Site-specific antibody-drug conjugates with peptide-containing linkers

Abstract

The present disclosure relates generally to antibody-drug conjugates comprising peptide-containing linkers, and methods of using these conjugates as therapeutic and / or diagnostic agents. Also disclosed herein are peptide-containing scaffolds useful for conjugating with a targeting moiety (e.g., an antibody), a drug, or both to generate antibody-drug conjugates.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and benefits of U.S. Provisional Application No. 62 / 958,916, filed on 9 January 2020, and U.S. Provisional Application No. 63 / 040,735, filed on 18 June 2020. The contents of each of these applications are incorporated herein by reference in their entirety.

[0002] Inclusion by referencing the sequence list The contents of the text file named "MRSN-029_001WO_SeqList.txt", created on January 6, 2021, with a size of 49KB, are incorporated in their entirety into this specification by reference. [Background technology]

[0003] background Traditionally, pharmaceuticals have primarily consisted of small molecules administered orally (as solid pills and liquids) or by injection. Over the past 30 years, formulations (i.e., compositions that control the route and / or rate of drug delivery, enabling the delivery of therapeutic agents to the site where they are needed) have become increasingly common and complex. Nevertheless, many questions and challenges regarding the development of new therapies and the mechanisms for administering them still need to be addressed. For example, many drugs generally have limited or otherwise diminished potency and therapeutic effect because they are partially broken down before reaching their desired target in the body, accumulate in non-target tissues, and / or have a short half-life.

[0004] Therefore, one objective in the field of drug delivery systems is to deliver intact drugs to specifically targeted areas of the body via systems that can stabilize the drug and / or extend its half-life, and control the in vivo movement of the therapeutic agent using either physiological or chemical mechanisms, or both.

[0005] Antibody-drug conjugates have been developed as target-specific therapeutic agents. Antibodies against various cancer cell surface antigens have been combined with microtubulin inhibitors (e.g., meitansinoids, auristatin, and taxanes), e.g., U.S. Patent No. 5,208,020 (Patent Document 1), U.S. Patent No. 5,416,064 (Patent Document 2), U.S. Patent No. 6,333,410 (Patent Document 3), U.S. Patent No. 6,441,163 (Patent Document 4), U.S. Patent No. 6,340,701 (Patent Document 5), U.S. Patent No. 6,372,738 (Patent Document 6), U.S. Patent No. 6,436,931 (Patent Document 7), U.S. Patent No. 6,596,757 (Patent Document 8) and U.S. It is conjugated with a variety of cytotoxic agents, including, but not limited to, U.S. Patent No. 7,276,497 (Patent Document 9) and DNA interaction therapeutics (e.g., calicheamicin, doxorubicin, and CC-1065 analogs, e.g., U.S. Patent No. 5,475,092 (Patent Document 10), U.S. Patent No. 5,585,499 (Patent Document 11), U.S. Patent No. 5,846,545 (Patent Document 12), U.S. Patent No. 6,534,660 (Patent Document 13), U.S. Patent No. 6,756,397 (Patent Document 14), and U.S. Patent No. 6,630,579 (Patent Document 15)). Antibody-drug conjugates with some of these cytotoxic drugs are being actively investigated clinically for cancer treatment (see, e.g., Ricart, AD, and Tolcher, AW, 2007, Nature Clinical Practice, 4, 245-255 (Non-Patent Literature 1); Krop et al., 2010, J. Clin. Oncol., 28, 2698-2704 (Non-Patent Literature 2)). However, existing antibody-drug conjugates have limitations. The main limitation is the inability to deliver sufficient concentrations of the drug to the target site, due to the limited number of target antigens, as well as / or the relatively moderate cytotoxicity of cancer drugs such as auristatin, methotrexate, daunorubicin, maytansinoids, taxanes, and vincristine. One approach to achieving significant cytotoxicity is to directly or indirectly conjugate multiple drug molecules to an antibody.However, such highly modified antibodies often exhibit impaired binding to target antigens and / or rapid in vivo clearance from the bloodstream. Therefore, it is necessary to improve the ability to deliver sufficient concentrations of the drug to the target so that maximum cytotoxicity with respect to the drug is achieved.

[0006] Conjugating a drug moiety to an antibody via covalent bonding generally results in a heterogeneous mixture of molecules, where the drug moiety is bound to several sites on the antibody. In some embodiments, cytotoxic drugs are typically conjugated to antibodies via lysine or cysteine ​​residues, thereby generating a heterogeneous antibody-drug conjugate mixture. Depending on the reaction conditions, the heterogeneous mixture typically contains a distribution of 0 to approximately 8 drug moieties bound to various sites on the antibody. Analytical and parsing methods are insufficient to isolate and characterize these antibody-drug conjugate species molecules within the heterogeneous mixture resulting from the conjugation reaction. Furthermore, the conjugation process can be irreproducible due to the difficulty in controlling the reaction conditions. Therefore, there is a need to reproducibly generate antibody-drug conjugates that possess site specificity (with respect to the conjugation site in the antibody) and / or stoichiometry (with respect to the ratio of antibody to drug). [Prior art documents] [Patent Documents]

[0007] [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]

[0008] [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 [Overview of the Initiative]

[0009] overview This disclosure features site-specific antibody-drug conjugates. These site-specific, target-directed moiety-drug conjugates exhibit controlled drug loading and strong binding to target antigens. In some embodiments, the target-directed moiety is a protein-recognizing molecule (PBRM). This disclosure also features peptide-containing scaffolds useful for conjugating with PBRMs, drugs, or both to obtain target-directed moiety-drug conjugates.

[0010] In some aspects, the present disclosure provides an antibody-drug conjugate of formula (I’): TIFF0007846624000001.tif34128 is provided, wherein, a2 is an integer from 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 12, ANTIBODY is a modified antibody, L P’ is a bivalent linker moiety that connects the modified antibody to M P and its corresponding monovalent moiety L P contains a functional group W that can form a covalent bond with a functional group of the modified antibody, P including, M P is a stretcher unit, L M is a linker, or a trivalent or tetravalent linker, and when L M is a linker, a2 is 1, or when L M is a trivalent linker, a2 is 2, or when L M is a tetravalent linker, a2 is 3, L 3 is a carbonyl-containing moiety, M A includes a peptide moiety containing at least two amino acids, T 1 is a hydrophilic group, and the 1 between T A and M TIFF0007846624000002.tif12128 indicates a direct or indirect bond between T 1 and M A showing, each occurrence of D is, independently, a therapeutic agent having a molecular weight of about 5 kDa or less, L D each occurrence of which is, independently, D to M AIt is a divalent linker portion that connects to and contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in its active form for its intended therapeutic effect.

[0011] In some aspects, this disclosure relates to an antibody-drug conjugate of formula (XXX): Provide TIFF0007846624000003.tif32160, In the formula, each R A teeth The filename is TIFF0007846624000004.tif160150, In the formula, d 13 It is 2, The antibody contains one or more asparagine groups at N297, which is connected to the remainder of the conjugate.

[0012] This disclosure also provides compositions comprising conjugates, methods for preparing them, and methods for using them in the treatment of various disorders, including cancer, without limit.

[0013] In some embodiments, this disclosure further relates to pharmaceutical compositions comprising a scaffold or conjugate described herein and a pharmaceutically acceptable carrier.

[0014] In some aspects, this disclosure relates to methods for preparing antibody-drug conjugates.

[0015] In some respects, this disclosure relates to a method for preparing an antibody-drug conjugate, comprising one or more steps described herein.

[0016] In some aspects, this disclosure is approximately 7 mg / m² 2 ~Approx. 162mg / m 2 The present invention relates to a conjugate or method in which an effective amount of the conjugate is administered to the subject at a dose such as on the first day of treatment and thereafter every three or four weeks.

[0017] In some aspects, the disclosure relates to a method for treating a disorder (e.g., cancer) in a subject requiring the treatment, comprising the step of administering an effective amount of the conjugate disclosed herein to the subject.

[0018] In some embodiments, the present disclosure relates to a method for treating NaPi2b-expressing cancer in a subject requiring such treatment, comprising the step of administering an effective amount of the conjugate disclosed herein to the subject.

[0019] In some aspects, the disclosure provides the use of the conjugates disclosed herein in the manufacture of pharmaceuticals for treating a disorder in which such conjugates are needed (e.g., cancer).

[0020] In some embodiments, the disclosure provides the use of the conjugates disclosed herein in the manufacture of pharmaceuticals for treating NaPi2b-expressing cancers of subjects requiring such use.

[0021] In some aspects, the disclosure provides the use of the conjugates disclosed herein for treating a disorder in which such treatment is required (e.g., cancer).

[0022] In some embodiments, the disclosure provides the use of the conjugates disclosed herein for treating NaPi2b-expressing cancers of subjects requiring such treatment.

[0023] In some embodiments, the Disclosure provides a conjugate for use in treating a disorder (e.g., cancer) in a subject requiring it, comprising the step of administering an effective amount of the conjugate disclosed herein to the subject.

[0024] In some embodiments, the present disclosure provides a conjugate for use in treating NaPi2b-expressing cancer in a subject requiring such treatment, comprising the step of administering an effective amount of the conjugate disclosed herein to the subject.

[0025] In some embodiments, the Disclosure relates to a method for diagnosing a disorder in an object suspected to have the disorder. The method includes administering an effective dose of the conjugate described herein to the object suspected to have the disorder, or performing an assay to detect a target antigen / receptor in a sample obtained from the object, to determine whether the object expresses a target antigen or receptor.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. In this specification, the singular form includes the plural form unless the context clearly indicates otherwise. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference. References cited herein are not considered prior art to the claimed invention. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to be limiting.

[0027] [Invention 1001] Antibody-drug conjugate of formula (XXX): TIFF0007846624000005.tif30150 In the formula, each R A teeth TIFF0007846624000006.tif157150 And, In the formula, d 13 It is 2, The antibody contains one or more asparagine groups at N297, which is connected to the remainder of the conjugate. [Invention 1002] Any of the above-mentioned conjugates of formula (XXX): TIFF0007846624000007.tif30152 In the formula, each R A teeth TIFF0007846624000008.tif112160TIFF0007846624000009.tif211126TIFF0007846624000010.tif213132TIFF0007846624000011.tif201137TIFF0007846624000012.tif171150 And, In the formula, d 13 It is 2, The antibody contains one or more asparagine groups at N297, which is connected to the remainder of the conjugate. [Invention 1003] Each R A but TIFF0007846624000013.tif104128 The conjugate of any of the present inventions described above. [Invention 1004] Each R A but TIFF0007846624000014.tif85128 The conjugate of any of the present inventions described above. [Invention 1005] Each R A but TIFF0007846624000015.tif96128 The conjugate of any of the present inventions described above. [Invention 1006] Each R A but TIFF0007846624000016.tif92128 The conjugate of any of the present inventions described above. [Invention 1007] Each R A but TIFF0007846624000017.tif94128 The conjugate of any of the present inventions described above. [Invention 1008] Each R A but TIFF0007846624000018.tif82128 The conjugate of any of the present inventions described above. [Invention 1009] Each R A but TIFF0007846624000019.tif84129 The conjugate of any of the present inventions described above. [Invention 1010] Each R A but TIFF0007846624000020.tif89128 The conjugate of any of the present inventions described above. [Invention 1011] Each R A but TIFF0007846624000021.tif86128 The conjugate of any of the present inventions described above. [Invention 1012] The conjugate is one of the following: TIFF0007846624000022.tif100135 During the ceremony, d 13 It is 2, ANTIBODY is amino acid sequence TIFF0007846624000023.tif4128 CDRH1, which includes, amino acid sequence TIFF0007846624000024.tif4128 CDRH2, which includes, amino acid sequence TIFF0007846624000025.tif4128 CDRH3, which includes, amino acid sequence TIFF0007846624000026.tif4128 CDRL1, which includes, amino acid sequence TIFF0007846624000027.tif4128 CDRL2, which includes, amino acid sequence TIFF0007846624000028.tif4128 CDRL3 including It is a NaPi2b antibody that contains, The antibody contains one or more asparagine groups at N297, which is connected to the remainder of the conjugate. TIFF0007846624000029.tif5128 It is GlcNAc, TIFF0007846624000030.tif5128 It is Fuc, TIFF0007846624000031.tif4128 It is GalNAc. [Invention 1013] The NaPi2b antibody is a conjugate of the present invention 1012, comprising the heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2. [Invention 1014] The conjugate is one of the following: TIFF0007846624000032.tif80160 During the ceremony, d 13 It is 2, ANTIBODY is amino acid sequence TIFF0007846624000033.tif4128 CDRH1, which includes, amino acid sequence TIFF0007846624000034.tif4128 CDRH2, which includes, amino acid sequence TIFF0007846624000035.tif4128 CDRH3, which includes, amino acid sequence TIFF0007846624000036.tif4128 CDRL1, which includes, amino acid sequence TIFF0007846624000037.tif4128 CDRL2, which includes, amino acid sequence TIFF0007846624000038.tif4128 CDRL3 including It is a NaPi2b antibody that contains, The antibody contains one or more asparagine groups at N297, which is connected to the remainder of the conjugate. TIFF0007846624000039.tif5128 It is GlcNAc, TIFF0007846624000040.tif5128 It is Fuc, TIFF0007846624000041.tif4128 It is GalNAc. [Invention 1015] The NaPi2b antibody is a conjugate of the present invention 1014, comprising the heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2. [Invention 1016] A method for preparing an antibody-drug conjugate, comprising the step of reacting a modified antibody with a scaffold selected from the scaffolds listed in Table B, thereby forming a site-specific antibody-drug conjugate, The modified antibody is obtained by contacting a glycoprotein containing an antibody and a core-GlcNAc moiety connected to site N297 of the antibody with endoglycosidase Endo SH to form an intermediate antibody containing the terminal GlcNAc moiety, and by contacting the intermediate antibody with 4-AzGalNAc-UDP in the presence of the enzyme β-(1,4)-GalNAcT to form a modified antibody containing the modified GlcNAc moiety. A method in which steps (a) and (b) are performed simultaneously. [Invention 1017] A pharmaceutical composition comprising any of the conjugates of the present invention described above and a pharmaceutically acceptable carrier. [Invention 1018] Any of the above-described conjugates of the present invention for treating cancer in a subject requiring cancer treatment, wherein an effective amount of the conjugate is administered to the subject. [Invention 1019] A method for treating cancer in a target subject requiring cancer treatment, comprising the step of administering an effective amount of any of the conjugates of the present invention described above. [Invention 1020] The method of the present invention 1019, wherein the cancer is a NaPi2b-expressing cancer selected from ovarian cancer, non-small cell lung cancer (NSCLC), endometrial cancer, papillary renal cell carcinoma, salivary duct cancer, papillary thyroid cancer, renal clear cell carcinoma, breast cancer, kidney cancer, cervical cancer, and cholangiocarcinoma. [Invention 1021] The method of the present invention 1020, wherein the NaPi2b-expressing cancer is ovarian cancer, non-small cell lung cancer (NSCLC), endometrial cancer, papillary renal cell carcinoma, salivary duct cancer, or papillary thyroid cancer. [Invention 1022] The method of the present invention 1020, wherein the NaPi2b-expressing cancer is ovarian cancer or non-small cell lung cancer (NSCLC). [Invention 1023] The effective dose of the conjugate is approximately 7 mg / m². 2 ~Approx. 162mg / m 2 The method of the present invention 1020, wherein the subject is administered the following dose on the first day of treatment and thereafter every three or four weeks. [Invention 1024] The dosage is approximately 7 mg / m². 2 ~Approx. 162mg / m 2 (For example, 7 mg / m²) 2 , 14 mg / m² 2 , 28 mg / m²2 , 56 mg / m² 2 , 84 mg / m² 2 , 112 mg / m² 2 , 135 mg / m² 2 or 162 mg / m² 2 The method of the present invention 1020. [Invention 1025] The method of the present invention 1020, wherein the ovarian cancer is platinum-resistant ovarian cancer. [Invention 1026] The method of the present invention 1020, wherein the ovarian cancer is a highly malignant serous ovarian cancer. [Invention 1027] The method of the present invention 1020, wherein the ovarian cancer is platinum-resistant, highly malignant serous ovarian cancer. [Invention 1028] The method of the present invention 1020 for ovarian cancer in which the patient has previously received monotherapy. [Invention 1029] The method of the present invention 1020, wherein the subject has ovarian cancer and has received three or fewer prior treatment lines. [Invention 1030] The method of the present invention 1020, wherein a subject with ovarian cancer has received three or fewer prior treatment lines, including at least one line of platinum-containing regimen. [Invention 1031] The method of the present invention 1020, wherein a subject with ovarian cancer has received four or fewer prior treatment lines, with or without the presence of at least one line of platinum-containing regimen. [Invention 1032] The method of the present invention 1020, wherein NSCLC is subtyped as adenocarcinoma. [Invention 1033] The method of the present invention 1020, wherein the subject has NSCLC and has previously received prior treatment with platinum-based chemotherapy (cisplatin or carboplatin) and a PD-1 monoclonal antibody or a PD-L1 monoclonal antibody. Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0028] [Figure 1] This graph shows different glycoforms of antibody glycans (G0, G1, G2, G0F, GIF, G2F, and M5). [Figure 2] This scheme demonstrates the deglycosylation of a mixture of glycoforms G0, G1, G2, G0F, GIF, G2F, and M5 in the presence of endoglycosidase. [Figure 3]This scheme illustrates the process for preparing azide-modified antibodies, in which an intermediate antibody containing a terminal GlcNAc moiety is reacted with an azide-modified UDP-GalNAc derivative molecule in the presence of glycosyltransferase. [Figure 4] This scheme illustrates one aspect of the process for preparing azide-modified antibodies. [Figure 5] This scheme illustrates one aspect of a process for preparing an antibody-drug conjugate in which an azide-modified antibody is conjugated to a linker-drug moiety containing a strained cycloalkynyl group. [Figure 6] This is a graph showing modified antibodies. [Figure 7] This graph shows the antitumor effects of XMT-1535 antibody-drug conjugates at payload levels of 0.05 mg / kg or 0.1 mg / kg, conjugate 11, conjugate 7 at payload levels of 0.025 mg / kg, 0.05 mg / kg, or 0.1 mg / kg, and unconjugated control conjugate 10 at payload level of 0.1 mg / kg in an OVCAR3 tumor-bearing mouse model. [Figure 8] This graph shows the antitumor effects of XMT-1535 antibody-drug conjugates at payload levels of 0.025 mg / kg, 0.05 mg / kg, or 0.1 mg / kg, conjugate 7, and unconjugated control conjugate 10 at payload levels of 0.05 mg / kg or 0.1 mg / kg in the NSCLC PDX CTG-0852 mouse model. [Figure 9] This graph shows the antitumor effects of HER2 antibody-drug conjugates, conjugate 9, and conjugate 13 at payload levels of 0.067 mg / kg and 0.199 mg / kg in a JIMT-1 tumor-bearing mouse model. [Figure 10]This graph shows the antitumor effects of HER2 antibody-drug conjugates, conjugate 8 and conjugate 12, and unconjugated control antibody-drug conjugate and conjugate 10, each at payload levels of 0.017 mg / kg, 0.033 mg / kg, and 0.067 mg / kg, respectively, in a JIMT-1 tumor-bearing mouse model. [Figure 11] The study shows elevated toxicological parameters (AST, ALT, ALP, RBC, WBC, neutrophils, lymphocytes, and hemoglobin, respectively) in rats in response to exposure to conjugate 11 or conjugate 7. [Figure 12] See the explanation in Figure 11. [Figure 13] See the explanation in Figure 11. [Figure 14] See the explanation in Figure 11. [Figure 15] See the explanation in Figure 11. [Figure 16] See the explanation in Figure 11. [Figure 17] See the explanation in Figure 11. [Figure 18] See the explanation in Figure 11. [Figure 19] The study shows elevated key toxicological parameters (AST, ALT, and ALP, respectively) in rats in response to exposure to Conjugate 8 or Conjugate 12. [Figure 20] See the explanation in Figure 19. [Figure 21] See the explanation in Figure 19. [Modes for carrying out the invention]

[0029] Detailed explanation This disclosure provides novel target-directed moiety-drug conjugates, scaffolds for preparing conjugates, synthesis methods for preparing conjugates or scaffolds, pharmaceutical compositions containing scaffolds and / or conjugates, and various uses thereof.

[0030] definition In some embodiments, the definitions of the compounds and specific functional groups of this disclosure are also described in further detail herein. For the purposes of this disclosure, Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Identify the chemical elements according to the ed. and inside cover, and generally define specific functional groups as described therein. In addition, the general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in their entirety in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, which is incorporated herein by reference. Furthermore, it will be understood by those skilled in the art that the synthetic methods described herein utilize a variety of protecting groups.

[0031] In both the following description and the claims, the use of the articles “a,” “an,” and “the” should be interpreted as encompassing both singular and plural unless otherwise specified herein or unless it is clearly inconsistent with the context. The terms “comprising,” “having,” “being,” “including,” and “containing,” as in “being a chemical formula,” should be interpreted as open terms (i.e., meaning “including without limitation”) unless otherwise specified, allowing but not requiring the inclusion of additional elements or processes. In some embodiments, a scaffold of a particular formula may include all components shown in that formula, and additional components not shown in that formula. Furthermore, whenever “comprising” or another open-ended term is used in an embodiment, it should be understood that the same embodiment may be further narrowed by using the intermediate term “essentially consisting of” or the closed term “consisting of.”

[0032] As used herein, expressions such as “one or more of A, B, or C,” “one or more A, B, or C,” “one or more of A, B, and C,” and “one or more A, B, and C” are used without distinction and refer to any selection from A, B, and / or C, i.e., one or more A's, one or more B's, one or more C's, or any combination thereof.

[0033] The terms “about,” “approximately,” or “nearly,” when used in relation to a number, mean that a set or range of values ​​is included. For example, “about X” includes the range of values ​​that are ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a number.

[0034] The enumeration of value ranges is intended solely as a convenient way to refer individually to each distinct value that falls within that range, unless otherwise specified herein, and each distinct value is incorporated herein as if it were individually enumerated herein. The ranges used herein, unless otherwise specified, include two limits to that range. For example, the expressions "x being an integer between 1 and 6" and "x being an integer of 1 to 6" both mean "x is 1, 2, 3, 4, 5, or 6," that is, the terms "X~Y" and "the range of X~Y" include X and Y as well as integers in between them.

[0035] "Protective group": As used herein, the term protecting group means that a particular functional moiety, such as O, S, or N, is temporarily blocked so that a reaction can be selectively carried out at another reaction site in a polyfunctional compound. As detailed herein, oxygen protecting groups, sulfur protecting groups, nitrogen protecting groups, and carbon protecting groups may be used. In some embodiments, exemplary oxygen protecting groups may be used. In some embodiments, nitrogen protecting groups may be used. In some embodiments, exemplary sulfur protecting groups may be used. Furthermore, various protecting groups are described in "Protective Groups in Organic Synthesis" Third Ed. Greene, TWand Wuts, PG, Eds., John Wiley & Sons, New York: 1999, the entire contents of which are incorporated herein by reference.

[0036] A "leaving group" refers to a molecular fragment that is eliminated by a pair of electrons during unequal bond cleavage. Leaving groups can be anions or neutral molecules. Leaving groups are not limited to halides, such as Cl. - , Br - and I - , sulfonic acid esters, for example, p-toluenesulfonate ("tosylate", TsO - The formula includes ) and RC(O)O-, where R is H, an aliphatic moiety, a heteroaliphatic moiety, a carbocyclic moiety, or a heterocycloalkyl moiety.

[0037] "Sugar" refers to monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). The term "sugar derivative" refers to a derivative of a monosaccharide, i.e., a monosaccharide containing substituents and / or functional groups. Examples of sugar derivatives include, but are not limited to, amino sugars and sugar acids. Other examples of sugar derivatives include S'(F') X1 Compounds represented as shown are also examples, where S' is a sugar or sugar derivative, F' is a functional group, and x1 indicates the number of functional groups.

[0038] As used herein, the term “core-GlcNAc moiety” refers to a monosaccharide, polysaccharide, or oligosaccharide moiety containing GlcNAc (e.g., core-GlcNAc) bound to an antibody (e.g., via the C1 position of GlcNAc). In some embodiments, GlcNAc is bound to the antibody via an N-glycosidic bond to the amide nitrogen atom of the side chain of the asparagine amino acid of the antibody. In some embodiments, the core-GlcNAc moiety is present at the native glycosylation site of the antibody or is introduced at a different site of the antibody. In some embodiments, the core-GlcNAc moiety is a monosaccharide (e.g., the core-GlcNAc moiety is also the terminal GlcNAc moiety). In some embodiments, the core-GlcNAc moiety further contains fucose, for example, the core-GlcNAc moiety is a disaccharide core-GlcNAc-(α1-6-Fuc) moiety (which may be called GlcNAc(Fuc)). Therefore, if an antibody contains a core-GlcNAc moiety, the antibody may contain a monosaccharide core-GlcNAc moiety or a disaccharide core-GlcNAc moiety, and the core-GlcNAc moiety may further contain fucose (e.g., a disaccharide core-GlcNAc(Fuc) moiety). If the core-GlcNAc moiety further contains fucose, the fucose may be bound to the α-1,6 to O-6 of the core-GlcNAc moiety. A core-GlcNAc moiety further containing fucose may be called core-GlcNAc(Fuc).

[0039] The term "core-GlcNAc" refers to internal GlcNAc, which is part of a polysaccharide or oligosaccharide, and which binds to the antibody via the internal GlcNAc.

[0040] As used herein, the term “terminal GlcNAc moiety” refers to a portion comprising GlcNAc that binds to an antibody and has terminal functional groups available for further modification (e.g., by a P''-S''-A'' compound). In some embodiments, the terminal GlcNAc moiety further comprises fucose. In some embodiments, the terminal GlcNAc moiety is formed by reacting a core-GlcNAc moiety of a glycoprotein (e.g., antibody glycan) with an endoglycosidase.

[0041] The term "nucleotide," used in its usual scientific sense, refers to a molecule composed of a nucleic acid base, a five-carbon sugar (either ribose or 2-deoxyribose), and one, two, or three phosphate groups. Without the phosphate groups, the nucleic acid base and sugar form a nucleoside. Therefore, a nucleotide can also be called a nucleoside monophosphate, nucleoside diphosphate, or nucleoside triphosphate. The nucleic acid base can be adenine, guanine, cytosine, uracil, or thymine.

[0042] The term "protein," used in its usual scientific sense, refers to polypeptides containing approximately 10 or more amino acids. Proteins may contain either natural or non-natural amino acids.

[0043] In this specification, “glycoprotein” is used in its usual scientific sense and refers to a protein containing one or more monosaccharides or oligosaccharides ("glycans") covalently bonded to the protein. Glycans may be bonded to a hydroxyl group on the protein (O-linked glycans), to an amide function on the protein (N-glycoproteins), or to a carbon on the protein (C-glycoproteins). A glycoprotein may contain multiple glycans, may contain a combination of one or more monosaccharide glycans and one or more oligosaccharide glycans, or may contain a combination of N-linked glycans, O-linked glycans, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and are therefore eligible to be glycoproteins.

[0044] In this specification, "glycan" is used in its usual scientific sense and refers to a monosaccharide or oligosaccharide chain bound to a protein. Therefore, glycan refers to the carbohydrate portion of a glycoprotein. A glycan is bound to a protein via the C-1 carbon of one sugar, and may not involve additional substitutions (monosaccharide), or it may be further substituted with one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain 1 to about 10 sugar units. However, if longer sugar chains are bound to a protein, these are also considered glycans. Glycans of glycoproteins may be monosaccharides. Glycans may also be oligosaccharides. Oligosaccharide chains of glycoproteins may be linear or branched. In oligosaccharides, the sugar directly bound to the protein is called the core sugar. In oligosaccharides, the sugar that is not directly bound to the protein but is bound to at least two other sugars is called the internal sugar. In oligosaccharides, sugars that are not directly bound to a protein but are bound to a single other sugar, i.e., sugars that do not have additional sugar substituents on one or more of the other hydroxyl groups, are called terminal sugars. To avoid misunderstanding, oligosaccharides in glycoproteins can have multiple terminal sugars, but there is only one core sugar. Glycans can be O-linked glycans, N-linked glycans, or C-linked glycans. In delinked glycans, monosaccharide glycans or oligosaccharide glycans are bound to the carbon atom of an amino acid in a protein.

[0045] "Glycosyltransferase" refers to a superfamily of enzymes involved in the synthesis of complex carbohydrates present on glycoproteins and glycolipids.

[0046] N-acetylgalactosamine transferase (GalNAc-T) is an N-acetyl-D-galactosamine transferase enzyme that catalyzes the addition of N-acetyl-D-galactosamine to proteins.

[0047] As used herein, “biocompatibility” is intended to describe compounds that exert minimal destructive or host response effects while in contact with bodily fluids or living cells or tissues. Therefore, as used herein, a biocompatible group refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl, or heteroaryl moiety included in the definition of the term biocompatibility as defined above and herein. The term “biocompatible” as used herein is also interpreted to mean that a compound exhibits minimal interaction with recognition proteins, such as naturally occurring antibodies, cellular proteins, cells, and other components of a biological system, unless such interaction is particularly desirable. Therefore, substances and functional groups specifically intended to induce the minimal interactions described above, such as drugs and prodrugs, are considered biocompatible. In some embodiments, a compound is considered "biocompatible" if, when added in vitro to normal cells at concentrations similar to the intended systemic in vivo concentration (with the exception of compounds intended to be cytotoxic, e.g., antineoplastic agents), it results in less than 1% cell death within a time equivalent to the in vivo half-life of the compound (e.g., the time required for 50% of the in vivo administered compound to be eliminated / removed), and when administered in vivo, it induces minimal medically acceptable inflammation, xenobiotic reaction, immunotoxicity, chemotoxicity, and / or other such adverse effects. In the above sentence, the term "normal cells" refers to cells that are not intended to be destroyed by the compound being tested, or otherwise not significantly affected.

[0048] "Biodegradable": As used herein, a "biodegradable" compound or part is one that, upon being taken up by a cell, can be degraded by lysosomal mechanisms or other chemical mechanisms, or by hydrolysis into components that can be reused or processed by the cell without significant toxicity to the cell. As used herein, the term "biocleavable" has the same meaning as "biodegradable". The biodegradation of some conjugates (or their components, e.g., peptide-containing scaffolds, and linkers between the scaffolds and antibody or drug molecules) can also be enhanced extracellularly, for example, in low pH areas of an animal's body, e.g., in the immediate vicinity of inflammatory areas, activated macrophages, or other cells releasing degradation-promoting factors. The integrity of the conjugates or scaffolds disclosed herein can be measured, for example, by size exclusion HPLC or LC / MS. While faster degradation may be preferred in some cases, generally, it is relatively desirable for the conjugates or scaffolds disclosed herein to be degraded intracellularly at a rate not exceeding the rate of metabolism or elimination of their fragments by the cell. In some embodiments, the biodegradable by-products of the conjugate or scaffold disclosed herein are biocompatible.

[0049] "Hydrophilic": The term "hydrophilic" is not essentially different from its general meaning in the art, and refers to a chemical moiety that contains ionizable, polar, or polarizable atoms, or otherwise can be solvated by water molecules. Thus, as used herein, a hydrophilic moiety or hydrophilic group refers to an aliphatic moiety, cycloalkyl moiety, heteroaliphatic moiety, heterocycloalkyl moiety, aryl moiety, or heteroaryl moiety included in the definition of the term hydrophilic as defined above. The hydrophilicity of the compounds disclosed herein (including drugs, conjugates, and scaffolds) can be measured directly by determining the hydration energy, or by investigation between two liquid phases, HIC chromatography, or chromatography of a known hydrophobic solid phase.

[0050] "Polysaccharide," "carbohydrate," or "oligosaccharide": The terms "polysaccharide," "carbohydrate," or "oligosaccharide" are known in the art and generally refer to a chemical formula (CH2O) where n > 2. n The term "polysaccharide" generally refers to substances having polyhydroxyaldehydes or polyhydroxyketones, or to substances that are converted into such substances through simple chemical transformations such as hydrolysis, oxidation, or reduction. These cyclic units (monosaccharides) can link together to form molecules having a small number (oligosaccharides) or several (polysaccharides) monosaccharide units. Often, carbohydrates with a distinct number, type, and position of monosaccharide units are called oligosaccharides, while carbohydrates consisting of a variable number of molecules and / or positions of monosaccharide units are called polysaccharides. The terms "polysaccharide," "carbohydrate," and "oligosaccharide" are used interchangeably herein. Polysaccharides may include natural sugars and / or derivatives of naturally occurring sugars.

[0051] "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 that requires it.

[0052] "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 undergoes in vivo processing to convert the drug into a physiologically active form. In some cases, the prodrug itself may have the desired physiological effect. The desired physiological effect may be, for example, therapeutic, cytotoxic, or immunomodulatory.

[0053] "Cytotoxic": As used herein, the term "cytotoxic" means toxic to cells or selected cell populations (e.g., cancer cells). Toxicity may result in cell death and / or lysis. In some embodiments, toxicity may be sublethal destructive effects on cells, such as delayed or cessation of cell proliferation. To achieve a cytotoxic effect, the drug or prodrug may, among other things, be a DNA damage agent, a microtubule disruptor, or a cytotoxic protein or cytotoxic polypeptide.

[0054] "Cell growth inhibitory": As used herein, the term "cell growth inhibitory" refers to a drug or other compound that inhibits or halts cell growth and / or cell multiplication.

[0055] "Small molecule": As used herein, the term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or artificially produced (e.g., via chemical synthesis). In some embodiments, small molecules are biologically active in that they produce a topical or systemic effect on animals (e.g., mammals, humans). In some embodiments, small molecules are drugs and are referred to as "drug molecules," "drugs," or "therapeutic agents." In some embodiments, drug molecules have a molecular weight (MW) of about 5 kDa or less (e.g., about 1.5 kDa or less). In some embodiments, drug molecules are selected from compounds found in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and "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 some embodiments, the drugs used in this disclosure are therapeutic agents having antiproliferative (inhibitory and / or cytotoxic) activity against target cells or target pathways.

[0056] As used herein, “active form” refers to the form of a compound that exhibits the intended pharmaceutical effect in vivo or in vitro. In particular, if a drug molecule intended to be delivered by the conjugate of this disclosure is released from the conjugate, the active form may be the drug itself or a derivative thereof that exhibits the intended therapeutic properties. The release of the drug from the conjugate may be achieved by cleaving the biodegradable bond of the linker that binds the drug to the scaffold or conjugate of this disclosure.

[0057] "Diagnostic Label": As used herein, the term "diagnostic label" refers to an atom, group of atoms, part or functional group, nanocrystal, or other individual element of a composition that can be detected in vivo or ex vivo using analytical methods known in the art. When associated with a conjugate of this disclosure, such a diagnostic label enables in vivo monitoring of the conjugate. Alternatively or additionally, constructs and compositions containing diagnostic labels can be used to monitor biological function or biological structure.

[0058] "Animal": As used herein, the term "animal" means humans, as well as non-human animals at any stage of development, including, for example, mammals, birds, reptiles, amphibians, fish, insects, and single-celled organisms. In some embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, primates, or pigs). Animals may be transgenic animals or human clones. The term "subject" encompasses animals.

[0059] "Efficient amount": The term "efficient amount" generally refers to the amount of an activator or drug delivery device required to induce a desired biological response. As those skilled in the art will understand, the efficient amount of a drug or device can vary depending on factors such as the desired biological endpoint, the drug being delivered, the composition of the encapsulation matrix, and the target tissue.

[0060] As used herein, “natural amino acid” refers to any of the common naturally occurring L-amino acids found in naturally occurring proteins or their stereoisomers. Unless otherwise specified, references to amino acids include the amino acid itself and its stereoisomers.

[0061] As used herein, "non-natural amino acids" refers to any amino acid that is not a natural amino acid. This includes, for example, amino acids containing α-, β-, γ-, D-, and L-aminoacyl residues. More generally, non-natural amino acids are those in which the side chain R is not a naturally occurring amino acid side chain. Contains residues from TIFF0007846624000042.tif18128.

[0062] When used herein, either by itself or as part of another term, “alkyl” refers to a substituted or unsubstituted linear or branched, saturated or unsaturated hydrocarbon having the number of carbon atoms indicated (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. In some embodiments, the alkyl group is unsubstituted. The alkyl group may be substituted with one or more groups. In some embodiments, the alkyl group may be saturated.

[0063] When used herein, either by itself or as part of another term, “alkylene” refers to a substituted or unsubstituted saturated or unsaturated branched or linear or cyclic hydrocarbon radical of the number of carbon atoms described, typically 2 to 10 carbon atoms, having two monovalent radical centers obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane. In some embodiments, alkylene is a branched or linear hydrocarbon (i.e., not a cyclic hydrocarbon). In any embodiment provided herein, alkylene may be a saturated alkylene.

[0064] When used herein, either by itself or as part of another term, “aryl” refers to a monovalent carbocyclic aromatic hydrocarbon radical with 6 to 20 carbon atoms (e.g., 6 to 14 carbon atoms) obtained by removing one hydrogen atom from a single carbon atom of an aromatic ring system. Some aryl groups are represented as “Ar” in exemplary structures.

[0065] When used herein, either by itself or as part of another term, "arylene" refers to the aryl group as defined above, in which one of the hydrogen atoms of the aryl group is substituted by a bond (i.e., it is divalent) and which may be ortho, meta, or para oriented.

[0066] In some embodiments, for example, when the polyfunctional linker or drug unit contains an arylene, the arylene is the aryl group as defined above, in which one or two of the hydrogen atoms of the aryl group are substituted by a bond (i.e., the arylene may be divalent or trivalent).

[0067] When the term "heterogenetic ring" is used herein, either by itself or as part of another term, it refers to a specific number of elements (e.g., 3 to 8 or C). 3~8 A heterocyclic ring system refers to a monovalent substituted or unsubstituted aromatic ("heteroaryl") or unaromatic ("heterocycloalkyl") monocyclic, dicyclic, tricyclic, or tetracyclic ring system that independently has one carbon atom (also called a ring member) and one to four heteroatom ring members N, O, P, or S, obtained by removing one hydrogen atom from the ring atom of the parent ring system. One or more N, C, or S atoms in the heterocyclic ring can be oxidized. The ring containing heteroatoms can be aromatic or unaromatic. Unless otherwise specified, heterocyclic rings are bonded to their pendant group by any heteroatom or carbon atom, thereby resulting in a stable structure.

[0068] When used herein, "heterocyclo" or "heterocyclo-" refers to a heterocyclic group (e.g., C) defined above, in which one or more of the additional hydrogen atoms of the heterocycle are substituted by a bond (i.e., it is polyvalent, e.g., divalent or trivalent). 3~8 This refers to a heterocyclic group. In some embodiments, if the hydrophilic group, polyfunctional linker, or linker-drug moiety contains a heterocyclo, then the heterocyclo is a heterocyclic group as defined above, in which one or two of the hydrogen atoms of the heterocyclic group are substituted by a bond (i.e., the heterocyclo can be divalent or trivalent).

[0069] When used herein, either by itself or as part of another term, "carbocyclic ring" refers to a ring obtained by removing one hydrogen atom from the ring atoms of a parent ring system, with a certain number (e.g., 3 to 8 or C) of atoms. 3~8 A monovalent, substituted or unsubstituted aromatic ("aryl") or saturated or unsaturated non-aromatic ("cycloalkyl"), monocyclic, bicyclic, tricyclic, or tetracyclic carbocyclic ring system having carbon atoms (also called ring members). The carbocyclic ring may have 3, 4, 5, 6, 7, or 8 members.

[0070] When "carbocyclo" or "carbocyclo-" is used herein, either by itself or as part of another term, it refers to a C as defined above, in which another hydrogen atom of the carbocyclic group is substituted by a bond (i.e., it is divalent). 3~8 This refers to a carbocyclic group. In some embodiments, for example, when a hydrophilic group, polyfunctional linker, or linker-drug moiety contains a carbocyclo, the carbocyclo is the carbocyclic group as defined above, in which one or two of the hydrogen atoms of the carbocyclic group are substituted by a bond (i.e., the carbocyclo can be divalent or trivalent).

[0071] When "heteroalkyl" is used herein, either by itself or in combination with other terms, unless otherwise specified, it means a stable linear or branched hydrocarbon, or a combination thereof, that is fully saturated or contains 1 to 3 degrees of unsaturation, comprising the number of carbon atoms described and 1 to 10 (e.g., 1 to 3 heteroatoms O, N, Si, or S), the nitrogen and sulfur atoms may be oxidized, and the nitrogen heteroatom may be quaternized. The heteroatoms O, N, and S may be located at any internal position of the heteroalkyl group or at positions where the alkyl group is bonded to the remainder of the molecule. The heteroatom Si may be located at any position of the heteroalkyl group, including positions where the alkyl group is bonded to the remainder of the molecule. In some embodiments, up to two heteroatoms may be consecutive. In some embodiments, C 1~4Heteroalkyl or C 1~4 Heteroalkylenes have 1 to 4 carbon atoms and 1 or 2 heteroatoms, C 1~3 Heteroalkyl or C 1~3 Heteroalkylenes have 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some embodiments, the heteroalkyl or heteroalkylene is saturated.

[0072] When used herein, “heteroalkylene” means a divalent group derived from a heteroalkyl group, as exemplified by -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2- (as described above), either by itself or as part of another substituent. In the case of a heteroalkylene group, the heteroatom may also occupy one or both of the chain ends. Furthermore, in the case of alkylene and heteroalkylene bonding groups, the orientation of the bonding group is not suggested. In a selected embodiment, for example, when a hydrophilic group, polyfunctional linker, or linker-drug moiety contains a heteroalkylene, the heteroalkylene is the heteroalkyl group as defined above, in which one or two of the hydrogen atoms of the heteroalkyl group are substituted by the bond (i.e., the heteroalkylene may be divalent or trivalent).

[0073] As used herein, “may be substituted” means that the chemical moiety (e.g., alkyl, heteroalkyl, carbocyclic, and heterocyclic) is either substituted or unsubstituted. Unless otherwise specified, the chemical moieties disclosed herein may be substituted. When a chemical moiety is substituted, one or more hydrogen atoms are independently substituted by the substituent. Typical substituents include, but are not limited to, -X', -R', -O, -OR', -SR', and -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, -Cl, -Br or -I, and each R' is independently -H, -C 1~20 Alkyl, -C 6~20 Ariel, -C3~C 14 These are heterocyclic compounds, protecting groups, or prodrug moieties. Typical substituents include oxo (=O).

[0074] As used herein, “linker-drug portion” refers to the non-target-directed (e.g., non-antibody) portion of a conjugate disclosed herein. The linker component of the linker-drug portion has a release mechanism called a releaseable aggregate unit, which interposes between a polyfunctional linker and a drug unit. In some embodiments, the linker-drug portion is the non-antibody (e.g., non-target-directed) portion of a conjugate.

[0075] As used herein, “polyfunctional linker” refers to a linker that connects one or more hydrophilic groups, one or more drug units, and a target-directing moiety (e.g., an antibody) to form a conjugate or scaffold disclosed herein. The connection of these components to the polyfunctional linker may be in parallel or in series. In some embodiments, the polyfunctional linker includes a peptide moiety between the target-directing moiety and the hydrophilic group, and the peptide moiety includes at least two amino acids. In other embodiments, the polyfunctional linker does not need to include a peptide moiety of at least two amino acids if the hydrophilic group is a polyalcohol or a derivative thereof. In other embodiments, the polyfunctional linker does not need to include a peptide moiety of at least two amino acids if the hydrophilic group is a glucosylamine, di-glucosylamine, tri-glucosylamine or a derivative thereof.

[0076] As used herein, “free drug” refers to a biologically active form of a drug moiety that is not directly or indirectly covalently bonded to a hydrophilic group or to a breakdown product of a ligand unit. A free drug may refer to a drug that is present immediately after cleavage from a polyfunctional linker via a release mechanism provided by a releaseable aggregate unit within the linker-drug moiety, or that remains present until subsequent intracellular conversion or metabolism. In some embodiments, the free drug may have a morphological HD or exist as a charged moiety. In some embodiments, the pharmacologically active species may not be the parent drug and may include a linker component that encapsulates the drug in a target-directed moiety that has not undergone subsequent intracellular metabolism.

[0077] Hydrophobicity can be measured using clogP, or clogP can be defined as the logarithm of the octanol / water partition coefficient (including implicit hydrogen) and calculated using the Chemical Computing Group program MOE® (clogP 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).

[0078] In some embodiments, the Disclosure provides a target-directed moiety-drug conjugate composition comprising a group of target-directed moiety-drug conjugates. A target-directed moiety-drug conjugate comprises a target-directed moiety unit and a plurality of linker-drug moieties coupled thereto. In some embodiments, each target-directed moiety in the conjugate contains, on average, about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 4, or about 1 to about 2 linker-drug moieties (e.g., d of formula (I')). 13 Exemplary binding to specific sites on the target-directed moiety is achieved by modifying the target-directed moiety N-glycan to include an azide group, a keto group, or an alkynyl group.

[0079] This disclosure is intended to include all isotopes of atoms present in the compound (e.g., isotopes of hydrogen and isotopes of carbon).

[0080] The compounds, scaffolds, or conjugates of this disclosure may exist in multiple isomeric forms. Where a compound, scaffold, or conjugate is described herein, this disclosure is understood to refer to any isomer of the compound, scaffold, or conjugate. Such disclosures refer to positional isomers, optical isomers, and tautomers, where applicable. Optical isomers include enantiomers and diastereomers, chiral isomers, and non-chiral isomers. Optical isomers include isolated optical isomers, as well as mixtures of optical isomers, including racemic and non-racemic mixtures. Isomers may be in isolated forms or in mixtures with one or more other isomers. Unless otherwise specified, any compound, scaffold, or conjugate described herein means any isomer of the compound, scaffold, or conjugate, or any mixture thereof. Where a compound, scaffold, or conjugate is shown as a specific isomer, it should be understood that this disclosure is not limited to that specific isomer, but may refer to any specific isomer in any aspect.

[0081] The compounds, scaffolds, or conjugates of this disclosure may exist as cis and / or trans isomers. Unless otherwise indicated, any compound, scaffold, or conjugate described herein means the cis or trans isomer of such compound, scaffold, or conjugate, and any mixture thereof. Where a compound, scaffold, or conjugate is shown as a cis or trans isomer, it should be understood that this disclosure is not limited to that particular cis or trans isomer, but may refer to any particular cis or trans isomer in any aspect.

[0082] The compounds, scaffolds, or conjugates of this disclosure may exist as positional isomers. Unless otherwise specified, any compound, scaffold, or conjugate described herein means any positional isomer of such compound, scaffold, or conjugate, or any mixture thereof. Where a compound, scaffold, or conjugate is shown as a specific positional isomer, it should be understood that this disclosure is not limited to that specific positional isomer and may refer to that specific positional isomer in any manner. Any enumeration or description of a compound, scaffold, or conjugate of this disclosure that does not have a specific stereochemical designation, or has such a designation with respect to fewer chiral centers than any chiral center, is intended to include racemates, racemic mixtures, individual enantiomers, diastereoisomer mixtures, and individual diastereomers of such compound, where such form is possible due to the presence of one or more chiral centers for any chiral center not such designated.

[0083] Antibody-drug conjugates and scaffolds In some embodiments, the disclosure provides site-specific antibody-drug conjugates. In some embodiments, the conjugates are biodegradable and biocompatible, and / or exhibit high drug loading and strong binding to target antigens.

[0084] In some embodiments, the disclosure provides an antibody-drug conjugate comprising a target-directed moiety (e.g., an antibody) and one or more linker-drug moieties, wherein the target-directed moiety is covalently bound to one or more linker-drug moieties.

[0085] In some embodiments, the present disclosure provides a scaffold useful for conjugating with a target-directed moiety (e.g., an antibody) to form the conjugates disclosed herein.

[0086] In some embodiments, the target-directing portion is an antibody.

[0087] In some aspects, the present disclosure relates to an antibody-drug conjugate comprising a target-directed moiety (e.g., an antibody) and one or more linker-drug moies covalently bound to the target-directed moiety, Each linker-drug portion includes a polyfunctional linker that connects a target-directing portion to one or more drug units (e.g., one or more therapeutic agents (D)) via a releaseable aggregate unit for each drug unit, and connects a hydrophilic group to the drug unit of each linker-drug portion. The releaseable aggregate unit can release the free drug in close proximity to the target site targeted by the target-directing portion. The polyfunctional linker provides an antibody-drug conjugate comprising a peptide moiety between a target-directing moiety and a hydrophilic group, wherein the peptide moiety comprises at least two amino acids.

[0088] In some aspects, the present disclosure relates to an antibody-drug conjugate comprising a target-directed moiety (e.g., an antibody) and one or more linker-drug moies covalently bound to the target-directed moiety, Each linker-drug portion includes a polyfunctional linker that connects a target-directing portion to one or more drug units (e.g., one or more therapeutic agents (D)) via a releaseable aggregate unit for each drug unit, and connects a hydrophilic group to the drug unit of each linker-drug portion. The present invention provides an antibody-drug conjugate in which releaseable aggregate units can release a free drug in proximity to a target site targeted by a target-directed portion.

[0089] In some aspects, this disclosure relates to the antibody-drug conjugate of formula (I'): Provide TIFF0007846624000043.tif34128, During the ceremony, a2 is an integer between 1 and 3. a3 is an integer between 0 and 1. a4 is an integer between 1 and approximately 5. a5 is an integer between 1 and 3. d 13 These are integers from 1 to approximately 12. The ANTIBODY is a modified antibody, L P’ is a divalent linker moiety that connects the modified antibody to M P and its corresponding monovalent moiety L P contains a functional group W that can form a covalent bond with a functional group of the modified antibody, P and M P is a stretcher unit, L M is a linker, or a trivalent or tetravalent linker, and when L M is a linker (i.e., a divalent linker), a2 is 1, or when L M is a trivalent linker, a2 is 2, or when L M is a tetravalent linker, a2 is 3, L 3 is a carbonyl-containing moiety, M A contains a peptide moiety comprising at least two amino acids, T 1 is a hydrophilic group, and the 1 between T A and M TIFF0007846624000044.tif8128 indicates a direct or indirect bond between T 1 and M A and each occurrence of D is, independently, a therapeutic agent having a molecular weight of about 5 kDa or less, L D each occurrence of which is, independently, a divalent linker moiety that connects D to M A and contains at least one cleavable bond such that upon cleavage of the bond, D is released in an active form for its intended therapeutic effect.

[0090] In some aspects, the disclosure provides an antibody-drug conjugate of formula (IV), or a scaffold of any one of formulas (II)-(III) and (V)-(VI): TIFF0007846624000045.tif147128, wherein, a2 is an integer from 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 12, ANTIBODY is a modified antibody, L P’ is a divalent linker moiety that connects the modified antibody to M P and its corresponding monovalent moiety L P is a functional group W that can form a covalent bond with the reactive moiety of the modified antibody, P and includes, M P is a stretcher unit, L M if present, is a linker, or a trivalent or tetravalent linker, and if L M is a linker (i.e., a divalent linker), a2 is 1, or if L M is a trivalent linker, a2 is 2, or if L M is a tetravalent linker, a2 is 3, L 3 is a carbonyl-containing moiety, M A includes a peptide moiety containing at least two amino acids, T 1 is a hydrophilic group, and T 1 and M A between TIFF0 and M 1 and M A indicates a direct or indirect bond between, W D each presence of which, if present, 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 or less, L D each presence of which is independently a divalent linker moiety that connects W D or D to M A and L DIt contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in an active form for its intended therapeutic effect.

[0091] In some aspects, this disclosure relates to an antibody-drug conjugate of formula (IV'), or any one of the scaffolds of formulas (II')-(III') and (V')-(VI'): Provide TIFF0007846624000047.tif138128, During the ceremony, a2 is an integer between 1 and 3. a3 is an integer between 0 and 1. a4 is an integer between 1 and approximately 5. a5 is an integer between 1 and 3. d 13 These are integers from 1 to approximately 12. ANTIBODY is a modified antibody, L P’ M P This is the divalent linker portion that connects to the corresponding unvalent portion L. P This is a functional group W that can form a covalent bond with the reactive portion of the modified antibody. P Includes, M P This is a stretcher unit, L M If present, it is a bond, or a trivalent linker or tetravalent linker, L M If it is a bond (i.e., a divalent linker), then a2 is either 1 or L M If it is a trivalent linker, then a2 is 2 or L M If it is a tetravalent linker, then a2 is 3, L 3 This is the carbonyl-containing portion, M A It contains a peptide moiety that includes at least two amino acids, T 1 It is a hydrophilic group, T 1 and M A Between TIFF0007846624000048.tif8128 is T1 and M A This indicates a direct or indirect connection with, W D Each of these entities, when present, is a functional group that can independently form a covalent bond with a functional group of a therapeutic agent ("D") having a molecular weight of approximately 5 kDa or less. L D Each of these beings is independent of W D Or D to M A This is the divalent linker portion that connects to L D It contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in an active form for its intended therapeutic effect.

[0092] In some aspects, this disclosure relates to a scaffold containing any one of the peptides of formulas (VII) to (XII): Provide TIFF0007846624000049.tif153128, During the ceremony, a2 is an integer between 1 and 3. a3 is an integer between 0 and 1. a4 is an integer between 1 and approximately 5. a5 is an integer between 1 and 3. L P This is a functional group W that can form a covalent bond with the reactive portion of the modified antibody. P It is a monovalent linker portion that includes, M P This is a stretcher unit, L M If present, it is a bond, or a trivalent linker or tetravalent linker, L M If it is a bond (i.e., a divalent linker), then a2 is either 1 or L M If it is a trivalent linker, then a2 is 2 or L M If it is a tetravalent linker, then a2 is 3, L 3 If present, this is the carbonyl-containing portion. M A It contains a peptide moiety that includes at least two amino acids, T 1It is a hydrophilic group, T 1 and M A Between TIFF0007846624000050.tif9128 is T 1 and M A This indicates a direct or indirect connection with, W D Each of these entities is a functional group that can independently form a covalent bond with a functional group of a therapeutic agent ("D") having a molecular weight of approximately 5 kDa or less. L D Each of these beings is independent of W D Or D to M A This is the divalent linker portion that connects to L D It contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in an active form for its intended therapeutic effect.

[0093] In some aspects, this disclosure relates to a scaffold containing any one of the peptides of formulas (VII') to (XII'): Provide TIFF0007846624000051.tif140128, During the ceremony, a2 is an integer between 1 and 3. a3 is an integer between 0 and 1. a4 is an integer between 1 and approximately 5. a5 is an integer between 1 and 3. L P This is a functional group W that can form a covalent bond with the reactive portion of the modified antibody. P It is a monovalent linker portion that includes, M P This is a stretcher unit, L M If present, it is a bond, or a trivalent linker or tetravalent linker, L M If it is a bond, then a2 is 1 or L M If it is a trivalent linker, then a2 is 2 or L M If it is a tetravalent linker, then a2 is 3, L 3 If present, this is the carbonyl-containing portion. M A It contains a peptide moiety that includes at least two amino acids, T 1 It is a hydrophilic group, T 1 and M A Between TIFF0007846624000052.tif9128 is T 1 and M A This indicates a direct or indirect connection with, W D Each of these entities is a functional group that can independently form a covalent bond with a functional group of a therapeutic agent ("D") having a molecular weight of approximately 5 kDa or less. L D Each of these beings is independent of W D Or D to M A This is the divalent linker portion that connects to L D It contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in an active form for its intended therapeutic effect.

[0094] The conjugates and scaffolding of this disclosure may include one or more of the following features, where applicable:

[0095] In some aspects, d 13 These are integers between 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 1 and 2, 4 and 10, 4 and 8, 4 and 6, 6 and 12, 6 and 10, 6 and 8, 8 and 14, 8 and 12, or 8 and 10.

[0096] In some aspects, d 13 is an integer in the range of 1 to 2 (for example, d 13 (is 1 or 2). In some embodiments, d 13 is an integer in the range of 2 to 4 (for example, d 13 (is 2, 3 or 4). In some embodiments, d 13 is an integer in the range of 4 to 6 (for example, d 13 (is 4, 5, or 6). In some embodiments, d 13 is an integer in the range of 6 to 8 (for example, d 13(is 6, 7, or 8). In some embodiments, d 13 is an integer in the range of 6 to 10 (for example, d 13 (is 6, 7, 8, 9 or 10). In some embodiments, d 13 is 1 or 2. In some embodiments, d 13 is 1. In some embodiments, d 13 The answer is 2.

[0097] In some embodiments, each L 3 If present, independently, *-C 1~12 Alkyl-C(O)-**, *-NH-C 1~12 Alkyl-C(O)-**, or *-C 1~12 Alkyl-C(O)-NH-C 1~12 Alkyl-C(O)-**, where * indicates another L if present. 3 , or L M This indicates a connection to **, and if present, another L 3 , or M A This indicates a connection to [the specified location].

[0098] In some embodiments, at least one L 3 is either *-CH2CH2-C(O)-** or *-NH-CH2CH2-C(O)-**, where * is another L if present. 3 , or L M This indicates a connection to **, and if present, another L 3 , or M A This indicates a connection to [the specified location].

[0099] In some embodiments, a3 is 2 or more, and at least one L 3 is *-C 1~12 It is alkyl-C(O)-** and has at least one L 3 is *-NH-C 1~12 It is alkyl-C(O)-**.

[0100] In some aspects, (L 3 ) a3This is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-** or *NH-CH2CH2-C(O)-CH2CH2-C(O)-**, where * is L M This indicates a binding to M A This indicates a connection to [the specified location].

[0101] In some embodiments, a4 is 1. In some embodiments, a4 is 2. In some embodiments, a4 is 3.

[0102] Variable L P and L P’ In some embodiments, L P’ L P Functional groups (e.g., W P It is formed by a reaction between the ) and the reactive portion of the modified antibody (for example, the modified GlcNAc portion of *-GlcNAc-S''-A'').

[0103] In some embodiments, L P’ L P Functional groups (e.g., W P It contains a triazolyl formed between the ) and the reactive portion of the modified antibody (for example, the modified GlcNAc portion of *-GlcNAc-S''-A'').

[0104] In some embodiments, each L P If not attached to an antibody, the terminal group W P Includes.

[0105] In some embodiments, at least one W P teeth It is TIFF0007846624000053.tif110128, During the ceremony, R 8j Hydrogen, halogen, C 1~24 Alkyl (for example, C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 It is an alkyl)-(6-24 member heteroaryl) and C 1~24 The alkyl group may be interrupted by one or more O, N, or S atoms, and C 1~24 Alkyl (for example, C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 Alkyl)-(6-24 member heteroaryl) is one or more C1-C 12 Alkyl, C2~C 12 Alkenyl, C2~C 12 Alkinyl, C3~C 12 Cycloalkyl, -O(C1~C 12 Alkyl), -O(C2~C 12 Alkenyl), -O(C2~C 12 Alkinyl), -O(C3~C 12 They may be substituted with cycloalkyl, halogen, amino, oxo, or silyl, C1~C 12 Alkyl, C2~C 12 Alkenyl, C2~C 12 Alkinyl, C3~C 12 Cycloalkyl, -O(C1~C 12 Alkyl), -O(C2~C 12 Alkenyl), -O(C2~C 12 Alkinyl), -O(C3~C 12 The cycloalkyl group may be substituted, C1-C 12 Alkyl, C3~C 12Cycloalkyl, -O(C1~C 12 Alkyl) or -O(C3~C 12 The cycloalkyl group may be interrupted by one or more O, N, or S atoms. R 10j is hydrogen, halogen, C 1~24 Alkyl (for example, C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 It is an alkyl)-(6-24 member heteroaryl) and C 1~24 Alkyl (for example, C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 The alkyl)-(6-24 member heteroaryl) may be substituted, Each R 11j These are, independently, hydrogen and C 1~24 Alkyl (for example, C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24(aryl), or -(C 1~24 It is an alkyl)-(6-24 member heteroaryl) and Each R 12j These are, independently, halogen, -OR 10j -NO2, -CN, -S(O)2R 10j , C 1~24 Alkyl (for example, C 1~6 Alkyl), C 6~24 Cycloalkyl, 6-24 member heterocycloalkyl, C 6~24 Aryl, 6-24 member heteroaryl, -(C 1~24 Alkyl)-(C 6~24 Cycloalkyl), -(C 1~24 Alkyl)-(6-24 member heterocycloalkyl),-(C 1~24 Alkyl)-(C 6~24 (aryl), or -(C 1~24 It is an alkyl)-(6-24 member heteroaryl) and u2 is an integer in the range of 0 to 8.

[0106] In some embodiments, at least one W P teeth The filename is TIFF0007846624000054.tif32128.

[0107] In some embodiments, at least one W P teeth The filename is TIFF0007846624000055.tif34128.

[0108] In some embodiments, each R 11j is hydrogen. In some embodiments, u2 is 0. In some embodiments, R 8j It is hydrogen.

[0109] In some embodiments, at least one W P teeth The filename is TIFF0007846624000056.tif34128.

[0110] In some embodiments, at least one W P teeth The filename is TIFF0007846624000057.tif31128.

[0111] In some embodiments, at least one R 12j is an electron-withdrawing group, for example, a group whose Hammett substituent constant σ is positive. In some embodiments, suitable electron-withdrawing groups are known in the art. In some embodiments, at least one R 12j is a halogen (e.g., F or Cl), -OR 10j -NO2, -CN, -S(O)2R 7j , substitution C1~C 12 Alkyl or substituted C6-C 12 It is an aryl group, and at least one of its substituents is an electron-withdrawing group. In some embodiments, at least one R 12j Fluorinated C1~C 12 Alkyl (e.g., -CF3), fluorinated C5~C 12 Aryl (e.g., -C6F5) or haloalkylated C5~C 12 It is an aryl group (for example, -[3,5-(CF3)2(C6H3)]).

[0112] In some embodiments, at least one W P teeth The filename is TIFF0007846624000058.tif34128.

[0113] In some embodiments, at least one W P teeth The filename is TIFF0007846624000059.tif73138.

[0114] In some embodiments, at least one W P teeth The filename is TIFF0007846624000060.tif36128.

[0115] In some embodiments, each R 11j is hydrogen. In some embodiments, u2 is 0.

[0116] In some embodiments, at least one W P teeth The filename is TIFF0007846624000061.tif31128.

[0117] In some embodiments, each W P If present, independently, The filename is TIFF0007846624000062.tif35128.

[0118] In some embodiments, each W P teeth The filename is TIFF0007846624000063.tif29128.

[0119] In some embodiments, each L P’ When it is attached to an antibody, the binding group W P’ Includes.

[0120] In some embodiments, at least one W P’ teeth The filename is TIFF0007846624000064.tif43128.

[0121] In some embodiments, at least one W P’ teeth The filename is TIFF0007846624000065.tif45128.

[0122] In some embodiments, at least one W P’ teeth The filename is TIFF0007846624000066.tif44128.

[0123] In some embodiments, at least one W P’ teeth The filename is TIFF0007846624000067.tif44128.

[0124] In some embodiments, at least one W P’ teeth The filename is TIFF0007846624000068.tif43128.

[0125] Stretcher unit M P In some embodiments, M P teeth It is TIFF0007846624000069.tif162138, In the formula, * represents L P’ or L P This indicates a connection to L M or M A This shows a connection to, Each R 66 Each R3 is independently NH or O, and each R3 is independently -C(O)-NR5- or -NR5-C(O)-. Each R5 independently produces hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, COOH, or COO-C 1~6 It is alkyl, R4 is bound or -NR5-(CR 20 R 21 )-C(O)-, Each R 20 and R 21 These are, independently, hydrogen and 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 A cycloalkyl group, or a side chain of a natural or unnatural amino acid, Each b1 is an independent integer in the range of 0 to 6. Each e1 is an independent integer in the range of 0 to 8. Each f1 is an independent integer in the range of 1 to 6. Each g2 is an independent integer in the range of 1 to 4.

[0126] In some embodiments, b1 is 0. In some embodiments, b1 is 1.

[0127] In some embodiments, each f1 is independently 1 or 2. In some embodiments, f1 is 1. In some embodiments, f1 is 2.

[0128] In some embodiments, g2 is 1 or 2. In some embodiments, g2 is 1. In some embodiments, g2 is 2.

[0129] M P Regarding the manner, * is L P’ or L P This indicates a connection to L M or M A It is understood that this indicates a connection to.

[0130] In some embodiments, M P teeth The filename is TIFF0007846624000070.tif169144.

[0131] In some embodiments, M P teeth The filename is TIFF0007846624000071.tif11128.

[0132] In some embodiments, M P teeth The filename is TIFF0007846624000072.tif13128.

[0133] In some embodiments, M P teeth The filename is TIFF0007846624000073.tif15130.

[0134] In some embodiments, M P teeth The filename is TIFF0007846624000074.tif18128.

[0135] In some embodiments, MP teeth The filename is TIFF0007846624000075.tif19128.

[0136] In some embodiments, M P teeth The filename is TIFF0007846624000076.tif20128.

[0137] In some embodiments, M P teeth The filename is TIFF0007846624000077.tif15128.

[0138] Variable L M and W M In some embodiments, L M The linker is either a divalent linker (i.e., having two arms) or a multi-arm linker (e.g., trivalent or tetravalent, or having three or four arms), where each arm may be the same or different.

[0139] In some embodiments, L M The linker is either a divalent linker (i.e., having two arms) or a multi-arm linker (e.g., tetravalent, having four arms, or trivalent with three arms), where each arm may be the same or different.

[0140] As used herein, the term "arm" means (1) M P (2) If it is attached to L 3 It is either bound to L 3 If it does not exist, then M A L is connected M It is understood that this refers to the part;

[0141] In some embodiments, L M This is a bond (i.e., a divalent linker, or having two arms).

[0142] In some embodiments, L M The linker is a multi-arm linker (for example, trivalent or tetravalent, or having three or four arms), where each arm may be the same or different. In some embodiments, L M This is a multi-arm linker (for example, trivalent or tetravalent, or having three or four arms).

[0143] In some embodiments, L M This is a trivalent linker having three arms, each of which may be the same or different.

[0144] In some embodiments, L M This is a tetravalent linker having four arms, each of which may be the same or different.

[0145] In some embodiments, a2 is 2, L M teeth It is TIFF0007846624000078.tif94136, During the ceremony, TIFF0007846624000079.tif7128 is M P This shows a connection to, Y1 is L if it exists. 3 Binding to, or L 3 If it does not exist, then M A This shows a connection to, R2 and R'2 are, independently, hydrogen or substituted carbon. 1~6 Alkyl, possibly substituted C 2~6 Alkenyl, C may be substituted 2~6 Alkynyl, C may be substituted. 3~19 Branched alkyl, possibly substituted C 3~8 Cycloalkyl, may be substituted C 6~10 Aryl, possibly substituted heteroaryl, possibly 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, C may be substituted. 2~6 Alkanoyl, C may be substituted. 2~6 Alkanoyloxy, C may be substituted. 2~6 Substituting alkanoyloxy, -COOH, or -COO-C 1~6 It is alkyl, Each of c1, c2, c3, c4, c5, c7, and c8, if present, is an independent integer in the range of 0 to 10. Each of d1, d2, d3, d4, d5, and d7, if present, is an independent integer in the range of 0 to 10.

[0146] In some embodiments, a2 is 2, L M teeth It is TIFF0007846624000080.tif97134, During the ceremony, TIFF0007846624000081.tif9128 is M P This shows a connection to, Y1 is L if it exists. 3 Binding to, or L 3 If it does not exist, then M A This shows a connection to, R2 and R'2 are, independently, hydrogen or substituted carbon. 1~6 Alkyl, possibly substituted C 2~6 Alkenyl, C may be substituted 2~6 Alkynyl, C may be substituted. 3~19 Branched alkyl, possibly substituted C 3~8 Cycloalkyl, may be substituted C 6~10 Aryl, possibly substituted heteroaryl, possibly substituted C 1~6 Heteroalkyl, C 1~6 Alkoxy, aryloxy, C 1~6 Heteroalkoxy, C 2~6Alkanoyl, optionally substituted arylcarbonyl, C 2~6 Alkoxycarbonyl, C 2~6 Alkanoyloxy, arylcarbonyloxy, C may be substituted. 2~6 Alkanoyl, C may be substituted. 2~6 Alkanoyloxy, C may be substituted. 2~6 Substituting alkanoyloxy, -COOH, or -COO-C 1~6 It is alkyl, Each of c1, c2, c3, c4, c5, c7, and c8, if present, is an independent integer in the range of 0 to 10. Each of d1, d2, d3, d4, d5, and d7, if present, is an independent integer in the range of 0 to 10.

[0147] In some embodiments, a2 is 2, L M teeth The filename is TIFF0007846624000082.tif25128.

[0148] In some embodiments, a2 is 2, L M teeth The filename is TIFF0007846624000083.tif26128.

[0149] In some embodiments, c1, c2, c3, c4, c5, c7, and c8, if present, are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 0 or 1, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 0, 1, or 2, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 0, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are independently 1, respectively. In some embodiments, c1, c2, c3, c4, c5, c7, and c8 are each independently 2.

[0150] In some embodiments, d1, d2, d3, d4, d5, and d7, if present, are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 0 or 1. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 1, 2, 3, or 4. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 1. In some embodiments, d1, d2, d3, d4, d5, and d7 are independently 2. In some embodiments, d1, d2, d3, d4, d5, and d7 are each independently 3. In some embodiments, d1, d2, d3, d4, d5, and d7 are each independently 4.

[0151] In some embodiments, R2 and R'2 are, independently, hydrogen and C. 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, -COOH, or -COO-C1~6 It is alkyl. In some embodiments, R2 and R'2 are independently hydrogen or C 1~6 It is alkyl. In some embodiments, R2 and R'2 are each independently hydrogen. In some embodiments, R2 and R'2 are each independently C 1~6 It is alkyl.

[0152] In some embodiments, L M teeth The filename is TIFF0007846624000084.tif81154.

[0153] In some embodiments, a2 is 3, L M teeth It is TIFF0007846624000085.tif229159, During the ceremony, TIFF0007846624000086.tif8128 is M P This shows a connection to, Y1 is L if it exists. 3 Binding to, or L 3 If it does not exist, then M A This shows a connection to, R2 and R'2 are, independently, hydrogen or substituted carbon. 1~6 Alkyl, possibly substituted C 2~6 Alkenyl, C may be substituted 2~6 Alkynyl, C may be substituted. 3~19 Branched alkyl, possibly substituted C 3~8 Cycloalkyl, may be substituted C 6~10 Aryl, possibly substituted heteroaryl, possibly 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, C may be substituted.2~6 Alkanoyl, C may be substituted. 2~6 Alkanoyloxy, C may be substituted. 2~6 Substituting alkanoyloxy, -COOH, or -COO-C 1~6 It is alkyl, Each of c1, c2, c3, c4, c5, c6, c7, and c8 is an independent integer in the range of 0 to 10. Each of d1, d2, d3, d4, d5, d6, d7, and d8 is an independent integer in the range of 0 to 10. Each of e1, e2, e3, e4, e5, e6, e7, and e8 is an independent integer in the range of 0 to 10.

[0154] In some embodiments, a2 is 3, L M teeth It is TIFF0007846624000087.tif226155, During the ceremony, TIFF0007846624000088.tif7128 is M P This shows a connection to, Y1 is L if it exists. 3 Binding to, or L 3 If it does not exist, then M A This shows a connection to, R2 and R'2 are, independently, hydrogen or substituted carbon. 1~6 Alkyl, possibly substituted C 2~6 Alkenyl, C may be substituted 2~6 Alkynyl, C may be substituted. 3~19 Branched alkyl, possibly substituted C 3~8 Cycloalkyl, may be substituted C 6~10 Aryl, possibly substituted heteroaryl, possibly 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 2~6Alkanoyloxy, arylcarbonyloxy, C may be substituted. 2~6 Alkanoyl, C may be substituted. 2~6 Alkanoyloxy, C may be substituted. 2~6 Substituting alkanoyloxy, -COOH, or -COO-C 1~6 It is alkyl, Each of c1, c2, c3, c4, c5, c6, c7, and c8 is an independent integer in the range of 0 to 10. Each of d1, d2, d3, d4, d5, d6, d7, and d8 is an independent integer in the range of 0 to 10. Each of e1, e2, e3, e4, e5, e6, e7, and e8 is an independent integer in the range of 0 to 10.

[0155] In some embodiments, a2 is 3, L M teeth The filename is TIFF0007846624000089.tif27128.

[0156] In some embodiments, a2 is 3, L M teeth The filename is TIFF0007846624000090.tif27128.

[0157] In some embodiments, -L M -(L 3 ) a2 -teeth The filename is TIFF0007846624000091.tif46128.

[0158] In some embodiments where an amino acid unit has two binding sites (i.e., terminal drug units), one of the binding sites shown above is, for example, H, OH, or C. 1~3 It may be substituted with an unsubstituted alkyl group.

[0159] In some embodiments, L M It is a multi-arm linker, and stretcher unit M P If you are not yet connected to W M LM It is the end of W M Each of these entities independently forms a hydrogen atom, a protecting group, a leaving group, or a covalent bond with L M to M P It is a functional group that can be connected to [something].

[0160] In some embodiments, W M is an amine protecting group. In some embodiments, W M It is BOC.

[0161] In some embodiments, W M L is an amine protecting group, M teeth The filename is TIFF0007846624000092.tif25128.

[0162] In some embodiments, W M L is an amine protecting group, M teeth The filename is TIFF0007846624000093.tif23128.

[0163] In some embodiments, W M is BOC, L M teeth The filename is TIFF0007846624000094.tif24128.

[0164] In some embodiments, W M L is an amine protecting group, M teeth The filename is TIFF0007846624000095.tif23128.

[0165] In some embodiments, W M is BOC, L M teeth The filename is TIFF0007846624000096.tif23128.

[0166] In some embodiments, W M It contains an amine group. In some embodiments, W Mis -C(O)-(CH2) w -NH2 is included in the formula, where w is an integer from 1 to 6. In some embodiments, W M It is -C(O)-CH2-NH2.

[0167] In some embodiments, W M It is -C(O)-CH2-NH2, and L M teeth The filename is TIFF0007846624000097.tif26128.

[0168] In some embodiments, W M It is -C(O)-CH2-NH2, and L M teeth The filename is TIFF0007846624000098.tif24128.

[0169] In some embodiments, W M H is H.

[0170] Variable L 3 In some embodiments, each L 3 If present, this is the carbonyl-containing portion.

[0171] L 3 Regarding the aspect, * indicates another L if present. 3 , or L M This indicates a connection to **, and if present, another L 3 , or M A It is understood that this indicates a connection to.

[0172] In some embodiments, each L 3 If present, independently, *-C 1~12 Alkyl-C(O)-**, *-NH-C 1~12 It is alkyl-C(O)-** or *-C1~12alkyl-C(O)-NH-C1~12alkyl-C(O)-**.

[0173] In some embodiments, at least one L 3 is *-C1~12 It is alkyl-C(O)-**.

[0174] In some embodiments, at least one L 3 It is *-CH2CH2-C(O)-**.

[0175] In some embodiments, L 3 It is *-CH2CH2-C(O)-**.

[0176] In some aspects, (L 3 ) a3 It is *-CH2CH2-C(O)-**.

[0177] In some embodiments, at least one L 3 is *-NH-C 1~12 It is alkyl-C(O)-**.

[0178] In some embodiments, at least one L 3 It is *-NH-CH2CH2-C(O)-**.

[0179] In some embodiments, L 3 It is *-NH-CH2CH2-C(O)-**.

[0180] In some aspects, (L 3 ) a3 It is *-NH-CH2CH2-C(O)-**.

[0181] In some embodiments, at least one L 3 is *-C 1~12 Alkyl-C(O)-NH-C 1~12 It is alkyl-C(O)-**.

[0182] In some embodiments, at least one L 3 The formula is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-**.

[0183] In some embodiments, L 3The formula is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-**.

[0184] In some aspects, (L 3 ) a3 The formula is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-**.

[0185] In some embodiments, a3 is 2 or more, and at least one L 3 is *-C 1~12 It is alkyl-C(O)-** and has at least one L 3 is *-NH-C 1~12 It is alkyl-C(O)-**.

[0186] In some aspects, (L 3 ) a3 The formula is *-CH2CH2-C(O)-NH-CH2CH2-C(O)-**.

[0187] In some aspects, (L 3 ) a3 It is *NH-CH2CH2-C(O)-CH2CH2-C(O)-**.

[0188] Variable M A In some embodiments, M A L contains one or more drugs and one or more hydrophilic groups. P or L P’ This is a linker section that can be connected. In some embodiments, M A It comprises a peptide moiety of at least two amino acids. In some embodiments, a singular amino acid is referred to herein as "AA," and a plural amino acid is referred to herein as "AA's."

[0189] In some embodiments, the peptide portion is -L D-A portion that can form a covalent bond with a D unit, enabling the binding of multiple drugs. In some embodiments, the peptide portion contains a single AA unit or has two or more AA units (e.g., 2-10, 2-6, or 2, 3, 4, 5, or 6), where each AA unit is independently a native or non-native amino acid, an amino alcohol, an amino aldehyde, a diamine, a polyamine, or a combination thereof. In some embodiments, in order to have the required number of bonds, at least one of the AA units is -L D -It has a functionalized side chain that provides a bond for a D unit. In some embodiments, exemplary functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes) include, for example, azide-functionalized AA units or alkyne-functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes modified to have an azide group or an alkyne group). In some embodiments, the azide group or alkyne group is for bonding using click chemistry.

[0190] In some embodiments, the peptide portion has 2 to 12 AA units. In some embodiments, the peptide portion has 2 to 10 AA units. In some embodiments, the peptide portion has 2 to 6 AA units. In some embodiments, the peptide portion has 2, 3, 4, 5, or 6 AA units.

[0191] In some embodiments, the peptide portion has 2 AA units. In some embodiments, the peptide portion has 3 AA units. In some embodiments, the peptide portion has 4 AA units. In some embodiments, the peptide portion has 5 AA units. In some embodiments, the peptide portion has 6 AA units.

[0192] In some embodiments, the binding within the peptide moiety, or the binding to the conjugate, its intermediate, or other components of the scaffold, may be via, for example, amino, carboxy, or other functional groups. In some embodiments, each amino acid in the peptide moiety may independently be a D-isomer or L-isomer of a thiol-containing amino acid. In some embodiments, each amino acid in the peptide moiety may independently be a D-isomer of a thiol-containing amino acid. In some embodiments, each amino acid in the peptide moiety may independently be an L-isomer of a thiol-containing amino acid. In some embodiments, the thiol-containing amino acid may be, for example, cysteine, homocysteine, or penicillamine.

[0193] In some embodiments, each amino acid containing the peptide portion may independently be one 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, aminoalkynic acid, aminoalkanediic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, its stereoisomer, or an L-isomer or D-isomer of its derivative.

[0194] In some embodiments, each amino acid containing 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.

[0195] In some embodiments, the peptide portion comprises a monopeptide, dipeptide, tripeptide, tetrapeptide, or pentapeptide. In some embodiments, the peptide portion comprises a pentapeptide.

[0196] In some embodiments, the peptide portion contains at least about 5 amino acids (e.g., 5, 6, 7, 8, 9, or 10 amino acids). In some embodiments, the peptide portion contains up to about 10 amino acids.

[0197] In some embodiments, the amino acids independently comprising the peptide moiety are glycine, serine, glutamic acid, lysine, aspartic acid, and cysteine.

[0198] In some embodiments, the peptide portion comprises at least four glycine molecules and at least one serine molecule, for example, (glycine)4 and serine, where the serine molecule is located at any position along the peptide chain, for example, (serine)-(glycine)4;(glycine)-(serine)-(glycine)3;(glycine)2-(serine)-(glycine)2;(glycine)3-(serine)-(glycine); or (glycine)4-(serine), etc.

[0199] In some embodiments, the peptide portion comprises (glycine)4-(serine) or (serine)-(glycine)4. In some embodiments, the peptide portion comprises (glycine)4-(serine). In some embodiments, the peptide portion comprises (serine)-(glycine)4.

[0200] In some embodiments, the peptide portion comprises at least four glycine molecules and at least one glutamic acid molecule, for example, (glycine)4 and glutamic acid, where the glutamic acid molecule is at any position along the peptide chain.

[0201] In some embodiments, the peptide portion comprises (glutamic acid)-(glycine)4 or (glycine)4-(glutamic acid).

[0202] In some embodiments, the peptide portion comprises (β-alanine)-(glycine)4-(serine), where the serine is at any position along the peptide chain.

[0203] In some embodiments, the peptide moiety comprises (glycine)4-(serine)-(glutamic acid), with serine at any position along the peptide chain. In some embodiments, the peptide moiety comprises (β-alanine)-(glycine)4-(serine)-(glutamic acid), with serine at any position along the peptide chain.

[0204] In some embodiments, the peptide portion is (glycine) 1~4 -(Serine) The peptide portion is transmitted via one of the glycine molecules, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1 It is bound, and the peptide portion is connected via serine, if present, L D It is connected.

[0205] In some embodiments, the peptide portion is (serine)-(glycine) 1~4 Includes, The peptide portion is transmitted via serine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound to the peptide portion, and if present, via glycine, T 1 It is bound, and the peptide portion is connected via serine, if present, L D It is connected.

[0206] Regarding the form of the peptide portion, * indicates L if present. 3 to, or L 3 If it does not exist, then L M It is understood that this indicates a coupling to. In some aspects, ** is T if present. 1 to, or T 1 If not present, it indicates a bond to -OH. In some embodiments, *** is L if present. D to, or L DIf it is not present, it shows a bond to hydrogen.

[0207] In some embodiments, the peptide portion is Includes TIFF0007846624000099.tif17128.

[0208] In some embodiments, the peptide portion includes (glycine)-(serine), The peptide portion is transmitted via glycine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1 It is bound, and the peptide portion is connected via serine, if present, L D It is connected.

[0209] In some embodiments, the peptide portion includes (glycine)-(serine), The peptide portion is transmitted via serine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound to the peptide portion, and if present, via glycine, T 1 It is bound, and the peptide portion is connected via serine, if present, L D It is connected.

[0210] In some embodiments, the peptide portion is Includes TIFF0007846624000100.tif18128.

[0211] In some embodiments, the peptide portion includes (glycine)4-(serine),

[0212] The peptide portion is transmitted via one of the glycine molecules, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T1 It is bound, and the peptide portion is connected via serine, if present, L D It is bound to. In some embodiments, the peptide portion is Includes TIFF0007846624000101.tif18128.

[0213] In some embodiments, the peptide portion is (serine)-(glycine) 1~4 Includes, The peptide portion is transmitted via serine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T 1 It is bound, and the peptide portion is connected via serine, if present, L D It is connected.

[0214] In some embodiments, the peptide portion is Includes TIFF0007846624000102.tif18128.

[0215] In some embodiments, the peptide portion is (serine)-(glycine) -4 Includes, The peptide portion is transmitted via serine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T 1 It is bound, and the peptide portion is connected via serine, if present, L D It is connected.

[0216] In some embodiments, the peptide portion is Includes TIFF0007846624000103.tif17128.

[0217] In some embodiments, the peptide portion is (β-alanine)-(glycine) 1~4-(Serine) The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1 It is bound, and the peptide portion is connected via serine, if present, L D It is connected.

[0218] In some embodiments, the peptide portion is Includes TIFF0007846624000104.tif19128.

[0219] In some embodiments, the peptide portion comprises (β-alanine)-(glycine)4-(serine), The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bound, and the peptide portion is via serine, if present, T 1 It is bound, and the peptide portion is connected via serine, if present, L D It is connected.

[0220] In some embodiments, the peptide portion is Includes TIFF0007846624000105.tif18128.

[0221] In some embodiments, the peptide portion is (glycine) 1~4 -Contains glutamic acid, The peptide portion is transmitted via one of the glycine molecules, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0222] In some embodiments, the peptide portion is Includes TIFF0007846624000106.tif28128.

[0223] In some embodiments, the peptide portion includes (glycine)-(glutamic acid), The peptide portion is transmitted via glycine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0224] In some embodiments, the peptide portion is Includes TIFF0007846624000107.tif28128.

[0225] In some embodiments, the peptide portion includes (glycine)4-(glutamic acid), The peptide portion is transmitted via one of the glycine molecules, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0226] In some embodiments, the peptide portion is Includes TIFF0007846624000108.tif28128.

[0227] In some embodiments, the peptide portion is (glutamic acid)-(glycine) 1~4 Includes, The peptide portion, via glutamic acid, if present, is L 3to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0228] In some embodiments, the peptide portion is Includes TIFF0007846624000109.tif27128.

[0229] In some embodiments, the peptide portion comprises (glutamic acid)-(glycine)4, The peptide portion, via glutamic acid, if present, is L 3 to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0230] In some embodiments, the peptide portion is Includes TIFF0007846624000110.tif26128.

[0231] In some embodiments, the peptide portion includes (glutamic acid)-(glycine), The peptide portion, via glutamic acid, if present, is L 3 to, or L 3 If it does not exist, then L M It is bound to, and the peptide portion is transmitted via one of the glycines, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0232] In some embodiments, the peptide portion is Includes TIFF0007846624000111.tif26128.

[0233] In some embodiments, the peptide portion is (β-alanine)-(glycine) 1~4 -Contains glutamic acid, The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0234] In some embodiments, the peptide portion is Includes TIFF0007846624000112.tif28128.

[0235] In some embodiments, the peptide portion includes (β-alanine)-(glycine)4-(glutamic acid), The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then L M It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0236] In some embodiments, the peptide portion is Includes TIFF0007846624000113.tif28128.

[0237] In some embodiments, the peptide portion includes (β-alanine)-(glycine)-(glutamic acid), The peptide portion is transmitted via β-alanine, if present, through L 3 to, or L 3 If it does not exist, then LM It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to T 1 It is bonded, and the peptide portion is transmitted via glutamic acid, if present, to L D It is connected.

[0238] In some embodiments, the peptide portion is Includes TIFF0007846624000114.tif28128.

[0239] Variable L D and W D In some embodiments, L D Each of these entities independently controls D to M A It is a divalent linker portion that connects to and contains at least one cleavable bond, and as a result, when the bond is cleaved, D is released in its active form for its intended therapeutic effect.

[0240] In some embodiments, L D L is a component of a releaseable aggregate unit. In some embodiments, L D L is a releaseable aggregate unit. In some embodiments, L D It includes one severable bond. In some embodiments, L D This includes multiple severable parts or joints.

[0241] In some embodiments, functional groups for forming cleavable bonds may include, for example, sulfhydryl groups for forming disulfide bonds, aldehyde groups, ketone groups or hydrazine groups for forming hydrazone bonds, hydroxylamine groups for forming oxime bonds, carboxyl groups or amino groups for forming peptide bonds, carboxyl groups or hydroxyl groups for forming ester bonds, and sugars for forming glycosidic bonds. In some embodiments, L D This includes a disulfide bond that can be cleaved by disulfide exchange, an acid-unstable bond that can be cleaved at an acidic pH, and / or a bond that can be cleaved by a hydrolase. In some embodiments, LD It contains a carbamate bond (i.e., -OC(O)-NR- where R is hydrogen or alkyl, etc.).

[0242] In some embodiments, L D The structure and arrangement of the cleavable bonds within may be such that the bonds are cleaved by the action of enzymes present at the target site. In some embodiments, the cleavable bonds may be cleavable by other mechanisms.

[0243] In some embodiments, L D The structure and arrangement of the cleavable bonds within may be such that the bonds are cleaved by the action of enzymes present at the target site. In some embodiments, the cleavable bonds may be cleavable by other mechanisms.

[0244] In some embodiments, the cleavable bond can be enzymatically cleaved by one or more enzymes, including tumor-associated proteases, to release drug units or D, and the conjugate or intermediate or scaffold of the present disclosure is protonated in vivo upon release to provide drug units or D.

[0245] In some embodiments, L D It may contain one or more amino acids. In some embodiments, L D Each amino acid within may be a native isomer or a non-native isomer and / or a D isomer or an L isomer, insofar as cleavable bonds exist. In some embodiments, L D It contains alpha amino acids, beta amino acids, or gamma amino acids, which may be natural or unnatural. In some embodiments, L D It contains 1 to 12 amino acids (for example, 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) in a continuous sequence.

[0246] In some embodiments, L D It may contain natural amino acids. In some embodiments, L DIt may contain non-natural amino acids. In some embodiments, L D It does not contain natural amino acids. In some embodiments, L D It does not contain unnatural amino acids. In some embodiments, L D It may include natural amino acids bonded to non-natural amino acids. In some embodiments, L D This may include natural amino acids bonded to the D-isomer of a natural amino acid. In some embodiments, L D This includes dipeptides, such as -Val-Cit-, -Phe-Lys-, or -Val-Ala-.

[0247] In some embodiments, L D This includes monopeptide units, dipeptide units, tripeptide units, tetrapeptide units, pentapeptide units, hexapeptide units, heptapeptide units, octapeptide units, nonapeptide units, decapeptide units, undecapeptide units, or dodecapeptide units.

[0248] In some embodiments, L D It contains a peptide (e.g., 1 to 12 amino acids) directly conjugated to a drug unit. In some such embodiments, the peptide is a single amino acid. In some such embodiments, the peptide is a dipeptide.

[0249] In some embodiments, L D Each amino acid in the compound is independently selected from alanine, β-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 acid, aminoalkynic acid, aminoalkanedioic acid, aminobenzoic acid, aminoheterocycloalkanoic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid and its derivatives.

[0250] In some embodiments, each amino acid is independently selected from alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, citrulline, and their derivatives.

[0251] In some embodiments, each amino acid is selected from proteinogenic and non-proteinogenic amino acids.

[0252] In some embodiments, L D Each amino acid in the compound can be independently selected from 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, aminoalkynic acid, aminoalkanediic acid, heterocyclocarboxylic acid, citrulline, statin, diaminoalkanoic acid, valine, and L-isomers or D-isomers of citrulline and its derivatives.

[0253] In some embodiments, L D Each amino acid in the compound 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.

[0254] In some embodiments, L D Each amino acid in the compound is independently selected from the following amino acids, namely, alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, and the L-isomers of valine.

[0255] In some embodiments, L D Each amino acid in the compound is independently selected from the following amino acids, namely alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, and the D-isomers of valine.

[0256] In some embodiments, L D The amino acids in it are alanine, β-alanine, glutamine, glutamic acid, isoglutamic acid, isoaspartic acid, valinecitrulline, or aspartic acid.

[0257] In some embodiments, L D It contains β-alanine. In some embodiments, L D It contains (β-alanine)-(alanine). In some embodiments, L D It contains (β-alanine) and optionally alanine, glutamic acid, glutamine, isoglutamic acid, aspartic acid, isospartic acid, valine, (valine)-(alanine), (alanine)-(alanine), or (valine)-(citrulline).

[0258] In some embodiments, L D It contains (glutamic acid)-(alanine).

[0259] In some embodiments, L D It contains (β-alanine)-(glutamine).

[0260] In some embodiments, L D It contains (β-alanine)-(glutamine)-(alanine).

[0261] In some embodiments, L DIt contains glutamic acid and optionally alanine, glycine, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)-(alanine), (alanine)-(alanine), or (valine)-(citrulline).

[0262] In some embodiments, L D It contains 2,3-diaminopropanoic acid. In some embodiments, W w It contains (R)-2,3-diaminopropanoic acid. In some embodiments, W w It contains glutamic acid. In some embodiments, W w It contains (glutamic acid)-(alanine). In some embodiments, L D It contains (glutamic acid)-(glycine)-(alanine).

[0263] In some embodiments, L D This includes L-glutamic acid, D-glutamic acid, (L-glutamic acid)-(L-alanine), (L-glutamic acid)-(D-alanine), (D-glutamic acid)-(L-alanine), (D-glutamic acid)-(D-alanine), (L-glutamic acid)-(glycine)-(L-alanine), (D-glutamic acid)-(glycine)-(D-alanine), (L-glutamic acid)-(glycine)-(D-alanine), or (D-glutamic acid)-(glycine)-(L-alanine). In some embodiments, L D It contains one or more amino acids in addition to a carbamate bond.

[0264] In some embodiments, L D The selectivity of enzymatic cleavage by a specific enzyme can be designed and optimized. In some embodiments, the specific enzyme is a tumor-associated protease. In some embodiments, L D This includes a bond whose cleavage is catalyzed by cathepsins B, C, and D, or plasmin proteases.

[0265] In some embodiments, L D It includes a sugar cleavage site. In some embodiments, L DIt contains a sugar moiety (Su) bonded to a self-immolative group via an oxygen glycosidic bond. In some embodiments, the “self-immolative group” consists of three separate chemical moieties (i.e., a sugar moiety (via a glycosidic bond), a drug unit (directly or indirectly), and M A It may be a trifunctional chemical moiety that can be covalently bonded together (directly or indirectly). In some embodiments, the glycosidic bond can be cleaved at the target site to initiate a self-sacrificing reaction sequence that results in drug release.

[0266] Therapeutic agent, drug unit or D In some embodiments, the therapeutic agent is a small molecule having a molecular weight of about 5 kDa or less (for example, having a molecular weight of about 4 kDa or less, about 3 kDa or less, about 1.5 kDa or less, or about 1 kDa or less).

[0267] In some embodiments, the therapeutic agent has an IC of less than approximately 1 nM. 50 It has. In some embodiments, the therapeutic agent has an IC of less than 1 nM. 50 It has.

[0268] In some embodiments, the therapeutic agent has an IC of more than approximately 1 nM. 50 (For example, the therapeutic agent has an IC of about 1 to about 50 nM) 50 (Having) In some embodiments, the therapeutic agent has an IC of more than about 1 nM. 50 It has. In some embodiments, the therapeutic agent has an IC of greater than 1 nM. 50 (For example, the therapeutic agent has an IC of 1-50 nM) 50 (having). In some embodiments, the therapeutic agent has an IC of greater than 1 nM. 50 It has.

[0269] In some embodiments, ICs exceeding approximately 1 nM 50Some therapeutic agents (e.g., “low-potency drugs”) are unsuitable for conjugation with antibodies using conjugation techniques recognized in the art. While we do not wish to be bound by theory, such therapeutic agents have insufficient potency for use in conventional targeted antibody-drug conjugates because it is not possible to conjugate a sufficient number of copies of the drug (i.e., more than 8) using techniques recognized in the art without degrading the pharmacokinetic and physicochemical properties of the conjugate. In some embodiments, the conjugation strategies described herein can be used to achieve sufficiently high loadings of these low-potency drugs, thereby resulting in a high loading of the therapeutic agent while maintaining desirable pharmacokinetic and physicochemical properties. In some embodiments, the present disclosure provides an antibody-drug conjugate comprising an antibody, a scaffold, and at least 8 therapeutic agent moieties, wherein the therapeutic agent has an IC50 greater than approximately 1 nM. 50 This relates to antibody-drug conjugates having the following properties.

[0270] In some embodiments, the drug is a derivative of an immunomodulatory compound, as described in U.S. Patent No. 2018 / 0154018, whose entire contents are incorporated herein by reference: (a) auristatin compounds, (b) calicheamycin compounds, (c) duocalmycin compounds, (d) SN38, (e) pyrrolobenzodiazepines, (f) vinca compounds, (g) tubulisin compounds, (h) non-natural camptothecin compounds, (i) meitansinoid compounds, (j) DNA binding agents, (k) kinase inhibitors, (l) MEK inhibitors, (m) KSP inhibitors, (n) topoisomerase inhibitors, (o) DNA alkylating agents, (p) RNA polymerases, (q) PARP inhibitors, (r) NAMPT inhibitors, (s) topoisomerase inhibitors, (t) protein synthesis inhibitors, (u) DNA binding agents, (v) DNA intercalation agents, or (w) immunomodulatory compounds.

[0271] In some embodiments, the drug used in this disclosure is auristatin F-hydroxypropylamide-L-alanine.

[0272] In some embodiments, auristatin is a compound of formula (X): It is TIFF0007846624000115.tif21128, During the ceremony, R 31 and R 32 Each of them independently contains hydrogen or C 1~8 It is alkyl, R 31 and R 32 The maximum of these is H, R 33 is hydrogen, C 1~8 Alkyl, C 3~8 carbocycle, C 6~10 Ariel, C 1~8 Alkyl-C 6~10 Ariel, X 1 -(C 3~8 carbon ring), C 3~8 A complex algebra, or X 1 -(C 3~8 It is a complex algebra, R 34 is hydrogen, C 1~8 Alkyl, C 3~8 carbocycle, C 6~10 Ariel, X 1 -C 6~10 Ariel, X 1 -(C 3~8 carbon ring), C 3~8 A complex algebra, or X 1 -(C 3~8 It is a complex algebra, R 35 is hydrogen or methyl, Or, R 34 and R 35 Together with the carbon atoms to which they are bonded, they form the formula -(CR 55 R 41 ) b - forms a carbon cyclic ring having, in the formula, R 55 and R 41 Each of them independently contains hydrogen or C 1~8 It is an alkyl group, and b is an integer between 3 and 7. R 36 is hydrogen or C 1~8It is alkyl, R 37 is hydrogen, C 1~8 Alkyl, C 3~8 carbocycle, C 6~10 Ariel, -X 1 -C 6~10 Ariel, -X 1 -(C 3~8 carbon ring), C 3~8 A complex algebra, or -X 1 -(C 3~8 It is a complex algebra, Each R 38 These are independently hydrogen, OH, and C. 1~8 Alkyl, C 3~8 A carbocyclic ring, or O-(C 1~8 It is alkyl, R 53 teeth, TIFF0007846624000116.tif19128 or R 54 And, R 39 is hydrogen, C 1~8 Alkyl, C 6~10 Ariel, -X 1 -C 6~10 Ariel, C 3~8 carbocycle, C 3~8 The complex algebra, -X 1 -C 3~8 Complex algebra, -C 1~8 It is alkylene-NH2 or (CH2)2SCH3, each X 1 Independently, C 1~10 Alkylene or C 3~10 It is a cycloalkylene, R 44 is hydrogen or C 1~8 It is alkyl, R 45 X 3 -R 42 or NH-R 19 And, X 3 is either O or S, R 19 is hydrogen, OH, amino group, C 1~8 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 21 Each of them independently contains hydrogen and 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 A cycloalkyl group, or a 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 These are, independently, hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl, X 2 These are side chains of natural or unnatural amino acids. R 77 is hydrogen or X 2 And, NR 77 It forms nitrogen-containing cyclic compounds, R 82 -NR 23 or oxygen, R 54 -C(R 56 )2--C(R 56 )2-C 6~10 Aryl, -C(R 56 )2--C(R 56 )2-C 3~8 Heterogeneous ring, or -C(R 56 )2--C(R 56 )2-C 3~8 It is a carbon ring, R 56 These are independently H, OH, and C. 1~8 Alkyl, C 3~8 carbocycle, -OC 1~8 Alkyl, -OC(O)-R 29 , or -OR 23 -OC 1~6 It is alkyl-NH2, R 29 It is an amino group, a 5-12 member heterocycloalkyl group, -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 is or R 29 R is defined herein. 47 And, R 28 It does not exist, or NR 23 or oxygen, a is an integer between 1 and 6, c is an integer between 0 and 3, d is an integer between 1 and 3, and f is an integer between 1 and 12.

[0273] In some embodiments, in the auristatin compound of formula (X): R 39 is benzyl or TIFF0007846624000117.tif21128 and R 44 is hydrogen.

[0274] In some embodiments, the auristatin is a compound of formula (Xa): TIFF0007846624000118.tif22128, wherein R 33 ~R 38 , and R 44 are as defined herein, R 31 and R 32 one of which is hydrogen or C 1~8 alkyl and the other is TIFF0007846624000119.tif26128, wherein R 83 is hydrogen or CH3, R 84 is C 1~6 alkyl or C 6~10 aryl, each R[[ID=5']] 12 ’ is independently halogen, -C 1~8 alkyl, -O-C 1~8 alkyl, nitro, or cyano, h is an integer from 0 to 4, u is the integer 0 or 1, R 53 is TIFF0007846624000120.tif21128 or R 54 and R 39 is hydrogen, C 1~8 alkyl, C 6~10 aryl, -X 1 -C 6~10 aryl, C 3~8 carbocycle, C 3~8 heterocycle, -X 1 -C3~8 Complex algebra, -C 1~8 It is alkylene-NH2 or (CH2)2SCH3, each X 1 Independently, C 1~10 Alkylene or C 3~10 It is a cycloalkylene, R 45 X 3 -R 42 or NH-R 19 And, X 3 is either O or S, R 19 is hydrogen, OH, amino group, C 1~8 Alkylamino, or -[C(R 20 R 21 )] a -R 22 And, R 42 is hydrogen, amino group, C 1~6 Alkylamino, or -[C(R 20 R 21 )] a -R 22 And, R 20 and R 21 Each of them independently contains hydrogen and 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 A cycloalkyl group, or a 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)(R23 )、 or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 wherein each R 23 is independently hydrogen, C 1~6 alkyl, C 6~10 aryl, C 3~8 cycloalkyl, -COOH, or -COO-C 1~6 alkyl, X​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​)] a -R 22 , or -R 28 -C 1~6 Alkyl-C 6~12 Aryl-C 1~6 Alkyl-R 22 is or R 29 R as defined herein 47 And, R 28 It does not exist, or NR 23 or oxygen, a is an integer between 1 and 6, c is an integer between 0 and 3, d is an integer between 1 and 3, and f is an integer between 1 and 12.

[0275] In some embodiments, the auristatin compound of formula (Xa) is a compound of formula (XIa) or formula (XIb): It is TIFF0007846624000121.tif51139, During the ceremony, R 92 teeth, It is TIFF0007846624000122.tif19128, R 83 It is either hydrogen or CH3.

[0276] In some embodiments, auristatin of formula (X) is a compound of formula (XI), formula (XII), or formula (XIII), Here, the compound of formula (XI) It is TIFF0007846624000123.tif23128, In the formula, R 31 is hydrogen or CH3, and R 42 is either -CH3 or one of the following structures: It is TIFF0007846624000124.tif184155, During the ceremony, a is an integer between 1 and 6, c is an integer between 0 and 3, and g is an integer between 2 and 6; Here, the compound of formula (XII) It is TIFF0007846624000125.tif24128, In the formula, R 31 is hydrogen or CH3, and R 40 is hydrogen, -OH, -NH2, or one of the following structures: It is TIFF0007846624000126.tif234155, During the ceremony, a is an integer between 1 and 6, g is an integer between 2 and 6, and c is an integer between 0 and 3; Here, the compound of formula (XIII) It is TIFF0007846624000127.tif28134, During the ceremony, R 31 is hydrogen or CH3, R 29 It is an amino group, a 5-12 member heterocycloalkyl group, -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 is or R 29 R as defined herein 47 And, R 20 and R 21 Each of them independently contains hydrogen and 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 3~8A cycloalkyl group, or a 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 These are, independently, hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C 3~8 Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl, X 2 These are side chains of natural or unnatural amino acids. R 77 is hydrogen or X 2 And, NR 77 It forms nitrogen-containing cyclic compounds, R 82 -NR 23 or oxygen, R 28 It does not exist, or NR 23 or oxygen, a is an integer between 1 and 6, c is an integer between 0 and 3, d is an integer between 1 and 3, and f is an integer between 1 and 12.

[0277] In some aspects of equation (XII), R 40 teeth The filename is TIFF0007846624000128.tif60161.

[0278] In some embodiments, the compound of formula (XII) is a compound of formula (XIIa), (XIIb), (XIIc), (XIId), (XIIe), (XIIf), (XIIg), or (XIIh): These are TIFF0007846624000129.tif168128TIFF0007846624000130.tif205110TIFF0007846624000131.tif64128.

[0279] In some embodiments of the compound of formula (XIII), R 29 -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 is or R 29 R as defined herein 47 And, R 28 It does not exist, or 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 -(O CH2-CH2) f -N(H)(R 23 ), or -R 82 -(C(O)-CH(X 2 )-NH) d -R 77 And, Each R 23 These are, independently, hydrogen and C 1~6 Alkyl, C 6~10 Ariel, C3~8 Cycloalkyl, -COOH, or -COO-C 1~6 It is alkyl, X 2 These are side chains of natural or unnatural amino acids. R 77 is hydrogen or X 2 And, NR 77 It forms nitrogen-containing cyclic compounds, R 82 -NR 23 or oxygen, c is an integer between 0 and 3, d is an integer between 1 and 3, and f is an integer between 1 and 12.

[0280] In some embodiments, R 29 It is one of the following structures: TIFF0007846624000132.tif60149TIFF0007846624000133.tif210164, During the ceremony, a is an integer between 1 and 6, c is an integer between 0 and 3, and g is an integer between 2 and 6.

[0281] TIFF0007846624000134.tif24128In formula, R 42 H, -CH3 (m / z=760), In the formula TIFF0007846624000135.tif11141TIFF0007846624000136.tif26128, R40 is H, TIFF0007846624000137.tif188160 In the formula, -C(O)-R 29 teeth, The filename is TIFF0007846624000138.tif48153.

[0282] In some embodiments, D is The filename is TIFF0007846624000139.tif45128.

[0283] In some embodiments, D is The filename is TIFF0007846624000140.tif30128.

[0284] Hydrophilic group or T 1 In some embodiments, the hydrophilic groups contained in the conjugate or scaffold of the present disclosure are water-soluble and substantially non-antigenic polymers. In some embodiments, examples of hydrophilic groups include, but are not limited to, polyalcohols, polyethers, polyanions, polycations, polyphosphates, polyamines, polysaccharides, polyhydroxy compounds, polylysine and its derivatives. In some embodiments, one end of the hydrophilic group is connected to a polyfunctional linker or M by an inseparable bond or via a cleavable bond. A Linker (for example, M A The hydrophilic group may be functionalized to be covalently bonded to an amino acid in the linker. In some embodiments, the functionalization may be via, for example, an amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or other functional group. In some embodiments, the other end (terminus) (or termini) of the hydrophilic group is free and unbonded. In some embodiments, "unbonded" means that the hydrophilic group is not bonded to another part, such as a D or drug unit, a releaseable aggregate unit, or other components of the conjugate or scaffold of the disclosure. In some embodiments, the free and unbonded end of the hydrophilic group may include a methoxy, carboxylic acid, alcohol, or other suitable functional group. In some embodiments, the methoxy, carboxylic acid, alcohol, or other suitable functional group acts as a cap for the terminus or termini of the hydrophilic group.

[0285] In some embodiments, a cleavable bond refers to a bond that is substantially insensitive to cleavage while circulating in plasma but sensitive to cleavage in an intracellular or intratumoral environment. In some embodiments, an uncleavable bond is substantially insensitive to cleavage in any biological environment. In some embodiments, chemical hydrolysis of hydrazones, reduction of disulfides, and enzymatic cleavage of peptide or glycosidic bonds are examples of cleavable bonds. In some embodiments, exemplary bonds of hydrophilic groups are mediated by amide bonds, ether bonds, ester bonds, hydrazone bonds, oxime bonds, disulfide bonds, peptide bonds, or triazole bonds. In some embodiments, a polyfunctional linker or M A (For example, M) A The attachment of hydrophilic groups to amino acids in the linker is via amide bonds.

[0286] In some embodiments of the present disclosure, the conjugate or scaffold comprises multiple hydrophilic groups, the multiple hydrophilic groups may be the same or different chemical moieties. In some embodiments, the multiple hydrophilic groups may form a polyfunctional linker or M at a single bonding site or at different sites. A It can be linked to a linker.

[0287] In some embodiments, the addition of hydrophilic groups can have two potential effects on the pharmacokinetics of the resulting conjugate. In some embodiments, the desired effect is a decrease in clearance (and consequently, an increase in exposure) resulting from a reduction in nonspecific interactions induced by the exposed hydrophobic elements of the drug or drug-linker. In some embodiments, the undesirable effect is a decrease in volume and distribution velocity, which may result from an increase in the molecular weight of the conjugate. In some embodiments, increasing the molecular weight of the hydrophilic group may increase the hydrodynamic radius of the conjugate and decrease its diffusivity, thereby reducing the conjugate's ability to penetrate tumors. In some embodiments, a hydrophilic group that is large enough but not large enough to significantly reduce its diffusivity may be used to reduce conjugate clearance and thus increase plasma exposure, thereby reducing the conjugate's ability to reach the intended target cell population.

[0288] In some embodiments, the hydrophilic group includes, but is not limited to, sugar alcohols (also known as polyalcohols, polyhydric alcohols, alditols or glycitols) or their derivatives (e.g., aminopolyalcohols), carbohydrates (e.g., sugars), polyvinyl alcohols, carbohydrate polymers (e.g., dextran), hydroxypropyl methacrylamide (HPMA), polyalkylene oxides and / or copolymers thereof.

[0289] In some embodiments, the hydrophilic group includes a portion that incorporates multiple hydroxyl groups, such as monosaccharides, oligosaccharides, polysaccharides, etc. In some embodiments, the hydrophilic group includes multiple -(CR 58 It contains an OH)- group, and in the formula, R 58 is -H or C 1~8 It is alkyl.

[0290] In some embodiments, the hydrophilic group is given by formula: TIFF0007846624000141.tif6128 contains one or more of the following fragments, where n1 is an integer from 0 to approximately 6, and each R 58These are, independently, hydrogen or C 1~8 It is alkyl, R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 - and in the formula, R 59 R is hydrogen, alkyl, cycloalkyl or arylalkyl, 61 CH2OR 62 COOR 62 ,-(CH2) n2 COOR 62 , or a heterocycloalkyl substituted with one or more hydroxyls, R 62 is hydrogen or C 1~8 It is an alkyl group, and n² is an integer between 1 and approximately 5.

[0291] In some embodiments, R 58 is hydrogen, R 60 is a combination or C 1~6 It is an alkyl linker, where n1 is an integer from 1 to approximately 6, and R 61 is CH2OH or COOH. In some embodiments, R 58 is hydrogen, R 60 ha-CHR 59 - and n1 is 0, R 61 This is a heterocycloalkyl group, such as a monosaccharide, that is substituted with one or more hydroxyls.

[0292] In some embodiments, the hydrophilic group includes a glucosylamine, a diamine, or a triamine.

[0293] In some embodiments, the hydrophilic group is one or more of the following fragments or stereoisomers: Includes TIFF0007846624000142.tif216167, During the ceremony, R 59 is hydrogen, C 1~8 They are alkyl, cycloalkyl, or arylalkyl. n1 is an integer between 1 and approximately 6, n2 is an integer between 1 and approximately 5, and n3 is an integer between approximately 1 and approximately 3.

[0294] In this specification, it is understood that all stereochemical forms of hydrophilic groups are intended. In the above formulas, the hydrophilic group may be derived from ribose, xylose, glucose, mannose, galactose, or other sugars, and may retain the stereochemical configuration of the pendant hydroxyl group and alkyl group present on those molecules. In some embodiments, it is understood that various deoxy compounds are also intended in the above formulas. Illustratively, where applicable, one or more of the following characteristics are intended for the hydrophilic group:

[0295] In some embodiments, n3 is 2 or 3. In some embodiments, n3 is 2. In some embodiments, n3 is 3. In some embodiments, n1 is 1, 2 or 3. In some embodiments, n1 is 1. In some embodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments, n2 is 1.

[0296] In some embodiments, R 59 H is H.

[0297] In some embodiments, the hydrophilic group is Includes TIFF0007846624000143.tif16128.

[0298] In some embodiments, the hydrophilic group is Includes TIFF0007846624000144.tif24128.

[0299] In some embodiments, the hydrophilic group is Includes TIFF0007846624000145.tif18128.

[0300] In some embodiments, the hydrophilic group is Including TIFF0007846624000146.tif14128, in the formula, n4 is an integer between 1 and approximately 25. Each R 63These are, independently, hydrogen or C 1~8 It is alkyl, R 64 is a combination or C 1~8 It is an alkyl linker, R 65 is hydrogen, C 1~8 Alkyl, or -(CH2) n2 COOR 62 And, R 62 is hydrogen or C 1~8 It is alkyl, n² is an integer between 1 and approximately 5.

[0301] In some embodiments, the hydrophilic group is Includes TIFF0007846624000147.tif11128.

[0302] In some embodiments, n4 is an integer between approximately 2 and approximately 20, approximately 4 and approximately 16, approximately 6 and approximately 12, or approximately 8 and approximately 12.

[0303] In some embodiments, n4 is an integer between approximately 2 and approximately 20. In some embodiments, n4 is an integer between approximately 4 and approximately 16. In some embodiments, n4 is an integer between approximately 6 and approximately 12. In some embodiments, n4 is an integer between approximately 8 and approximately 12.

[0304] In some embodiments, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0305] In some embodiments, n4 is 8 or 12.

[0306] In some embodiments, n4 is 8.

[0307] In some aspects, T' is Includes TIFF0007846624000148.tif50128, In the formula, n4 is an integer between approximately 2 and 20, approximately 4 and 16, approximately 6 and 12, or approximately 8 and 12.

[0308] In some embodiments, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0309] In some embodiments, n4 is 8 or 12.

[0310] In some embodiments, n4 is 8.

[0311] In some embodiments, the hydrophilic group includes a polyether, such as polyalkylene glycol (PAO). In some embodiments, the PAO may be, without limitation, C 1~6 This includes polymers of alkylene oxides, particularly polymers of ethylene oxides. In some embodiments, the polyalkylene glycol is polyethylene glycol (PEG). In some embodiments, the polyethylene glycol is mPEG.

[0312] In some embodiments, the hydrophilic group comprises a PEG unit containing one or more PEG chains. In some embodiments, the PEG chains can be linked to each other in, for example, linear, branched, or star-shaped configurations. In some embodiments, the PEG unit may include non-PEG material in addition to repeating PEG subunits (for example, to facilitate the coupling of multiple PEG chains to each other or to facilitate coupling to amino acids).

[0313] In some embodiments, the PEG unit is a polyfunctional linker or M via a reactive group. A Linker (for example, M A The linker may be covalently bonded to the amino acids. In some embodiments, the reactive group is one to which the activated PEG molecule can be bound (e.g., a free amino group or a carboxyl group). In some embodiments, the N-terminal amino acid and lysine (K) have a free amino group, 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 binding PEG.

[0314] In some embodiments, the PEG unit is used by methoxylated PEG ("mPEG") having various reactive moieties, including but not limited to succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imidate, para-nitrophenyl carbonate (NPC), succinimidyl propionate (SPA), and cyanuryl chloride, thereby creating a polyfunctional linker or M A Linker (for example, M A It can be bound to amino acids in the linker. In some embodiments, various PEG species can be used, and substantially any suitable reactive PEG reagent can be used. In some embodiments, the reactive PEG reagent is a polyfunctional linker or M A Linker (for example, M A When it binds to an amino acid in the linker, it forms a carbamate bond or an amide bond.

[0315] In some embodiments, a PEG unit includes 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 embodiments, a PEG unit includes approximately 72 or fewer subunits.

[0316] 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.

[0317] In some embodiments, the PEG unit includes at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, or at least 12 subunits.

[0318] In some embodiments, the PEG unit comprises at least six subunits, at least seven subunits, or at least eight subunits.

[0319] In some embodiments, the PEG unit includes at least six subunits. In some embodiments, the PEG unit includes at least seven subunits. In some embodiments, the PEG unit includes at least eight subunits.

[0320] In some embodiments, a PEG unit comprises one or more linear PEG chains, each having at least two subunits, at least three subunits, at least four subunits, at least five subunits, at least six subunits, at least seven subunits, at least eight subunits, at least nine subunits, at least ten subunits, at least eleven subunits, at least twelve subunits, at least thirteen subunits, at least fourteen subunits, at least fifteen subunits, at least sixteen subunits, at least seventeen subunits, at least eighteen subunits, at least nineteen subunits, at least twenty subunits, at least twenty-one subunits, at least twenty-two subunits, at least twenty-three subunits, or at least twenty-four subunits. In some embodiments, a PEG unit comprises a total of at least six subunits, at least eight, at least ten, or at least twelve subunits. In some such embodiments, a PEG unit comprises a total of about 72 or fewer subunits. In some such embodiments, a PEG unit contains a total of approximately 36 or fewer subunits.

[0321] In some embodiments, a PEG unit has a total of 4-72, 4-60, 4-48, 4-36, or 4-24 subunits, 5-72, 5-60, 5-48, 5-36, or 5-24 subunits, 6-72, 6-60, 6-48, 6-36, or 6-24 subunits, 7-72, 7-60, 7-48, 7-36, or 7-24 subunits, 8-72, 8-60, 8-48, 8-36, or 8-24 subunits, 9-72, 9-60, 9- 48, 9-36, or 9-24 subunits, 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- It includes 24 subunits, 20-72, 20-60, 20-48, 20-36, or 20-24 subunits, 21-72, 21-60, 21-48, 21-36, or 21-24 subunits, 22-72, 22-60, 22-48, 22-36, or 22-24 subunits, 23-72, 23-60, 23-48, 23-36, or 23-24 subunits, or 24-72, 24-60, 24-48, or 24-36 subunits.

[0322] In some embodiments, a PEG unit may consist of a total of 4-72, 4-60, 4-48, 4-36, or 4-24 subunits; 5-72, 5-60, 5-48, 5-36, or 5-24 subunits; 6-72, 6-60, 6-48, 6-36, or 6-24 subunits; 7-72, 7-60, 7-48, 7-36, or 7-24 subunits; 8-72, 8-60, 8-48, 8-36, or 8-24 subunits; 9-72, 9-60, 9-48, 9 ~36 or 9~24 subunits, 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 of 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- It comprises one or more linear PEG chains having 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, or 24-36 subunits.

[0323] 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.

[0324] In some embodiments, a PEG unit consists of 6-72, 6-60, 6-48, 6-36, or 6-24 subunits, 7-72, 7-60, 7-48, 7-36, or 7-24 subunits, 8-72, 8-60, 8-48, 8-36, or 8-24 subunits, 9-72, 9-60, 9-48, 9-36, or 9-24 subunits, or 10-72, 10-60, 10-48, 10-36, or 10-24 subunits. Knit, 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, Alternatively, it is a derivatized linear single PEG chain having 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, or 24-36 subunits.

[0325] In some embodiments, examples of hydrophilic groups suitable for the conjugates, scaffolds and methods disclosed herein can be found, for example, in U.S. Patent No. 8,367,065, Column 13, U.S. Patent No. 8,524,696, Column 6, International Publication No. 2015 / 057699, and International Publication No. 2014 / 062697, the entire contents of which are incorporated herein by reference.

[0326] antibody As used herein, the term “antibody” refers to a protein produced by the immune system that can recognize and bind to a specific antigen. In some embodiments, an antibody is a glycoprotein. In some embodiments, an antibody is, for example, a polyclonal antibody, a monoclonal antibody, a camelid single-domain antibody, an intracellular antibody ("intrabody"), a recombinant antibody, an anti-idiotype antibody, a domain antibody, a linear antibody, a multispecific antibody, an antibody fragment (e.g., Fv, Fab, F(ab)2, F(ab)3, Fab', Fab'-SH, F(ab')2), a single-chain variable fragment antibody (scFv), a tandem / bis-scFv, Fc, pFc', scFvFc (or scFv-Fc), a disulfide Fv (dsFv), a bispecific antibody (bc-scFv), such as a BiTE antibody; a camelid antibody. These antibodies may exist in a variety of forms, including resurfaced antibodies, humanized antibodies, fully human antibodies, single-domain antibodies (sdAb, also known as NANOBODY®), chimeric antibodies, chimeric antibodies containing at least one human constant region, biaffinity antibodies, such as biaffinity retargeting proteins (DART®), divalent (or bivalent) single-stranded variable fragments (di-scFv, bi-scFv) containing minibodies, diabodies, triabodies or tribodies, tetrabodies, etc., and multivalent antibodies.

[0327] As used herein, the term “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. In some embodiments, the term includes genetically engineered antibodies, antibody derivatives, and antibody fragments, which can be obtained by methods known in the art. In some embodiments, antibodies can be engineered to include at least one chemically reactive group.

[0328] In some embodiments, a glycoprotein containing a core-N-acetylglucosamine substituent (core-GlcNAc moiety) is an antibody containing a core-N-acetylglucosamine substituent (core-GlcNAc moiety). In some embodiments, the glycoprotein is a monoclonal antibody (mAb) IgA antibody, IgD antibody, IgE antibody, IgG antibody, or IgM antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, if the antibody is a whole antibody, the antibody contains one or more (e.g., one) core-GlcNAc moieties on each heavy chain, and the core-GlcNAc moieties may be fucosylated. In some embodiments, the whole antibody contains two or more (e.g., two) fucosylated core-GlcNAc moieties. In some embodiments, if the antibody is a single-chain antibody or antibody fragment, e.g., a Fab fragment or an Fc fragment, the antibody contains one or more fucosylated core-GlcNAc moieties. In some embodiments, within an antibody containing a core-GlcNAc moiety, the core-GlcNAc moiety may be located anywhere on the antibody, provided that the substituent does not interfere with the antibody's antigen-binding site. In some embodiments, the core-GlcNAc moiety is located at the antibody's natural N-glycosylation site. In some embodiments, the antibody contains, or is engineered to contain, at least one chemically reactive group or chemically reactive amino acid moiety or chemically reactive side chain.

[0329] In some embodiments, antibodies can direct conjugates to specific tissues, cells, or intracellular locations. In some embodiments, antibodies can direct conjugates in a culture, in a whole organism, or both. In some embodiments, antibodies contain ligands present on the cell surface of target cells to which the antibody binds with effective specificity, affinity, and avidity. In some embodiments, antibodies direct conjugates to tissues other than the liver. In some embodiments, antibodies direct conjugates to specific tissues, such as the liver, kidney, lung, or pancreas. In some embodiments, antibodies direct conjugates to target cells (e.g., cancer cells), receptors expressed on cells (e.g., cancer cells), matrix tissue, or cancer-related proteins (e.g., tumor antigens). In some embodiments, cells including tumor vascular systems may be targeted. In some embodiments, antibodies can direct conjugates to specific types of cells, for example, specifically targeting hepatocytes in the liver rather than Kupffer cells. In some embodiments, the antibody can deliver the conjugate to reticular endothelial or lymphoid cells, or to professional phagocytic cells such as macrophages or eosinophils. In some embodiments, the conjugate itself is an effective delivery system that does not require specific targeting.

[0330] In some embodiments, antibodies can guide conjugates to a location within the cell (e.g., the nucleus, cytoplasm, or endosomes). In some embodiments, antibodies enhance cell binding to receptors, or cytoplasmic transport to the nucleus, and nuclear entry or release from endosomes or other intracellular vesicles.

[0331] In some embodiments, the conjugate comprises a Her-2 antibody or a NaPi2b antibody. In some embodiments, the Her-2 antibody suitable for the conjugate is trastuzumab. In some embodiments, the Her-2 antibodies suitable for the conjugate include those described in International Publication No. 2015 / 195917 and PCT / US2018 / 019873, each of which is incorporated herein by reference in its entirety.

[0332] NaPi2b antibody In some embodiments, NaPi2b antibodies suitable for conjugation bind to the extracellular domain of SLC34A2. In some embodiments, the disclosure provides NaPi2b target-directed monoclonal antibodies that specifically recognize NaPi2b, also known as sodium-dependent phosphate transport protein 2B. In some embodiments, NaPi2b antibodies used in the conjugates disclosed herein can modulate, for example, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one biological activity of NaPi2b, and are useful for modulating, for example, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with at least one biological activity of NaPi2b. In some embodiments, the antibodies disclosed herein also include antibodies that bind to soluble NaPi2b. In some embodiments, NaPi2b antibodies specifically bind to an epitope on the extracellular domain (ECD) of human NaPi2b. These antibodies are collectively referred to as "NaPi2b" antibodies in this specification.

[0333] In some embodiments, the NaPi2b antibody-drug conjugates provided herein have an equilibrium dissociation constant (K) of 1 μM or less (e.g., 100 nM or less, 10 nM or less, and 1 nM or less). d or K D ) comprises an antibody that binds to the NaPi2b epitope. In some embodiments, the NaPi2b antibody used in the antibody-drug conjugate disclosed herein is in the range of about 1 nM or less to about 1 pM. d This indicates.

[0334] In some embodiments, the NaPi2b antibody-drug conjugates provided herein may include antibodies that help modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the functional activity of NaPi2b. In some embodiments, the functional activity of NaPi2b may include, for example, involvement in transcellular inorganic phosphate (Pi) absorption, thereby contributing to the maintenance of phosphate homeostasis in the body. In some embodiments, the NaPi2b antibody may completely or partially inhibit the functional activity of NaPi2b by partially or completely modulating, block, inhibit, reduce, antagonize, neutralize, or otherwise interfering with transcellular inorganic phosphate absorption.

[0335] In some embodiments, a NaPi2b antibody is considered to completely modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPi2b functional activity if the level of NaPi2b functional activity in the presence of the NaPi2b antibody is reduced by at least 95%, for example, 96%, 97%, 98%, 99%, or 100%, compared to the level of NaPi2b functional activity in the absence of binding to the NaPi2b antibody described herein. In some embodiments, a NaPi2b antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPi2b functional activity if the level of NaPi2b activity in the presence of the NaPi2b antibody is reduced by less than 95%, for example, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, compared to the level of NaPi2b activity in the absence of binding to the NaPi2b antibody described herein.

[0336] In some embodiments, the exemplary antibodies disclosed herein include XMT-1535 antibodies. These antibodies have been shown to exhibit specificity for human NaPi2b and to inhibit NaPi2b activity.

[0337] The NaPi2b human monoclonal antibody or NaPi2b humanized monoclonal antibody XMT-1535 contains a heavy chain (HC), heavy chain variable region (VH), light chain (LC), and light chain variable region (VL), as shown in the amino acid sequences and corresponding nucleic acid sequences in Table 1 below. In the following amino acid sequences, the variable heavy chain region and variable light chain region of each antibody are shaded. In the following amino acid sequences, the complementarity-determining regions (CDRs) of the heavy and light chains are underlined. The amino acids comprising the complementarity-determining regions (CDRs) of the XMT-1535 antibody are as defined by E. Kabat et al. (see Kabat, E., et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)), disclosed in U.S. Patent No. 8,603,474.

[0338] (Table 1) Sequence of NaPi2b human monoclonal antibody or NaPi2b humanized monoclonal antibody XMT-1535 TIFF0007846624000149.tif89134

[0339] The antibodies disclosed herein specifically bind to epitopes on the extracellular domain (ECD) of human NaPi2b.

[0340] In some embodiments, those skilled in the art will recognize that it is possible to determine, without excessive experimentation, whether a monoclonal antibody has the same specificity as the monoclonal antibodies disclosed herein (e.g., XMT-1535, 10H1.11.4B) by checking whether the former prevents the latter from binding to its natural binding partner or to other molecules known to associate with NaPi2b. When a monoclonal antibody under test competes with a monoclonal antibody disclosed herein, as indicated by the reduced binding by the monoclonal antibodies disclosed herein, the two monoclonal antibodies bind to the same epitope or closely related epitopes.

[0341] An alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibodies disclosed herein is to pre-incubate the monoclonal antibodies disclosed herein with soluble NaPi2b (which is typically reactive with it), and then add the monoclonal antibody under test to determine whether the ability of the monoclonal antibody under test to bind to NaPi2b is inhibited. If the monoclonal antibody under test is inhibited, it almost certainly has the same or functionally equivalent epitope specificity as the monoclonal antibodies disclosed herein.

[0342] Furthermore, the monoclonal antibodies disclosed herein can be screened, for example, by measuring NaPi2b-mediated activity and determining whether the test monoclonal antibody can modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPi2b activity.

[0343] In some embodiments, the antibodies disclosed herein include a heavy chain variable region having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the same amino acid sequence as a sequence selected from SEQ ID NO:3, and a light chain variable region having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the same amino acid sequence as a sequence selected from SEQ ID NO:4.

[0344] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:1, and a light chain amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:2.

[0345] In some embodiments, the antibodies disclosed herein include a heavy chain variable region amino acid sequence of SEQ ID NO:3 and a light chain variable region amino acid sequence of SEQ ID NO:4.

[0346] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence of SEQ ID NO:2.

[0347] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:5, the CDRH2 amino acid sequence of SEQ ID NO:6, the CDRH3 amino acid sequence of SEQ ID NO:7, the CDRL1 amino acid sequence of SEQ ID NO:8, the CDRL2 amino acid sequence of SEQ ID NO:9, and the CDRL3 amino acid sequence of SEQ ID NO:10.

[0348] In some embodiments, the antibodies disclosed herein include an amino acid sequence that is identical to that of SEQ ID NO: 5 by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more; CDRH2 including an amino acid sequence that is identical to that of SEQ ID NO: 6 by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more; SEQ ID CDRH3; SEQ ID NO:7 contains at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the same amino acid sequence as CDRL1; SEQ ID NO:8 contains at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the same amino acid sequence as CDRL1; SEQ ID NO:7 CDRL2 containing at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the same amino acid sequence as NO:9;CDRL3 containing at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical amino acid sequences to SEQ ID NO:10.

[0349] In some embodiments, the antibodies disclosed herein include one or more conserved amino acid substitutions in the variable domain sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more conserved substitutions in the variable domain sequence. In some embodiments, these conserved amino acid substitutions are located within the CDR region, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more conserved substitutions are cumulatively present across the entire CDR, and in some specific embodiments, each CDR sequence, for example, SEQ ID NO: 5-10, may contain up to one, up to two, up to three, or up to four conserved amino acid substitutions.

[0350] In some embodiments, those skilled in the art will recognize that it is possible to determine, without excessive experimentation, whether a monoclonal antibody has the same specificity as the monoclonal antibody XMT-1535 by checking whether the former prevents the latter from binding to its natural binding partner or to other molecules known to associate with NaPi2b. When a monoclonal antibody under test competes with a monoclonal antibody disclosed herein, as indicated by the reduced binding by the monoclonal antibody disclosed herein, the two monoclonal antibodies bind to the same epitope or closely related epitopes.

[0351] In some embodiments, an alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody disclosed herein is to pre-incubate the monoclonal antibody disclosed herein with soluble NaPi2b (which is typically reactive with it), and then add the monoclonal antibody under test to determine whether the ability of the monoclonal antibody under test to bind to NaPi2b is inhibited. In some embodiments, if the monoclonal antibody under test is inhibited, it has the same or functionally equivalent epitope specificity as the monoclonal antibody disclosed herein.

[0352] Furthermore, the monoclonal antibodies disclosed herein can be screened, for example, by measuring NaPi2b-mediated activity and determining whether the test monoclonal antibody can modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPi2b activity.

[0353] In some embodiments, NaPi2b antibodies suitable for conjugation can be produced and purified by known techniques, such as International Publication No. 2009 / 097128, International Publication No. 2017 / 160754, and U.S. Patent No. 16 / 136,706, each of which is incorporated herein by reference in its entirety.

[0354] HER2 antibody In some embodiments, HER2 antibodies suitable for conjugation bind to human HER2 in a soluble form or in a membrane-bound state (i.e., when expressed on the cell surface). In some embodiments, the disclosure provides monoclonal antibodies that bind to HER2 and are humanized or fully human. In some embodiments, the disclosure provides monoclonal antibodies that specifically bind to HER2. These antibodies are collectively referred to herein as “HER2” antibodies.

[0355] In some embodiments, the HER2 antibody suitable for conjugation has an equilibrium dissociation constant (K) of 1 μM or less (e.g., 100 nM or less; 10 nM or less; 1 nM or less). d or K D ) binds to the HER2 epitope. In some embodiments, the present disclosure provides monoclonal antibodies that bind to HER2 and are humanized or fully human. For example, the HER2 antibodies provided herein have a K content in the range of about 1 nM or less to about 1 pM. d This indicates.

[0356] In some embodiments, the HER2 antibodies disclosed herein help to modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the functional activity of HER2. In some embodiments, the functional activity of HER2 includes, for example, the modulation of PI3K-Akt pathway activity. In some embodiments, the HER2 antibodies completely or partially inhibit HER2 functional activity by partially or completely modulating, block, inhibit, reduce, antagonize, neutralize, or otherwise interfering with PI3K-Akt pathway activity. PI3K-Akt pathway activity is evaluated using any method recognized in the art for detecting PI3K-Akt pathway activity, including, but not limited to, the detection of phosphorylated Akt levels in the presence and absence of the antibodies or antigen-binding fragments disclosed herein.

[0357] In some embodiments, a HER2 antibody is considered to completely modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with HER2 functional activity if the level of HER2 functional activity in the presence of the HER2 antibody is reduced by at least 80%, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, compared to the level of HER2 functional activity in the absence of binding to the HER2 antibody described herein. In some embodiments, a HER2 antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with HER2 functional activity if the level of HER2 activity in the presence of the HER2 antibody is reduced by less than 95%, for example, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, compared to the level of HER2 activity in the absence of binding to the HER2 antibody described herein.

[0358] In some embodiments, the exemplary antibodies disclosed herein include the XMT-1519 antibody, which exhibits specificity for human HER2 and has been shown to inhibit the functional activity of HER2 in vitro.

[0359] The HER-2 monoclonal antibody XMT-1519 contains a heavy chain (HC), a variable heavy chain region (VH), a light chain (LC), and a variable light chain region (VL), as shown in the amino acid sequence and corresponding nucleic acid sequence in Table 2 below. In the following amino acid sequences, the variable heavy chain region and variable light chain region of each antibody are shaded. In the following amino acid sequences, the complementarity-determining regions (CDRs) of the heavy and light chains are underlined. The amino acids that comprise the complementarity-determining regions (CDRs) are as defined by E. Kabat et al. (See Kabat, E., et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).

[0360] (Table 2) Sequence of HER2 human monoclonal antibody or HER2 humanized monoclonal antibody XMT-1519 TIFF0007846624000150.tif94138

[0361] The antibodies and antigen-binding fragments disclosed herein specifically bind to an epitope on the full-length human HER2 receptor containing the amino acid sequence of SEQ ID NO:16.

[0362] The antibodies and antigen-binding fragments disclosed herein specifically bind to an epitope on the extracellular domain (ECD) of the human HER2 receptor containing the amino acid sequence SEQ ID NO:31.

[0363] In some embodiments, the antibodies of this disclosure exhibit HER2-binding characteristics different from those of antibodies described in the Art. In some embodiments, the antibodies disclosed herein bind to different epitopes of HER2, such that they cross-block each other but do not cross-block trastuzumab, pertuzumab, Fab37, or chA21 from binding to HER2. Furthermore, in contrast to known antibodies, the antibodies disclosed herein can be efficiently internalized into HER2-expressing cells without promoting cell proliferation.

[0364] In some embodiments, the antibodies disclosed herein are fully human monoclonal antibodies that conjugate to novel epitopes and / or possess other desirable properties for therapeutic use. In some embodiments, exemplary properties include, but are not limited to, desirable binding properties to cancer cells expressing high or low levels of human HER2, specific binding to recombinant human HER2 and recombinant cynomolgus monkey HER2, efficient internalization upon binding to HER2, high ability to kill cancer cells expressing high or low levels of HER2 when administered as an antibody-drug conjugate (ADC), substantial agonist activity against the proliferation of HER2-expressing cancer cells, and / or effective antibody-dependent cytotoxicity (ADCC)-mediated killing of HER2-expressing cells, as well as any combination of the aforementioned properties.

[0365] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to the epitope of the human HER2 receptor, comprising residues 452-531 of the extracellular domain of the human HER2 receptor, residues 474-553 of SEQ ID NO:16, or residues 452-531 of SEQ ID NO:31.

[0366] In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that bind to at least a portion of the N-terminus of domain IV of the human HER2 receptor but do not cross-compete with antibodies that bind to epitope 4D5 of the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments described herein do not cross-compete with trastuzumab for binding to the human HER2 receptor, since trastuzumab is known to bind to epitope 4D5 of the human HER2 receptor. As used herein, the term epitope 4D5 of the human HER2 receptor refers to amino acid residues 529-627 of the extracellular domain of the human HER2 receptor, residues 551-649 of SEQ ID NO:16, or residues 529-627 of SEQ ID NO:31. In some embodiments, the antibodies or antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0367] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to the epitope of the human HER2 receptor, comprising residues 452-500 of the extracellular domain of the human HER2 receptor, residues 474-522 of SEQ ID NO:16, or residues 452-500 of SEQ ID NO:31.

[0368] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to the epitope of the human HER2 receptor, comprising at least one amino acid residue selected from the extracellular domain amino acid residues E521, L525, and R530 of the human HER2 receptor, for example, residues 543, 547, and 552 of SEQ ID NO: 16, and residues 521, 525, and 530 of SEQ ID NO: 31. In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to the epitope of the extracellular domain of the human HER2 receptor, comprising at least two amino acid residues selected from the extracellular domain amino acid residues E521, L525, and R530 of the human HER2 receptor. In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to the epitope of the human HER2 receptor, comprising at least the amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these antibodies or their antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0369] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that bind to at least a portion of domain III and at least a portion of the N-terminus of domain IV of the human HER2 receptor, but do not cross-compete with Fab37 monoclonal antibodies or antibodies that bind to epitope 4D5 of the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments described herein do not cross-compete with Fab37 monoclonal antibodies and / or trastuzumab for binding to the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0370] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to the epitope of the human HER2 receptor, comprising residues 520-531 of the extracellular domain of the human HER2 receptor, residues 542-553 of SEQ ID NO:16, or residues 520-531 of SEQ ID NO:31.

[0371] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to an epitope of the human HER2 receptor, comprising at least one amino acid residue selected from the extracellular domain residues C453, H456, H473, N476, R495, G496, H497, and W499 of the human HER2 receptor, for example, residues 475, 478, 495, 498, 517, 518, 519, and 521 of SEQ ID NO:16, or residues 453, 456, 473, 476, 495, 496, 497, and 499 of SEQ ID NO:31. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that specifically bind to an epitope in the extracellular domain of the human HER2 receptor, comprising at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, or at least six amino acid residues selected from the amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that specifically bind to an epitope in the extracellular domain of the human HER2 receptor, comprising at least the amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these antibodies or antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0372] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments that specifically bind to an epitope of the human HER2 receptor, comprising at least one amino acid residue selected from the extracellular domain residues C453, H473, N476, R495, H497, and W499 of the human HER2 receptor, for example, residues 475, 495, 498, 517, 519, and 521 of SEQ ID NO:16, or residues 453, 473, 476, 495, 497, and 499 of SEQ ID NO:31. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that specifically bind to an epitope in the extracellular domain of the human HER2 receptor, comprising at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, or at least six amino acid residues selected from the amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments that specifically bind to an epitope in the extracellular domain of the human HER2 receptor, comprising at least the amino acid residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these antibodies or antigen-binding fragments also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0373] In some embodiments, these antibodies have been shown to exhibit specificity for human HER2 and to modulate the PI3K-Akt pathway, which promotes cell survival by reducing the level of phosphorylated AKT, e.g., by blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with it. In some embodiments, these antibodies internalize from the cell surface of HER2-expressing cells at the same or substantially similar rate as trastuzumab or its biosimilars. In some embodiments, these antibodies and antigen-binding fragments have an internalization rate at which approximately 50% of the total surface bound at time 0 is internalized by time 4.

[0374] In some embodiments, the antibodies disclosed herein include a sequence selected from SEQ ID NO:17 and a heavy chain variable region having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the same amino acid sequence, and SEQ ID It includes a sequence selected from NO:24 and a light chain variable region having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical amino acid sequences.

[0375] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:19, and a light chain amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of SEQ ID NO:26.

[0376] In some embodiments, the antibodies disclosed herein include the heavy chain variable region amino acid sequence of SEQ ID NO:17 and the light chain variable region amino acid sequence of SEQ ID NO:24.

[0377] In some embodiments, the antibodies disclosed herein include a heavy chain amino acid sequence of SEQ ID NO:19 and a light chain amino acid sequence of SEQ ID NO:26.

[0378] In some embodiments, the antibodies disclosed herein include the CDRH1 amino acid sequence of SEQ ID NO:20, the CDRH2 amino acid sequence of SEQ ID NO:21, the CDRH3 amino acid sequence of SEQ ID NO:22, the CDRL1 amino acid sequence of SEQ ID NO:27, the CDRL2 amino acid sequence of SEQ ID NO:28, and the CDRL3 amino acid sequence of SEQ ID NO:29.

[0379] In some embodiments, the antibodies disclosed herein include one or more conserved amino acid substitutions in the variable domain sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more conserved substitutions in the variable domain sequence. In some embodiments, these conserved amino acid substitutions are located within the CDR region, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more conserved substitutions are cumulatively present across the entire CDR. In some embodiments, each CDR sequence, for example, SEQ ID NO: 20-22 and 27-29, may contain up to one, up to two, up to three, or up to four conserved amino acid substitutions.

[0380] Those skilled in the art will recognize that, without excessive experimentation, it is possible to determine whether a monoclonal antibody has the same specificity as the monoclonal antibody XMT-1519 by checking whether the former prevents the latter from binding to its natural binding partner or other molecules known to associate with HER2. In some embodiments, when the monoclonal antibody under test competes with the monoclonal antibody disclosed herein, as indicated by the reduced binding by the monoclonal antibody disclosed herein, the two monoclonal antibodies bind to the same epitope or closely related epitopes.

[0381] In some embodiments, an alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody disclosed herein is to pre-incubate the monoclonal antibody disclosed herein with soluble HER2 (which is typically reactive with it), and then add the monoclonal antibody under test to determine whether the ability of the monoclonal antibody under test to bind to HER2 is inhibited. If the monoclonal antibody under test is inhibited, it almost certainly has the same or functionally equivalent epitope specificity as the monoclonal antibody disclosed herein.

[0382] In some embodiments, the monoclonal antibodies disclosed herein can be screened, for example, by measuring HER2-mediated PI3K-Akt pathway activity and determining whether the test monoclonal antibody can modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with PI3K-Akt pathway activity. In some embodiments, HER2 antibodies suitable for conjugation can be produced and purified by well-known techniques, such as International Publication No. 2015 / 195917 and PCT / US2018 / 019873, each of which is incorporated herein by reference in its entirety.

[0383] modified antibody In some embodiments, the antibody is a modified antibody.

[0384] In some embodiments of the modified antibody, * indicates direct or indirect binding to the remainder of the modified antibody. In some embodiments, S'' is a sugar or derivatized sugar. In some embodiments, A'' is a functional group that can form a covalent bond with the functional group of the linker-drug moiety.

[0385] In some embodiments, the modified antibody before conjugation contains a sugar-derivative moiety of *-S''-A''.

[0386] In some embodiments, the modified antibody contains an asparagine group within region 290-305 (e.g., at N297). In some embodiments, the sugar-derivative moiety is directly or indirectly bound to this asparagine group (e.g., at N297).

[0387] In some embodiments, the modified antibody before conjugation contains a modified GlcNAc moiety, *-GlcNAc-S''-A'', where GlcNAc is N-acetylglucosamine.

[0388] In some embodiments, the modified GlcNAc moiety is connected to the remainder of the modified antibody via the C1 position of GlcNAc. In some embodiments, the modified GlcNAc moiety further comprises fucose.

[0389] In some embodiments, the modified GlcNAc moiety is directly or indirectly bonded to the asparagine group (for example, to N297).

[0390] In some embodiments, the modified antibody is conjugated to the linker-drug moiety via a covalent bond formed between A'' and the functional group of the linker-drug moiety.

[0391] In some embodiments, the modified antibody of this disclosure is (a) A step of contacting a glycoprotein (e.g., antibody glycan) containing an antibody and a core-GlcNAc moiety with an endoglycosidase to form an intermediate antibody containing an antibody and a terminal GlcNAc moiety, wherein optionally the terminal GlcNAc moiety further contains fucose, and (b) Obtained by a process comprising contacting an intermediate antibody with a compound having the structure P''-S''-A'' in the presence of a glycosyltransferase to form a modified antibody comprising the antibody and a modified GlcNAc moiety, *-GlcNAc-S''-A'', wherein optionally the modified GlcNAc moiety is bound to the remainder of the modified antibody via the C1 position of GlcNAc, GlcNAc is N-acetylglucosamine, S'' is a sugar or a derivatized sugar, A'' is azid, keto, or alkinyl, P'' is uridine diphosphate (UDP), guanosine diphosphate (GDP), or cytidine diphosphate (CDP).

[0392] In some embodiments, steps (a) and (b) are performed sequentially. In some embodiments, steps (a) and (b) are performed simultaneously.

[0393] In some embodiments, the antibody glycan comprises a mixture of glycoforms G0, G1, G2, G0F, GIF, G2F, and M5 (for example, the glycoforms shown in Figure 1).

[0394] In some embodiments, the antibody is a monoclonal antibody (mAb).

[0395] In some embodiments, the antibody is an IgA antibody, an IgD antibody, an IgE antibody, an IgG antibody, or an IgM antibody.

[0396] In some embodiments, the antibody is an IgG antibody, such as an IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody.

[0397] In some embodiments, the antibody is a full-length antibody, and the antibody glycan comprises one or more core-GlcNAc moieties.

[0398] In some embodiments, the antibody is a full-length antibody, and the antibody glycan comprises one or more core-GlcNAc moieties attached to each heavy chain of the antibody.

[0399] In some embodiments, the core-GlcNAc portion further includes fucose.

[0400] In some embodiments, the antibody is a full-length antibody, and the antibody glycan comprises two or more core-GlcNAc moieties attached to the full-length antibody.

[0401] In some embodiments, the antibody is a full-length antibody, and the antibody glycan contains two core-GlcNAc moieties attached to the full-length antibody.

[0402] In some embodiments, at least one of two or more core-GlcNAc portions further includes fucose.

[0403] In some embodiments, each of two or more core-GlcNAc portions further includes fucose.

[0404] In some embodiments, the antibody is a single-chain antibody or antibody fragment (e.g., a Fab fragment or an Fc fragment), and the antibody glycan comprises one or more core-GlcNAc moieties (optionally further comprising fucose) attached to the antibody.

[0405] In some embodiments, the core-GlcNAc portion is attached to a site on the antibody, and the core-GlcNAc portion does not substantially interfere with the antigen-binding site of the antibody.

[0406] In some embodiments, the core-GlcNAc moiety is attached to the Fc fragment of the antibody. In some embodiments, the core-GlcNAc moiety is attached to the CH domain. In some embodiments, the core-GlcNAc moiety is attached to the Fab or Fc fragment of the antibody. In some embodiments, the core-GlcNAc moiety is attached to the antibody via an N-glycosidic bond to the amide nitrogen atom of the side chain of the asparagine amino acid of the antibody. In some embodiments, the core-GlcNAc moiety is attached to the native N-glycosylation site of the antibody.

[0407] In some embodiments, the antibody is an IgG antibody, and the core-GlcNAc moiety is connected to the natural N-glycosylation site of IgG.

[0408] In some embodiments, the antibody is an IgG antibody, and the core-GlcNAc moiety is connected to a native N-glycosylation site of IgG (e.g., the N297 N-glycosylation site of IgG). In some embodiments, the N297 N-glycosylation site is located in a conserved Fc region of the heavy chain of the IgG antibody in asparagine within region 290-305 (e.g., at N297).

[0409] In some embodiments, the intermediate antibody is of formula (XXII): It is TIFF0007846624000151.tif21128, During the ceremony, Ab is an antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, u3 is 0 or 1, and u4 is an integer in the range of 1 to 16.

[0410] In some embodiments, u4 is an integer in the range of 1 to 10. In some embodiments, u4 is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, u4 is 1, 2, 3, 4, 5, or 6. In some embodiments, u4 is 1, 2, 3, or 4. In some embodiments, u4 is 2 or 4. In some embodiments, u4 is 1 or 2. In some embodiments, u4 is 1. In some embodiments, u4 is 2.

[0411] In some embodiments, the antibody contains one core-GlcNAc moiety (e.g., u4 is 1). In some embodiments, the antibody contains two core-GlcNAc moieties (e.g., u4 is 2).

[0412] In some embodiments, modified antibodies are obtained by the process outlined in Scheme 1. As shown below, contact of an intermediate antibody of formula (XXIII) containing one terminal GlcNAc moiety with a compound having the structure P''-S''-A'' in the presence of glycosyltransferase yields a modified antibody containing one modified GlcNAc moiety (e.g., a modified antibody of formula (XXIIIa)).

[0413] In some embodiments, the modified antibody is obtained by contacting an intermediate antibody of formula (XXIV) containing two terminal GlcNAc moieties with a compound having a P''-S''-A'' structure in the presence of a glycosyltransferase, providing a modified antibody containing two modified GlcNAc moieties (e.g., a modified antibody of formula (XXIVa)).

[0414] Scheme 1 In formula TIFF0007846624000152.tif49153, u3, Ab, S'', A'', and P'' are as defined herein.

[0415] In some embodiments, the antibody glycan modified by the process according to the present disclosure comprises a glycan, the glycan comprising a core-GlcNAc moiety, i.e., a GlcNAc moiety located at the non-reducing end of the glycan. In some embodiments, the glycan comprises one or more sugar moieties and may be linear or branched.

[0416] In some embodiments, reaction with endoglycosidase may form an intermediate antibody containing a terminal GlcNAc moiety (e.g., an intermediate antibody of formula (XXIII) or (XXIV)).

[0417] In some embodiments, step (a) of the process (deglycosylation or trimming) is as shown in Figure 2, where a mixture of antibody glycoforms G2F, GIF, G0F, G2, G1, G0, and M5 (see, for example, Figure 1) and optionally additional glycoforms (e.g., triple-stranded glycans) is converted into an intermediate antibody containing a terminal GlcNAc moiety which may contain fucose (e.g., u3 is 0 or 1).

[0418] In some embodiments, the endoglycosidase is endoglycosidase Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, or a combination thereof.

[0419] In some embodiments, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, or a combination thereof.

[0420] In some embodiments, the endoglycosidase is Endo S, Endo SH, or a combination thereof. In some embodiments, the endoglycosidase is Endo SH.

[0421] In some embodiments, step (b) of the process (formation of modified antibody) is as shown in Figure 3, where the intermediate antibody comprises a monoclonal antibody (mAb) and terminal GlcNAc moieties on each heavy chain of the monoclonal antibody (mAb) (which may contain fucose (e.g., u3 is 0 or 1)). In some embodiments, in step (b), the terminal GlcNAc moieties are converted to modified GlcNAc moieties. In some embodiments, this conversion may be carried out by the reaction of the terminal GlcNAc moieties with a P''-S''-A'' compound in the presence of glycosyltransferase.

[0422] In some embodiments, the P''-S''-A'' compound is GalNAz-UDP (e.g., 4-AzGalNAc-UDP). In some embodiments, the terminal GlcNAc portion is *-GlcNAc-GalNAz or *-GlcNAc(Fuc)-GalNAz, where * represents binding to the remainder of the modified antibody.

[0423] In some embodiments, the deglycosylation / trimming step and the modification antibody formation step are performed sequentially.

[0424] In some embodiments, the deglycosylation / trimming step and the step of forming a modified antibody are performed simultaneously.

[0425] In some embodiments, the process for preparing modified antibodies is carried out in a suitable buffer, such as buffered saline (e.g., phosphate-buffered saline, Tris-buffered saline), citrate, HEPES, Tris, and glycine. In some embodiments, the buffer is phosphate-buffered saline (PBS) or Tris-buffered saline. In some embodiments, the buffer is phosphate-buffered saline (PBS).

[0426] In some embodiments, the process is carried out at a temperature in the range of approximately 4 to approximately 50°C. In some embodiments, the process is carried out at a temperature in the range of approximately 10 to approximately 45°C. In some embodiments, the process is carried out at a temperature in the range of approximately 20 to approximately 40°C. In some embodiments, the process is carried out at a temperature in the range of approximately 30 to approximately 37°C. In some embodiments, the process is carried out at a temperature of approximately 30°C. In some embodiments, the process is carried out at a temperature of 30°C.

[0427] In some embodiments, the process is carried out at a pH value in the range of about 5 to about 9 (e.g., about 5.5 to about 8.5, about 6 to about 8, or about 7 to about 8). In some embodiments, the process is carried out at a pH value of about 7.4.

[0428] In some embodiments, the process for preparing modified antibodies is as shown in Figure 4.

[0429] In some embodiments, the process for preparing modified antibodies is: A step of contacting a glycoprotein (e.g., antibody glycan) containing an antibody and a core-GlcNAc moiety connected to site N297 of the antibody with endoglycosidase Endo SH, thereby forming an intermediate antibody containing the terminal GlcNAc moiety, and The process includes the step of contacting an intermediate antibody with 4-AzGalNAc-UDP in the presence of the β-(1,4)-GalNAcT enzyme, thereby forming a modified antibody containing a modified GlcNAc moiety. Processes (a) and (b) are performed simultaneously.

[0430] In some embodiments, the endoglycosidase is Endo SH, which is a fusion of two endoglycosidases, Endo S and Endo H, linked by a glycy-rich spacer containing an internal 6xHis tag, resulting in a total molecular weight of 139 kDa.

[0431] In some embodiments, the β-(1,4)-GalNAcT enzyme contains an N-terminal 6xHis tag and has a total molecular weight of 45.7 kDa. In some embodiments, the β-(1,4)-GalNAcT enzyme containing an N-terminal 6xHis tag is derived from the nettle moth (Trichoplusia ni).

[0432] In some embodiments, the process is carried out in PBS buffer at a pH of approximately 7.4 and a temperature of approximately 30°C.

[0433] Endoglycosidase Endoglycosidases are enzymes that can cleave internal glycosidic bonds within a glycan structure, thereby reconstructing or trimming the glycan structure. For example, endoglycosidases can be used for the easy homogenization of heterogeneous glycan populations when cleaved at predictable sites within conserved glycan regions. One class of endoglycosidases includes endo-β-N-acetylglucosaminidase (EC 3.2.1.96, commonly known as Endo S or ENGase), a class of hydrolytic enzymes that remove N-glycans from glycoproteins by hydrolyzing the β-1,4-glycosidic bonds of the N,N'-diacetylchitobiose core, leaving a single-core N-linked GlcNAc residue, as described in Wong et al. Chem. Rev. 2011, 111, 4259 (whose entirety is incorporated herein by reference). Endo-β-N-acetylglucosaminidases are widely found in nature, along with common chemioenzyme variants including Endo D, which is specific to pausimannose; Endo A and Endo H, which are specific to high-mannose; the Endo F subtype, which extends from high-mannose to double-stranded complexes; and Endo M, which can cleave most N-glycan structures (high-mannose / complex / hybrid types) except for fucosylated glycans. The hydrolytic activity of high-mannose oligosaccharides is significantly higher than that of complex and hybrid oligosaccharides. In some embodiments, these ENGases exhibit specificity for distal N-glycan structures but not for proteins presenting distal N-glycan structures, and are useful for cleaving most N-linked glycans from glycoproteins under natural conditions.

[0434] In some embodiments, endoglycosidases F1, F2, and F3 are suitable for deglycosylating native proteins. The binding specificity of Endo F1, F2, and F3 suggests a general strategy for protein deglycosylation that can remove any class of N-linked oligosaccharides without denaturing the protein. In some embodiments, double-stranded and triple-stranded structures can be immediately removed by endoglycosidases F2 and F3, respectively. In some embodiments, oligomannoses and hybrid structures can be removed by Endo F1.

[0435] Endo S is an endoglycosidase secreted by Streptococcus pyogenes and belongs to the glycoside hydrolase family 18, as disclosed by Collin et al. (EMBO J., 2001, 20, 3046), whose entire work is incorporated herein by reference. In contrast to the above ENGase, Endo S has even clearer specificity, being specific to cleave only the conserved N-glycan within the Fc domain of human IgG (other substrates have not been identified to date), suggesting that the protein-protein interaction between the enzyme and IgG gives rise to this specificity.

[0436] Endo S49, also known as Endo S2, is a homolog of Endo S, isolated from Streptococcus pyogenes NZ131, and is described in its entirety in International Publication No. 2013 / 037824, which is incorporated herein by reference. Endo S49 has specific endoglycosidase activity for native IgG and cleaves a wider variety of Fc glycans than Endo S.

[0437] Endo SH is a fusion of two endoglycosidases, Endo S and Endo H, linked by a glycy-rich spacer. Endo SH specifically cleaves the N-linked glycan between two N-acetylglucosamine (GluNAc) moieties within the core region of the glycan chain.

[0438] In some embodiments, the endoglycosidase for deglycosylating antibodies is Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, Endo H, Endo M, Endo A, or a combination thereof. In some embodiments, the endoglycosidase for deglycosylating antibodies is Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, Endo H, or a combination thereof. In some embodiments, the endoglycosidase is Endo S, Endo SH, Endo S2, or Endo S49.

[0439] In some embodiments, when the glycan being trimmed is a complex bifurcated structure, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, or a combination thereof.

[0440] In some embodiments, when the glycoprotein is an antibody and the trimmed oligosaccharide is a complex bifurcated structure present at the IgG-conserved N-glycosylation site at N297, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, Endo F1, Endo F2, Endo F3, or a combination thereof. In some embodiments, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, or a combination thereof.

[0441] In some embodiments, if the glycoprotein is an antibody and the glycan being trimmed is a complex, bifurcated structure and is not present at the IgG-conserved N-glycosylation site at N297, the endoglycosidase is Endo F1, Endo F2, Endo F3, or a combination thereof.

[0442] In some embodiments, when the glycan being trimmed is high in mannose, the endoglycosidase is Endo H, Endo M, Endo A, Endo F1, or a combination thereof.

[0443] In some embodiments, when the glycoprotein is an antibody, and the trimmed oligosaccharide is high in mannose, in addition to having a complex bifurcated structure, and is present at the IgG-conserved N-glycosylation site at N297, the endoglycosidase is Endo S, Endo SH, Endo S2, Endo S49, or a combination thereof. In some embodiments, the endoglycosidase is Endo S or Endo SH. In some embodiments, the endoglycosidase is Endo SH.

[0444] In some embodiments, the endoglycosidase enzymes as defined herein include a sequence encoding a tag for facilitating purification. In some embodiments, the tag includes, but is not limited to, a FLAG tag, a poly(His)-tag, an HA-tag, a Myc-tag, a SUMO-tag, a GST-tag, an MBP-tag, or a CBP-tag. In some embodiments, the tag is a 6xHis tag. In some embodiments, the tag is covalently bound to the endoglycoside enzyme at the C-terminus or as an internal residue. In some embodiments, the tag is covalently bound to the endoglycoside enzyme at the N-terminus.

[0445] In some embodiments, Endo SH is a fusion of two endoglycosidases, Endo S and Endo H, linked by a glycy-rich spacer containing an internal 6xHis tag, resulting in a total molecular weight of 139 kDa.

[0446] Glycosyltransferase The process for forming a modified antibody includes treating a deglycosylated / trimmed antibody having a fucosylated terminal N-acetylglucosamine (Gal-NAc) moiety with a compound of formula S''(A'')-P'' in the presence of a glycosyltransferase to form a modified antibody having a GlcNAc-S''(A'') substituent bound to the antibody at C1 of the GalNAc moiety via a β-1,4-O-glycosidic bond.

[0447] In some embodiments, glycosyltransferases are β-1,4-galactosyltransferase (4Gal-T), β-(1,4)-acetylgalactosaminyltransferase (β-(1,4)-GalNAcT or GalNAcT), or variants thereof.

[0448] β-(1,4)-acetylgalactosaminyltransferase (β-(1,4)-GalNAcT or β-(1,4)-GalNAcT) has been identified in numerous organisms, including humans, the nematode Caenorhabditis elegans (Kawar et al, J. Biol. Chem. 2002, 277, 34924, whole text incorporated herein by reference), Drosophila melanogaster (Hoskins et al., Science 2007, 316, 1625, whole text incorporated herein by reference), and the nettle moth (Vadaie et al, J. Biol. Chem. 2004, 279, 33501, whole text incorporated herein by reference).

[0449] β-(1,4)-N-acetylgalactosaminyltransferase (β-(1,4)-GalNAcT) is known in the art. In some embodiments, β-(1,4)-GalNAcT is an enzyme that catalyzes the transfer of N-acetylgalactosamine (GalNAc) from uridine diphosphate-GalNAc (UDP-GalNAc, also called GalNAc-UDP) to the terminal GlcNAc portion of a glycoprotein glycan, where C1 of the GalNAc portion is bound to an antibody via a β-1,4-O-glycosidic bond. In some embodiments, the terminal GlcNAc portion is fucosylated.

[0450] In some embodiments, the β-(1,4)-GalNAcT enzyme used in the process of the present invention is an invertebrate β-(1,4)-GalNAcT enzyme or derived therefrom, for example, a β-(1,4)-GalNAcT from an invertebrate species or derived therefrom. The β-(1,4)-GalNAcT enzyme may be or be derived from any invertebrate β-(1,4)-GalNAcT enzyme known to those skilled in the art. In some embodiments, the β-(1,4)-GalNAcT enzyme may be a β-(1,4)-GalNAcT enzyme from the phylum Nematoda, for example, a class such as Chromadorea or Seernentea, or from the phylum Arthropoda, for example, a class such as Insecta, or derived therefrom. In some aspects, the β-(1,4)-GalNAcT enzyme is found in *Elegans* nematodes, *Caenorhabditis remanei*, *Caenorhabditis briggsae*, *Ascaris suum*, *Urtica dioica*, *Drosophila melanogaster*, *Wuchereria bancrofti*, *Loa loa*, *Cerapachys biroi*, *Zootermopsis nevadensis*, *Camponotus floridanus*, *Crassostrea gigas*, or *Danaus* The β-(1,4)-GalNAcT enzyme is derived from or is derived from *Plexippus*, (e.g., *Plexippus elegans*, *Ranunculus porcini*, *Galium saccharina*, or *Drosophila melanogaster*. In some embodiments, the β-(1,4)-GalNAcT enzyme is derived from or is derived from *Plexippus*, *Ranunculus porcini*, or *Galium saccharina*. In other embodiments, the β-(1,4)-GalNAcT enzyme is derived from or is derived from *Galium saccharina*.

[0451] The term “derived from” includes, for example, cleavage enzymes, mutant enzymes, enzymes with tags to facilitate purification, or combinations thereof. Therefore, “derived from” means having an amino acid sequence modified by substituting, inserting, deleting, or adding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more) amino acids from the naturally occurring β-(1,4)-GalNAcT enzyme. A β-(1,4)-GalNAcT enzyme derived from a β-(1,4)-GalNAcT enzyme is also referred to herein as an inducible β-(1,4)-GalNAcT enzyme, a modified β-(1,4)-GalNAcT enzyme, or a β-(1,4)-GalNAcT mutant enzyme.

[0452] In some embodiments, the inducible β-(1,4)-GalNAcT enzyme is modified by adding or deleting additional N-terminal or C-terminal amino acids or chemical moieties to increase stability, solubility, activity, and / or ease of purification.

[0453] In some embodiments, the β-(1,4)-GalNAcT enzyme is modified by deleting the N-terminal cytoplasmic domain and transmembrane domain, and is called a cleavage enzyme.

[0454] A β-(1,4)-GalNAcT enzyme in which one or more amino acids are substituted, added, or deleted is also referred to herein as a mutant β-(1,4)-GalNAcT enzyme or an inducible β-(1,4)-GalNAcT enzyme. In some embodiments, a β-(1,4)-GalNAcT enzyme is modified by deleting the N-terminal cytoplasmic domain and the transmembrane domain, and is mutated by substituting one or more amino acids. Substitution of one or more amino acids is also referred to herein as a mutation. An enzyme containing one or more substituted amino acids is also referred to as a mutant enzyme.

[0455] In some embodiments, if the glycosyltransferase is a β-(1,4)-GalNAcT enzyme or a cleavage-type β-(1,4)-GalNAcT enzyme, the enzyme further comprises one or more mutations. In some embodiments, these mutations include, but are not limited to, substitutions of isoleucine (I, also known as He) at position 257 by leucine (Leu, also known as L), methionine (Met, also known as M), or alanine (Ala, also known as A). In some embodiments, substitutions of methionine (Met, also known as M) at position 312 by histidine (His, also known as H). The amino acid position numbering herein is based on the amino acid position numbering in the wild-type β-(1,4)-GalNAcT enzyme. If the β-(1,4)-GalNAcT enzyme is, for example, a cleavage-type enzyme, the numbers used herein to indicate the amino acid substitution positions correspond to the numbering of amino acid positions in the corresponding wild-type β-(1,4)-GalNAcT enzyme.

[0456] In some embodiments, glycosyltransferase is a β(1,4)-GalT enzyme containing a mutant catalytic domain.

[0457] The catalytic domain may have an amino acid sequence similar to that found in the wild-type enzyme, or it may have an amino acid sequence different from the wild-type sequence. A catalytic domain having an amino acid sequence different from the wild-type sequence is referred to herein as a mutant catalytic domain. In some embodiments, the mutation may include a single amino acid change (e.g., a point mutation), multiple amino acid changes (e.g., 1 to 10, or 1 to 6, or 1, 2, 3, or 4, or 1 or 2 amino acids), or the deletion or insertion of one or more amino acids (e.g., 1 to 10, or 1 to 6, or 1, 2, 3, or 4, or 1 or 2 amino acids). In some embodiments, the mutant catalytic domain may be present in a full-length enzyme, such as β(1,4)-galactosyltransferase or α(1,3)-N-galactosyltransferase, but may also be present in polypeptide fragments or recombinant polypeptides containing the mutant catalytic domain optionally bound to additional amino acids.

[0458] β(1,4)-galactosyltransferase I is referred to herein as GalT. Such mutant GalT catalytic domains are disclosed, for example, in International Publication No. 2004 / 063344, which is incorporated herein in its entirety by reference. International Publication No. 2004 / 063344 also discloses Tyr-289 variants of GalT and methods for their preparation. These variants are referred to as Y289L, Y289N, or Y289I.

[0459] In some embodiments, the GalT mutation-catalyzing domain is Y289L, Y289N, Y289I, Y284L, or R228K. In some embodiments, the GalT mutation-catalyzing domain is Y289L.

[0460] In some embodiments, GalT Y289F, GalT Y289M, GalT Y289V, GalT Y289G, GalT Y289I, GalT Y289A, GalT Y289N, and GalT Y289L variants may be generated via site-directed mutagenesis processes described, for example, in International Publication No. 2004063344, Qasba et al, Prot.Expr.Pur.2003,30,219, and Qasba et al, J.Biol.Chem.2002,277,20833 (all of which are incorporated herein by reference in their entirety). In GalT Y289F, the tyrosine amino acid (Y) at position 289 is substituted with the amino acid phenylalanine (F); in GalT Y289M, the tyrosine is substituted with the amino acid methionine (M); in GalT Y289V, it is substituted with the amino acid valine (V); in GalT Y289G, it is substituted with the amino acid glycine (G); in GalT Y289I, it is substituted with the amino acid isoleucine (I); and in Y289A, it is substituted with the amino acid analine (A).

[0461] In some embodiments, the β-(1,4)-GalNAcT enzyme includes a sequence encoding a tag for facilitating purification. In some embodiments, the tag includes, but is not limited to, a FLAG tag, a poly(His)-tag, an HA-tag, a Myc-tag, a SUMO-tag, a GST-tag, an MBP-tag, or a CBP-tag. In other embodiments, the tag is a 6xHis tag. In some embodiments, the tag is covalently bound to the β-(1,4)-GalNAcT enzyme at its C-terminus. In some embodiments, the tag is covalently bound to the β-(1,4)-GalNAcT enzyme at its N-terminus.

[0462] In some embodiments, the β-(1,4)-GalNAcT enzyme contains an N-terminal 6xHis tag and has a total molecular weight of 45.7 kDa. In some embodiments, the β-(1,4)-GalNAcT enzyme containing an N-terminal 6xHis tag is derived from nettle moth.

[0463] P''-S''-A'' molecule In some embodiments, the P''-S''-A'' molecule for use in the process of preparing the modified antibody of this disclosure may be any sugar derivative nucleotide that is a substrate for a suitable galactosyltransferase catalyst.

[0464] In some embodiments, S''-A'' is a sugar derivative moiety, in the formula, S'' is a sugar or a derivatized sugar, and A'' is a functional group that can form a covalent bond with the functional group of the linker-drug moiety.

[0465] In some embodiments, A'' is an azid moiety, a keto moiety, or an alkynyl moiety. In some embodiments, A'' is an azid moiety or a keto moiety. In some embodiments, A'' is an azid moiety. In some embodiments, A'' is -N 3 In some aspects, A'' is the keto part.

[0466] In some embodiments, A'' is -[C(R 8k )2] x2 C(O)R 9k And in the formula, R 9k C is methyl or may be substituted. 2~24 It is alkyl, Each R 8k These are independently hydrogen, halogen, or R 9k And, x² is an integer in the range of 0 to 24.

[0467] In some embodiments, x2 is an integer in the range of 0 to 10. In some embodiments, x2 is 0, 1, 2, 3, 4, 5, or 6.

[0468] In some embodiments, each R 8k It is hydrogen.

[0469] In some embodiments, A'' is an alkynyl moiety. In some embodiments, A'' is a terminal alkynyl moiety, a cycloalkynyl moiety, or a heterocycloalkynyl moiety. In some embodiments, A'' is a terminal alkynyl moiety. In some embodiments, A'' is a cycloalkynyl moiety. In some embodiments, A'' is a heterocycloalkynyl moiety.

[0470] In some aspects, A'' is -[C(R 8k )2] x2 -C≡CR 8k It is a base, and in the formula, R 8k and x2 are as defined herein. In some embodiments, A'' is -[CH2] x2 -C ≡ CH

[0471] In some embodiments, S''-A'' is derived from a sugar or a derivatized sugar, such as an amino sugar or other derivatized sugar. In some embodiments, examples of sugars and derivatized sugars include, but are not limited to, galactose (Gal), mannose (Man), glucose (Glc), glucuronic acid (Gcu), and fucose (Fuc). An amino sugar is understood to be a sugar in which a hydroxyl (OH) group is substituted with an amine group. Examples of amino sugars include, but are not limited to, N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Examples of sugars derivatized by other means include, but are not limited to, glucuronic acid (Gcu) and N-acetylneuraminic acid (sialic acid).

[0472] In some embodiments, S''-A'' is derived from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc), or N-acetylneuraminic acid (sialic acid). In some embodiments, S''-A'' is derived from GlcNAc, Glc, Gal, or GalNAc. In some embodiments, S''-A'' is derived from GlcNAc. In some embodiments, S''-A'' is derived from Glc. In some embodiments, S''-A'' is derived from Gal or GalNAc. In some embodiments, S''-A'' is derived from Gal. In some embodiments, S''-A'' is derived from GalNAc.

[0473] In some embodiments, the functional group A'' can be bonded to S'' in various ways.

[0474] In some embodiments, A'' is directly bonded to the C2, C3, C4, or C6 carbon atoms of the sugar or derivatized sugar S'' (for example, instead of the hydroxyl at the corresponding position).

[0475] In some embodiments, S'' is fucose or derivatized fucose lacking any hydroxyl C6 position. In some embodiments, if A'' is bonded to the C6 position of fucose or derivatized fucose, A'' is directly bonded to the carbon atom at the C6 position.

[0476] In some embodiments, A'' is an azide moiety, and A'' is bonded to the C2, C4, or C6 position of the sugar or derivatized sugar of S''.

[0477] In some embodiments, A'' is an azide moiety, and A'' is directly bonded to the C2, C3, C4, or C6 carbon atom of the sugar or derivatized sugar S'' (e.g., instead of the hydroxyl at the corresponding position). In some embodiments, S''-A'' is 6-azidofucose (6-AzFuc). In some embodiments, A'' is an azide moiety, and A'' is bonded to the N-acetyl moiety of the amino sugar or derivatized amino sugar (e.g., by substituting the acetyl moiety with the azidoacetyl moiety). In some embodiments, S''-A'' is 2-azidoacetamidogalactose (GalNAz), 6-azido-6-deoxygalactose (6-AzGal), 6-azido-6-deoxy-2-acetamidogalactose (6-AzGalNAc), 4-azido-4-deoxy-2-acetamidogalactose (4-AzGalNAc), 6-azido-6-deoxy-2-azidoacetamidogalactose (6-A S''-A'' is GalNAz, 4-AzGalNAc, GlcNAz, 6-AzGlc, 6-AzGlcNAc, 4-AzGlcNAc, or 6-AzGlcNAz. In some embodiments, S''-A'' is GalNAz, 4-AzGalNAc, GlcNAz, or 6-AzGlcNAz.

[0478] In some embodiments, P''-S''-A'' is a compound or salt of formula (XXIVb), (XXXIVc), or (XXIVd).

[0479] In some embodiments, A'' keto and A'' are directly bonded to the carbon atom at the C2 position of the sugar or derivatized sugar S'' (for example, instead of the hydroxyl at the corresponding position).

[0480] In some embodiments, A'' is bonded to the nitrogen atom of an amino sugar or derivatized amino sugar, such as a C2-derivative amino sugar. In some embodiments, the derivatized amino sugar is -NC(O)-R 9k The formula includes the part, R 9k C is methyl or may be substituted. 2~24 It is an alkyl group (for example, ethyl).

[0481] In some embodiments, R 9k It is ethyl.

[0482] In some embodiments, S''-A'' is 2-deoxy-(2-oxopropyl)-galactose (2-keto-Gal), 2-N-propionyl-galactosamine (2-N-propionylGal-NAc), 2-N-(4-oxopentanoyl)-galactosamine (2-N-Lev-Gal), or 2-N-butyryl-galactosamine (2-N-butyryl-GalNAc). In some embodiments, S''-A'' is 2-ketoGalNAc or 2-N-propionyl-GalNAc.

[0483] In some embodiments, P''-S''-A'' is a compound of formula (XXIVe) or (XXIVf) or a salt thereof.

[0484] In some embodiments, A'' is a terminal alkynyl, cycloalkynyl, or heterocycloalkynyl. In some embodiments, A'' is bonded to the C2-derivative amino sugar of S''.

[0485] In some embodiments, S''-A'' is 2-(buta-3-iodine amide)-2-deoxy-galactose.

[0486] In some embodiments, P''-S''-A'' is a compound of formula (XXIVg) or a salt thereof. In some embodiments, P''-S''-A'' is a compound of formula (XXIVd) or a salt thereof.

[0487] In some embodiments, the P''-S''-A'' compounds can be synthesized according to various methods known in the art. In some embodiments, the compounds are synthesized by attaching a nucleoside monophosphate P'' or nucleoside diphosphate P'' to a sugar derivative S''-A'', as disclosed, for example, in Wang et al. (Chem.Eur.J.16:13343-13345 (2010)), Piller et al. (ACS Chem.Biol.7:753 (2012)), Piller et al. (Bioorg.Med.Chem.Lett.15:5459-5462 (2005)), and PCT Publication International Publication No. 2009 / 102820, each of which is incorporated herein by reference in its entirety.

[0488] In some embodiments, P'' is a nucleoside monophosphate or a nucleoside diphosphate. In some embodiments, P'' is uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP), or cytidine monophosphate (CMP). In some embodiments, P'' is uridine diphosphate (UDP).

[0489] In some embodiments, P''-S''-A'' is a compound of formula (XXIVb), (XXIVc), (XXIVd), (XXIVe), (XXIVf), or (XXIVg): TIFF0007846624000153.tif89128 or a salt thereof, where R 9k is C 2~24 It is an alkyl group.

[0490] In some embodiments, P''-S''-A'' is GalNAz-UDP (e.g., formula (XXIVb)), 6-AzGal-UDP (e.g., formula (XXIVc)), 6-AzGalNAc-UDP (e.g., formula (XXIVd)), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP, 2-ketoGal-UDP (e.g., formula (XXIVe)), 2-N-propionylGalNAc-UDP (e.g., formula (XXIVf), where R 9k (is ethyl), or 2-(buta-3-iodamide)-2-deoxy-galactose-UDP (e.g., formula (XXIVg)).

[0491] In some embodiments, P''-S''-A'' is GalNAz-UDP or 4-AzGalNAc-UDP. In some embodiments, P''-S''-A'' is a compound of formula (XXIVb) or (XXIVd). The synthesis of GalNAz-UDP (e.g., formula (XXIVb)) and 6-AzGalNAc-UDP (e.g., formula (XXIVd)) is disclosed in Piller et al. (Bioorg. Med. Chem. Lett. 15:5459-5462 (2005)) and Wang et al. (Chem. Eur. J. 16:13343-13345 (2010)), each of which is incorporated herein by reference in its entirety.

[0492] In some embodiments, P''-S''-A'' is 4-AzGalNAc-UDP. In some embodiments, P''-S''-A'' is the compound of formula (XXIVd) or a salt thereof. The synthesis of 2-ketoGal-UDP(XXIVe) is disclosed in Qasba et al. (J.Am.Chem.Soc.125:16162(2003)) and its supplementary information, which are incorporated herein by reference in their entirety.

[0493] The synthesis of 2-(buta-3-ino acid amide)-2-deoxy-galactose-UDP is disclosed in PCT Publication International 2009 / 102820, which is incorporated herein by reference in its entirety.

[0494] Antibody-drug conjugates In some embodiments, the antibody-drug conjugate of the disclosed herein comprises a modified antibody, a modified GlcNAc moiety within the modified antibody, and a functional group (e.g., W) capable of forming a covalent bond with the functional group A'' of *-GlcNAc-S''-A''. P It can be obtained by reacting a linker containing the drug portion with the drug portion.

[0495] In some embodiments, W P This includes alkynyls, such as cycloalkynyls, heterocycloalkynyls, or terminal alkynyls.

[0496] In some embodiments, the functional group A'' of the modified antibody is azide, keto, or alkynyl. In some embodiments, the functional group A'' of the modified antibody is azide. In some embodiments, the azide functional group A'' of the modified antibody is W of the linker-drug moiety. P It reacts with alkynyl groups (e.g., cycloalkynyl, heterocycloalkynyl, or terminal alkynyl) to form a triazole moiety (e.g., via cycloaddition). Cycloaddition reactions between azide groups and alkynyl groups are well known in the art as "click chemistry".

[0497] In some embodiments, the linker-drug portion W P The molecule contains a terminal alkynyl, and the cycloaddition reaction can be carried out in the presence of a catalyst (e.g., a Cu(I) catalyst).

[0498] In some embodiments, the linker-drug portion W P This includes cycloalkynyl or heterocycloalkynyl (e.g., strained cycloalkynyl or strained heterocycloalkynyl).

[0499] In some embodiments, the linker-drug portion W PThe cycloalkynyl or strained heterocycloalkynyl is included, and the cycloaddition reaction may occur in or without the catalyst. In some embodiments, the cycloaddition reaction may occur spontaneously by a reaction known in the art as strain-enhanced azide-alkyne cycloaddition (SPAAC), also known as "metal-free click chemistry." In some embodiments, the strained cycloalkynyl or strained heterocycloalkynyl is as described herein.

[0500] In some embodiments, during conjugation, the functional group A'' of the modified antibody and the W of the linker-drug moiety are combined. P This forms the triazole portion.

[0501] In some embodiments, during conjugation, the functional group A'' of the modified antibody and the W of the linker-drug moiety are combined. P This is expressed by equation (XXXV): Forms the triazole portion of TIFF0007846624000154.tif25128, In the formula, * represents direct or indirect binding to the remainder of the modified antibody, and ** represents M p This indicates a connection to [the specified location].

[0502] In some embodiments, when an azide-modified antibody of the present disclosure is reacted with a linker-drug moiety containing an alkynyl group to form an antibody-drug conjugate via a cycloaddition reaction, the triazole moiety formed within the antibody-drug conjugate may be resistant to hydrolysis and / or other degradation pathways.

[0503] In some embodiments, when an aldehyde-modified antibody or ketone-modified antibody of the present disclosure is reacted with a linker-drug moiety containing hydroxylamine or hydrazine, the resulting oxime or hydrazone moiety in the antibody-drug conjugate may be relatively inactive under neutral conditions.

[0504] In some embodiments, the antibody-drug conjugates of this disclosure may have high stability.

[0505] In some embodiments, the modified antibodies and antibody-drug conjugates of the present disclosure can be synthesized by practical synthetic routes, since the process for introducing the functional group A'' (e.g., azide, keto, or alkynyl) into the antibody is straightforward and generally applicable.

[0506] In some embodiments, the site-specific antibody-drug conjugate of the present disclosure is obtained by a process comprising the step of reacting a modified antibody with a linker-drug moiety. The linker-drug portion contains cycloalkynyl or heterocycloalkynyl. The modified antibody, prior to conjugation, consists of the antibody and a modified GlcNAc moiety of *-GlcNAc-S''-A'' bound to the antibody via the C1 position of GlcNAc, where GlcNAc is N-acetylglucosamine, S'' is a sugar or derivatized sugar, and A'' is an azide.

[0507] In some embodiments, A'' is a cycloalkynyl or heterocycloalkynyl. In some embodiments, A'' is a cycloalkynyl. In some embodiments, A'' is a heterocycloalkynyl.

[0508] In some embodiments, A'' is a strained cycloalkynyl or a strained heterocycloalkynyl. In some embodiments, A'' is a strained cycloalkynyl. In some embodiments, A'' is a strained heterocycloalkynyl.

[0509] In some embodiments, the site-specific antibody-drug conjugate of the present disclosure is (a) In the presence of galactosyltransferase, an intermediate antibody of formula (XXII) is used: TIFF0007846624000155.tif22128In formula, Ab is an antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, u3 is 0 or 1, and d 13However, intermediate antibodies are integers in the range of 1 to 12. of, The compound P''-S''-A'', in which, Compound P''-S''-A'' is a compound in which S'' is a sugar or a derivatized sugar, A'' is an azide, and P is uridine diphosphate (UDP), guanosine diphosphate (GDP), or cytidine diphosphate (CDP). The steps include: bringing it into contact with the modified GlcNAc moiety, thereby forming a modified antibody containing the modified GlcNAc moiety, *-GlcNAc-S''-A'' (optionally, the modified GlcNAc moiety is bound to the remainder of the modified antibody via the C1 position of GlcNAc), and (b) Obtained by a process comprising the step of reacting a modified antibody with a linker-drug moiety containing a strained cycloalkynyl or strained heterocycloalkynyl, thereby forming an antibody-drug conjugate.

[0510] In some embodiments, the process for preparing site-specific antibody-drug conjugates is as shown in Figure 5.

[0511] In some embodiments, modified antibodies containing an azide at each amino acid N297 of the antibody are conjugated by metal-free click chemistry with a linker-drug moiety containing a strained cycloalkynyl or strained heterocycloalkynyl to form the site-specific antibody-drug conjugate of the present disclosure.

[0512] In some embodiments, if the modified antibody contains at least one azide moiety and the linker-drug moiety contains a strained cycloalkynyl, the presence of a copper catalyst is not required for the cycloaddition reaction between the azide in the modified antibody and the strained cycloalkynyl or strained heterocycloalkynyl in the linker-drug moiety. In some embodiments, the cycloaddition reaction proceeds in the absence of a copper catalyst, thereby mitigating some of the potential drawbacks of using a copper catalyst in the process.

[0513] In some embodiments, Cu(I) catalysts are generally required for the cyclization and addition of the azide moiety to the terminal alkyne moiety of an antibody. In some embodiments, extensive optimization and fine-tuning of conditions may be necessary to find the optimal parameters for efficient conversion. Nevertheless, even under such conditions, the co-formation of reactive oxygen species is not always completely avoided, which can induce oxidative damage to the antibody / protein (e.g., oxidation of methionine, histidine, cysteine, or disulfide bonds). Other protocols use Cu(I) sources such as CuBr to label fixed cells and synthesize glycoproteins. In these cases, the instability of Cu(I) in air requires an extremely large excess of Cu (e.g., more than 4 mm) and ligands for efficient reaction, which can increase the risk of residual metals after purification, in addition to damage or precipitation of the antibody / protein. Therefore, conjugation of azide-containing antibodies with terminal alkynes in the presence of copper catalysts can result in extensive byproduct formation due to undesirable amino acid oxidation.

[0514] In some embodiments, a modified antibody containing an azide (for example, at each amino acid N297 of the antibody) is conjugated with a linker-drug moiety containing a strained cycloalkynyl or strained heterocycloalkynyl (for example, by metal-free click chemistry).

[0515] In some embodiments, during conjugation, the azide portion of the modified antibody and the strained cycloalkynyl or strained heterocycloalkynyl of the linker-drug portion are combined using formula (XXXV): Forms the triazole portion of TIFF0007846624000156.tif25128, In the formula, * represents direct or indirect binding to the remainder of the modified antibody, and ** represents M p This indicates a connection to [the specified location].

[0516] In some embodiments, the antibody-drug conjugate of the present disclosure comprises the presence of one or more D, each D independently being a therapeutic agent (e.g., a drug), and the one or more D may be the same or different.

[0517] In some embodiments, one or more specific sites of an antibody are bound to a linker-drug moiety, and the linker-drug moieties bound to one or more specific sites may be the same or different. In some embodiments, one or more linker-drug moieties containing one or more D are bound to one antibody.

[0518] In some embodiments, D is (a) an auristatin compound, (b) a calicheamycin compound, (c) a duocalmycin compound, (d) SN38, (e) a pyrrolobenzodiazepine, (f) a vinca compound, (g) a tubulisin compound, (h) a non-natural camptothecin compound, (i) a meitansinoid compound, (j) a DNA binding agent, (k) a kinase inhibitor, (l) a MEK inhibitor, (m) a KSP inhibitor, (n) a topoisomerase inhibitor, (o) a DNA alkylating agent, (p) an RNA polymerase, (q) a PARP inhibitor, (r) a NAMPT inhibitor, (s) a topoisomerase inhibitor, (t) a protein synthesis inhibitor, (u) a DNA binding agent, (v) a DNA intercalation agent, or (w) an immunomodulatory compound.

[0519] In some embodiments, D is (a) an auristatin compound, (b) a calicheamycin compound, (c) a duocalmycin compound, (d) a camptothecin compound, (e) a pyrrolobenzodiazepine compound, (f) a vinca compound or an analogue thereof.

[0520] In some embodiments, the auristatin compound is auristatin, drastatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F, AF-HPA, MMAF-HPA, or phenylenediamine (AFP).

[0521] In some embodiments, duocalmycin or its analogues are duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, CC-1065, adzelesin, biceresin, or calzelsin.

[0522] In some embodiments, the camptothecin compound is camptothecin, CPT-11 (irinotecan), SN-38, or topotecan.

[0523] In some embodiments, the pyrrolobenzodiazepine compound is a pyrrolobenzodiazepine monomer, a symmetric pyrrolobenzodiazepine dimer, or an asymmetric pyrrolobenzodiazepine dimer.

[0524] In some embodiments, the antibody-drug conjugate of the present disclosure has a nucleotide of approximately 40 kDa or greater (e.g., approximately 60 kDa or greater, approximately 80 kDa or greater, approximately 100 kDa or greater, approximately 120 kDa or greater, approximately 140 kDa or greater, approximately 160 kDa or greater, approximately 180 kDa or greater, or approximately 200 kDa or greater, or approximately 40 to approximately 200 kDa, approximately 40 to approximately 180 kDa). It contains modified antibodies having molecular weights of approximately Da, 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).

[0525] In some embodiments, the modified antibody has a nucleotide of approximately 40 kDa or greater (for example, approximately 60 kDa or greater, approximately 80 kDa or greater, approximately 100 kDa or greater, approximately 120 kDa or greater, approximately 140 kDa or greater, approximately 160 kDa or greater, approximately 180 kDa or greater, or approximately 200 kDa or greater, or approximately 40 to approximately 200 kDa, approximately 40 to approximately 180 kDa, approximately 40 to approximately 1 It has molecular weights of 40kDa, approximately 60-200kDa, approximately 60-180kDa, approximately 60-140kDa, approximately 80-200kDa, approximately 80-180kDa, approximately 80-140kDa, approximately 100-200kDa, approximately 100-180kDa, or approximately 100-140kDa), and is modified with amino acid N297.

[0526] In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the antibody is 12 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the antibody is 10 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the antibody is 8 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the antibody is 6 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the antibody is 4 or less. In some embodiments, the total number of specific bonds (or binding sites) formed between the linker-drug moiety and the antibody is 2 or less.

[0527] In some embodiments, the total number of specific bindings (or binding sites) formed between the linker-drug moiety and the antibody is 2.

[0528] In some embodiments (for example, for conjugation with one or more linker-drug moieties), the modified antibody has a molecular weight of approximately 140 kDa to approximately 180 kDa.

[0529] In some embodiments, antibodies within this molecular weight range include, but are not limited to, full-length antibodies such as IgG or IgM.

[0530] In some embodiments, the modified antibodies, linkers, or therapeutic agents described herein may be incorporated into the conjugates or scaffolds of this disclosure according to various techniques and methods known in the art. The conjugates of this disclosure and methods for producing them are described herein (e.g., by non-limiting embodiments and examples).

[0531] In some embodiments, the total number of bonds (or binding sites) formed between the linker-drug moiety and the modified antibody is 12 or less.

[0532] In some embodiments, the linker-drug moiety to modified antibody ratio is greater than 1:1 and less than or equal to 12:1. In some embodiments, the linker-drug moiety to modified antibody ratio is approximately 12:1, approximately 11:1, approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, or approximately 1:1. In some embodiments, the linker-drug moiety to modified antibody ratio is between 2:1 and 10:1. In some embodiments, the linker-drug moiety to modified antibody ratio is approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, or approximately 2:1. In some embodiments, the linker-drug moiety to modified antibody ratio is between approximately 2:1 and approximately 4:1. In some embodiments, the linker-drug moiety to modified antibody ratio is approximately 4:1, approximately 3:1, or approximately 2:1. In some embodiments, the linker-drug moiety to modified antibody ratio is approximately 2:1, or 1:1.

[0533] In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, or approximately 1:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, or approximately 1:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 6:1, approximately 5:1, approximately 4:1, or approximately 3:1. In some embodiments, a2 is 3, the linker-drug ratio to the modified antibody is 1:1, and the ratio of therapeutic agent (D) to the modified antibody is approximately 3:1, approximately 2:1, or approximately 1:1.

[0534] In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 8:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 6:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 5:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 4:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 3:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 2:1. In some embodiments, a2 is 3, the linker-drug moiety to modified antibody ratio is 2:1, and the ratio of therapeutic agent (D) to modified antibody is approximately 1:1.

[0535] In some embodiments, the linker-drug ratio to the modified antibody is approximately 2:1.

[0536] In some embodiments, the antibody contains an asparagine group at region 290-305 (e.g., at N297) attached to the sugar-derivative moiety containing functional group A'', and the modified antibody is conjugated to the linker-drug moiety by a covalent bond formed between A'' and the functional group of the linker-drug moiety.

[0537] In some embodiments, the linker-drug moiety comprises at least two functional groups, each of which can form a covalent bond with a functional group A'' of the sugar-derivative moiety of the modified antibody (for example, at amino acid N297 of the antibody) to form an antibody-drug conjugate.

[0538] In some embodiments (for example, for conjugation with a linker-drug moiety), the modified antibody 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 some embodiments, the ratio of the modified antibody to the linker-drug moiety is approximately 1:1 to approximately 1:2.

[0539] In some embodiments, antibodies within this molecular weight range include, but are not limited to, full-length antibodies (e.g., IgG and IgM).

[0540] In some embodiments (for example, for conjugation with one or more linker-drug moieties), the modified antibody has a molecular weight of 60 kDa to 120 kDa. In some embodiments, the ratio of the modified antibody to the linker-drug moiety is approximately 1:1 to approximately 1:2.

[0541] In some embodiments, antibodies within this molecular weight range include, but are not limited to, antibody fragments (e.g., Fab2, scFcFv, and camelid).

[0542] In some embodiments (for example, for conjugation with one or more linker-drug moieties), the modified antibody has a molecular weight of 40 kDa to 80 kDa. In some embodiments, the ratio of the modified antibody to the linker-drug moiety is about 1:1 to about 1:2.

[0543] In some embodiments, the antibody-drug conjugates and scaffolds of the present disclosure may be purified by extensive diafiltration (e.g., to remove any starting material). If necessary, additional purification by size exclusion chromatography may be performed to remove aggregated conjugates. In some embodiments, the purified conjugate or scaffold may contain less than 5% w / w (e.g., less than 2% w / w) of aggregated conjugate as determined by SEC; less than 0.5% w / w (e.g., less than 0.1% w / w) of free (unconjugated) drug as determined by RP-HPLC; less than 1% w / w of drug-carrying peptide-containing scaffold as determined by SEC; and / or less than 2% w / w (e.g., less than 1% w / w) of unconjugated antibody as determined by HIC-HPLC.

[0544] In some embodiments, the linker-drug portion is selected from the scaffolds listed in Table B below.

[0545] (Table B) TIFF0007846624000157.tif219158TIFF0007846624000158.tif223158TIFF000 7846624000159.tif214158TIFF0007846624000160.tif217158TIFF00078466240 00161.tif220158TIFF0007846624000162.tif220158TIFF0007846624000163.t if159158TIFF0007846624000164.tif157158TIFF0007846624000165.tif194158

[0546] In some embodiments, the antibody-drug conjugate is selected from the conjugates listed in Table C below.

[0547] (Table C) TIFF0007846624000166.tif209165TIFF0007846624000167.tif214165TIFF0007 846624000168.tif192165TIFF0007846624000169.tif205165TIFF000784662400 0170.tif188165TIFF0007846624000171.tif209165TIFF0007846624000172.tif 191165TIFF0007846624000173.tif209165TIFF0007846624000174.tif81165In the formula, TIFF0007846624000175.tif5128 is a GlcNAc, TIFF0007846624000176.tif5128 is Fuc, TIFF0007846624000177.tif5128 is GalNAc, d 13 This is as defined herein.

[0548] Unless otherwise specified, in this disclosure, The symbol TIFF0007846624000178.tif5128 is understood to refer to GlcNAc. Unless otherwise indicated, in this disclosure, The symbol TIFF0007846624000179.tif5128 is understood to refer to fucose. Unless otherwise indicated, in this disclosure, The symbol TIFF0007846624000180.tif5128 is understood to refer to GalNAc.

[0549] In some embodiments, the antibody-drug conjugate is of formula (XXX): The filename is TIFF0007846624000181.tif32160, During the ceremony, Each RA teeth The filename is TIFF0007846624000182.tif146150, d 13 It is 2, One or more linker-drug moieties are bound to the asparagine group at N297 of the antibody.

[0550] In some embodiments, the antibody-drug conjugate is of formula (XXX): It is TIFF0007846624000183.tif32155, In the formula, each R A teeth TIFF0007846624000184.tif220160TIFF0007846624000185.tif219133TIFF0007846624000186.tif214137TIFF0007846624000187.tif191150TIFF0007846624000188.tif79128, d 13 It is 2, The antibody contains one or more asparagine groups at N297, which is connected to the remainder of the conjugate.

[0551] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000189.tif107128.

[0552] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000190.tif85128.

[0553] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000191.tif96128.

[0554] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000192.tif90128.

[0555] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000193.tif90128.

[0556] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000194.tif87137.

[0557] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000195.tif87137.

[0558] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000196.tif98135.

[0559] In some embodiments, the antibody-drug conjugate is of formula (XXX), where each R A teeth The filename is TIFF0007846624000197.tif98135.

[0560] In some embodiments, the antibody-drug conjugate is of formula (XXXII-1), (XXXII-2), (XXXII-3), or (XXXII-4): The filename is TIFF0007846624000198.tif139161.

[0561] In some embodiments, the antibody-drug conjugate is of formula (XXXIII): It is TIFF0007846624000199.tif34147, In the formula, each R B teeth The filename is TIFF0007846624000200.tif197160.

[0562] In some embodiments, the antibody-drug conjugate is of formula (XXXII-1), (XXXII-2), (XXXII-3), (XXXII-4), or (XXXIII), where -L D -The part D The filename is TIFF0007846624000201.tif141164.

[0563] In some embodiments, the antibody-drug conjugate is of formula (XXXII-1), (XXXII-2), (XXXII-3), (XXXII-4), or (XXXIII), where -L D -The part D The filename is TIFF0007846624000202.tif74128.

[0564] In some embodiments, the antibody-drug conjugate is of formula (XXXIV): It is TIFF0007846624000203.tif22148, In the formula, each R A teeth These are TIFF0007846624000204.tif90128TIFF0007846624000205.tif179128TIFF0007846624000206.tif168131.

[0565] In some embodiments, the antibody-drug conjugate is a conjugate of formula (XXXIV), where each R A teeth It is TIFF0007846624000207.tif94128, Optionally, the antibody comprises one or more asparagine groups at N297 connected to the remainder of the conjugate, and optionally, the unmodified antibody is the NaPi2b antibody, XMT-1535, disclosed in U.S. Patent Application No. 15 / 457,574.

[0566] In some embodiments, the antibody-drug conjugate is a conjugate of formula (XXXV): It is TIFF0007846624000208.tif110137, During the ceremony, d 13 It is 2, ANTIBODY is an amino acid sequence CDRH1 containing TIFF0007846624000209.tif4128 and the amino acid sequence CDRH2 containing TIFF0007846624000210.tif4128 and the amino acid sequence CDRH3 containing TIFF0007846624000211.tif4128 and the amino acid sequence CDRL1 containing TIFF0007846624000212.tif4128 and the amino acid sequence CDRL2 containing TIFF0007846624000213.tif4128 and the amino acid sequence This is a NaPi2b antibody containing CDRL3 containing TIFF0007846624000214.tif4128, a heavy chain containing the amino acid sequence of SEQ ID NO:1, and a light chain containing the amino acid sequence of SEQ ID NO:2. The linker-drug portion is bound to the asparagine group at N297 of the antibody. TIFF0007846624000215.tif5128 is a GlcNAc, TIFF0007846624000216.tif5128 is Fuc, TIFF0007846624000217.tif5128 is GalNAc.

[0567] In some embodiments, the antibody-drug conjugate is a conjugate of formula (XXXVI): It is TIFF0007846624000218.tif87160, During the ceremony, d 13 is an integer 2, ANTIBODY is an amino acid sequence CDRH1 containing TIFF0007846624000219.tif4128 and the amino acid sequence CDRH2 containing TIFF0007846624000220.tif4128 and the amino acid sequence CDRH3 containing TIFF0007846624000221.tif4128 and the amino acid sequence CDRL1 containing TIFF0007846624000222.tif4128 and the amino acid sequence CDRL2 containing TIFF0007846624000223.tif4128 and the amino acid sequence This is a NaPi2b antibody containing CDRL3 containing TIFF0007846624000224.tif4128, a heavy chain containing the amino acid sequence of SEQ ID NO:1, and a light chain containing the amino acid sequence of SEQ ID NO:2. The linker-drug portion is bound to the asparagine group at N297 of the antibody. TIFF0007846624000225.tif5128 is a GlcNAc, TIFF0007846624000226.tif5128 is Fuc, TIFF0007846624000227.tif5128 is GalNAc.

[0568] Pharmaceutical composition In some aspects of this disclosure, pharmaceutical compositions are provided, comprising one or more conjugates disclosed herein in an acceptable carrier, such as a stabilizer or buffer. In some aspects, the conjugates can be administered and introduced subject to a pharmaceutical composition by standard means, with or without a stabilizer or buffer. In some aspects, administration may be parenteral administration including intravenous injection or infusion, intra-arterial injection or infusion, subcutaneous injection or infusion, intraperitoneal injection or infusion, or intramuscular injection or infusion, or intracranial administration, such as intrathecal or intraventricular administration. In some aspects, the conjugates can be formulated and used as sterile solutions and / or suspensions for injectable administration; as lyophilized powders for reconstitution before injection / infusion; as topical compositions; as tablets, capsules or elixirs for oral administration; or as suppositories for rectal administration, and as other compositions known in the art.

[0569] A pharmacological composition or preparation refers to a composition or preparation in a form suitable for administration to cells or subjects, including, for example, humans, such as systemic administration. The preferred form depends, in part, on the route of use or delivery, such as orally, by inhalation, transdermally, or by injection / infusion. Such a form should not prevent the composition or preparation from reaching target cells (i.e., cells to which the drug is desirable to be delivered).

[0570] "Systemic administration" means in vivo systemic absorption or accumulation of the conjugate in the bloodstream, followed by systemic distribution. Administration routes resulting in systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular. The use of the conjugates of this disclosure allows for the localization of drug delivery to specific cells, such as cancer cells, through the specificity of the antibody.

[0571] "Pharmacologically acceptable formulations" means compositions or formulations that enable the effective distribution of conjugates at the physical location most suitable for their desired activity. In some embodiments, effective delivery occurs before clearance by the reticuloendothelial system or the occurrence of off-target binding, which may result in reduced efficacy or toxicity. Non-limiting examples of active ingredients suitable for formulation with conjugates include P-glycoprotein inhibitors (such as Pluronic P85) that can enhance the entry of the activator into the CNS; biodegradable polymers for sustained-release delivery after intracerebral implantation, e.g., poly(DL-lactide-coglycolide) microspheres; and added nanoparticles that can deliver the activator across the blood-brain barrier and alter the neuronal uptake mechanism, e.g., nanoparticles made from polybutylcyanoacrylate.

[0572] In some embodiments of this disclosure, a pharmaceutical composition is prepared for storage or administration, comprising a pharmaceutically effective amount of a desired conjugate in a pharmaceutically acceptable carrier or diluent. In some embodiments, acceptable carriers, diluents and / or excipients for therapeutic use are well known in the pharmaceutical field. In some embodiments, buffers, preservatives, bulking agents, dispersants, stabilizers or dyes may be provided. In some embodiments, antioxidants and suspending agents may be used.

[0573] As used herein, the term "pharmaceutical effective dose" refers to the amount of a drug used to treat, improve, or prevent a specific disease or condition, or to produce a detectable therapeutic or inhibitory effect. The effect can be detected by any assay known in the art. The exact effective dose for a subject depends on the subject's weight, size, and health status; the nature and severity of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration.

[0574] In some embodiments, for any conjugate, the pharmaceutically effective dose can be initially estimated, for example, in a cell culture assay of neoplastic cells, or in an animal model, usually rat, mouse, rabbit, dog, or pig. In some embodiments, an animal model may be used to determine an appropriate concentration range and route of administration. In some embodiments, such information can be used to determine a useful dose and route for administration to humans. In some embodiments, therapeutic and / or prophylactic effects, as well as toxicity, can be determined using standard pharmaceutical procedures in cell culture or experimental animals, e.g., ED 50 (Therapeutic dose effective in 50% of the population) and LD 50 It can be determined by (a lethal dose for 50% of the population). In some embodiments, the dose ratio between the toxic effect and the therapeutic and / or prophylactic effect is the therapeutic index, and the ratio LD50. 50 / ED 50 It can be expressed as follows. In some embodiments, the pharmaceutical composition exhibits a large therapeutic index. In some embodiments, the dosage may vary within this range depending on the dosage form used, the patient's sensitivity, and the route of administration.

[0575] In some embodiments, Cell titer Glo can be used to evaluate the ability of drugs or their derivatives, drug-conjugates, or PBRM-drug conjugates to inhibit tumor growth in certain cell lines. Dose-response curves can be generated using SoftMax Pro software, and ICs can be obtained from 4-parameter curve fitting. 50 The value can be determined. The cell lines used may include cell lines that are targets of PBRM, and control cell lines that are not targets of PBRM and are included in the test conjugate.

[0576] In some embodiments, the conjugate is formulated for parenteral administration by injection, including the use of conventional catheterization techniques or infusions. In some embodiments, the injectable formulation may be provided in unit dosage forms, e.g., ampoules or multi-dose containers, with preservatives added. In some embodiments, the conjugate may be administered parenterally in sterile culture medium. In some embodiments, the conjugate may be suspended or dissolved in a vehicle, depending on the vehicle and concentration used. In some embodiments, adjuvants, e.g., local anesthetics, preservatives, and buffers may be dissolved in the vehicle. As used herein, the term “parenteral” includes transcutaneous injection techniques, subcutaneous injection techniques, intravascular (e.g., intravenous) injection techniques, intramuscular injection techniques, or intrathecal injection techniques. Furthermore, pharmaceutical formulations comprising a conjugate and a pharmaceutically acceptable carrier are provided. One or more of the conjugates may be present in association with one or more non-toxic, pharmaceutically acceptable carriers and / or diluents and / or adjuvants, and, if desired, other active ingredients.

[0577] In some embodiments, sterile injection preparations may also be sterile injection solutions or sterile injection suspensions in non-toxic, parenterally acceptable diluents or solvents. In some embodiments, acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In some embodiments, sterile fixatives have been conventionally used as solvents or suspension media. In some embodiments, non-irritating fixatives containing synthetic monoglycerides or synthetic diglycerides may be used. In some embodiments, fatty acids such as oleic acid are used in the preparation of injections.

[0578] In some embodiments, the conjugates and compositions described herein may be administered in a suitable form (e.g., parenterally or intravenously).

[0579] In some embodiments, the conjugate may be administered once a week for more than six weeks. In some embodiments, the conjugate may be administered once every two, three, or four weeks. In some embodiments, a bolus dose is administered using about 50 to 400 mL of saline solution to which about 5 to 10 mL of human serum albumin can be added. In some embodiments, continuous infusions are performed using about 250 to 500 mL of saline solution to which about 25 to 50 mL of human serum albumin can be added per 24 hours.

[0580] In some cases, patients may receive a second course of treatment approximately 1 to 4 weeks after the first course (for example, approximately 3 weeks or 4 weeks after the first course).

[0581] In some embodiments, the therapeutic dose may be delivered according to a different regular schedule, i.e., daily, weekly, monthly, or yearly, or according to an irregular schedule with different administration days, weeks, months, etc. In some embodiments, the therapeutic dose delivered may vary. In some embodiments, the therapeutic dose of the initial dose is higher than one or more therapeutic doses of subsequent doses. In some embodiments, the therapeutic dose of the initial dose is lower than one or more therapeutic doses of subsequent doses. In some embodiments, equivalent doses may be delivered over a variety of intervals, including, without limitation, every 2 hours, every 6 hours, every 8 hours, every 12 hours, every 24 hours, every 36 hours, every 48 hours, every 72 hours, every week, every 2 weeks, every 3 weeks, every month, and every 2 months. In some embodiments, the number and frequency of doses corresponding to a completed series of treatments are determined according to the recommendations of the relevant regulatory body and the judgment of the healthcare professional. In some embodiments, the therapeutic dose described herein refers to the total amount delivered over a given period. In other words, when multiple different conjugates described herein are administered, the therapeutically effective dose corresponds to the total amount administered. It is understood that a specific dose level for a particular subject depends on various factors, including the activity of the particular conjugate, age, weight, overall health, sex, diet, administration time, route of administration and excretion rate, combination with other activators, and the severity of the particular disease being treated.

[0582] In some embodiments, the therapeutically effective dose of the conjugate disclosed herein generally relates to the amount required to achieve a therapeutic objective. In some embodiments, this may be the binding interaction between the antibody and its target antigen, which in some cases interferes with the function of the target. In some embodiments, the amount required to be administered further depends on the binding affinity of the antibody to its specific antigen and on the rate at which the administered antibody is depleted from the free volume of the other target to which it is administered. In some embodiments, the range for therapeutically effective administration of the conjugate disclosed herein may, as non-limiting examples, be 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. In some embodiments, the frequency of administration may range, for example, from twice a day to once a month (e.g., once a day, once a week; once every two weeks; once every three weeks, or once a month). In some embodiments, the conjugates disclosed herein may be administered intravenously by infusion (for example, as a single dose weekly, every two weeks, every three weeks, or every four weeks) at doses of approximately 0.1 mg / kg to approximately 20 mg / kg (for example, 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).In some embodiments, the conjugates disclosed herein may be administered to treat cancer at doses of approximately 0.1 mg / kg to approximately 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., as a single dose weekly, every two weeks, every three weeks, or monthly).

[0583] In some embodiments, the conjugates disclosed herein are administered by injection (for example, as a single dose weekly, every two weeks, every three weeks, or every four weeks) at a dose of approximately 7 mg / m². 2 ~Approx. 162mg / m 2 (For example, 7 mg / m²) 2 , 14 mg / m² 2 , 28 mg / m² 2 , 56 mg / m² 2 , 84 mg / m² 2 , 112 mg / m² 2 , 135 mg / m² 2 or 162 mg / m² 2 It may be administered intravenously. Numerous packages or kits for dispensing pharmaceuticals for regular oral use are known in the art. In some embodiments, the package has an indicator for each period. In some embodiments, the package is a labeled blister package, a dial dispenser package or a bottle. In some embodiments, the packaging means of the kit itself may be adapted for administration such as a syringe, pipette, eye dropper or other such device, from which the formulation may be applied to a part of the body, injected into a subject, or applied to and mixed with other components of the kit.

[0584] How to use In some embodiments, the Disclosure provides a method for treating or preventing a disease or disorder in which such treatment is required, comprising the step of administering a therapeutically effective dose of the conjugate disclosed herein to the target.

[0585] In some embodiments, the Disclosure provides a method for treating a disease or disorder in a target area requiring such treatment, comprising the step of administering a therapeutically effective dose of the conjugate disclosed herein to the target area.

[0586] In some embodiments, the Disclosure relates to a method for treating a target cancer requiring the use of the Conjugate Disclosed herein, comprising the step of administering an effective amount of the Conjugate Disclosed herein to the target. In some embodiments, the Disclosure relates to a method for treating a target NaPi2b-expressing cancer requiring the use of the Conjugate Disclosed herein, comprising the step of administering an effective amount of the Conjugate Disclosed herein to the target.

[0587] In some embodiments, the disclosure provides conjugates disclosed herein for use in treating or preventing a disease or disorder of interest that requires such treatment.

[0588] In some embodiments, the present disclosure provides conjugates disclosed herein for use in the treatment of a disease or disorder in which such treatment is required.

[0589] In some embodiments, the Disclosure provides the use of the conjugates disclosed herein for treating target cancers that require such use. In some embodiments, the Disclosure provides the use of the conjugates disclosed herein for treating target NaPi2b-expressing cancers that require such use.

[0590] In some aspects, the disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating a target disease or disorder that requires such use.

[0591] In some aspects, the disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating or preventing a target disease or disorder that requires such use.

[0592] In some embodiments, the Disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating a target cancer that requires such use. In some embodiments, the Disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for treating a target NaPi2b-expressing cancer that requires such use.

[0593] In some embodiments, the disclosure provides a conjugate for use in treating a disease or disorder in which such treatment is required.

[0594] In some embodiments, the disclosure provides a conjugate for use in treating or preventing a disease or disorder in which it is needed.

[0595] In some embodiments, the Disclosure provides a conjugate for use in treating cancer in a target subject requiring its use, comprising the step of administering an effective amount of the conjugate disclosed herein to the target subject. In some embodiments, the Disclosure provides a conjugate for use in treating NaPi2b-expressing cancer in a target subject requiring its use, comprising the step of administering an effective amount of the conjugate disclosed herein to the target subject.

[0596] In some embodiments, the Disclosure provides a method for treating or preventing a disease or disorder in a subject requiring such treatment, comprising the step of administering an efficient amount of at least one conjugate of the Disclosure to the subject, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0597] In some embodiments, the Disclosure provides a method for treating a disease or disorder in a subject requiring such treatment, comprising the step of administering an efficient amount of at least one conjugate of the Disclosure to the subject, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0598] In some aspects, the disease is cancer.

[0599] In some embodiments, cancer therapies provided herein, comprising a NaPi2b-targeted antibody-drug conjugate, are administered in amounts sufficient to exert a therapeutically beneficial effect. Typically, the activator is administered in amounts that do not cause undesirable side effects in the treated patient, or minimize or reduce observed side effects. NaPi2b-expressing cancers include, for example, ovarian cancer, non-small cell lung cancer (NSCLC), endometrial cancer, papillary renal cell carcinoma, salivary duct cancer, papillary thyroid cancer, clear cell renal carcinoma, breast cancer, kidney cancer, cervical cancer, and cholangiocarcinoma.

[0600] Determining the precise amount of an activator containing a NaPi2b target-directed polymer antibody-drug conjugate to be administered to a target is within the scope of the art. For example, such agents and their uses for treating cancer and solid tumors are well known in the art. Therefore, the dosage of such an agent can be selected based on a standard administration regimen for that agent under a given route of administration.

[0601] It is understood that the precise dosage and duration of treatment are functions of the tissue or tumor being treated and may be determined empirically using known test protocols, or by extrapolation from in vivo or in vitro test data, and / or from known dosing regimens for a particular drug. It should also be noted that concentration and dosage values ​​may vary depending on the age of the individual being treated, the individual's weight, the route of administration, and / or the stage or severity of the disease, as well as other factors within the level of expertise of the healthcare professional. Generally, dosing regimens are selected to limit toxicity. It should be noted that the attending physician knows how and when to terminate, interrupt, or adjust treatment to reduce the dosage due to toxicity or dysfunction of the bone marrow, liver, kidney, or other tissues. Conversely, the attending physician also knows how and when to adjust treatment to a higher level if the clinical response is inadequate (eliminating toxic side effects). It should be further understood that for any particular subject, a specific dosing regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the formulation, and that the concentration ranges described herein are illustrative only and not intended to limit the range.

[0602] For example, a NaPi2b targeted polymer antibody-drug conjugate is administered in a therapeutically effective dose to reduce tumor volume.

[0603] The amount of NaPi2b target-directed polymer antibody-drug conjugate administered for a disease or condition, such as cancer or solid tumors, can be determined by standard clinical techniques. Furthermore, in vitro assays and animal models can be used to help identify the optimal dose range. The exact dose may be determined empirically and may depend on the route of administration, the type of disease being treated, and the severity of the disease.

[0604] In some embodiments, the conjugates provided herein are administered intravenously. In some embodiments for intravenous administration, the conjugate may be administered by push or bolus, by infusion, or a combination thereof. In some embodiments, the infusion time may be about 1 minute to about 3 hours, for example, about 1 minute to about 2 hours, or about 1 minute to about 60 minutes, or at least 10 minutes, at least 40 minutes, or at least 60 minutes.

[0605] In some embodiments, the dosage is approximately 7 mg / m². 2 ~Approx. 162mg / m 2 (For example, 7 mg / m²) 2 , 14 mg / m² 2 , 28 mg / m² 2 , 56 mg / m² 2 , 84 mg / m² 2 , 112 mg / m² 2 , 135 mg / m² 2 or 162 mg / m² 2 ) In some embodiments, the dosage is approximately 6.5 mg / m². 2 ~about 7.5mg / m 2 , about 13.5mg / m 2 ~about 14.5mg / m 2 , about 27.5mg / m 2 ~about 28.5mg / m 2 , about 55.5mg / m 2 ~about 56.5mg / m 2 , about 83.5mg / m 2 ~about 84.5mg / m 2 , about 111.5mg / m 2 ~Approx. 112.5mg / m 2 , about 134.5mg / m 2 ~Approx. 135.5mg / m 2 , or approximately 161.5 mg / m² 2 ~Approx. 162mg / m 2 In some embodiments, the dose is administered intravenously once every three weeks (i.e., a 21-day cycle) or once every four weeks (i.e., a 28-day cycle).

[0606] The frequency and timing of administration, as well as the dosage, may be administered cyclically over a dosing cycle to maintain the continuous and / or long-term effects of the activator over a desired length of time. The provided composition of the NaPi2b target-directed antibody-drug conjugate may be administered hourly, daily, weekly, monthly, yearly, or once. The length of the dosing cycle can be determined empirically and depends on the disease being treated, the severity of the disease, the specific patient, and other considerations within the skill level of the treating physician. The length of treatment with the combination therapies provided herein may be one week, two weeks, one month, several months, one year, several years, or longer.

[0607] In some embodiments, the administration frequency of the NaPi2b targeted antibody-drug conjugate is once daily, every other day, twice weekly, once weekly, once every two weeks, once every three weeks, or once every four weeks. The dose may be divided into multiple administration cycles during the course of treatment. For example, the NaPi2b targeted antibody-drug conjugate may be administered at frequencies over a period of approximately one month, two months, three months, four months, five months, six months, one year, or longer. The administration frequency may be the same or different throughout the duration of the cycle. In some embodiments, the NaPi2b targeted antibody-drug conjugate is administered at least twice weekly for the first week of the administration cycle. After the first week, the frequency may continue at twice weekly, increase to more than twice weekly, or decrease to once weekly or less. Determining specific dosages, disease or condition being treated, severity of disease or condition, age of the subject, and other similar factors is within the scope of the skills of the art.

[0608] In some cases, if disease symptoms persist in the absence of the interrupted treatment, treatment may be continued for a longer period. Throughout the course of treatment, evidence of disease and / or treatment-related toxicity or side effects may be monitored.

[0609] The administration cycle of the NaPi2b targeted antibody-drug conjugate may be adjusted to include a period of interrupted treatment to provide a rest period from drug exposure. The length of the treatment interruption may be over a predetermined period of time or may be determined empirically depending on how the patient responds or on any observed side effects. For example, treatment may be interrupted for one week, two weeks, three weeks, one month, or several months. In some embodiments, the interrupted treatment period is incorporated into the cycle of the administration regimen for the patient.

[0610] Exemplary dosing regimens are 21-day or 28-day treatment or dosing cycles. In some embodiments, the dosing regimen is a treatment cycle, or the dosing cycle is 28 days long. In some embodiments, the NaPi2b targeted antibody-drug conjugate disclosed herein is administered on day 1 followed by no administration for 20 days, or administered on day 1 followed by no administration for 27 days. Determining the exact dosing cycle and dosing schedule is within the scope of the skill of the art.

[0611] In some embodiments, the administration cycle may span any desired length of time (for example, a 21-day or 28-day administration cycle may be repeated over any length of time). For example, a 21-day or 28-day administration cycle may be repeated over 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years or longer.

[0612] In some embodiments, NaPi2b-expressing cancers include, for example, ovarian cancer, non-small cell lung cancer (NSCLC), endometrial cancer, papillary renal cell carcinoma, salivary duct cancer, papillary thyroid cancer, clear cell renal carcinoma, breast cancer, kidney cancer, cervical cancer, and cholangiocarcinoma.

[0613] In some embodiments, NaPi2b-expressing cancers include, for example, ovarian cancer, non-small cell lung cancer (NSCLC), endometrial cancer, papillary renal cell carcinoma, salivary duct cancer, and papillary thyroid cancer.

[0614] In some aspects, the disclosure relates to a method for treating ovarian cancer or non-small cell lung cancer (NSCLC) in a subject requiring such treatment, comprising the step of administering an effective amount of the conjugate disclosed herein to the subject.

[0615] In some aspects, the disclosure provides the use of the conjugates disclosed herein in the manufacture of a pharmaceutical product for the treatment of ovarian cancer or non-small cell lung cancer (NSCLC) in a subject requiring such treatment.

[0616] In some aspects, the disclosure provides the use of the conjugates disclosed herein for treating ovarian cancer or non-small cell lung cancer (NSCLC) in a subject requiring such treatment.

[0617] In some embodiments, the Disclosure provides a conjugate for use in the treatment of ovarian cancer or non-small cell lung cancer (NSCLC) in a subject requiring such treatment, comprising the step of administering an effective amount of the conjugate disclosed herein to the subject.

[0618] In some aspects, the cancer is non-small cell lung cancer (NSCLC). In some aspects, NSCLC is subtyped as adenocarcinoma.

[0619] In some embodiments, ovarian cancer is platinum-resistant ovarian cancer. In some embodiments, ovarian cancer is high-grade serous ovarian cancer. In some embodiments, ovarian cancer is platinum-resistant high-grade serous ovarian cancer.

[0620] In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is papillary renal cell carcinoma. In some embodiments, the cancer is salivary duct cancer. In some embodiments, the cancer is papillary thyroid cancer.

[0621] In some embodiments, the subjects have epithelial ovarian cancer, ductal fallopian carcinoma, primary peritoneal cancer, platinum-resistant ovarian cancer, non-squamous NSCLC cancer, advanced, radioactive iodine-resistant, locally recurrent or metastatic papillary thyroid cancer, or epithelial endometrial cancer.

[0622] In some embodiments, subjects with epithelial ovarian cancer are subtyped as high-grade ovarian cancer, low-grade serous ovarian cancer, or clear cell ovarian cancer.

[0623] In some embodiments, subjects with ovarian cancer have previously received prior treatment with chemotherapy agents such as docetaxel, doxorubicin, cyclophosphamide, carboplatin, paclitaxel, nab-paclitaxel, gemcitabine, and cisplatin; angiogenesis inhibitors such as bevacizumab (Avastin); PARP inhibitors such as niraparib (Zejula), olaparib (Lynparza), and veliparib; olaparib (Lynparza) in combination with bevacizumab; or a combination thereof.

[0624] In some cases, subjects with ovarian cancer have previously received prior monotherapy, such as weekly treatment with pegylated liposomal doxorubicin, paclitaxel-topotecangemcitabine, or PARP inhibitors.

[0625] In some aspects, subjects with ovarian cancer have received three or fewer prior treatment lines, such as combination chemotherapy, for example, carboplatin + paclitaxel; pegylated liposomal doxorubicin; or weekly treatment with paclitaxel, docetaxel, topotecan, gemcitabine, or PARP inhibitors.

[0626] In some embodiments, subjects with ovarian cancer have received three or fewer prior treatment lines, including at least one line of platinum-containing regimen. In some embodiments, subjects with ovarian cancer have received four or fewer prior treatment lines, with or without at least one line of platinum-containing regimen.

[0627] In some embodiments, subjects with NSCLC cancer are subtyped as adenocarcinoma. In some embodiments, subjects with adenocarcinoma NSCLC cancer may be metastatic or recurrent.

[0628] In some embodiments, the subjects have NSCLC and have received prior treatment, such as platinum-based chemotherapy (cisplatin or carboplatin) and PD-1 monoclonal antibodies or PD-L1 monoclonal antibodies. In some embodiments, the subjects have NSCLC and have received prior treatment with carboplatin / paclitaxel, Abraxane nab-paclitaxel, docetaxel, premetrexed, gemcitabine, or a combination of docetaxel and ramucirumab.

[0629] In some aspects, the subjects had NSCLC and had received up to two prior lines of chemotherapy.

[0630] In some embodiments, subjects have NSCLC and have not received additional prior treatment with cytotoxic agents or immunotherapy. In other embodiments, subjects with NSCLC have demonstrated intolerance or disease progression due to a known oncogenic mutation for which there is an approved treatment (e.g., ALK translocation, EGFR mutation, or KRAS mutation).

[0631] In some embodiments, subjects with NSCLC cancer are treated with a NaPi2b antibody-drug conjugate and a PD-1 monoclonal antibody or PD-L1 monoclonal antibody, such as nivolumab, pembrolizumab, atezolizumab, or avelumab.

[0632] In some embodiments, subjects with NSCLC cancer are treated with a NaPi2b antibody-drug conjugate, a PD-1 monoclonal antibody or a PD-L1 monoclonal antibody, or pembrolizumab.

[0633] In some embodiments, the targets include PARP inhibitors, e.g., olaparib, niraparib, lucaparib, talazoparib; PD1 / PDL-1 inhibitors, e.g., nivolumab, pembrolizumab, atezolizumab, avelumab; chemotherapy, e.g., carboplatin, cisplatin, oxaliplatin, doxil, cyclophosphamide, gemcitabine, topotecan, premetrexe; and VEGF inhibitors, e.g. Treatment may involve using NaPi2b antibody-drug conjugates in combination with tyrosine kinase inhibitors such as bevacizumab and ramucirumab; tyrosine kinase inhibitors such as gefitinib, afatinib, erlotinib, dacomitinib, osimertinib, and pazopanib; ALK inhibitors such as alectinib, crizotinib, certinib, and brigatinib; or BRAF inhibitors such as dabrafenib and trametinib.

[0634] In some embodiments, the immune checkpoint inhibitor is pembrolizumab.

[0635] In some embodiments, the subject is treated with a NaPi2b antibody-drug conjugate in combination with pembrolizumab, carboplatin, doxil, bevacizumab, or a PARP inhibitor.

[0636] In some embodiments, the subjects have papillary thyroid carcinoma that is resistant to or intolerant to prior kinase inhibitor therapy, or have previously received treatment for low-grade hormone receptor-positive endometrioid adenocarcinoma.

[0637] In some aspects, endometrial cancer is not a stromal tumor or carcinosarcoma. In some aspects, the subject has endometrial cancer and has previously received treatment with carboplatin / paclitaxel or a similar regimen.

[0638] In some embodiments, the subjects have papillary renal cell carcinoma or clear cell carcinoma primarily exhibiting a papillary growth pattern. In one embodiment, the subjects have a histological diagnosis of salivary duct cancer that has progressed after standard systemic therapy.

[0639] In some embodiments, the subjects are refractory to chemotherapy, including standard first-line chemotherapy agents.

[0640] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. In this specification, the singular form includes the plural form unless the context clearly indicates otherwise. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described below. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are merely illustrative and not intended to limit the scope.

[0641] Throughout this specification, when compounds, scaffolds, and compositions are described as having, including, or comprising certain components, the compositions are also intended to be essentially composed of or consisting of the listed components. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process is also intended to be essentially composed of or consisting of the listed processing steps. Furthermore, it should be understood that the order or sequence of steps for performing a particular operation is not important as long as the invention remains operational. Moreover, two or more steps or operations may be performed simultaneously.

[0642] All methods described herein may be performed in any preferred order, unless otherwise specified herein or unless it is clearly inconsistent with the context. The use of any examples or illustrative language provided herein (e.g., "etc.") is solely intended to further illustrate the invention and should not be construed as a limitation on the claims unless expressly asserted otherwise. No language herein should be construed as indicating that any element not asserted is essential to what is asserted.

[0643] Synthesis method Using any available technique, conjugates or their compositions, as well as intermediates and components (e.g., scaffolds) useful for producing them, can be prepared. In some embodiments, semi-synthetic and fully synthetic methods may be used.

[0644] General methods for generating conjugates or scaffolds disclosed herein are shown in Schemes 2 and 3 below, as well as in concurrently pending U.S. Patent No. 15 / 819,650, the entire disclosure of which is incorporated herein. These schemes involve variables (e.g., M P M A , L 3 , W D , W M , L D and L P’ Unless otherwise specified, terms such as (etc.) have the same definitions as those given herein.

[0645] Scheme 2 TIFF0007846624000228.tif128149

[0646] Scheme 3 TIFF0007846624000229.tif149151 Here, the antibody is the modified antibody of this disclosure.

[0647] The synthesis process of this disclosure can accommodate a wide variety of functional groups. Therefore, a variety of substituted starting materials can be used. The process generally provides the desired final compound at or near the end of the overall process, but in some cases it may be desirable to further convert the compound to its pharmaceutically acceptable salt, ester, or prodrug.

[0648] The drug compounds used in the conjugates of this disclosure can be prepared in various ways using commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by using standard synthetic methods and procedures that are known to those skilled in the art or are apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and the transformation and manipulation of functional groups can be obtained from relevant scientific literature or from standard textbooks in the art. Classical textbooks incorporated herein by reference, but not limited to one or more sources, include, for example, Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th edition, John Wiley & Sons: New York, 2001; and Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3 rd John Wiley & Sons, New York, 1999, is a useful and well-known reference textbook on organic synthesis known to those skilled in the art. The following description of the synthetic methods is designed to illustrate, but is not limited to, general procedures for preparing the compounds of this disclosure.

[0649] The conjugates and drug compounds contained herein can be prepared by various methods well known to those skilled in the art. Each of the conjugates or compounds of this disclosure having any of the formulas described herein can be prepared from commercially available starting materials or starting materials that can be prepared using procedures described in the literature, following the procedures outlined below. These procedures illustrate the preparation of representative conjugates of this disclosure.

[0650] Conjugates designed, selected, and / or optimized by the methods described above, once generated, can be characterized using a variety of assays known to those skilled in the art to determine whether the conjugates have biological activity. In some embodiments, conjugates can be characterized by conventional assays, including, but not limited to, the assays described below, to determine whether they have the expected activity, binding activity, and / or binding specificity.

[0651] Furthermore, high-throughput screening can be used to expedite analyses using such assays. Common methods for performing high-throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker, and U.S. Patent No. 5,763,263. High-throughput assays may employ one or more different assay techniques, including, but not limited to, those described below.

[0652] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure. [Examples]

[0653] The following examples illustrate linkers, drug molecules, and antibodies or antibody fragments, as well as methods for preparing them. These are not intended to be limiting, and it will be readily apparent to those skilled in the art that other reagents or methods may be used.

[0654] Abbreviation The following abbreviations are used in the reaction schemes and synthesis examples below. This list is not intended to be a comprehensive list of abbreviations used in this application as additional standard abbreviations readily understood by those skilled in the art of organic synthesis, and may also be used in the synthesis schemes and examples. Abbreviation: TIFF0007846624000230.tif90128TIFF0007846624000231.tif218110TIFF0007846624000232.tif40128

[0655] General information Unless otherwise specified, all reagents were purchased from the relevant suppliers.

[0656] XMT-1535 (anti-NaPi2b antibody) is disclosed in U.S. Patent No. 15 / 457,574, a concurrently pending application filed on 13 March 2017, which is incorporated herein by reference in its entirety. XMT-1519 (anti-Her2 antibody) is disclosed in U.S. Patent No. 9,555,112, issued on 31 January 2017, and U.S. Patent No. 9,738,720, issued on 22 August 2017, which are incorporated herein by reference in their entirety.

[0657] Endo SH was prepared as described in its entirety in PCT International Publication No. 2017137459, which is incorporated herein by reference. UDP-azido sugar and GalNAcT were prepared as described in its entirety in U.S. Patent No. 9,988,662, which is incorporated herein by reference.

[0658] Tumor growth inhibition (%TGI) was defined as the percentage difference in median tumor volume (MTV) between the treatment group and the control group. Tumor size was measured throughout each efficacy trial to determine TGI.

[0659] HPLC purification was performed using a Phenomenex Gemini 5μm C18 110Å, 250×10mm half-part column. Where applicable, the drug content of the conjugate was determined spectrophotometrically; otherwise, RP-HPLC, LC / MS, or quantification of the drug content was performed. 1 1H-NMR was performed.

[0660] The protein content of antibody-drug conjugates was determined spectrophotometrically at 280 nm or by ELISA.

[0661] If necessary, the antibody-drug conjugate, drug-loaded scaffold, or antibody scaffold was purified by extensive diafiltration, HIC, or Protein A (i.e., removal of residual unreacted drugs, non-conjugate antibodies, enzymes, or starting materials). If necessary, additional purification by SEC or HIC was performed to remove aggregated antibody-drug conjugates. Generally, after purification, antibody-drug conjugates contained less than 5% (w / w) (e.g., less than 2% (w / w)) of aggregated antibody-drug conjugate as determined by SEC; less than 0.5% (w / w) (e.g., less than 0.1% (w / w)) of free (unconjugated) drug as determined by RP-HPLC and / or LC-MS / MS; less than 1% (w / w) of free drug conjugate as determined by SEC and / or RP-HPLC; and less than 2% (w / w) (e.g., less than 1% (w / w)) of unconjugated antibody or unconjugated antibody fragment as determined by HIC-HPLC and / or RP-HPLC. Reductive or partially reductive antibodies were prepared using procedures described in the literature. See, for example, Francisco et al., Blood 102(4):1458-1465 (2003). The total drug (conjugated and unconjugated) concentration was determined by inverse calculation from the DAR measured by RP-HPLC or CE-SDS.

[0662] To determine the concentration of free AF-HPA drug in biological samples, acidified samples were treated with ACN. The free drug was extracted, and the ACN supernatant was analyzed. To determine the concentration of conjugated AF-HPA in nonclinical samples, samples were subjected to immunocapture using magnetic beads coated with anti-IgG1 antibody, followed by thorough basic hydrolysis. The ACN supernatant containing the released AF-HPA drug was analyzed by LC-MS / MS. The total antibody concentration in nonclinical samples was measured by LC-MS / MS after immunocapture using anti-IgG1 antibody via detection of peptide sequences specific to the antibody after trypsin digestion. For clinical samples, the same procedure could be followed, except that anti-idiotype antibodies were used for immunocapture to avoid interference from endogenous antibodies.

[0663] Free AF ​​and free AF-HPA were analyzed by RP-HPLC using a C4 column, ACN gradient, and UV detection. Peak areas were combined and compared to AF and AF-HPA standards. The method is quantitative for AF and AF-HPA in plasma and tissue homogenates and is linear over the concentration range of 0.1 ng / mL to 150 ng / mL. Total drug (AF-HPA) released after hydrolysis with NaOH (aqueous solution) was measured under the same conditions with a dynamic range of 1 ng / mL to 5,000 ng / mL. Total antibody standards ranged from 0.1 μg / mL to 100 μg / mL.

[0664] The hydrophobicity of antibody-drug conjugates was determined by HIC-HPLC using a Shimadzu Prominence HPLC system equipped with a diode array detector (DAD). For these analyses, a TSK gel-butyl-NPR column (2.5 μm particle size) was maintained at 35°C. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate, pH 7.0, and mobile phase B was 25 mM sodium phosphate, 10% isopropyl alcohol, pH 7.0. Separation was performed over 25 minutes using a linear gradient of 0–100% for mobile phase B. The flow rate was 1 mL / min. Sample injection ranged from approximately 10 μg to 100 μg.

[0665] The drug-to-antibody ratio (DAR) was determined by subjecting the antibody-drug conjugate to thorough nucleotide hydrolysis. Subsequently, the released AF-HPA was quantified from a standard curve using RP-HPLC. The DAR was determined by correlating the measured AF-HPA concentration with the antibody content.

[0666] Example 1: Synthesis of scaffolding 6 TIFF0007846624000233.tif104141

[0667] Step 1. compound 3 TIFF0007846624000234.tif129160

[0668] Compound 1 (548 mg, 0.165 mmol, prepared as described in U.S. Patent No. 15 / 819,650, incorporated herein by reference), water (14 mL), NMP (1.4 mL), EDC (158 mg, 0.824 mmol), and HOAt (112 mg, 0.824 mmol) were stirred in an ice bath, and the pH was adjusted to approximately 6.5 using 1N NaHCO3 (aqueous solution). Compound 2 (696 mg, 0.577 mmol) was added, and the pH was adjusted to approximately 6.5. The resulting mixture was stirred at low temperature for 3 hours. Additional EDC, HOAt, and Compound 2 (198 mg, 0.164 mmol) were added, and stirring was continued overnight. The reaction mixture was purified on a C18 cartridge (275g) using a step gradient of ACN / H2O (0.1% TFA) in concentrations of 10%~50%~90% v / v. The desired fraction was freeze-dried to obtain compound 3 as a white amorphous solid (825mg, yield 76%). MS:2120.04(3 + ),1590.27(4 + ),1272.41(5 + ).

[0669] Step 2. compound 4 TIFF0007846624000235.tif115160

[0670] A mixture of compound 3 (825 mg, 0.126 mmol), EtOH (90 mL), and water (9.00 mL) in a glass Parr bottle was mixed with acetic acid (0.288 mL, 5.04 mmol). Argon was passed through the mixture to allow bubbling, and then Pd / C (134 mg, 0.126 mmol) was added. The bottle was mounted in a hydrogenation apparatus, then continuously vacuum-fed, filled with argon, and then filled with hydrogen (0.762 mg, 0.378 mmol) to 30 psi, and the mixture was vigorously stirred overnight. The reaction mixture was filtered through a silica gel plug and concentrated into an oil. The oil was dissolved in ACN / H2O (0.1% TFA), freeze-dried, and compound 4 was obtained as a white amorphous solid (790 mg, 100% yield). MS:2094.65(3 + ), 1570.98(4 + ),1257.18(5 + ).

[0671] Step 3. compound 5 TIFF0007846624000236.tif115160

[0672] To an ice-cold solution of compound 4 (100 mg, 0.016 mmol) dissolved in THF (1.5 mL) and water (1.5 mL), formic acid (4.5 mL, 117 mmol) was added. The solution was cooled and stirred for 15 minutes, then stirred overnight at room temperature. The THF was removed, and the resulting mixture was diluted with water and purified on a C18 cartridge (150 g) using ACN / H2O (0.1% TFA) as the mobile phase. The desired fraction was lyophilized to obtain compound 5 as a white amorphous solid (71 mg, yield 72%). MS:2060.97(3 + ),1545.98(4 + ),1236.99(5 + ).

[0673] Process 4. Scaffolding 6 TIFF0007846624000237.tif122154

[0674] To an ice-cold solution of compound 5 (71 mg, 0.011 mmol) in DCM (5 mL) and DMF (0.500 mL), ((1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-yl)methyl(2,5-dioxopyrrolidine-1-yl) carbonate (5.02 mg, 0.017 mmol) and DIPEA (0.018 mL, 0.103 mmol), with a final pH of approximately 8 to 9, were added. After 18 hours, DMF (1.5 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was then concentrated and purified by preparative HPLC using ACN / H2O (0.1% AcOH) as the mobile phase. The desired fraction was lyophilized to obtain scaffold 6 as a white amorphous solid (34 mg, yield 47%). MS:2120.04(3 + ),1590.27(4 + ),1272.41(5 + ).

[0675] Example 2: Synthesis of XMT-1535 drug conjugate (conjugate 7, DAR 6.0) of scaffold 6 TIFF0007846624000238.tif114153In formula, TIFF0007846624000239.tif5128 is a GlcNAc, TIFF0007846624000240.tif5128 is Fuc, TIFF0007846624000241.tif5128 is GalNAc.

[0676] Step 1. Azide modified XMT-1535 antibody To a solution of XMT-1535 antibody (910.6 mg, 6.22 μmole) in TBS, pH 7.6, TBS (249 μL, pH 7.6), Endo SH (9.10 mg, 0.065 μmole), GalNAcT (77.9 mg, 1.69 μmole), UDP-azide sugar (144 mg, 227 μmole), and MnCl2 (76 mg, 604 μmole) were added in that order to achieve a final antibody concentration of 15 g / L. The reaction mixture was stirred overnight at 30 rpm at 30°C. The crude azide-modified XMT-1535 antibody was purified by protein A chromatography and dialysis to obtain azide-modified XMT-1535 antibody (880 mg, yield 97%).

[0677] Step 2. XMT-1535 drug conjugate on scaffold 6 (conjugate 7, DAR 6.0) A solution of azide-modified XMT-1535 antibody (300 mg, 2.05 μmole) in PBS, pH 7.4 (5.5 mL) was gently mixed with an aqueous solution of scaffold 6 (127.2 mg, 20.0 μmole), and then left to stand at 30°C for 20 hours without shaking or agitation. The crude product was purified by UF / DF and HIC to obtain conjugate 7 (132 mg, 44% yield), which had a DAR of 6.6 as determined by hydrolysis and subsequent RP-HPLC.

[0678] Example 3: Synthesis of XMT-1519 conjugate (conjugate 8, DAR 7.3) on scaffolding 6 TIFF0007846624000242.tif112152In formula, TIFF0007846624000243.tif5128 is a GlcNAc, TIFF0007846624000244.tif5128 is Fuc, TIFF0007846624000245.tif5128 is GalNAc.

[0679] Conjugate 8 was synthesized as described in Example 2, except that azide-modified XMT-1519 (400 mg, 2.78 μmole) was used in step 2 instead of azide-modified XMT-1535 antibody. Purified conjugate 8 (163 mg, 41% yield) had a drug-to-DAR of 7.3 as determined by hydrolysis and subsequent RP-HPLC.

[0680] Example 4: Synthesis of trastuzumab conjugate (conjugate 9, DAR 7.3) of scaffold 6 TIFF0007846624000246.tif108147In formula, TIFF0007846624000247.tif5128 is a GlcNAc, TIFF0007846624000248.tif5128 is Fuc, TIFF0007846624000249.tif5128 is GalNAc.

[0681] Conjugate 9 was the same as described in Example 2, except that azide-modified trastuzumab (35 mg, 0.239 μmol) was used in step 2 instead of azide-modified XMT-1535 antibody. The purified conjugate 9 had a DAR of 7.3 as determined by hydrolysis and subsequent RP-HPLC.

[0682] Example 5: Synthesis of rituximab conjugate (conjugate 10, DAR 6.8) of scaffold 6 TIFF0007846624000250.tif110151In formula, TIFF0007846624000251.tif5128 is a GlcNAc, TIFF0007846624000252.tif5128 is Fuc, TIFF0007846624000253.tif5128 is GalNAc.

[0683] Conjugate 10 was prepared as described in Example 2, except that azide-modified rituximab (40 mg, 0.28 μmole) was used in step 2 instead of azide-modified XMT-1535 antibody. The purified conjugate 10 had a drug-to-DAR of 6.8, as determined by hydrolysis and subsequent RP-HPLC.

[0684] Example 6: Synthesis of XMT-1535 conjugate 11 (DAR 6.5) TIFF0007846624000254.tif114148

[0685] To a solution of XMT-1535 (200 mg, 1.37 μmol) in sodium acetate buffer (25 mM, pH 5.5, 21.1 mL) at +4°C, TEAA (aqueous solution, 50 mM) and EDTA (1 mM, pH 7.0, 18.95 mL) were added. The pH of the resulting mixture was adjusted to pH 7.0 using NaHCO3 (aqueous solution, 1.0 M), and then TCEP (1.37 mg, 4.77 μmole) in a solution of TEAA (aqueous solution, 50 mM) and EDTA (1 mM, pH 7.0) was added. After 90 minutes, the pH of the reaction mixture was adjusted to pH 6.0 using acetic acid (aqueous solution, 1.0 M). Scaffold 6 (79.8 mg, 12.3 μmol, prepared as described in U.S. ...

Claims

1. The antibody-drug conjugate or optical isomer thereof, as shown in the following formula: During the ceremony, It is GlcNAc, It is Fuc, It is GalNAc, d 13 It is 2, The antibody is attached to the remainder of the conjugate by one or more asparagine residues within the amino acid region 290–305 of the antibody's heavy chain.

2. Antibodies, amino acid sequence CDRH1, which includes, amino acid sequence CDRH2, which includes, amino acid sequence CDRH3, which includes, amino acid sequence CDRL1, which includes, amino acid sequence CDRL2, which includes, amino acid sequence CDRL3 including The conjugate according to claim 1, which is a NaPi2b antibody containing the above.

3. The conjugate according to claim 2, wherein the NaPi2b antibody comprises a heavy chain amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence of SEQ ID NO:

2.

4. Modified antibodies, as follows: A method for preparing a conjugate according to any one of claims 1 to 3, comprising the step of reacting with a scaffold which is either or an optical isomer thereof, thereby forming a site-specific antibody-drug conjugate, Modified antibodies are, (a) A step of contacting a glycoprotein containing an antibody and a core-GlcNAc moiety connected to an asparagine residue in the amino acid region 290-305 of the antibody's heavy chain with endoglycosidase Endo SH, thereby forming an intermediate antibody containing the terminal GlcNAc moiety, and (b) A step of contacting an intermediate antibody with 4-AzGalNAc-UDP in the presence of the β-(1,4)-GalNAcT enzyme to form a modified antibody containing a modified GlcNAc moiety. Obtained by, The method wherein steps (a) and (b) are performed simultaneously.

5. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 3, or an optical isomer thereof, and one or more pharmaceutically acceptable carriers.

6. The pharmaceutical composition according to claim 5 for treating a target cancer.

7. The pharmaceutical composition according to claim 5 for use in the manufacture of a pharmaceutical for treating a target cancer.

8. The pharmaceutical composition according to claim 6 or 7, wherein the cancer is selected from ovarian cancer, non-small cell lung cancer, endometrial cancer, papillary renal cell carcinoma, salivary duct cancer, papillary thyroid cancer, clear cell renal carcinoma, breast cancer, kidney cancer, cervical cancer, and bile duct cancer.

9. The pharmaceutical composition according to claim 8, wherein the non-small cell lung cancer is subtyped as adenocarcinoma.

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