Improved CD30-targeting antibody drug conjugates and methods of use thereof

Abstract

The present invention relates to an antibody drug conjugate (ADC), comprising: (a) brentuximab, the ADC comprising a tubulin tyrosine ligase recognition sequence and an unnatural amino acid at the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of all of the heavy and light chains of brentuximab; and (b) at least one drug moiety, wherein the drug moiety is linked to each of the unnatural amino acids via a linker. The present invention further relates to methods of making the ADC, pharmaceutical compositions comprising the ADC, and uses thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to European Patent Application No. 20216838.1, filed December 23, 2020, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] TECHNICAL FIELD OF THEINVENTION The present invention relates to antibody drug conjugates (ADCs) comprising brentuximab and at least one drug moiety, methods of making said ADCs, pharmaceutical compositions comprising said ADCs, and uses thereof. [Background technology]

[0003] background Lymphomatous malignancies, accounting for approximately 4-5% of all cancers, are of lymphatic origin. In classical Hodgkin lymphoma as well as some non-Hodgkin lymphomas, such as anaplastic large cell lymphoma and peripheral T-cell lymphoma, CD30 has recently proven to be a valuable tumor biomarker for targeted treatment. With the first-in-class approval of Brentuximab vedotin in 2011, the first CD30-specific biological drug entered the market and has since become an essential treatment option showing good overall responses. Brentuximab vedotin consists of a tumor-targeting chimeric IgG1 antibody element, brentuximab, and a linker-payload element containing a cathepsin B-cleavable valine-citrulline linker moiety chemically connected with a potent payload moiety, monomethyl auristatin E, which efficiently induces apoptosis after intracellular delivery and release.

[0004] Brentuximab vedotin is an antibody-drug conjugate (ADC), a relatively new treatment that has attracted great interest in recent years. These biologics deliver potent cytotoxic drugs directly to tumor cells, which may therefore broaden the therapeutic window compared to conventional chemotherapy. To date, nine stochastically conjugated ADCs have been approved. These molecules are based on conjugation with activated carboxylic acids (e.g., Mylotarg, Besponsa, Kadcyla) that react randomly with exposed lysine residues. Alternatively, maleimides have become the most important conjugation reagents for conjugating linker payload moieties to free cysteines after intrachain disulfide reduction (e.g., Brentuximab vedotin (available as ADCETRIS®), Porayvy, Padceb, Enhartz, Trodelvy, Brenrep).

[0005] However, brentuximab vedotin is known to cause hypersensitivity and long-term toxicity as side effects such as thrombocytopenia in clinical routine.

[0006] Thus, there remains a need for improved brentuximab-containing ADCs, preferably with reduced side effects. The present invention aims to address this need. Summary of the Invention

[0007] This need is addressed by the subject matter defined in the claims and embodiments described herein.

[0008] Thus, the present invention relates to an antibody drug conjugate (ADC), said ADC comprising: (a) brentuximab, comprising a tubulin tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of all of the heavy and light chains of brentuximab; and (b) at least one drug moiety; Including, Here, the drug moiety is linked to each of the unnatural amino acids via a linker.

[0009] The heavy chain of brentuximab may have an amino acid sequence comprising or has at least 95% sequence identity to SEQ ID NO: 1, and / or the light chain of brentuximab has an amino acid sequence comprising or has at least 95% sequence identity to SEQ ID NO: 2, preferably brentuximab consists of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain consisting of the amino acid sequence of SEQ ID NO: 2.

[0010] The drug moiety may be selected from the group consisting of camptothecins, maytansinoids, calicheamicins, duocarmycins, tubulysins, amatoxins, dolastatins and auristatins such as monomethylauristatin E (MMAE), pyrrolobenzodiazepine dimers, indolinobenzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof, preferably the drug moiety is MMAE.

[0011] The recognition sequence for tubulin tyrosine ligase may have at least the amino acid sequence X1X2X3X4 (SEQ ID NO:3), where X1 and X2 are any amino acid, X3 is E, D, or C, and X4 is E, preferably where X2 is G, S, A, V, or F, and / or X1 is E, D, A, K, or P.

[0012] The recognition sequence may be EGEE (SEQ ID NO: 4), and preferably the recognition sequence is The file is TIFF2024500922000001.tif18165.

[0013] The unnatural amino acid may be a 2-, 3- or 4-substituted tyrosine or derivative substituted at the benzylic position. The 3- or 4-substituted tyrosine derivative may be 3-nitrotyrosine, 3-aminotyrosine, 3-azidotyrosine, 3-formyltyrosine, 3-acetyltyrosine or 4-aminophenylalanine, preferably the unnatural amino acid is 3-formyltyrosine.

[0014] The linker may be preferably cleavable by a protease, more preferably by a cathepsin, such as cathepsin B. The linker may comprise a valine-citrulline moiety. The linker may comprise a hydroxylamine group and the unnatural amino acid comprises a formyl group ortho to the hydroxyl group in the aromatic ring, such as 3-formylthiolsine, where the hydroxylamine group of the linker forms an oxime with the formyl group of the unnatural amino acid after conjugation.

[0015] Brentuximab may be conjugated to 2, 4, 6 or 8, preferably 2 or 4, drug moieties, more preferably 2 drug moieties.

[0016] The linker, prior to being linked to the unnatural amino acid, has the structure 1: and may have the structure shown in TIFF2024500922000002.tif14128, wherein R is one or more drug moieties, optionally linked to the hydroxylamine of Structure 1 by one or more cleavage sites, preferably wherein the hydroxylamine of Structure 1 is conjugated to an unnatural amino acid.

[0017] The linker may have the structure 2 or 3: and may have the structure shown in TIFF2024500922000003.tif34128, wherein Z is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; where D is one or more drug moieties; and wherein Y is a cleavage site, such as the cleavage site of a cathepsin, such as cathepsin B; Preferably, herein, the hydroxylamine of structure 2 or 3 is conjugated to an unnatural amino acid.

[0018] The linker may have the structure 4 or 5: It may have the structure shown in TIFF2024500922000004.tif87140, where D is a drug moiety, preferably MMAE.

[0019] The unnatural amino acid may be 3-formyl tyrosine, and the hydroxylamine group of the linker may form an oxime with the 3-formyl group of the unnatural amino acid.

[0020] The present invention further relates to a method of making an ADC as defined herein, said method comprising: (a) introducing or appending a recognition sequence for tubulin tyrosine ligase to the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of both the light and heavy chains of brentuximab; (b) contacting the brentuximab from step (a) in the presence of tubulin tyrosine ligase and an unnatural amino acid under conditions suitable for tubulin tyrosine ligase to ligate the brentuximab to the unnatural amino acid; and (c) conjugating an optionally cleavable linker comprising a drug moiety to the linked brentuximab obtained in step (b). Includes.

[0021] The invention further relates to an ADC obtained by the method of making an ADC as defined herein.The invention further relates to an ADC obtained by the method of making an ADC as defined herein.

[0022] The invention further relates to pharmaceutical compositions comprising the ADCs of the invention.

[0023] The present invention further relates to the ADC of the present invention or the pharmaceutical composition of the present invention for use in a method for treating a disease.Preferably, the disease is associated with overexpression of CD30.More preferably, the disease is selected from the group consisting of lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemia, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell cancer, graft-versus-host disease (GvHD), and lupus, particularly systemic lupus erythematosus (SLE), preferably Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). [Brief description of the drawings]

[0024] The invention will be better understood with reference to the detailed description, taken in conjunction with the non-limiting examples and the accompanying drawings, in which: [Figure 1] Chromatograms of analytical size-exclusion chromatography (SEC) after Protein A chromatography (PAC) are shown. Figure 1A shows the SEC of unmodified brentuximab, and Figures 1B-D show the SEC of a Tub-tagged variant of brentuximab. The monoclonal antibody (mAb) has a high monomer percentage after expression and purification. [Diagram 2]Analysis of Brentuximab (black) and Tub tag analogs of Brentuximab (abbreviated as "Bren") shown as Bren. LC-Tub (grey, dotted line), Bren. HC-Tub (grey), Bren. LCHC-Tub (black, dotted line) are shown. Figure 2A shows a chromatogram of hydrophilic interaction chromatography (HIC) with normalized absorption spectra recorded at 220 nm. Retention time is a measure of hydrophobicity. Figure 2B shows differential scanning fluorimetry (DSF) for the determination of melting temperature (Tm). [Diagram 3] Analysis of brentuximab Tub-tag ADC by analytical SEC and HIC is shown. Each of the brentuximab variants (brentuximab is abbreviated as "Bren", and Figures 3A and D show Bren. HC-vc-PAB-MMAE DAR2 (also referred to herein as "Bren. HC-2"), Figures 3B and E show Bren. LC-vc-PAB-MMAE DAR2 (also referred to herein as "Bren. LC-2"), and Figures 3C and F show Bren. LCHC-vc-PAB-MMAE DAR4 (also referred to herein as "Bren. LCHC-2")) was expressed, purified by PAC, and conjugated to payload 2 for the generation of DAR2 and payload 2 or payload 3 for the generation of DAR4 ADC. After final polishing by HIC and buffer exchange, the Tub-tag ADC was analyzed for aggregate content and DAR homogeneity. All Tub-tagged ADCs contained very low contents of HMWS (<1%) and showed excellent DAR homogeneity. [Figure 4]Analysis of Brentuximab Tub tag ADC by middle-up protein MS after deconvolution of crude spectrum. Figure 4A shows the results of Bren. HC-Tub, Figure 4B shows the results of Bren. LCHC-Tub, Figure 4C shows the results of Bren-LC-Tub, Figure 4D shows the results of Bren. HC-vc-PAB-MMAE (also referred to herein as "Bren. HC-2"), Figure 4E shows the results of Bren-LCHC-vc-PAB-MMAE (also referred to herein as "Bren. LCHC-2"), and Figure 4F shows the results of Bren. LC-2 (vc-PAB-MMAE) (also referred to herein as "Bren. LC-3"). The mass shift of 1369 m / z is due to the incorporation of 3-formyl-L-tyrosine 1 and oxime linkage with payload 2. After oxime linkage with payload 3, a mass difference of 2604 m / z was observed. These results are consistent with the calculated values. [Diagram 5] In Figure 5A, RP-HPLC analysis of valine-citrulline (vc) containing payloads 2 and 3 during digestion reaction with protease cathepsin B (1:1000 for each vc moiety) is shown. Before analysis, the reaction was stopped with E-64. Chromatograms were recorded at 220 nm after 150 min reaction time. The plot in Figure 5B shows the increase in free MMAE (black) and the decrease in payload 2 (grey, circles) over time. Figure 5C shows the cleavage of payload 3. During reaction with cathepsin B, an intermediate containing one MMAE moiety is formed (grey, triangles). [Figure 6]The results of storage at elevated temperatures for brentuximab vedotin and brentuximab Tub-tag ADC are shown. Figure 6A shows the increase in HMWS over the course of the study (Bren.LC-2(vc-PAB-MMAE) (also referred to herein as "Bren. LC-3"); Bren. LC-vc-PAB-MMAE (also referred to herein as "Bren. LC-2"); Bren. HC-vc-PAB-MMAE (also referred to herein as "Bren. HC-2"). The highest increase in aggregate content can be seen for brentuximab vedotin. In comparison, the HMWS content of the Tub-tag ADC remains nearly constant. Figures 6B and 6C show SEC chromatograms and normalized absorption spectra recorded at 220 nm for brentuximab vedotin (average drug-to-antibody ratio (DARav) 4) and Bren. LC conjugated with payload 3 (Bren. LC-3, DAR 4). [Figure 7] 7A and 7B show the results of Brentuximab Vedotin storage in mouse plasma at 37° C., and FIG. 7C and 7D show the results of Bren. LC-3 DAR 4 storage in mouse plasma at 37° C. [Figure 8] Shows in vitro efficacy of brentuximab Tub-tag ADC in the CD30-overexpressing cell line Karpas299 and the CD30-negative cell line HL60. [Figure 9] Figure 1 shows the in vivo efficacy of Bren. LC-2 expressed in terms of tumor volume (cm3). For evaluation of the in vivo efficacy of Bren. LC-2, mice with tumor volumes between 100-150 mm3 were randomized and treated with two injections of 1.5 mg / kg on days 7 and 10 after tumor implantation. [Figure 10-1]Figure 10 shows the in vivo efficacy of Bren. LC-2 expressed in terms of tumor volume (cm3) and survival percentage of treated animals. For evaluation of the in vivo efficacy of Bren. LC-2, mice with tumor volumes between 100 and 150 mm3 were randomized and treated with a single 1.0 mg / kg injection. [Figure 10-2] See description of Figure 10-1. [Figure 11] Figure 1 shows the in vivo efficacy of Bren. LC-3 expressed in terms of tumor volume (cm3) and survival percentage of treated animals (Kaplan-Meier plot). For evaluation of the in vivo efficacy of Bren. LC-3, mice with tumor volumes between 100-150 mm3 were randomized and treated with two injections of 0.5 mg / kg on days 8 and 11 after tumor implantation. [Figure 12] PK analysis of Brentuximab Tub-tagged MMAE (Bren LC-2). Figure 12A shows the amount of intact ADC compared to Brentuximab Vedotin. Figure 12B shows the amount of MMAE transferred to blood proteins as analyzed by mass spectrometry. [Figure 13] 1 shows the mean serum concentrations of intact ADC and total antibodies in male and female rats following intravenous (bolus) administration of brentuximab Tub Tag ADC at 10 mg / kg compared to brentuximab vedotin on days 1, 8, 15, and 22. [Figure 14]14 shows an exemplary toxicity profile of Brentuximab Tub Tag ADC (Bren LC-2) (grey bars) compared to Brentuximab Vedotin (black bars) in male and female rats. The parameters shown in FIG. 14 are reticulocytes in counts / microliter (RET in K / μl), red blood cells in million / microliter (RBC in M / μl), hemoglobin in million / microliter (Hb in M / μl), hematocrit in volume % (HTC in %), eosinophils in counts / microliter (EOS in K / μl), activated partial thromboplastin time (APPT) in seconds (sec), glucose level in millimoles / liter (mmol / L), thymus weight in grams (g), and testis weight in grams (g). [Figure 15] The chromatogram of purified HA-VC-PAB-MMAE is shown. [Figure 16] The chromatogram of purified Boc-Glu-(VC-PAB-MMAE)2 is shown. [Figure 17] The chromatogram of purified Boc-HA-Glu-(VC-PAB-MMAE)2 is shown. [Figure 18] The chromatogram of purified HA-Glu-(VC-PAB-MMAE)2 is shown. [Figure 19] Toxicokinetic analysis of brentuximab Tub-tag MMAE in cynomolgus monkeys dosed at 12 and 15 mg / kg. Total mAb and intact ADC were assessed by ELISA. The high degree of overlap of the intact ADC and total mAb curves indicates high stability of brentuximab Tub-tag MMAE at both dose levels. [Figure 20]Figure 20 shows body weight and selected different blood values ​​collected throughout a repeat dose study of Brentuximab Tub-Tagged MMAE (Bren. LC-2) in cynomolgus monkeys. The top left figure in Figure 20 shows body weight over time. The top right figure in Figure 20 shows lymphocyte concentration over time. The bottom left figure in Figure 20 shows neutrophil concentration over time. The bottom right figure in Figure 20 shows white blood cell concentration over time. Data are shown as mean and error for three groups of two females receiving repeat doses of Brentuximab Tub-Tagged MMAE (Bren. LC-2) at 6, 12, and 15 mg / kg in each case, compared to literature values ​​from a similar study with Brentuximab Vedotin (Adcetris). Body weight is shown in kilograms, lymphocytes in billion / liter, neutrophils in billion / liter, and white blood cells in billion / liter. [Figure 21] The amino acid sequences of the heavy and light chains of brentuximab are shown in the 2013 Report of the Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau Ministry of Health, Labour and Welfare on the Deliberation Results for Brentuximab Vedotin, available at the following link: https: / / www.pmda.go.jp / files / 000229787.pdf. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention The present invention is described in detail below and is further illustrated by the accompanying examples and figures.

[0026] Lymphomatous malignancies account for approximately 4-5% of all cancers and are of lymphatic origin. In classical Hodgkin's lymphoma as well as some non-Hodgkin's lymphomas, such as anaplastic large cell lymphoma and peripheral T-cell lymphoma, CD30 has recently proven to be a valuable tumor biomarker for targeted treatment. With the first-in-class approval of brentuximab vedotin (Adcetris®) in 2011, the first CD30-specific biological drug entered the market and has since become an essential treatment option showing good overall responses. Brentuximab vedotin consists of a tumor-targeting chimeric IgG1 antibody element, brentuximab, and a linker-payload element containing a cathepsin B-cleavable valine-citrulline linker moiety chemically connected with a potent payload moiety, monomethyl auristatin E, which efficiently induces apoptosis after intracellular delivery and release.

[0027] However, brentuximab vedotin is known to cause hypersensitivity and long-term toxicity as side effects such as thrombocytopenia in clinical routine.

[0028] The inventors have been able to show that the use of tubulin tyrosine ligase surprisingly allows for highly specific and stoichiometrically defined conjugation of drug moieties to brentuximab modified by adding a TTL recognition sequence to the C-terminus of the light and / or heavy chain. TTL can be used to conjugate unnatural amino acids such as 3-formyl tyrosine to the TTL recognition sequence. Drug moieties, such as MMAE, can be specifically conjugated to unnatural amino acids by linkers, such as linkers that contain an active group that can react with an active group on the unnatural amino acid. For example, the linker may contain a hydroxylamine that reacts with the formyl group of the unnatural amino acid to form an oxime (see Example 1). In summary, the inventors have shown that brentuximab vedotin can be surprisingly improved by combining its functional elements, brentuximab and MMAE, by the conjugation strategy described herein. The ADC of the present invention surprisingly exhibits higher in vitro stability (see Example 2), higher in vivo efficacy (see Example 4), higher in vivo stability (see Example 5), and has an improved toxicity profile (see Example 6), thereby surprisingly reducing the side effects of brentuximab vedotin.

[0029] Thus, the present invention relates to an antibody drug conjugate (ADC), said ADC comprising: (a) brentuximab, comprising a tubulin tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of all of the heavy and light chains of brentuximab; and (b) at least one drug moiety; Including, Here, the drug moiety is linked to each of the unnatural amino acids via a linker.

[0030] The term "antibody drug conjugate" (ADC), as generally used herein, refers to the linkage of an antibody, such as brentuximab or an antigen-binding fragment thereof, to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc. The linkage can be a covalent bond or a non-covalent interaction, such as through electrostatic forces, preferably a covalent bond. A variety of linkers known in the art and described herein can be used to form antibody drug conjugates. The ADCs of the present invention have the formula Ab-(L-(D) x ) y "Ab" represents an antibody, such as brentuximab or an antigen-binding fragment thereof, "L" represents a linker, and "D" represents a drug moiety. "x" may be an integer from 1 to 10. "y" may be an integer from 1 to 10. Thus, x may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Thus, y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Purely by way of example, x or y may be an integer from 1 to 8, 1 to 6, 1 to 4, or 1 to 2.

[0031] "Ab": ​​Antibody Brentuximab As outlined herein, the ADCs of the invention have the formula Ab-(L-(D) x ) y The "Ab portion" of the ADC of the present invention is brentuximab. Brentuximab, also known as cAC10 or SGN-30, is an antibody well known to those skilled in the art (see, for example, Wahl et al. (2002), Cancer Res, 62:3736-3742). Brentuximab is a monoclonal antibody that specifically binds to CD30 on the cell surface of target cells. Binding to CD30 initiates the internalization of brentuximab, which then traffics to the lysosomal compartment. Inside the cell, the drug portion of the ADC can be released from the monoclonal antibody, for example, via cleavage or degradation of the linker. Brentuximab is included in the ADC of the present invention.

[0032] CD30, also known as TNFRSF8, is a cell membrane protein of the tumor necrosis factor receptor family and a tumor marker. This receptor is expressed by activated T and B cells, but not resting T and B cells. TRAF2 and TRAF5 can interact with this receptor and mediate signaling leading to activation of NF-κB. It is a positive regulator of apoptosis and has also been shown to limit the proliferative capacity of autoreactive CD8 effector T cells, protecting the body from autoimmunity. Two alternatively spliced ​​transcript variants of this gene encoding different isoforms have been reported. CD30 is associated with anaplastic large cell lymphoma. It is expressed in embryonal carcinomas but not seminomas, and is therefore a useful marker to distinguish these germ cell tumors. CD30 and CD15 are also expressed in Reed-Sternberg cells, typical of Hodgkin's lymphoma.

[0033] Each heavy chain of Brentuximab as an approved drug and used herein has the amino acid sequence of SEQ ID NO: 1 (also shown in FIG. 21). Antibodies whose heavy chains have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 may also be used herein. For example, as shown in FIG. 21, the last residue K at position 447 can be removed as needed. Such heavy chains can be considered variants of the heavy chain of Brentuximab. Similarly, for example, the first two or three N-terminal residues of the heavy chain of Brentuximab can be deleted. Such heavy chains have three or four mutations compared to Brentuximab, and therefore have about 99.2% (four amino acid differences) to 99.8% (one amino acid difference) sequence identity to the Brentuximab heavy chain.

[0034] Each light chain of Brentuximab as approved drug and used herein has the amino acid sequence of SEQ ID NO: 2 (also shown in FIG. 21). Antibodies whose light chains have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 can also be used herein. For example, the last residue C at position 218 can be removed as needed. Such light chains can be considered as variants of the light chain of Brentuximab. Similarly, for example, the first two or three N-terminal residues of the light chain of Brentuximab can be deleted. Such light chains have three or four mutations compared to Brentuximab, and therefore have 98.2% (four amino acid differences) to 99.5% (one amino acid difference) sequence identity to the Brentuximab light chain.

[0035] In one particular embodiment, brentuximab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1, and a light chain comprising the amino acid sequence of SEQ ID NO: 2. In a further particular embodiment, brentuximab consists of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain consisting of the amino acid sequence of SEQ ID NO: 2.

[0036] Generally, as used herein, the term "percent identical" or "percent identity" refers to the degree to which sequences or subsequences are the same in the context of multiple nucleic acid or polypeptide sequences. Two sequences are "identical" if they have the same sequence of amino acids or nucleotides over the region being compared. Two sequences are "substantially identical" if they have a specified percentage of the same amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or the entire sequence if not specified), when compared and aligned for maximum correspondence over a comparison window, or over a specified region, using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.

[0037] Percentages of sequence homology or sequence identity can be determined herein, for example, using the program BLASTP, version blastp 2.2.5 (November 16, 2002) (see Altschul et al., Nucleic Acids Res, 1997). In this embodiment, the percentage of homology is preferably determined using an alignment of the entire polypeptide sequence, including the propeptide sequence, using the wild-type protein backbone as the basis in the pairwise comparison (matrix: BLOSUM 62; gap cost: 11.1; cutoff value 10 -3 It is based on the alignment set to 0. It is calculated as the percentage of the number of "positives" (homologous amino acids) that result from dividing the BLASTP program output by the total number of amino acids selected by the program for the alignment.

[0038] TTL recognition sequence and linking of unnatural amino acids to brentuximab In the ADCs of the present invention, brentuximab can be fused to one or more TTL recognition sequences, for example, at the C-terminus of the light chain, the heavy chain, or both the light and heavy chains. This TTL recognition sequence allows TTL to conjugate an unnatural amino acid to the TTL recognition sequence at the C-terminus of the light and / or heavy chain of brentuximab. As a result, brentuximab contained in the ADCs of the present invention comprises a recognition sequence for tubulin tyrosine ligase ("TTL recognition sequence") at the C-terminus of its light chain, the C-terminus of its heavy chain, or all of the C-terminus of its heavy and light chains. In the ADCs of the present invention, an unnatural amino acid is linked to this TTL recognition sequence. A drug moiety is linked to this unnatural amino acid via a linker. As used herein, "Tub tag" refers to a recognition sequence for TTL or a TTL recognition sequence, preferably SEQ ID NO: 4 or SEQ ID NO: 5.

[0039] The term "tubulin tyrosine ligase", as used herein and throughout the description, may also be abbreviated as "TTL", and includes polypeptides capable of functionalizing a polypeptide, i.e., covalently attaching a non-natural amino acid, as defined herein, to a polypeptide. For that action, the polypeptide preferably includes a recognition sequence for TTL. The term includes TTL from a eukaryote, preferably a mammal, more preferably from the gray wolf (Canis Lupus). A preferred TTL is shown in SEQ ID NO: 13. Also included in the term is a TTL that has 70%, 80%, 90%, or 95% or more identity over its entire amino acid sequence to the amino acid sequence of TTL shown in SEQ ID NO: 13. Preferably, such polypeptides having an amino acid sequence sharing the aforementioned identity have TTL activity. TTL activity can be tested as known in the art or as described herein. The percentage of sequence identity can, for example, be determined herein as described above. Preferably, the amino acid sequence shown in SEQ ID NO:13 is used as a reference in the pairwise comparison.

[0040] As used throughout this specification and description, the term "functionalization" in all its grammatical forms as used herein means "covalently attaching a non-natural amino acid" to a polypeptide such as brentuximab. Without wishing to be bound by a particular theory, it is assumed that TTL adds a non-natural amino acid, as defined herein, to the final C-terminal amino acid of the TTL recognition sequence.

[0041] Methods for modifying proteins using tubulin tyrosine ligase (TTL) are described in WO 2016 / 066749 and WO 2017 / 186855, which are incorporated herein by reference. TTL requires a recognition sequence in order for TTL to bind unnatural amino acids to a protein of interest, such as the antibody of the ADC described herein. The ADC described herein may be modified to include a recognition sequence for tubulin tyrosine ligase (TTL) at the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of both the light chain and the heavy chain of brentuximab, comprising at least the amino acid sequence X4X3X2X1. The terms "recognition sequence" or "recognition motif" are used interchangeably herein and refer to a stretch of amino acids recognized by TTL. Such recognition sequences are known in the art; see, for example, Ruediger et al. (1994), Eur. J. Biochem. 220, 309-320 or Prota e al. (2013), J. Cell. Biol. 200, No. 3, 259-270. Moreover, the skilled artisan can easily test whether an amino acid sequence of interest is a TTL recognition sequence, for example by applying the test "TTL-mediated peptide tyrosination" described in Ruediger et al. "Recognized" by TTL includes the binding of TTL to the recognition motif. The recognition motif advantageously comprises at least four amino acids, designated herein as X4, X3, X2, and X1. In general, "X" may represent any amino acid, unless otherwise stated herein.Amino acids include, but are not limited to, the 20 "standard" amino acids: isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophan (Trp, W), valine (Val, V), alanine (Ala, A), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), proline (Pro, P), serine (Ser, S), tyrosine (Tyr, Y), arginine (Arg, R), and histidine (His, H). The present invention also includes, but is not limited to, D-configured amino acids, β-amino acids, amino acids with side chains, as well as all unnatural amino acids known to those of skill in the art.

[0042] The recognition sequence for tubulin tyrosine ligase may have at least the amino acid sequence X1X2X3X4 (SEQ ID NO: 3), where X1 and X2 are any amino acid, X3 is E, D, or C, and X4 is E, preferably where X2 is G, S, A, V, or F, and / or X1 is E, D, A, K, or P. The recognition sequence may be EGEE (SEQ ID NO: 4), preferably the recognition sequence is TIFF2024500922000005.tif18165. In one embodiment, the recognition sequence is The file is TIFF2024500922000006.tif4128.

[0043] The number of TTL recognition sequences present in the brentuximab contained in the ADC of the invention determines the drug-antibody ratio (DAR). The drug-antibody ratio (DAR) is a defined stoichiometric value (e.g., the formula Ab-(L-(D))) for a particular conjugate molecule. x ) y(y multiplied by x in (x)), it is understood that the value may be an average value when used to describe a sample containing many molecules, typically due to some degree of heterogeneity associated with the conjugation step. Thus, in the context of any one of the antibody drug conjugates described hereinbefore and hereinafter, the average loading of the antibody drug conjugate to a sample is referred to as the drug-antibody ratio, or "DAR". However, preferably, the present invention provides ADCs in which substantially all or all ADCs have the required DAR, or in other words, are stoichiometrically defined.

[0044] The ADC of the present invention is based on the monoclonal IgG antibody brentuximab. IgG antibodies contain or consist of two "light chains" and two "heavy chains". The maximum number of TTL recognition sequences in brentuximab is limited to four, since the TTL recognition sequence can only be condensed to the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of both the light and heavy chains. If either the heavy or light chain contains a TTL recognition sequence at the C-terminus, then the brentuximab contained in the ADC of the present invention has two TTL recognition sequences. Heavy chain and When the light chain contains a TTL recognition sequence at the C-terminus, the brentuximab contained in the ADC of the invention has four TTL recognition sequences.

[0045] In the ADCs of the present invention, the TTL recognition sequence may be directly fused to the C-terminus of the chain of the antibody Brentuximab. In this context, see the Brentuximab heavy chain of (SEQ ID NO: 12), where the TTL recognition sequence is directly fused to the C-terminus of the heavy chain (i.e., there are no amino acid residues in between). Alternatively, a (peptide) linker may be placed between the TTL recognition sequence and the chain of the antibody. Thus, the Brentuximab light chain, the heavy chain, or both the light and heavy chains may comprise an amino acid linker sequence placed between the TTL recognition sequence and their respective C-terminus. As an illustration, see the light chain of SEQ ID NO: 11, where the TTL recognition sequence is fused to the C-terminus of the Brentuximab light chain via a GGGGS (G4S) linker. The amino acid linker may have any suitable length, as long as the function of the TTL recognition sequence is maintained. For example, the amino acid linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues. See again the light chain of SEQ ID NO: 11, where a GGGGS (G4S) linker is placed between the TTL recognition sequence and the C-terminus of brentuximab. It is also possible to use other standard linkers such as (G4S)2 or (G4S)3, which are commonly used as linkers for condensing different chains of recombinant antibody molecules, such as single-chain Fv fragments, to each other. Thus, in these examples, the linker may have a length of 10 amino acid residues ((G4S)2) or 15 amino acid residues ((G4S)3). However, the linker is by no means limited to a length of 15 amino acid residues, although in accordance with the above disclosure, the amino acid linker sequence may comprise more than 15 amino acids.

[0046] Also in accordance with the foregoing disclosure, the ADCs of the invention may be constructed such that none of the antibody chains contain a peptide linker between the TTL recognition sequence and the respective antibody chain. In such ADCs, the TTL recognition sequence may be directly fused to both the light and heavy chains of brentuximab. However, the ADCs of the invention may be constructed such that one of the antibody chains contains a peptide linker between the TTL recognition sequence and the respective antibody chain. In such ADCs, the TTL recognition sequence may be fused to each of the light chains via a linker (e.g., a GGGGS linker, as in the light chain of SEQ ID NO: 11), but the TTL recognition sequence may be directly fused to each of the two heavy chains of brentuximab. Finally, the ADCs of the invention may be constructed such that both the antibody light chain and the antibody heavy chain contain a peptide linker between the TTL recognition sequence and the respective antibody chain. In such ADCs, the TTL recognition sequence may be fused to each of the two light chains and each of the two heavy chains of brentuximab via a linker (e.g., a GGGGS linker, or a (G4S)2 linker).

[0047] In one embodiment, Brentuximab comprises a TTL recognition sequence at the C-terminus of the light chain, i.e., a total of two. The TTL recognition sequence may be fused to the C-terminus of the light chain via an amino acid linker sequence (e.g., GGGGS). The TTL recognition sequence may be directly attached to the C-terminus of the light chain. The Brentuximab heavy chain may comprise a polypeptide having a sequence as set forth in SEQ ID NO: 1, and the Brentuximab light chain may comprise a polypeptide having a sequence as set forth in SEQ ID NO: 11. The Brentuximab heavy chain may comprise a polypeptide having a sequence as set forth in SEQ ID NO: 1, and the Brentuximab light chain may comprise a polypeptide having a sequence as set forth in SEQ ID NO: 14. The Brentuximab heavy chain may consist of a polypeptide having a sequence as set forth in SEQ ID NO: 1, and the Brentuximab light chain may consist of a polypeptide having a sequence as set forth in SEQ ID NO: 11. The brentuximab heavy chain may consist of a polypeptide having the sequence shown in SEQ ID NO:1, and the brentuximab light chain may consist of a polypeptide having the sequence shown in SEQ ID NO:14.

[0048] In one embodiment, Brentuximab comprises TTL recognition sequences at the C-terminus of the light chain and the heavy chain, i.e., a total of four. The TTL recognition sequence may be condensed to the C-terminus of the light chain via an amino acid linker sequence (e.g., GGGGS), or the TTL recognition sequence may be directly attached to the C-terminus of the heavy chain. The TTL recognition sequence may be attached to the C-terminus of the light chain via an amino acid linker (e.g., GGGGS), or the TTL recognition sequence may be attached to the C-terminus of the heavy chain via an amino acid linker (e.g., GGGGS). The TTL recognition sequence may be directly attached to the C-terminus of the light chain, or the TTL recognition sequence may be directly attached to the C-terminus of the heavy chain. The Brentuximab heavy chain may comprise a polypeptide having a sequence as shown in SEQ ID NO: 12, and the Brentuximab light chain may comprise a polypeptide having a sequence as shown in SEQ ID NO: 11. The Brentuximab heavy chain may comprise a polypeptide having a sequence as shown in SEQ ID NO: 12, and the Brentuximab light chain may comprise a polypeptide having a sequence as shown in SEQ ID NO: 14. The brentuximab heavy chain may consist of a polypeptide having a sequence as set forth in SEQ ID NO: 12, and the brentuximab light chain may consist of a polypeptide having a sequence as set forth in SEQ ID NO: 11. The brentuximab heavy chain may consist of a polypeptide having a sequence as set forth in SEQ ID NO: 12, and the brentuximab light chain may consist of a polypeptide having a sequence as set forth in SEQ ID NO: 14.

[0049] In one embodiment, Brentuximab comprises a TTL recognition sequence at the C-terminus of the heavy chain, i.e., a total of two. The TTL recognition sequence may be directly attached to the C-terminus of the heavy chain. The TTL recognition sequence may be attached to the C-terminus of the heavy chain via an amino acid linker sequence (e.g., GGGGS). The Brentuximab heavy chain may comprise a polypeptide having a sequence as shown in SEQ ID NO: 12, and the Brentuximab light chain may comprise a polypeptide having a sequence as shown in SEQ ID NO: 2. The Brentuximab heavy chain may consist of a polypeptide having a sequence as shown in SEQ ID NO: 12, and the Brentuximab light chain may consist of a polypeptide having a sequence as shown in SEQ ID NO: 2.

[0050] However, the number of TTL recognition sequences is not necessarily equal to the number of drug moieties conjugated to the non-natural amino acid, i.e., it is not necessarily equal to the DAR. Also included in the present invention is linking multiple drug moieties to the non-natural amino acid (via linkers). For example, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2 drug moieties may be linked to one non-natural amino acid via linkers. In this case, the linker may act as a scaffold to which 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2 drug moieties may be linked. The linker is then linked to the non-natural amino acid. An exemplary linker that acts as a scaffold for two drug moieties is structure 3 or 5. Thus, an antibody may be conjugated to 2, 4, 6, or 8, preferably 2 or 4 drug moieties, more preferably 2 drug moieties.

[0051] Unnatural Amino Acids The unnatural amino acid is the anchor point of the linker in the "Ab" portion of the ADC of the invention. In this regard, the "ADC formula" Ab-(L-(D) x ) y Therefore, the unnatural amino acid that can be linked by TTL to the C-terminus of a polypeptide having a TTL recognition sequence at its C-terminus, such as brentuximab, included in the ADC of the invention contains a functional group that can react with a functional group of the linker, L, to form a covalent bond.

[0052] In general, the unnatural amino acid can be considered a tyrosine derivative. Preferably, the tyrosine derivative contains an active group that allows for conjugation of a drug moiety via a linker.

[0053] The tyrosine derivatives may (further) contain unnatural (non-natural) functional groups, which are preferably used for chemoselective or bioorthogonal modifications. The functional groups may be suitable for click chemistry. The term "click chemistry" refers to a chemical philosophy introduced by Kolb, Finn and Sharpless in 2001 and includes a group of powerful ligation reactions that can rapidly and reliably generate covalent bonds by combining small units containing reactive groups. Click chemistry reactions are typically modular, broad in scope, have high chemical yields, generate harmless by-products, are stereospecific, and / or can be carried out under simple, physiological reaction conditions using readily available starting materials and reagents. In addition, click chemistry reactions preferably do not use toxic solvents or use harmless or easily removed solvents (preferably water) and / or provide simple product isolation by non-chromatographic methods (crystallization or distillation). The well-defined exothermic reaction makes the reactants "spring-loaded".

[0054] Click chemistry reactions include, for example, hetero-Diels-Alder reactions, which are cycloaddition reactions, especially from the 1,3-dipolar family; nucleophilic ring-opening reactions of strained heterocyclic electrophiles, such as epoxides, aziridines, cyclic sulfates, cyclic sulfamidates, aziridinium ions and episulfonium ions; non-aldol type carbonyl chemistry (e.g., formation of oxime ethers, hydrazones and aromatic heterocycles); and additions to carbon-carbon multiple bonds; for example, epoxidation, dihydroxylation, aziridination, and oxidation reactions such as nitrosyl and sulfenyl halide additions, as well as certain Michael addition reactions. The general principles of click chemistry reactions are described by Kolb, Finn and Sharpless (2001). It is within the knowledge of one of ordinary skill in the art to select a suitable click chemistry reaction for linking the desired drug moiety via a linker to the tyrosine derivative or unnatural amino acid covalently attached to brentuximab contained in the ADC of the present invention.

[0055] The term "click chemistry handle" as used herein refers to a reactant or reactive group that can participate in a click chemistry reaction. Such reactants or reactive groups are preferably unnatural (non-natural) functional groups for chemoselective or bioorthogonal modification; however, they may alternatively be natural functional groups for chemoselective or bioorthogonal modification. For example, a strained alkyne, such as cyclooctyne, is a click chemistry handle because it can participate in strain-promoted cycloaddition, such as strain-promoted azide-alkyne cycloaddition (SPAAC). In general, a click chemistry reaction requires at least two molecules that contain click chemistry handles that can react with each other. Such pairs of click chemistry handles that are reactive with each other are sometimes referred to herein as "partner click chemistry handles". For example, azide is a partner click chemistry handle for cyclooctyne or any other alkyne. In the context of the present invention, the click chemistry handle may preferably be selected from the group consisting of terminal alkynes, azides, strained alkynes, dienes, dienophiles, alkoxyamines, carbonyls, β-arylethylamines, phosphines, hydrazides, hydrazines, thiols, tetrazines, alkenes, cyclooctenes, norbornenes, tetrazines, nitrones, cyanobenzothiazoles, and cyclooctynes. Other suitable click chemistry handles are readily available to the skilled artisan.

[0056] In the context of click chemistry conjugation, the conjugation is via a covalent bond formed by reaction of the click chemistry handles. In certain embodiments, the association is covalent and the entities are said to be "conjugated" to one another.

[0057] It should be noted that the present invention is not limited to the foregoing, and exemplary click chemistry handles, as well as additional click chemistry handles, pairs of reactive click chemistry handles, and reaction conditions for such pairs of click chemistry handles will be apparent to one of skill in the art.

[0058] Other methods suitable for conjugating a linker to an unnatural amino acid in brentuximab in an ADC of the invention include the Staudinger reaction (e.g., Staudinger ligation, Staudinger-phosphite reaction, Staudinger-phosphonite reaction), strain-promoted cycloaddition, tetrazine ligation, inverse electron demand Diels-Alder reaction, thiazolidine-forming reaction of an aldehyde or ketone with a 1,2-aminothiol, oxazolidine-forming reaction of an aldehyde, oxime formation, hydrazone formation, or acetal-forming reaction of a ketone with a 1,2-aminoalcohol, an aldehyde or ketone with a 1,2-diol, the Pictet-Spengler reaction, Trapp-Knoevenagel ligation, tandem-Knoevenagel condensation, thiol addition to an alkene or alkyne, cross-metathesis, metal catalyzed, particularly Pd, Cu, Ni and Fe catalyzed cross-coupling with tyrosine derivatives substituted with electron-withdrawing groups, preferably oxime formation.

[0059] It is envisioned that the drug moiety may be linked to the non-natural amino acid covalently attached to brentuximab via a linker, for example, by click chemistry or any other suitable method described herein. Thus, the drug moiety may be conjugated to the non-natural amino acid via a linker by a non-peptide bond, but may alternatively be conjugated to the non-natural amino acid via a linker by a peptide bond.

[0060] The non-natural amino acid may be a 2-, 3-, or 4-substituted tyrosine or 2-, 3-, or 4-substituted tyrosine derivative substituted at the benzylic position. The 3- or 4-substituted tyrosine derivative may be 3-nitrotyrosine, 3-aminotyrosine, 3-azidotyrosine, 3-formyltyrosine, 3-acetyltyrosine, or 4-aminophenylalanine. The 3-substituted tyrosine may be 3-nitrotyrosine. The 3-substituted tyrosine may be 3-aminotyrosine. The 3-substituted tyrosine may be 3-azidotyrosine. The 3-substituted tyrosine may be 3-formyltyrosine. The 3-substituted tyrosine may be 3-acetyltyrosine. The substituted tyrosine may be substituted phenylalanine. The 4-substituted tyrosine may be 4-aminophenylalanine. A particularly preferred unnatural amino acid is 3-formyltyrosine.

[0061] As used throughout this specification and description, the term "optionally substituted" or "substituted" refers to one or more (e.g., 1 to the maximum number of hydrogen atoms bonded to the group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms being substituted with an alkyl (preferably, C 1~6 Alkyl), alkenyl (preferably C 2~6 alkenyl), alkynyl (preferably C 2~6 alkynyl), aryl (preferably 3- to 14-membered aryl), heteroaryl (preferably 3- to 14-membered heteroaryl), cycloalkyl (preferably 3- to 14-membered cycloalkyl), heterocyclyl (preferably 3- to 14-membered heterocyclyl), halogen, CN, azide, NO2, OR 71 , N(R 72 )(R 73 ), ON(R 72 )(R 73 ), N + (O - )(R 72 )(R 73 ), S(O) 02 R71 , S(O) 02 OR 71 , OS(O) 02 R 71 , OS(O) 02 OR 71 , S(O) 02 N(R 72 )(R 73 ), OS(O) 02 N(R 72 )(R 73 ), N(R 71 )S(O) 02 R 71 , N.R. 71 S(O) 02 OR 71 , N.R. 71 S(O) 02 N(R 72 )(R 73 ), C(=W 1 )R 71 , C(=W 1 )W 1 R 71 , W 1 C(=W 1 )R 71 , and -W 1 C(=W 1 )W 1 R 71 where R 71 , R 72 , and R 73 , H, C 1~6 Alkyl, C 2~6 Alkenyl, C 26 independently selected from the group consisting of alkynyl, 3- to 7-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 3- to 7-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is selected from the group consisting of C 1~3 Alkyl, halogen, CF3, CN, azide, NO2, OH, O(C 1~3 alkyl), S(C 1~3 alkyl), NH2, NH(C 1~3 alkyl), N(C 1~3 alkyl)2, NHS(O)2(C 1~3alkyl), S(O)2NH 2-z (C 1~3 Alkyl) z , C(=O)OH, C(=O)O(C 1~3 alkyl), C(=O)NH 2z (C 1~3 Alkyl) z , NHC(=O)(C 1~3 alkyl), NHC(=NH)NH z2 (C 1~3 Alkyl) z , and N(C 1~3 Alkyl)C(=NH)NH 2-z (C 1~3 Alkyl) z where z is 0, 1, or 2, and C 1~3 Alkyl is methyl, ethyl, propyl or isopropyl; 1 are O, S, and NR 84 are independently selected from, where R 84 is -H or C 1~3 It is an alkyl.

[0062] The term "alkyl" as used herein and throughout the description refers to a monoradical of a saturated straight or branched chain hydrocarbon. Preferably, the alkyl group contains 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. In some embodiments, the alkyl group used in the present invention contains 1 to 20 carbon atoms (C 1~20 In another embodiment, the alkyl group used contains 1 to 15 carbon atoms (C 1~15 In another embodiment, the alkyl group used contains 1 to 10 carbon atoms (C 1~10 In another embodiment, the alkyl group used contains 1 to 8 carbon atoms (C 1~8In another embodiment, the alkyl group used contains 1 to 6 carbon atoms (C 1~6 In another embodiment, the alkyl group used contains 1 to 5 carbon atoms (C 1~5 In another embodiment, the alkyl group used contains 1 to 4 carbon atoms (C 1~4 In another embodiment, the alkyl group used contains 1 to 3 carbon atoms (C 1~3 In another embodiment, the alkyl group used contains 1 to 2 carbon atoms (C 1~2 alkyl). Examples of alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like, which may bear one or more substituents. Alkyl group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety. In some embodiments, the alkyl chain is linear. In some embodiments, the alkyl chain is branched. In some embodiments, the alkyl chain is substituted. In some embodiments, the alkyl chain is unsubstituted. In some embodiments, the alkyl chain is linear and substituted or unsubstituted. In some embodiments, the alkyl chain is branched and substituted or unsubstituted.

[0063] The term "alkylene" as used throughout this specification and description refers to a saturated straight or branched chain hydrocarbon diradical. Preferably, the alkylene group contains 1 to 10 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 1 to 8 carbon atoms, e.g. 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH)CH-), 2,2-propylene (-C(CH)-), and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-isobutylene, , 1,2-isobutylene, and 1,3-isobutylene), pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-isopentylene, 1,1-sec-pentyl, 1,1-neopentyl), hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1-isohexylene), and the like. Alkylene groups can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. Alkylene group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety.

[0064] As used herein and throughout the description, the term "halogen" or "halo" refers to fluoro, chloro, bromo, or iodo.

[0065] As used throughout this specification and discussion, the term "azido" refers to N3.

[0066] The term "alkenyl" as used herein and throughout the description refers to a monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. In general, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to an integer calculated by dividing the number of carbon atoms in the alkenyl group by 2, and rounding down the result of the division to the next integer if the number of carbon atoms in the alkenyl group is odd. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 4, i.e., 1, 2, 3, or 4 carbon-carbon double bonds. Preferably, the alkenyl group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, e.g., 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, an alkenyl group contains 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. In certain embodiments, an alkenyl group for use in the present invention contains 2 to 20 carbon atoms (C 2~20 In some embodiments, the alkenyl group used in the invention contains 2 to 15 carbon atoms (C 2~15 In another embodiment, the alkenyl group used contains 2 to 10 carbon atoms (C 2~10 In yet other embodiments, the alkenyl group contains 2 to 8 carbon atoms (C 2~8 In yet other embodiments, the alkenyl group contains 2 to 6 carbons (C 2~6 In yet other embodiments, the alkenyl group contains 2 to 5 carbons (C 2~5 In yet other embodiments, the alkenyl group contains 2 to 4 carbons (C 2~4 In yet other embodiments, the alkenyl group contains 2 to 3 carbons (C2~3 alkenyl). In yet other embodiments, the alkenyl group contains two carbons (C2 alkenyl). The carbon-carbon double bond may be in the cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl , 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, and the like. When an alkenyl group is connected to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. In some embodiments, the alkenyl chain is straight-chained. In some embodiments, the alkenyl chain is branched. In some embodiments, the alkenyl chain is substituted. In some embodiments, the alkenyl chain is unsubstituted. In some embodiments, the alkenyl chain is straight-chained and substituted or unsubstituted. In some embodiments, the alkenyl chain is branched, and is substituted or unsubstituted. Alkenyl group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety.

[0067] The term "alkenylene" as used herein and throughout the description refers to an unsaturated straight-chain or branched hydrocarbon diradical having at least one carbon-carbon double bond. In general, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to an integer calculated by dividing the number of carbon atoms in the alkenylene group by 2, and rounding down the result of the division to the next integer if the number of carbon atoms in the alkenylene group is odd. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 4, i.e., 1, 2, 3, or 4 carbon-carbon double bonds. Preferably, the alkenylene group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, the alkenylene group contains from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably from 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as from 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or from 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds, which may be in the cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethene-1,2-diyl, vinylidene, 1-propene-1,2-diyl, 1-propene-1,3-diyl, 1-propene-2,3-diyl, allylidene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, etc. In the case where an alkenylene group is connected to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. Alkenylene groups can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted.Alkenylene group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety.

[0068] The term "alkynyl" as used herein and throughout the description refers to a monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon triple bond. In general, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to an integer calculated by dividing the number of carbon atoms in the alkynyl group by 2, and rounding down the result of the division to the next integer if the number of carbon atoms in the alkynyl group is odd. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 4, i.e., 1, 2, 3, or 4, more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the alkynyl group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, an alkynyl group contains 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 (preferably 1, 2, or 3) carbon-carbon triple bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 (preferably 1 or 2) carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. In certain embodiments, an alkynyl group for use in the present invention contains 2 to 20 carbon atoms (C 2~20 In some embodiments, the alkynyl group used in the invention contains 2 to 15 carbon atoms (C 215 In another embodiment, the alkynyl group used contains 2 to 10 carbon atoms (C 2~10 In yet other embodiments, the alkynyl group contains 2 to 8 carbon atoms (C 2~8 In yet other embodiments, the alkynyl group contains 2 to 6 carbons (C 2~6In yet other embodiments, the alkynyl group contains 2 to 5 carbons (C 2~5 In yet other embodiments, the alkynyl group contains 2 to 4 carbons (C 2~4 In yet other embodiments, the alkynyl group contains 2 to 3 carbons (C 2~3 In yet other embodiments, the alkynyl group contains two carbons (C2 alkynyl). Exemplary alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl , 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl, 7-octynyl, 1-nonylyl, 2-nonylyl, 3-nonyl, 4-nonyl, 5-nonyl, 6-nonyl, 7-nonyl, 8-nonyl, 1-decynyl, 2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, 9-decynyl, and the like, which may bear one or more substituents. Alkynyl group substituents include, but are not limited to, any substituent described herein that results in the formation of a stable moiety. When an alkynyl group is connected to a nitrogen atom, the triple bond cannot be alpha to the nitrogen atom. In some embodiments, the alkynyl chain is straight. In some embodiments, the alkynyl chain is branched. In some embodiments, the alkynyl chain is substituted. In some embodiments, the alkynyl chain is unsubstituted. In some embodiments, the alkynyl chain is straight and substituted or unsubstituted, hi some embodiments, the alkynyl chain is branched and substituted or unsubstituted.

[0069] The term "alkynylene" as used herein and throughout the description refers to an unsaturated linear or branched hydrocarbon diradical having at least one carbon-carbon triple bond. In general, the maximum number of carbon-carbon triple bonds in an alkynylene group can be equal to an integer calculated by dividing the number of carbon atoms in the alkynylene group by 2, and rounding down the result of the division to the next integer if the number of carbon atoms in the alkynylene group is odd. For example, for an alkynylene group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynylene group has 1 to 4, i.e., 1, 2, 3, or 4, more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the alkynylene group contains 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, the alkynylene group contains 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 (preferably 1, 2, or 3) carbon-carbon triple bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 (preferably 1 or 2) carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon triple bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Exemplary alkynylene groups include ethyne-1,2-diyl, 1-propyne-1,3-diyl, 1-propyne-3,3-diyl, 1-butyne-1,3-diyl, 1-butyne-1,4-diyl, 1-butyne-3,4-diyl, 2-butyne-1,4-diyl, and the like. When the alkynylene group is linked to a nitrogen atom, the triple bond cannot be alpha to the nitrogen atom. Alkynylene groups can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. Alkynylene group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety.

[0070] The term "cycloalkyl" or "alicyclic" or "carbocyclic" or "carbocycle" as used throughout this specification and description refers to cyclic non-aromatic versions of "alkyl" and "alkenyl", preferably having 3 to 14 carbon atoms, e.g., 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 3 to 8 carbon atoms, and even more preferably 3 to 7 carbon atoms. In certain embodiments, cycloalkyl groups used in the present invention contain 3 to 14 carbon atoms (C3-7 14 In certain embodiments, the cycloalkyl groups used in the present invention contain 3 to 12 carbon atoms (C 3~12 In another embodiment, the cycloalkyl group used in the present invention contains 3 to 10 carbon atoms (C 3~10 In another embodiment, the cycloalkyl group used in the present invention contains 3 to 8 carbon atoms (C 3~8 In another embodiment, the cycloalkyl group used in the present invention contains 3 to 7 carbon atoms (C 3~7 In another embodiment, the cycloalkyl group used in the present invention contains 3 to 6 carbon atoms (C 3~6 In another embodiment, the cycloalkyl group used in the present invention contains 3 to 5 carbon atoms (C 3~5 In another embodiment, the cycloalkyl group used in the present invention contains 3 to 4 carbon atoms (C 3~4Cycloalkyl). In another embodiment, the cycloalkyl group used in the present invention contains three carbon atoms (C3 cycloalkyl). Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. The term "cycloalkyl" is also intended to include its bicyclic and tricyclic versions. When a bicyclic ring is formed, it is preferred that each ring is connected to each other at two adjacent carbon atoms, however, alternatively, the two rings are connected through the same carbon atom, i.e., forming a spirocyclic or "bridged" ring system. Preferred examples of cycloalkyl include C3-C8-cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, bicyclo[4.1.0]heptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl, and bicyclo[4.2.0]octyl. Cycloalkyl group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety.

[0071] The term "cyclopropylene" as used throughout this specification and description refers to a cyclopropyl group as defined above in which one hydrogen atom is removed to give a diradical. Cyclopropylene may link two atoms or moieties through the same carbon atom (1,1-cyclopropylene, i.e., a geminal diradical) or through two carbon atoms (1,2-cyclopropylene).

[0072] As used herein, the term "aryl" or "aromatic ring" as used throughout this specification and description refers to an aromatic monocyclic or polycyclic ring system having 3 to 20 ring atoms, all of whose ring atoms are carbon and may be substituted or unsubstituted. In certain embodiments of the invention, "aryl" refers to a monocyclic, bicyclic, or tricyclic C4-C6 ring system having 1, 2, or 3 aromatic rings. 20 It refers to an aromatic ring system, where the aromatic ring includes, but is not limited to, phenyl, biphenyl, naphthyl, and the like, and may bear one or more substituents. Preferably, the aryl group contains 3 to 14 (e.g., 5 to 10, e.g., 5, 6, or 10) carbon atoms, more preferably 6 to 10 carbon atoms, which may be arranged in one ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. In certain embodiments, the aryl groups used in the present invention contain 3 to 20 carbon atoms (C 3~20 In certain embodiments, the aryl group used in the present invention contains 3 to 18 carbon atoms (C 3~18 In another embodiment, the aryl group used in the present invention contains 3 to 16 carbon atoms (C 3~16 In another embodiment, the aryl group used in the present invention contains 6 to 16 carbon atoms (C 6~16 In another embodiment, the aryl group used in the present invention contains 7 to 16 carbon atoms (C 7~16 In another embodiment, the aryl group used in the present invention contains 6 to 14 carbon atoms (C 6~14 In another embodiment, the aryl group used in the present invention contains 7 to 14 carbon atoms (C 7~14 In another embodiment, the aryl group used in the present invention contains 6 to 12 carbon atoms (C6~12 In another embodiment, the aryl group used in the present invention contains 7 to 12 carbon atoms (C 7~12 In another embodiment, the aryl group used in the present invention contains 6 to 11 carbon atoms (C 6~11 In another embodiment, the aryl group used in the present invention contains 7 to 11 carbon atoms (C 7~11 In another embodiment, the aryl group used in the present invention contains 6 to 10 carbon atoms (C 6~10 In another embodiment, the aryl group used in the present invention contains 7 to 10 carbon atoms (C 7~10 In another embodiment, the aryl group used in the present invention contains 6 to 8 carbon atoms (C 6~8 In another embodiment, the aryl group used in the present invention contains 6 carbon atoms (C6 aryl). In another embodiment, the aryl group used in the present invention contains 10 carbon atoms (C 10 In some embodiments, Z is not a substituted monocyclic 6-membered aryl or an unsubstituted monocyclic 6-membered aryl. In some embodiments, Z is not a substituted phenyl or an unsubstituted phenyl. In some embodiments, Z is -NO2, -N3, halogen, -NH2, hydroxyl, -OR 11 and -C(=O)R 11 where R is not phenyl substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of 11 is hydrogen, substituted alkyl, or substituted alkynyl. Aryl substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety.

[0073] The term "arylene" as used herein, as used throughout this specification and description, refers to an aryl biradical derived from an aryl group as defined herein by removing two hydrogen atoms. The arylene group can be substituted or unsubstituted. Arylene group substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety. In addition, the arylene group can be incorporated into an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene group as defined herein as a linker group.

[0074] The term "heteroaryl" or "heteroaromatic ring" as used throughout this specification and description refers to an aryl group as defined above, in which one or more carbon atoms in the aryl group are replaced by a heteroatom of O, S, or N. Preferably, the heteroaryl group contains 3 to 14 carbon atoms. Preferably, heteroaryl refers to a 5- or 6-membered aromatic monocyclic ring in which 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Or, it refers to an aromatic bicyclic or tricyclic ring system in which 1, 2, 3, 4, or 5 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. In certain embodiments, the heteroaryl group used in the present invention is a 5-membered aromatic monocyclic ring in which 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. In certain embodiments, the heteroaryl group used in the present invention is a 5-membered aromatic monocyclic ring, in which 1, 2 or 3 carbon atoms are replaced by the same or different heteroatoms of O. In certain embodiments, the heteroaryl group used in the present invention is a 5-membered aromatic monocyclic ring, in which 1, 2 or 3 carbon atoms are replaced by the same or different heteroatoms of O and N. In certain embodiments, the heteroaryl group used in the present invention is a 5-membered aromatic monocyclic ring, in which 1, 2 or 3 carbon atoms are replaced by the same or different heteroatoms of O and S. In certain embodiments, the heteroaryl group used in the present invention is a 5-membered aromatic monocyclic ring, in which 1, 2 or 3 carbon atoms are replaced by the same or different heteroatoms of N and S. In certain embodiments, the heteroaryl group used in the present invention is a 6-membered aromatic monocyclic ring, in which 1, 2 or 3 carbon atoms are replaced by the same or different heteroatoms of O, S or N. In certain embodiments, heteroaryl groups employed in the invention are 6-membered aromatic monocyclic rings in which 1, 2, or 3 carbon atoms are replaced with N.In certain embodiments, the heteroaryl group used in the present invention is an aromatic bicyclic ring system in which 1, 2, 3, 4, or 5 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. In certain embodiments, the heteroaryl group used in the present invention is an aromatic bicyclic ring system in which one carbon atom is replaced by O. In certain embodiments, the heteroaryl group used in the present invention is an aromatic bicyclic ring system in which one carbon atom is replaced by N. In some embodiments, the heteroaryl group is a substituted or unsubstituted indolyl. In certain embodiments, the heteroaryl group used in the present invention is an aromatic bicyclic ring system in which two carbon atoms are replaced by N. In some embodiments, the heteroaryl group is a substituted or unsubstituted 7-azaindolyl. In some embodiments, the heteroaryl group is a substituted or unsubstituted 6-azaindolyl. In some embodiments, the heteroaryl group is a substituted or unsubstituted 5-azaindolyl. In some embodiments, the heteroaryl group is a substituted or unsubstituted 4-azaindolyl. In some embodiments, the heteroaryl group is a substituted or unsubstituted imidazolyl. In certain embodiments, heteroaryl groups for use in the invention are aromatic bicyclic ring systems in which three carbon atoms are replaced with N, preferably substituted or unsubstituted diazaindolyl groups.Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), benzofuranyl (1- and 2-), indolyl, azaindolyl (4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, 40-, 41-, 42-, 43-, 44-, 45-, 46-, 47-, 48-, 49-, 50-, 51-, 52-, 53-, 54-, 55-, 56-, 57-, 58-, 59-, 60-, 61-, 62-, 63-, 64-, 65-, 66-, 67-, 6- and 7-), diazaindolyl, isoindolyl, benzothienyl (1- and 2-), 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl (1,2,3- and 1,2,4-benzotriazinyl), pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, Heteroaryl groups include xanthenyl, phenoxathiinyl, pyrrolidinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl (1,5-, 1,6-, 1,7-, 1,8-, and 2,6-), cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (1,7-, 1,8-, 1,10-, 3,8-, and 4,7-), phenazinyl, oxazolopyridinyl, isoxazolopyridinyl, pyrrolooxazolyl, pyrrolopyrrolyl, and the like, which may bear one or more substituents. Heteroaryl substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety.Exemplary 5- or 6-membered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), and pyridazinyl. Exemplary bicyclic heteroaryl groups include 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, 4-azaindolyl, diazaindolyl, and indolyl.

[0075] The term "diazaindolyl" or "diazaindole" as used throughout this specification and description refers to a compound having an indole nucleus structure in which two carbon atoms of the cyclized phenyl ring are replaced with N. Preferably, carbon atoms 4, 5, 6 and / or 7 of the indole nucleus are replaced with N. Preferably, carbon atoms 4 and 5 of the indole nucleus are replaced with N. Preferably, carbon atoms 4 and 6 of the indole nucleus are replaced with N. Preferably, carbon atoms 4 and 7 of the indole nucleus are replaced with N. Preferably, carbon atoms 5 and 6 of the indole nucleus are replaced with N. Preferably, carbon atoms 6 and 7 of the indole nucleus are replaced with N. Preferably, carbon atoms 5 and 7 of the indole nucleus are replaced with N. In some embodiments, the diazaindolyl is substituted. In some embodiments, the diazaindolyl is unsubstituted.

[0076] The term "heteroarylene" as used herein, as used throughout this specification and description, refers to a biradical derived from a heteroaryl group as defined herein by removing two hydrogen atoms. Heteroarylene groups can be substituted or unsubstituted. In addition, heteroarylene groups can be incorporated into alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene groups as defined herein as linker groups. Heteroarylene group substituents include, but are not limited to, any substituents described herein that result in the formation of a stable moiety.

[0077] As used herein and throughout the description, the terms "arylalkyl" and "heteroarylalkyl" include radicals in which an aryl group and a heteroaryl group, respectively, are linked to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced, for example, by an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.). Preferably, the arylalkyl is a substituted or unsubstituted (C6-C 14 )aryl(C1-C6)alkyl. Preferably, the arylalkyl is a substituted or unsubstituted (C6-C 10 )aryl(C1-C6)alkyl. Preferably, the heteroarylalkyl is a substituted or unsubstituted (C3-C 14 )heteroaryl(C1-C6)alkyl. Preferably, the heteroarylalkyl is a substituted or unsubstituted (C3-C 10 )heteroaryl(C1-C6)alkyl. In some embodiments, the alkyl chain is linear. In some embodiments, the alkyl chain is branched. In some embodiments, the alkyl chain is substituted. In some embodiments, the alkyl chain is unsubstituted. In some embodiments, the alkyl chain is linear and substituted or unsubstituted. In some embodiments, the alkyl chain is branched and substituted or unsubstituted.

[0078] The terms "heterocyclyl" or "heterocyclic ring" or "heterocycle" as used herein and throughout the description refer to cyclic heteroaliphatic groups. Heterocyclic groups refer to non-aromatic, partially unsaturated or fully saturated, 3- to 10-membered ring systems, including monocyclic rings of 3-8 atoms in size, as well as bicyclic and tricyclic ring systems that may contain an aromatic 5- or 6-membered aryl or heteroaryl group fused to a non-aromatic ring. Heterocyclic groups can be substituted or unsubstituted. These heterocyclic groups include those having 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, where the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. In certain embodiments, the term heterocyclic refers to non-aromatic 5-, 6-, or 7-membered rings or heterocyclic rings in which at least one ring atom is a heteroatom selected from O, S, and N (where the nitrogen and sulfur heteroatoms may be optionally oxidized), and the remaining ring atoms are carbon. It refers to a polycyclic group, where the radical is linked to the remainder of the molecule through any ring atom. Heterocycyl groups include, but are not limited to, bicyclic or tricyclic groups, including fused 5-, 6-, or 7-membered rings, having between 1 and 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, where (i) each 5-membered ring has 0-2 double bonds, each 6-membered ring has 0-2 double bonds, and each 7-membered ring has 0-3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may be optionally quaternized, and (iv) any of the foregoing heterocyclic rings may be fused to an aryl or heteroaryl ring. Preferably, in each ring of a heterocyclyl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" also encompasses partially or fully hydrogenated forms (e.g., dihydro, tetrahydro, or perhydro forms) of the foregoing heteroaryl groups. Exemplary heterocyclyl groups include morpholino, isochromanyl, chromanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, indolinyl, isoindolinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydrooxazolyl,Di- and tetrahydroisoxazolyl, di- and tetrahydrooxadiazolyl (1,2,5- and 1,2,3-), dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1,2,3- and 1,2,4-), di- and tetrahydrothiazolyl, di- and tetrahydrothiazolyl, di- and tetrahydrothiadiazolyl (1,2,3- and 1,2,5-), di- and tetrahydropyridyl, di- and tetrahydropyrimidinyl, di- and tetrahydropyrazinyl, di- and tetrahydrotriazinyl di- and tetrahydrobenzofuranyl (1- and 2-), di- and tetrahydroindolyl, di- and tetrahydroisoindolyl, di- and tetrahydrobenzothienyl (1- and 2-), di- and tetrahydro-1H-indazolyl, di- and tetrahydrobenzimidazolyl, di- and tetrahydrobenzoxazolyl, di- and tetrahydroindoxazinyl, di- and tetrahydrobenzisoxazolyl, di- and tetrahydrobenzothiazolyl, di- and tetrahydrobenziizolyl, Sothiazolyl, di- and tetrahydrobenzotriazolyl, di- and tetrahydroquinolinyl, di- and tetrahydroisoquinolinyl, di- and tetrahydrobenzodiazinyl, di- and tetrahydroquinoxalinyl, di- and tetrahydroquinazolinyl, di- and tetrahydrobenzotriazinyl (1,2,3- and 1,2,4-), di- and tetrahydropyridazinyl, di- and tetrahydrophenoxazinyl, di- and tetrahydrothiazolopyridinyl (e.g. 4,5,6-7-tetrahydro[1,3]thiazolo[5,4-c]pyridinyl or 4 , 5,6-7-tetrahydro[1,3]thiazolo[4,5-c]pyridinyl, for example 4,5,6-7-tetrahydro[1,3]thiazolo[5,4-c]pyridin-2-yl or 4,5,6-7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-2-yl), di- and tetrahydropyrrolothiazolyl, for example 5,6-dihydro-4H-pyrrolo[3,4-d][1,3]thiazolyl, di- and tetrahydrophenothiazinyl, di- and tetrahydroisobenzofuranyl, di- and tetrahydrochromenyl, di- and tetrahydroxanthenyl,Di- and tetrahydrophenoxathiinyl, di- and tetrahydropyrrolidinyl, di- and tetrahydroindolizinyl, di- and tetrahydroindazolyl, di- and tetrahydropurinyl, di- and tetrahydroquinolizinyl, di- and tetrahydrophthalazinyl, di- and tetrahydronaphthyridinyl (1,5-, 1,6-, 1,7-, 1,8-, and 2,6-), di- and tetrahydrocinnolinyl, di- and tetrahydropteridinyl, di- and tetrahydrocarba These include zolyl, di- and tetrahydrophenanthridinyl, di- and tetrahydroacridinyl, di- and tetrahydroperimidinyl, di- and tetrahydrophenanthrolinyl (1,7-, 1,8-, 1,10-, 3,8-, and 4,7-), di- and tetrahydrophenazinyl, di- and tetrahydrooxazolopyridinyl, di- and tetrahydroisoxazolopyridinyl, di- and tetrahydropyrrolooxazolyl, and di- and tetrahydropyrrolopyrrolyl. Exemplary 5- or 6-membered heterocyclyl groups include morpholino, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydrooxazolyl, di- and tetrahydroisoxazolyl, di- and tetrahydrooxadiazolyl (1,2,5- and 1,2,3-), dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1,2, 3- and 1,2,4-), di- and tetrahydrothiazolyl, di- and tetrahydroisothiazolyl, di- and tetrahydrothiadiazolyl (1,2,3- and 1,2,5-), di- and tetrahydropyridyl, di- and tetrahydropyrimidinyl, di- and tetrahydropyrazinyl, di- and tetrahydrotriazinyl (1,2,3-, 1,2,4-, and 1,3,5-), di- and tetrahydropyridazinyl, and the like, which may have one or more substituents. Preferable are 2H-1-benzopyranyl (2H-chromenyl), benzodihydropyranyl (chromanyl), 4H-1-benzopyranyl (4H-chromenyl), 1H-2-benzopyranyl (1H-isochromanyl), isochromanyl, 3H-2-benzopyranyl (3H-isochromenyl),1-benzopyran-4-one-yl (chromonyl), 4-chromanonyl, 1-benzopyran-2-one-yl (coumarinyl), dihydrocoumarinyl, 3-isochromanonyl, 2-coumaranon-yl. In some embodiments, the heterocyclyl group is substituted or unsubstituted 2H-1-benzopyranyl (2H-chromenyl). In some embodiments, the heterocyclyl group is substituted or unsubstituted benzodihydropyranyl (chromanyl). In some embodiments, the heterocyclyl group is substituted or unsubstituted 4H-1-benzopyranyl (4H-chromenyl). In some embodiments, the heterocyclyl group is substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl). In some embodiments, the heterocyclyl group is substituted or unsubstituted isochromanyl. In some embodiments, the heterocyclyl group is substituted or unsubstituted 3H-2-benzopyranyl (3H-isochromenyl). In some embodiments, the heterocyclyl group is substituted or unsubstituted 1-benzopyran-4-one-yl (chromonyl). In some embodiments, the heterocyclyl group is substituted or unsubstituted 4-chromanonyl. In some embodiments, the heterocyclyl group is substituted or unsubstituted 1-benzopyran-2-one-yl (coumarinyl). In some embodiments, the heterocyclyl group is substituted or unsubstituted dihydrocoumarinyl. In some embodiments, the heterocyclyl group is substituted or unsubstituted 3-isochromanonyl. In some embodiments, the heterocyclyl group is substituted or unsubstituted 2-coumaranon-yl. In some embodiments, the heterocyclyl group is substituted or unsubstituted (C3-C, 14 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C-C) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with O. 14 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C-C) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with N. 14In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C-C) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with S. 14 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C9-C10) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. 10 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C9-C10) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with O. 10 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C9-C10) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with N. 10 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C9-C10) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with S. 10 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. 10 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C ) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with O. 10 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C ) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with N. 10 In some embodiments, the heterocyclyl group is a substituted or unsubstituted (C ) heterocyclyl group in which 1, 2, 3, 4, or 5 carbon atoms are replaced with S. 10 ) heterocyclyl group.

[0079] As used herein, the term "heteroalkyl" as used throughout the specification and description refers to an alkyl moiety, as defined herein, that contains one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) between the carbon atoms. Heteroalkyl can be substituted or unsubstituted. In certain embodiments, heteroalkyl groups contain 1 to 20 carbon atoms and 1 to 6 heteroatoms (C 1~20 In certain embodiments, a heteroalkyl group contains 1 to 10 carbon atoms and 1 to 4 heteroatoms (C 1~10 In certain embodiments, a heteroalkyl group contains 1 to 6 carbon atoms and 1 to 3 heteroatoms (C 1~6 In certain embodiments, a heteroalkyl group contains 1 to 5 carbon atoms and 1 to 3 heteroatoms (C 1~5 In certain embodiments, a heteroalkyl group contains 1 to 4 carbon atoms and 1 to 2 heteroatoms (C 1~4 In certain embodiments, a heteroalkyl group contains 1 to 3 carbon atoms and 1 heteroatom (C 1~3 In certain embodiments, a heteroalkyl group contains 1 to 2 carbon atoms and 1 heteroatom (C 1~2 Heteroalkyl). As used herein, the term "heteroalkylene" refers to a biradical derived from a heteroalkyl group, as defined herein, by the removal of two hydrogen atoms. A heteroalkylene group can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. In certain embodiments, a heteroalkyl group is a substituted heteroalkyl group containing 1-6 carbon atoms and 1-3 heteroatoms (C 1~6 In certain embodiments, the heteroalkyl group is an unsubstituted heteroalkyl group containing 1 to 6 carbon atoms and 1 to 3 heteroatoms (C 1~6In some embodiments, the heteroalkyl is an alkyl moiety replaced with S at the methylene group. In some embodiments, the heteroalkyl is an alkyl moiety replaced with O at the methylene group. In some embodiments, the heteroalkyl is an alkyl moiety replaced with NR at the methylene group. 1 where the alkyl moiety is replaced by hydrogen, substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C2-C6) alkenyl, substituted or unsubstituted (C2-C6) alkynyl, substituted or unsubstituted (C3-C8) cycloalkyl, substituted or unsubstituted (C6-C 14 ) Aryl and substituted or unsubstituted (C3-C 14 ) heteroaryl. In some embodiments, the heteroalkyl is -CH2SCH3. In some embodiments, the heteroalkyl is -CH2OCH3.

[0080] As used herein and throughout the description, heteroalkylene group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety.

[0081] As used herein and throughout the specification and description, the term "heteroalkenyl" refers to an alkenyl moiety, as defined herein, further comprising one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) between the carbon atoms. In certain embodiments, heteroalkenyl groups contain 2 to 20 carbon atoms and 1 to 6 heteroatoms (C 2~20 In certain embodiments, a heteroalkenyl group contains 2 to 10 carbon atoms and 1 to 4 heteroatoms (C 2~10 In certain embodiments, a heteroalkenyl group contains 2 to 6 carbon atoms and 1 to 3 heteroatoms (C 2~6 In certain embodiments, a heteroalkenyl group contains 2 to 5 carbon atoms and 1 to 3 heteroatoms (C 2~5In certain embodiments, a heteroalkenyl group contains 2 to 4 carbon atoms and 1 to 2 heteroatoms (C 2~4 In certain embodiments, a heteroalkenyl group contains 2 to 3 carbon atoms and 1 heteroatom (C 2~3 Heteroalkenyl). As used herein, the term "heteroalkenylene" refers to a biradical derived from a heteroalkenyl group, as defined herein, by removal of two hydrogen atoms. A heteroalkenylene group can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. In certain embodiments, a heteroalkenyl group is a substituted heteroalkenyl group containing 1-6 carbon atoms and 1-3 heteroatoms (C 1~6 In certain embodiments, the heteroalkenyl group is an unsubstituted heteroalkenyl group containing 1 to 6 carbon atoms and 1 to 3 heteroatoms (C 1~6 heteroalkenyl).

[0082] As used herein and throughout the specification and description, the term "heteroalkynyl" refers to an alkynyl moiety, as defined herein, further comprising one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) between the carbon atoms. In certain embodiments, heteroalkynyl groups contain 2 to 20 carbon atoms and 1 to 6 heteroatoms (C 2~20 In certain embodiments, a heteroalkynyl group contains 2 to 10 carbon atoms and 1 to 4 heteroatoms (C 2~10 In certain embodiments, a heteroalkynyl group contains 2 to 6 carbon atoms and 1 to 3 heteroatoms (C 2~6 In certain embodiments, a heteroalkynyl group contains 2 to 5 carbon atoms and 1 to 3 heteroatoms (C 2~5 In certain embodiments, a heteroalkynyl group contains 2 to 4 carbon atoms and 1 to 2 heteroatoms (C 2~4In certain embodiments, a heteroalkynyl group contains 2 to 3 carbon atoms and 1 heteroatom (C 2~3 Heteroalkynyl). As used herein, the term "heteroalkynylene" refers to a biradical derived from a heteroalkynyl group, as defined herein, by removal of two hydrogen atoms. A heteroalkynylene group can be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. In certain embodiments, a heteroalkynyl group is a substituted heteroalkynyl group containing 1-6 carbon atoms and 1-3 heteroatoms (C 1~6 In certain embodiments, the heteroalkynyl group is an unsubstituted heteroalkynyl group containing 1 to 6 carbon atoms and 1 to 3 heteroatoms (C 1~6 heteroalkynyl).

[0083] The unnatural amino acids described herein have Formula I: The image may be represented by having TIFF2024500922000007.tif32128.

[0084] In some embodiments, the compound having the formula of Formula I is 1 or S; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl; Z is a substituted or unsubstituted (C1-C6) alkyl, a substituted or unsubstituted (C2-C6) alkenyl, a substituted or unsubstituted (C2-C6) alkynyl, a substituted or unsubstituted (C3-C8) cycloalkyl, a substituted or unsubstituted (C6 ... 14 ) Aryl, substituted or unsubstituted (C6-C 14 )Aryl(C1-C6)Alkyl, substituted or unsubstituted (C3-C 14 ) Heteroaryl, substituted or unsubstituted (C3-C 14 )Heteroaryl(C1-C6)Alkyl, substituted or unsubstituted (C3-C 14 ) heterocyclyl, substituted or unsubstituted (C1-C6) heteroalkyl, substituted or unsubstituted (C2-C6) heteroalkenyl, and substituted or unsubstituted (C2-C6) heteroalkynyl; R 1is hydrogen or a substituted or unsubstituted (C1-C6) alkyl, where A is a methylene group, and n is 0 or 1, preferably n is 1.

[0085] In some embodiments, the compound having the formula of Formula I is a compound having the formula 1 or S; Y is hydrogen or substituted or unsubstituted (C1-C6) alkyl; Z is substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C6-C 14 ) Aryl, substituted or unsubstituted (C3-C 14 ) Heteroaryl, substituted or unsubstituted (C3-C 14 ) heterocyclyl and substituted or unsubstituted (C1-C6)heteroalkyl; R 1 is hydrogen or a substituted or unsubstituted (C1-C6) alkyl, where A is a methylene group, and n is 0 or 1, preferably n is 1.

[0086] In some embodiments, the compound having the formula of Formula I is a compound in which X is O; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl; Z is a substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C6-C 14 ) Aryl, substituted or unsubstituted (C3-C 14 ) Heteroaryl, substituted or unsubstituted (C3-C 14 )heterocyclyl and substituted or unsubstituted (C1-C6)heteroalkyl, where A is a methylene group and n is 0 or 1, preferably n is 1.

[0087] In some embodiments, the compound having the formula of Formula I is 1 or S; Y is hydrogen or substituted or unsubstituted (C1-C6) alkyl; Z is substituted or unsubstituted (C6-C 14 )Aryl, unsubstituted (C3-C 14 ) Heteroaryl and substituted or unsubstituted (C3-C 14 ) heterocyclyl; R1 is hydrogen or a substituted or unsubstituted (C1-C6) alkyl, where A is a methylene group, and n is 0 or 1, preferably n is 1.

[0088] In some embodiments, the compound having the formula of Formula I is a compound in which X is O; Y is hydrogen or a substituted or unsubstituted (C1-C6) alkyl; Z is a substituted or unsubstituted (C6-C 14 ) Aryl, substituted or unsubstituted (C3-C 14 ) Heteroaryl and substituted or unsubstituted (C3-C 14 ) heterocyclyl, where A is a methylene group and n is 0 or 1, preferably n is 1.

[0089] In some embodiments, the compound having the formula of Formula I is 1 or S; Y is hydrogen or substituted or unsubstituted (C1-C6) alkyl; Z is substituted or unsubstituted 2H-1-benzopyranyl (2H-chromenyl), substituted or unsubstituted benzodihydropyranyl (chromanyl), substituted or unsubstituted 4H-1-benzopyranyl (4H-chromenyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl), substituted or unsubstituted isochromanyl, substituted or unsubstituted 3H-2-benzopyranyl (3H-isochromenyl), substituted or unsubstituted 1-benzopyran-4-on-yl (chromonyl), substituted or unsubstituted 4-chloropyranyl, substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl), substituted or unsubstituted 1H-2-benzopyran-4-on-yl (chromon ... manonyl, substituted or unsubstituted 1-benzopyran-2-on-yl (coumarinyl), substituted or unsubstituted dihydrocoumarinyl, substituted or unsubstituted 3-isochromanonyl, substituted or unsubstituted 2-coumaranon-yl, substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C1-C6) heteroalkyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted azaindolyl including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl and 4-azaindolyl, substituted or unsubstituted diazaindolyl and substituted or unsubstituted indolyl; R 1is hydrogen or a substituted or unsubstituted (C1-C6) alkyl, where A is a methylene group, and n is 0 or 1, preferably n is 1.

[0090] In some embodiments, the compound having the formula of Formula I is a compound in which X is O; Y is hydrogen or substituted or unsubstituted (C1-C6) alkyl; Z is substituted or unsubstituted 2H-1-benzopyranyl (2H-chromenyl), substituted or unsubstituted benzodihydropyranyl (chromanyl), substituted or unsubstituted 4H-1-benzopyranyl (4H-chromenyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl), substituted or unsubstituted isochromanyl, substituted or unsubstituted 3H-2-benzopyranyl (3H-isochromenyl), substituted or unsubstituted 1-benzopyran-4-on-yl (chromonyl), substituted or unsubstituted 4-chromanonyl, substituted or unsubstituted 5H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 6H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 7H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 8H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 9H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 10H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 11H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 12H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 13H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 14H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 15H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 16H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 17H-benzopyran-4-onyl (chromonyl), substituted or unsubstituted 18H-benzopyran-4-onyl ( and n is 0 or 1, preferably n is 1.

[0091] In some embodiments, the compound having the formula of Formula I is 1 or S; Y is hydrogen or substituted or unsubstituted (C1-C6) alkyl; Z is selected from the group consisting of substituted or unsubstituted 1-benzopyran-2-on-yl (coumarinyl), substituted or unsubstituted dihydrocoumarinyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted azaindolyl including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl and 4-azaindolyl, and substituted or unsubstituted indolyl; R 1is hydrogen or a substituted or unsubstituted (C1-C6) alkyl, where A is a methylene group, and n is 0 or 1, preferably n is 1.

[0092] In some embodiments, the compound having the formula of Formula I may be characterized in that X is O; Y is hydrogen or substituted or unsubstituted (C1-C6) alkyl; Z is selected from the group consisting of substituted or unsubstituted 1-benzopyran-2-on-yl (coumarinyl), substituted or unsubstituted dihydrocoumarinyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted azaindolyl including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, and 4-azaindolyl, and substituted or unsubstituted indolyl, where A is a methylene group, and n is 0 or 1, preferably n is 1.

[0093] In some embodiments of the compounds having the formula of Formula I, X is O. In some embodiments of the compounds having the formula of Formula I, X is S. In some embodiments of the compounds having the formula of Formula I, X is NR 1 where R 1 is hydrogen, substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C2-C6) alkenyl, substituted or unsubstituted (C2-C6) alkynyl, substituted or unsubstituted (C3-C8) cycloalkyl, substituted or unsubstituted (C6-C 14 ) Aryl and substituted or unsubstituted (C3-C 14 ) heteroaryl, preferably hydrogen or substituted or unsubstituted (C1-C6) alkyl, more preferably hydrogen, more preferably substituted or unsubstituted (C1-C6) alkyl, even more preferably unsubstituted (C1-C6) alkyl.

[0094] In some embodiments of the compounds having the formula of Formula I, Y is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl. In some embodiments of the compounds having the formula of Formula I, Y is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, and substituted or unsubstituted heteroalkyl. In some embodiments of the compound having the formula of Formula I, Y is a substituted or unsubstituted (C1-C6) alkyl, a substituted or unsubstituted (C2-C6) alkenyl, a substituted or unsubstituted (C2-C6) alkynyl, a substituted or unsubstituted (C3-C8) cycloalkyl, a substituted or unsubstituted (C6-C8) aryl ... 14 ) Aryl, substituted or unsubstituted (C6-C 14 )Aryl(C1-C6)Alkyl, substituted or unsubstituted (C3-C 14 ) Heteroaryl, substituted or unsubstituted (C3-C 14 )Heteroaryl(C1-C6)Alkyl, substituted or unsubstituted (C3-C 14)heterocyclyl, substituted or unsubstituted (C1-C6)heteroalkyl, substituted or unsubstituted (C2-C6)heteroalkenyl, and substituted or unsubstituted (C2-C6)heteroalkynyl. In some embodiments of the compound having the formula of formula I, Y is hydrogen or substituted or unsubstituted (C1-C6)alkyl. In some embodiments of the compound having the formula of formula I, Y is hydrogen. In some embodiments of the compound having the formula of formula I, Y is substituted or unsubstituted alkyl, preferably substituted or unsubstituted (C1-C6)alkyl. In some embodiments of the compound having the formula of formula I, Y is substituted or unsubstituted alkenyl, preferably substituted or unsubstituted (C2-C6)alkenyl. In some embodiments of the compound having the formula of formula I, Y is substituted or unsubstituted alkynyl, preferably substituted or unsubstituted (C2-C6)alkynyl. In some embodiments of the compound having the formula of formula I, Y is substituted or unsubstituted cycloalkyl, preferably substituted or unsubstituted (C3-C8)cycloalkyl. In some embodiments of the compounds having the formula of Formula I, Y is a substituted or unsubstituted aryl, preferably a substituted or unsubstituted (C6-C 14 In some embodiments of the compounds having the formula of Formula I, Y is a substituted or unsubstituted arylalkyl, preferably a substituted or unsubstituted (C6-C 14 In some embodiments of the compounds having the formula of Formula I, Y is a substituted or unsubstituted heteroaryl, preferably a substituted or unsubstituted (C3-C 14 In some embodiments of the compounds having the formula of Formula I, Y is a substituted or unsubstituted heteroarylalkyl, preferably a substituted or unsubstituted (C3-C 14 In some embodiments of the compounds having the formula of Formula I, Y is a substituted or unsubstituted heterocyclyl, preferably a substituted or unsubstituted (C3-C 14)heterocyclyl. In some embodiments of the compound having the formula of Formula I, Y is substituted or unsubstituted heteroalkyl, preferably substituted or unsubstituted (C1-C6)heteroalkyl. In some embodiments of the compound having the formula of Formula I, Y is substituted or unsubstituted heteroalkenyl, preferably substituted or unsubstituted (C2-C6)heteroalkenyl. In some embodiments of the compound having the formula of Formula I, Y is substituted or unsubstituted heteroalkynyl, preferably substituted or unsubstituted (C2-C6)heteroalkynyl.

[0095] In some embodiments of the compounds having the formula of Formula I, Z is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; with the proviso that Z is not substituted or unsubstituted monocyclic 6-membered aryl. In some embodiments of the compounds having the formula of Formula I, Z is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, and substituted or unsubstituted heteroalkyl; with the proviso that Z is not substituted or unsubstituted monocyclic 6-membered aryl. In some embodiments of the compound having the formula of Formula I, Z is a substituted or unsubstituted (C1-C6) alkyl, a substituted or unsubstituted (C2-C6) alkenyl, a substituted or unsubstituted (C2-C6) alkynyl, a substituted or unsubstituted (C3-C8) cycloalkyl, a substituted or unsubstituted (C6-C8) aryl ... 14 ) Aryl, substituted or unsubstituted (C6-C 14 )Aryl(C1-C6)Alkyl, substituted or unsubstituted (C3-C 14 ) Heteroaryl, substituted or unsubstituted (C3-C 14)Heteroaryl(C1-C6)Alkyl, substituted or unsubstituted (C3-C 14 )heterocyclyl, substituted or unsubstituted (C1-C6)heteroalkyl, substituted or unsubstituted (C2-C6)heteroalkenyl, and substituted or unsubstituted (C2-C6)heteroalkynyl; provided that Z is not substituted or unsubstituted monocyclic 6-membered aryl. In some embodiments of the compound having the formula of Formula I, Z is hydrogen or substituted or unsubstituted (C1-C6)alkyl. In some embodiments of the compound having the formula of Formula I, Z is substituted or unsubstituted alkyl, preferably substituted or unsubstituted (C1-C6)alkyl. In some embodiments of the compound having the formula of Formula I, Z is substituted or unsubstituted alkenyl, preferably substituted or unsubstituted (C2-C6)alkenyl. In some embodiments of the compound having the formula of Formula I, Z is substituted or unsubstituted alkynyl, preferably substituted or unsubstituted (C2-C6)alkynyl. In some embodiments of the compound having the formula of Formula I, Z is a substituted or unsubstituted cycloalkyl, preferably a substituted or unsubstituted (C3-C8) cycloalkyl. In some embodiments of the compound having the formula of Formula I, Z is a substituted or unsubstituted aryl, preferably a substituted or unsubstituted (C6-C 14 )aryl, with the proviso that preferably Z is not a substituted or unsubstituted monocyclic 6-membered aryl. In some embodiments of the compounds having the formula of Formula I, Z is a substituted or unsubstituted arylalkyl, preferably a substituted or unsubstituted (C6-C 14 )aryl(C1-C6)alkyl, provided that preferably Z is not a substituted or unsubstituted monocyclic 6-membered aryl. In some embodiments of the compounds having the formula of Formula I, Z is a substituted or unsubstituted heteroaryl, preferably a substituted or unsubstituted (C3-C 14 In some embodiments of the compounds having the formula of Formula I, Z is a substituted or unsubstituted heteroarylalkyl, preferably a substituted or unsubstituted (C3-C 14In some embodiments of the compounds having the formula of Formula I, Z is substituted or unsubstituted heterocyclyl, preferably substituted or unsubstituted (C3-C 14)heterocyclyl. In some embodiments of the compound having the formula of Formula I, Z is substituted or unsubstituted heteroalkyl, preferably substituted or unsubstituted (C1-C6)heteroalkyl. In some embodiments of the compound having the formula of Formula I, Z is substituted or unsubstituted heteroalkenyl, preferably substituted or unsubstituted (C2-C6)heteroalkenyl. In some embodiments of the compound having the formula of Formula I, Z is substituted or unsubstituted heteroalkynyl, preferably substituted or unsubstituted (C2-C6)heteroalkynyl. In some embodiments of the compound having the formula of Formula I, Z is substituted or unsubstituted 2H-1-benzopyranyl (2H-chromenyl), substituted or unsubstituted benzodihydropyranyl (chromanyl), substituted or unsubstituted 4H-1-benzopyranyl (4H-chromenyl), substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl), substituted or unsubstituted isochromanyl, substituted or unsubstituted 3H-2-benzopyranyl (3H-isochromenyl), substituted or unsubstituted 1-benzopyran-4-on-yl (chromonyl), substituted or unsubstituted 4-chloropyranyl. Manonyl, substituted or unsubstituted 1-benzopyran-2-on-yl (coumarinyl), substituted or unsubstituted dihydrocoumarinyl, substituted or unsubstituted 3-isochromanonyl, substituted or unsubstituted 2-coumaranon-yl, substituted or unsubstituted (C1-C6) alkyl, substituted or unsubstituted (C1-C6) heteroalkyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted azaindolyl including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl and 4-azaindolyl, and substituted or unsubstituted indolyl. In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted 2H-1-benzopyranyl (2H-chromenyl). In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted benzodihydropyranyl (chromanyl). In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted 4H-1-benzopyranyl (4H-chromenyl). In some embodiments of the compounds having the formula of Formula I, Z is substituted or unsubstituted 1H-2-benzopyranyl (1H-isochromenyl). In some embodiments of the compounds having the formula of Formula I, Z is substituted or unsubstituted isochromanyl.In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted 3H-2-benzopyranyl (3H-isochromenyl). In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted 1-benzopyran-4-one-yl (chromonyl). In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted 4-chromanonyl. In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted 1-benzopyran-2-one-yl (coumarinyl). In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted dihydrocoumarinyl. In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted 3-isochromanonyl. In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted 2-coumaranon-yl. In some embodiments of the compound having the formula of formula I, Z is substituted or unsubstituted (C1-C6) alkyl. In some embodiments of the compound having the formula of Formula I, Z is a substituted or unsubstituted (C1-C6)heteroalkyl. In some embodiments of the compound having the formula of Formula I, Z is a substituted or unsubstituted imidazolyl. In some embodiments of the compound having the formula of Formula I, Z is a substituted or unsubstituted azaindolyl, including 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, and 4-azaindolyl. In some embodiments of the compound having the formula of Formula I, Z is a substituted or unsubstituted indolyl. In some embodiments of the compound having the formula of Formula I, Z is not a substituted monocyclic 6-membered aryl or an unsubstituted monocyclic 6-membered aryl. In some embodiments of the compound having the formula of Formula I, Z is not a substituted phenyl or an unsubstituted phenyl. In some embodiments of the compound having the formula of Formula I, Z is -NO2, -N3, halogen, -NH2, hydroxyl, -OR. 11 and -C(=O)R 11 where R is not phenyl substituted with 1, 2, 3, 4 or 5 substituents selected from the group consisting of 11 is hydrogen, substituted alkyl or substituted alkynyl.

[0096] In some embodiments of the compounds having the formula of Formula I, A is a methylene group and n is 1. In some embodiments of the compounds having the formula of Formula I, A is a methylene group and n is 2.

[0097] As used herein and throughout the description, the term "aliphatic" includes both saturated and unsaturated, non-aromatic, straight-chain (i.e., unbranched), branched, acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, which may be substituted with one or more functional groups. As will be appreciated by those skilled in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, the term "alkyl" as used herein includes straight-chain, branched, and cyclic alkyl groups. A similar convention applies to other general terms, such as "alkenyl," "alkynyl," and the like. Furthermore, the terms "alkyl," "alkenyl," "alkynyl," and the like, as used herein, include both substituted and unsubstituted groups. In certain embodiments, "aliphatic" as used herein refers to an aliphatic group (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1 to 20 carbon atoms (C 1~20 In certain embodiments, an aliphatic group has 1 to 10 carbon atoms (C 1~10 In certain embodiments, the aliphatic group has 1 to 6 carbon atoms (C 1~6 In certain embodiments, the aliphatic group has 1 to 5 carbon atoms (C 1~5 In certain embodiments, the aliphatic group has 1 to 4 carbon atoms (C 1~4 In certain embodiments, the aliphatic group has 1 to 3 carbon atoms (C 1~3 In certain embodiments, the aliphatic group has 1 to 2 carbon atoms (C 1~2Aliphatic). Aliphatic group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety. In some embodiments, the aliphatic group is a saturated or unsaturated, unbranched or branched alkyl, preferably (C1-C 20 ) alkyl, more preferably (C1-C 10 ) alkyl, and even more preferably (C1-C6) alkyl.

[0098] The term "heteroaliphatic" as used throughout this specification and description refers to an aliphatic moiety as defined herein, including both saturated and unsaturated, non-aromatic, straight-chain (i.e., unbranched), branched, non-cyclic, cyclic (i.e., heterocyclic), or polycyclic hydrocarbons, which may be substituted with one or more functional groups, and further including one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, or silicon atoms) between carbon atoms. In certain embodiments, the heteroaliphatic moiety is substituted by independently replacing one or more of the hydrogen atoms thereon with one or more substituents. As will be understood by those skilled in the art, "heteroaliphatic" is intended herein to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl moieties. Thus, the term "heteroaliphatic" as used herein includes terms such as "heteroalkyl", "heteroalkenyl", and "heteroalkynyl". Additionally, as used herein, terms such as "heteroalkyl," "heteroalkenyl," "heteroalkynyl," and the like, include both substituted and unsubstituted groups. In certain embodiments, as used herein, "heteroaliphatic" refers to a heteroaliphatic group (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-20 carbon atoms and 1-6 heteroatoms (C 1~20 In certain embodiments, heteroaliphatic groups contain 1-10 carbon atoms and 1-4 heteroatoms (C 1~10In certain embodiments, heteroaliphatic groups contain 1-6 carbon atoms and 1-3 heteroatoms (C 1~6 In certain embodiments, heteroaliphatic groups contain 1-5 carbon atoms and 1-3 heteroatoms (C 1~5 In certain embodiments, heteroaliphatic groups contain 1-4 carbon atoms and 1-2 heteroatoms (C 1~4 In certain embodiments, heteroaliphatic groups contain 1-3 carbon atoms and 1 heteroatom (C 1~3 In certain embodiments, heteroaliphatic groups contain 1-2 carbon atoms and 1 heteroatom (C 1~2 Heteroaliphatic). Heteroaliphatic group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety. In some embodiments, an aliphatic group is a saturated or unsaturated, unbranched or branched alkyl, preferably (C1-C 20 ) heteroalkyl, more preferably (C1-C 10 )heteroalkyl, and even more preferably (C1-C6)heteroalkyl.

[0099] "L": Linker The present disclosure provides antibody drug conjugates in which brentuximab modified as described herein is linked to a drug moiety. According to the present disclosure, brentuximab may be linked to a drug moiety via a covalent bond by a linker. As used herein, a "linker" is any chemical moiety capable of linking an antibody, such as brentuximab, an antibody fragment (e.g., an antigen-binding fragment), or a functional equivalent, to another moiety, such as a drug moiety. In this regard, the "ADC formula" described herein: Ab-(L-(D) x ) yReferring again to Figure 1, the drug moiety D can be linked to brentuximab through a linker L. L is any chemical moiety capable of linking brentuximab Ab to drug moiety D. Preferably, linker L links brentuximab Ab to drug moiety D through a covalent bond. A linker reagent is a bifunctional or multifunctional moiety that can be used to link drug moiety D and brentuximab Ab to form an antibody drug conjugate. The antibody drug conjugate can be prepared with a linker having a reactive functional group for binding to drug moiety D and antibody Ab. The terms "linker reagent", "crosslinking reagent", "linker derived from crosslinking reagent" and "linker" may be used interchangeably throughout this disclosure.

[0100] The linker may be susceptible to cleavage, such as enzymatic cleavage, acid-induced cleavage, photoinduced cleavage and disulfide bond cleavage (cleavable linker). Enzymatic cleavage includes, but is not limited to, protease-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphatase-induced cleavage, and sulfatase-induced cleavage, preferably under conditions under which the compound or antibody remains active. Alternatively, the linker may be substantially resistant to cleavage (e.g., stable linker or non-cleavable linker). In some aspects, the linker may be a procharged linker, a hydrophilic linker, a PEG-based linker, or a dicarboxylic acid-based linker. Thus, in some embodiments of any one of the antibody drug conjugates disclosed herein, the linker (L) is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a PEG-based linker, a procharged linker, and a dicarboxylic acid-based linker. In some embodiments, L is a cleavable linker. In some embodiments, L is a non-cleavable linker. In some embodiments, L is a linker that is susceptible to enzymatic cleavage. In some embodiments, L is an acid-labile linker, a photolabile linker, a peptidase-cleavable linker, a protease-cleavable linker, an esterase-cleavable linker, a glycosidase-cleavable linker, a phosphatase-cleavable linker, a sulfatase-cleavable linker, a disulfide bond-reducing linker, a hydrophilic linker, a procharged linker, a PEG-based linker, or a dicarboxylic acid-based linker. Preferably, it is a peptidase-cleavable linker. Other preferred linkers are cleavable by proteases.

[0101] Non-cleavable linker is any chemical moiety that can link drug to antibody in a stable covalent manner, and does not fall into the above category of cleavable linker.Therefore, non-cleavable linker is substantially resistant to acid-induced cleavage, peptidase-induced cleavage, protease-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.Furthermore, non-cleavable refers to the ability of the chemical bond in or adjacent to the linker to withstand the cleavage induced by acid, photolabile cleavage agent, peptidase, protease, esterase, or chemical or physiological compound that cleaves disulfide bond under the condition that drug or antibody does not lose its activity.

[0102] An acid labile linker is a linker that is cleavable at an acidic pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), providing suitable conditions for cleaving an acid labile linker.

[0103] Some linkers can be cleaved by peptidases, i.e., peptidase-cleavable linkers. In this regard, certain peptides are easily cleaved intracellularly or extracellularly, see, for example, Trout et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989). Peptides are composed of α-amino acids and peptide bonds, which are chemically amide bonds between the carboxylate of one amino acid and the amino group of the second amino acid.

[0104] Some linkers can be cleaved by esterases, i.e., esterase-cleavable linkers. In this regard, certain esters can be cleaved by esterases present inside or outside the cell. Esters are formed by condensation of a carboxylic acid with an alcohol. Simple esters are esters formed with simple alcohols, such as aliphatic alcohols, as well as small cyclic and small aromatic alcohols.

[0105] Procharged linkers are derived from charged cross-linking reagents that retain their charge after incorporation into an antibody drug conjugate. Examples of procharged linkers are described in US 2009 / 0274713.

[0106] The linker may be preferably cleavable by a protease, more preferably by a cathepsin, such as cathepsin B. The linker may comprise a valine-citrulline moiety that may be cleaved by a cathepsin, such as cathepsin B. The linker may comprise a hydroxylamine group, and the non-natural amino acid comprises a formyl group at the ortho position of the hydroxyl group of the aromatic ring, such as 3-formylthiolsine, where the hydroxylamine group of the linker forms an oxime with the formyl group of the non-natural amino acid after conjugation. The linker may comprise a cleavage site. A "cleavage site" is a sequence of amino acids that is recognized by a protease or peptidase and hydrolyzed by said protease or peptidase. Preferably, the cleavage site is a valine-citrulline moiety.

[0107] In one embodiment, the linker of the ADC of the invention has the formula: a -X x -W w -Y y wherein: -A- is a Stretcher unit; a is 0 or 1; where -X- is a second Spacer unit; x is independently an integer ranging from 0 to 12; each -W- is independently an Amino Acid unit; w is independently an integer ranging from 0 to 12; -Y- is a first Spacer unit; and y is independently an integer ranging from 0 to 12, preferably 0, 1 or 2.

[0108] The Stretcher unit (-A-), when present, can link the Ligand unit, i.e., the unnatural amino acid of Brentuximab contained in the ADC of the invention, to the Amino Acid unit (-W-). In this regard, the unnatural amino acid has a functional group that can form a bond with the functional group of the Stretcher. Useful functional groups that can be present on the unnatural amino acid, either naturally or through chemical manipulation, include, but are not limited to, sulfhydryl (-SH), amino, azido, alkynyl, hydroxyl, carboxy, anomeric hydroxyl group of carbohydrates, formyl, and carboxyl. In one aspect, the unnatural amino acid functional group is sulfhydryl and amino. Preferably, the functional group of the unnatural amino acid is formyl. The sulfhydryl group can be generated by reduction of the intramolecular disulfide bond of the Ligand. Alternatively, the sulfhydryl group can be generated by reaction of the amino group of the lysine moiety of the unnatural amino acid with 2-iminothiolane (Traut's Reagent) or another sulfhydryl generating reagent.

[0109] The reactive group of the Stretcher may comprise a reactive site that is reactive to the (-CHO) group of a modified carbohydrate that may be present on the unnatural amino acid. For example, the carbohydrate may be mildly oxidized using a reagent such as sodium periodate, or the unnatural formyl tyrosine amino acid may be linked to a ligand using TTL and the resulting (-CHO) unit may be condensed with a Stretcher that contains functional groups such as hydrazides, oximes, pyrazolones, thiopyrazolones, primary or secondary amines, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides, such as those described in Kaneko, T. et al. (1991) Bioconjugate Chem 2:133-41. Exemplary Stretcher units after attachment to the unnatural amino acids of this embodiment are represented by structures VIa, VIb, and VIc: where -W-, -Y-, -D, w and y are as defined above, and L is an unnatural amino acid in brentuximab comprised in an ADC of the invention, Here, R 17 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl.

[0110] The stretcher unit consists of: TIFF2024500922000009.tif142157, where G is selected from -Cl, -Br, -I, -O-mesyl, and -O-tosyl; and where J is -Cl, -Br, -I, -F, -OH, -ON-succinimide, -O(4-nitrophenyl), -O-pentafluorophenyl, -O-tetrafluorophenyl, and -OC(O)-OR. 18 Selected from; R 18 is -C1-C8 alkyl or -aryl, and where R 17 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl. The wavy line indicates a covalent bond to the amino acid unit W, if present, to the Spacer unit W, if no amino acid is present and the Spacer unit is present, or to the drug moiety if neither the amino acid unit nor the Spacer unit is present.

[0111] The Stretcher unit may be linked to an unnatural amino acid in the form of 1: It may comprise or consist of the structure shown in TIFF2024500922000010.tif14128, Here, R may represent a covalent bond to the second spacer unit X, the amino acid unit W, the first spacer unit W, or the drug moiety.

[0112] In the linkers described herein, the Stretcher unit may be joined to an unnatural amino acid by a 6 or 7: It may comprise or consist of the structure shown in TIFF2024500922000011.tif29128, wherein Z is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; z is independently an integer ranging from 0 to 12; wherein X is preferably substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl, or PEG. i wherein i is an integer ranging from 2 to 12, preferably selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 2; x is independently an integer ranging from 0 to 12; wherein Y is any first spacer unit as defined herein; y is independently an integer ranging from 0 to 12; where W is any amino acid unit as defined herein; w is independently an integer ranging from 0 to 12; and where D is a drug moiety, Preferably, herein, the hydroxylamine of structure 6 or 7 is conjugated to an unnatural amino acid of brentuximab included in the ADC of the invention, thereby forming an oxime after linkage.

[0113] The Amino Acid unit (-W-), when present, links the Stretcher unit to the Spacer unit if the Spacer unit is present, the Stretcher unit to the Drug moiety if the Spacer unit is absent, and the Ligand unit to the Stretcher unit and the Drug moiety if the Spacer unit is absent.

[0114] W w - is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit. Each -W- unit is independently one of the following: TIFF2024500922000012.tif28128 has the formula shown in square brackets, where w is an integer in the range of 0 to 12, In the formula, R 19are hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, The file is TIFF2024500922000013.tif95145.

[0115] The amino acid unit is enzymatically cleaved by one or more enzymes, including tumor-associated proteases, to liberate the drug moiety (-D), which in one embodiment is protonated in vivo after release to provide the drug (D). w The units are represented by formulae (VII) to (IX): TIFF2024500922000014.tif23128In the above formula, R 20 and R 21 is the following: As per TIFF2024500922000015.tif64128; TIFF2024500922000016.tif23128In the above formula, R 20 , R 21 , and R 22 is the following: As per TIFF2024500922000017.tif22128; TIFF2024500922000018.tif23128In the above formula, R 20 , R 21 , R 22 , and R 23 is the following: As shown in TIFF2024500922000019.tif21128.

[0116] Further suitable linkers are disclosed in Salomon et al., Mol. Pharmaceutics 2019, 16, 12, 4817-4825, which is incorporated herein by reference.

[0117] Exemplary amino acid units include, but are not limited to, units of formula (VII), where: 20 is benzyl and R 21 is -(CH2)4NH2; R 20 is isopropyl and R 21 is -(CH2)4NH2; R 20 is isopropyl, and R 21 is -(CH2)3NHCONH2. Another exemplary amino acid unit is a unit of formula (VIII), where R 20 is benzyl, and R 21 is benzyl and R 22 is -(CH2)4NH2.

[0118] Useful -W w - units can be designed and optimized for their selectivity for enzymatic cleavage by specific enzymes, e.g., tumor-associated proteases. In one embodiment, -W w -units, the cleavage of which is catalyzed by cathepsins B, C and D, or plasmin proteases ("tumor-associated proteases").

[0119] In one embodiment, -W w - is a dipeptide, tripeptide, tetrapeptide or pentapeptide.

[0120] R 19 , R 20 , R 21 , R 22 , or R 23 If is other than hydrogen, R 19 , R20 , R 21 , R 22 , or R 23 The carbon atom to which is attached is chiral.

[0121] R 19 , R 20 , R 21 , R 22 , or R 23 Each carbon atom to which is linked is independently in the (S) or (R) configuration.

[0122] In one aspect of the amino acid unit, the amino acid unit is valine-citrulline. In another aspect, the amino acid unit is phenylalanine-lysine (i.e., fk). In yet another aspect of the amino acid unit, the amino acid unit is N-methylvalinecitrulline. In yet another aspect, the amino acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinoline carboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, β-alanine lysine, glycine serine valine glutamine, and isonepecotic acid.

[0123] In certain embodiments, the Amino Acid units may comprise natural amino acids, while in other embodiments, the Amino Acid units may comprise unnatural amino acids.

[0124] The first spacer unit (-Y-), when present, may link the amino acid unit to the drug moiety if the amino acid unit is present. Alternatively, the first spacer unit may link the stretcher unit to the drug moiety if the amino acid unit is absent. The first spacer unit may link the drug moiety to the ligand unit if both the amino acid unit and the stretcher unit are absent. The second spacer unit (-X-), when present, may link the stretcher unit to the amino acid unit. Alternatively, the second spacer unit may link the stretcher unit to the first spacer unit if no amino acid unit is present. Alternatively, the second spacer unit may link the stretcher unit to the drug moiety if no first spacer unit and no amino acid unit are present. Both spacer units may be the same or not the same. The disclosure about a "spacer unit" generally applies to both the first and second spacer units. The first and second spacer units may be the same. The first and second spacer units may be different, where each is independently selected from the following examples of spacer units:

[0125] Spacer units are of two general types: self-immolative and non-self-immolative. A non-self-immolative spacer unit is one in which some or all of the spacer unit remains attached to the drug moiety after the amino acid unit is cleaved, particularly enzymatically, from the drug-linker-ligand conjugate or drug-linker compound. Examples of non-self-immolative spacer units include, but are not limited to, the (glycine-glycine) spacer unit and the glycine spacer unit (both shown in Scheme 1) (below). When an exemplary compound containing a glycine-glycine spacer unit or a glycine spacer unit undergoes enzymatic cleavage by a tumor cell-associated protease, a cancer cell-associated protease, or a lymphocyte-associated protease, the glycine-glycine-drug moiety or the glycine-drug moiety becomes an LA a -Ww - In one embodiment, an independent hydrolysis reaction occurs within the target cell to cleave the glycine-drug moiety bond and liberate the drug. Scheme 1 TIFF2024500922000020.tif44128

[0126] In another embodiment, the spacer unit is a phenylene moiety Q m and p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3), substituted with Q, where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; and m is an integer ranging from 0 to 4.

[0127] In one embodiment, the non-self-immolative spacer unit is -Gly-Gly-. In another embodiment, the non-self-immolative spacer unit is -Gly-.

[0128] In one embodiment, an ADC is provided in which the first Spacer unit is absent (y=0), or a pharma- ceutically acceptable salt or solvate thereof. In one embodiment, an ADC is provided in which the second Spacer unit is absent (x=0), or a pharma- ceutically acceptable salt or solvate thereof.

[0129] Alternatively, exemplary compounds containing a self-immolative Spacer unit can release -D without the need for a separate hydrolysis step. In this embodiment, the Spacer unit is linked to -W via the amino nitrogen atom of the PAB group. w - and a PAB group directly linked to -D via a carbonate, carbamate, or ether group. Without being bound to any particular theory or mechanism, Scheme 2 shows a possible drug release mechanism of a PAB group directly linked to -D via a carbamate or carbonate group as proposed by Toki et al. (2002) J Org. Chem. 67:1866-1872. Scheme 2 TIFF2024500922000021.tif95128 where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; and p is in the range of 1 to about 20.

[0130] Without being bound to a particular theory or mechanism, Scheme 3 illustrates a possible drug release mechanism for a PAB group directly linked to -D via an ether or amine bond. Scheme 3 TIFF2024500922000022.tif103128 where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; and p is ranging from 1 to about 20.

[0131] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that cyclize after hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815), and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867). Elimination of amine-containing drugs substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) is also an example of a self-immolative spacer useful in the exemplary compounds.

[0132] In one embodiment, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, as shown in Scheme 4, which can be used to incorporate and release multiple drugs. Scheme 4 TIFF2024500922000023.tif36128 where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; n is 0 or 1; and p is ranging from 1 to about 20.

[0133] In one embodiment, the -D moieties are the same. In yet another embodiment, the -D moieties are different.

[0134] In one aspect, the spacer unit is represented by formula (X)-(XII): TIFF2024500922000024.tif27128In the above formula, Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; TIFF2024500922000025.tif43128.

[0135] The first spacer unit (-X-) is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl, or PEG. i and the like, where i is an integer ranging from 2 to 12, preferably selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 2; and x is independently an integer ranging from 0 to 12.

[0136] The second spacer unit (-Y-) is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl, or PEG. i and the like, where i is an integer ranging from 2 to 12, preferably selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 2; and x is independently an integer ranging from 0 to 12.

[0137] The linker may be selected from the group consisting of 1: It may have or include the structure shown in TIFF2024500922000026.tif14128, wherein R is one or more drug moieties, said drug moieties are linked to the hydroxylamine of 1, optionally by one or more cleavage sites, preferably where hydroxylamine 1 is conjugated to an unnatural amino acid.

[0138] The linker may be joined to the unnatural amino acid by 6 or 7: It may comprise or consist of the structure shown in TIFF2024500922000027.tif28128, wherein Z is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; z is independently an integer ranging from 0 to 12; wherein X is preferably substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl, or PEG. i wherein i is an integer ranging from 2 to 12, preferably selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 2; x is independently an integer ranging from 0 to 12; where Y is any (first) Spacer unit as defined herein; y is independently an integer ranging from 0 to 12; where W is any amino acid unit as defined herein; w is independently an integer ranging from 0 to 12; and where D is a drug moiety, Preferably, herein, the hydroxylamine of structure 6 or 7 is conjugated to an unnatural amino acid of brentuximab included in the ADC of the invention, thereby forming an oxime after linkage.

[0139] The linker may be modified by adding 2 or 3: It may have or include the structure shown in TIFF2024500922000028.tif35128, wherein Z is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; where D is one or more drug moieties; and wherein Y is a cleavage site, such as the cleavage site of a cathepsin, such as cathepsin B; Preferably, wherein the hydroxylamine of structure 2 or 3 is conjugated to an unnatural amino acid.

[0140] The linker may have the structure 4 or 5: It may have the structure shown in TIFF2024500922000029.tif76128, where D is a drug moiety described herein, preferably MMAE.

[0141] As used herein, "prior to being linked to a non-natural amino acid" refers to a chemical entity, such as a linker, prior to forming a covalent bond with its partner non-natural amino acid. However, the same is true for linkers or drug moieties herein that are not shown to be covalently attached to each other. One of ordinary skill in the art can readily recognize the structure that is formed as a result of the reaction of two chemical entities. In the case of structures 1-5 as defined herein, the partner is a non-natural amino acid that contains a formyl group. The non-natural amino acid may be 3-formyl tyrosine, and the hydroxylamine group of the linker having any one of structures 1-5 may form an oxime / hydroxyimine with the 3-formyl group of the non-natural amino acid. The reaction mechanism is shown below: TIFF2024500922000030.tif63169. In this context, mAb corresponds to brentuximab and payload refers to a drug moiety, as defined herein, that is linked via a linker, as defined herein, to an unnatural amino acid, here 3-formyltyrosine.

[0142] Suitable cross-linking reagents for forming a non-cleavable linker between the drug moiety D and the brentuximab Ab are well known in the art and can form a non-cleavable linker containing a sulfur atom (such as SMCC) or a linker that does not contain a sulfur atom. The cross-linking reagent for forming a non-cleavable linker between the drug moiety D and the brentuximab Ab includes a maleimide-based or haloacetyl-based moiety. According to the present disclosure, such a non-cleavable linker is said to be derived from a maleimide-based or haloacetyl-based moiety.

[0143] Cross-linking reagents containing a maleimide-based moiety include, but are not limited to, N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), the "long chain" analogue of SMCC (LC-SMCC) N-succinimidyl-4-(maleimidomethyl)cyclohexa-1-carboxy-(6-amidocaproate), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-succinimidyl ester (E-MA), and the like. These include succinimidyl esters (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide esters (MBS), N-(α-maleimidoacetoxy)-succinimide esters (AMSA), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB), N-(-p-maleimidophenyl)isocyanate (PMIP) and maleimide-based cross-linking reagents containing a polyethylene glycol spacer, such as maleimide-PEG-NHS, also referred to herein as MAL-PEG-NHS. These cross-linking reagents form non-cleavable linkers derived from maleimide-based moieties. Representative structures of maleimide-based cross-linking reagents are shown below: TIFF2024500922000031.tif57128.

[0144] In some embodiments, the linker L is derived from N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexanene-1-carboxylate (sulfo-SMCC), or MAL-PEG-NHS.

[0145] Cross-linking reagents containing a haloacetyl moiety include N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido) propionate (SBAP). These cross-linking reagents form non-cleavable linkers derived from the haloacetyl moiety. Representative structures of haloacetyl cross-linking reagents are shown below: TIFF2024500922000032.tif29128.

[0146] In some embodiments, the linker L is derived from N-succinimidyl iodoacetate (SIA) or N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB).

[0147] Suitable cross-linking reagents that form a cleavable linker between the drug moiety D and the brentuximab Ab are well known in the art. A disulfide-containing linker is a linker that can be cleaved by disulfide exchange that can occur under physiological conditions. According to the present disclosure, such a cleavable linker is said to be derived from a disulfide-based moiety. Suitable disulfide cross-linking reagents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)2-sulfo-butanoate (sulfo-SPDB), the structures of which are shown below. These disulfide cross-linking reagents form cleavable linkers that are derived from disulfide-based moieties. TIFF2024500922000033.tif161133

[0148] In some embodiments, the linker L is derived from N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB).

[0149] Suitable cross-linking reagents that form a charged linker between the drug moiety D and the brentuximab Ab are known as pre-charged cross-linking reagents. In one embodiment, the linker L is derived from a pre-charged cross-linking reagent that is CX1-1. The structure of CX1-1 is shown below: TIFF2024500922000034.tif39155.

[0150] Additional linkers that may be suitable for use in the brentuximab drug conjugates disclosed herein are maleimidocaproyl (MC), maleimidocaproyl (MC) with a self-cleaving peptide, maleimidodiaminopropionyl (mDPR) with a self-cleaving peptide, and 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC).

[0151] In some embodiments, the linker is maleimidocaproyl (MC). Maleimidocaproyl has the following structure: TIFF2024500922000035.tif22128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an MC linker, brentuximab or an antigen-binding fragment thereof (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula.

[0152] The maleimidocaproyl linker may, for example, have the following structure: The cross-linking reagent may be derived from an NHS ester having the formula: TIFF2024500922000036.tif25128.

[0153] In some embodiments, the linker is 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC). MCC has the following structure: TIFF2024500922000037.tif22128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or antigen-binding fragment (Ab) or drug moiety (D), preferably brentuximab or antigen-binding fragment (Ab). As a non-limiting example, in a brentuximab drug conjugate comprising an MCC linker, brentuximab or antigen-binding fragment (Ab) may be covalently attached to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula, and the drug moiety (D) may be covalently attached to the maleimide moiety.

[0154] In some embodiments, the linker is maleimidocaproyl (MC) with a self-cleaving peptide. Non-limiting examples of maleimidocaproyl linkers with self-cleaving peptides are described below.

[0155] In some embodiments, the linker is maleimidodiaminopropionyl (mDPR) with a self-cleaving peptide. Non-limiting examples of maleimidodiaminopropionyl (mDPR) linkers with a self-cleaving peptide are described below.

[0156] In some embodiments, the brentuximab drug conjugate Ab-(L-(D) x ) yAny one of the linkers (L) is derived from a cross-linking reagent selected from the group consisting of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)2-sulfo-butanoate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), maleimido-PEG-NHS, maleimidocaproyl (MC), self-cleaving peptides. Maleimidocaproyl (MC) with a self-cleaving peptide, maleimidodiaminopropionyl (mDPR) with a self-cleaving peptide, 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC), N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) and 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazahepta-decane-1-oate (CX1-1). In preferred embodiments, the linker is derived from a cross-linking reagent selected from the group consisting of N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), maleimidocaproyl (MC), maleimidocaproyl with a self-cleaving peptide (MC), maleimidodiaminopropionyl with a self-cleaving peptide (mDPR), 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC), N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Thus, in some embodiments, the linker is derived from N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB).

[0157] In some embodiments, the linker is derived from maleimidocaproyl (MC). In some embodiments, the linker is derived from maleimidocaproyl (MC) with a self-cleaving peptide. In some embodiments, the linker is derived from maleimidodiaminopropionyl (mDPR) with a self-cleaving peptide. In some embodiments, the linker is derived from 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC). In some embodiments, the linker is derived from N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0158] As previously described herein, the linker may be maleimidocaproyl (MC) having a self-cleaving peptide. In some embodiments, the linker having a self-cleaving peptide is selected from the group consisting of maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), maleimidocaproyl-valine-alanine-p-aminobenzyloxycarbonyl (MC-VA-PAB), maleimidocaproyl-lysine-phenylalanine-p-aminobenzyloxycarbonyl (MC-KF-PAB), maleimidocaproyl-valine-lysine-p-aminobenzyloxycarbonyl (MC-VK-PAB). Maleimidocaproyl (MC) linkers with self-cleaving peptides are disclosed, for example, in U.S. Patent Application Publication No. US 2006 / 0074008, GM Dubowchik et al., Bioconjuate Chem. 2002, 13, 855-869, or SO Doronina et al., Nature Biotechnology, vol. 21, 778-784 (2003), the entire disclosures of which are incorporated herein by reference.

[0159] In some embodiments, the linker having a self-cleaving peptide is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB). The MC-VC-PAB linker has the following structure: TIFF2024500922000038.tif26128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an MC-VC-PAB linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula. This linker, which has a self-cleaving peptide containing a para-aminobenzyloxycarbonyl group, and the maleimidocaproyl linker below, can be derived, for example, from a cross-linking reagent in which a para-nitrophenoxy group is attached at the position indicated by the wavy line (see, for example, GM Dubowchik et al., Bioconjuate Chem. 2002, 13, 855-869, or SO Doronina et al., Nature Biotechnology, vol. 21, 778-784 (2003)). In the case of the MC-VC-PAB linker, the cross-linking reagent thus has the structure: For example, when reacting such cross-linking reagents with a drug moiety, the para-nitrophenoxy group may be replaced by the drug moiety.

[0160] Without wishing to be bound by any theory, in the MC-VC-PAB linker and maleimidocaproyl linker with a self-cleaving peptide containing a para-aminobenzyloxycarbonyl group described below, cleavage by a protease may occur between the peptide moiety and the para-aminobenzyloxycarbonyl moiety (see, for example, GM Dubowchik et al., Bioconjuate Chem. 2002, 13, 855-869, or SO Doronina et al., Nature Biotechnology, vol. 21, 778-784 (2003)). The para-aminobenzyloxycarbonyl moiety may then act as a self-immolative group. In this regard, the term "self-immolative group" refers to a bifunctional chemical moiety that can covalently link two spaced apart chemical moieties into a typically stable tripartite molecule that spontaneously separates from the second chemical moiety upon cleavage of the bond to the first moiety.

[0161] In some embodiments, the linker having a self-cleaving peptide is maleimidocaproyl-valine-alanine-p-aminobenzyloxycarbonyl (MC-VA-PAB). The MC-VA-PAB linker has the following structure: TIFF2024500922000040.tif26128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an MC-VA-PAB linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula.

[0162] In some embodiments, the linker having a self-cleaving peptide is maleimidocaproyl-lysine-phenylalanine-p-aminobenzyloxycarbonyl (MC-KF-PAB). The MC-KF-PAB linker has the following structure: TIFF2024500922000041.tif26128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an MC-KF-PAB linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula.

[0163] In some embodiments, the linker having a self-cleaving peptide is maleimidocaproyl-valine-lysine-p-aminobenzyloxycarbonyl (MC-VK-PAB). The MC-VK-PAB linker has the following structure: TIFF2024500922000042.tif26128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an MC-VK-PAB linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula.

[0164] As previously described herein, the linker may be maleimidodiaminopropionyl (mDPR) having a self-cleaving peptide. Such a linker may have the following structure: TIFF2024500922000043.tif22128 containing a maleimidodiaminopropionyl moiety (mDPR).

[0165] In some embodiments, the maleimide diaminopropionyl (mDPR) linker having self-cleaving peptide is selected from the group consisting of maleimide diaminopropionyl-valine-citrulline-p-aminobenzyloxycarbonyl (mDPR-VC-PAB), maleimide diaminopropionyl-valine-alanine-p-aminobenzyloxycarbonyl (mDPR-VA-PAB), maleimide diaminopropionyl-lysine-phenylalanine-p-aminobenzyloxycarbonyl (mDPR-KF-PAB), maleimide diaminopropionyl-valine-lysine-p-aminobenzyloxycarbonyl (mDPR-VK-PAB). Maleimide diaminopropionyl (mDPR) linker having self-cleaving peptide is disclosed, for example, in US Patent Application Publication No. US 2013 / 0309256, the entire disclosure of which is incorporated herein by reference.

[0166] In some embodiments, the linker having a self-cleaving peptide is maleimidodiaminopropionyl-valine-citrulline-p-aminobenzyloxycarbonyl (mDPR-VC-PAB). The mDPR-VC-PAB linker has the following structure: TIFF2024500922000044.tif29128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an mDPR-VC-PAB linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula. Without wishing to be bound by any theory, in the mDPR-VC-PAB linker with a self-cleaving peptide containing a para-aminobenzyloxycarbonyl group and the following maleimidodiaminopropionyl linker, cleavage by a protease may occur between the peptide moiety and the para-aminobenzyloxycarbonyl moiety. The para-aminobenzyloxycarbonyl moiety can then act as a self-immolative group.

[0167] In some embodiments, the linker having a self-cleaving peptide is maleimidodiaminopropionyl-valine-alanine-p-aminobenzyloxycarbonyl (mDPR-VA-PAB). The mDPR-VA-PAB linker has the following structure: TIFF2024500922000045.tif29128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising the mDPR-VA-PAB linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula.

[0168] In some embodiments, the linker having a self-cleaving peptide is maleimidodiaminopropionyl-lysine-phenylalanine-p-aminobenzyloxycarbonyl (mDPR-KF-PAB). The mDPR-KF-PAB linker has the following structure: TIFF2024500922000046.tif29128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising the mDPR-KF-PAB linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula.

[0169] In some embodiments, the linker having a self-cleaving peptide is maleimidodiaminopropionyl-valine-lysine-p-aminobenzyloxycarbonyl (mDPR-VK-PAB). The mDPR-VK-PAB linker has the following structure: TIFF2024500922000047.tif29128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an mDPR-VK-PAB linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula.

[0170] In some embodiments, the linker of the brentuximab drug conjugate disclosed herein comprises glucuronic acid modification.For linkers with glucuronic acid modification, see, for example, SC Jeffrey et al., Bioconjugate Chem. 2006, 17, 831-840; RP Lyon et al., Nature Biotechnology, vol. 33, 733-736 (2015); US Patent Application Publication No. US 2013 / 0309256; and International Publication No. WO 2015 / 057699, the entire disclosures of which are incorporated herein by reference.

[0171] In some embodiments, the linker containing a glucuronic acid modification may be maleimidocaproyl-glucuronide (MC-G) or maleimidodiaminopropionyl-glucuronide (mDPR-G).

[0172] The maleimidocaproyl-glucuronide (MC-G) linker has the following structure: TIFF2024500922000048.tif42128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an MC-G linker, brentuximab or an antigen-binding fragment thereof (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as indicated by the wavy line in the above structural formula.

[0173] The maleimidodiaminopropionyl-glucuronide (mDPR-G) linker has the following structure: TIFF2024500922000049.tif46128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or an antigen-binding fragment (Ab) or a drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an mDPR-G linker, brentuximab or an antigen-binding fragment (Ab) may be covalently attached to the maleimide moiety, and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker, as shown by the wavy line in the above structural formula.

[0174] In some embodiments, the linker comprising glucuronic acid modification further comprises polyethylene glycol modification.For linkers with glucuronic acid modification and polyethylene glycol modification, see, for example, RP Lyon et al., Nature Biotechnology, vol. 33, 733-736 (2015) and International Publication No. WO 2015 / 057699, the entire disclosures of which are incorporated herein by reference.

[0175] In some embodiments, the linker comprising a glucuronic acid modification and further comprising a polyethylene glycol modification may be maleimidocaproyl-glucuronide-polyethylene glycol (MC-G-PEG), maleimidodiaminopropionyl-glucuronide-polyethylene glycol (mDPR-G-PEG), or maleimidopropionyl-glucuronide-polyethylene glycol (MP-G-PEG).

[0176] The maleimidocaproyl-glucuronide-polyethylene glycol (MC-G-PEG) linker has the following structure: TIFF2024500922000050.tif53128, where n is an integer ranging from 6 to 72, 8 to 72, 12 to 72, 10 to 72, 12 to 36 or 38, 6 to 24 or 8 to 24; and where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or antigen binding fragment (Ab) or drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an MC-G-PEG linker, brentuximab or antigen binding fragment (Ab) may be covalently attached to the maleimide moiety and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker as indicated by the wavy line in the above structural formula.

[0177] The maleimidodiaminopropionyl-glucuronide-polyethylene glycol (mDPR-G-PEG) linker has the following structure: TIFF2024500922000051.tif57128, where n is an integer ranging from 6 to 72, 8 to 72, 10 to 72, 12 to 72, 12 to 36 or 38, 6 to 24 or 8 to 24; and where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or antigen binding fragment (Ab) or drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising an mDPR-G-PEG linker, brentuximab or antigen binding fragment (Ab) may be covalently attached to the maleimide moiety and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker as indicated by the wavy line in the above structural formula.

[0178] The maleimidopropionyl-glucuronide-polyethylene glycol (MP-G-PEG) linker has the following structure: TIFF2024500922000052.tif50128, where n is an integer ranging from 6 to 72, 8 to 72, 10 to 72, 12 to 72, 12 to 36 or 38, 6 to 24 or 8 to 24; and where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or antigen binding fragment (Ab) or drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising a MP-G-PEG linker, brentuximab or antigen binding fragment (Ab) may be covalently attached to the maleimide moiety and the drug moiety (D) may be covalently attached to the carbonyl carbon atom of the linker as indicated by the wavy line in the above structural formula.

[0179] Without wishing to be bound by any theory, release of drug from brentuximab drug conjugates containing the MC-G, mDPR-G, MC-G-PEG, mDPR-G-PEG or MP-G-PEG linkers may be initiated by cleavage of the glucuronic acid moiety by glucuronidase (see, e.g., S.C. Jeffrey et al., Bioconjugate Chem. 2006, 17, 831-840).

[0180] In some embodiments, the linker is a platinum complex linker.For platinum complex class linkers, see, for example, NJ Sijbrandi et al, Cancer Res. 2016, 77(2), 257-267, D. Walboer et al., ChemMedChem 2015, 10, 797-803 or US Patent Application Publication No. US 2014 / 377174, the entire disclosures of which are incorporated herein by reference.

[0181] In some embodiments, the platinum complex linker has the following structure: TIFF2024500922000053.tif19128, where R independently represents a halogen atom, e.g., Cl, Br, F or I, preferably Cl. As a non-limiting example, in a brentuximab drug conjugate comprising a platinum complex linker, one brentuximab or an antigen-binding fragment thereof (Ab) and one drug moiety (D) may be attached to the platinum complex.

[0182] In some embodiments, the platinum complex linker, particularly the ethylenediamine platinum (II) linker, and another part of the brentuximab drug conjugate, particularly the brentuximab or antigen-binding fragment (Ab) or the drug moiety (D), preferably the drug moiety (D), may be separated by a spacer. Suitable spacers may have different properties in length, composition, or cleavability. In some embodiments, the spacer has the following formula: TIFF2024500922000054.tif23128, wherein the NH2 group of the spacer is attached to another moiety of the brentuximab drug conjugate, particularly brentuximab or antigen-binding fragment (Ab) or drug moiety (D), preferably drug moiety (D), and the nitrogen atom of the piperidine group is attached to said platinum complex linker. TIFF2024500922000055.tif35128 shows a preferred embodiment of the use of such a spacer, where the NH2 group is attached to another part of the brentuximab drug conjugate, particularly brentuximab or antigen-binding fragment (Ab) or drug moiety (D), preferably drug moiety (D), and R represents attachment to another part of the brentuximab drug conjugate, particularly brentuximab or antigen-binding fragment (Ab) or drug moiety (D), preferably brentuximab or antigen-binding fragment (Ab). Preferably, the NH2 group is covalently attached to the drug moiety (D) and said platinum complex linker is attached to brentuximab or antigen-binding fragment (Ab), indicated by R. In some embodiments, the spacer has the following structure: and 4-oxo-4-((piperidin-4-ylmethyl)amino)butanoyl having the following structure: TIFF2024500922000056.tif21128, where the wavy line indicates the covalent attachment of the carbonyl carbon atom of the spacer to another moiety of the brentuximab drug conjugate, particularly brentuximab or antigen binding fragment (Ab) or drug moiety (D), preferably drug moiety (D), and where the nitrogen atom of the piperidine group is attached to said platinum complex linker. TIFF2024500922000057.tif34128 shows a preferred embodiment of the use of such a spacer, where the wavy line indicates the covalent attachment of the carbonyl carbon atom to another part of the brentuximab drug conjugate, particularly brentuximab or antigen binding fragment (Ab) or drug moiety (D), preferably drug moiety (D), and R represents the attachment to another part of the brentuximab drug conjugate, particularly brentuximab or antigen binding fragment (Ab) or drug moiety (D), preferably brentuximab or antigen binding fragment (Ab). Preferably, the carbonyl carbon is covalently attached to the drug moiety (D), as shown by the wavy line, and the platinum complex linker is attached to brentuximab or antigen binding fragment (Ab), as shown by R.

[0183] In some embodiments, the linker is a disulfide bridge replacement linker. The term "disulfide bridge replacement linker" used throughout this disclosure refers to a linker that can replace the disulfide bridge that arises from two cysteines of brentuximab (Ab). Thus, when such a disulfide bridge replacement linker is attached to a drug moiety (D), the drug moiety (D) is introduced into the space between the two cysteines of brentuximab (Ab). For disulfide bridge replacement linkers, see, for example, MEB Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965, and WO 2013 / 173393, the entire disclosures of which are incorporated herein by reference.

[0184] In some embodiments, the disulfide bridge replacement linker has the following structure: TIFF2024500922000058.tif31128, where X represents a halogen atom, e.g., Cl, Br, F or I, preferably Cl or Br, more preferably Br, and where the wavy line indicates the covalent attachment of the nitrogen atom to the drug moiety (D). For such linkers, see MEB Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965. A preferred embodiment has the following structure: TIFF2024500922000059.tif29128, where the wavy line indicates the covalent attachment of the drug moiety (D) to the maleimide moiety. One or both bromine atoms may react with a thiol group, for example, as shown in the following reaction scheme: As shown in TIFF2024500922000060.tif48128, the disulfide bridge of brentuximab or antigen-binding fragment (Ab) is obtained by reduction, where AA1, AA2, AA3 and AA4 comprise the peptide chain of brentuximab or antigen-binding fragment (Ab). AA1, AA2, AA3 and AA4 may or may not be part of a single peptide chain. In a preferred embodiment, the drug moiety is covalently attached to the maleimide moiety, as shown by R. Before reacting with dibromomaleimide, the disulfide bridge of brentuximab or antigen-binding fragment (Ab) is reduced with a reducing agent to obtain a free thiol group. Suitable reducing agents are, for example, dithiothreitol (DTT), sodium dithionite, sodium thiosulfate, sodium sulfite or tris(2-carboxyethyl)phosphine (TCEP), with tris(2-carboxyethyl)phosphine (TCEP) being preferred. After a reduction step, dibromomaleimide is added to brentuximab (Ab), which reacts with free thiol groups to form a cross-link between the two thiol groups of cysteines of the peptide chain.

[0185] In some embodiments, the disulfide bridge replacement linker is TIFF2024500922000061.tif70150, where Ab indicates attachment of brentuximab or an antigen-binding fragment to the peptide chain and the wavy line indicates attachment to the drug moiety (D) of the brentuximab drug conjugate. For such linkers, see WO 2013 / 173393. Thus, in some embodiments, the disulfide bridge replacement linker comprises a fragment selected from the group consisting of: In some embodiments, the disulfide bridge replacement linker comprises the fragment In some embodiments, the disulfide bridge replacement linker comprises the fragment In some embodiments, the disulfide bridge replacement linker comprises the fragment In some embodiments, the disulfide bridge replacement linker comprises the fragment In some embodiments, the disulfide bridge replacement linker comprises the fragment In some embodiments, the disulfide bridge replacement linker comprises the fragment With respect to the attachment of the drug moiety (D) shown with a wavy line, in these embodiments, the drug may be attached to the fragment through an optional spacer. Such a spacer may be, for example, an ethylene glycol oligomer, e.g., It can be derived from TIFF2024500922000069.tif11128.

[0186] In some embodiments, the linker is a glycolic linker. The term "glycolic linker" used throughout this disclosure generally refers to a linker that comprises a monosaccharide or oligosaccharide fragment. Glycolinkers are described, for example, in FS Ekholm et al., ChemMedChem 2016, 11, 2501-2505, US Patent Application No. US 2016 / 0820797, US Patent Application No. 2016 / 0257764, International Publication No. WO 2016 / 053107, US Patent Application No. US 2016 / 0106860 and International Publication No. WO 2016 / 001485, the entire disclosures of which are incorporated herein by reference.

[0187] Thus, a glycolic acid as used herein may comprise a fragment-G-, where G is a monosaccharide or a linear or branched oligosaccharide comprising 2 to 20, preferably 2 to 12, more preferably 2 to 10, even more preferably 2 to 8, and most preferably 2 to 6 sugar moieties. The sugar moieties that may be present in the fragment-G- are known to those skilled in the art and include, for example, glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc) or sialic acid, xylose (Xyl).

[0188] In some embodiments, the glycolic acid linker is a fragment TIFF2024500922000070.tif18128, where the wavy line indicates attachment of the brentuximab drug conjugate to brentuximab or an antigen-binding fragment (Ab) and a drug moiety (D). For example, in some embodiments, the fragment Glycolinkers from galactose, including TIFF2024500922000071.tif17128, may be used. In some embodiments, the fragment may be linked to brentuximab or an antigen-binding fragment (Ab) and / or a drug moiety (D) through an optional spacer. For example, a glycolic linker comprising a monosaccharide fragment and a spacer may have the following structure: TIFF2024500922000072.tif38128, where the wavy line indicates the attachment of the brentuximab drug conjugate to brentuximab or antigen binding fragment (Ab) and drug moiety (D). For example, brentuximab or antigen binding fragment (Ab) may be attached to a glycolic acid at the carbonyl carbon atom, and drug moiety (D) may be attached to a monosaccharide fragment, thus having the following structure: TIFF2024500922000073.tif40128, where Ab is brentuximab or an antigen-binding fragment and D is a drug moiety. In some embodiments, when the monosaccharide fragment is galactose, the glycolinker has the following structure: TIFF2024500922000074.tif37128, where the wavy line indicates the attachment of the brentuximab drug conjugate to brentuximab or antigen binding fragment (Ab) and drug moiety (D). For example, brentuximab or antigen binding fragment (Ab) may be attached to a glycolipid at the carbonyl carbon atom, and drug moiety (D) may be attached to a monosaccharide fragment, thus having the following structure: TIFF2024500922000075.tif42128, where Ab is brentuximab or an antigen-binding fragment (Ab) and D is a drug moiety. Glycolinkers of these embodiments are described, for example, in FS Ekholm et al., ChemMedChem 2016, 11, 2501-2505.

[0189] In some embodiments, the glycolic acid linker has the structure TIFF2024500922000076.tif15128, where the wavy line indicates binding to brentuximab or an antigen-binding fragment (Ab), where Su(A) xis a sugar derivative Su containing x functional groups A, where A is independently selected from the group consisting of thiol groups or precursors thereof, halogens, sulfonyloxy groups, halogenated acetamide groups, mercaptoacetamide groups and sulfonated hydroxyacetamide groups, where x is 1, 2, 3 or 4, and where b is 0 or 1. x is derived from a sugar or sugar derivative Su, such as an amino sugar or an otherwise derivatized sugar. Examples of sugars and sugar derivatives include galactose (Gal), mannose (Man), glucose (Glc), N-acetylneuraminic acid or sialic acid (Sial) and fucose (Fuc). Sugar derivative Su(A) x is preferably derived from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), fucose (Fuc) and N-acetylneuraminic acid (sialic acid Sia or NeuNAc), preferably from the group consisting of GlcNAc, Glc, Gal and GalNAc. More preferably, Su(A) x is derived from Gal or GalNac, most preferably Su(A) x is derived from GalNac. The drug moiety (D) of the brentuximab drug conjugate may be attached to the glycolic acid linker by reaction with functional group A. The drug moiety may be attached to the glycolic acid linker via any spacer, such as, for example, a spacer that includes a maleimide moiety. Glycolic acids of these embodiments are described, for example, in U.S. Patent Application No. US 2016 / 0280797.

[0190] In some embodiments, the glycolic acid linker has the structure TIFF2024500922000077.tif15128, where the wavy line indicates binding to brentuximab or an antigen-binding fragment (Ab), where Su(A) xis a sugar derivative Su containing x functional groups A, where A is independently selected from the group consisting of a thiol group or a precursor thereof, a halogen, a sulfonyloxy group, a halogenated acetamide group, a mercaptoacetamide group and a sulfonated hydroxyacetamide group, where x is 1, 2, 3 or 4, where b is 0 or 1, where d is 0 or 1, where e is 0 or 1 and where G is a monosaccharide or a linear or branched oligosaccharide containing 2-20 sugar moieties. x is derived from a sugar or sugar derivative Su, such as an amino sugar or an otherwise derivatized sugar. Examples of sugars and sugar derivatives include galactose (Gal), mannose (Man), glucose (Glc), N-acetylneuraminic acid or sialic acid (Sial) and fucose (Fuc). Sugar derivative Su(A) x is preferably derived from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), fucose (Fuc) and N-acetylneuraminic acid (sialic acid Sia or NeuNAc), preferably from the group consisting of GlcNAc, Glc, Gal and GalNAc. More preferably, Su(A) x is derived from Gal or GalNac, most preferably Su(A) xis derived from GalNac. G represents a monosaccharide or a linear or branched oligosaccharide containing 2-20, preferably 2-12, more preferably 2-10, even more preferably 2-8, and most preferably 2-6 sugar moieties. The sugar moieties that may be present in the fragment G are known to the skilled artisan and include, for example, glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc) or sialic acid, xylose (Xyl). The drug moiety (D) of the brentuximab drug conjugate may be attached to the glycolic acid by reaction with the functional group A. The drug moiety (D) may be attached to the glycolic acid through any spacer, such as, for example, a spacer containing a maleimide moiety. Glycolinkers of these embodiments are described, for example, in U.S. Patent Application No. US 2016 / 0280797.

[0191] In some embodiments, the linker is a methylene alkoxycarbamate linker. For methylene alkoxycarbamate linkers, see, for example, RV Kolakowski et al., Angew. Chem. Int. Ed. 2016, 55, 7948-7951 or WO 2015 / 095755, the entire disclosures of which are incorporated herein by reference.

[0192] In some embodiments, the methylene alkoxycarbamate linker has the following structure: TIFF2024500922000078.tif31128, where the wavy line indicates the attachment of the brentuximab drug conjugate to brentuximab or an antigen binding fragment (Ab) and the drug moiety (D), and the R group is C1-C4 alkyl, TIFF2024500922000079.tif14128. Preferably, R is C1-C4 alkyl, such as ethyl, or TIFF2024500922000080.tif13128. More preferably, R is TIFF2024500922000081.tif13128. In some embodiments, the brentuximab or antigen-binding fragment (Ab) and the drug moiety (D) have the following structure: and attached to a methylene alkoxycarbamate linker, as shown in TIFF2024500922000082.tif27128, where Ab is brentuximab or an antigen-binding fragment, and D is a drug moiety. In these embodiments, the R group is C1-C4 alkyl, TIFF2024500922000083.tif14128. Preferably, R is C1-C4 alkyl, such as ethyl, or TIFF2024500922000084.tif13128. More preferably, R is TIFF2024500922000085.tif13128. In some embodiments, the brentuximab or antigen-binding fragment (Ab) and / or the drug moiety (D) are linked to the methylene alkoxycarbamate linker through an optional spacer. Such spacers include, for example, the following structures: TIFF2024500922000086.tif24128, where the wavy line indicates the bond to a methylene alkoxycarbamate linker, which has the following structure: TIFF2024500922000087.tif34128, where the wavy line indicates the covalent attachment of the linker to another moiety of the brentuximab drug conjugate, particularly brentuximab or antigen binding fragment (Ab) or drug moiety (D), preferably drug moiety (D). As a non-limiting example, in a brentuximab drug conjugate comprising such a methylene alkoxycarbamate linker, brentuximab or antigen binding fragment (Ab) may be attached to the maleimide moiety and drug moiety (D) may be attached at the position indicated by the wavy line. In these embodiments, the R group is a C1-C4 alkyl, TIFF2024500922000088.tif14128. Preferably, R is C1-C4 alkyl, such as ethyl, or TIFF2024500922000089.tif13128. More preferably, R is The file is TIFF2024500922000090.tif13128.

[0193] In some embodiments, the linker (L) is -(succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)- , -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, and -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, preferably -(succinimide-3-yl-N)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0194] "D": Drug portion The present disclosure provides antibody drug conjugates comprising a drug moiety. As used herein, the terms "drug moiety" or "payload" can be used interchangeably and refer to a chemical or biochemical moiety that is conjugated to an antibody or antigen-binding fragment. In this regard, the "ADC-type" Ab-(L-(D) x )y Refer again to. Brentuximab can be conjugated to several identical or different drug moieties using any method described herein or known in the art. In some preferred embodiments, the drug moiety is an anti-cancer drug. Thus, the drug can be selected from the group consisting of camptothecins, topoisomerase inhibitors, maytansinoids, calicheamycins, duocarmycins, tubulysins, amatoxins, dolastatins and auristatins, such as monomethylauristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof. In a preferred embodiment, the drug moiety is MMAE.

[0195] "Camptothecin" (CPT) as used herein refers to a topoisomerase poison. It was discovered in 1966 by ME Wall and MC Wani in a systematic screening of natural products for anticancer drugs. CPT was isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China and used as a cancer treatment in traditional Chinese medicine. The term camptothecin may also include CPT analogs. Four CPT analogs have been approved and are used in cancer chemotherapy today: topotecan, irinotecan, belotecan, and trastuzumab deruxtecan. Camptothecin has the following structure: I have TIFF2024500922000091.tif29128.

[0196] The following CPT analogs are also contemplated by the term CPT: TIFF2024500922000092.tif252161

[0197] In some embodiments, the drug moiety has the following structure: maytansinoid drug moieties, including those having the formula: TIFF2024500922000093.tif51128, where the wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid to the linker of the antibody drug conjugate. R is independently at each occurrence H or C1-C6 alkyl. The alkylene chain linking the amide group to the sulfur atom may be methanyl, ethanyl, or propanyl, i.e., m is 1, 2, or 3. (U.S. Patent No. 633,410, U.S. Patent No. 5,208,020, Chari et al. (1992) Cancer Res. 52; 127-131, Lui et al. (1996) Proc. Natl. Acad. Sci. 93:8618-8623).

[0198] All stereoisomers of the maytansinoid drug moiety, i.e., any combination of R and S configurations of the chiral carbon of the maytansinoid, are contemplated for the antibody drug conjugates disclosed herein. In some embodiments, the maytansinoid drug moiety has the following stereochemistry: I have TIFF2024500922000094.tif52128.

[0199] In some embodiments, the maytansinoid drug moiety is N 2' -Deacetyl-N 2' -(3-mercapto-1-oxopropyl)-maytansine (also known as DM1). DM1 has the following structure: Represented by TIFF2024500922000095.tif52128.

[0200] In some embodiments, the maytansinoid drug moiety is N 2' -Deacetyl-N 2' -(4-mercapto-1-oxopentyl)-maytansine (also known as DM3). DM3 has the following structure: Represented by TIFF2024500922000096.tif58128.

[0201] In some embodiments, the maytansinoid drug moiety is N 2' -Deacetyl-N 2' -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (also known as DM4). DM4 has the following structure: Represented by TIFF2024500922000097.tif48128.

[0202] Preferably, the antibody drug conjugates disclosed herein that include a maytansinoid drug moiety are N 2' -Deacetyl-N 2' -(3-mercapto-1-oxopropyl)-maytansine (DM1) or N 2' -Deacetyl-N 2' -(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).

[0203] The drug moiety may be a calicheamicin. As used herein, "calicheamicin" refers to a class of enediyne antitumor antibiotics derived from the bacterium Micromonospora echinospora, of which calicheamicin γ1 is the most well-known. It was originally isolated from the chalky soil or "caliche pit" in Kerrville, Texas in the mid-1980s. It is highly toxic to all cells. Thus, the drug moiety may have the following structure: It may also be calicheamicin gamma 1, exemplified by TIFF2024500922000098.tif45128.

[0204] The drug moiety may be a duocarmycin. As used herein, "duocarmycin" refers to a small molecule, synthetic, DNA minor groove binding alkylating agent. Duocarmycin may be suitable for targeting solid tumors. Duocarmycin binds to the minor groove of DNA and alkylates the nucleobase adenine at the N3 position. Irreversible alkylation of DNA disrupts the nucleic acid structure, which ultimately causes tumor cell death. Examples of duocarmycin include, but are not limited to, CC-1065, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, and taxol and their derivatives.

[0205] The drug moiety may be a platinum-based antitumor agent, such as cisplatin or a derivative thereof.

[0206] The drug moiety may be tubulysin. Tubulysin has functions as an anti-microtubule agent, an anti-mitotic agent, an apoptosis inducer, an anti-cancer agent, an anti-angiogenic agent, and an anti-proliferative agent. Tubulysin is a cytotoxic peptide and includes nine members (A-I). Preferably, the tubulysin is tubulysin A. Tubulysin A has the potential application as an anti-cancer agent. It arrests cells in the G2 / M phase. Tubulysin A has the following structure: I have TIFF2024500922000099.tif41128.

[0207] The drug moiety may be an amatoxin. Amatoxins are a collective term for a subgroup of at least eight related toxic compounds found in several genera of poisonous mushrooms, particularly Amanita phalloides and several other members of the Amanita genus, as well as in some Conocybe, Galerina, and Lepiota mushroom species. Amatoxins are lethal even at low doses. The compounds have a similar structure, with eight amino acid residues arranged in a conserved macrobicyclic motif (a pentacyclic structure overall, counting the rings unique to the proline and tryptophan derived residues). All amatoxins are oligopeptides synthesized as 35-amino acid proproteins from which the final eight amino acids are cleaved by prolyl oligopeptidase. The approximate amino acid sequence of amatoxins is Ile-Trp-Gly-Ile-Gly-Cys-Asn-Pro, with a bridge between Trp and Cys via a sulfoxide (S=O) moiety and a hydroxylation in the variant of the molecule. Currently, there are 10 known amatoxins, which can be drug moieties. TIFF2024500922000100.tif20179

[0208] The drug moiety may be a dolastatin, such as dolastatin 10 or dolastatin 15. Both are marine natural products isolated from the Indian Ocean sea hare Dollabella auricularia. This potent antitumor agent has also been isolated from the Palauan marine cyanobacterium Symploca sp. VP642. Dolastatins 10 and 15, small linear peptide molecules, are considered anticancer drugs with potential against breast cancer, liver cancer, solid tumors, and some leukemias. Preclinical studies have shown efficacy in experimental antineoplastic and tubulin assembly systems. Dolastatins are mitotic inhibitors. They inhibit microtubule assembly by disrupting the formation of tubulin, thereby disrupting cell division by mitosis and inducing apoptosis and Bcl-2 phosphorylation in some malignant cell types. Dolostatin 10 (N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]-1-pyrrolidinyl}-5-methyl-1-oxo-4-heptanyl]-N-methyl-L-valinamide) has the following structure: I have TIFF2024500922000101.tif40131.

[0209] Dolastatin 15 ((2S)-1-[(2S)-2-benzyl-3-methoxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl]-3-methyl-1-oxo-2-butanyl N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-prolyl) has the following structure: I have TIFF2024500922000102.tif67136.

[0210] In some embodiments of the antibody drug conjugates disclosed herein, the drug moiety is an auristatin. Preferably, the auristatin is monomethylauristatin F (MMAF) or monomethylauristatin E (MMAE).

[0211] In some embodiments of the antibody drug conjugates disclosed herein, the drug moiety is monomethyl auristatin F (also known as MMAF). MMAF has the following structural formula: Represented in TIFF2024500922000103.tif45128. Monomethylauristatin F (MMAF) may be attached to the linker via the nitrogen atom marked with an asterisk (*).

[0212] In some embodiments, the auristatin drug moiety is monomethylauristatin E (also known as MMAE). MMAE has the following structural formula: Represented in TIFF2024500922000104.tif49128. Monomethylauristatin E (MMAE) may be attached to the linker via the nitrogen atom marked with an asterisk (*).

[0213] These molecules have been shown to non-competitively inhibit the binding of vincristine to tubulin (at a location known as the vinca / peptide domain), but bind to the RZX / MAY domain.

[0214] The drug moiety has the following structure: The compound may be a pyrrolobenzodiazepine dimer, such as the compound having the formula: TIFF2024500922000105.tif32139.

[0215] The drug moiety has the following structure: The compound may be an indolinobenzodiazepine dimer, such as the compound having TIFF2024500922000106.tif47156.

[0216] The drug moiety may be a radioisotope. Exemplary radioisotopes as described herein may be brachysources, typically gamma or beta emitters such as iodine-125, iodine-131, iridium-192 or palladium-103.

[0217] The drug moiety may be a therapeutic protein or peptide or a fragment thereof. Typical examples are cytokines such as interleukins, ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin PE38, etc.

[0218] The drug moiety is a kinase inhibitor, preferably an inhibitor of a kinase associated with a pro-tumorigenic function. Exemplary kinase inhibitors include imatinib, nilotinib, dasatinib, bosutinib, ponatinib, gefitinib, erlotinib, afatinib, osimertinib, lapatinib, neratinib, sorafenib, sunitinib, pazopanib, axitinib, lenvatinib, cabozatinib, vandetanib, regorafenib, vemurafenib, dabrafenib, trametinib, cobimetinib, crizotinib, certinib, alectinib, brigatinib, loratinib, ibrutinib, acalibrutinib, midostaurin, ruxolitinib, idelalisib, copanlisib, palbociclib, ribociclib, or abemaciclib.

[0219] The drug moiety may be a MEK inhibitor. As used herein, MEK inhibitor refers to a chemical or drug that inhibits mitogen-activated protein kinase, kinase enzymes MEK1 and / or MEK2. MEK inhibitors can be used to affect the MAPK / ERK pathway, which is often overactive in some cancers. Thus, MEK inhibitors are promising for the treatment of some cancers, especially BRAF-mutated melanoma, and KRAS / BRAF-mutated colorectal cancer. Exemplary MEK inhibitors include trametinib (GSK1120212), cobimetinib or XL518, binimetinib (MEK162), selumetinib, PD-325901, CI-1040, PD035901, or TAK-733.

[0220] The drug moiety may be a KSP (kinesin spindle protein) inhibitor. Examples of KSP inhibitors include ispinesib (SB-715992), SB743921, AZ 3146, GSK923295, BAY 1217389, MPI-0479605, and ARQ 621.

[0221] The drug moiety may be a nucleic acid. When used as a drug moiety, the nucleic acid may be associated with a DNA / RNA molecule, for example, a DNA / RNA molecule with an immunomodulatory function (preferably against innate immunity). Immunomodulation refers to increasing or decreasing, preferably decreasing, the immune response. The DNA / RNA molecule may also be an siRNA, preferably designed to (specifically) regulate / manipulate (e.g., decrease) target protein expression (an exemplary target may be myotonic dystrophy protein kinase (DMPK)).

[0222] The drug moiety may be a PROTAC. Proteolysis-inducing chimeric molecules (PROTACs) are heterobifunctional small molecules that contain two active domains and a linker that can remove certain unwanted proteins. PROTACs do not act as conventional enzyme inhibitors, but rather preferably act by inducing selective intracellular protein degradation. PROTACs generally contain two covalently linked protein-binding molecules, one of which can bind to an E3 ubiquitin ligase and the other of which binds to a target protein for degradation. Recruitment of the E3 ligase to the target protein causes ubiquitination and subsequent degradation of the target protein by the proteasome. Exemplary PROTACS are described in Sakamoto et a. (2001), PNAS 98(15):8554-9, incorporated herein by reference.

[0223] How to Make ADCs The method of making the ADC of the present invention is known in the art.In particular, introducing or adding a recognition sequence of tubulin tyrosine ligase to the C-terminus of a polypeptide, contacting the polypeptide (here, brentuximab) with natural amino acid in the presence of tubulin tyrosine ligase, and conjugating a linker comprising a drug moiety to said linked brentuximab has already been described in WO 2016 / 066749, WO 2017 / 186855, Schumacher et al., Angew. Chem. Int. Ed. 2015, 54, 13787-13791, all of which are incorporated herein by reference.The material and method section of the examples also includes guidance on the method of making or obtaining the ADC of the present invention.

[0224] Thus, the present invention further relates to a method of making an ADC as defined herein, said method comprising: (a) introducing or appending a recognition sequence for tubulin tyrosine ligase to the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of both the light and heavy chains of brentuximab; (b) contacting the brentuximab from step (a) in the presence of tubulin tyrosine ligase and an unnatural amino acid under conditions suitable for tubulin tyrosine ligase to ligate the brentuximab to the unnatural amino acid; and (c) conjugating an optionally cleavable linker comprising a drug moiety to the linked brentuximab obtained in step (b). Includes.

[0225] The introduction or addition of the recognition sequence of TTL to the C-terminus of brentuximab is carried out as described herein.For example, such a recognition sequence may be introduced or added by genetic engineering or synthesis, chemical protein synthesis or synthesis via synthetic biology.

[0226] The invention further relates to an ADC obtained by the method of making an ADC as defined herein.The invention further relates to an ADC obtained by the method of making an ADC as defined herein.

[0227] Pharmaceutical Compositions The present invention further relates to pharmaceutical compositions comprising the ADC of the present invention. Pharmaceutical compositions according to the present invention may further comprise one or more pharmaceutically acceptable carriers. In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopoeias for use in animals, particularly humans. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water, 5% dextrose, or physiological buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters, suitable for administration to human or non-human subjects. Certain exemplary pharmaceutically acceptable carriers include (biodegradable) liposomes; microspheres of biodegradable polymer poly(D,L-lactic-co-glycolic acid) (PLGA), albumin microspheres; synthetic polymers (soluble); nanofibers, protein-DNA complexes; protein conjugates; red blood cells; or virosomes. Various carrier-based dosage forms include solid lipid nanoparticles (SLN), polymeric nanoparticles, ceramic nanoparticles, hydrogel nanoparticles, co-peptide nanoparticles, nanocrystals and nanosuspensions, nanocrystals, nanotubes and nanowires, functionalized nanocarriers, nanospheres, nanocapsules, liposomes, lipid emulsions, lipid microtubules / microcylinders, lipid microbubbles, lipospheres, lipopolyplexes, reverse lipid micelles, dendrimers, ethosomes, multicomposite ultrathin capsules, aquasomes, pharmacosomes, colloidosomes, niosomes, discomes, proniosomes, microspheres, microemulsions, and polymeric micelles. Other suitable pharma- ceutically acceptable carriers and excipients are described in, among others, Remington's Pharmaceutical Sciences, 15 thEd., Mack Publishing Co., New Jersey (1991). Also see, for example, Remington: The Science and Practice of Pharmacy, 21 st see also: Lippincott Williams & Wilkins, 2005.

[0228] In some embodiments, pharmaceutically acceptable carrier or composition is sterile.In addition to active agent, pharmaceutical composition can contain physiologically acceptable compound, which acts as, for example, bulking agent, filler, solubilizer, stabilizer, penetrant, absorption enhancer, etc. Physiologically acceptable compound includes, for example, carbohydrate, such as glucose, sucrose, lactose; dextran; polyol, such as mannitol; antioxidant, such as ascorbic acid or glutathione; preservative; chelating agent; buffer; or other stabilizer or excipient.

[0229] The selection of a pharma- ceutically acceptable carrier and / or a physiologically acceptable compound may depend, for example, on the properties of the active agents, e.g., solubility, compatibility (meaning that the substances may coexist in a composition without interacting in a manner that substantially reduces the pharmacological effectiveness of the pharmaceutical composition under normal conditions of use), and / or the route of administration of the composition.

[0230] The pharmaceutical compositions of the invention contain a therapeutically effective amount of the ADCs described herein and can be configured in various forms, for example, solid, liquid, gaseous or lyophilized forms, in particular in the form of ointments, creams, transdermal patches, gels, powders, tablets, liquids, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, solutions, elixirs, extracts, tinctures, or liquid extracts, or in a form particularly suitable for topical or oral administration. Various routes are applicable for administration of the polypeptides of the invention, including, but not limited to, oral, topical, transdermal, subcutaneous, intravenous, intraperitoneal, intramuscular, or intraocular. However, any other route may be easily selected by one skilled in the art, if necessary.

[0231] Use in treatment As shown in Examples 4 and 5, the ADCs of the invention can be used for treatment, particularly for the treatment of cancer. Thus, the present invention further relates to the ADCs of the invention or the pharmaceutical compositions of the invention for use in a method of treating a disease, optionally comprising administering to a subject or patient in need thereof an effective amount of the ADCs of the invention or the pharmaceutical compositions of the invention. Preferably, the disease is associated with overexpression of CD30. The disease may be a cancer associated with overexpression of CD30. More preferably, the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE), preferably Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). The disease may be selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. The disease may be peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL). The disease may be cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL). The disease may be Hodgkin's lymphoma (HL).

[0232] The present invention also relates to the use of the ADC of the present invention for the manufacture of a medicament for treating a disease. The present invention also relates to the use of the pharmaceutical composition of the present invention for the manufacture of a medicament for treating a disease. Preferably, the disease is associated with overexpression of CD30. The disease may be a cancer associated with overexpression of CD30. More preferably, the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell cancer, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE), preferably Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). The disease may be selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. The disease may be peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL). The disease may be cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL). The disease may be Hodgkin's lymphoma (HL).

[0233] The present invention also relates to a method of treating a disease, comprising administering to a subject or patient in need thereof an effective amount of an ADC of the present invention. The present invention also relates to a method of treating a disease, comprising administering to a subject or patient in need thereof an effective amount of a pharmaceutical composition of the present invention. Preferably, the disease is associated with overexpression of CD30. The disease may be a cancer associated with overexpression of CD30. More preferably, the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE), preferably Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). The disease may be, but is not limited to, peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, or follicular T-cell lymphoma. The disease may be peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL), or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL). The disease may be Hodgkin's lymphoma (HL).

[0234] The phrase "effective amount", when used alone or in combination with another therapeutic agent, refers to an amount of a therapeutic agent (e.g., an ADC of the present invention) that protects a subject from developing a disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those skilled in the art, such as in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays. The exact amount will depend on the purpose of the treatment and can be ascertained by those skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0235] Furthermore, the present invention relates to an antibody drug conjugate disclosed herein for use in a method for treating cancer in a patient. The term "patient" according to the present invention means a mammal, such as a human, a non-human primate or another animal, in particular a cow, a horse, a pig, a sheep, a goat, a dog, a cat, or a rodent, such as a mouse and a rat. In a particularly preferred embodiment, the patient is a human. Unless otherwise stated, the terms "patient" or "control" are used interchangeably herein. The term "treatment" includes therapeutic or prophylactic treatment in all its grammatical forms. "Therapeutic or prophylactic treatment" includes prophylactic treatment aimed at complete prevention of clinical and / or pathological symptoms, or therapeutic treatment aimed at improvement or amelioration of clinical and / or pathological symptoms. Thus, the term "treatment" also includes improvement or prevention of disease.

[0236] The ADC of the present invention may be administered at any therapeutically effective dose. The upper limit is usually a dose that is safe to administer in terms of side effects. Typically, the ADC of the present invention may be administered at an (effective) dose of 0.5 to 20 mg / kg. The ADC of the present invention may be administered at an (effective) dose of 1 to 10 mg / kg. The ADC of the present invention may be administered at an (effective) dose of 1 to 9 mg / kg. The ADC of the present invention may be administered at an (effective) dose of 1.2 to 9 mg / kg. The ADC of the present invention may be administered at an (effective) dose of 1.8 to 8 mg / kg. The ADC of the present invention may be administered at an (effective) dose of 2 to 6 mg / kg. By way of illustration, the ADCs of the invention may be administered at (effective) doses of 20 mg / kg, 18 mg / kg, 16 mg / kg, 14 mg / kg, 12 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg. Other doses are possible if deemed advantageous or necessary.

[0237] The ADC of the present invention may be co-administered with other agents, particularly anti-cancer drugs, or compounds that enhance the effects of such agents. Co-administration includes sequential administration and simultaneous administration. Suitable anti-cancer drugs include, for example, one or more drug moieties described herein. The ADC of the present invention may be co-administered with cyclophosphamide, doxorubicin and prednisone (CHP scheme). The ADC of the present invention may be co-administered with anti-cancer drugs, such as immunomodulatory drugs, checkpoint inhibitors, chemotherapeutic drugs (e.g., cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone (CHOP scheme) etoposide, vincristine, chlormethine), protein inhibitors, among others.

[0238] "Tumor" refers to a group of cells or tissues formed by misregulated cell proliferation, especially cancer. Tumors may partially or completely lack structural organization and functional coordination with normal tissues, and usually form a distinct mass of tissue, which may be benign or malignant. In particular, the term "tumor" refers to malignant tumors. According to one embodiment, the term "tumor" or "tumor cell" also refers to non-solid cancers and cells of non-solid cancers such as leukemia cells. According to another embodiment, each non-solid cancer or its cells are not included in the terms "tumor" and "tumor cell".

[0239] "Metastasis" means the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process that usually involves cancer cells breaking off from the primary tumor, entering the systemic circulation, and establishing and growing in other normal tissues in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells are usually similar to the cells of the original tumor. That is, for example, if breast cancer metastasizes to the lungs, the secondary tumor will consist of abnormal breast cells, not abnormal lung cells. Thus, the lung tumor is called metastatic breast cancer, not lung cancer.

[0240] Exemplary ADCs of the Invention The following table discloses an overview of exemplary ADCs, in which the unnatural amino acid is 3-formyltyrosine and the drug moiety (D) is a drug moiety as defined herein, preferably MMAE. TIFF2024500922000107.tif144168

[0241] Structures 4 and 5 are repeated below: TIFF2024500922000108.tif72128.

[0242] In some embodiments, the present invention relates to an antibody drug conjugate (ADC), the ADC comprising: (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain, each light chain containing a recognition sequence has SEQ ID NO: 11; and Each heavy chain of brentuximab has SEQ ID NO: 1, Brentuximab and (b) The C-terminus of each light chain recognition sequence is linked via an amide bond to the following structure: TIFF2024500922000109.tif38162, Here, the wavy lines indicate the binding to the C-terminus of the recognition sequence of each light chain.

[0243] In some embodiments, the present invention relates to an antibody drug conjugate (ADC), the ADC comprising: (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each heavy chain, Each heavy chain containing a recognition sequence has SEQ ID NO: 12; and Each light chain of brentuximab has SEQ ID NO: 2; Brentuximab and (b) The C-terminus of the recognition sequence of each heavy chain is linked via an amide bond to the following structure: TIFF2024500922000110.tif38162, Here, the wavy lines indicate the binding of the recognition sequence of each heavy chain to the C-terminus.

[0244] In some embodiments, the present invention relates to an antibody drug conjugate (ADC), the ADC comprising: (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each heavy chain; Each heavy chain containing a recognition sequence has SEQ ID NO: 12; and Brentuximab contains a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain; Each light chain containing a recognition sequence has SEQ ID NO: 11; Brentuximab and (b) The C-terminus of each heavy chain recognition sequence and the C-terminus of each light chain recognition sequence are connected via an amide bond to the following structure: TIFF2024500922000111.tif39163, Here, the wavy lines indicate the binding to the C-terminus of each heavy chain recognition sequence and to the C-terminus of each light chain recognition sequence.

[0245] In some embodiments, the present invention relates to an antibody drug conjugate (ADC), the ADC comprising: (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain, each light chain containing a recognition sequence has SEQ ID NO: 11; and Each heavy chain of brentuximab has SEQ ID NO: 1, Brentuximab and (b) The C-terminus of each light chain recognition sequence is linked via an amide bond to the following structure: TIFF2024500922000112.tif79153, Here, the wavy lines indicate the attachment of the recognition sequence of each light chain to the C-terminus. In some embodiments, the present invention relates to an antibody drug conjugate (ADC), the ADC comprising: (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain, Each light chain containing a recognition sequence has SEQ ID NO: 14; and Each heavy chain of brentuximab has SEQ ID NO: 1, Brentuximab and (b) The C-terminus of each light chain recognition sequence is linked via an amide bond to the following structure: TIFF2024500922000113.tif35150 Here, the wavy lines indicate the binding to the C-terminus of the recognition sequence of each light chain.

[0246] In some embodiments, the present invention relates to an antibody drug conjugate (ADC), the ADC comprising: (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each heavy chain; Each heavy chain containing a recognition sequence has SEQ ID NO: 12; and Brentuximab contains a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain; Each light chain containing a recognition sequence has SEQ ID NO: 14; Brentuximab and (b) The C-terminus of each heavy chain recognition sequence and the C-terminus of each light chain recognition sequence are connected via an amide bond to the following structure: TIFF2024500922000114.tif36151, Here, the wavy lines indicate the binding to the C-terminus of each heavy chain recognition sequence and to the C-terminus of each light chain recognition sequence.

[0247] In some embodiments, the present invention relates to an antibody drug conjugate (ADC), the ADC comprising: (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain, Each light chain containing a recognition sequence has SEQ ID NO: 14; and Each heavy chain of brentuximab has SEQ ID NO: 1, Brentuximab and (b) The C-terminus of each light chain recognition sequence is linked via an amide bond to the following structure: TIFF2024500922000115.tif80153, Here, the wavy lines indicate the binding to the C-terminus of the recognition sequence of each light chain.

[0248] array This disclosure specifically refers to the following sequences: Brentuximab / cAC10 heavy chain (SEQ ID NO: 1): TIFF2024500922000116.tif132170 Brentuximab / cAC10 light chain (SEQ ID NO: 2): TIFF2024500922000117.tif64170TTL recognition sequence (SEQ ID NO: 3): Xaa1-Xaa2-Xaa3-Glu, Where: Xaa1 is any amino acid; illustratively, Xaa1 can be Glu, Asp, Ala, Lys, or Pro; Xaa2 is any amino acid; illustratively, Xaa2 can be Gly, Ser, Ala, Val, or Phe; and Xaa3 is Glu, Asp or Cys. TTL Recognition Sequence (SEQ ID NO: 4): Glu-Gly-Glu-Glu TTL Recognition Sequence (SEQ ID NO: 5): TIFF2024500922000118.tif4128TTL recognition sequence (SEQ ID NO: 6): TIFF2024500922000119.tif4128TTL recognition sequence (SEQ ID NO: 7): TIFF2024500922000120.tif4128TTL recognition sequence (SEQ ID NO: 8): TIFF2024500922000121.tif4128TTL recognition sequence (SEQ ID NO: 9): TIFF2024500922000122.tif4128TTL recognition sequence (SEQ ID NO: 10): TIFF2024500922000123.tif4128 Brentuximab light chain (SEQ ID NO: 11), containing a Gly-Gly-Gly-Gly-Ser-linker and a TTL recognition sequence at the C-terminus: TIFF2024500922000124.tif71170 Brentuximab heavy chain (SEQ ID NO: 12) linked to a TTL recognition sequence at the C-terminus: TIFF2024500922000125.tif138170TTL from gray wolf (canis lupus) (SEQ ID NO: 13): TIFF2024500922000126.tif111170 Brentuximab light chain (SEQ ID NO: 14), containing a TTL recognition sequence at the C-terminus: TIFF2024500922000127.tif71170

[0249] Item of the present invention The present invention further relates to the following items: 1. (a) brentuximab, comprising a tubulin tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of all of the heavy and light chains of brentuximab; and (b) at least one drug moiety; Including, wherein a drug moiety is linked to each of the unnatural amino acids via a linker. Antibody drug conjugates (ADCs). 2. The ADC of item 1, wherein the heavy chain of brentuximab has an amino acid sequence comprising or has at least 95% sequence identity to SEQ ID NO: 1, and / or the light chain of brentuximab has an amino acid sequence comprising or has at least 95% sequence identity to SEQ ID NO: 2, preferably brentuximab consists of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain consisting of the amino acid sequence of SEQ ID NO: 2. 3. The ADC of any of the preceding items, wherein the drug moiety is selected from the group consisting of camptothecins, maytansinoids, calicheamicins, duocarmycins, tubulysins, amatoxins, dolastatins, and auristatins such as monomethylauristatin E (MMAE), pyrrolobenzodiazepine dimers, indolinobenzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof, preferably the drug moiety is MMAE. 4. The ADC of any of the preceding items, wherein the recognition sequence for tubulin tyrosine ligase has at least the amino acid sequence X1X2X3X4 (SEQ ID NO:3), where X1 and X2 are any amino acid, X3 is E, D, or C, and X4 is E, preferably where X2 is G, S, A, V, or F and / or X1 is E, D, A, K, or P. 5. The recognition sequence is EGEE (SEQ ID NO: 4), preferably the recognition sequence is The ADC of any of the preceding items, wherein the ADC is TIFF2024500922000128.tif24165. 6. The unnatural amino acid is 2-, 3-, or 4-substituted tyrosine or 2-, 3-, or 4-substituted tyrosine derivatives substituted at the benzyl position The ADC of any of the preceding items, 7. The ADC of item 6, wherein the 3-substituted tyrosine derivative or 4-substituted tyrosine derivative is 3-nitrotyrosine, 3-aminotyrosine, 3-azidotyrosine, 3-formyltyrosine, 3-acetyltyrosine, or 4-aminophenylalanine, preferably the unnatural amino acid is 3-formyltyrosine. 8. The ADC of any of the preceding items, wherein the linker is preferably cleavable by a protease, more preferably by a cathepsin, such as cathepsin B. 8a. The ADC of any of items 1 to 7, wherein the linker is a non-cleavable linker. 9. The ADC of any of the preceding items, wherein the linker comprises a valine-citrulline moiety. 10. The ADC of any of the preceding items, wherein the linker comprises a hydroxylamine group and the unnatural amino acid comprises a formyl group ortho to the hydroxyl group in the aromatic ring, such as 3-formylthiolsine, where the hydroxylamine group of the linker forms an oxime with the formyl group of the unnatural amino acid after conjugation. 11. The ADC of any of the preceding items, wherein brentuximab is conjugated to 2, 4, 6, or 8, preferably 2 or 4, drug moieties, more preferably 2 drug moieties. 12. Before the linker is linked to the unnatural amino acid, it has the structure 1: It has the structure shown in TIFF2024500922000129.tif14128, wherein R is one or more drug moieties, said drug moieties are optionally linked to the hydroxylamine of Structure 1 by one or more cleavage sites, preferably wherein the hydroxylamine of Structure 1 is conjugated to a non-natural amino acid. Any of the ADCs listed above. 13. The linker has the structure 2 or 3: It has the structure shown in TIFF2024500922000130.tif34128, wherein Z is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; where D is one or more drug moieties; and wherein Y is a cleavage site, such as the cleavage site of a cathepsin, such as cathepsin B; Preferably, wherein the hydroxylamine of structure 2 or 3 is conjugated to a non-natural amino acid. Any of the ADCs listed above. 14. The linker has the structure 4 or 5: before being linked to the unnatural amino acid: TIFF2024500922000131.tif95153, where D is a drug moiety, preferably MMAE; Any of the ADCs listed above. 15. The ADC of item 14, wherein D is MMAE, the unnatural amino acid is 3-formyltyrosine, and the hydroxylamine group of the linker forms an oxime with the 3-formyl group of the unnatural amino acid. 16. (a) A brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain; each light chain containing a recognition sequence has SEQ ID NO: 11; and Each heavy chain of brentuximab has SEQ ID NO: 1, Brentuximab and (b) The C-terminus of each light chain recognition sequence is linked via an amide bond to the following structure: TIFF2024500922000132.tif33138, Here, the wavy lines indicate the binding of the recognition sequence of each light chain to the C-terminus. Antibody drug conjugates (ADCs). 17. (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each heavy chain; Each heavy chain containing a recognition sequence has SEQ ID NO: 12; and Each light chain of brentuximab has SEQ ID NO: 2; Brentuximab and (b) The C-terminus of the recognition sequence of each heavy chain is linked via an amide bond to the following structure: TIFF2024500922000133.tif36152, Here, the wavy lines indicate the binding of the recognition sequence of each heavy chain to the C-terminus. Antibody drug conjugates (ADCs). 18. (a) Brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each heavy chain; Each heavy chain containing a recognition sequence has SEQ ID NO: 12; and Brentuximab contains a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain; Each light chain containing a recognition sequence has SEQ ID NO: 11; Brentuximab and (b) The C-terminus of each heavy chain recognition sequence and the C-terminus of each light chain recognition sequence are connected via an amide bond to the following structure: TIFF2024500922000134.tif33139, where the wavy lines indicate the binding to the C-terminus of each heavy chain recognition sequence and to the C-terminus of each light chain recognition sequence. Antibody drug conjugates (ADCs). 19. (a) Brentuximab, comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain, each light chain containing a recognition sequence has SEQ ID NO: 11; and Each heavy chain of brentuximab has SEQ ID NO: 1, Brentuximab and (b) The C-terminus of each light chain recognition sequence is linked via an amide bond to the following structure: TIFF2024500922000135.tif79153, Here, the wavy lines indicate the binding of the recognition sequence of each light chain to the C-terminus. Antibody drug conjugates (ADCs). 20. (a) introducing or adding a recognition sequence for tubulin tyrosine ligase to the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of both the light chain and the heavy chain of brentuximab; (b) contacting the brentuximab from step (a) in the presence of tubulin tyrosine ligase and an unnatural amino acid under conditions suitable for tubulin tyrosine ligase to ligate the brentuximab to the unnatural amino acid; and (c) conjugating an optionally cleavable linker comprising a drug moiety to the linked brentuximab obtained in step (b). 20. A method for producing an ADC as defined in any one of items 1 to 19, comprising: 21. An ADC obtainable or obtained by the method of item 20. 22. A pharmaceutical composition comprising the ADC of any of items 1 to 19 or 21. 23. The ADC of any of items 1 to 19 or 21 or the pharmaceutical composition of item 22 for use in a method for treating a disease. 24. The ADC or pharmaceutical composition for use according to item 23, wherein the disease is associated with overexpression of CD30. 25. The ADC or pharmaceutical composition for use according to item 23 or 24, wherein the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE), preferably Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). 25a. The ADC or pharmaceutical composition for use of item 23 or 24, wherein the disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 25b. The ADC or pharmaceutical composition for use of item 23 or 24, wherein the disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL). 26. Use of an ADC of any of items 1 to 19 or 21 for the manufacture of a medicament for treating a disease associated with overexpression of CD30. 27. The use of item 26, wherein the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE). 28. The use of item 27, wherein the disease is Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). 28a. The use of item 26, wherein the disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 28b. The use of item 26, wherein the disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL). 29. Use of the pharmaceutical composition of item 22 for the manufacture of a medicament for treating a disease associated with overexpression of CD30. 30. The use of item 29, wherein the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell cancer, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE). 31. The use of item 30, wherein the disease is Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). 31a. The use of item 29, wherein the disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 31b. The use of item 29, wherein the disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL). 32. A method for treating a disease associated with overexpression of CD30, comprising administering to a subject or patient in need thereof an effective amount of an ADC of any of items 1 to 19. 33. The method of item 32, wherein the disease is a cancer associated with overexpression of CD30. 34. The method of item 32 or 33, wherein the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE), preferably Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). 35. The method of item 34, wherein the disease is Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). 35a. The method of items 32 or 33, wherein the disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 35b. The method of items 32 or 33, wherein the disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL). 36. Any of the methods of items 32 to 35b, wherein the ADC is administered at a dose of 14 mg / kg, 12 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg or 1 mg / kg; or at a dose of 2 to 6 mg / kg. 37. A method for treating a disease associated with overexpression of CD30, comprising administering to a subject or patient in need thereof an effective amount of the pharmaceutical composition of item 22. 38. The method of item 37, wherein the disease is a cancer associated with overexpression of CD30. 39. The method of item 37 or 38, wherein the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE), preferably Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). 40. The method of item 39, wherein the disease is Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL). 40a. The method of items 37 or 38, wherein the disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma. 40b. The method of items 37 or 38, wherein the disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL). 41. Any of the methods of items 37 to 40b, wherein the ADC is administered at a dose of 14 mg / kg, 12 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg or 1 mg / kg; or at a dose of 2 to 6 mg / kg.

[0250] It is noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such different reagents, and reference to a "method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or substituted for the methods described herein.

[0251] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0252] The term "and / or" as used anywhere in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0253] The terms "less than" or "more than" do not include specific numbers.

[0254] For example, less than 20 means less than the indicated number. Similarly, more than or greater than means more than or greater than the indicated number, for example, more than 80% means more than or greater than 80% of the indicated number.

[0255] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises," "comprising" and the like are understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," or, as sometimes used herein, with the term "having." As used herein, "consisting of" excludes any element, step, or ingredient not specified.

[0256] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0257] As used herein, the terms "about," "approximately," or "essentially" mean within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. Also, a specific numerical value, i.e., "about 20," includes the numerical value 20.

[0258] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0259] All publications cited throughout the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manuals, etc., are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. In the event that material incorporated by reference conflicts or is inconsistent with this specification, the present specification supersedes any such material.

[0260] The contents of all publications and patent documents cited herein are incorporated herein by reference in their entirety. EXAMPLES

[0261] A more complete understanding of the present invention and its advantages will become apparent from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0262] material and method Chemicals, Materials, Reagents and Solvents Chemicals and solvents were purchased from Merck (Merck group, Germany), TCI (Tokyo chemical industry CO., LTD., Japan), Acros Organics (Thermo Fisher scientific, USA), Sigma-Aldrich (Merck group, Germany), Carl Roth (Carl Roth GmbH, Germany), and Cytiva (Cytiva UK Limited, England) and used without further purification. Anhydrous solvents were purchased from Acros Organics (Thermo Fisher scientific, USA).

[0263] Expression and purification of TTLs Tubulin-tyrosine ligase (TTL) was expressed in Escherichia coli (E. coli) BL21(DE3) with N-terminal His- and SUMO3 solubility tags as previously described (Schumacher et al. (2015), Angewandte Chemie (iNTERNATIONAL ED. IN eNGLISH), 54:13787-13791, WO 2016 / 066749, WO 2017 / 186855). Cultures were grown in LB medium supplemented with kanamycin (30 μg / mL) at 37 °C, 180 rpm, and 40 °C to an OD of 0.6–0.8. 600 After cooling the culture to 18°C, the cells were induced with 0.5 mM IPTG and protein expression was carried out at 18°C ​​for 18 hours.

[0264] Cultures were harvested by centrifugation and then subjected to sonication in TTL binding buffer (20 mM Tris / HCl pH 8.2, 250 mM NaCl, 20 mM imidazole, 3 mM β-mercaptoethanol) supplemented with lysozyme (100 μg / ml), DNase (25 μg / ml) and PMSF (1 mM), followed by centrifugation of the debris at 20.000 g for 30 min and filtration of the supernatant.

[0265] His-SUMO3-TTL was purified using 5 ml of His-Trap HP (Cytiva) applying a linear gradient to elution buffer (20 mM Tris / HCl pH 8.2, 250 mM NaCl, 500 mM imidazole, 3 mM β-mercaptoethanol) over 4 column volumes (CV). The purified protein was subsequently desalted on a PD-10 Sephadex G-25M column (Cytiva) by buffer exchange into TTL storage buffer (20 mM MOPS / KCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 50 mM L-arginine, 3 mM β-mercaptoethanol). Protein aliquots were flash frozen and stored at -80°C.

[0266] Expression and purification of brentuximab Tub tag Recombinant expression of brentuximab Tub-tag was performed in suspension-adapted CHO cells. Seeds were grown in chemically defined, animal component-free medium and the supernatant was harvested by centrifugation followed by filtration. Purification was performed using Protein A affinity chromatography (MabSelect SuRe, Cytiva). The pH of the HCCF was adjusted to 7.5 and the column was equilibrated with binding buffer (20 mM NaH2PO4, 50 mM NaCl, 1 mM EDTA pH 7.5). The flow rate was adjusted according to the titer of the culture to achieve optimal residence time on the column. Elution was performed stepwise up to elution buffer (100 mM trisodium citrate, pH 3.0). After neutralization of the eluate and buffer exchange into storage buffer (PBS, 100 mM L-arginine), the antibody was stored at 8–15 mg / mL, 2–8 °C (short-term storage) or flash frozen and stored at -80 °C (long-term storage).

[0267] Applying TTL to Brentuximab Tub-tag conjugation reaction TTL-catalyzed ligation was performed according to previous publications (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791, WO 2016 / 066749, WO 2017 / 186855). Briefly, 3-formyl-L-tyrosine was ligated to Brentuximab Tub tag mAbs in various amounts of 20 mM MOPS / KCl, 100 mM KCl, 10 mM MgCl2, 5 mM ATP, 5 mM 3-formyl-L-tyrosine, pH 7.0. After adjusting the pH value, Brentuximab Tub tag and TTL were added and the reaction was carried out at 18°C ​​for 12 hours to tyrosinate the antibody.

[0268] Purification of tyrosinated brentuximab Tub tag The crude tyrosination reaction was diluted to reduce the conductivity (<10 mS / cm). The mixture was then loaded onto a 5 mL HiTrap Capto Q ImpRes column (Cytiva). After washing the column with 5 CV of AEX binding buffer (14.4 mM Na2HPO4, 5.6 mM NaH2PO4, pH 4.5) and 5 CV of 10% AEX elution buffer (14.4 mM Na2HPO4, 5.6 mM NaH2PO4, 1 M NaCl pH 4.5), elution of the tyrosinylated brentuximab Tub tag was achieved by a linear gradient to 50% elution buffer in 10 CV. The protein concentration of the pooled product-containing fractions was measured by UV280 method (NanoPhotometer NP80, IMPLEN).

[0269] Oxime ligation after TTL reaction The protein concentration of tyrosinated brentuximab Tub tag after purification by anion exchange chromatography was adjusted and DMSO was added as a co-solvent (final 2% by volume). Oxime coupling was carried out with a slight excess of 2 (HA-vc-PAB-MMAE) or 3 (HA-(VC-PAB-MMAE)2) at 18°C ​​until reaction completion. The structures of payloads 2 and 3 are shown in Figure 23.

[0270] Purification of brentuximab Tub-tag ADC The crude oxime conjugate was diluted with CEX binding buffer (1:1 volume ratio, 14.4 mM Na2HPO4, 5.6 mM NaH2PO4, pH 4.5) and loaded onto a 5 mL HiTrap Capto S ImpAct column (Cytiva). After washing the column with 10 CV of CEX wash buffer (14.4 mM Na2HPO4, 5.6 mM NaH2PO4, 0.1% Triton-x-114, pH 4.5) and 5 CV of CEX binding buffer, elution of TUB-010 was achieved by a linear gradient to 100% CEX elution buffer (14.4 mM Na2HPO4, 5.6 mM NaH2PO4, 1 M NaCl pH 4.5) in 20 CV. The pH of the pooled fractions was adjusted to 7.2. Buffer exchange into formulation buffer was performed with TFF (Vivaflow 50R).

[0271] Synthesis of 3-formyl-L-tyrosine N-[(tert-butyloxy)carbonyl]-L-tyrosine TIFF2024500922000136.tif34128To a solution of 20 g L-tyrosine (0.110 mol, 1 eq.) in 300 ml H2O and 300 ml 1,4-dioxane, 24.5 ml triethylamine (0.176 mol, 1.6 eq.) was added slowly. The reaction mixture was cooled to 0°C and 26.4 g di-tert-butyl dicarbonate (0.121 mol, 1.1 eq.) was added in portions. The reaction mixture was then allowed to warm slowly to room temperature overnight. The organic solvents were removed under reduced pressure and the solution was acidified to pH 1 with 2N HCl. The aqueous phase was extracted 3 times with 200 ml EtOAc, the organic fractions were combined, dried over MgSO4 and all volatiles were removed under reduced pressure. The desired compound was obtained as a white solid (30.6 g, 0.109 mmol, 99%). Analytical data were consistent with previously reported results (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791).

[0272] N-[(tert-butyloxy)carbonyl]-L-formyltyrosine TIFF2024500922000137.tif38128To a suspension of 20 g N-[(tertbutyloxy)carbonyl]-L-tyrosine (0.071 mol, 1 eq.) in 300 ml chloroform, 2.56 ml H2O (0.142 mol, 2 eq.) and 17.1 mg powdered sodium hydroxide (0.427 mol, 6 eq.) were added in portions. The reaction mixture was heated to reflux overnight with vigorous stirring. Afterwards, the mixture was cooled to room temperature and diluted with 300 ml H2O. The organic layer was separated and discarded. The aqueous layer was acidified to pH 1 with 2N HCl and extracted three times with 200 ml EtOAc. The organic fractions were combined, dried over MgSO4, and all volatiles were removed under reduced pressure. The crude product was purified by column chromatography on silica (5% MeOH + 0.5% formic acid in CH2Cl2) to give a yellowish solid (5.26 g, 0.017 mol, 24%). Analytical data were consistent with previously reported results (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791).

[0273] L-Formyltyrosine TFA salt TIFF2024500922000138.tif45128500 mg of N-[(tertbutyloxy)carbonyl]-L-formyltyrosine (1.616 mmol, 1 equiv.) was suspended in 1 ml of CH2Cl2 and cooled to 0° C. 0.1 ml of H2O was added followed by dropwise addition of 1 ml of trifluoroacetic acid. The reaction mixture was stirred at 0 °C for 3 h, all volatiles were removed under a stream of nitrogen, and the crude product was purified by preparative HPLC (Gilson Inc) on a VP 250 / 32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH) with the following gradient: (A = HO + 0.1% TFA, B = MeCN (acetonitrile) + 0.1% TFA, flow rate 30 ml / min, 5% B 0-5 min, 5-30% B 5-50 min, 99% B 50-60 min. The desired product was obtained as a yellowish powder after lyophilization as the TFA salt (397 mg, 1.228 mmol, 76%). Analytical data were consistent with previously reported results (Schumacher et al. (2015), Angewandte Chemie (International ed. in English), 54:13787-13791).

[0274] Synthesis of hydroxylamine-MMAE (HA-VC-PAB-MMAE) (Fmoc-aminooxy)acetic acid (FMOC-HA-COOH) TIFF2024500922000139.tif29128To a 50 ml round bottom flask was added 100 mg (0.915 mmol, 1.0 equiv.) of O-(carboxymethyl)hydroxylamine hemihydrochloride (Sigma-Aldrich), dissolved in 4 ml of water, and 240 mg (2.264 mmol, 2.1 equiv.) of sodium carbonate was added in portions. The solution was cooled to 0°C and a solution of 260 mg (1.005 mmol, 1.1 equiv.) of fluorenylmethyloxycarbonyl chloride (TCI-chemicals) in 2 ml of 1,4-dioxane was added dropwise. The solution was allowed to warm to room temperature overnight. Dioxane was removed under reduced pressure, 50 ml of water was added, and the solution was acidified with 2 mol / l HCl. The aqueous layer was extracted three times with ethyl acetate and the combined organic fractions were dried over MgSO4. All volatiles were removed under reduced pressure to give the desired product as a white solid (283 mg, 99.0%). Analytical data were consistent with previously reported results (Clave et al. (2008), Organic & Biomolecular Chemistry, 6:3065-3078).

[0275] Fmoc-VC-PAB-PNP TIFF2024500922000140.tif34161To a 10 ml screw cap vial were added 200 mg of Fmoc-VC-PAB (0.333 mmol, 1.0 equiv), 202 mg of bis(4-nitrophenyl)carbonate (0.333 mmol, 1.0 equiv) and 1 ml of DMF. 232 μl of DIPEA (1.332 mmol, 4.0 equiv) were added and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was added to ice-cold diethyl ether and the filtrate was collected by centrifugation. The filtrate was redissolved in 1 ml of DMF and precipitated a second time with ice-cold diethyl ether. The precipitate was collected by centrifugation and dried under reduced pressure to give the desired product as a yellowish powder. (247 mg, 97.0%). Analytical data were consistent with previously reported results (WO2004010957).

[0276] H 2 N-VC-PAB-MMAE TIFF2024500922000141.tif27167To a 10 ml screw cap vial was added 60 μl of a 1 mol / l solution of MMAE TFA salt in DMSO (50 mg, 0.06 mmol, 1.0 equiv), 180 μl of a 0.4 mol / L solution of Fmoc-VC-PAB-PNP in DMSO (0.072 mmol, 1.2 equiv) and 60 μl of a 1 mol / l solution of hydroxybenzotriazole hydrate in DMSO (0.06 mmol, 1.0 equiv). 105 μl of DIPEA (0.6 mmol, 10.0 equiv) was added and the yellow solution was stirred for 3 h at room temperature. After complete consumption of the MMAE starting material, as monitored by UPLC / MS, 120 μl of a 50 wt % solution of diethanolamine in DMSO was added and the yellow solution was stirred for another 1 h. 1.5 ml of acetonitrile and 3 ml of water were added and the solution was purified by preparative HPLC (Gilson Inc) on a VP 250 / 32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH) with the following gradient: (A=HO+0.1% TFA, B=MeCN(acetonitrile)+0.1% TFA, flow rate 30 ml / min, 30% B 0-5 min, 30-60% B 5-25 min, 99% B 25-35 min. The desired product was obtained as a white powder after lyophilization as the TFA salt (29.5 mg, 39.7%). Analytical data were consistent with previously reported results (Tang et al. (2016), Organic & Biomolecular Chemistry, 14(40): 9501-9518).

[0277] HA-VC-PAB-MMAE TIFF2024500922000142.tif38136To a 10 ml screw cap vial was added 23.3 mg of H2N-VC-PAB-MMAE (0.0188 mmol, 1.0 equiv), 38 μl of a 0.6 mol / L solution of Fmoc-HA-COOH in DMSO (0.0226 mmol, 1.2 equiv) and 38 μl of a 0.6 mol / L solution of Pybop in DMSO (0.0226 mmol, 1.2 equiv). 33 μl of DIPEA (0.1880 mmol, 10.0 equiv) was added and the solution was stirred for 2 h at room temperature. 150 μl of a 50 wt% solution of diethanolamine in DMSO was added and the solution was stirred for an additional 1 h. 1.5 ml acetonitrile and 3 ml water were added and the solution was purified by preparative HPLC (Gilson Inc) on a VP 250 / 32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH) with the following gradient: (A=HO+0.1% TFA, B=MeCN (acetonitrile)+0.1% TFA, flow rate 30 ml / min, 30% B 0-5 min, 30-99% B 5-60 min, 99% B 60-70 min. The desired product was obtained as a white powder after lyophilization as the TFA salt (20.1 mg, 81.6%). HR-MS for C 60 H 98 N 11 O 14 + [M+H] + Calculated: 1196.7290, found 1196.7278. See Figure 15 for the HPLC chromatogram of the purified material.

[0278] Synthesis of branched MMAE hydroxylamine (HA-(VC-PAB-MMAE)2) BOC-Glu(OSu) 2 TIFF2024500922000143.tif29128A 50 mL round bottom flask was charged with 1.00 g Boc-Glu-OH (4.04 mmol, 1.00 equiv), 0.93 g N-hydroxysuccinimide (8.08 mmol, 2.00 equiv) and 25 mg DMAP (0.20 mmol, 0.05 equiv) dissolved in 20 mL anhydrous THF. The suspension was cooled to 0° C. and 1.84 g dicyclohexylcarbodiimide (8.88 mmol, 2.2 equiv) dissolved in 10 mL anhydrous THF was added dropwise. The white suspension was allowed to warm to room temperature overnight. The mixture was filtered, the filtrate was evaporated and the residue was purified by flash column chromatography on silica gel. The desired product was obtained as a colorless oil. (460 mg, 1.04 mmol, 25.8%). The analytical data were consistent with previously reported results (Koshi et al., J. Am. Chem. Soc. 2008, 130, 1, 245-251).

[0279] Boc-Glu-(VC-PAB-MMAE) 2 TIFF2024500922000144.tif79167To a 10 ml screw cap vial was added 15.83 mg of H2N-VC-PAB-MMAE TFA salt (0.012 mmol, 2.00 equiv), 2.84 mg of Boc-Glu(OSu)2 (0.06 mmol, 1.00 equiv) and 150 μL of DMF. 4.5 μl of DIPEA (0.0256 mmol, 4.0 equiv) was added and the solution was stirred at room temperature for 2 h. 1.5 ml of acetonitrile and 3 ml of water were added and the solution was purified by preparative HPLC using a Gilson PLC 2020 system (Gilson Inc, Middleton, WI, USA) and a VP 250 / 21 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) with the following gradient: (A=HO+0.1% TFA, B=MeCN (acetonitrile)+0.1% TFA, flow rate 10 ml / min, 30% B 0-5 min, 30-99% B 5-60 min, 99% B 60-70 min. The desired product was obtained as a white powder after lyophilization. (8.84 mg, 0.0036 mmol, 55.8%). HR-MS for C 126 H 203 N 21 O 28 2+ [M+2H] 2+ Calculated: 1229,7565, Found: 1229,7567. See Figure 16 for the HPLC chromatogram of the purified material.

[0280] Boc-HA-Glu-(VC-PAB-MMAE) 2 TIFF2024500922000145.tif731663.99 mg of Boc-Glu-(VC-PAB-MMAE)2 (1.63 μmol, 1.00 equiv.) dissolved in 100 μL of CH2Cl2 was added to a 10 ml screw cap vial and cooled to -20°C. 100 μL of trifluoroacetic acid was added dropwise. After 3 h at -20°C, the solution was warmed to 0°C and all volatiles were removed under a stream of N2. 0.94 mg of Boc-HA-OSu (3.26 μmol, 2.00 equiv.) dissolved in 100 μL of DMF was added, followed by 2.8 μl of DIPEA (0.163 μmol, 10.0 equiv.) and the solution was stirred at room temperature for 2 h. 1.5 ml of acetonitrile and 3 ml of water were added and the solution was purified by preparative HPLC using a Gilson PLC 2020 system (Gilson Inc, Middleton, WI, USA) and a VP 250 / 10 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) with the following gradient: (A=HO+0.1% TFA, B=MeCN (acetonitrile)+0.1% TFA, flow rate 5 ml / min, 30% B 0-5 min, 30-99% B 5-60 min, 99% B 60-70 min. The desired product was obtained as a white powder after lyophilization. (2.11 mg, 0.748 μmol, 46%). HR-MS for C 128 H 206 N 22 O 30 2+ [M+2H] 2+ Calculated: 1266,2647 Found: 1266,2644. See Figure 17 for the HPLC chromatogram of the purified material.

[0281] HA-(VC-PAB-MMAE)2 TIFF2024500922000146.tif71168To a 10 ml screw cap vial, 2.11 mg of Boc-Glu-(VC-PAB-MMAE)2 (0.75 μmol) dissolved in 100 μL of CH2Cl2 was added and cooled to -20°C. 100 μL of trifluoroacetic acid was added dropwise. After 3 h at -20°C, the solution was warmed to 0°C and all volatiles were removed under a stream of N2. The desired product was obtained as a white powder after lyophilization from 50% MeCN / H2O. (1.90 mg, 0.748 μmol, quantitative). HR-MS for C 123 H 198 N 22 O 28 2+ [M+2H] 2+ Calculated: 1216,2384 Found: 1216,2326. See Figure 18 for the HPLC chromatogram of the purified material.

[0282] Flash and thin layer chromatography Flash column chromatography was performed using NORMASIL 60® silica gel 40-63 μm (VWR international, USA). Glass TLC plates, silica gel 60W, coated with fluorescent indicator F254s were purchased from Merck (Merck Group, Germany). Spots were visualized by fluorescence quenching with a 254 nm lamp or by manganese staining (10 g K2CO3, 1.5 g KMnO4, 0.1 g NaOH in 200 ml H2O) followed by heating.

[0283] Preparative HPLC Preparative HPLC was performed on a Gilson PLC 2020 system (Gilson Inc, Middleton, WI, USA) using a VP 250 / 32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany). The following gradients were used: Method C: (A=H2O+0.1% TFA (trifluoroacetic acid), B=MeCN (acetonitrile)+0.1% TFA, flow rate 30 ml / min, 5% B 0-5 min, 5-90% B 5-60 min, 90% B 60-65 min. Method D: (A=H2O+0.1% TFA, B=MeCN++0.1% TFA), flow rate 30 ml / min, 5% B 0-5 min, 5-25% B 5-10 min, 25%-45% B 10-50 min, 45-90% 50-60 min, 90% B 60-65 min. Method E: 0.1% TFA, flow rate 18 ml / min, 5% B 0-5 min, 5-90% B 5-60 min, 90% B 60-65 min, VP 250 / 21 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) was used.

[0284] HR-MS High-resolution ESI-MS spectra were recorded on a Waters H-class instrument equipped with a quaternary solvent manager, Waters Sample Manager-FTN, Waters PDA detector and Waters column manager using an Acquity UPLC Protein BEH C18 column (1.7 μm, 2.1 mm × 50 mm). Samples were eluted at a flow rate of 0.3 mL / min. The following gradient was used: A: 0.01% FA in HO; B: 0.01% FA in MeCN. 5% B: 0–1 min; 5–95% B: 1–7 min; 95% B: 7–8.5 min. Mass spectrometry was performed on a Waters XEVO G2-XS QTof analyzer.

[0285] UPLC-UV / MS UPLC-UV / MS traces were recorded on a Waters H-class instrument equipped with a quaternary solvent manager, Waters autosampler, Waters TUV detector and Waters Acquity QDa detector using an Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm RP column at a flow rate of 0.6 mL / min (Waters Corp., USA). The following gradient was used for purity analysis: A: 0.1% TFA in H2O; B: 0.1% TFA in MeCN. 5% B 0–1.5 min, 5–95% B 1.5–11 min, 95% B 11–13 min, 5% B 13–15 min.

[0286] Intact Protein MS Intact proteins were analyzed on an Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1 mm × 50 mm) on a Waters H-class instrument equipped with a quaternary solvent manager, Waters Sample Manager-FTN, Waters PDA detector, and Waters column manager. Proteins were eluted at a flow rate of 0.3 mL / min. The following gradient was used: A: 0.01% FA in H2O; B: 0.01% FA in MeCN. 5–95% B 0–6 min. Mass spectrometry was performed on a Waters XEVO G2-XS QTof analyzer. Proteins were ionized in positive ion mode with a cone voltage of 40 kV. Raw data was analyzed with MaxEnt 1.

[0287] Resazurin assay HL60 and Karpas cell lines were cultured in RPMI-1640 supplemented with 10% FCS and 0.5% penicillin-streptomycin. Cells were plated in 96-well cell culture microplates at 5 x 10 3Cells were seeded at a density of 1000 x 1000 cells / well. ADCs or antibodies were serially diluted 1:4 in cell culture medium starting at a final concentration of 3 μg / mL and transferred in duplicate to each well of the microplate. Plates were incubated at 37 °C and 5% CO2 for 96 h. Resazurin was then added to a final concentration of 50 μM, followed by incubation at 37 °C and 5% CO2 for 3–4 h. The metabolic conversion of resazurin to resorufin was monitored by monitoring the fluorescent signal of resorufin (λ) on a Tecan Infinite M1000 microplate reader. EX = 560 nm and λ EM = 590 nm). Means and standard deviations were calculated from duplicates, normalized to untreated controls, and plotted against antibody concentration. Data analysis was performed with MatLab R2016 software.

[0288] A-SEC characterization and stability assessment of ADCs ADCs were adjusted to a protein concentration of 1 mg / mL in PBS (Dulbeccos Phosphate Buffered Saline, Sigma-Aldrich Merck KGaA) and sterile filtered (Ultrafree-MC Centrifugal filter units, Merck Millipore). Samples were stored at 4-8°C, 37°C, and 40°C for up to 14 days. For samples stored at higher temperatures, condensation was prevented from forming. Before analysis by A-SEC, samples were centrifuged at 4000 x g for 4 min at 4°C.

[0289] In vivo xenograft model In vivo efficacy experiments were conducted in accordance with the Animal Welfare Act and approved by local authorities. Briefly, Karpas 299 cells were subcutaneously injected into CB17-Scid mice on day 0. Treatment was initiated when tumors reached a certain mean tumor volume. Mice were randomized into treatment and control groups and then administered ADCs via intravenous injection. Tumor volume, body weight, and general health were recorded throughout the study.

[0290] Pharmacokinetic analysis The experiments were performed in accordance with the Animal Welfare Act and approved by the local government. Test items were administered to Sprague-Dawley rats by a single intravenous (bolus) injection on day 1. Blood was collected from the jugular vein at various time points up to 504 hours with a target volume of 1 mL for bioanalysis. Samples were kept at room temperature for at least 1 hour to allow clotting. Samples were centrifuged at 1500 g for 10 minutes at 4°C. Resulting serum was stored at -20°C. PK parameters were calculated by Phoenix (WinNonlin) pharmacokinetic software 1.4 (Certara, 6.4) using a non-compartmental approach consistent with the intravenous (bolus) route of administration and for free payload using an in vivo formation profile approach (extravascular).

[0291] Repeated dose toxicity study The experiments were conducted in accordance with the Animal Welfare Act and approved by the local government. Male and female Sprague-Dawley rats (10 rats / sex / group) were administered control (vehicle), brentuximab Tub-tag ADC (10 mg / kg) or brentuximab vedotin (10 mg / kg) once a week (days 1, 8, 15 and 22), with main dosing necropsies performed on day 26 (5 rats / sex / group) and recovery necropsies performed on day 51 (5 rats / sex / group). Clinical observations, body weights, and food intake were checked daily during the study. Pharmacokinetic samples were collected from each animal before each dose, 15 min after each dose, and at necropsy. All animals were necropsied and organs were weighed and held in fixative. Clinical pathology (hematology, clinical chemistry and coagulation endpoints) samples were also collected at necropsy.

[0292] Example 1: Brentuximab Tub Tag ADC for treatment of CD30+ indications Tub-tag technology utilizes a naturally occurring α-tubulin-derived peptide that is highly polar and serves as a recognition sequence for the enzyme tubulin tyrosine ligase (TTL), ie, is a recognition sequence for tubulin tyrosine ligase within the meaning of the present disclosure.

[0293] TTL recognizes short hydrophilic peptide tags (Tub tags) and catalyzes peptide bond formation with tyrosine derivatives and amino acid-like building blocks. Here we show that Tub tag technology can be used to conjugate payloads to the antibody brentuximab (cAC10) and facilitate CD30 binding to an ADC that surprisingly outperforms the approved brentuximab-based ADC brentuximab vedotin in terms of stability, efficacy, and toxicity.

[0294] Figure 22 shows the site-specific condensation of a drug moiety (payload) via a linker to a monoclonal antibody such as brentuximab by Tubuli's proprietary Tub-Tag® ("TTL recognition sequence") technology. TTL: Tubulin tyrosine ligase.

[0295] The inventors began their investigations by condensing (at the nucleic acid level) a recognition sequence for tubulin tyrosine ligase to the light chain (light chain SEQ ID NO: 11, heavy chain: SEQ ID NO: 1), heavy chain (light chain SEQ ID NO: 2, heavy chain: SEQ ID NO: 12) or light and heavy chains (light chain SEQ ID NO: 11, heavy chain: SEQ ID NO: 12) of monoclonal antibody cAC10 (brentuximab, a Tub tag condensed to the light chain as exemplarily shown in FIG. 23), optionally with a Gly4Ser amino acid linker sequence placed between the recognition sequence for tubulin tyrosine ligase and the respective antibody chain.

[0296] Figure 23 shows chemoenzymatic conjugation for ADC production. (Blue) Tub tag recognition sequence, (Black) tyrosine derivative, (Yellow) payload with bioorthogonal handle. Tub tags can be placed on the light and / or heavy chain, allowing for homogenous ADCs with drug-antibody ratios (DAR) ranging from 2 to 8, depending on the number of Tub tags used. Payloads 2 and 3 are also shown.

[0297] These brentuximab variants were recombinantly expressed using established cell culture methods and subsequently successfully purified by protein A chromatography. Analysis by size exclusion chromatography (SEC) showed very high purity of the Tub-tagged antibody, with only small amounts of high molecular weight species (HMWS) observed (HMWS = 1.5-7%). The results are shown in Figure 1. These results indicate that the antibody after expression and purification has a high monomer fraction. As can be seen in Figure 1B, linking the Tub-tag recognition sequence to the heavy chain of brentuximab reduces the high molecular weight species from 11% to only 1.5%, a ten-fold decrease.

[0298] Figure 1A shows an analytical size exclusion chromatogram of unmodified brentuximab, also referred to herein simply as "brentuximab", after Protein A chromatography (PAC). "HMWC" stands for "high molecular weight component".

[0299] FIG. 1B shows an analytical size-exclusion chromatogram of brentuximab containing a TTL recognition sequence (Tub tag) fused to the heavy chain (light chain: SEQ ID NO: 2, heavy chain: SEQ ID NO: 12), also designated herein as "Bren. HC-Tub", after Protein A chromatography (PAC).

[0300] FIG. 1C shows an analytical size-exclusion chromatogram of brentuximab containing a TTL recognition sequence (Tub tag) fused to the light chain (light chain: SEQ ID NO: 11, heavy chain: SEQ ID NO: 1), also denoted herein as "Bren. LC-Tub", after Protein A chromatography (PAC).

[0301] FIG. 1D shows an analytical size-exclusion chromatogram of brentuximab containing a TTL recognition sequence (Tub tag) fused to the light and heavy chains (light chain: SEQ ID NO: 11, heavy chain: SEQ ID NO: 12), also designated herein as "Bren. LCHC-Tub", after Protein A chromatography (PAC).

[0302] After demonstrating the manufacturability of brentuximab containing a recognition sequence for tubulin tyrosine ligase at the C-terminus of the light chain, the C-terminus of the heavy chain, or at the C-terminus of all of the heavy and light chains of brentuximab ("Tub tag variants" or "brentuximab tub tag"), we determined retention times by hydrophilic interaction chromatography (HIC), a common measure of hydrophobicity. This study revealed that all Tub tag variants had increased hydrophilicity and surface polarity compared to unmodified cAC10 (brentuximab) (Figure 2A).

[0303] FIG. 2A shows analytical hydrophobic interaction chromatograms of brentuximab (black), Bren. LC-Tub (grey, dotted line), Bren. HC-Tub (grey), and Bren. LCHC-Tub (black, dotted line). The chromatograms show normalized absorbance spectra recorded at 220 nm. Retention time is a measure of hydrophobicity. Longer retention times on the hydrophobic interaction chromatograms indicate greater hydrophobicity. Shorter retention times indicate greater hydrophilicity.

[0304] Because modifications to the antibody backbone can cause perturbations in structure and stability, the melting temperatures (Tm) of brentuximab and the Tub-tag variants were measured by differential scanning fluorometry (DSF). These experiments demonstrated that the Tub-tag variants have the same melting temperatures (Tm = 70.9-71.8 °C) as the unmodified mAb (Tm = 71.0 °C) and therefore the same stability to heat stress (Figure 2B).

[0305] FIG. 2B shows differential scanning fluorimetry curves for the determination of melting temperature (Tm) of Brentuximab, Bren. LC-Tub, Bren. HC-Tub and Bren. LCHC-Tub.

[0306] We tested different tyrosine derivatives for enzymatic modification of Tub tag antibodies, and found that 3-formyl-L-tyrosine was optimal for ADC production. Briefly, we incubated different brentuximab Tub tag variants with TTL, and covalently attached 3-formyl-L-tyrosine to the terminal glutamic acid residue of the Tub tag. In particular, the amino group of 3-formyl-L-tyrosine was attached to the C-terminus of the Tub tag. The excess 3-L-formyl-tyrosine and residual TTL were then removed by protein A chromatography or anion exchange chromatography (AEX). The linker payload ("linker drug moiety") was then covalently attached by a bioorthogonal reaction. Purification of the resulting ADC was performed by cation exchange chromatography (CEX) to remove excess linker payload and increase the purity of the ADC. The final step of the process involved formulation by tangential flow filtration (TFF) and sterile filtration. For the generation of ADCs with a drug-antibody ratio (DAR) of 2, a linear payload 2 was applied (shown in FIG. 23, conjugated with MMAE, “structure 4” as defined in the description and claims). For the generation of ADCs with a DAR of 4, a branched payload 3 was applied (shown in FIG. 23, conjugated with MMAE, “structure 5” as defined in the description and claims). In particular, the H2N-O- moiety of payload 2 and payload 3 reacted with the formyl group of 3-formyl-L-tyrosine bound to a TTL recognition sequence to obtain an oxime with a -C=NO- structure. This procedure yielded pure and homogeneous ADCs, which were quality controlled, for example, by SEC, HIC (FIG. 3) and mid-up MS (FIG. 4), showing excellent purity and homogeneity.

[0307] The following ADCs were prepared and further investigated in the Examples (Payloads 2 and 3 are shown in Figure 23). TIFF2024500922000147.tif138169

[0308] FIG. 3 shows the analysis of brentuximab Tub-tag ADCs by SEC (FIG. 3A-C) and HIC (FIG. 3D-F). FIG. 3A and D show the results for Bren. HC-vc-PAB-MMAE DAR2 (also referred to herein as "Bren. HC-2"), FIG. 3B and E show the results for Bren. LC-vc-PAB-MMAE DAR2 (also referred to herein as "Bren. LC-2"), and FIG. 3C and F show the results for Bren. LCHC-vc-PAB-MMAE DAR4 (also referred to herein as "Bren. LCHC-2"). Each of the brentuximab variants was expressed, purified with PAC, and conjugated to payload 2 for the generation of DAR2 and payload 2 or payload 3 for the generation of DAR4 ADCs. After final polishing by HIC and buffer exchange, the Tub-tag ADCs were analyzed for aggregate content and DAR homogeneity. All Tub-tagged ADCs contained very low contents of HMWS (<1%) and showed excellent DAR homogeneity.

[0309] Figure 4 shows the analysis of Brentuximab Tub tag ADC by middle-up protein MS after deglycosylation. Figure 4A shows the results of Bren. HC-Tub; Figure 4B shows the results of Bren. LCHC-Tub; Figure 4C shows the results of Bren-LC-Tub, Figure 4D shows the results of Bren. HC-vc-PAB-MMAE (also referred to herein as "Bren. HC-2"); Figure 4E shows the results of Bren. LCHC-vc-PAB-MMAE (also referred to herein as "Bren. LCHC-2"); and Figure 4F shows the results of Bren. LC-2 (vc-PAB-MMAE) (also referred to herein as "Bren. LC-3"). The mass shift of 1369 m / z is due to the incorporation of 3-formyl-L-tyrosine and oxime linkage with payload 2. After oxime linkage with payload 3, a mass difference of 2604 m / z was observed. These results are consistent with the calculated values.

[0310] Prior to conjugation with antibodies, payloads 2 and 3 were analyzed for susceptibility to cleavage by the protease cathepsin B (FIG. 5). The results show that payloads 2 and 3 are cleaved by cathepsin B to release the free drug monomethylauristatin E (MMAE).

[0311] FIG. 5 shows in FIG. 5A RP-HPLC analysis of valine-citrulline (vc) containing payload 2 during digestion reaction with protease cathepsin B (1:1000 for each vc moiety). Before analysis, the reaction was stopped with E-64. Chromatograms were recorded at 220 nm after 150 min reaction time. The plot in FIG. 5B shows the increase in free MMAE (black) and the decrease in payload 2 (grey, circles) over time during reaction with cathepsin B. FIG. 5C shows the cleavage of payload 3. During reaction of payload 3 with cathepsin B, an intermediate containing one MMAE moiety is formed (grey, triangles).

[0312] Example 2: In Vitro Stability of ADCs To elucidate the stability of the Brentuximab ADC described in Example 1, the inventors performed a thermal stress study based on the fluorescent dye SYPRO Orange. DSF experiments revealed that the Brentuximab Tub tag ADC has a similar melting point as the corresponding mAb (Brentuximab Tm = 71.0 °C, Brentuximab Tub tag ADC Tm = 70.9-72.9 °C). This indicates the antibody-like stability of the Brentuximab Tub tag ADC. Brentuximab vedotin, used for comparison, showed a lower melting point of 68.5 °C, with the melting curve covering a higher temperature range. In addition, stability studies of the ADC were performed in buffer and mouse plasma. Samples stored in buffer were separated by SEC and analyzed for the formation of HMWS (high molecular weight species). No significant differences were observed in the chromatograms after 2 weeks of storage at 4 °C. However, differences became evident during storage at elevated temperatures. After dissolving Brentuximab Vedotin according to the manufacturer's guidelines, the HMWS was determined to be 0.74%. This value increased to 11.44% during storage at 40 °C for 14 days, indicating a 15.5-fold increase in HMWS. In comparison to these values, the Brentuximab Tub-tag ADC was very stable, with a lower increase in HMWS content of 1.0-5.0-fold (Figure 6A). Brentuximab LC-3 DAR 4 contains the same drug amount as Brentuximab Vedotin (DARav 4) and is therefore characterized by a high hydrophobicity derived from the payload, but with a lower increase in HMWS content. After preparation of the Tub-tag ADC Bren. LC-3 DAR 4, the HMWS was 0.47%. Only a slight increase in HMWS to 2.34% was observed during storage at 40 °C. (Figure 6C, for comparison the results for Brentuximab Vedotin are shown in Figure 6B).

[0313] FIG. 6 shows the results of storage at elevated temperatures for Brentuximab Vedotin and Brentuximab Tub Tag ADC. FIG. 6A shows the increase in HMWS over the course of the study for Bren.LC-2(vc-PAB-MMAE) (also referred to herein as "Bren. LC-3"); Bren. LC-vc-PAB-MMAE (also referred to herein as "Bren. LC-2"); Bren. HC-vc-PAB-MMAE (also referred to herein as "Bren. HC-2"); and Brentuximab Vedotin. The highest increase in aggregate content can be seen for Brentuximab Vedotin. In comparison, the HMWS content of the Tub Tag ADC remains nearly constant. FIG. 6B and 6C show SEC chromatograms showing the increase in HMWS over the course of the study for Brentuximab Vedotin (mean drug-antibody ratio (DAR) av ) 4) and normalized absorption spectra recorded at 220 nm for Bren. LC conjugated with payload 3 (Bren. LC-3, DAR 4).

[0314] In addition, the stability of the brentuximab Tub-tag ADC was evaluated in mouse plasma, showing excellent stability of the Tub-tag ADC. In contrast, brentuximab vedotin was rapidly degraded (Figure 7, Table 1). Rapid degradation of maleimide-linked ADCs such as brentuximab vedotin has been recently demonstrated, and reverse Michael addition may be one explanation for our findings.

[0315] Table 1. Storage of brentuximab vedotin and brentuximab Tub-tag ADC in mouse plasma at 37°C for 7 days. TIFF2024500922000148.tif44165

[0316] Figure 7 shows a diagram illustrating the stability of Brentuximab Tub Tag ADC in mouse plasma compared to Brentuximab Vedotin. Figures 7A and 7B show the results of Brentuximab Vedotin storage in mouse plasma at 37°C. Figures 7C and 7D show the results of Bren. LC-3 DAR 4 storage in mouse plasma at 37°C.

[0317] Example 3: In Vitro Efficacy of ADCs We then evaluated the in vitro efficacy of Brentuximab Tub-tag ADC in a cell viability assay and compared the results with Brentuximab Vedotin. CD30-overexpressing cell line Karpas299 as well as CD30-negative cell line HL60 were used in this resazurin assay (Figure 8). The IC50 value of Brentuximab Vedotin determined in the antigen-positive cell line Karpas299 (IC50=2.08) was consistent with literature data. Overall, Brentuximab Tub-tag ADC showed low IC50 values ​​and good in vitro efficacy (Table 2).

[0318] Table 2. IC of brentuximab vedotin and brentuximab Tub-tag ADC as determined by resazurin assay 50 value TIFF2024500922000149.tif39128

[0319] FIG. 8 shows the in vitro efficacy of brentuximab Tub-tag ADC in the CD30-overexpressing cell line Karpas299 and the CD30-negative cell line HL60.

[0320] Example 4: In vivo efficacy of brentuximab Tub tag ADC The in vivo efficacy of Brentuximab Tub-tag ADC was evaluated in a Karpas299-derived tumor model in immunodeficient CB17-SCID mice. As seen in Figures 9 and 10, Bren. LC-2 showed unexpectedly high in vivo efficacy similar to that of Brentuximab Vedotin. This finding was obtained despite the fact that Brentuximab LC-2 is a DAR 2 MMAE ADC and therefore has half the drug dose compared to Brentuximab Vedotin, which is conjugated to four MMAE molecules. Furthermore, unexpectedly, Bren. LC-3 significantly outperformed the in vivo efficacy of Brentuximab Vedotin, with a 2.5-fold increase in median survival (Figure 11).

[0321] Figure 9 shows the tumor volume (cm 3LC-2 in vivo efficacy, expressed in terms of the amount of 100-150 mm 3 Mice with tumor volumes between were randomized and treated with two injections of 1.5 mg / kg on days 7 and 10 after tumor implantation.

[0322] Figure 10 shows the tumor volume (cm 3 LC-2 in vivo, expressed in terms of survival rate (Kaplan-Meier plot) and survival percentage of treated animals. 3 Mice with tumor volumes between were randomized and treated with a single 1.0 mg / kg injection.

[0323] Figure 11 shows the tumor volume (cm 3 LC-3) and the in vivo efficacy of Bren. LC-3 expressed in terms of survival percentage of treated animals. 3 Mice with tumor volumes between were randomized and treated with two injections of 0.5 mg / kg on days 8 and 11 after tumor implantation.

[0324] Example 5: Pharmacokinetics of Brentuximab Tub Tag ADC Motivated by the surprising finding that the Brentuximab Tub-tag ADC features high in vitro stability and excellent in vivo efficacy, we wanted to understand whether the in vitro stability also applies to living organisms, and performed a pharmacokinetic analysis in Sprague-Dawley rats. The test items were administered to the animals as a single intravenous (bolus) injection on day 1. To analyze the ADC content over time, an ELISA assay was developed to detect antibody and intact ADC content (by measuring the payload linked to the antibody). The total antibody assay provides useful information on antibody clearance, and the detection difference compared to the intact ADC assay may explain the payload loss from the antibody. Furthermore, we developed a mass spectrometry assay that facilitates the determination of payload loss and payload migration to blood proteins. This study showed that the Brentuximab Tub-tag ADC has excellent in vivo stability (Figure 12A). Furthermore, in contrast to brentuximab vedotin, the brentuximab Tub tag ADC does not feature a mechanism that allows the transfer and covalent attachment of the payload to blood proteins (Figure 12B).

[0325] Figure 12 shows a pharmacokinetic (PK) analysis of Brentuximab Tub-tagged MMAE (Bren LC-2). Figure 12A shows the amount of intact ADC compared to Brentuximab Vedotin. Figure 12B shows the amount of MMAE transferred to blood proteins as analyzed by mass spectrometry.

[0326] Example 6: Repeated dose toxicity study of TUB-010 So far, Brentuximab Tub-tag ADC has been shown to be characterized by high efficacy and stability in vivo. Therefore, we investigated whether the increased stability in vivo would lead to a beneficial toxicity profile of Brentuximab Tub-tag ADC. This is advantageous because the combination of improved toxicity profile and improved efficacy could lead to a significant expansion of the therapeutic window and therefore a great advantage for the treatment of patients. Therefore, we evaluated the toxicity and toxicokinetics of Brentuximab Tub-tag ADC in Sprague-Dawley male and female rats and compared its profile with Brentuximab Vedotin. Male and female Sprague-Dawley rats were dosed weekly (days 1, 8, 15, and 22) with control (vehicle), brentuximab Tub-tag ADC (Bren LC-2) (10 mg / kg), or brentuximab vedotin (10 mg / kg), with main dosing necropsies performed on day 26 (5 animals / sex / group) and recovery necropsies performed on day 51 (5 animals / sex / group). Clinical observations, body weights, and food intake were monitored daily throughout the study. Pharmacokinetic samples were collected from each animal prior to each dose, 15 minutes after each dose, and at necropsy. All animals were necropsied, and organs were weighed and held in fixative. Clinical pathology (hematology, clinical chemistry, and coagulation endpoints) samples were also collected at necropsy. Mean serum concentrations of intact ADC and total antibody are shown in Figure 13. The graph clearly shows that administration of Bren LC-2 results in reasonable serum concentrations in vivo after repeated dosing. The high degree of overlap between the intact ADC and the whole antibody of Bren LC-2 again highlights the superior stability of the construct in vivo, differentiating it from the Brentuximab Vedotin group.

[0327] FIG. 13 shows the mean serum concentrations of intact ADC and total antibodies in male and female rats following intravenous (bolus) administration of brentuximab Tub Tag ADC at 10 mg / kg compared to brentuximab vedotin on days 1, 8, 15, and 22.

[0328] In contrast to Brentuximab Vedotin, no effects on erythrocyte populations were observed with the Brentuximab Tub-tag ADC (Bren LC-2). This may offset the anemia (and possible thrombocytopenia) observed with Brentuximab Vedotin in clinical practice. The expected MMAE-induced testicular changes were observed with both Bren LC-2 and Brentuximab Vedotin, but with a delayed onset with Bren LC-2, suggesting that the stability of the Brentuximab Tub-tag ADC plays a role in the delayed onset of testicular toxicity. An exemplary toxicity profile for Bren. LC-2 is shown in Figure 14.

[0329] Figure 14 shows an exemplary toxicity profile of Brentuximab Tub Tag ADC (Bren LC-2) (left bar) compared to Brentuximab Vedotin (right bar) in male and female rats. The parameters shown in Figure 14 for Brentuximab Tub Tag ADC Bren LC-2 are reticulocytes in counts / microliter (RET in K / μl), red blood cells in million / microliter (RBC in M / μl), hemoglobin in million / microliter (Hb in M / μl), hematocrit in volume % (HTC in %), eosinophils in counts / microliter (EOS in K / μl), activated partial thromboplastin time (APPT) in seconds (sec), glucose level in millimoles / liter (mmol / L), thymus weight in grams (g), and testis weight in grams (g). As a reference, rats were administered a control (vehicle) (data not shown).

[0330] In particular, FIG. 14 shows: Reticulocytes: Treatment with brentuximab vedotin significantly impacts reticulocyte counts, whereas no effect is observed with brentuximab Tub Tag ADC treatment. Thus, treatment with brentuximab Tub Tag ADC is expected to reduce clinical anemia compared to treatment with brentuximab vedotin (consistent with red blood cell, hemoglobin and hematocrit data). Red blood cells: Treatment with brentuximab vedotin significantly impacts red blood cell counts, whereas no significant effect is observed with treatment with brentuximab Tub Tag ADC. Therefore, treatment with brentuximab Tub Tag ADC is expected to reduce clinical anemia compared to treatment with brentuximab vedotin (consistent with reticulocyte, hemoglobin and hematocrit data). hemoglobin: Treatment with brentuximab vedotin reduces hemoglobin levels (mainly in males), while no significant effect is observed with treatment with brentuximab Tub Tag ADC. Thus, treatment with brentuximab Tub Tag ADC is expected to reduce clinical anemia compared to treatment with brentuximab vedotin (consistent with red blood cell, reticulocyte and hematocrit data). Hematocrit: Treatment with brentuximab vedotin reduces hematocrit (mainly in males), while no significant effect is observed with treatment with brentuximab Tub Tag ADC. Thus, treatment with brentuximab Tub Tag ADC is expected to reduce clinical anemia compared to treatment with brentuximab vedotin (consistent with red blood cell, hemoglobin and reticulocyte data). Eosinophils: Treatment with brentuximab vedotin reduces eosinophil counts compared to treatment with brentuximab Tub-tag ADC. Assuming this is a general effect on white blood cells, treatment with brentuximab Tub-tag ADC is expected to reduce clinical neutropenia compared to treatment with brentuximab vedotin. APTT: Treatment with brentuximab vedotin reduces APTT values ​​compared to treatment with brentuximab Tub Tag ADC. Brentuximab vedotin does not have any effect on blood clotting time / APTT in clinical practice, so based on the data provided, brentuximab Tub Tag ADC is not expected to pose any concerns regarding blood clotting time. glucose:Treatment with brentuximab vedotin increases glucose levels more than treatment with brentuximab Tub tag ADC.Therefore, treatment with brentuximab Tub tag ADC reduces glucose levels compared to treatment with brentuximab vedotin.Therefore, treatment with brentuximab Tub tag ADC is expected to be less likely to cause the hyperglycemia observed in clinical practice with treatment with brentuximab vedotin. Thymus: Treatment with brentuximab vedotin reduces thymus weight compared to treatment with brentuximab Tub tag ADC. The changes in thymus weight observed with treatment with MMAE-containing ADC are typical target-independent / free toxin-related toxicity and are due to cell depletion caused by MMAE. Therefore, brentuximab Tub tag ADC is expected to have a more favorable clinical safety profile in terms of less target-independent / free MMAE-related toxicity compared to brentuximab vedotin. Testes: Treatment with brentuximab vedotin reduces testis weight compared to treatment with brentuximab Tub tag ADC. The changes in testis weight observed in treatment with ADC containing MMAE are typical target-independent / free toxin-related toxicity. Therefore, brentuximab Tub tag ADC is expected to have a more favorable clinical safety profile in terms of less target-independent / free MMAE-related toxicity compared to brentuximab vedotin.

[0331] Overall, the data presented indicate that the brentuximab Tub-tag ADC has a better safety profile in rats compared to brentuximab vedotin. Thus, in the clinic, the brentuximab Tub-tag ADC is less likely to cause certain dose-limiting toxicities of brentuximab vedotin, including anemia, neutropenia, and hyperglycemia. The lack of target-independent / free toxin-related toxicities associated with brentuximab vedotin also offsets characteristic changes such as peripheral neuropathy seen with many MMAE-containing ADCs, including brentuximab vedotin.

[0332] Example 7: Repeated-dose toxicity study of Brentuximab Tub-Tagged MMAE in cynomolgus monkeys Next, the toxicity of Brentuximab Tub-Tagged MMAE (Bren. LC-2) was evaluated in cynomolgus monkeys. The objective of the study was to evaluate the toxicity and toxicokinetics (TK) of Brentuximab Tub-Tagged MMAE (Bren. LC-2) in cynomolgus monkeys. Brentuximab Tub-Tagged ADC was administered at 6, 12 and 15 mg / kg (6 mg / kg Q3W×4, 12 and 15 mg / kg Q3W×2). Clinical status (including body weight, food consumption and clinical findings), clinical pathology and immunophenotyping (T, B and NK cells) were evaluated throughout the study, with frequent sampling for TK and anti-drug antibody measurements. All animals were subjected to full necropsy and detailed gross observations. Brentuximab Tub-Tagged MMAE (Bren. LC-2) was clinically well tolerated at 6, 12 and 15 mg / kg with no effect on body weight. ADCETRIS was poorly tolerated at 6 mg / kg (MTD 3 mg / kg) in cynomolgus monkeys based on FDA PharmTox review, with deaths and early euthanasia occurring between 11 and 15 days after the first dose. In contrast, no mortality or gross or sever microscopic findings were observed within this study of Brentuximab Tub-Tagged MMAE (Bren. LC-2). Red blood cell (RBC), hemoglobin, and neutrophil reductions were observed with Brentuximab Tub-Tagged MMAE (Bren. LC-2) at 12 and 15 mg / kg, which were relatively consistent with those observed with ADCETRIS at 3 mg / kg (historical data). Additionally, Brentuximab Tub-Tagged MMAE (Bren. LC-2) was characterized by high stability in toxicokinetic analysis, as shown in Figure 19.

[0333] To analyze the ADC content in cynomolgus serum over time, an ELISA assay was developed to detect antibody and intact ADC content (by measuring the payload linked to the antibody; the same ELISA assay setup is used in the rat pharmacokinetic and repeat-dose toxicity studies, with the difference being that this assay has been further validated for use in cynomolgus serum). The whole antibody assay provides useful information on antibody clearance, and the detection difference compared to the intact ADC assay may explain the payload loss from the antibody. The high degree of overlap between the intact ADC and whole mAb curves in Figure 19 indicates minimal payload loss and high ADC stability for brentuximab Tub-tag MMAE.

[0334] FIG. 19 shows the toxicokinetic analysis of Brentuximab Tub-tag MMAE (i.e., Bren. LC-2) in cynomolgus monkeys administered at 12 and 15 mg / kg. The total amount of mAb and intact ADC was evaluated by ELISA. The high degree of overlap of the curves of intact ADC and total mAb indicates the high stability of Brentuximab Tub-tag MMAE at both dose levels. During the repeated dose study of Brentuximab Tub-tag MMAE (Bren. LC-2), the body weight and different blood values ​​of cynomolgus monkeys were monitored, namely lymphocyte, neutrophil and leukocyte concentrations.

[0335] FIG. 20 shows values ​​obtained for body weight and selected different blood values ​​collected throughout a repeat dose study of Brentuximab Tub tagged MMAE (i.e., Bren. LC-2) in cynomolgus monkeys. More specifically, the top left plot of FIG. 20 shows body weight over time. The top right plot of FIG. 20 shows lymphocyte concentration over time. The bottom left plot of FIG. 20 shows neutrophil concentration over time. The bottom right plot of FIG. 20 shows white blood cell concentration over time. Data are presented as the mean and error for three groups of two females receiving repeated doses of 6, 12, and 15 mg / kg brentuximab Tub Tag MMAE (Bren. LC-2), in each case, compared to toxicity results for brentuximab vedotin (Adcetris) as described in the FDA report "Clinical Pharmacology And Biopharmaceutics Review" in application number 125388Orig1S00, available at: https: / / www.accessdata.fda.gov / drugsatfda_docs / nda / 2011 / 125388orig1s000clinpharmr.pdf. Body weight is presented in kilograms, lymphocytes in billion / liter, neutrophils in billion / liter, and white blood cells in billion / liter.

[0336] One of the dose-limiting toxicities of Brentuximab Vedotin in clinical practice is neutropenia. The data set shown in Figure 20, bottom left panel, shows that Brentuximab Tub-tag MMAE (Bren. LC-2) advantageously does not cause more neutropenia in direct comparison with Brentuximab Vedotin, even when applied at twice (6 mg / kg), four times (12 mg / kg) and five times (15 mg / kg) the dose of Brentuximab Vedotin (3 mg / kg). In addition, an increase in body weight was observed (Figure 20, top left panel). No significant changes were observed in lymphocyte and leukocyte concentrations (Figure 20, top right and bottom right panels).

[0337] The combination of the findings of no significant changes in neutrophil concentration, weight gain in all animals throughout the study period, and white blood cell and lymphocyte concentrations clearly indicates that the ACD of the present disclosure is well tolerated, which is greater than that of brentuximab vedotin. Thus, the data clearly suggests that the ACD is well tolerated in the clinic, which is greater than that of brentuximab vedotin.

[0338] Taken together, the results presented demonstrate that brentuximab Tub-tag ADCs are characterized by unexpectedly high stability in vitro and in vivo, which explains their superior in vivo efficacy ...

Claims

1. (a) brentuximab, comprising a tubulin tyrosine ligase recognition sequence and a non-natural amino acid at the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of all of the heavy and light chains of brentuximab; and (b) at least one drug moiety; Including, wherein a drug moiety is linked to each of the unnatural amino acids via a linker. Antibody drug conjugates (ADCs).

2. the heavy chain of brentuximab has an amino acid sequence comprising or consisting of SEQ ID NO: 1 or has at least 95% sequence identity to SEQ ID NO: 1, optionally with the last residue K at position 447 of the heavy chain deleted; and / or The light chain of brentuximab has an amino acid sequence comprising or consisting of SEQ ID NO: 2, or has at least 95% sequence identity to SEQ ID NO: 2; The ADC of claim 1.

3. Brentuximab, a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1, optionally with the last residue K at position 447 of said heavy chain deleted; and A light chain consisting of the amino acid sequence of SEQ ID NO: 2 The ADC of claim 2, comprising:

4. The ADC of any one of the preceding claims, wherein the drug moiety is selected from the group consisting of camptothecins, maytansinoids, calicheamicins, duocarmycins, tubulysins, amatoxins, dolastatins and auristatins such as monomethylauristatin E (MMAE), pyrrolobenzodiazepine dimers, indolinobenzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof.

5. 5. The ADC of claim 4, wherein the drug moiety is MMAE.

6. The recognition sequence for tubulin tyrosine ligase comprises at least the amino acid sequence X 1 X 2 X 3 X 4 (SEQ ID NO: 3), wherein X 1 and X 2 is any amino acid, and X 3 is E, D, or C, and X 4 is E.

7. X 2 is G, S, A, V, or F, and / or X 1 is E, D, A, K, or P.

8. The ADC of claim 7, wherein the recognition sequence is EGEE (SEQ ID NO: 4).

9. The recognition sequence is The ADC of claim 7,

10. the unnatural amino acid being 2-, 3- or 4-substituted tyrosine or derivatives thereof substituted at the benzyl position 6. The ADC of any one of the preceding claims,

11. 11. The ADC of claim 10, wherein the 3-substituted tyrosine derivative or 4-substituted tyrosine derivative is 3-nitrotyrosine, 3-aminotyrosine, 3-azidotyrosine, 3-formyltyrosine, 3-acetyltyrosine, or 4-aminophenylalanine.

12. 12. The ADC of claim 11, wherein the 3-substituted tyrosine derivative or 4-substituted tyrosine derivative is 3-formyltyrosine.

13. The ADC of any one of the preceding claims, wherein the linker is cleavable.

14. The ADC of claim 13, wherein the linker is cleavable by a protease.

15. 15. The ADC of claim 14, wherein the protease is a cathepsin, such as cathepsin B.

16. The ADC of any one of the preceding claims, wherein the linker comprises a valine-citrulline moiety.

17. 6. The ADC of any one of the preceding claims, wherein the linker comprises a hydroxylamine group and the unnatural amino acid comprises a formyl group ortho to the hydroxyl group on the aromatic ring, wherein the hydroxylamine group of the linker forms an oxime with the formyl group of the unnatural amino acid after conjugation.

18. The ADC of any one of the preceding claims, wherein brentuximab is conjugated to 2, 4, 6, or 8 drug moieties.

19. 19. The ADC of claim 18, wherein brentuximab is conjugated to two or four drug moieties.

20. 20. The ADC of claim 19, wherein brentuximab is conjugated to two drug moieties.

21. Before the linker is linked to the unnatural amino acid, it has the structure 1: It has a structure shown in wherein R is one or more drug moieties, said drug moieties being optionally linked to the hydroxylamine of Structure 1 by one or more cleavage sites. The ADC of any one of the preceding claims.

22. 22. The ADC of claim 21, wherein the hydroxylamine of structure 1 is conjugated to said unnatural amino acid.

23. The linker has the structure 2 or 3: It has a structure shown in wherein Z is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, and substituted or unsubstituted heteroalkynyl; where D is one or more drug moieties; and wherein Y is a cleavage site, such as the cleavage site of a cathepsin, such as cathepsin B; The ADC of any one of the preceding claims.

24. 24. The ADC of claim 23, wherein a hydroxylamine of structure 2 or 3 is conjugated to the unnatural amino acid.

25. The linker has the structure 4 or 5: where D is a drug moiety. The ADC of any one of the preceding claims.

26. 26. The ADC of claim 25, wherein said unnatural amino acid is 3-formyl tyrosine, and said hydroxylamine group of said linker forms an oxime with the 3-formyl group of said unnatural amino acid.

27. 27. The ADC of claim 26, wherein the drug moiety is MMAE.

28. (a) a brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain; each light chain containing the recognition sequence has SEQ ID NO: 11; and Each heavy chain of brentuximab has SEQ ID NO: 1, optionally with the last residue K at position 447 of the heavy chain deleted; Brentuximab and (b) the C-terminus of the recognition sequence of each light chain is connected via an amide bond to the following structure: and is bonded to a group having the formula wherein the wavy lines indicate the attachment of the recognition sequence of each light chain to the C-terminus. Antibody drug conjugates (ADCs).

29. (a) brentuximab comprising a recognition sequence for tubulin tyrosine ligase at the C-terminus of each light chain, each light chain containing the recognition sequence has SEQ ID NO: 11; and Each heavy chain of brentuximab has SEQ ID NO: 1, optionally with the last residue K at position 447 of the heavy chain deleted; Brentuximab and (b) the C-terminus of the recognition sequence of each light chain is connected via an amide bond to the following structure: and is bonded to a group having the formula wherein the wavy lines indicate the attachment of the recognition sequence of each light chain to the C-terminus. Antibody drug conjugates (ADCs). (a) an anti-CD30 IgG antibody comprising two light chains and two heavy chains, wherein the heavy chain has the amino acid sequence set forth in SEQ ID NO: 1, optionally with the last residue K at position 447 of the heavy chain deleted, and wherein the light chain has the amino acid sequence set forth in SEQ ID NO: 11; and (b) the C-terminus of each light chain is linked via an amide bond to the following structure: and is bonded to a group having the formula Here, the wavy lines indicate the binding of the recognition sequence of each light chain to the C-terminus. Antibody drug conjugates (ADCs).

31. (a) introducing or adding a recognition sequence for tubulin tyrosine ligase to the C-terminus of the light chain, the C-terminus of the heavy chain, or the C-terminus of both the light and heavy chains of brentuximab; (b) contacting the brentuximab from step (a) in the presence of tubulin tyrosine ligase and the unnatural amino acid under conditions suitable for the tubulin tyrosine ligase to ligate the brentuximab to the unnatural amino acid; and (c) conjugating an optionally cleavable linker comprising a drug moiety to the linked brentuximab obtained in step (b).

31. A method of making an ADC as defined in any one of claims 1 to 30, comprising:

32. 32. An ADC obtainable or obtained by the method of claim 31.

33. 33. A pharmaceutical composition comprising the ADC of any one of claims 1 to 30 or 32.

34. 33. The ADC of any one of claims 1 to 30 or 32 for use in a method of treating a disease.

35. 35. The ADC for use according to claim 34, wherein the disease is associated with overexpression of CD30.

36. the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE); or The disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma; 36. An ADC for use according to claim 34 or 35.

37. the disease is Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL); or The disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL); 37. The ADC for use according to claim 36.

38. 34. The pharmaceutical composition of claim 33 for use in a method of treating a disease.

39. 39. The pharmaceutical composition for use according to claim 38, wherein the disease is associated with overexpression of CD30.

40. the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE); or The disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma; 40. A pharmaceutical composition for use according to claim 38 or 39.

41. the disease is Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL); or The disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL); 41. A pharmaceutical composition for use according to claim 40.

42. Use of the ADC of any one of claims 1 to 30 or 32 for the manufacture of a medicament for treating a disease associated with overexpression of CD30.

43. the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE); or The disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma; 43. The use according to claim 42.

44. the disease is Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL); or The disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL); 44. The use according to claim 43.

45. 34. Use of the pharmaceutical composition of claim 33 for the manufacture of a medicament for treating a disease associated with overexpression of CD30.

46. the disease is selected from the group consisting of lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B-cell lymphoma, childhood lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukemias, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and mast cell leukemia, germ cell carcinoma, graft-versus-host disease (GvHD), and lupus, in particular systemic lupus erythematosus (SLE); or The disease is selected from the group consisting of peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), enteropathy-associated T-cell lymphoma (EATL), adult T-cell leukemia / lymphoma (ATLL), extranodal natural killer / T-cell lymphoma (ENKTCL), hepatosplenic and intestinal gamma / delta-T-cell lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, and follicular T-cell lymphoma; 46. ​​The use according to claim 45.

47. the disease is Hodgkin's lymphoma (HL) or anaplastic large cell lymphoma (ALCL); or The disease is peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (ALCL); or cutaneous T-cell lymphoma (CTCL), including primary cutaneous anaplastic large cell lymphoma (pcALCL); 47. The use according to claim 46.

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