including racemic and non-racemic mixtures; where an isomer may be present in isolated form or in mixture with one or more other isomers.

[0112] Conjugates and peptide-containing scaffolds In one aspect, the present disclosure relates to a conjugate of formula (I) having a protein-based recognition molecule (PBRM): TIFF2025114641000073.tif43128 formula, a1 is an integer of 0 to 1; a2 is an integer of 1 to 3; a3 is an integer from 0 to 1; a4 is an integer from 1 to about 5; a5 is an integer from 1 to 3; d 13 is an integer from 1 to about 14; PBRM stands for protein-based recognition molecule; L P’ PBRM M P and its corresponding monovalent moiety L P is a functional group W that can form a covalent bond with the functional group of the PBRM. P Contains; M P is the stretcher unit; L M is a bond or a trivalent or tetravalent linker, and L M is a bond, a2 is 1, and L M is a trivalent linker, a2 is 2, or L M is a tetravalent linker, a2 is 3; L 3 is a carbonyl-containing moiety; M A comprises a peptide portion containing at least two amino acids; T 1 is a hydrophilic group, and T 1 and M A Between TIFF2025114641000074.tif12128 is T 1 and M A means a direct or indirect connection with; each occurrence of D is independently a therapeutic agent having a molecular weight of ≦about 5 kDa; and L D Each occurrence of independently converts D to M A and contains at least one cleavable bond, such that upon bond rupture D is released in an active form for its intended therapeutic effect.

[0113] In another aspect, the present disclosure relates to a peptide-containing scaffold of any one of formulas (II) to (XIV): TIFF2025114641000075.tif170128TIFF2025114641000076.tif20899In the formula, a1 is an integer of 0 to 1; a2 is an integer of 1 to 3; a3 is an integer from 0 to 1; a4 is an integer from 1 to about 5; a5 is an integer from 1 to 3; d 13 is an integer from 1 to about 10; PBRM stands for protein-based recognition molecule; L P’ PBRM M P and its corresponding monovalent moiety L P is a functional group W that can form a covalent bond with the functional group of the PBRM. P Contains; M P is the stretcher unit; L M is a bond or a trivalent or tetravalent linker, and L M is a bond, a2 is 1, and L M is a trivalent linker, a2 is 2, or L M is a tetravalent linker, a2 is 3; L 3 is a carbonyl-containing moiety; M A comprises a peptide portion containing at least two amino acids; T 1 is a hydrophilic group, and T 1 and M A Between TIFF2025114641000077.tif12128 is T 1 and M A means a direct or indirect connection with; W MEach occurrence of independently may be replaced by forming a hydrogen, a protecting group, a leaving group, or a covalent bond to L M M P is a functional group capable of linking to; W D is independently a functional group capable of forming a covalent bond with a functional group of a therapeutic agent (“D”) having a molecular weight of ≦about 5 kDa; and L D Each occurrence of is independently D Or D to M A and L D contains at least one cleavable bond, and upon bond rupture D is released in an active form for its intended therapeutic effect.

[0114] The conjugates and scaffolds of the present disclosure can include one or more of the following features, where applicable.

[0115] In one embodiment, d 13 is an integer from 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 10, 4 to 8, 4 to 6, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 14, 8 to 12, or 8 to 10.

[0116] In one embodiment, d 13 is an integer between 2 and 4 (e.g., d 13 is 2, 3, or 4).

[0117] In one embodiment, d 13 is an integer between 4 and 6 (e.g., d 13 is 4, 5, or 6).

[0118] In one embodiment, d 13 is an integer between 6 and 8 (e.g., d 13 is 6, 7, or 8).

[0119] In one embodiment, d 13 is an integer between 6 and 10 (e.g., d 13is 6, 7, 8, 9, or 10).

[0120] In certain embodiments, d 13 is 4 or 5.

[0121] In some embodiments, L 3 If present, -XC 1~10 alkylene-C(O)-, and X is L M wherein X is CH, O, or NR, and R is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, COOH, or COO-C 1~6 It is alkyl.

[0122] In some embodiments, L 3 is -NR5-(CH2) v -C(O)- or -CH2-(CH2) v -C(O)-NR5-(CH2) v -C(O)-, where each v is independently an integer from 1 to 10. For example, L 3 is, if present, —NH—(CH2)2—C(O)— or —(CH2)2—C(O)—NH—(CH2)2—C(O)—.

[0123] In one embodiment, each v is independently an integer from 1 to 6 or from 2 to 4, or v is 2.

[0124] In one embodiment, a4 is 1.

[0125] In one embodiment, a4 is 2.

[0126] In one embodiment, a4 is 3.

[0127] L P and L P’ L P When not linked to a PBRM, the terminal group W P where each WP are independently: TIFF2025114641000078.tif78128TIFF2025114641000079.tif218145TIFF2025114641000080.tif174166, where Ring A is cycloalkyl or heterocycloalkyl; Ring B is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 1K is a leaving group; R 1A is a sulfur protecting group; R 1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; R 2J is hydrogen, an aliphatic, aryl, heteroaliphatic, or carbocyclic moiety; R 3J is C 1~6 alkyl, and each of Z1, Z2, Z3, and Z7 is independently a carbon or nitrogen atom; R 4j are hydrogen, halogen, OR, -NO2, -CN, -S(O)2R, C 1~24 Alkyl (e.g., C 1~6 alkyl), or 6- to 24-membered aryl or heteroaryl, where C 1~24 Alkyl (e.g., C 1~6 alkyl), or 6- to 24-membered aryl or heteroaryl, optionally substituted with one or more aryl or heteroaryl; or two R 4j together form a fused cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R is hydrogen, alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl; R 5j is C(R 4j )2, O, S, or NR; and z1 is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0128] L P’ PBRM M P and its corresponding monovalent moiety is L P is.

[0129] For example, each R 1K is halo or RC(O)O—, where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0130] For example, each R 1A is, independently, TIFF2025114641000081.tif24128, where r is 1 or 2, and R s1 , R s2 , and R s3 Each of is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0131] For example, ring A is C 3~8 It can be a cycloalkyl or a 5- to 19-membered heterocycloalkyl.

[0132] For example, ring A is TIFF2025114641000082.tif33128; where R 6j is hydrogen, halogen, C 1~24 Alkyl (e.g., C 1~6 alkyl), or 6- to 24-membered aryl or heteroaryl, where C 1~24 Alkyl (e.g., C 1~6 alkyl), or 6- to 24-membered aryl or heteroaryl may be substituted with one or more aryls or heteroaryls.

[0133] For example, ring A is It could be TIFF2025114641000083.tif29128.

[0134] For example, ring A or B may be C 3~8It can be a cycloalkyl or a 3- to 12-membered heterocycloalkyl.

[0135] For example, ring A or B can be piperazinyl or piperidinyl.

[0136] For example, R s1 , R s2 , and R s3 Each of 1~6 It can be alkyl.

[0137] In some embodiments, W P teeth, TIFF2025114641000084.tif26128.

[0138] In some embodiments, W P teeth, TIFF2025114641000085.tif30128.

[0139] In some embodiments, W P but If it is TIFF2025114641000086.tif29128, L P’ teeth, Includes TIFF2025114641000087.tif27128.

[0140] In some embodiments, W P but If it is TIFF2025114641000088.tif14128.

[0141] In some embodiments, W P but If it is TIFF2025114641000089.tif28128.

[0142] In some embodiments, W P but If it is TIFF2025114641000090.tif33128.

[0143] In some embodiments, W P teeth, The file is TIFF2025114641000091.tif18128.

[0144] In some embodiments, W P but If it is TIFF2025114641000092.tif18128, L P’ teeth, Includes TIFF2025114641000093.tif18128.

[0145] In some embodiments, W P teeth, TIFF2025114641000094.tif22128, where X a and X b wherein one of the maleimide groups is H and the other is a maleimide blocking moiety. For example, a maleimide blocking compound (i.e., a compound that can react with a maleimide and convert it to a succinimide) can be used, for example, to quench the reaction of a linker-drug moiety with a PBRM, where the maleimide blocking moiety refers to the chemical moiety that is attached to the succinimide upon conversion. For example, a maleimide blocking moiety is a moiety that can be covalently attached to one of the two olefinic carbon atoms upon reaction of a maleimide group with a thiol-containing compound of formula (II'): R 90 -(CH2) d -SH (II') During the ceremony, R 90 is NHR 91 ,OH,COOR 93 , CH(NHR 91 )COOR 93 or a substituted phenyl group; R 93 is hydrogen or C 1~4 is alkyl; R 91 is hydrogen, CH3, or CH3CO, and d is an integer of 1 to 3.

[0146] For example, the maleimide blocking compound can be cysteine, N-acetylcysteine, cysteine methyl ester, N-methylcysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e., HOCHSH), benzylthiol (wherein the phenyl is substituted with one or more hydrophilic substituents), or 3-aminopropane-1-thiol. The one or more hydrophilic substituents on the phenyl can be OH, SH, methoxy, ethoxy, COOH, CHO, COC. 1~4 Includes alkyl, NH2, F, cyano, SO3H, PO3H, etc.

[0147] For example, the maleimide blocking group is -S-(CH2) d -R 90 where: R 90 is OH, COOH, or CH(NHR 91 )COOR 93 and; R 93 is hydrogen or CH3; R 91 is hydrogen or CHCO; and d is 1 or 2.

[0148] For example, a maleimide blocking group is -S-CH2-CH(NH2)COOH.

[0149] Stretcher Unit M P M P If present, -(Z4)-[(Z5)-(Z6)] z - and Z4 is L P’ or L P The Z6 is connected to the L M where: z is 1, 2, or 3; The Z4 is TIFF2025114641000095.tif104161, where * is L P’ or L Pand ** means connection to Z5 or Z6, if present, or to L if both Z5 and Z6 are absent. M means connection to; b1 is an integer of 0 to 6; e1 is an integer from 0 to 8, R 17 is C 1~10 Alkylene, C 1~10 Heteroalkylene, C 3~8 Cycloalkylene, O-(C 1~8 Alkylene, arylene, -C 1~10 Alkylene-arylene-, -arylene-C 1~10 Alkylene-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-, -(C 3~8 Cycloalkylene-C 1~10 Alkylene-, 4- to 14-membered heterocycloalkylene, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-, -C 1~10 Alkylene-C(=O)-, -C 1~10 Heteroalkylene-C(=O)-, -C 3~8 Cycloalkylene -C(=O)-, -O-(C 1~8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1~10 Alkylene-arylene-C(=O)-, -arylene-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-C(=O)-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-C(=O)-, -4 to 14-membered heterocycloalkylene-C(=O)-, -C 1~10 Alkylene-(4-14 membered heterocycloalkylene)-C(=O)-, -(4-14 membered heterocycloalkylene)-C 1~10 Alkylene-C(=O)-, -C 1~10 Alkylene-NH-, -C 1~10 Heteroalkylene-NH-, -C 3~8Cycloalkylene-NH-, -O-(C 1~8 alkyl)-NH-, -arylene-NH-, -C 1~10 Alkylene-arylene-NH-, -arylene-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-NH-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-NH-, -4 to 14-membered heterocycloalkylene-NH-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-NH-, -(4- to 14-membered heterocycloalkylene)-C 1~10 Alkylene-NH-, -C 1~10 Alkylene-S-, -C 1~10 Heteroalkylene -S-, -C 3~8 Cycloalkylene -S-, -OC 1~8 Alkyl)-S-, -arylene-S-, -C 1~10 Alkylene-arylene-S-, -arylene-C 1~10 Alkylene-S-, -C 1~10 Alkylene-(C 3~8 Cycloalkylene)-S-, -(C 3~8 Cycloalkylene)-C 1~10 Alkylene-S-, -4 to 14-membered heterocycloalkylene-S-, -C 1~10 Alkylene-(4- to 14-membered heterocycloalkylene)-S-, or -(4- to 14-membered heterocycloalkylene)-C1-C 10 alkylene-S-; Each Z5 is independently absent, R 57 -R 17 or a polyether unit; Each R 57 are independently bonded, NR 23 , S, or O; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; and Each Z6 may independently be absent, -C 1~10 Alkyl-R3-, -C 1~10 Alkyl-NR5-, -C 1~10 Alkyl-C(O)-, -C 1~10 Alkyl-O-, -C 1~10 Alkyl-S-, or -(C 1~10 alkyl-R3) g1 -C 1~10 alkyl-C(O)-; each R3 is independently -C(O)-NR5- or -NR5-C(O)-; Each R5 is independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, COOH, or COO-C 1~6 is alkyl; and g1 is an integer of 1 to 4.

[0150] In one embodiment, Z4 is TIFF2025114641000096.tif16128, for example, where b1 is 1 or 4.

[0151] In another embodiment, Z4 is TIFF2025114641000097.tif14128, for example, where b1 is 4.

[0152] In another embodiment, Z4 is TIFF2025114641000098.tif14128, for example, where b1 is 0.

[0153] In another embodiment, Z4 is TIFF2025114641000099.tif13128.

[0154] In some embodiments, each Z5 is independently a polyalkylene glycol (PAO), including, but not limited to, a polymer of a lower alkylene oxide, such as a polymer of propylene oxide, particularly ethylene oxide, polypropylene glycol, polyethylene glycol (PEG), polyoxyethylenated polyols, copolymers thereof, and block copolymers thereof. In other embodiments, the polyalkylene glycol is polyethylene glycol (PEG), including, but not limited to, polydisperse PEG, monodisperse PEG, and discrete PEG. 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. In another embodiment, the PEG unit is discrete PEG, providing a single molecule with a defined and specified chain length. In some embodiments, the polyethylene glycol is mPEG.

[0155] As used herein, subunit, when referring to a PEG unit, has the formula TIFF2025114641000100.tif12128. In some such embodiments, the PEG unit comprises multiple PEG subunits.

[0156] In some embodiments, when z is 2 or 3, at least one Z5 is a polyalkylene glycol (PAO), eg, a PEG unit.

[0157] In one embodiment, the PEG unit comprises 1 to 6 subunits.

[0158] In another embodiment, the PEG unit comprises 1 to 4 subunits.

[0159] In other embodiments, the PEG unit comprises 1 to 3 subunits.

[0160] In one embodiment, the PEG unit comprises two subunits.

[0161] In another embodiment, the PEG unit comprises one subunit.

[0162] In other embodiments, the PEG unit comprises one or more PEG subunits linked together by a PEG linking unit. The PEG linking unit that links one or more chains of repeating CH2CH2O- subunits can be Z6. For example, Z6 can be -C 1~10 Alkyl-R3-, -C 2~10 Alkyl-NH-, -C 2~10 Alkyl-C(O)-, -C 2~10 Alkyl-O- or -C 1~10 alkyl-S, where R3 is -C(O)-NR5- or -NR5-C(O)-.

[0163] In some embodiments, the PEG linking unit is -C 1~10 Alkyl-C(O)-NH- or -C 1~10 alkyl-NH-C(O)-. In one embodiment, the PEG linking unit is -(CH2)2-C(O)-NH-.

[0164] In some embodiments, each Z5 is absent (absent).

[0165] In some embodiments, when z is 2 or 3, at least one Z5 is absent.

[0166] In some embodiments, each Z5 is -(CH2-CH2-O-)2-.

[0167] In some embodiments, when z is 2 or 3, at least one Z5 is -(CH2-CH2-O-)2-.

[0168] In some embodiments, each Z is independently R 57 -R 17For example, each Z5 can independently be R 17 , NHR 17 , OR 17 , or SR 17 is.

[0169] In some embodiments, when z is 2 or 3, at least one Z is R 57 -R 17 , e.g., R 17 , NHR 17 , OR 17 , or SR 17 is.

[0170] In some embodiments, each Z6 is absent.

[0171] In some embodiments, when z is 2 or 3, at least one Z6 is absent.

[0172] In some embodiments, at least one of Z5 and Z6 is not absent.

[0173] In some embodiments, each Z6 is independently -C 1~10 Alkyl-R3-, -C 1~10 Alkyl-NH-, -C 1~10 Alkyl-C(O)-, -C 1~10 Alkyl-O-, -C 1~10 Alkyl-S-, or -(C 1~10 alkyl-R3) g1 -C 1~10 For example, g1 is an integer of 1 to 4.

[0174] In some embodiments, when z is 2 or 3, at least one Z6 is -C 1~10 Alkyl-R3-, -C 1~10 Alkyl-NH-, -C 1~10 Alkyl-C(O)-, -C 1~10 Alkyl-O-, -C 1~10 Alkyl-S-, or -(C 1~10 alkyl-R3) g1 -C1~10 For example, g1 is an integer of 1 to 4.

[0175] In some embodiments, each Z6 independently, or at least one Z6 is -C 2~10 Alkyl-C(O)-, for example, -(CH2)2-C(O)-.

[0176] In some embodiments, each Z6 independently, or at least one Z6 is -C 2~10 Alkyl-R3-C 2~10 Alkyl-C(O)-, for example, -(CH2)2-C(O)NH-(CH2)2-C(O)-.

[0177] In some embodiments, each Z6 independently, or at least one Z6 is -(C 2~10 alkyl-R3) g1 -C 2~10 Alkyl-C(O)-, for example, -(CH2)2-C(O)NH-(CH2)2-NHC(O)-(CH2)-C(O)-.

[0178] In one embodiment, -[(Z5)-(Z6)] z - is not non-existence.

[0179] In one embodiment, -[(Z5)-(Z6)] z - is a bond.

[0180] In one embodiment, -[(Z5)-(Z6)] z - is -(CH2CH2O)2-(CH2)2-C(O)-NH-(CH2CH2O)2-.

[0181] In some embodiments, M P If exists, TIFF2025114641000101.tif148154, where * is L P’ or L P and ** means connection to L M means connection to; R3, R5, R 17 , and R 23 is as defined herein; R4 is a bond or -NR5-(CR 20 R 21 )-C(O)-; Each R 20 and R 21 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; each b1 is independently an integer from 0 to 6; e1 is an integer from 0 to 8, each f1 is independently an integer from 1 to 6; and g2 is an integer of 1 to 4.

[0182] In some embodiments, b1 is 1.

[0183] In some embodiments, each f1 is independently 1 or 2.

[0184] In some embodiments, f1 is 2.

[0185] In some embodiments, g2 is 1 or 2.

[0186] In some embodiments, g2 is 2.

[0187] In some embodiments, R 17 is non-substituted.

[0188] In some embodiments, R 17 may be substituted.

[0189] In some embodiments, R 17 is a base unit, e.g., -(CH2) x NH2, -(CH2) x NHR a , and -(CH2) x N(R a )2, where x is an integer from 1 to 4, and each R a independently, C 1~6 Alkyl and C 1~6 haloalkyl, or two R a The groups combine with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl, or piperidinyl group.

[0190] In some embodiments, R 17 -C 2~5 alkylene -C(=O)-, where alkylene is a base unit, e.g., -(CH) x NH2, -(CH2) x NHR a , and -(CH2) x N(R a )2, where x and R a is as defined herein.

[0191] In some embodiments, M P If exists, TIFF2025114641000102.tif89150, where * is L P’ or L P and ** means connection to L M means connection to

[0192] In some embodiments, M P If exists, TIFF2025114641000103.tif11128, where * is L P’ or L P and ** means connection to L M means connection to

[0193] In some embodiments, M P If exists, TIFF2025114641000104.tif14128, where * is L P’ or L P and ** means connection to L M means connection to

[0194] L M and W M L M is a bond, or a multi-armed linker (e.g., trivalent or tetravalent, or having three or four arms), where each arm can be the same or different.

[0195] In some embodiments, a2 is 2 and L M teeth, TIFF2025114641000105.tif82128TIFF2025114641000106.tif145128, where TIFF2025114641000107.tif12128, if it exists, P Connection to or M P If there is no L P or L P’ means connection to; Y1, if present, is L 3 Connection to or L 3 If there is no M A means connection to; R and R′ are each independently hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, optionally substituted C 3~19 Branched alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10 Aryl, optionally substituted heteroaryl, optionally substituted C 1~6 Heteroalkyl, C1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, optionally substituted C 2~6 Alkanoyl, optionally substituted C 2~6 Alkanoyloxy, optionally substituted C 2~6 Substituted alkanoyloxy, -COOH, or -COO-C 1~6 is alkyl; each of c1, c2, c3, c4, c5, c7, and c8 is independently an integer ranging from 0 to 10; and Each of d1, d2, d3, d4, d5, and d7 is independently an integer in the range of 0 to 10.

[0196] In some embodiments, a2 is 2 and L M teeth, TIFF2025114641000108.tif31128 For example, The file is TIFF2025114641000109.tif33128.

[0197] In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are each independently 0 or 1.

[0198] In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0199] In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are each independently 0, 1, or 2.

[0200] In some embodiments, d1, d2, d3, d4, d5, and d7 are each independently 0 or 1.

[0201] In some embodiments, d1, d2, d3, d4, d5, and d7 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0202] In some embodiments, d1, d2, d3, d4, d5, and d7 are each independently 1, 2, 3, or 4.

[0203] In some embodiments, R and R′ are each independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl.

[0204] In some embodiments, R and R′ are each independently hydrogen or C 1~6 It is alkyl.

[0205] In some embodiments, R2 and R'2 are each independently hydrogen.

[0206] In some embodiments, R and R′ are each independently selected from the group consisting of C 1~6 It is alkyl.

[0207] In some embodiments, L M teeth, TIFF2025114641000110.tif179143TIFF2025114641000111.tif54128.

[0208] In some embodiments, a2 is 3 and L M teeth, TIFF2025114641000112.tif154134TIFF2025114641000113.tif208140TIFF2025114641000114.tif93135; where: TIFF2025114641000115.tif12128, if it exists, is PConnection to or M P If there is no L P or L P’ means connection to; Y1, if present, is L 3 Connection to or L 3 If there is no M A means connection to; R and R′ are each independently hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, optionally substituted C 3~19 Branched alkyl, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 6~10 Aryl, optionally substituted heteroaryl, optionally substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6 Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, optionally substituted C 2~6 Alkanoyl, optionally substituted C 2~6 Alkanoyloxy, optionally substituted C 2~6 Substituted alkanoyloxy, -COOH, or -COO-C 1~6 is alkyl; each of c1, c2, c3, c4, c5, c6, c7, and c8 is independently an integer ranging from 0 to 10; each of d1, d2, d3, d4, d5, d6, d7, and d8 is independently an integer ranging from 0 to 10; and Each of e1, e2, e3, e4, e5, e6, e7, and e8 is independently an integer in the range of 0 to 10.

[0209] In some embodiments, a2 is 3 and L M teeth, TIFF2025114641000116.tif33128, for example, The file is TIFF2025114641000117.tif33128.

[0210] In some embodiments, -L M -(L 3 ) a2 -teeth, TIFF2025114641000118.tif47128.

[0211] In some embodiments, a2 is 2 and L M teeth, Selected from TIFF2025114641000119.tif166140, where: The wavy line indicates the site of attachment within a conjugate of the present disclosure or an intermediate thereof; R 110 teeth, TIFF2025114641000120.tif93158TIFF2025114641000121.tif146158, where the asterisk indicates the connection to the carbon marked with an x, and the wavy line indicates one of the three connection sites; R 100 are independently hydrogen or -C 1~3 alkyl; Y is N or CH; each occurrence of Y' is independently selected from NH, O, or S; and Each occurrence of c' is independently an integer from 1 to 10.

[0212] In some embodiments, R 100 is independently selected from hydrogen and CH3.

[0213] In some embodiments, Y is N.

[0214] In some embodiments, Y is CH.

[0215] In some embodiments, R 100is H or CH3.

[0216] In some embodiments, each c' is independently an integer from 1 to 3.

[0217] In some embodiments, R 110 teeth, Not TIFF2025114641000122.tif12128.

[0218] In some embodiments, when an AA unit has two attachment sites (i.e., terminal drug units), one of the attachment sites shown above is selected from, for example, H, OH, or C. 1~3 It may be replaced by an unsubstituted alkyl group.

[0219] L M is a multi-arm drinker, and the stretcher unit M P If it is not already connected to W M L M and W M Each occurrence of independently may be replaced by forming a hydrogen, a protecting group, a leaving group, or a covalent bond to L M M P is a functional group that can be linked to

[0220] In some embodiments, W M is an amine protecting group, e.g., BOC. In some embodiments, W M is an amine protecting group, e.g., BOC, and L M teeth, TIFF2025114641000123.tif31128, for example, TIFF2025114641000124.tif33128.

[0221] In some embodiments, W M represents an amine group, e.g., -C(O)-(CH2) w -NH2, where w is an integer from 1 to 6.

[0222] In some embodiments, W M is —C(O)—CH—NH. In some embodiments, W M is -C(O)-CH2-NH2, and L M teeth, TIFF2025114641000125.tif33128, for example, TIFF2025114641000126.tif33128.

[0223] In some embodiments, W M is hydrogen.

[0224] M A M A is a compound containing one or more drugs and one or more hydrophilic groups. P or L P’ In some embodiments, M A comprises a peptide moiety of at least two amino acids (AA).

[0225] The peptide portion is -L D -AA units are moieties capable of forming covalent bonds with -D units, allowing for the attachment of multiple drugs. In some embodiments, the peptide moiety comprises a single AA unit, or has two or more AA units (e.g., 2 to 10, preferably 2 to 6, e.g., 2, 3, 4, 5, or 6), where each AA unit is independently a natural or unnatural amino acid, amino alcohol, amino aldehyde, diamine, or polyamine, or a combination thereof. Optionally, to achieve the required number of attachments, at least one of the AA units may be a -L D The AA units have functionalized side chains to provide for attachment of the -D units. Exemplary functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes) include, for example, azide- or alkyne-functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes modified using click chemistry to have an azide or alkyne group for attachment).

[0226] In some embodiments, the peptide moiety has 2 to 12 AA units.

[0227] In some embodiments, the peptide moiety has 2 to 10 AA units.

[0228] In another embodiment, the peptide moiety has 2 to 6 AA units.

[0229] In yet other embodiments, the peptide moiety has 2, 3, 4, 5, or 6 AA units.

[0230] In some embodiments, the AA units are (e.g., L M , a hydrophilic group or another AA unit, and -L D -D units) has three attachment sites. For example, the AA unit has the formula: TIFF2025114641000127.tif31128, where the wavy line indicates the site of attachment within a conjugate of the present disclosure or an intermediate thereof; and R 100 and R 110 is as defined herein.

[0231] In some embodiments, the AA unit has two attachment sites (i.e., terminal units), and one of the attachment sites shown above is, for example, H, OH, or unsubstituted C. 1~3 It may be substituted by an alkyl group.

[0232] In some embodiments, the peptide moiety comprises at least two AA units of the following formula: TIFF2025114641000128.tif31128, wherein: Each R 111are independently H, p-hydroxybenzyl, methyl, isopropyl, isobutyl, sec-butyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, TIFF2025114641000129.tif30156; The wavy line indicates the site of attachment within the conjugate or its intermediate; and R 100 and R 110 is as defined herein.

[0233] In some embodiments, the peptide moiety comprises at least two AA units, for example, cysteine-alanine as shown below: TIFF2025114641000130.tif46143, where the wavy line and asterisk indicate the site of attachment within the conjugate or its intermediate. For example, the asterisk represents -L D -D unit or hydrophilic group attachment site. For example, the wavy line adjacent to the carbonyl group represents -L D -D unit or hydrophilic group attachment site. For example, the wavy line adjacent to the amine group indicates the -L D -D units or hydrophilic groups. For example, one or two of the wavy lines and asterisks may represent one or more -L D - indicates the attachment site of the D unit or one or more hydrophilic groups.

[0234] In some embodiments, the peptide moiety comprises at least two AA units, such as cysteine-alanine, as shown below, providing two connection sites: TIFF2025114641000131.tif46156, where the wavy line and asterisk indicate the site of attachment within the conjugate or its intermediate. For example, the asterisk represents -L D -D unit or hydrophilic group attachment site. For example, the wavy line indicates -L D -Indicates the attachment site of the D unit or hydrophilic group.

[0235] One or more AA units (e.g., amino acids, amino alcohols, amino aldehydes, or polyamines) of the peptide moiety may be optionally substituted C 1~20 Heteroalkylene (e.g., optionally substituted C 1~12 heteroalkylene), optionally substituted C 3~8 Heterocyclo, optionally substituted C 6~14 Arylene or optionally substituted C 3~8 The optionally substituted heteroalkylene, heterocycle, arylene, or carbocyclo may have one or more functional groups for connection within the conjugate or its intermediate. Suitable substituents include (=O), -R 1C , -R 1B , -OR 1B , -SR 1B , -N(R 1B )2, -N(R 1B )3, =NR 1B , C(R 1C )3, CN, OCN, SCN, N=C=O, NCS, NO, NO2, =N2, N3, NR 1B C(=O)R 1B , -C(=O)R 1B , -C(=O)N(R 1B )2, SO3 - , SO3H, S(=O)2R 1B , -OS(=O)2OR 1B , -S(=O)2NR 1B , -S(=O)R 1B,-OP(=O)(OR 1B )2, -P(=O)(OR 1B )2, PO3 - , PO3H2, AsO2H2, C(=O)R 1B , C(=O)R 1C , C(=S)R 1B , CO2R 1B , CO2-, C(=S)OR 1B , C(=O)SR 1B , C(=S)SR 1B , C(=O)N(R 1B )2, C(=S)N(R 1B )2, and C(=NR 1B )N(R 1B ) 2, where each R 1C is independently a halogen (e.g., —F, —CI, —Br, or —I), and each R 1B are independently -H, -C 1~20 Alkyl, -C 6~20 Aryl, -C 3~14 It is a heterocycle, a protecting group, or a prodrug moiety.

[0236] In some embodiments, one or more substituents on the heteroalkylene, heterocycle, arylene, or carbocyclo is (═O), R 1C , R 1B , OR 1B , S.R. 1B , and N(R 1B )2 is selected.

[0237] In some embodiments, the peptide moiety is linear or has the formula: TIFF2025114641000132.tif16128, wherein Each B-B' is independently an amino acid, optionally substituted C 1~20 Heteroalkylene (e.g., optionally substituted C 1~12 heteroalkylene), optionally substituted C 3~8 Heterocyclo, optionally substituted C 6~14 arylene, or optionally substituted C3-C8 carbocyclo; d 12 is an integer between 1 and 10; and The wavy lines indicate the site of covalent attachment within the conjugate or its intermediate.

[0238] In some embodiments, d 12 is an integer between 2 and 10.

[0239] In some embodiments, d 12 is an integer between 2 and 6.

[0240] In some embodiments, d 12 is an integer 4, 5, or 6.

[0241] In some embodiments, d 12 is an integer between 5 and 6.

[0242] In some embodiments, the optionally substituted heteroalkylene, heterocycle, arylene, or carbocyclo has functional groups for connecting between BB' subunits and / or for connecting within a conjugate or intermediate thereof disclosed herein.

[0243] In some embodiments, the peptide moiety comprises no more than two optionally substituted C 1~20 Heteroalkylene, optionally substituted C 3~18 Heterocyclo, optionally substituted C 6~14 Arylene or optionally substituted C 3~8 Contains carbocyclo.

[0244] In other embodiments, the peptide moiety contains no more than one optionally substituted C 1~20 Heteroalkylene, optionally substituted C 3~18 Heterocyclo, optionally substituted C 6~14 Arylene or optionally substituted C 3~8The optionally substituted heteroalkylene, heterocycle, arylene, or carbocyclo has a functional group for connecting between BB' subunits and / or within the conjugates or intermediates thereof disclosed herein.

[0245] In some embodiments, at least one BB ’ is an amino acid. For example, the amino acid may be an alpha, beta, or gamma amino acid, which may be natural or unnatural. The amino acid may be a D or L isomer.

[0246] In some embodiments, the connection within the peptide moiety or with other components of the conjugate (or its intermediate, or scaffold) can be, for example, by amino, carboxy, or other functionality.

[0247] In one embodiment, each amino acid of the peptide moiety can independently be the D or L isomer of a thiol-containing amino acid, which can be, for example, cysteine, homocysteine, or penicillamine.

[0248] In another embodiment, each amino acid comprising the peptide moiety can independently be the L or D isomer of the following amino acids: alanine (including β-alanine), arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynols, aminoalkanedioics, heterocyclo-carboxylic acids, citrulline, statins, diaminoalkanoic acids, stereoisomers thereof (e.g., isoaspartic acid and isoglutamic acid), and derivatives thereof.

[0249] In one embodiment, each amino acid comprising the peptide moiety is independently cysteine, homocysteine, penicillamine, ornithine, lysine, serine, threonine, glycine, glutamine, alanine, aspartic acid, glutamic acid, selenocysteine, proline, glycine, isoleucine, leucine, methionine, valine, alanine, or a stereoisomer thereof (e.g., isoaspartic acid and isoglutamic acid).

[0250] In some embodiments, the peptide moiety comprises a monopeptide, dipeptide, tripeptide, tetrapeptide, or pentapeptide.

[0251] In some embodiments, the peptide portion contains at least about 5 amino acids (eg, 5, 6, 7, 8, 9, or 10 amino acids).

[0252] In some embodiments, the peptide portion contains at most about 10 amino acids.

[0253] In one embodiment, the peptide moiety comprises a pentapeptide.

[0254] In one embodiment, each amino acid comprising the peptide moiety is independently glycine, serine, glutamic acid, lysine, aspartic acid, and cysteine.

[0255] In another embodiment, the peptide moiety contains at least four glycines and at least one serine, e.g., (glycine)4 and serine (wherein the serine is at any position along the peptide chain), e.g., (serine)-(glycine)4; (glycine)-(serine)-(glycine)3; (glycine)2-(serine)-(glycine)2; (glycine)3-(serine)-(glycine); or (glycine)4-(serine).

[0256] In another embodiment, the peptide moiety comprises (glycine)4-(serine) or (serine)-(glycine)4.

[0257] In another embodiment, the peptide moiety contains at least four glycines and at least one glutamic acid, e.g., (glycine)4 and glutamic acid (wherein the glutamic acid is at any position along the peptide chain), e.g., (glutamic acid)-(glycine)4; (glycine)-(glutamic acid)-(glycine)3; (glycine)2-(glutamic acid)-(glycine)2; (glycine)3-(glutamic acid)-(glycine); or (glycine)4-(glutamic acid).

[0258] In another embodiment, the peptide moiety comprises (glutamic acid)-(glycine)4 or (glycine)4-(glutamic acid).

[0259] In another embodiment, the peptide moiety comprises (β-alanine)-(glycine)-(serine), where serine is at any position along the peptide chain, e.g., (β-alanine)-(serine)-(glycine)-; (β-alanine)-(glycine)-(serine)-(glycine)-; (β-alanine)-(glycine)-(serine)-(glycine)-; (β-alanine)-(glycine)-(serine)-(glycine); or (β-alanine)-(glycine)-(serine).

[0260] In another embodiment, the peptide moiety comprises (glycine)4-(serine)-(glutamic acid) (where serine is at any position along the peptide chain), e.g., (serine)-(glycine)4-(glutamic acid); (glycine)-(serine)-(glycine)3-(glutamic acid); (glycine)2-(serine)-(glycine)2-(glutamic acid); (glycine)3-(serine)-(glycine)-(glutamic acid); or (glycine)4-(serine)-(glutamic acid). In another embodiment, the peptide moiety comprises (β-alanine)-(glycine)4-(serine)-(glutamic acid) (where serine is at any position along the peptide chain), e.g., (β-alanine)-(serine)-(glycine)4-(glutamic acid); (β-alanine)-(glycine)-(serine)-(glycine)3-(glutamic acid); (β-alanine)-(glycine)2-(serine)-(glycine)2-(glutamic acid); (β-alanine)-(glycine)3-(serine)-(glycine)-(glutamic acid); or (β-alanine)-(glycine)4-(serine)-(glutamic acid).

[0261] In other embodiments, the hydrophilic group (or T 1 ) is a polyalcohol or a derivative thereof (e.g., an aminopolyalcohol) or a glucosylamine or a di-glucosylamine or a tri-glucosylamine, A does not need to contain a peptide moiety, e.g., M A L M For example, M A includes one or more of the following: TIFF2025114641000133.tif136147 where, The wavy line indicates the site of attachment within a conjugate of the present disclosure or an intermediate thereof; and R 100 and R 110 is as defined herein.

[0262] In some embodiments, R 110 teeth, TIFF2025114641000134.tif251158, where the asterisk indicates the connection to the carbon marked with an x, and the wavy line indicates one of the three connection sites.

[0263] In some embodiments, R 100 is independently selected from hydrogen and CH3.

[0264] In some embodiments, Y is N.

[0265] In some embodiments, Y is CH.

[0266] In some embodiments, R 100 is H or CH3.

[0267] In some embodiments, each c' is independently an integer from 1 to 3.

[0268] In some embodiments, R 110 teeth, Not TIFF2025114641000135.tif12128.

[0269] L D and W D L D Each occurrence of independently converts D to M A and contains at least one cleavable bond, such that upon bond rupture D is released in an active form for its intended therapeutic effect.

[0270] In some embodiments, L D is a component of a releasable assembly unit. D is a releasable assembly unit.

[0271] In some embodiments, L D contains one cleavable bond.

[0272] In some embodiments, L D contains multiple cleavage sites or bonds.

[0273] Functional groups for forming cleavable bonds can include, for example, sulfhydryl groups for forming disulfide bonds, aldehyde, ketone, or hydrazine groups for forming hydrazone bonds, hydroxylamine groups for forming oxime bonds, carboxyl or amino groups for forming peptide bonds, carboxyl or hydroxy groups for forming ester bonds, and sugars for forming glycosidic bonds. D contains a disulfide bond that is cleavable by disulfide exchange, an acid-labile bond that is cleavable at acidic pH, and / or a bond that is cleavable by hydrolytic enzymes (e.g., peptidases, esterases, and glucuronidases). D contains a carbamate bond (i.e., -OC(O)-NR-, where R is H or alkyl, etc.).

[0274] L D The structure and sequence of the cleavable bond in the can be such that the bond is cleaved by the action of an enzyme present at the target site. In other embodiments, the cleavable bond can be cleavable by other mechanisms.

[0275] In some embodiments, the cleavable bond can be enzymatically cleaved by one or more enzymes, including tumor-associated proteases, to liberate the Drug unit or D, which, in one embodiment, becomes protonated in vivo upon release to provide the Drug unit or D.

[0276] In one embodiment, L D can contain one or more amino acids, provided that a cleavable bond is present, e.g., L D Each amino acid in may be natural or unnatural and / or D or L isomer. Dcomprises an alpha, beta, or gamma amino acid, which may be natural or unnatural. D comprises a contiguous sequence of 1 to 12 (e.g., 1 to 6, or 1 to 4, or 1 to 3, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acids.

[0277] In one embodiment, L D In other embodiments, L may contain only natural amino acids. D In some embodiments, L can contain only unnatural amino acids. D may comprise a natural amino acid linked to an unnatural amino acid. D can include a natural amino acid linked to a D isomer of a natural amino acid. D includes dipeptides such as -Val-Cit-, -Phe-Lys-, or -Val-Ala-.

[0278] In some embodiments, L D comprises a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit.

[0279] In some embodiments, L D comprises a peptide (e.g., of 1 to 12 amino acids) directly conjugated to a Drug Unit. In some such embodiments, the peptide is a single amino acid or a dipeptide.

[0280] In some embodiments, L DEach amino acid in is independently selected from alanine, beta-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, penicillamine, aminoalkanoic acids, aminoalkynic acids, aminoalkanedioic acids, aminobenzoic acids, amino-heterocyclo-alkanoic acids, heterocyclo-carboxylic acids, citrulline, statins, diaminoalkanoic acids, and derivatives thereof.

[0281] In some embodiments, each amino acid is independently selected from alanine, beta-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, citrulline, and selenocysteine.

[0282] In some embodiments, each amino acid is independently selected from the group consisting of alanine, beta-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, citrulline, and derivatives thereof.

[0283] In some embodiments, each amino acid is selected from a proteinogenic amino acid or a non-proteinogenic amino acid.

[0284] In some embodiments, L DEach amino acid in may be independently selected from the L or D isomer of the following amino acids: alanine, β-alanine, arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynol, aminoalkanedioic acid, heterocyclo-carboxylic acid, citrulline, statin, diaminoalkanoic acid, valine, citrulline, or a derivative thereof.

[0285] In some embodiments, L D Each amino acid in is independently cysteine, homocysteine, penicillamine, ornithine, lysine, serine, threonine, glycine, glutamine, alanine, aspartic acid, glutamic acid, selenocysteine, proline, glycine, isoleucine, leucine, methionine, valine, citrulline, or alanine.

[0286] In some embodiments, L D Each amino acid in is independently selected from the L-isomer of the following amino acids: alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, or valine.

[0287] In some embodiments, L D Each amino acid in is independently selected from the D-isomer of the following amino acids: alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, or valine.

[0288] In some embodiments, L DEach amino acid in is alanine, beta-alanine, glutamic acid, isoglutamic acid, isoaspartic acid, valine, citrulline, or aspartic acid.

[0289] In one embodiment, L D contains β-alanine.

[0290] In another embodiment, L D contains (β-alanine)-(alanine).

[0291] In another embodiment, L D contains (β-alanine)-(glutamic acid).

[0292] In another embodiment, L D contains (β-alanine)-(isoglutamic acid).

[0293] In another embodiment, L D contains (β-alanine)-(aspartic acid).

[0294] In another embodiment, L D contains (β-alanine)-(isoaspartic acid).

[0295] In another embodiment, L D contains (β-alanine)-(valine).

[0296] In another embodiment, L D contains (β-alanine)-(valine)-(alanine).

[0297] In another embodiment, L D contains (β-alanine)-(alanine)-(alanine).

[0298] In another embodiment, L D contains (β-alanine)-(valine)-(citrulline).

[0299] In some embodiments, L Dcontains one or more amino acids plus a carbamate bond.

[0300] In one embodiment, L D can be designed and optimized in selectivity for enzymatic cleavage by a particular enzyme, such as a tumor-associated protease.

[0301] In one embodiment, L D contains a bond whose cleavage is catalyzed by cathepsins B, C, and D, or plasmin proteases.

[0302] In another embodiment, L D In some such embodiments, L D comprises a sugar moiety (Su) linked via an oxygen glycosidic bond to a self-immolative group. A "self-immolative group" is a group consisting of three spaced chemical moieties: a sugar moiety (via a glycosidic bond), a drug unit (directly or indirectly), and an M A The glycosidic bond may be a trifunctional chemical moiety capable of covalently linking (directly or indirectly) the glycosidic bond together, which can be cleaved at the target site to initiate a self-immolative reaction sequence leading to the release of the drug.

[0303] For example, L D is the expression: TIFF2025114641000136.tif32128, wherein the self-immolative group (K) forms a covalent bond (directly or indirectly) with a Drug unit; and further comprising: M A Examples of self-immolative groups are described, for example, in WO 2015 / 057699, the contents of which are incorporated herein by reference in their entirety.

[0304] When not linked to a drug or before being linked to a drug, L D is the functional group W D Each WD is independently, W P For example, each W D is, independently, TIFF2025114641000137.tif189143TIFF2025114641000138.tif169154, where R 1A is a sulfur protecting group, each of rings A and B is independently cycloalkyl or heterocycloalkyl, and R W is an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; Ring D is a heterocycloalkyl; R 1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; and R 1K is a leaving group (e.g., a halide or RC(O)O-, where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety).

[0305] In some embodiments, W D teeth, TIFF2025114641000139.tif18128.

[0306] In some embodiments, W D teeth, TIFF2025114641000140.tif22128, where X a and X b One of the is H and the other is a maleimide blocking moiety.

[0307] In some embodiments, W D teeth, The file is TIFF2025114641000141.tif14128.

[0308] Therapeutic Agent, Drug Unit, or D In certain embodiments, the therapeutic agent is a small molecule, preferably having a molecular weight of ≦about 5 kDa, more preferably ≦about 4 kDa, more preferably ≦about 3 kDa, and most preferably ≦about 1.5 kDa or ≦about 1 kDa.

[0309] In some embodiments, the therapeutic agent has an IC of less than about 1 nM 50 It has.

[0310] In another embodiment, the therapeutic agent has an IC of greater than about 1 nM 50 For example, the therapeutic agent has an IC of about 1-50 nM. 50 It has.

[0311] IC greater than approximately 1 nM 50 Some therapeutic agents having an IC of 0.05 or less (e.g., "low-potency drugs") are unsuitable for conjugation to antibodies using art-recognized conjugation techniques. Without wishing to be bound by theory, such therapeutic agents have insufficient potency for use in targeting antibody-drug conjugates using conventional techniques because sufficient copies of the drug (i.e., more than eight) cannot be conjugated using art-recognized techniques without compromising the pharmacokinetic and physiochemical properties of the conjugate. However, using the conjugation strategies described herein, sufficiently high loading of these low-potency drugs can be achieved, thereby resulting in high loading of the therapeutic agent while maintaining desirable pharmacokinetic and physiochemical properties. Accordingly, the present disclosure also relates to antibody-drug conjugates comprising an antibody, a scaffold, and at least eight therapeutic agent moieties, wherein the therapeutic agent has an IC of greater than about 1 nM. 50 It has.

[0312] Small molecule therapeutics (e.g., antiproliferative (cytotoxic and cytostatic) agents that can be linked to a targeting moiety via a linker of the present disclosure) for use in the present disclosure include cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PI3K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperone inhibitors, HDAC inhibitors, PARP inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, Wnt / hedgehog signaling pathway inhibitors, and RNA polymerase inhibitors.

[0313] Broad spectrum cytotoxins include, but are not limited to, DNA-binding intercalating or alkylating drugs, microtubule stabilizing and destabilizing agents, platinum compounds, topoisomerase I inhibitors, and protein synthesis inhibitors.

[0314] Exemplary DNA-binding intercalating or alkylating agents include CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin, nemorubicin, and its derivatives, PNU-159682), bisnapthalimide compounds such as elinafide (LU79553) and its analogs, alkylating agents such as calicheamicins, dactinomycins, mitomycins, pyrrolobenzodiazepines, and the like. Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin A, duocarmycin C1, duocarmycin C2, duocarmycin B1, duocarmycin B2, duocarmycin D, DU-86, KW-2189, adozelesin, bizelesin, carzelesin, seco-adozelesin, and related analogs and prodrug forms, examples of which are described in U.S. Patent Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and 7,049,316. Doxorubicin and its analogs include those described in U.S. Patent No. 6,630,579. Calicheamicins include, for example, enediynes, such as esperamicin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116. Duocarmycins include those described in U.S. Patent Nos. 5,070,092; 5,101,038; 5,187,186; 6,548,530; 6,660,742; and 7,553,816 B2; and Li et al., Tet Letts., 50:2932-2935 (2009).

[0315] Pyrrolobenzodiazepines (PBDs) and their analogs include those described in Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000) and Antonow and Thurston, Chem Rev., 2815-2864 (2010).

[0316] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and cabazitaxel; maytansinoids, auristatins and their analogs, vinca alkaloid derivatives, epothilones, and cryptophycins.

[0317] Exemplary maytansinoids or maytansinoid analogs include maytansinol and maytansinol analogs, maytansine or DM-1 and DM-4, and those described in U.S. Patent Nos. 5,208,020; 5,416,064; 6,333,410; 6,441,163; 6,716,821; RE39,151; and 7,276,497. In certain embodiments, the cytotoxic agent is a maytansinoid, another group of antitubulin agents (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131), maytansinoids, or maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Suitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

[0318] Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F, auristatin F phenylenediamine (AFP), auristatin F hydroxylpropylamide (AF HPA), monomethyl auristatin F hydroxylpropylamide (MMAF HPA), and dolastatin. Suitable auristatins also include those described in U.S. Patent Application Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248; PCT Application Publication Nos. WO 09 / 117531, WO 2005 / 081711, WO 04 / 010957; WO 02 / 088172 and WO 01 / 24763, and U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065; 5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; Nos. 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entireties.

[0319] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). Suitable vinca alkaloids that can be used in the present disclosure are also disclosed in U.S. Patent Application Publication Nos. 2002 / 0103136 and 2010 / 0305149, and U.S. Patent No. 7,303,749 B1, the disclosures of which are incorporated herein by reference in their entireties.

[0320] Exemplary epothilone compounds include epothilones A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Pat. Nos. 6,956,036; 6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145; and 5,886,026; and WO 97 / 19086; WO 98 / 08849; WO 98 / 22461; WO 98 / 25929; WO 98 / 38192; WO 99 / 01124; WO 99 / 02514; WO 99 / 03848; WO 99 / 07692; WO 99 / 27890; and WO 99 / 28324; the disclosures of which are incorporated herein by reference in their entireties.

[0321] Exemplary cryptophycin compounds are described in US Pat. Nos. 6,680,311 and 6,747,021.

[0322] Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin (PARAPLATIN®), oxaliplatin (ELOXATINE®), iproplatin, ormaplatin, and tetraplatin.

[0323] Still other classes of compounds or compounds with these or other cytotoxic modes of action may be selected, including, for example, mitomycin C, mitomycin A, daunorubicin, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, aminopterin, bleomycin, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol, pyrrolobenzodiazepine (PBD) polyamides and dimers thereof. Other suitable cytotoxic agents include, for example, puromycins, topotecan, rhizoxin, echinomycin, combretastatin, netropsin, estramustine, cryptophycins, cemadotin, discodermolide, eleutherobin, and mitoxantrone.

[0324] Exemplary topoisomerase I inhibitors include camptothecin, camptothecin derivatives, camptothecin analogs, and unnatural camptothecins, such as CPT-11 (irinotecan), SN-38, GI-147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, rubitecan, gimatecan, karenitecin, ciratecan, lurtotecan, ixatecan, diflomotecan, belotecan, lurtotecan, and S39625. Other camptothecin compounds that can be used in the present disclosure include, for example, those described in J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30:1774 (1987).

[0325] Angiogenesis inhibitors include but are not limited to MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors.Exemplary VGFR and PDGFR inhibitors include sorafenib (Nexavar), sunitinib (Sutent) and vatalanib.Exemplary MetAP2 inhibitors include fumagillol analogues, which means any compound that contains fumagillin core structure, including fumagylamine, which inhibits the ability of MetAP-2 to remove NH2-terminal methionine from protein, as described in Rodeschini et al., J. Org. Chem., 69, 357-373, 2004 and Liu et al., Science 282, 1324-1327, 1998. Non-limiting examples of "fumagillol analogs" are described in J. Org. Chem., 69, 357, 2004; J.Org. Chem., 70, 6870, 2005; European Patent Application 0 354 787; J. Med. Chem., 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg. Med. Chem., 14, 91, 2004; Tet. Lett. 40, 4797, 1999; WO99 / 61432; and 6,207,704.

[0326] Exemplary cell cycle progression inhibitors include CDK inhibitors, such as BMS-387032 and PD0332991; Rho-kinase inhibitors, such as GSK429286; checkpoint kinase inhibitors, such as AZD7762; Aurora kinase inhibitors, such as AZD1152, MLN8054, and MLN8237; PLK inhibitors, such as BI 2536, BI6727 (vorasertib), GSK461364, ON-01910 (Estybon); and KSP inhibitors, such as SB 743921, SB 715992 (ispinesib), MK-0731, AZD8477, AZ3146, and ARRY-520.

[0327] Exemplary PI3K / m-TOR / AKT signaling pathway inhibitors include phosphoinositide 3 kinase (PI3K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors, and PDK-1 inhibitors.

[0328] Exemplary PI3 kinase inhibitors are disclosed in U.S. Pat. No. 6,608,053 and include BEZ235, BGT226, BKM120, CAL101, CAL263, demethoxyviridine, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PI-103, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, wortmannin, XL147, and XL765.

[0329] Exemplary AKT inhibitors include, but are not limited to, AT7867.

[0330] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf, and p38 MAPK inhibitors.

[0331] Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,994 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, RO5126766 and RO4987655, PD0325901, AZD6244, AZD 8330, and GDC-0973.

[0332] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0333] Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820, and SB 202190.

[0334] Receptor tyrosine kinase (RTK) is a cell surface receptor that is often involved in the signal transduction pathway that stimulates the unlimited proliferation and angiogenesis of cancer cells.Many RTKs have been identified that are overexpressed or have mutations that lead to the constitutive activation of receptors, including but not limited to VEGFR, EGFR, FGFR, PDGFR, EphR and RET receptor family receptors.Exemplary specific RTK targets include ErbB2, FLT-3, c-Kit and c-Met.

[0335] Exemplary inhibitors of the ErbB2 receptor (EGFR family) include, but are not limited to, AEE788 (NVP-AEE 788), BIBW2992, (afatinib), lapatinib, erlotinib (Tarceva), and gefitinib (Iressa).

[0336] Exemplary RTK inhibitors that target more than one signaling pathway (multiple-targeted kinase inhibitors) include AP24534 (ponatinib), which targets FGFR, FLT-3, VEGFR-PDGFR, and Bcr-Abl receptors; ABT-869 (linifanib), which targets FLT-3 and VEGFR-PDGFR receptors; AZD2171, which targets VEGFR-PDGFR, Flt-1, and VEGF receptors; CHR-258 (dovitinib), which targets VEGFR-PDGFR, FGFR, Flt-3, and c-Kit receptors; sunitinib (Sutent), which targets VEGFR, PDGFR, KIT, FLT-3, and CSF-IR; and sorafenib (Nexavar) and vatalanib, which target intracellular serine / threonine kinases in the VEGFR, PDGFR, and Raf / Mek / Erk pathways.

[0337] Exemplary protein chaperone inhibitors include HSP90 inhibitors.Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922, and KW-2478.

[0338] Exemplary HDAC inhibitors include belinostat (PXD101), CUDC-101, droxinostat, ITF2357 (gibinostat, gabinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, dacinostat), LBH-589 (panobinostat), MC1568, MGCD0103 (mosetinostat), MS-275 (entinostat), PCI-24781, pyroxamide (NSC 696085), SB939, trichostatin A, and vorinostat (SAHA).

[0339] Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (veliparib), AG014699, CEP 9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

[0340] Exemplary NAMPT inhibitors include FK866 (APO866) and CHS828, GPP78, GMX1778 (CHS828), STF-118804, STF-31, CB 300919, CB 30865, GNE-617, IS001, TP201565, Nampt-IN-1, P7C3, MPC-9528, CB30865, MPI0479883, and (E)-N-(5-((4-(((2-(1H-indol-3-yl)ethyl)(isopropyl)amino)methyl)phenyl)amino)pentyl)-3-(pyridin-3-yl)acrylamide.

[0341] Exemplary Wnt / hedgehog signaling pathway inhibitors include vismodegib (RG3616 / GDC-0449), cyclopamine (11-deoxojervine) (a hedgehog pathway inhibitor), and XAV-939 (a Wnt pathway inhibitor).

[0342] Exemplary RNA polymerase inhibitors include amatoxins, including α-amanitins, β-amanitins, γ-amanitins, ε-amanitins, amanulin, amanuric acid, amaninamide, amanine, and proamanulin.

[0343] Exemplary protein synthesis inhibitors include trichothecene compounds.

[0344] In one embodiment, the drug is a topoisomerase inhibitor (e.g., a non-natural camptothecin compound), a vinca alkaloid, a kinase inhibitor (e.g., a PI3 kinase inhibitor (GDC-0941 and PI-103)), a MEK inhibitor, a KSP inhibitor, an RNA polymerase inhibitor, a protein synthesis inhibitor, a PARP inhibitor, a NAMPT inhibitor, docetaxel, paclitaxel, doxorubicin, a duocarmycin, an auristatin, a dolastatin, a calicheamicin, topotecan, SN38, camptothecin, ixatecan, nemorubicin and its derivatives, PNU-159682, CC1065, elinafide, a trichothecene, a pyrrolobenzodiazepine, a maytansinoid, a DNA binding drug, or a platinum compound, and analogs thereof. In certain embodiments, the drug is a derivative of SN-38, camptothecin, topotecan, ixatecan, calicheamicin, nemorubicin, PNU-159682, anthracycline, maytansinoid, taxane, trichothecene, CC1065, elinafide, vindesine, vinblastine, PI-103, AZD 8330, dolastatin, auristatin E, auristatin F, duocarmycin compounds, ispinesib, pyrrolobenzodiazepine, ARRY-520, and stereoisomers, isosteres, and analogs thereof.

[0345] In another embodiment, the drug used in the present disclosure is a combination of two or more drugs, such as, for example, a PI3 kinase inhibitor and a MEK inhibitor; a broad-spectrum cytotoxic compound and a platinum compound; a PARP inhibitor, a NAMPT inhibitor, and a platinum compound; a broad-spectrum cytotoxic compound and a PARP inhibitor.

[0346] In yet another embodiment, the drug used in the present disclosure is auristatin F-hydroxypropylamido-L-alanine.

[0347] In one embodiment, the vinca alkaloid is a compound of formula (V1) TIFF2025114641000142.tif84128, During the ceremony, R14 is hydrogen, -C(O)-C 1~3 Alkyl, or -C(O)-chloro substituted C 1~3 is alkyl; R 15 is hydrogen, -CH3, or -CHO; R 17 and R 18 If are chosen independently, R 18 is hydrogen and R 16 and R 17 one of which is ethyl and the other is hydroxyl; R 17 and R 18 When they are taken together with the carbon to which they are attached to form an oxirane ring, R 16 is ethyl; R 19 is hydrogen, OH, amino group, alkylamino, or -[C(R 20 R 21 )] a -R 22 and; R 20 and R 21 each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; R 22 -OH, -NH2, -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2)-NH) d -R 77 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms a nitrogen-containing heterocyclic moiety; R 82 is -NR 23 or oxygen; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; and f is an integer from 1 to 12.

[0348] Further examples of vinca alkaloids are described in US8524214B2 and US 2002 / 0103136.

[0349] In one embodiment, the vinca alkaloid of formula (V1) is a compound of formula (VI1): TIFF2025114641000143.tif83128, During the ceremony, R 40 is hydrogen, -OH, -NH2, or one of the following structures: TIFF2025114641000144.tif125129TIFF2025114641000145.tif160157, where a is an integer from 1 to 6; g is an integer from 2 to 6; and c is an integer of 0 to 3.

[0350] In one embodiment, in formula (VI1), R 40 teeth, The file is TIFF2025114641000146.tif63167.

[0351] In another embodiment, R 40 teeth, The file is TIFF2025114641000147.tif9128.

[0352] In another embodiment, R 40 teeth, TIFF2025114641000148.tif16128.

[0353] In another embodiment, R 40 teeth, TIFF2025114641000149.tif21128.

[0354] In another embodiment, R 40 teeth, The file is TIFF2025114641000150.tif23128.

[0355] In another embodiment, the compound of formula (VI1) can be prepared from a compound of formula (VIa), (VIb), (VIc), (VId), (VIe), or (VIf): TIFF2025114641000151.tif23484TIFF2025114641000152.tif183128TIFF2025114641000153.tif84128

[0356] In another embodiment, the topoisomerase inhibitor is a camptothecin compound of formula (VII1): TIFF2025114641000154.tif57128, During the ceremony, R 24 is —H, —Cl, —F, —OH, or alkyl; or R 24 and R 25 may be taken together to form an optionally substituted five- or six-membered ring; R 25are -H, -F, -OH, -CH3, -CH=NOt-butyl, -CH2CH2Si(CH3)3, -Si((CH3)2)-t-butyl, -OC(O)-R 29 and; R 29 is -NH2, -R 28 -C 1~6 Alkyl-R 22 , 5- to 12-membered heterocycloalkyl, R 28 -C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 or R 29 is R as defined herein 47 and; R 26 is -H, -CH2-N(CH3)2, NH2, or NO2; R 27 is -H, ethyl, N-methylpiperidine, cycloalkyl, -CH2OH, -CH2CH2NHCH(CH3)2, or -N-4-methylcyclohexylamine; R 79 is -H or -C(O)-R 28 -[C(R 20 R 21 )] a -R 22 and; R 20 and R 21 each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; R 22-OH, -NH2, -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; Alternatively, R 26 and R 27 when taken together with the two carbon atoms to which they are attached and a third carbon atom connecting those two carbon atoms, form an optionally substituted six-membered ring; R 28 is non-existence, NR 23 or oxygen; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; f is an integer from 1 to 12; u is an integer 0 or 1; w is an integer 0 or 1; and However, the compound of formula (VII1) is R 29 and R 79It must contain at least one of the following:

[0357] In one embodiment, the camptothecin compound of formula (VII1) is a compound of formula (VIII1), (VIIIa), or (VIIIb), or of formula (XXV) or (XXVa): TIFF2025114641000155.tif20695TIFF2025114641000156.tif39128, During the ceremony, R 30 is -NH2, -R 28 -[C(R 20 R 21 )] a -R 22 , -R 28 -C 1~6 Alkyl-R 22 , 5- to 12-membered heterocycloalkyl, R 28 -C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 22 or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 and; R 28 is non-existence, NR 23 , or oxygen; R 20 and R 21 each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; R 22 -OH, -NH2, -COOH, -R 82 -C(O)(CH2) c -C(H)(R23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NR 23 ) d -R 77 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; and f is an integer from 1 to 12.

[0358] In some embodiments, R 30 is one of the following structures: TIFF2025114641000157.tif118150, where a is an integer from 1 to 6; c is an integer from 0 to 3; and g is an integer of 2 to 6.

[0359] In one embodiment, in formula (VII1), R 30 teeth, TIFF2025114641000158.tif50128.

[0360] In another embodiment, the compound of formula (VII1) can be a compound of formula (VIIa), (VIIb), (VIIc), (VIId), (VIIe), (VIIf), (VIIg), (VIIh), (VIIi), or (VIIj): TIFF2025114641000159.tif206101TIFF2025114641000160.tif201106TIFF2025114641000161.tif192137

[0361] In another embodiment, the PI3 kinase inhibitor is a compound of formula (IX1): TIFF2025114641000162.tif46128, During the ceremony, R 47 is an amino group, -R9-[C(R 20 R 21 )] a -R 10 , -R9-C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 10 , 5- to 12-membered heterocycloalkyl, or -R9-C 6~10 is aryl; R 20 and R 21 each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; R 10 -OH, -NHR 83 , -N-(R 83 )R 11 , -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 , or -R 82 -C(O)-[C(R 20 R 21 )] a -R 82 -R 83 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; R9 is absent, N-(R 83 ) or oxygen; R 83 is hydrogen or CH3; or R 11 : TIFF2025114641000163.tif35153 each R 12 are independently hydrogen, chloride, -CH3, or -OCH3; R 13 is hydrogen or -C(O)-(CH2) d -(O-CH2-CH2) f -NH2; R 82 is -NR 23 or oxygen, X4 is the side chain of lysine, arginine, citrulline, alanine, or glycine; X5 is the side chain of phenylalanine, valine, leucine, isoleucine, or tryptophan; each of X6 and X7 is independently a side chain of glycine, alanine, serine, valine, or proline; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; f is an integer from 1 to 12; and each u is independently an integer 0 or 1; Alternatively, R 11 Ha-Y u -W q -R 88 and where: Y is one of the following structures: TIFF2025114641000164.tif33168; in each of these, the terminal NR of Y 83 The group is R 88 Located proximal to; R 83 is hydrogen or CH3; Each W is an amino acid unit; Each R 12 ' are independently halogen, -C 1~8 Alkyl, -OC 1~8 alkyl, nitro, or cyano; R 88 is hydrogen or -C(O)-(CH2) ff -(NH-C(O)) aa -E j -(CH2) bb -R 85 and R 85 is NH2 or OH; E is -CH2- or -CH2CH2O-; u is an integer 0 or 1; q is an integer from 0 to 12; aa is an integer 0 or 1; bb is an integer 0 or 2; ff is an integer from 0 to 10; h is an integer from 0 to 4; j is an integer from 0 to 12; and when E is -CH2-, bb is 0 and j is an integer from 0 to 10; and when E is -CH2CH2-O-, bb is 2 and j is an integer from 1 to 12; Alternatively, R 11 teeth, TIFF2025114641000165.tif35128; where R 83 is hydrogen or CH3; R 84 is C 1~6 Alkyl or C 6~10 is aryl; Each R 12 ' are independently halogen, -C 1~8 Alkyl, -OC 1~8 alkyl, nitro, or cyano; h is an integer from 0 to 4; and u is an integer 0 or 1.

[0362] In some embodiments, R 11 teeth, TIFF2025114641000166.tif33142, where Each R 12 ' is independently chloride, -CH3, or -OCH3; R 88 is hydrogen or -C(O)-(CH2) ff -(CH2-CH2O) j -CH2-CH2-NH2; R 82 is -NR 23 or oxygen, X4 is the side chain of lysine, arginine, citrulline, alanine, or glycine; X5 is the side chain of phenylalanine, valine, leucine, isoleucine, or tryptophan; each of X6 and X7 is independently a side chain of glycine, alanine, serine, valine, or proline; ff is an integer from 1 to 3; j is an integer from 1 to 12, h is an integer from 0 to 4; and Each u is independently an integer 0 or 1.

[0363] In some embodiments, TIFF2025114641000167.tif23128 is citrulline-valine; lysine-phenylalanine; citrulline-phenylalanine; citrulline-leucine; citrulline-valine-glycine-glycine; glycine-phenylalanine-glycine-glycine; valine; proline; leucine or isoleucine.

[0364] In another embodiment, R 11 is one of the following structures: TIFF2025114641000168.tif136128TIFF2025114641000169.tif181128TIFF2025114641000170.tif50150.

[0365] In some embodiments, R 47 is one of the following structures: TIFF2025114641000171.tif143155TIFF2025114641000172.tif226124TIFF2025114641000173.tif49128; where: a is an integer from 1 to 6; c is an integer from 0 to 3; and g is an integer of 2 to 6.

[0366] In another embodiment, the auristatin is a compound of formula (X): TIFF2025114641000174.tif38148, During the ceremony, R 31 and R 32 each independently represents hydrogen or C 1~8alkyl, and R 31 and R 32 at most one of which is hydrogen; R 33 is hydrogen, C 1~8 Alkyl, C 3~8 carbocycle, C 6~10 Aryl, C 1~8 Alkyl-C 6~10 Aryl, X 1 -(C 3~8 carbon ring), C 3~8 Heterocycle, or X 1 -(C 3~8 heterocycle); R 34 is hydrogen, C 1~8 Alkyl, C 3~8 carbocycle, C 6~10 Aryl, X 1 -C 6~10 Aryl, X 1 -(C 3~8 carbon ring), C 3~8 Heterocycle, or X 1 -(C 3~8 heterocycle); R 35 is hydrogen or methyl; Alternatively, R 34 and R 35 together with the carbon atoms to which they are attached, form the formula -(CR 55 R 41 ) b -, forming a carbocyclic ring having the formula: 55 and R 41 each independently represents hydrogen or C 1~8 alkyl and b is an integer from 3 to 7; R 36 is hydrogen or C 1~8 is alkyl; R 37 is hydrogen, C 1~8 Alkyl, C 3~8 carbocycle, C 6~10 Aryl, -X 1 -C 6~10 Aryl, -X 1 -(C 3~8 carbon ring), C 3~8 Heterocycle, or -X1 -(C 3~8 heterocycle); Each R 38 are independently hydrogen, OH, C 1~8 Alkyl, C 3~8 Carbocyclic ring, or O-(C 1~8 alkyl); R 53 teeth, TIFF2025114641000175.tif25128 or R 54 and; R 39 is H, C 1~8 Alkyl, C 6~10 Aryl, -X 1 -C 6~10 Aryl, C 3~8 carbocycle, C 3~8 Heterocycle, -X 1 -C 3~8 Heterocycle, -C 1~8 alkylene-NH2, or (CH2)2SCH3; each X 1 independently, C 1~10 Alkylene or C 3-10 is cycloalkylene; R 44 is hydrogen or C 1~8 is alkyl; R 45 is X 3 -R 42 or NH-R 19 and; X 3 is O or S; R 19 is hydrogen, OH, amino group, alkylamino, or -[C(R 20 R 21 )] a -R 22 and; R 42 is an amino group, C 1~6 alkylamino, or -[C(R 20 R 21 )] a -R 22 and; R 20 and R 21each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; R 22 -OH, -NHR 23 , -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; R 54 is -C(R 56 )2--C(R 56 )2-C 6~10 Aryl, -C(R 56 )2--C(R 56 )2-C 3~8 Heterocycle, or -C(R56 )2--C(R 56 )2-C 3~8 It is a carbocyclic ring; R 56 are independently H, OH, C 1~8 Alkyl, C 3~8 Carbocycle, -OC 1~8 Alkyl, -OC(O)-R 29 , and -OR 23 -OC 1~6 alkyl-NH2; R 29 is an amino group, a 5- to 12-membered heterocycloalkyl, -R 28 -C 1~6 Alkyl-R 22 , R 28 -C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 22 , -[C(R 20 R 21 )] a -R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 or R 29 is R as defined herein 47 and; R 28 is non-existence, NR 23 , or oxygen; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; and f is an integer from 1 to 12.

[0367] In some embodiments, in the auristatin compound of formula (X), R 39 is benzyl or TIFF2025114641000176.tif31128; and R 44 is hydrogen.

[0368] In another embodiment, the auristatin is a compound of formula (Xa): TIFF2025114641000177.tif38148, During the ceremony, R 33 ~R 38 and R 44 is as defined herein; R 31 and R 32 One of the two is hydrogen or C 1~8 alkyl, and the other is TIFF2025114641000178.tif35128, where R 83 is hydrogen or CH3; R 84 is C 1~6 Alkyl or C 6~10 is aryl; Each R 12 ' are independently halogen, -C 1~8 Alkyl, -OC 1~8 alkyl, nitro, or cyano; h is an integer from 0 to 4; and u is an integer 0 or 1; R 53 teeth, TIFF2025114641000179.tif25128 or R 54 and R 39 is H, C 1~8 Alkyl, C 6~10 Aryl, -X 1 -C 6~10 Aryl, C 3~8 carbocycle, C 3~8 Heterocycle, -X 1 -C 3~8 Heterocycle, -C 1~8 alkylene-NH2, or (CH2)2SCH3; each X 1 independently, C 1~10 Alkylene or C 3-10 is cycloalkylene; R 45 is X3 -R 42 or NH-R 19 and; X 3 is O or S; R 19 is hydrogen, OH, amino group, alkylamino, or -[C(R 20 R 21 )] a -R 22 and; R 42 is H, amino group, C 1~6 alkylamino, or -[C(R 20 R 21 )] a -R 22 and; R 20 and R 21 each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; R 22 -OH, -NHR 23 , -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(O-CH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; R 54 is -C(R 56 )2--C(R 56 )2-C 6~10 Aryl, -C(R 56 )2--C(R 56 )2-C 3~8 Heterocycle, or -C(R 56 )2--C(R 56 )2-C 3~8 It is a carbocyclic ring; R 56 are independently H, OH, C 1~8 Alkyl, C 3~8 Carbocycle, -OC 1~8 Alkyl, -OC(O)-R 29 , and -OR 23 -OC 1~6 alkyl-NH2; R 29 is an amino group, a 5- to 12-membered heterocycloalkyl, -R 28 -C 1~6 Alkyl-R 22 , R 28 -C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 22 , -[C(R 20 R 21 )] a -R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 or R 29 is R as defined herein 47 and; R 28 is non-existence, NR 23 , or oxygen; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; and f is an integer from 1 to 12.

[0369] In one embodiment, the auristatin compound of Formula (Xa) is a compound of Formula (XIa) or Formula (XIb): TIFF2025114641000180.tif85164, During the ceremony, R 92 teeth, TIFF2025114641000181.tif27134, and R 83 is hydrogen or CH3.

[0370] In one embodiment, the auristatin of formula (X) is a compound of formula (XI), formula (XII), or formula (XIII): wherein the compound of formula (XI) is TIFF2025114641000182.tif37141, In the formula, R 31 is H or CH3, and R 42 is -CH3 or one of the following structures: TIFF2025114641000183.tif150148TIFF2025114641000184.tif111156; where: a is an integer from 1 to 6; c is an integer from 0 to 3; and g is an integer from 2 to 6; wherein the compound of formula (XII) is TIFF2025114641000185.tif39147, In the formula, R 31 is H or CH3, and R 40is hydrogen, -OH, -NH2, or one of the following structures: TIFF2025114641000186.tif14128TIFF2025114641000187.tif198150TIFF2025114641000188.tif69168; where: a is an integer from 1 to 6; g is an integer from 2 to 6; and c is an integer from 0 to 3; wherein the compound of formula (XIII) is TIFF2025114641000189.tif43164, where: R 31 is H or CH3; R 29 is an amino group, a 5- to 12-membered heterocycloalkyl, -R 28 -C 1~6 Alkyl-R 22 , R 28 -C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 22 , -R 28 -[C(R 20 R 21 )] a -R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 or R 29 is R as defined herein 47 and; R 20 and R 21 each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8cycloalkyl, or the side chain of a natural or unnatural amino acid; R 22 -OH, -NHR 23 , -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; R 28 is non-existence, NR 23 , or oxygen; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; and f is an integer from 1 to 12.

[0371] In one embodiment, in formula (XII), R 40 teeth, The file is TIFF2025114641000190.tif61169.

[0372] In another embodiment, the compound of formula (XII) is a compound of formula (XIIa), (XIIb), (XIIc), (XIId), (XIIe), (XIIf), (XIIg), or (XIIh): TIFF2025114641000191.tif42128TIFF2025114641000192.tif176128TIFF2025114641000193.tif165128TIFF2025114641000194.tif157128.

[0373] In one embodiment, in the compound of formula (XIII), R 29 is -NH2, 5-membered heterocycloalkyl, -R 28 -C 1~6 Alkyl-R 22 , R 28 -C 5~12 Heterocycloalkyl-C 1~6 Alkyl-R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 or R 29 is R as defined herein 47 and; R 28 is non-existence, NR 23 , or oxygen; R 22 -OH, -NHR 23 , -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 and; Each R 23 are independently hydrogen, C 1~6Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; c is an integer from 0 to 3; d is an integer from 1 to 3; and f is an integer from 1 to 12.

[0374] In yet another embodiment, R 29 is one of the following structures: TIFF2025114641000195.tif149155TIFF2025114641000196.tif216116TIFF2025114641000197.tif116128; where: a is an integer from 1 to 6; c is an integer from 0 to 3; and g is an integer of 2 to 6.

[0375] In one embodiment, the MEK inhibitor is a compound of formula (XIV): TIFF2025114641000198.tif44128, During the ceremony, R 43 is H or -R 46 -R 47 and; R 20 and R 21 each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; R 22 -OH, -NH2, -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; R 46 is -C(O)-; -C(O)-O-; -C(O)-NH-, or absent; R 47 is as defined herein; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; and f is an integer from 1 to 12.

[0376] Further examples of MEK inhibitors are disclosed in US Pat. No. 7,517,994 B2.

[0377] In some embodiments, R 43 is -C(O)-(CH2) a -NH2 or -C(O)-C(H)(CH3)-(CH2) c -NH2; where a is an integer of 1 to 6; and c is an integer of 0 to 3.

[0378] In another embodiment, the duocarmycin compound is a compound of formula (XV): TIFF2025114641000199.tif69128, During the ceremony, R 47 is as defined herein; R 48 is hydrogen, -COOC 1~6 alkyl, -COOH, -NH2, or -CH3; R 49 is Cl, Br, or -OH; R 50 is hydrogen, -OCH3, TIFF2025114641000200.tif37145; R 51 and R 52 each is independently hydrogen or —OCH; and Ring AA is either a phenyl or a pyrrolyl ring.

[0379] Further examples of duocarmycin compounds are disclosed in US Pat. No. 7,553,816.

[0380] In one embodiment, the duocarmycin compound of formula (XV) is a compound of formula (XVI), (XVII), (XVIII), or (XIX): TIFF2025114641000201.tif64128TIFF2025114641000202.tif212142; During the ceremony, R 49 is Cl, Br, or —OH; and R47 is as defined herein.

[0381] In another embodiment, the duocarmycin compound is a duocarmycin SA compound of formula (XX) or (XXI): TIFF2025114641000203.tif115128, During the ceremony, R 42 is C 1~6 Alkylamino or -[C(R 20 R 21 )] a -R 22 and; R 20 and R 21 each independently represents hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycle, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl, or the side chain of a natural or unnatural amino acid; R 22 -OH, -NH2, -COOH, -R 82 -C(O)(CH2) c -C(H)(R 23 )-N(H)(R 23 ), -R 82 -C(O)(CH2) d -(OCH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 and; Each R 23 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 is alkyl; X 2 is the side chain of a natural or unnatural amino acid; R 77 is hydrogen or X 2 and N.R. 77 forms nitrogen-containing cyclic compounds; R 82 is -NR 23 or oxygen; a is an integer from 1 to 6; c is an integer from 0 to 3; d is an integer from 1 to 3; and f is an integer from 1 to 12.

[0382] In some embodiments, R 42 is one of the following structures: TIFF2025114641000204.tif171148TIFF2025114641000205.tif96151, where: a is an integer from 1 to 6; g is an integer from 2 to 6; and c is an integer of 0 to 3.

[0383] In another embodiment, the KSP inhibitor compound is a compound of formula (XXVI): TIFF2025114641000206.tif69128, where R 30 is as defined herein.

[0384] In some embodiments, R 30 teeth, TIFF2025114641000207.tif131150, where: a is an integer from 1 to 6; c is an integer from 0 to 3; and g is an integer of 2 to 6.

[0385] In another embodiment, the duocarmycin compound is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, CC-1065, adozelesin, bizelesin, or carzelesin. Additional duocarmycin compounds suitable for the conjugates, scaffolds, and methods of the present disclosure are described in US 5,101,038.

[0386] In another embodiment, the KSP inhibitor compound is a compound of formula (XXVII), (XXVIII), or (XXIX): TIFF2025114641000208.tif67150, During the ceremony, R 51 is a bond, -C(O)-(CH2)-C(O)NH-(CH2)2-NH-, -C(O)-(CHO-CH2)-C(O)NH-(CH2)2-NH-, or R 11 is as defined herein.

[0387] Those skilled in the art of therapeutic agents will readily understand that each of the therapeutic agents described herein can be modified in such a way that the resulting compound still retains the specificity and / or activity of the original compound. Those skilled in the art will also understand that many of these compounds can be used in place of the therapeutic agents described herein. Thus, the therapeutic agents disclosed herein encompass analogs and derivatives of the compounds described herein.

[0388] Table A below provides additional examples of therapeutic agents and their derivatives suitable for conjugation to form the antibody-drug conjugates or drug-loaded scaffolds of the present disclosure. Spectral data for specific compounds is also provided (ND in the table means "not determined"). These examples may also be the active forms of the drugs when released from the conjugates in vitro or in vivo.

[0389] (Table A) TIFF2025114641000209.tif21281TIFF2025114641000210.tif203115TIFF2025 114641000211.tif227108TIFF2025114641000212.tif207101TIFF20251146410 00213.tif202154TIFF2025114641000214.tif212150TIFF2025114641000215.t if194150TIFF2025114641000216.tif209161TIFF2025114641000217.tif195134

[0390] Hydrophilic group or T 1 In one embodiment, the hydrophilic group contained in the conjugate or scaffold of the present disclosure is a water-soluble and substantially non-antigenic polymer. Examples of hydrophilic groups include, but are not limited to, polyalcohols, polyethers, polyanions, polycations, polyphosphates, polyamines, polysaccharides, polyhydroxy compounds, polylysines, and derivatives thereof. One end of the hydrophilic group can be connected to a multifunctional linker or M via a non-cleavable bond or a cleavable bond. A In the linker (e.g., M A The hydrophilic group may be functionalized to allow covalent bonding (to an amino acid in a linker). Functionalization can be, for example, via an amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or other functional group. The other end(s) of the hydrophilic group are free and unconstrained. "Unconstrained" means that the hydrophilic group is not connected to another moiety, such as a D or drug unit, a releasable assembly unit, or other component of a conjugate or scaffold of the present disclosure. The free, unconstrained end of the hydrophilic group may comprise 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 end(s) of the hydrophilic group.

[0391] A cleavable bond refers to a bond that is not substantially susceptible to cleavage while circulating in plasma, but is susceptible to cleavage in an intracellular or intratumoral environment. A non-cleavable bond is one that is not substantially susceptible to cleavage in any biological environment. Chemical hydrolysis of hydrazones, reduction of disulfides, and enzymatic cleavage of peptide or glycosidic bonds are examples of cleavable bonds. Exemplary connections of hydrophilic groups are via amide, ether, ester, hydrazone, oxime, disulfide, peptide, or triazole bonds. In some embodiments, a multifunctional linker or M A to the linker (e.g., M A The connection of the hydrophilic group (to an amino acid in the linker) is by an amide bond.

[0392] For embodiments in which a conjugate or scaffold of the present disclosure comprises two or more hydrophilic groups, the multiple hydrophilic groups can be the same or different chemical moieties (e.g., hydrophilic groups of different molecular weight, number of subunits, or chemical structure). The multiple hydrophilic groups can be attached at a single attachment site or at different sites by a multifunctional linker or M A It can be connected to a linker.

[0393] The addition of hydrophilic groups can have two potential effects on the pharmacokinetics of the resulting conjugate. The desirable effect is a decrease in clearance (and consequently an increase in exposure) due to a decrease in nonspecific interactions caused by exposed hydrophobic elements on the drug or drug-linker. The second effect is an undesirable effect: a decrease in the amount and rate of distribution, which can be attributed to an increase in the molecular weight of the conjugate. Increasing the molecular weight of the hydrophilic group increases the hydrodynamic radius of the conjugate and decreases its diffusivity, which may reduce the conjugate's ability to penetrate tumors. Because of these two competing pharmacokinetic effects, it is desirable to use hydrophilic groups that are large enough to reduce the conjugate's clearance and therefore increase its plasma exposure, but not so large that they significantly reduce its diffusivity (which may reduce the conjugate's ability to reach its intended target cell population).

[0394] In some embodiments, hydrophilic groups include, but are not limited to, sugar alcohols (also known as polyalcohols, polyhydric alcohols, alditols, or glycitols, e.g., inositol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, galactitol, mannitol, sorbitol, etc.) or derivatives thereof (e.g., aminopolyalcohols), carbohydrates (e.g., sugars), polyvinyl alcohol, carbohydrate-based polymers (e.g., dextran), hydroxypropyl methacrylamide (HPMA), polyalkylene oxides, and / or copolymers thereof.

[0395] In one embodiment, the hydrophilic group comprises a moiety incorporating multiple hydroxyl ("-OH") groups, e.g., monosaccharides, oligosaccharides, polysaccharides, etc. In yet another embodiment, the hydrophilic group comprises a moiety incorporating multiple -(CR 58 OH)-group, where R 58 is hydrogen or C 1~8 It is alkyl.

[0396] In some embodiments, the hydrophilic group comprises one or more of the fragments of the following formula: TIFF2025114641000218.tif6128 where, n1 is an integer from 0 to about 6; Each R 58 are independently hydrogen or C 1~8 is alkyl; R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 -, where R 59 is H, alkyl, cycloalkyl, or arylalkyl; R 61 is CH2OR 62 , COOR 62 , -(CH2) n2 COOR 62 or heterocycloalkyl substituted with one or more hydroxyl; R 62 is H or C 1~8 is alkyl; and n2 is an integer of 1 to about 5.

[0397] For example, R 58 is hydrogen and R 60 is a bond or C 1~6 is an alkyl linker, n1 is an integer from 1 to about 6, and R 61 is CH2OH or COOH. For example, R 58 is hydrogen and R 60 Ha-CHR 59 -, n1 is 0, and R 61 is a heterocycloalkyl substituted with one or more hydroxyls, e.g., a monosaccharide.

[0398] In some embodiments, the hydrophilic group comprises a glucosylamine, a diamine, or a triamine.

[0399] In some embodiments, the hydrophilic group comprises one or more of the following fragments or stereoisomers thereof: TIFF2025114641000219.tif199168 where, R 59 is H, alkyl, cycloalkyl, or arylalkyl; n1 is an integer from 1 to about 6; n2 is an integer from 1 to about 5; and n3 is an integer of about 1 to about 3.

[0400] It is understood that all stereochemical forms of hydrophilic groups are contemplated herein.For example, in the above formula, the hydrophilic group can be derived from ribose, xylose, glucose, mannose, galactose, or other sugars, and can retain the stereochemical configuration of the pendant hydroxyl and alkyl groups present in those molecules.Furthermore, it should be understood that various deoxy compounds are also contemplated in the above formula.By way of example, for the hydrophilic group, one or more of the following characteristics are contemplated, where applicable:

[0401] For example, n3 is 2 or 3.

[0402] For example, n1 is 1, 2, or 3.

[0403] For example, n2 is 1.

[0404] For example, R 59 is hydrogen.

[0405] For example, the hydrophilic group is Includes TIFF2025114641000220.tif16128.

[0406] For example, the hydrophilic group is Includes TIFF2025114641000221.tif25128.

[0407] For example, the hydrophilic group is Includes TIFF2025114641000222.tif25128.

[0408] In some embodiments, the hydrophilic group is TIFF2025114641000223.tif16128, where: n4 is an integer from 1 to about 25; Each R 63 are independently hydrogen or C 1~8 is alkyl; R 64 is a bond or C 1~8 is an alkyl linker; R 65 is H, C 1~8 Alkyl, or -(CH2) n2 COOR 62 and; R 62 is H or C 1~8 is alkyl; and n2 is an integer of 1 to about 5.

[0409] In some embodiments, the hydrophilic group is Includes TIFF2025114641000224.tif12128.

[0410] For example, n4 is an integer of about 2 to about 20, about 4 to about 16, about 6 to about 12, or about 8 to about 12.

[0411] For example, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0412] In other embodiments, the hydrophilic group comprises a polyether, such as a polyalkylene glycol (PAO). PAOs include, but are not limited to, polymers of lower alkylene oxides, such as propylene oxide, particularly ethylene oxide, polypropylene glycol, polyethylene glycol (PEG), polyoxyethylenated polyols, copolymers thereof, and block copolymers thereof. In other embodiments, the polyalkylene glycol is polyethylene glycol (PEG), including, but not limited to, polydisperse PEG, monodisperse PEG, and discrete PEG. 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. In another embodiment, the PEG unit is discrete PEG, providing a single molecule with a defined and specified chain length. In some embodiments, the polyethylene glycol is mPEG.

[0413] In some embodiments, the hydrophilic group comprises a PEG unit comprising one or more polyethylene glycol chains. The polyethylene glycol chains can be linked together, for example, in a linear, branched, or star configuration. In addition to comprising repeating polyethylene glycol subunits, the PEG unit may also contain non-PEG material (e.g., to facilitate coupling of multiple PEG chains to each other or to an amino acid). Non-PEG material refers to atoms in the PEG chain that are not part of the repeating -CH2CHO- subunits. In one embodiment, the PEG chain can comprise two monomeric PEG chains linked to each other via a non-PEG element. In another embodiment, the PEG unit can comprise two linear PEG chains attached to a central core that is attached to an amino acid (i.e., the PEG unit itself is branched).

[0414] The PEG unit is attached to a multifunctional linker or M via a reactive group. A In the linker (e.g., M A The reactive group can be covalently attached to an amino acid in the linker. The reactive group is one (e.g., a free amino or carboxyl group) to which an activated PEG molecule can be attached. For example, the N-terminal amino acid and lysine (K) have free amino groups, and the C-terminal amino acid residue has a free carboxyl group. Sulfhydryl groups (e.g., as found in cysteine residues) can also be used as reactive groups for attaching PEG.

[0415] In some embodiments, the PEG units are linked to multifunctional linkers or MPEGs by using methoxylated PEGs ("mPEGs") with different reactive moieties, including but not limited to: A to the linker (e.g., M AThe following may be attached to an amino acid in the linker: succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imidate, para-nitrophenyl carbonate (NPC), succinimidyl propionate (SPA), and cyanuric chloride. Examples of mPEG include mPEG-succinimidyl succinate (mPEG-SS), mPEG2-succinimidyl succinate (mPEG2-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG2-succinimidyl carbonate (mPEG2-SC), mPEG-imidate, mPEG-para-nitrophenyl carbonate (mPEG-NPC), mPEG-imidate, mPEG2-para-nitrophenyl carbonate (mPE). Examples of reactive PEG reagents include, but are not limited to, mPEG-NPC, mPEG-succinimidyl propionate (mPEG-SPA), mPEG2-succinimidyl propionate (mPEG2-SPA), mPEG-N-hydroxy-succinimide (mPEG-NHS), mPEG2-N-hydroxy-succinimide (mPEG2-NHS), mPEG-cyanuric chloride, mPEG2-cyanuric chloride, mPEG2-lysinol-NPC, and mPEG2-Lys-NHS. A wide variety of PEG species can be used, and virtually any suitable reactive PEG reagent can be used. In some embodiments, the reactive PEG reagent is a multifunctional linker or MPEG-NHS. A to the linker (e.g., M AThe attachment of the PEG to an amino acid in the linker results in the formation of a carbamate or amide bond. Reactive PEG reagents include mPEG2-N-hydroxysuccinimide (mPEG2-NHS), bifunctional PEG propionaldehyde (mPEG2-ALD), multi-arm PEG, and maleimide-containing PEG. (mPEG(MAL)2, mPEG2(MAL)), mPEG-NH2, mPEG-succinimidyl propionate (mPEG-SPA), mPEG butanoate acid succinimide (mPEG-SBA), mPEG-thioester, mPEG-double ester, mPEG-BTC, mPEG-ButyrALD, mPEG-acetaldehyde diethyl acetal (mPEG-ACET), heterofunctional PEGs (e.g., NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-vinyl sulfone (NHS-PEG-VS), or NHS-PEG-MAL), PEG acrylate (ACRL-PEG-NHS), PEG-phospholipids (e.g., mPEG-DSPE), the SUNBRITE™ series of multi-armed PEGs, such as glycerin-based PEGs activated by a chemistry selected by one of skill in the art, any SUNBRITE-activated PEG, including, but not limited to, carboxyl-PEG, p-NP-PEG, tresyl-PEG, aldehyde-PEG, acetal-PEG, amino-PEG, thiol-PEG, maleimide-PEG, hydroxyl-PEG-amine, amino-PEG-COOK hydroxyl-PEG-aldehyde, carboxylic acid anhydride-PEG, functionalized PEG-phospholipids, and other similar and / or suitable reactive PEGs.

[0416] In some embodiments, the PEG unit comprises 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 some such embodiments, the PEG unit comprises up to about 72 subunits.

[0417] In some embodiments, the PEG unit comprises 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.

[0418] In some embodiments, the PEG unit comprises 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, or at least 18 subunits.

[0419] In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, or at least 12 subunits.

[0420] In some embodiments, the PEG unit comprises at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, or at least 12 subunits.

[0421] In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, or at least 8 subunits.

[0422] In some embodiments, the PEG unit comprises one or more linear PEG 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 other embodiments, the PEG unit comprises a total of at least 6 subunits, at least 8, at least 10 subunits, or at least 12 subunits. In some such embodiments, the PEG unit comprises a total of up to about 72 subunits, preferably a total of up to about 36 subunits.

[0423] In some embodiments, the PEG units may be a total of 4 to 72, 4 to 60, 4 to 48, 4 to 36, or 4 to 24 subunits, 5 to 72, 5 to 60, 5 to 48, 5 to 36, or 5 to 24 subunits, 6 to 72, 6 to 60, 6 to 48, 6 to 36, or 6 to 24 subunits, 7 to 72, 7 to 60, 7 to 48, 7 to 36, or 7 to 24 subunits, 8 to 72, 8 to 60, 8 to 48, 8 to 36, or 8 to 24 subunits, 9 to 72, 9 to 60, 9 to 48, 9 to 36, or 9 to 24 subunits, 10 to 72, 10 to 60, 10 to 48, 10 to 36, or 10 to 24 subunits, 11 to 72, 11 to 60, 11 to 48, 11 to 36, or 11 to 24 subunits, 12 to 72, 12 to 60, 12 to 48, 12 to 36, or 12 to 24 subunits, 13 to 72, 13 to 60, 13 to 48, 13 to 36, or 13 to 24 subunits, 14 to 72, 14 to 60, 14 to 48, 14 to 36, or 14 to 24 subunits, 15 to 72, 15 to 60, 15 to 48, 15 to 36, or 15 to 24 subunits, , 9-48, 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 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.

[0424] In some embodiments, the PEG units may be a total of 4 to 72, 4 to 60, 4 to 48, 4 to 36, or 4 to 24 subunits, 5 to 72, 5 to 60, 5 to 48, 5 to 36, or 5 to 24 subunits, 6 to 72, 6 to 60, 6 to 48, 6 to 36, or 6 to 24 subunits, 7 to 72, 7 to 60, 7 to 48, 7 to 36, or 7 to 24 subunits, 8 to 72, 8 to 60, 8 to 48, 8 to 36, or 8 to 24 subunits, 9 to 72, 9 to 60, 9 to 48, , 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, 21-24 It comprises one or more linear PEG chains having 0 to 60, 20 to 48, 20 to 36, or 20 to 24 subunits, 21 to 72, 21 to 60, 21 to 48, 21 to 36, or 21 to 24 subunits, 22 to 72, 22 to 60, 22 to 48, 22 to 36, or 22 to 24 subunits, 23 to 72, 23 to 60, 23 to 48, 23 to 36, or 23 to 24 subunits, or 24 to 72, 24 to 60, 24 to 48, 24 to 36, or 24 subunits.

[0425] In some embodiments, the PEG unit is a derivatized linear single PEG chain 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.

[0426] In some embodiments, the PEG units are 6 to 72, 6 to 60, 6 to 48, 6 to 36, or 6 to 24 subunits, 7 to 72, 7 to 60, 7 to 48, 7 to 36, or 7 to 24 subunits, 8 to 72, 8 to 60, 8 to 48, 8 to 36, or 8 to 24 subunits, 9 to 72, 9 to 60, 9 to 48, 9 to 36, or 9 to 24 subunits, 10 to 72, 10 to 60, 10 to 48, 10 to 36, or 10 to 24 subunits. 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 or a derivatized linear single PEG chain having 20 to 24 subunits, 21 to 72, 21 to 60, 21 to 48, 21 to 36, or 21 to 24 subunits, 22 to 72, 22 to 60, 22 to 48, 22 to 36, or 22 to 24 subunits, 23 to 72, 23 to 60, 23 to 48, 23 to 36, or 23 to 24 subunits, or 24 to 72, 24 to 60, 24 to 48, 24 to 36, or 24 subunits.

[0427] In some embodiments, the PEG unit is a derivatized linear single PEG chain having from 2 to 72, from 2 to 60, from 2 to 48, from 2 to 36, or from 2 to 24 subunits, from 2 to 72, from 2 to 60, from 2 to 48, from 2 to 36, or from 2 to 24 subunits, from 3 to 72, from 3 to 60, from 3 to 48, from 3 to 36, or from 3 to 24 subunits, from 3 to 72, from 3 to 60, from 3 to 48, from 3 to 36, or from 3 to 24 subunits, from 4 to 72, from 4 to 60, from 4 to 48, from 4 to 36, or from 4 to 24 subunits, or from 5 to 72, from 5 to 60, from 5 to 48, from 5 to 36, or from 5 to 24 subunits.

[0428] For example, a linear PEG unit may be TIFF2025114641000225.tif74128, where: The wavy line represents a multifunctional linker or M A to the linker (e.g., M A indicates the site of attachment (to an amino acid in the linker); Y 71 is the PEG connection unit; Y 72 is a PEG capping unit; Y 73 is a PEG coupling unit (i.e., for coupling multiple PEG subunit chains together); d9 is an integer of 2 to 72, preferably 4 to 72, more preferably 6 to 72, 8 to 72, 10 to 72, 12 to 72, or 6 to 24; each d 10 are independently an integer from 1 to 72, d 11 is an integer between 2 and 5.

[0429] In some embodiments, there are at least 6, preferably at least 8, at least 10, or at least 12 PEG subunits in the PEG unit, and in some embodiments, there are up to 72 or 36 PEG subunits in the PEG unit.

[0430] In some embodiments, d9 is 8 or about 8, 12 or about 12, 24 or about 24.

[0431] In some embodiments, each Y 72 independently, -C 1~10 Alkyl, -C 2~10 Alkyl-CO2H, -C 2~10 Alkyl-OH, -C 2~10 Alkyl-NH2, -C 2~10 Alkyl-NH(C 1~3 alkyl), or C 2~10 Alkyl-N(C 1~3 alkyl)2.

[0432] In some embodiments, Y 72 -C 1~10 Alkyl, -C 2~10 Alkyl-CO2H, -C 2~10 Alkyl-OH, or -C 2~10 It is alkyl-NH2.

[0433] A PEG coupling unit is a non-PEG material that is part of a PEG unit and acts to link two or more chains of repeating CH2CH2O- subunits. In some embodiments, the PEG coupling unit Y 73 -C 2~10 Alkyl-C(O)-NH-, -C 2~10 Alkyl-NH-C(O)-, -C 2~10 Alkyl-NH-, -C 2~10 Alkyl-C(O)-, -C 2~10 Alkyl-O- or -C 2~10 It is alkyl-S-.

[0434] In some embodiments, each Y 73 independently, -C 1~10 Alkyl-C(O)-NH-, -C 1~10 Alkyl-NH-C(O)-, -C 2~10 Alkyl-NH-, -C 2~10 Alkyl-O-, -C 1~10 Alkyl-S- or -C 1~10It is alkyl-NH-.

[0435] The PEG linking unit is a portion of the PEG unit that connects the PEG unit to a multifunctional linker or M A to the linker (e.g., M A The PEG unit acts to link to an amino acid in the linker. For example, the amino acid has a functional group that forms a bond with the PEG unit. Functional groups for connecting the PEG unit to an amino acid include a sulfhydryl group for forming a disulfide or thioether bond, an aldehyde, ketone, or hydrazine group for forming a hydrazone bond, a hydroxylamine for forming an oxime bond, a carboxyl or amino group for forming a peptide bond, a carboxyl or hydroxyl group for forming an ester bond, a sulfonic acid for forming a sulfonamide bond, an alcohol for forming a carbamate bond, and an amine for forming a sulfonamide, carbamate, or amide bond. Thus, the PEG unit can be connected to the amino acid, for example, via a disulfide, thioether, hydrazone, oxime, peptide, ester, sulfonamide, carbamate, or amide bond. Typically, the reaction for connecting the PEG unit can be a cycloaddition, addition, addition / elimination, or substitution reaction, or a combination thereof, if applicable.

[0436] In some embodiments, the PEG connecting unit Y 71 is a bond, -C(O)-, -O-, -S-, -S(O)-, -S(O)2-, -NR5-, -C(O)O-, -C(O)-C 1~10 Alkyl, -C(O)-C 1~10 Alkyl-O-, -C(O)-C 1~10 Alkyl-CO2-, -C(O)-C 1~10 Alkyl-NR5-, -C(O)-C 1~10 Alkyl-S-, -C(O)-C 1~10 Alkyl-C(O)-NR5-, -C(O)-C 1~10 Alkyl-NR5-C(O)-, -C 1~10 Alkyl, -C 1~10Alkyl-O-, -C 1~10 Alkyl-CO2-, -C 1~10 Alkyl-NR5-, -C 1~10 Alkyl-S-, -C 1~10 Alkyl-C(O)-NR5-, -C 1~10 Alkyl-NR5-C(O)-, -CH2CH2SO2-C 1~10 Alkyl-, -CH2C(O)-C 1~10 Alkyl-, =N-(O or N)-C 1~10 Alkyl-O-, =N-(O or N)-C 1~10 Alkyl-NR5-, =N-(O or N)-C 1~10 Alkyl-CO2-, =N-(O or N)-C 1~10 Alkyl-S-, The file is TIFF2025114641000226.tif34128.

[0437] In some embodiments, Y 71 is -NH-, -C(O)-, a triazole group, -S-, or a maleimide group, e.g., TIFF2025114641000227.tif33128, where the wavy line represents a multifunctional linker or M A to the linker (e.g., M A The linker indicates the attachment point (to an amino acid in the linker), and the asterisk indicates the site of attachment within the PEG unit.

[0438] Examples of linear PEG units include: TIFF2025114641000228.tif119150 is mentioned; where the wavy line represents a multifunctional linker or M A to the linker (e.g., M A and each d9 independently represents an integer from 4 to 24, 6 to 24, 8 to 24, 10 to 24, 12 to 24, 14 to 24, or 16 to 24.

[0439] In some embodiments, d9 is about 8, about 12, or about 24.

[0440] In other embodiments, 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; or from about 300 daltons to about 1 kilodalton. In some such aspects, the PEG unit has at least 6 subunits or at least 8, 10, or 12 subunits. In some embodiments, the PEG unit has at least 6 subunits or at least 8, 10, or 12 subunits, but up to 72 subunits, preferably up to 36 subunits.

[0441] Suitable polyethylene glycols may have a free hydroxy group at each end of the polymer molecule, or one hydroxy group etherified with a lower alkyl, e.g., methyl, group. Derivatives of polyethylene glycol having an esterifiable carboxy group are also suitable for the practice of the present disclosure. Polyethylene glycols are typically commercially available under the trademark PEG as a mixture of polymers characterized by average molecular weight. Polyethylene glycols having an average molecular weight of about 300 to about 5,000 are preferred, with those having an average molecular weight of about 600 to about 1,000 being particularly preferred.

[0442] Other examples of hydrophilic groups suitable for the conjugates, scaffolds, and methods disclosed herein can be found, for example, in US 8,367,065 column 13; US 8524696 column 6; WO2015 / 057699 and WO 2014 / 062697, the contents of each of which are incorporated herein by reference in their entirety.

[0443] Protein-based recognition molecules (PBRMs) Protein-based recognition molecules target conjugates containing peptide linkers to specific tissues, cells, or subcellular locations. Protein-based recognition molecules can target conjugates to cultures, whole organisms, or both. In either case, the protein-based recognition molecule has a ligand present on the cell surface of the target cells and binds to them with effective specificity, affinity, and avidity. In some embodiments, protein-based recognition molecules target conjugates to tissues other than the liver. In other embodiments, protein-based recognition molecules target conjugates to specific tissues such as the liver, kidney, lung, or pancreas. Protein-based recognition molecules can target conjugates to target cells such as cancer cells, for example, receptors expressed on cells such as cancer cells, matrix tissue, or cancer-associated proteins such as tumor antigens. Alternatively, they can target cells containing tumor vasculature. Protein-based recognition molecules can target conjugates to specific cell types, such as hepatocytes in the liver rather than Kupffer cells. In other cases, protein-based recognition molecules can target the conjugate to cells of the reticuloendothelium or lymphatic system, or to professional phagocytes such as macrophages or eosinophils (in such cases, the conjugate itself can also be an effective delivery system without the need for specific targeting).

[0444] In yet other embodiments, the protein-based recognition molecule can target the conjugate to a location within the cell, such as, for example, the nucleus, cytoplasm, or endosome. In certain embodiments, the protein-based recognition molecule can increase cellular binding to a receptor or cytoplasmic transport to the nucleus, and nuclear entry or release from endosomes or other intracellular vesicles.

[0445] In certain embodiments, protein-based recognition molecules include antibodies, proteins, and peptides or peptidomimetics.

[0446] In a preferred embodiment, the protein-based recognition molecule contains a sulfhydryl group and the protein-based recognition molecule is conjugated to the linker-drug moiety by forming a covalent bond via the sulfhydryl group and a functional group on the linker-drug moiety.

[0447] Exemplary antibodies derived from Fab, Fab2, scFv, or camelid antibody heavy chain fragments specific for cell surface markers include 5T4, AOC3, ALK, AXL, C242, C4.4a, CA-125, CCL11, CCR 5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CDH6, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44, CD44 v6, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD74, CD79-B, CD80, CD125, CD138, CD141, CD147, CD152, CD 154, CD326, CEA, CEACAM-5, clumping factor, CTLA-4, CXCR2, EGFR (HER1), ErbB2, ErbB3, EpCAM, EPHA2, EPHB2, EPHB4, FGFR (i.e., FGFR1, FGFR2, FGFR3, FGFR4), FLT3, folate receptor, FAP, GD2, GD3, GPNMB, GCC (GUCY2C), HGF, HER2, HER3, HMI.24, ICAM, ICOS-L, IGF-1 receptor, VEGFR1, EphA2, TRPV1, CFTR, gpNMB, CA9, Cripto, c-KIT, c-MET, ACE, APP, adrenergic receptor-β2, claudin 3, LIV1, LY6E, mesothelin, MUC1, MUC13, NaPi2b, NOTCH1, NOTCH2, NOTCH3, NOTCH4, RON, ROR1, PD-L1, PD-L2, PTK7, B7-H3, B7-B4, IL-2 receptor, IL-4 receptor, IL-13 receptor, TROP-2, Frizzled-7, integrin (α4, α v β3, α v β5, α vβ6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIb β3 integrin), IFN-α, IFN-γ, IgE, IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), L-selectin (CD62L), mucin, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostate cancer cells, Pseudomonas aeruginosa aeruginosa), rabies, RANKL, respiratory syncytial virus, rhesus factor, SLAMF7, sphingosine-1-phosphate, TAG-72, T cell receptor, tenascin-C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, and the like.

[0448] In one embodiment, the antibody derived from a Fab, Fab2, scFv, or camelid antibody heavy chain fragment specific for a cell surface marker, such as CA-125, C242, CD3, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD138, CD141, CD326, CEA, CTLA-4, EGFR (HER1), ErbB2, ErbB3, FAP, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, TAG-72, tenascin-C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFR α, phosphatidylserine, prostate cancer cells, adrenergic receptor-β2, claudin 3, mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, frizzled-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, and integrin (α v β3, α vβ5, α v β6, α1β4, α4β1, α5β1, and α6β4 integrins), tenascin-C, TRAIL-R2, and vimentin.

[0449] Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO), adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab (CEA-SCAN), acelizumab, atlizumab (tocilizumab, Actemra, RoActemra), atrolimumab, bapinoysumab, basiliximab (Simulect), bavituximab, bectumomab (LYMPH OSCAN), belimumab (BENLYSTA), benralizumab, bertilimumab, besilesomab (SCINITIMUN), bevacizumab (AVASTIN), biciromab (FIBRISCINT), bivatuzumab, blinatumomab, brentuximab, briakinumab, canakinumab (ILARIS), cantuzumab, capromab, catumaxomab (REMOVAB), CC49, cedelizumab, certolizumab, cetuximab (ERBITUX), sitatuzumab, sixutumumab, clenoliximab, clivatuzumab, conatumumab, CR6261 , dacetuzumab, daclizumab (ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab, dorlixizumab, ecromeximab, eculizumab (SOLIRIS), edovacomab, edrecolomab (PANOREX), efalizumab (RAPTIVA), efungumab (MYCOGRAB), elotuzumab, ersilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab (REXOMUN), etaracizumab (ABEGRIN), exibirumab, fanolesomab (NEUT ROSPEC), faralimomab, farletuzumab, felvizumab, fezakinumab, figitumumab, hontolizumab (HuZAF), folavirumab, fresolimumab, galiximab, gantenerumab, gavilimomab, gemtuzumab, girentuximab, glenbatumumab, golimumab (SIMPONI), golimumab, ibalizumab, ibritumomab, igovomab (INDIMACIS-125), imuciromab (MYOSCINT), infliximab (REMICADE), intetumumab, inolimomab, inotuzumab, ipilimumab,Iratumumab, keliximab, labetuzumab (CEA-CIDE), lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, lintuzumab, lucatumumab, rumiliximab, mapatumumab, maslimomab, matuzumab, mepolizumab (BOSATRIA), metelimumab, milatuzumab, minletumomab, mitumomab, morolimumab, motavizumab (NUMAX), muromonab-CD3 (ORTHOCLONE) OKT3), nacolomab, naptumomab, natalizumab (TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM), nofetumomab, ocrelizumab, ozlimomab, ofatumumab (ARZERRA), olaratumab, omalizumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX), otelixizumab, pagibaximab, palivizumab (SYNAGIS), panitumumab Tutumumab (VECTIBIX), panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab (OMNITARG), pexelizumab, pintumomab, priliximab, pritumumab, PRO140, rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), lobatumumab, lontalizumab, robelizumab (L EUKARREST), ruplizumab (ANTOVA), satumomab pendetide, cevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab, solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab (LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptokis, tefibazumab (AUREXIS), terimomab, Tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tocilizumab (atlizumab, ACTEMRA), toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN), tremelimumab, tucotuzumab, tuvilumab, urtoxazumab, ustekinumab (STELERA), bapaliximab, vedolizumab, veltuzumab, bepalimomab,These include visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), diralimumab, and zolimomab.

[0450] In some embodiments, the antibody is selected from the group consisting of 5T4, CA-125, CEA, CDH6, CD3, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD51, CTLA-4, CEACAM5, EpCAM, HER2, EGFR (HER1), FAP, folate receptor, GCC (GUCY2C), HGF, integrin alpha v β3, integrin α5β1, IGF-1 receptor, GD3, GPNMB, mucin, LIV1, LY6E, mesothelin, MUC1, MUC13, PTK7, phosphatidylserine, prostate cancer cells, PDGFR α, TAG-72, tenascin-C, TRAIL-R2, VEGF-A, and VEGFR2. In this embodiment, the antibody is selected from the group consisting of abagovomab, adecatumumab, aracizumab, altumomab, anatumomab, arcitumomab, bavituximab, bevacizumab (AVASTIN), bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab, sitatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, gemtuzumab, glenbatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, labetuzumab These include rilotumumab, lexatumumab, lintuzumab, lucatumumab, matuzumab, mitumomab, naptumomab, estafenatoxin, necitumumab, oportuzumab, oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab (RITUXAN), rilotumumab, lobatumumab, satumomab, sibrotuzumab, taplitumomab, tenatumomab, tenatumomab, ticilimumab (tremelimumab), tigatuzumab, trastuzumab (HERCEPTIN), tositumomab, tremelimumab, tucotuzumab, celmoleukin, volociximab, and zalutumumab.

[0451] In particular embodiments, for HER2, the antibody against the cell surface marker is pertuzumab or trastuzumab, for EGFR (HER1), the antibody is cetuximab or panitumumab; for CD20, the antibody is rituximab, for VEGF-A, the antibody is bevacizumab, for CD-22, the antibody is epratuzumab or veltuzumab, and for CEA, the antibody is labetuzumab.

[0452] Exemplary peptides or peptidomimetics include integrin targeting peptides (RGD peptides), LHRH receptor targeting peptides, ErbB2 (HER2) receptor targeting peptides, prostate-specific membrane-bound antigen (PSMA) targeting peptides, lipoprotein receptor LRP1 targeting ApoE protein-derived peptides, ApoA protein peptides, somatostatin receptor targeting peptides, chlorotoxin-derived peptides, and bombesin.

[0453] In particular embodiments, the peptides or peptidomimetics are LHRH receptor targeting peptides and ErbB2 (HER2) receptor targeting peptides.

[0454] Exemplary proteins include insulin, transferrin, fibrinogen gamma fragment, thrombospondin, claudins, apoprotein E, affibody molecules such as ABY-025, ankyrin repeat proteins, ankyrin-like repeat proteins, and synthetic peptides.

[0455] In some embodiments, the protein-drug conjugate comprises a broad-spectrum cytotoxin in combination with the following cell surface markers: for HER2, e.g., pertuzumab or trastuzumab; for EGFR, e.g., cetuximab and panitumumab; for CEA, e.g., labetuzumab; for CD20, e.g., rituximab; for VEGF-A, e.g., bevacizumab; for CD-22, e.g., epratuzumab or veltuzumab.

[0456] In other embodiments, the protein-drug conjugate or protein conjugate used in the present disclosure comprises a combination of two or more protein-based recognition molecules, for example, a combination of bispecific antibodies against EGF receptor (EGFR) on tumor cells and CD3 and CD28 on T cells; a combination of an antibody derived from a Fab, Fab2, scFv, or camelid antibody heavy chain fragment with a peptide or peptidomimetic; a combination of an antibody derived from a Fab, Fab2, scFv, or camelid antibody heavy chain fragment with a protein; or a combination of two bispecific antibodies, for example, CD3xCD19 and CD28xCD22 bispecific antibodies.

[0457] In other embodiments, the protein-drug conjugate or protein conjugate used in the present disclosure comprises a protein-based recognition molecule that is an antibody against an antigen: for example, trastuzumab, cetuximab, rituximab, bevacizumab, epratuzumab, veltuzumab, labetuzumab, B7-H4, B7-H3, CA125, CDH6, CD33, CXCR2, CEACAM5, EGFR, FGFR1, FGFR2, FGFR3, FGFR4, GCC (GUCY2C), HER2, LIV1, LY6E, NaPi2b, c-Met, mesothelin, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1, PTK7, c-Kit, MUC1, MUC13, and 5T4.

[0458] In certain embodiments, the protein-drug conjugate or protein conjugate of the present disclosure comprises a protein-based recognition molecule that is an antibody against 5T4, for example, a humanized anti-5T4 scFvFc antibody.

[0459] Examples of suitable 5T4 targeting ligands or immunoglobulins include those commercially available or described in patent or non-patent literature, such as U.S. Pat. No. 8,044,178, U.S. Pat. No. 8,309,094, U.S. Pat. No. 7,514,546, EP 1036091 (commercially available as TroVax™, Oxford Biomedica), EP 2368914 A1, WO 2013041687 A1 (Amgen), U.S. Pat. No. 2010 / 0173382, and P. Sapra, et al., Mol. Cancer Ther. 2013, 12:38-47. Anti-5T4 antibodies are disclosed in U.S. Provisional Application No. 61 / 877,439, filed September 13, 2013, and U.S. Provisional Application No. 61 / 835,858, filed June 17, 2013. The contents of each of the patent and scientific publications are incorporated herein by reference in their entirety.

[0460] As used herein, the term "5T4 antigen-binding moiety" refers to a polypeptide sequence capable of selectively binding to the 5T4 antigen. In exemplary conjugates, the 5T4 antigen-binding moiety generally comprises a single-chain scFv-Fc form engineered from an anti-5T4 antibody. The single-chain variable fragment (scFv-Fc) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, linked by a linker peptide and further linked to the Fc region, including the hinge region and CH2 and CH3 regions of the antibody (any such combination of antibody moieties with each other or other peptide sequences may be referred to herein as an "immunofusion" molecule). Within such scFvFc molecules, the scFv moiety may be linked at its C-terminus to the N-terminus of the Fc moiety by a linker peptide.

[0461] In other specific embodiments, the protein-drug conjugate or protein conjugate of the present disclosure comprises a protein-based recognition molecule that is a Her-2 or NaPi2b antibody.

[0462] For example, Her-2 antibodies suitable for the conjugates or scaffolds of the present disclosure include: Amino acid sequence variable heavy chain complementarity-determining region 1 (CDRH1) containing TIFF2025114641000229.tif4128; Amino acid sequence variable heavy chain complementarity-determining region 2 (CDRH2) containing TIFF2025114641000230.tif4128; Amino acid sequence variable heavy chain complementarity-determining region 3 (CDRH3) containing TIFF2025114641000231.tif5128; Amino acid sequence variable light chain complementarity-determining region 1 (CDRL1) containing TIFF2025114641000232.tif5128; Amino acid sequence variable light chain complementarity-determining region 2 (CDRL2) containing TIFF2025114641000233.tif4128; and Amino acid sequence Variable light chain complementarity determining region 3 (CDRL3) including TIFF2025114641000234.tif4128 (see, e.g., US20150366987(A1) published December 24, 2015).

[0463] For example, NaPi2b antibodies suitable for the conjugates or scaffolds of the present disclosure include: Amino acid sequence variable light chain complementarity-determining region 1 (CDRL1) containing TIFF2025114641000235.tif4128; Amino acid sequence variable light chain complementarity-determining region 2 (CDRL2) containing TIFF2025114641000236.tif4128; Amino acid sequence variable light chain complementarity-determining region 3 (CDRL3) containing TIFF2025114641000237.tif4128; Amino acid sequence variable heavy chain complementarity-determining region 1 (CDRH1) containing TIFF2025114641000238.tif4128; Amino acid sequence variable heavy chain complementarity-determining region 2 (CDRH2), including TIFF2025114641000239.tif4128; and Amino acid sequence Variable heavy chain complementarity determining region 3 (CDRH3) including TIFF2025114641000240.tif4128 (see, e.g., co-pending application US15 / 457,574, filed March 13, 2017).

[0464] PBRM-drug conjugates The conjugates of the present disclosure include one or more occurrences of D, where D is a therapeutic agent, e.g., a drug, and where the one or more occurrences of D can be the same or different.

[0465] In certain other embodiments, one or more occurrences of PBRM are connected to a linker-drug moiety, where the one or more occurrences of PBRM can be the same or different. In certain other embodiments, one or more linker-drug moieties containing one or more occurrences of D are linked to one PBRM (e.g., an antibody).

[0466] In one embodiment, D is a) an auristatin compound; (b) a calicheamicin compound; (c) a duocarmycin compound; (d) a topoisomerase inhibitor, (e) a pyrrolobenzodiazepine compound; (f) a vinca compound; (g) a protein synthesis inhibitor; (h) an RNA polymerase inhibitor; (i) a tubulin-binding compound; (j) a NAMPT inhibitor, or an analog thereof.

[0467] In some embodiments, D is (a) an auristatin compound; (b) a calicheamicin compound; (c) a duocarmycin compound; (d) a camptothecin compound, (e) a pyrrolobenzodiazepine compound; (f) a vinca compound; or an analog thereof.

[0468] For example, the auristatin compound is an auristatin, a dolastatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin F, AF HPA, MMAF HPA, or phenylenediamine (AFP).

[0469] For example, the duocarmycin or analog thereof is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin, bizelesin, or carzelesin.

[0470] For example, the camptothecin compound is camptothecin, CPT-11 (irinotecan), SN-38, or topotecan.

[0471] For example, the pyrrolobenzodiazepine compound is a pyrrolobenzodiazepine monomer, a symmetrical pyrrolobenzodiazepine dimer, or an asymmetrical pyrrolobenzodiazepine dimer.

[0472] The PBRM-drug conjugates of the present disclosure include a PBRM having a molecular weight of about 40 kDa or more (e.g., 60 kDa or more; 80 kDa or more; 100 kDa or more; 120 kDa or more; 140 kDa or more; 160 kDa or more; 180 kDa or more; or 200 kDa or more, or about 40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180 kDa, or 100-140 kDa).

[0473] For example, the PBRM has a molecular weight of about 40 kDa or more (e.g., 60 kDa or more; 80 kDa or more; 100 kDa or more; 120 kDa or more; 140 kDa or more; 160 kDa or more; 180 kDa or more; or 200 kDa or more, or about 40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180 kDa, or 100-140 kDa) and has a sulfhydryl (i.e., -SH or thiol) group.

[0474] For example, the total number of sulfide bonds (or the total number of connection points) formed between the PHF and the PBRM is 10 or less.

[0475] For example, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater, 80 kDa or greater, 100 kDa or greater, 120 kDa or greater, 140 kDa or greater, 160 kDa or greater, or 180 kDa or greater, or about 40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180 kDa, or 100-140 kDa).

[0476] For example, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 40 kDa to 200 kDa.

[0477] For example, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 40 kDa to 80 kDa.

[0478] PBRMs within this molecular weight range include, but are not limited to, antibody fragments, such as Fab.

[0479] For example, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 60 kDa to 120 kDa.

[0480] PBRMs within this molecular weight range include, but are not limited to, camelids, Fab2, scFvFc, and the like.

[0481] For example, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 140 kDa to 180 kDa.

[0482] PBRMs within this molecular weight range include, but are not limited to, full-length antibodies, such as IgG and IgM.

[0483] These targeting ligands, linkers, and drug or prodrug fragments described herein can be assembled into the conjugates or scaffolds of the present disclosure, for example, according to the disclosed techniques and methods. The therapeutic and targeting conjugates of the present disclosure, and methods for their production, are described below by way of non-limiting examples.

[0484] For example, the total number of sulfide bonds (or total number of connection points) formed between the linker-drug moiety and the PBRM is 14 or less.

[0485] For example, the ratio of linker-drug moiety to PBRM is greater than 1:1 and less than or equal to 14:1.

[0486] For example, the linker-drug moiety ratio is about 14:1, 12:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0487] For example, the ratio of linker-drug moiety to PBRM is 2:1 to 10:1.

[0488] For example, the ratio of linker-drug moiety to PBRM is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0489] For example, the ratio of linker-drug moiety to PBRM is 2:1 to 4:1.

[0490] For example, the ratio of linker-drug moiety to PBRM is about 4:1, 3:1, or 2:1.

[0491] In some embodiments, linker-drug moiety is conjugated with PBRM by utilizing cysteine-based bioconjugation strategy.See, for example, WO2010100430 and US 7,595,292; the contents of which are incorporated herein by reference in their entirety.In one embodiment, one or more linker-drug moieties are conjugated with PBRM (e.g., antibody) through cysteine in antibody hinge region.Without wishing to be bound by theory, the resulting conjugate is stabilized by the formation of interchain crosslink structure.

[0492] Thus, the present disclosure also relates to linker-drug moieties comprising at least two moieties, where each moiety is capable of conjugating to a thiol group derived from an amino acid (e.g., cysteine) in the PBRM to form a protein-linker-drug conjugate.

[0493] In some embodiments, one or more free thiol groups of the PBRM are generated by reducing a protein. The one or more free thiols of the PBRM are then reacted with one or more linker-drug moieties that can be conjugated to the thiol groups derived from amino acids, resulting in the PBRM being conjugated to the linker-drug moieties. In one embodiment, at least two moieties linked to the PBRM are maleimide groups.

[0494] In some embodiments, the free thiol group of the PBRM used for conjugation is derived from a disulfide bridge of a natural protein or a disulfide bridge of a protein complex consisting of two or more protein chains linked by disulfide bridges. The disulfide bridge can be an intrachain or interchain crosslink. Alternatively, the free thiol group of the PBRM is derived from an unpaired thiol group or cysteine of a natural protein that is not involved in interchain or intrachain disulfide bridge formation.

[0495] Disulfide bonds can be reduced, for example, using conventional methods with dithiothreitol, mercaptoethanol, tris-carboxyethylphosphine, dehydroascorbic acid, or copper sulfate. Proteins can contain one or more disulfide bridges. Reduction to produce free thiol groups can be controlled to reduce one or more specific disulfide bridges in a protein. Depending on the degree of disulfide reduction and the stoichiometry of the moieties in the linker-drug moiety, one or more linker-drug moieties can be conjugated to a protein. If it is desired to reduce fewer disulfides than the total number, immobilized reducing agents can be used, since partial reduction is possible using various reducing conditions or the addition of denaturing agents.

[0496] For example, for linker-drug moiety conjugation, the PBRM has a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater; 80 kDa or greater; or 100 kDa or greater; 120 kDa or greater; 140 kDa or greater; 160 kDa or greater, or 180 kDa or greater). In this embodiment, the ratio of PBRM to linker-drug moiety is about 1:1 to about 1:10, about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:1 to about 1:2, about 1:2 to about 1:6, about 1:2 to about 1:5, about 1:2 to about 1:4, or about 1:2 to about 1:3.

[0497] PBRMs within this molecular weight range include, but are not limited to, full-length antibodies, such as IgG and IgM.

[0498] For example, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 60 kDa to 120 kDa. In this embodiment, the ratio of PBRM to linker-drug moiety is about 1:1 to about 1:10, about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:1 to about 1:2, about 1:2 to about 1:6, about 1:2 to about 1:5, about 1:2 to about 1:4, or about 1:2 to about 1:3.

[0499] PBRMs within this molecular weight range include, but are not limited to, antibody fragments such as Fab2, scFcFv, and camelids.

[0500] For example, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 40 kDa to 80 kDa. In this embodiment, the ratio of PBRM to linker-drug moiety is about 1:1 to about 1:10, about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:1 to about 1:2, about 1:2 to about 1:6, about 1:2 to about 1:5, about 1:2 to about 1:4, or about 1:2 to about 1:3.

[0501] PBRMs within this molecular weight range include, but are not limited to, antibody fragments, such as Fab.

[0502] In another aspect, the present disclosure features a scaffold useful for conjugating either or both of a protein-based recognition molecule (PBRM) and a therapeutic agent (D), such as a scaffold of any of Formulas (II)-(IX) disclosed herein.

[0503] In some embodiments, each of the drug-loaded scaffolds described herein (i.e., without linkage to a PBRM) typically has a polydispersity index (PDI) of 1.

[0504] The conjugates and scaffolds disclosed herein can be purified (i.e., removed from starting materials) by extensive diafiltration. If necessary, additional purification by size exclusion chromatography can be performed to remove aggregated conjugates. In general, purified conjugates typically contain less than 5% (e.g., <2% w / w) aggregated conjugates as determined by SEC; less than 0.5% (e.g., <0.1% w / w) free (unconjugated) drug as determined by RP-HPLC; less than 1% drug-loaded peptide-containing scaffolds as determined by SEC; and less than 2% (e.g., <1% w / w) unconjugated PBRM as determined by HIC-HPLC.

[0505] Tables B and C below provide examples of drug-loaded peptide-containing scaffolds and conjugates, respectively, of the present disclosure.

[0506] (Table B) TIFF2025114641000241.tif144168TIFF2025114641000242.tif207168TIFF2025114641000243.t if181168TIFF2025114641000244.tif169168TIFF2025114641000245.tif175168TIFF20251146410 00246.tif210168TIFF2025114641000247.tif213168TIFF2025114641000248.tif194168TIFF202 5114641000249.tif205168TIFF2025114641000250.tif211168TIFF2025114641000251.tif235168

[0507] (Table C) TIFF2025114641000252.tif205166TIFF2025114641000253.tif173166TIFF2025114641000254.t if206166TIFF2025114641000255.tif168166TIFF2025114641000256.tif214166TIFF20251146410 00257.tif207166TIFF2025114641000258.tif180166TIFF2025114641000259.tif222166TIFF202 5114641000260.tif199166TIFF2025114641000261.tif197166TIFF2025114641000262.tif222166

[0508] In some embodiments, the protein-drug conjugate is a conjugate of formula (XXX): TIFF2025114641000263.tif21153In formula, each R A teeth, TIFF2025114641000264.tif111132.

[0509] In other embodiments, the protein-drug conjugate is a conjugate of formula (XXX): TIFF2025114641000265.tif27140, each R A teeth, TIFF2025114641000266.tif212155TIFF2025114641000267.tif209164TIFF2025114641000268.tif203163TIFF2025114641000269.tif190159TIFF2025114641000270.tif80145.

[0510] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, The file is TIFF2025114641000271.tif101139.

[0511] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, The file is TIFF2025114641000272.tif92135.

[0512] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, TIFF2025114641000273.tif108148.

[0513] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, The file is TIFF2025114641000274.tif105137.

[0514] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, The file is TIFF2025114641000275.tif105137.

[0515] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, TIFF2025114641000276.tif80145.

[0516] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, The file is TIFF2025114641000277.tif80156.

[0517] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, The file is TIFF2025114641000278.tif92161.

[0518] For example, the protein-drug conjugate is of formula (XXX), where each R A teeth, The file is TIFF2025114641000279.tif82141.

[0519] In some embodiments, the protein-drug conjugate is a conjugate of formula (XXXII-1), (XXXII-2), (XXXII-3), or (XXXII-4): The file is TIFF2025114641000280.tif178165.

[0520] In some embodiments, the protein-drug conjugate is a conjugate of formula (XXXIII): TIFF2025114641000281.tif19128 In the formula, each R B teeth, TIFF2025114641000282.tif215121.

[0521] In some embodiments, the protein-drug conjugate is a conjugate of formula (XXXII-1), (XXXII-2), (XXXII-3), (XXXII-4), or (XXXIII), wherein the variable -L D -D is TIFF2025114641000283.tif167128TIFF2025114641000284.tif86128.

[0522] In other embodiments, the protein-drug conjugate is a conjugate of formula (XXXIV-1), (XXXIV-2), (XXXXIV-3), or (XXXIV-4): TIFF2025114641000285.tif135165TIFF2025114641000286.tif132164TIFF2025114641000287.tif144159TIFF2025114641000288.tif119154.

[0523] Pharmaceutical Compositions Pharmaceutical compositions comprising one or more conjugates disclosed herein in an acceptable carrier, such as stabilizers, buffers, etc., are also included. The conjugates can be administered and introduced into a subject by standard means, with or without stabilizers, buffers, etc. to form a pharmaceutical composition. Administration can be topical (including administration to the mucous membranes, including the eye and vaginal and rectal delivery), pulmonary administration, for example, by inhalation or injection of powders or aerosols, including administration by a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral administration, or parenteral administration, including intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, for example, intrathecal or intraventricular administration. The conjugates can be formulated and used as sterile solutions and / or suspensions for injection; lyophilized powders for reconstitution before injection / infusion; topical compositions; tablets, capsules, or elixirs for oral administration; or suppositories for rectal administration, as well as other compositions known in the art.

[0524] A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., to a cell or systemically to a subject, e.g., a human. Suitable forms depend, in part, on the use or route of entry, e.g., oral, inhalation, transdermal, or injection / infusion. Such forms should not prevent the composition or formulation from reaching the target cells (i.e., cells to which the drug is desired to be delivered). For example, a pharmacological composition injected into the bloodstream should be soluble. Other factors are known in the art and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

[0525] "Systemic administration" refers to in vivo systemic absorption or accumulation of the conjugate in the bloodstream, followed by distribution throughout the body. Routes of administration that result in systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular. Each of these routes of administration exposes the conjugate to accessible diseased tissue. The rate of entry of active agents into the circulation has been shown to be a function of molecular weight or size. By using the conjugates of the present disclosure, drug delivery can be localized to specific cells, such as cancer cells, via the specificity of the PBRM.

[0526] A "pharmaceutically acceptable formulation" refers to a composition or formulation that allows for effective distribution of the conjugate to the physical location most suitable for its desired activity. In one embodiment, effective delivery occurs before clearance by the reticuloendothelial system or before off-target binding occurs, which can result in reduced efficacy or toxicity. Non-limiting examples of agents suitable for formulation with conjugates include: P-glycoprotein inhibitors (such as Pluronic P85), which can increase the entry of active agents into the CNS; biodegradable polymers such as poly(DL-lactide-coglycolide) microspheres for sustained-release delivery after intracerebral implantation; and loaded nanoparticles, such as those made from polybutylcyanoacrylate, which can deliver active drugs across the blood-brain barrier and alter neural uptake mechanisms.

[0527] Also encompassed herein are pharmaceutical compositions prepared for storage or administration, containing a pharmaceutically effective amount of the desired conjugate in a pharmaceutically acceptable carrier or diluent. Carriers, diluents, and / or excipients acceptable for therapeutic use are well known in the pharmaceutical arts. For example, buffers, preservatives, bulking agents, dispersing agents, stabilizers, and dyes may be added. Additionally, antioxidants and suspending agents may be used. Examples of suitable carriers, diluents, and / or excipients include, but are not limited to, (1) Dulbecco's phosphate-buffered saline, pH about 6.5 (containing about 1 mg / ml to 25 mg / ml human serum albumin), (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose.

[0528] The term " pharmaceutically effective amount " as used herein refers to the amount of pharmaceutical agent that treats, improves, or prevents identified disease or condition, or shows detectable therapeutic or inhibitory effect.Can detect effect by any assay method known in the art.The exact effective amount for a subject depends on the subject's weight, size, and health condition; the nature and degree of condition; and the therapeutic agent or combination of therapeutic agents selected for administration.The pharmaceutically effective amount in a given situation can be determined by routine experimentation, which is within the skill and judgment of a clinician.In a preferred aspect, disease or condition can be treated through gene silencing.

[0529] For any conjugate, the pharmaceutically effective amount can be estimated first in cell culture assays, for example, in tumor cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.Animal models can also be used to determine the appropriate concentration range and administration route.These information can then be used to determine the useful dose and administration route in humans.Therapeutic / prophylactic efficacy and toxicity, such as ED, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. 50 (the dose that is therapeutically effective in 50% of the population) and LD 50The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD ratio can be determined. 50 / ED 50 Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage can vary within this range depending on the dosage form used, the sensitivity of the patient, and the route of administration.

[0530] For example, drugs or their derivatives, drug-conjugates, or PBRM-drug conjugates can be evaluated for their ability to inhibit tumor growth in several cell lines using Cell titer Glo. Dose-response curves can be generated using SoftMax Pro software, and IC 50 Values can be determined from a four-parameter curve fit. The cell lines used can include a cell line that is the target of the PBRM and a control cell line that is not the target of the PBRM contained in the test conjugate.

[0531] In one embodiment, the conjugate is formulated for parenteral administration by injection, including the use of conventional catheterization or infusion. The formulation for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers, with added preservatives. The conjugate can be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the conjugate can be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives, and buffering agents can be dissolved in the vehicle. As used herein, the term "parenteral" includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection, or infusion techniques. In addition, a pharmaceutical formulation comprising the conjugate and a pharmaceutically acceptable carrier is provided. One or more of the conjugates may be present together with one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants, and optionally other active ingredients.

[0532] Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol.Particularly acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution.In addition, sterile, fixed oils are conventionally used as solvents or suspending media.For this purpose, bland, fixed oils, including synthetic mono- or diglycerides, can be used.In addition, fatty acids such as oleic acid are used in injectable preparations.

[0533] The conjugates and compositions described herein can be administered in suitable forms, preferably parenterally, more preferably intravenously.For parenteral administration, the conjugates or compositions can be aqueous or non-aqueous sterile solutions, suspensions, or emulsions.Propylene glycol, vegetable oils, and injectable organic esters, such as ethyl oleate, can be used as solvents or vehicles.The compositions can also contain adjuvants, emulsifiers, or dispersants.

[0534] Dosage levels on the order of between about 0.001 mg / kg and about 140 mg / kg of body weight per day are useful in treating the conditions indicated above (about 0.05 mg / kg to about 7 g / subject per day). In some embodiments, the dosage administered to a patient is about 0.001 mg / kg to about 100 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 0.01 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 0.1 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 0.1 mg / kg to about 20 mg / kg of the subject's body weight. In some embodiments, the dosage administered is about 0.1 mg / kg to about 5 mg / kg or about 0.1 mg / kg to about 10 mg / kg of the subject's body weight. In some embodiments, the administered dosage is about 1 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the administered dosage is about 1 mg / kg to about 10 mg / kg of the subject's body weight. The amount of conjugate that can be combined with a carrier material to produce a single dosage form varies depending on the host treated and the particular method of administration. Dosage unit forms may generally contain about 0.001 mg to about 100 mg; about 0.01 mg to about 75 mg; or about 0.01 mg to about 50 mg; or about 0.01 mg to about 25 mg of conjugate.

[0535] For intravenous administration, dosage levels can range from the ranges described in the previous paragraph, or from about 0.01 to about 200 mg of conjugate per kg of animal body weight. In one aspect, the composition can contain from about 1 to about 100 mg of conjugate per kg of animal body weight. In another aspect, the amount administered ranges from about 0.1 to about 25 mg of compound per kg of body weight.

[0536] In some embodiments, the conjugate can be administered as follows: The conjugate can be given daily for about 5 days as a daily intravenous injection, a bolus, or as a continuous infusion for about 5 days.

[0537] Alternatively, the conjugate may be administered once a week for six weeks or more. Alternatively, the conjugate may be administered once every two or three weeks. A bolus dose is administered in about 50 to about 400 ml of saline, to which about 5 to about 10 ml of human serum albumin may be added. A continuous infusion is administered in about 250 to about 500 ml of saline, to which about 25 to about 50 ml of human serum albumin may be added, per 24 hours.

[0538] In some embodiments, the patient can receive a second course of treatment about 1 to about 4 weeks after treatment. Specific clinical trial protocols regarding route of administration, excipients, diluents, dosages, and times can be determined by one skilled in the art based on the clinical situation.

[0539] In other embodiments, the therapeutically effective amount may be provided based on another regular schedule, i.e., daily, weekly, monthly, or yearly, or based on an irregular schedule with varying administration days, weeks, months, etc. Alternatively, the therapeutically effective amount to be administered may vary. In one embodiment, the therapeutically effective amount for the first administration is higher than the therapeutically effective amount for one or more of the subsequent administrations. In another embodiment, the therapeutically effective amount for the first administration is lower than the therapeutically effective amount for one or more of the subsequent administrations. Equivalent dosages may be administered over various time periods, including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every 2 weeks, about every 3 weeks, about every month, and about every 2 months. The number and frequency of doses corresponding to a completed course of treatment will be determined according to the recommendations of relevant regulatory bodies and the judgment of a medical professional. The therapeutically effective amount described herein refers to the total amount administered during a given period of time; that is, if more than one different conjugate described herein is administered, the therapeutically effective amount corresponds to the total amount administered. It is understood that the specific dose level for a particular subject will depend on various factors, including the specific conjugate activity, age, body weight, overall health, sex, diet, administration time, administration route and excretion rate, combination with other active agents, and the severity of the particular disease being treated.

[0540] In some embodiments, a therapeutically effective amount of a conjugate disclosed herein generally refers to the amount necessary to achieve a therapeutic goal. As described above, this may be the binding interaction between an antibody and its target antigen, which, in some cases, interferes with the function of the target. The amount required to be administered further depends on the binding affinity of the antibody for its specific antigen and the rate at which the administered antibody is removed from the free volume of the subject to which it is administered. Typical ranges for therapeutically effective doses of the conjugates disclosed herein may be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight, about 0.1 mg / kg body weight to about 100 mg / kg body weight, or about 0.1 mg / kg body weight to about 150 mg / kg body weight. Typical dosing frequencies may range, for example, from twice daily to once monthly (e.g., once daily, once weekly; once every other week; every three weeks, or once monthly). For example, the conjugates disclosed herein can be administered (e.g., weekly, every two weeks, every three weeks, or monthly as a single dose) at about 0.1 mg / kg to about 20 mg / kg (e.g., 0.2 mg / kg, 0.5 mg / kg, 0.67 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg).For example, the conjugates disclosed herein can be administered at about 0.1 mg / kg to about 20 mg / kg (e.g., 0.2 mg / kg, 0.5 mg / kg, 0.67 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 19 mg / kg, or 20 mg / kg) (e.g., weekly, every two weeks, every three weeks, or monthly as a single dose) to treat cancer.

[0541] For administration to non-human animals, the conjugate can also be added to the animal's feed or drinking water. It may be convenient to formulate the animal's feed and drinking water so that the animal ingests a therapeutically appropriate amount of the conjugate with its diet. It may also be convenient to provide the conjugate as a premix for addition to the feed or drinking water.

[0542] The conjugates can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. Using multiple compounds to treat an indication can increase beneficial effects while reducing the presence of side effects. In some embodiments, the conjugates are used in combination with chemotherapeutic agents, such as those disclosed in U.S. Patent No. 7,303,749. In other embodiments, the chemotherapeutic agents include, but are not limited to, letrozole, oxaliplatin, docetaxel, 5-FU, lapatinib, capecitabine, leucovorin, erlotinib, pertuzumab, bevacizumab, and gemcitabine. The present disclosure also provides pharmaceutical kits containing one or more containers containing one or more conjugates and / or compositions of the present disclosure, including one or more chemotherapeutic agents. Such kits can also include, for example, other compounds and / or compositions, devices for administering the compounds and / or compositions, and written instructions in a format prescribed by a government agency having jurisdiction over the manufacture, use, or sale of pharmaceuticals or biological products. The compositions described herein can be packaged as a single dose or for continuous or periodic discontinuous administration.For continuous administration, the package or kit can include the conjugate in each dosage unit (for example, a solution, or other unit described above or used in drug delivery), and optionally instructions for administering the dose daily, weekly, or monthly for a predetermined period or as prescribed.If it is desired to change the composition, the concentration of the components of the composition, or the relative ratio of the conjugate or drug in the composition over time, the package or kit can contain a series of dosage units that provide the desired variability.

[0543] Packages or kits for dispensing pharmaceutical agents for periodic oral use are known in the art. In one embodiment, the package has an indicator for each period. In another embodiment, the package is a labeled blister package, a dial dispenser package, or a bottle. The packaging means of the kit may itself be intended for administration, such as a syringe, pipette, eye dropper, or other such device, from which the formulation can be applied to the affected area of the body, injected into a subject, or even applied to and mixed with other components of the kit.

[0544] How to use Treatment method In certain preferred embodiments, the protein-drug conjugates of the present disclosure are used in methods of treating animals (preferably mammals, most preferably humans, including men, women, infants, children, and adults). In one embodiment, the conjugates of the present disclosure may be used in methods of treating animals, comprising administering to the animal a biodegradable, biocompatible conjugate of the present disclosure. For example, the conjugates of the present disclosure can be administered in the form of soluble linear polymers, copolymers, conjugates, colloids, particles, gels, solids, fibers, films, and the like. The biodegradable, biocompatible conjugates disclosed herein can be used as drug carriers and drug carrier components in controlled drug release systems, preparations for minimally invasive surgical procedures, and the like. The pharmaceutical formulations can be injectable, implantable, and the like.

[0545] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of at least one conjugate of the present disclosure; wherein the conjugate releases one or more therapeutic agents after biodegradation.

[0546] In another embodiment, the conjugates can be administered in vitro, in vivo, and / or ex vivo to treat patients and / or modulate the growth of selected cell populations, including, for example, cancer. Specific types of cancer that can be treated with the conjugates include, but are not limited to, the following: (1) fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, and prostate cancer. , esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma, pleomorphic astrocytoma, medulloblastoma, craniopharyngioma, (2) solid tumors, including, but not limited to, acute lymphoblastic leukemia "ALL", acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia "AML", acute promyelocytic leukemia "APL", acute monoblastic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonocytic ... Leukemias, acute nonlymphocytic leukemia, acute anaplastic leukemia, chronic myeloid leukemia "CML", chronic lymphocytic leukemia "CLL", hairy cell leukemia, multiple myeloma, acute and chronic leukemias, e.g., lymphoblastic, myeloid, and lymphocytic, myeloid leukemia, including blood-bone cancers; and (3) lymphomas, e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and polycythemia vera.

[0547] In another embodiment, the conjugates can be administered in vitro, in vivo, and / or ex vivo to modulate the proliferation of selected cell populations in and / or treat patients with anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, mouth cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, pharyngeal cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, renal cancer, or gastric cancer.

[0548] In another embodiment, the cancer is selected from the group consisting of breast cancer, gastric cancer, non-small cell lung cancer (NSCLC), prostate cancer, and ovarian cancer.

[0549] In another embodiment, the conjugate can be administered in vitro, in vivo, and / or ex vivo to treat, prevent, reduce the risk of developing, and / or delay the onset of a certain condition or disorder, such as cancer. For example, the conjugate of the present disclosure is useful in treating, preventing, delaying the progression of, or otherwise ameliorating the symptoms of cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, mouth cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, pharyngeal cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, kidney cancer, and gastric cancer.

[0550] In another embodiment, the conjugates can be administered in vitro, in vivo, and / or ex vivo to treat autoimmune diseases such as systemic lupus, rheumatoid arthritis, psoriasis, and multiple sclerosis; transplant rejection such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection; graft-versus-host disease; viral infections such as CMV infection, HIV infection, and AIDS; and parasitic infections such as giardiasis, amebiasis, schistosomiasis, and the like.

[0551] In certain embodiments, the conjugates can also be used for the manufacture of a medicament useful for treating or lessening the severity of disorders characterized by abnormal cell proliferation (eg, cancer).

[0552] In certain embodiments, the therapeutic agent is delivered locally to a specific target cell, tissue, or organ.

[0553] In some embodiments, in the implementation of the method of the present disclosure, the conjugate further comprises or is combined with a diagnostic label.In some exemplary embodiments, the diagnostic label is selected from the group consisting of radiopharmaceuticals or radioisotopes for gamma scintigraphy and PET, contrast agents for magnetic resonance imaging (MRI), contrast agents for CT examination, contrast agents for X-ray imaging examination, agents for ultrasound diagnosis, agents for neutron activation, moieties that can reflect, scatter or affect X-rays, ultrasound, radio waves and microwaves, and fluorophores.In some exemplary embodiments, the conjugate is further monitored in vivo.

[0554] Examples of diagnostic labels include, but are not limited to, diagnostic radiopharmaceuticals or radioisotopes for gamma scintigraphy and PET, contrast agents for magnetic resonance imaging (MRI) (e.g., paramagnetic atoms and superparamagnetic nanocrystals), contrast agents for CT, contrast agents for X-ray imaging, agents for ultrasound diagnosis, agents for neutron activation, and moieties that can reflect, scatter, or affect X-rays, ultrasound, radio waves, and microwaves, fluorophores in various optical procedures, etc. Diagnostic radiopharmaceuticals include □-emitting radionuclides, such as indium-111, technetium-99m, and iodine-131. Contrast agents for MRI (magnetic resonance imaging) include magnetic compounds, such as paramagnetic ions, iron, manganese, gadolinium, lanthanides, organic paramagnetic moieties, and superparamagnetic, ferromagnetic, and antiferromagnetic compounds, such as iron oxide colloids and ferrite colloids. Contrast agents for CT and other X-ray imaging methods include compounds that absorb X-rays, such as iodine, barium, etc. Contrast agents for ultrasound-based methods include compounds that can absorb, reflect, and scatter ultrasound, such as emulsions, crystals, bubbles, etc. Still other examples include substances useful for neutron activation, such as boron and gadolinium. Furthermore, labels that can reflect, refract, scatter, or otherwise affect X-rays, ultrasound, radio waves, microwaves, and other radiation useful in diagnostic procedures can also be used. Fluorescent labels can be used for photographic imaging. In some embodiments, the modifier comprises a paramagnetic ion or group.

[0555] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising preparing an aqueous formulation of at least one conjugate of the present disclosure and parenterally injecting the formulation into the subject.

[0556] In another aspect, the present disclosure provides a method for treating a disease or disorder in a subject, the method comprising preparing an implant comprising at least one conjugate of the present disclosure and implanting the implant into the subject. In an exemplary embodiment, the implant is a biodegradable gel matrix.

[0557] In another aspect, the present disclosure provides a method for treating an animal in need thereof, comprising administering a conjugate according to the methods described above.

[0558] In another aspect, the present disclosure provides a method for eliciting an immune response in an animal, comprising administering a conjugate according to the method described above.

[0559] In another aspect, the present disclosure provides a method of diagnosing a disease in an animal, comprising: administering a conjugate according to the method described above, wherein the conjugate comprises a detectable molecule; and Detecting the detectable molecule The present invention provides a method comprising:

[0560] In certain exemplary embodiments, detecting the detectable molecule is performed non-invasively, hi certain exemplary embodiments, detecting the detectable molecule is performed using suitable imaging equipment.

[0561] In one embodiment, a method for treating an animal includes administering to the animal a biodegradable, biocompatible conjugate of the present disclosure as a filler for a surgical wound where a tumor or growth has been removed. The biodegradable, biocompatible conjugate filler replaces the tumor site during recovery and degrades as the wound heals.

[0562] In some embodiments, the conjugate is coupled to a diagnostic label for in vivo monitoring.

[0563] The conjugates described above can be used for therapeutic, prophylactic, and analytical (diagnostic) treatment of animals. The conjugates are generally for parenteral administration, although in some cases they may be administered by other routes.

[0564] In one embodiment, the soluble or colloidal conjugate is administered intravenously. In another embodiment, the soluble or colloidal conjugate is administered by local (e.g., subcutaneous, intramuscular) injection. In another embodiment, the solid conjugate (e.g., particle, implant, drug delivery system) is administered by implantation or injection.

[0565] In another embodiment, a conjugate containing a detectable label is administered to study the pattern and kinetics of label distribution within an animal.

[0566] In certain embodiments, any one or more of the conjugates disclosed herein may be used in the practice of any of the methods described herein.

[0567] Pharmaceutical compositions of the conjugates described herein can be included in a container, pack, or dispenser together with instructions for administration.

[0568] In some embodiments, the composition may contain two or more active compounds necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitor. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0569] In one embodiment, the active compound (e.g., the conjugate or drug of the present disclosure) is administered in combination therapy, i.e., in combination with other agents, for example, therapeutic agents useful for treating pathological conditions or disorders such as various forms of cancer, autoimmune diseases, and inflammatory diseases. The term "in combination" in this context means that the agents are given substantially contemporaneously, either simultaneously or sequentially. When given sequentially, at the start of administration of the second compound, the first of the two compounds is preferably still detectable at an effective concentration at the treatment site.

[0570] For example, the combination therapy can include one or more conjugates disclosed herein co-formulated and / or co-administered with one or more additional antibodies, which can be the same or different antibodies as those used to form the conjugate.

[0571] For example, the combination therapy can include one or more therapeutic agents and / or adjuvants. In certain embodiments, the additional therapeutic agent is a small molecule inhibitor, another antibody-based therapy, a polypeptide or peptide-based therapy, a nucleic acid-based therapy, and / or other biologic.

[0572] In certain embodiments, the additional therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a growth inhibitor, an angiogenesis inhibitor, a PARP (poly(ADP)-ribose polymerase) inhibitor, an alkylating agent, an antimetabolite, an anti-microtubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic, any other nucleic acid damaging agent, or an immune checkpoint inhibitor. In one embodiment, therapeutic agents used in the treatment of cancer include platinum compounds (e.g., cisplatin or carboplatin); taxanes (e.g., paclitaxel or docetaxel); topoisomerase inhibitors (e.g., irinotecan or topotecan); anthracyclines (e.g., doxorubicin (ADRIAMYCIN®) or liposomal doxorubicin (DOXIL®)); antimetabolites (e.g., gemcitabine, pemetrexed); cyclophosphamide; vinorelbine (NAVELBINE®); hexamethylmelamine; ifosfamide; etoposide; angiogenesis inhibitors (e.g., bevacizumab (Avastin®)), thalidomide, TNP-470, platelet factor 4, interferon, or endostatin); PARP inhibitors (e.g., olaparib (Lynparza) (TM)); immune checkpoint inhibitors, such as monoclonal antibodies targeting either PD-1 or PD-L (pembrolizumab (Keytruda®), atezolizumab (MPDL3280A), or nivolumab (Opdivo®)), or CTA-4 (ipilimumab (Yervoy®)), kinase inhibitors (e.g., sorafenib or erlotinib), These include, but are not limited to, proteasome inhibitors (e.g., bortezomib or carfilzomib), immunomodulatory agents (e.g., lenalidomide or IL-2), radiation agents, ALK inhibitors (e.g., crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa), dalantercept (ACE-041), brigatinib (AP26113), entrectinib (NMS-E628), PF-06463922 TSR-011, CEP-37440, and X-396), and / or biosimilars thereof and / or combinations thereof.Other suitable agents include those considered standard of care by those of skill in the art and / or chemotherapeutic agents known to those of skill in the art.

[0573] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an antibody against CTLA-4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against CTLA-4. In other embodiments, the immune checkpoint inhibitor is a human or humanized antibody against CTLA-4. In one embodiment, the anti-CTLA-4 antibody blocks binding of CTLA-4 to CD80 (B7-1) and / or CD86 (B7-2) expressed on antigen-presenting cells. Exemplary antibodies against CTLA-4 include, but are not limited to, Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016, and MDX-101); Millipore's anti-CTLA4 antibody, clone 9H10; Pfizer's tremelimumab (CP-675,206, ticilimumab); and Abcam's anti-CTLA4 antibody clone BNI3.

[0574] In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO 2001014424; WO 2004035607; US2005 / 0201994; EP 1212422 B1; WO2003086459; WO2012120125; WO2000037504; WO2009100140; WO200609649; WO2005092380; WO2007123737; WO2006029219; WO20100979597; WO200612168; and WO1997020574. Additional CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publication Nos. WO 01 / 14424 and WO 00 / 37504; and U.S. Patent Application Publication Nos. 2002 / 0039581 and 2002 / 086014; and / or U.S. Patent Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281 (incorporated herein by reference). In some embodiments, the anti-CTLA-4 antibody is one disclosed in, for example, WO 98 / 42752; U.S. Patent Nos. 6,682,736 and 6,207,156; Hurwitz et al, Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al, J. Clin. Oncol., 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al, Cancer Res., 58:5301-5304 (1998) (incorporated herein by reference).

[0575] In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO1996040915.

[0576] In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, anti-CTLA4 RNAi molecules are disclosed by Mello and Fire in PCT Publication Nos. WO 1999 / 032619 and WO 2001 / 029058; U.S. Patent Application Publication Nos. 2003 / 0051263, 2003 / 0055020, 2003 / 0056235, 2004 / 265839, 2005 / 0100913, 2006 / 0024798, 2008 / 0050342, 2008 / 0081373, 2008 / 0248576, and 2008 / 055443; and / or U.S. Patent Nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438, which are incorporated herein by reference. In some cases, anti-CTLA4 RNAi molecule is in the form of the double-stranded RNAi molecule described by Tuschl in European Patent No. EP 1309726 (incorporated herein by reference).In some cases, anti-CTLA4 RNAi molecule is in the form of the double-stranded RNAi molecule described by Tuschl in United States Patent No. 7,056,704 and United States Patent No. 7,078,196 (incorporated herein by reference).In some embodiments, CTLA4 inhibitor is the aptamer described in PCT Publication No. WO2004081021.

[0577] Additionally, the anti-CTLA4 RNAi molecules of the present disclosure may take the form of RNA molecules described by Crooke in U.S. Patent Nos. 5,898,031, 6,107,094, 7,432,249, and 7,432,250, and European Application No. EP 0928290 (incorporated herein by reference).

[0578] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-L1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-L1. In other or additional embodiments, the immune checkpoint inhibitor is a human or humanized antibody against PD-L1. In one embodiment, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-L1. In another embodiment, the immune checkpoint inhibitor reduces the interaction between PD-1 and PD-L1. Exemplary immune checkpoint inhibitors include antibodies (e.g., anti-PD-L1 antibodies), RNAi molecules (e.g., anti-PD-L1 RNAi), antisense molecules (e.g., anti-PD-L1 antisense RNA), dominant-negative proteins (e.g., dominant-negative PD-L1 proteins), and small molecule inhibitors. Antibodies include monoclonal antibodies, humanized antibodies, deimmunized antibodies, and Ig fusion proteins. An exemplary anti-PD-L1 antibody includes clone EH12. Exemplary antibodies against PD-L1 include: MPDL3280A (RG7446) from Genentech; anti-mouse PD-L1 antibody clone 10F.9G2 (Cat #BE0101) from BioXcell; anti-PD-L1 monoclonal antibodies MDX-1105 (BMS-936559) and BMS-935559 from Bristol-Meyer's Squibb; MSB0010718C; mouse anti-PD-L1 clone 29E.2A3; and MEDI4736 from AstraZeneca.In some embodiments, the anti-PD-L1 antibody is an anti-PD-L1 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO2013079174; CN101104640; WO2010036959; WO2013056716; WO2007005874; WO2010089411; WO2010077634; WO2004004771; WO2006133396; WO201309906; US 20140294898; WO2013181634 or WO2012145493.

[0579] In some embodiments, the PD-L1 inhibitor is a nucleic acid inhibitor of PD-L1 expression. In some embodiments, the PD-L1 inhibitor is disclosed in one of the following patent publications (incorporated herein by reference): WO2011127180 or WO2011000841. In some embodiments, the PD-L1 inhibitor is rapamycin.

[0580] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L2. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-L2. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-L2. In other or additional embodiments, the immune checkpoint inhibitor is a human or humanized antibody against PD-L2. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-L2. In other embodiments, the immune checkpoint inhibitor reduces the interaction between PD-1 and PD-L2. Exemplary immune checkpoint inhibitors include antibodies (e.g., anti-PD-L2 antibodies), RNAi molecules (e.g., anti-PD-L2 RNAi), antisense molecules (e.g., anti-PD-L2 antisense RNA), dominant-negative proteins (e.g., dominant-negative PD-L2 proteins), and small molecule inhibitors. Antibodies include monoclonal antibodies, humanized antibodies, deimmunized antibodies, and Ig fusion proteins.

[0581] In some embodiments, the PD-L2 inhibitor is GlaxoSmithKline's AMP-224 (Amplimmune). In some embodiments, the PD-L2 inhibitor is rHIgM12B7.

[0582] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-1. In other embodiments, the immune checkpoint inhibitor is a human or humanized antibody against PD-1. For example, inhibitors of PD-1 biological activity (or their ligands) disclosed in U.S. Patent Nos. 7,029,674; 6,808,710; or U.S. Patent Application Publication Nos. 20050250106 and 20050159351 can be used in the combinations provided herein. Exemplary antibodies against PD-1 include: anti-mouse PD-1 antibody clone J43 (Cat #BE0033-2) from BioXcell; anti-mouse PD-1 antibody clone RMP1-14 (Cat #BE0146) from BioXcell; mouse anti-PD-1 antibody clone EH12; MK-3475 anti-mouse PD-1 antibody from Merck (Keytruda®, pembrolizumab, lambrolizumab, h409A1 1); and anti-PD-1 antibody from AnaptysBio (known as ANB011); antibody MDX-1 106 (ONO-4538); Bristol-Myers These include Squibb's human IgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106); AstraZeneca's AMP-514, and AMP-224; and pidilizumab (CT-011 or hBAT-1), CureTech Ltd.

[0583] Additional exemplary anti-PD-1 antibodies are described by Goldberg et al, Blood 1 10(1): 186-192 (2007), Thompson et al, Clin. Cancer Res. 13(6): 1757-1761 (2007), and Korman et al, International Application No. PCT / JP2006 / 309606 (Publication No. WO 2006 / 121168 A1), each of which is expressly incorporated herein by reference. In some embodiments, the anti-PD-1 antibody is an anti-PD-1 antibody disclosed in any of the following patent publications (incorporated herein by reference): W0014557; WO2011110604; WO2008156712; US2012023752; WO2011110621; WO2004072286; WO2004056875; WO20100036959; WO2010029434; WO201213548; WO2002078731; WO2012145493; WO2010089411; WO2001014557; WO2013022091; WO2013019906; WO2003011911; US20140294898; and WO2010001617.

[0584] In some embodiments, the PD-1 inhibitor is a PD-1 binding protein as disclosed in WO200914335 (incorporated herein by reference).

[0585] In some embodiments, the PD-1 inhibitor is a peptidomimetic inhibitor of PD-1, such as those disclosed in WO2013132317 (incorporated herein by reference).

[0586] In some embodiments, the PD-1 inhibitor is anti-mouse PD-1 mAb: clone J43, BioXCell (West Lebanon, NH).

[0587] In some embodiments, the PD-1 inhibitor is a PD-L1 protein, a PD-L2 protein, or fragment, as well as the antibody MDX-1 106 (ONO-4538), which has been tested in clinical studies for the treatment of certain malignancies (Brahmer et al., J Clin Oncol. 2010 28(19): 3167-75, Epub 2010 Jun. 1). Other blocking antibodies can be readily identified and generated by those skilled in the art based on known domains of interaction between PD-1 and PD-L1 / PD-L2, as discussed above. For example, peptides corresponding to the IgV region of PD-1 or PD-L1 / PD-L2 (or a portion of this region) can be used as antigens to develop blocking antibodies, using methods well known in the art.

[0588] In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO1. In some embodiments, the immune checkpoint inhibitor is a small molecule against IDO1. Exemplary small molecules against IDO1 include INCB024360 from Incyte, NSC-721782 (also known as 1-methyl-D-tryptophan), and F001287 from Bristol Meyers Squibb.

[0589] In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3 (CD223). In some embodiments, the immune checkpoint inhibitor is an antibody against LAG3. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against LAG3. In other or additional embodiments, the immune checkpoint inhibitor is a human or humanized antibody against LAG3. In additional embodiments, the antibody against LAG3 blocks the interaction of LAG3 with major histocompatibility complex (MHC) class II molecules. Exemplary antibodies against LAG3 include anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience; anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences; IMP321 (ImmuFact) from Immutep; anti-Lag3 antibody BMS-986016; and LAG-3 chimeric antibody A9H12. In some embodiments, the anti-LAG3 antibody is an anti-LAG3 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO2010019570; WO2008132601; or WO2004078928.

[0590] In some embodiments, the immune checkpoint inhibitor is an antibody against TIM3 (also known as HAVCR2). In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against TIM3. In other or additional embodiments, the immune checkpoint inhibitor is a human or humanized antibody against TIM3. In additional embodiments, the antibody against TIM3 blocks the interaction of TIM3 with galectin-9 (Gal9). In some embodiments, the anti-TIM3 antibody is an anti-TIM3 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO2013006490; WO201155607; WO2011159877; or WO200117057. In another embodiment, the TIM3 inhibitor is a TIM3 inhibitor disclosed in WO2009052623.

[0591] In some embodiments, the immune checkpoint inhibitor is an antibody against B7-H3. In one embodiment, the immune checkpoint inhibitor is MGA271.

[0592] In some embodiments, the immune checkpoint inhibitor is an antibody against MR. In one embodiment, the immune checkpoint inhibitor is lirilumab (IPH2101). In some embodiments, the antibody against MR blocks the interaction between KIR and HLA.

[0593] In some embodiments, the immune checkpoint inhibitor is an antibody against CD137 (also known as 4-1BB or TNFRSF9). In one embodiment, the immune checkpoint inhibitor is urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor). In one embodiment, the anti-CD137 antibody is an antibody disclosed in U.S. Patent Application Publication No. US 2005 / 0095244; an antibody disclosed in issued U.S. Patent No. 7,288,638 (e.g., 20H4.9-IgG4 [1007 or BMS-663513] or 20H4.9-IgG1 [BMS-663031]); antibodies disclosed in issued U.S. Patent No. 6,887,673 [4E9 or BMS-554271]; antibodies disclosed in issued U.S. Patent No. 7,214,493; antibodies disclosed in issued U.S. Patent No. 6,303,121; antibodies disclosed in issued U.S. Patent No. 6,569,997; antibodies disclosed in issued U.S. Patent No. 6,905,685; antibodies disclosed in issued U.S. Patent No. 6,355,476; antibodies disclosed in issued U.S. Patent No. 6,362,325 [1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1]; an antibody disclosed in issued U.S. Patent No. 6,974,863 (e.g., 53A2); or an antibody disclosed in issued U.S. Patent No. 6,210,669 (e.g., 1D8, 3B8, or 3E1). In a further embodiment, the immune checkpoint inhibitor is one disclosed in WO 2014036412. In another embodiment, the antibody against CD137 blocks the interaction between CD137 and CD137L.

[0594] In some embodiments, the immune checkpoint inhibitor is an antibody against PS. In one embodiment, the immune checkpoint inhibitor is bavituximab.

[0595] In some embodiments, the immune checkpoint inhibitor is an antibody against CD52. In one embodiment, the immune checkpoint inhibitor is alemtuzumab.

[0596] In some embodiments, the immune checkpoint inhibitor is an antibody against CD30. In one embodiment, the immune checkpoint inhibitor is brentuximab vedotin. In another embodiment, the antibody against CD30 blocks the interaction between CD30 and CD30L.

[0597] In some embodiments, the immune checkpoint inhibitor is an antibody against CD33. In one embodiment, the immune checkpoint inhibitor is gemtuzumab ozogamicin.

[0598] In some embodiments, the immune checkpoint inhibitor is an antibody against CD20. In one embodiment, the immune checkpoint inhibitor is ibritumomab tiuxetan. In another embodiment, the immune checkpoint inhibitor is ofatumumab. In another embodiment, the immune checkpoint inhibitor is rituximab. In another embodiment, the immune checkpoint inhibitor is tositumomab.

[0599] In some embodiments, the immune checkpoint inhibitor is an antibody against CD27 (also known as TNFRSF7). In one embodiment, the immune checkpoint inhibitor is CDX-1127 (Celldex Therapeutics). In another embodiment, the antibody against CD27 blocks the interaction between CD27 and CD70.

[0600] In some embodiments, the immune checkpoint inhibitor is an antibody against OX40 (also known as TNFRSF4 or CD134). In one embodiment, the immune checkpoint inhibitor is an anti-OX40 mouse IgG. In another embodiment, the antibody against OX40 blocks the interaction between OX40 and OX40L.

[0601] In some embodiments, the immune checkpoint inhibitor is an antibody against glucocorticoid-induced tumor necrosis factor receptor (GITR). In one embodiment, the immune checkpoint inhibitor is TRX518 (GITR, Inc.). In another embodiment, the antibody against GITR blocks the interaction between GITR and GITRL.

[0602] In some embodiments, the immune checkpoint inhibitor is an antibody against inducible T cell costimulatory molecule (ICOS, also known as CD278). In one embodiment, the immune checkpoint inhibitor is MEDI570 (MedImmune, LLC) or AMG557 (Amgen). In another embodiment, the antibody against ICOS blocks the interaction of ICOS with ICOSL and / or B7-H2.

[0603] In some embodiments, the immune checkpoint inhibitor is an inhibitor of BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM. As described elsewhere herein, the immune checkpoint inhibitor can be one or more binding proteins that bind to immune checkpoint molecules, antibodies (or fragments or variants thereof), nucleic acids that downregulate the expression of immune checkpoint molecules, or any other molecules that bind to immune checkpoint molecules (i.e., small organic molecules, peptidomimetics, aptamers, etc.). In some instances, the inhibitor of BTLA (CD272) is HVEM. In some instances, the inhibitor of CD160 is HVEM. In some instances, the inhibitor of 2B4 is CD48. In some instances, the inhibitor of LAIR1 is collagen. In some instances, the inhibitor of TIGHT is CD112, CD113, or CD155. In some instances, the inhibitor of CD28 is CD80 or CD86. In some cases, the inhibitor of LIGHT is HVEM. In some cases, the inhibitor of DR3 is TL1A. In some cases, the inhibitor of CD226 is CD155 or CD112. In some cases, the inhibitor of CD2 is CD48 or CD58. In some cases, SLAM is autoinhibitory and the inhibitor of SLAM is SLAM.

[0604] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of CTLA4 (cytotoxic T lymphocyte antigen 4, also known as CD152), PD-L1 (programmed cell death 1 ligand 1, also known as CD274), PDL2 (programmed cell death protein 2), PD-1 (programmed cell death protein 1, also known as CD279), B-7 family ligands (B7-H1, B7-H3, B7-H4), BTLA (B and T lymphocyte attenuator, also known as CD272), HVEM, TIM3 (T cell membrane protein 3), GAL9, LAG-3 (lymphocyte activation gene 3; CD223), VISTA, KIR (killer immunoglobulin receptor), 2B4 (also known as CD244), CD160, CGEN-15049, CHK1 (checkpoint kinase 1), CHK2 (checkpoint kinase 2), A2aR (adenosine A2a receptor), CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1 (indoleamine 2,3-dioxygenase 1), IDO2 (indoleamine 2,3-dioxygenase 2), ICOS (inducible T cell costimulatory molecule), LAIR1, LIGHT (TNF family member, also known as TNFSF14), MARCO (macrophage receptor for collagen-like structures), OX40 (tumor necrosis factor receptor superfamily, member 4, TNFRSF4, and also known as CD134) and its ligand OX40L (CD252), SLAM, TIGHT, VTCN1, or a combination thereof.

[0605] In some embodiments, the immune checkpoint inhibitor interacts with a ligand of a checkpoint protein, including CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulatory molecule), LAIR1, LIGHT, MARCO (collagenous structure macrophage receptor), OX-40, SLAM, TIGHT, VTCN1, or a combination thereof.

[0606] In some embodiments, the immune checkpoint inhibitor inhibits checkpoint proteins including CTLA-4, PDL1, PD1, or a combination thereof.

[0607] In some embodiments, the immune checkpoint inhibitor inhibits checkpoint proteins including CTLA-4 and PD1 or a combination thereof.

[0608] In some embodiments, immune checkpoint inhibitors comprise pembrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1 105, durvalumab (MEDI4736), MPDL3280A, BMS-936559, IPH2101, TSR-042, TSR-022, ipilimumab, lirilumab, atezolizumab, avelumab, tremelimumab, or a combination thereof.

[0609] In some embodiments, the immune checkpoint inhibitor is nivolumab (BMS-936558), ipilimumab, pembrolizumab, atezolizumab, tremelimumab, durvalumab, avelumab, or a combination thereof.

[0610] In some embodiments, the immune checkpoint inhibitor is pembrolizumab.

[0611] Throughout the description, when compounds, scaffolds, and compositions are described as having, including, or comprising specific components, it is intended that the compositions also consist essentially of, or consist of, the described components. Similarly, when a method or process is described as having, including, or comprising specific process steps, the process also consists essentially of, or consists of, the described processing steps. Furthermore, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be performed simultaneously.

[0612] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples provided herein, or the use of exemplary descriptions (e.g., "etc.") are intended merely to better explain the invention and should not be construed as limitations on the claims unless otherwise expressly claimed. Nothing in this specification should be construed as indicating any non-claimed element as an essential element of what is claimed.

[0613] Synthesis method Any available technique can be used to make the conjugates or compositions thereof, as well as intermediates and components (e.g., scaffolds) useful for making them. For example, semi-synthetic and fully synthetic methods can be used.

[0614] General methods for making the conjugates or scaffolds disclosed herein are shown below in Schemes 1 and 2. More specific synthetic methods are described in the Examples. Variables in these schemes (e.g., M P , M A , L3 , W D , W M , L D , and L P’ etc.) have the same definition as set forth herein unless otherwise specified.

[0615] Scheme 1 TIFF2025114641000289.tif123145

[0616] Scheme 2 TIFF2025114641000290.tif160160

[0617] The synthetic processes of the present disclosure can tolerate a wide variety of functional groups; therefore, a variety of substituted starting materials can be used. While these processes generally provide the desired final compound at or near the end of the overall process, in some cases it may be desirable to further convert the compound to a pharmaceutically acceptable salt, ester, or prodrug thereof.

[0618] The drug compounds used in the conjugates of the present disclosure can be prepared...

Claims

1. A conjugate comprising a targeting moiety and one or more linker-drug moieties covalently attached to the targeting moiety, each linker-drug moiety comprises a multifunctional linker that links a targeting moiety to one or more drug units via a releasable assembly unit for each drug unit, and links a hydrophilic group to the drug unit of each linker-drug moiety, the releasable assembly unit being capable of releasing free drug near a target site targeted by the targeting moiety; and The multifunctional linker comprises a peptide moiety between the targeting moiety and the hydrophilic group, the peptide moiety comprising at least two amino acids. Conjugates.

2. 2. The conjugate of claim 1, wherein the targeting moiety is a protein-based recognition molecule (PBRM).

3. The conjugate of any one of the preceding claims, wherein the PBRM is an antibody or an antibody fragment.

4. The conjugate according to any one of the preceding claims, wherein the peptide moiety comprises from 3 to about 10 amino acids.

5. 10. The conjugate according to claim 1, wherein the peptide moiety comprises at least 4 amino acids or at least 5 amino acids.

6. 10. The conjugate of claim 1, wherein the hydrophilic group comprises a polyalcohol or a derivative thereof, a polyether or a derivative thereof, or a combination thereof.

7. 10. The conjugate of claim 1, wherein the hydrophilic group comprises an aminopolyalcohol.

8. The aminopolyalcohol where: n 1 is an integer from 0 to about 6; Each R 58 are independently hydrogen or C 1～8 is alkyl; R 60 is a bond, C 1～6 Alkyl linker, or -CHR 59 -, where R 59 is H, alkyl, cycloalkyl, or arylalkyl; R 61 is CH 2 OR 62 , COOR 62 , -(CH 2 ) n2 COOR 62 or heterocycloalkyl substituted with one or more hydroxyl groups; and R 62 is H or C 1～8 is alkyl; and n 2 is an integer from 1 to about 5; A conjugate according to any one of the preceding claims.

9. 10. The conjugate of claim 1, wherein the hydrophilic group comprises glucamine.

10. The hydrophilic group is 10. The conjugate of any one of the preceding claims, comprising:

11. The hydrophilic group is where: n 4 is an integer from 1 to about 25; Each R 63 are independently hydrogen or C 1～8 is alkyl; R 64 is a bond or C 1～8 is an alkyl linker; R 65 is H, C 1～8 Alkyl, or -(CH 2 ) n2 COOR 62 and R 62 is H or C 1～8 is alkyl; and n 2 is an integer from 1 to about 5; A conjugate according to any one of the preceding claims.

12. 10. The conjugate of claim 1, wherein the hydrophilic group comprises polyethylene glycol.

13. 10. The conjugate of any one of the preceding claims, wherein the hydrophilic group comprises polyethylene glycol having from about 6 to about 24 PEG subunits, preferably from about 6 to about 12 PEG subunits or from about 8 to about 12 PEG subunits.

14. A conjugate comprising a targeting moiety and one or more linker-drug moieties covalently attached to the targeting moiety, Each linker-drug moiety comprises a multifunctional linker that links a targeting moiety to one or more drug units via a releasable assembly unit for each drug unit, and links a polyalcohol or a derivative thereof to the drug unit of each linker-drug moiety, wherein the releasable assembly unit is capable of releasing free drug near a target site targeted by the targeting moiety; Conjugates.

15. 15. The conjugate of claim 14, wherein the polyalcohol or derivative thereof comprises an aminopolyalcohol.

16. The aminopolyalcohol where: n 1 is an integer from 0 to about 6; Each R 58 are independently hydrogen or C 1～8 is alkyl; R 60 is a bond, C 1～6 Alkyl linker, or -CHR 59 -, where R 59 is H, alkyl, cycloalkyl, or arylalkyl; R 61 is CH 2 OR 62 , COOR 62 , -(CH 2 ) n2 COOR 62 , -(CH 2 ) 1～5 COOH, or heterocycloalkyl substituted with one or more hydroxyls; R 62 is H or C 1～8 is alkyl; and n 2 is an integer from 1 to about 5; 16. The conjugate of claim 15.

17. The conjugate of any one of claims 14 to 16, wherein the polyalcohol or derivative thereof comprises glucamine.

18. A conjugate of formula (I): During the ceremony, a 1 is an integer from 0 to 1; a 2 is an integer from 1 to 3; a 3 is an integer from 0 to 1; a 4 is an integer from 1 to about 5; a 5 is an integer from 1 to 3; d 13 is an integer from 1 to about 14; PBRM stands for protein-based recognition molecule; L P’ M PBRM P and its corresponding monovalent moiety L P is a functional group W that can form a covalent bond with the functional group of the PBRM. P Contains; M P is a stretcher unit; L M is a bond or a trivalent or tetravalent linker, and L M If is a bond, a 2 is 1 and L M If is a trivalent linker, a 2 is 2, or L M is a tetravalent linker, 2 is 3; L 3 is a carbonyl-containing moiety; M A comprises a peptide portion containing at least two amino acids; T 1 is a hydrophilic group, and T 1 and M A Between is T 1 and M A means a direct or indirect connection with; each occurrence of D is independently a therapeutic agent having a molecular weight of ≦about 5 kDa; and L D Each occurrence of independently converts D to M A and contains at least one cleavable bond, such that upon bond rupture D is released in an active form for its intended therapeutic effect. A conjugate according to any one of the preceding claims.

19. A peptide-containing scaffold having any of formulas (II) to (XIV): During the ceremony, a 1 is an integer from 0 to 1; a 2 is an integer from 1 to 3; a 3 is an integer from 0 to 1; a 4 is an integer from 1 to about 5; a 5 is an integer from 1 to 3; d 13 is an integer from 1 to about 14; PBRM means protein-based recognition molecule; L P’ M PBRM P and its corresponding monovalent moiety L P is a functional group W that can form a covalent bond with the functional group of the PBRM. P Contains; M P is a stretcher unit; L M is a bond or a trivalent or tetravalent linker, and L M If is a bond, a 2 is 1 and L M If is a trivalent linker, a 2 is 2, or L M is a tetravalent linker, 2 is 3; L 3 is a carbonyl-containing moiety; M A comprises a peptide portion containing at least two amino acids; T 1 is a hydrophilic group, and T 1 and M A Between is T 1 and M A means a direct or indirect connection with; W M Each occurrence of independently may be replaced by forming a hydrogen, a protecting group, a leaving group, or a covalent bond to L M M P is a functional group capable of linking to; W D is independently a functional group capable of forming a covalent bond with a functional group of a therapeutic agent (“D”) having a molecular weight of ≦about 5 kDa; and L D Each occurrence of is independently D Or D to M A and L D contains at least one cleavable bond, and upon bond rupture D is released in an active form for its intended therapeutic effect; Peptide-containing scaffolds.

20. L 3 If present, -XC 1～10 alkylene-C(O)-, and X is L M where X is CH 2 , O, or NR 5 and R 5 is hydrogen, C 1～6 Alkyl, C 6～10 Aryl, C 3～8 Cycloalkyl, COOH, or COO-C 1～6 10. The conjugate or scaffold of any one of the preceding claims, which is alkyl.

21. L 3 If present, -NR 5 -(CH 2 ) v -C(O)- or -CH 2 -(CH 2 ) v -C(O)-NR 5 -(CH 2 ) v 10. The conjugate or scaffold of any preceding claim, wherein each v is independently an integer from 1 to 10.

22. L 3 When present, -NH-(CH 2 ) 2 -C(O)- or -(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 10. The conjugate or scaffold of any preceding claim, which is -C(O)-.

23. 10. The conjugate or scaffold of any preceding claim, wherein each v is independently an integer from 1 to 6 or 2 to 4, or is 2.

24. a 4 10. The conjugate or scaffold of claim 9, wherein is 1, 2, or 3.

25. d 13 10. The conjugate or scaffold of any preceding claim, wherein is 4 or 5.

26. Each W P , if present, independently, where: Ring A is cycloalkyl or heterocycloalkyl; Ring B is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 1K is a leaving group; R 1A is a sulfur protecting group; R 1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; R 2J is hydrogen, an aliphatic, aryl, heteroaliphatic, or carbocyclic moiety; R 3J is C 1～6 Alkyl and Z 1 , Z 2 , Z 3 , and Z 7 each is independently a carbon or nitrogen atom; R 4j is hydrogen, halogen, OR, -NO 2 , -CN, -S(O) 2 R.C. 1～24 alkyl, or 6- to 24-membered aryl or heteroaryl, where C 1～24 The alkyl, or 6- to 24-membered aryl or heteroaryl may be substituted with one or more aryl or heteroaryl; or two R 4j together form a fused cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R is hydrogen, alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl; R 5j is C(R 4j ) 2 , O, S, or NR; and z 1 is the integer 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, A conjugate or scaffold according to any one of the preceding claims.

27. R 1K is halo or RC(O)O—, where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

28. R 1A but, where r is 1 or 2 and R s1 , R s2 , and R s3 each of which is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; A conjugate or scaffold according to any one of the preceding claims.

29. M P exists, -(Z 4 )-[(Z 5 )-(Z 6 )] z - and Z 4 is L P’ or L P It is connected to Z 6 is L M is connected to; z is 1, 2, or 3; Z 4 teeth, where * is L P’ or L P and ** means connection to Z if present 5 Or Z 6 to or Z 5 and Z 6 If neither of these exists, then L M means a connection to; b 1 is an integer from 0 to 6; e 1 is an integer between 0 and 8, R 17 is C 1～10 Alkylene, C 1～10 Heteroalkylene, C 3～8 Cycloalkylene, O-(C 1～8 Alkylene, arylene, -C 1～10 Alkylene-arylene-, -arylene-C 1～10 Alkylene-, -C 1～10 Alkylene-(C 3～8 Cycloalkylene)-, -(C 3～8 Cycloalkylene-C 1～10 Alkylene-, 4- to 14-membered heterocycloalkylene, -C 1～10 Alkylene-(4- to 14-membered heterocycloalkylene)-, -(4- to 14-membered heterocycloalkylene)-C 1～10 Alkylene-, -C 1～10 Alkylene-C(=O)-, -C 1～10 Heteroalkylene-C(=O)-, -C 3～8 Cycloalkylene -C(=O)-, -O-(C 1～8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1～10 Alkylene-arylene-C(=O)-, -arylene-C 1～10 Alkylene-C(=O)-, -C 1～10 Alkylene-(C 3～8 Cycloalkylene)-C(=O)-, -(C 3～8 Cycloalkylene)-C 1～10 Alkylene-C(=O)-, -4 to 14-membered heterocycloalkylene-C(=O)-, -C 1～10 Alkylene-(4- to 14-membered heterocycloalkylene)-C(=O)-, -(4- to 14-membered heterocycloalkylene)-C 1～10 Alkylene-C(=O)-, -C 1～10 Alkylene-NH-, -C 1～10 Heteroalkylene-NH-, -C 3～8 Cycloalkylene-NH-, -O-(C 1～8 alkyl)-NH-, -arylene-NH-, -C 1～10 Alkylene-arylene-NH-, -arylene-C 1～10 Alkylene-NH-, -C 1～10 Alkylene-(C 3～8 Cycloalkylene)-NH-, -(C 3～8 Cycloalkylene)-C 1～10 Alkylene-NH-, -4 to 14-membered heterocycloalkylene-NH-, -C 1～10 Alkylene-(4- to 14-membered heterocycloalkylene)-NH-, -(4- to 14-membered heterocycloalkylene)-C 1～10 Alkylene-NH-, -C 1～10 Alkylene-S-, -C 1～10 Heteroalkylene -S-, -C 3～8 Cycloalkylene -S-, -OC 1～8 Alkyl)-S-, -arylene-S-, -C 1～10 Alkylene-arylene-S-, -arylene-C 1～10 Alkylene-S-, -C 1～10 Alkylene-(C 3～8 Cycloalkylene)-S-, -(C 3～8 Cycloalkylene)-C 1～10 Alkylene-S-, -4 to 14-membered heterocycloalkylene-S-, -C 1～10 Alkylene-(4- to 14-membered heterocycloalkylene)-S-, or -(4- to 14-membered heterocycloalkylene)-C 1 ~C 10 alkylene-S-; each Z 5 is independently non-existent, R 57 -R 17 or a polyether unit; Each R 57 are independently bonded, NR 23 , S, or O; Each R 23 are independently hydrogen, C 1～6 Alkyl, C 6～10 Aryl, C 3～8 Cycloalkyl, -COOH, or -COO-C 1～6 is alkyl; and each Z 6 is independently absent, -C 1～10 Alkyl-R 3 -, -C 1～10 Alkyl-NR 5 -, -C 1～10 Alkyl-C(O)-, -C 1～10 Alkyl-O-, -C 1～10 Alkyl-S-, or -(C 1～10 Alkyl-R 3 ) g1 -C 1～10 alkyl-C(O)-; Each R 3 are independently -C(O)-NR 5 -or-NR 5 -C(O)-; Each R 5 are independently hydrogen, C 1～6 Alkyl, C 6～10 Aryl, C 3～8 Cycloalkyl, COOH, or COO-C 1～6 is alkyl; and g 1 is an integer between 1 and 4, A conjugate or scaffold according to any one of the preceding claims.

30. M P But if it exists, where * is L P’ or L P and ** means connection to L M means a connection to; R 3 is -C(O)-NR 5 or -NR 5 -C(O)-; R 4 is a bond or -NR 5 -(CR 20 R 21 )—C(O)—; R 5 is hydrogen, C 1～6 Alkyl, C 6～10 Aryl, C 3～8 Cycloalkyl, -COOH, or -COO-C 1～6 is alkyl; R 17 is C 1～10 Alkylene, C 1～10 Heteroalkylene, C 3～8 Cycloalkylene, O-(C 1～8 Alkylene, arylene, -C 1～10 Alkylene-arylene-, -arylene-C 1～10 Alkylene-, -C 1～10 Alkylene-(C 3～8 Cycloalkylene)-, -(C 3～8 Cycloalkylene-C 1～10 Alkylene-, 4- to 14-membered heterocycloalkylene, -C 1～10 Alkylene-(4- to 14-membered heterocycloalkylene)-, -(4- to 14-membered heterocycloalkylene)-C 1～10 Alkylene-, -C 1～10 Alkylene-C(=O)-, -C 1～10 Heteroalkylene -C(=O)-, -C 3～8 Cycloalkylene -C(=O)-, -O-(C 1～8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1～10 Alkylene-arylene-C(=O)-, -arylene-C 1～10 Alkylene-C(=O)-, -C 1～10 Alkylene-(C 3～8 Cycloalkylene)-C(=O)-, -(C 3～8 Cycloalkylene)-C 1～10 Alkylene-C(=O)-, -4 to 14-membered heterocycloalkylene-C(=O)-, -C 1～10 Alkylene-(4- to 14-membered heterocycloalkylene)-C(=O)-, -(4- to 14-membered heterocycloalkylene)-C 1～10 Alkylene-C(=O)-, -C 1～10 Alkylene-NH-, -C 1～10 Heteroalkylene-NH-, -C 3～8 Cycloalkylene-NH-, -O-(C 1～8 alkyl)-NH-, -arylene-NH-, -C 1～10 Alkylene-arylene-NH-, -arylene-C 1～10 Alkylene-NH-, -C 1～10 Alkylene-(C 3～8 Cycloalkylene)-NH-, -(C 3～8 Cycloalkylene)-C 1～10 Alkylene-NH-, -4 to 14-membered heterocycloalkylene-NH-, -C 1～10 Alkylene-(4- to 14-membered heterocycloalkylene)-NH-, -(4- to 14-membered heterocycloalkylene)-C 1～10 Alkylene-NH-, -C 1～10 Alkylene-S-, -C 1～10 Heteroalkylene -S-, -C 3～8 Cycloalkylene -S-, -OC 1～8 Alkyl)-S-, -arylene-S-, -C 1～10 Alkylene-arylene-S-, -arylene-C 1～10 Alkylene-S-, -C 1～10 Alkylene-(C 3～8 Cycloalkylene)-S-, -(C 3～8 Cycloalkylene)-C 1～10 Alkylene-S-, -4 to 14-membered heterocycloalkylene-S-, -C 1～10 Alkylene-(4- to 14-membered heterocycloalkylene)-S-, or -(4- to 14-membered heterocycloalkylene)-C 1 ~C 10 alkylene-S-; Each R 20 and R 21 are independently hydrogen, C 1～6 Alkyl, C 6～10 Aryl, Hydroxylated C 6～10 Aryl, Polyhydroxylated C 6～10 Aryl, 5- to 12-membered heterocycle, C 3～8 Cycloalkyl, Hydroxylated C 3～8 Cycloalkyl, Polyhydroxylated C 3～8 cycloalkyl, or the side chain of a natural or unnatural amino acid; Each R 23 are independently hydrogen, C 1～6 Alkyl, C 6～10 Aryl, C 3～8 Cycloalkyl, -COOH, or -COO-C 1～6 is alkyl; each b 1 are independently an integer from 0 to 6; e 1 is an integer between 0 and 8, each f 1 are independently an integer from 1 to 6; and g 2 is an integer between 1 and 4, A conjugate or scaffold according to any one of the preceding claims.

31. M P But if it exists, where * is L P’ or L P and ** means connection to L M means connection to A conjugate or scaffold according to any one of the preceding claims.

32. L M is a bond and a 2 10. The conjugate or scaffold of any preceding claim, wherein:

33. a 2 is 2 and L M but, where: If present, M P Connection to or M P If there is no L P or L P’ means a connection to; Y 1 If present, L 3 Connection to or L 3 If there is no M A means a connection to; R 2 and R' 2 are each independently hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, optionally substituted C 3～19 Branched alkyl, optionally substituted C 3～8 Cycloalkyl, optionally substituted C 6～10 Aryl, optionally substituted heteroaryl, optionally substituted C 1～6 Heteroalkyl, C 1～6 Alkoxy, aryloxy, C 1～6 Heteroalkoxy, C 2～6 Alkanoyl, optionally substituted arylcarbonyl, C 2～6 Alkoxycarbonyl, C 2～6 Alkanoyloxy, arylcarbonyloxy, optionally substituted C 2～6 Alkanoyl, optionally substituted C 2～6 Alkanoyloxy, optionally substituted C 2～6 Substituted alkanoyloxy, -COOH, or -COO-C 1～6 is alkyl; c 1 , c 2 , c 3 , c 4 , c 5 , c 7 , and c 8 are each independently an integer ranging from 0 to 10; and d 1 , d 2 , d 3 , d 4 , d 5 , and d 7 are each independently an integer ranging from 0 to 10; A conjugate or scaffold according to any one of the preceding claims.

34. a 2 is 2 and L M but That is, A conjugate or scaffold according to any one of the preceding claims.

35. a 2 is 3 and L M but and During the ceremony, If present, M P Connection to or M P If there is no L P or L P’ means a connection to; Y 1 If present, L 3 Connection to or L 3 If there is no M A means a connection to; R 2 and R' 2 are each independently hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, optionally substituted C 3～19 Branched alkyl, optionally substituted C 3～8 Cycloalkyl, optionally substituted C 6～10 Aryl, optionally substituted heteroaryl, optionally substituted C 1～6 Heteroalkyl, C 1～6 Alkoxy, aryloxy, C 1～6 Heteroalkoxy, C 2～6 Alkanoyl, optionally substituted arylcarbonyl, C 2～6 Alkoxycarbonyl, C 2～6 Alkanoyloxy, arylcarbonyloxy, optionally substituted C 2～6 Alkanoyl, optionally substituted C 2～6 Alkanoyloxy, optionally substituted C 2～6 Substituted alkanoyloxy, -COOH, or -COO-C 1～6 is alkyl; c 1 , c 2 , c 3 , c 4 , c 5 , c 6 , c 7 , and c 8 are each independently an integer ranging from 0 to 10; d 1 , d 2 , d 3 , d 4 , d 5 , d 6 , d 7 , and d 8 are each independently an integer ranging from 0 to 10; and e 1 , e 2 , e 3 , e 4 , e 5 , e 6 , e 7 , and e 8 are each independently an integer ranging from 0 to 10; A conjugate or scaffold according to any one of the preceding claims.

36. a 2 is 3 and L M but That is, A conjugate or scaffold according to any one of the preceding claims.

37. M A 10. The conjugate or scaffold of claim 9, wherein said conjugate or scaffold comprises a peptide portion containing at least about 5 amino acids.

38. M A 10. The conjugate or scaffold of claim 1, wherein the conjugate or scaffold comprises a peptide portion containing at most about 10 amino acids.

39. M A comprises a peptide moiety containing from 3 to about 10 amino acids selected from glycine, serine, glutamic acid, aspartic acid, lysine, cysteine, and combinations thereof.

40. M A 10. The conjugate or scaffold of any one of the preceding claims, wherein said conjugate or scaffold comprises a peptide moiety containing at least four glycines and at least one serine.

41. M A 10. The conjugate or scaffold of claim 9, wherein said peptide moiety contains at least four glycines and at least one glutamic acid.

42. M A 10. The conjugate or scaffold of claim 9, wherein said peptide moiety contains at least four glycines, at least one serine, and at least one glutamic acid.

43. 10. A conjugate according to any one of the preceding claims selected from those in Table B.

44. 10. The scaffold of any one of the preceding claims selected from those in Table C.

45. A conjugate of formula (XXX): In the formula, each R A but, That is, A conjugate according to any one of the preceding claims.

46. A conjugate of formula (XXX): In the formula, each R A but, That is, A conjugate according to any one of the preceding claims.

47. Each R A but, That is, A conjugate according to any one of the preceding claims.

48. Each R A but, That is, A conjugate according to any one of the preceding claims.

49. Each R A but, That is, A conjugate according to any one of the preceding claims.

50. Each R A but, That is, A conjugate according to any one of the preceding claims.

51. Each R A but, That is, A conjugate according to any one of the preceding claims.

52. Each R A but, That is, A conjugate according to any one of the preceding claims.

53. Each R A but, That is, A conjugate according to any one of the preceding claims.

54. Each R A but, That is, A conjugate according to any one of the preceding claims.

55. Each R A but, That is, A conjugate according to any one of the preceding claims.

56. A pharmaceutical composition comprising the conjugate of any one of the preceding claims and a pharmaceutically acceptable carrier.

57. 10. A method of treating a disorder in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate according to any one of the preceding claims.

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