Cereblon degrader conjugates and uses thereof

Abstract

Provided herein are cereblon degrader antibody conjugates (cDACs) that include a cereblon degrader moiety covalently attached to an antibody. The cDACs target proteins for intracellular degradation and may be useful in treating diseases and disorders.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 435,142, filed December 23, 2022, and U.S. Provisional Application No. 63 / 525,282, filed July 6, 2023, the contents of each of which are incorporated by reference in their entirety.

[0002] The present disclosure relates generally to cereblon degrading agent antibody conjugate (cDAC) compositions, intermediates for their production, and methods of their use. cDACs are useful for promoting the intracellular degradation of target proteins. [Background technology]

[0003] Cereblon is a 442-amino acid multifunctional protein present in the cytoplasm, nucleus, and peripheral membranes of the human brain and other tissues (Wada et al., Biochem. & Biophys. Res. Comm. 477:388-94 (2016)). Cereblon ensures the normal metabolic and physiological function of ion channels, which are important for maintaining cell growth and proliferation. Cereblon is also involved in the development of many diseases, including cancer (Shi et al., (2017) J. Immunol. Res. Article ID 9130608). Cereblon interacts with DNA damage-binding protein-1 (DDB1), cullin 4 (Cul4A and Cul4B), and regulator of cullin 1 (RoC1) to form a functional E3 ubiquitin ligase complex, known as the CRL4 / CRBNE3 ubiquitin ligase complex. Cereblon's role as part of this complex includes targeting several proteins for proteolysis (degradation) via the ubiquitin-proteasome pathway (Chang et al., (2011) Int. J. Biochem. Mol. Biol. 2(3):287-94). This complex ubiquitinates several other proteins. Cereblon is also involved in developing brain tissue and has been linked to memory and learning processes due to its expression in the hippocampus, among other regions (Higgins et al., (2004) Neurol. 63(10):1927-31).

[0004] Cereblon regulates immune responses and is a target for immunomodulatory drugs (IMiDs) that contain glutarimide functional groups (Kazantsev, A. et al., (2022) Expert Opinion on Therapeutic Patents, 32:2, 171-190; Kronke et al., (2015) Nature 523:183-8; Hagner et al., (2016) Blood 126(6):779-89). The IMiD class includes thalidomide and analogs: lenalidomide, pomalidomide, iveldomide, and apremilast. Thalidomide is approved by the FDA for the treatment of multiple myeloma. Lenalidomide (REVLIMID®) and pomalidomide (POMALYST®) are approved by the FDA for the treatment of multiple myeloma and other diseases. Cytokine regulation and T cell costimulation by IMiDs leads to interleukin-2 production in T cells (Schafer et al., (2003) J. Pharmacol. & Exper. Ther. 305:1222-32). IMiDs have pleiotropic effects on a wide range of immune cells, including activation of natural killer (NK) cells and inhibition of B cells and monocytes (Corral et al., (1999) J. Immunol. 163:380-6). Approved drugs, thalidomide and derivatives lenalidomide and pomalidomide, have been repurposed as immunomodulatory drugs (IMiDs) for hematological cancers (Ito T, et al. (2020) Proc Jpn Acad Ser B Phys Biol Sci. 96(6):189-203). Structural studies have shown that IMiDs such as thalidomide, lenalidomide, and pomalidomide bind to a shallow hydrophobic pocket on the surface of cereblon, and that binding is mediated by the glutarimide ring.

[0005] As a binding protein for MiDs, cereblon is responsible for the multiple effects of IMiDs such as thalidomide and its analogs (P. Ottis, et al. (2017) ACS Chem. Biol. 12(4):892-898; Shi Q, et al. (2017) J Immunol Res. 2017:9130608; Sperling AS, et al. (2019) Blood 134(2):160-170). Cereblon expression can affect cellular metabolism and cause disease even in the absence of IMiDs. Cereblon orthologs are highly conserved from plants to humans, highlighting its physiological importance (Zhihua H, et al. (2011) Annu Rev Plant Biol. 62(1):299-334).

[0006] The ATP-dependent ubiquitin-proteasome system (UPS) is a major pathway for intracellular protein degradation. The UPS system, which consists of ubiquitin (Ub), proteasomes, catalytic enzymes, and specific substrates, plays an important role in various biological processes. Ubiquitination occurs through a cascade of enzymatic events, particularly the synergistic action of Ub-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-ligase enzymes (E3). Once a substrate protein is polyubiquitinated, it is recognized and degraded by the proteasome, and the UPS can digest the substrate protein into small peptides. Because specific recognition of substrate proteins is the apparent function of E3s, E3s play an important role in determining the specificity of Ub-mediated proteolysis (B.E. Smith et al., (2019), Nat. Commun. 10(1):131; K.M. Sakamoto, (2010), Pediatr. Res. 67(5):505-508; M. Scheepstra et al., (2019), Comput. Struct. Biotechnol. J. 17:160-176; P. Ottis et al., (2017), ACS Chem. Biol. 12(4):892-898).

[0007] Proteolytic targeting chimeras (PROTACs) are heterobifunctional degrader constructs capable of targeted degradation of abnormally functioning proteins using the cellular ubiquitin-proteasome machinery. The primary mechanism of PROTAC technology is to degrade proteins of interest (POIs), such as target proteins that are themselves disease mediators, by using the UPS to bring E3 ubiquitin ligases into close proximity with the POI targeted for degradation, resulting in the degradation of the target protein (Lu et al., (2015) Cell Cancer 22(6):755-63; Wang, C. et al. (2021) Eur J Med Chem. 225:113749). The PROTAC's E3 ligase ligand can hijack the E3 ligase and tag the POI with ubiquitin. In this process, the PROTAC itself is not degraded, but is reused to promote the ubiquitination and degradation of other target proteins (M.L. Drummond, et al. (2019), J. Chem. Inf. Model. 59(4):1634-1644; S. An, et al. (2018), EBioMedicine 36:553-562; W. Farnaby, et al. (2019), Nat. Chem. Biol. 15(7):672-680; M.S. Gadd, et al. (2017), Nat. Chem. Biol. 13(5):514-521; R.P. Nowak, et al. (2018) Nat. Chem. Biol. 14(7):706-714). This catalytic event-driven modality acts in contrast to the function of traditional inhibitors, where sequential target binding is required to stimulate the desired effect. In the case of typical small molecule drugs, which are driven by standard occupancy, binding affinity is required for their efficacy.In contrast, PROTACs trigger the degradation of POIs via the UPS, an event-driven modality that can be used to overcome the common drawbacks of traditional occupancy-driven small molecule drugs (KM Sakomoto, et al., (2001), Proc. Natl. Acad. Sci. USA 98(15):8554-8559; P. Martin-Acosta, et al., (2021), Eur. J. Med. Chem. 210:112993; S. Zeng, et al., (2021) Eur. J. Med. Chem. 210:112981; M. Toure, et al., (2016) Angew Chem. Int. Ed. Engl. 55(6):1966-1973).

[0008] "Molecular glues" are degradation constructs that mediate proximity-induced protein degradation by interacting with a ligase (more frequently) or a target POI by inducing or stabilizing a protein-protein interaction between the E3 ubiquitin ligase and the POI, forming a ternary complex that induces ubiquitination and degradation of the target protein POI (den Besten, W. et al. (2020) Nature Chemical Biology 16:1158). Molecular glues can degrade proteins that would otherwise be unable to be ligated by coordinating direct interactions between the target and the ligase (Mayor-Ruiz, C. et al. (2020) Nature Chemical Biology 16:1199-1207; Dong G, et al. (2021) J Med Chem. 64(15):10606-10620). Molecular adhesive degraders can target the nuclear receptor GSPT1 (Huber, AD, et al. (2022) ACS Med. Chem. Lett. 13:1311-1320).

[0009] Although molecular adhesives and PROTACs are both bifunctional proteolytic agents, their mechanisms of action and structural requirements differ (den Besten, W., et al. (2020) Nat Chem Biol 16:1157-1158). However, cereblon ligands can be components of both PROTACs and molecular adhesive degraders that recruit target POIs to CRL4 / CRBNE 3 ubiquitin ligase for degradation (Lu et al., (2015) Cell Cancer 22(6):755-63; Wang, C. et al. (2021) Eur J Med Chem. 225:113749). Certain glutarimide compounds, such as thalidomide, lenalidomide, and pomalidomide, function as molecular adhesives that enhance or induce the interaction between E3 ligases and target proteins, thereby causing ubiquitination and degradation (Dong G, et al. (2021) J Med Chem. 64(15):10606-10620).

[0010] One protein of interest is bromodomain-containing protein 4 (BRD4). Certain small molecule BRD4 inhibitors disrupt protein-protein interactions and have been the subject of antitumor drug development. Limitations include the reversible binding of BRD4 inhibitors (e.g., JQ1, OTX015), which requires large systemic drug concentrations and sustained exposure to ensure sufficient functional inhibition (J. Shi, et al. (2018) Mol. Pharm. 15(9):4139-4147).

[0011] The target protein ligand has been used in PROTACs using pomalidomide via a PEG linker to various BRD4 target protein ligands, which induced significant degradation of BRD4 in BL (Burkitt's lymphoma) cells with a DC50 value of less than 1 nM (J. Lu, et al. (2015) Chem. Biol. 22(6):755-763). PROTACs with other BRD4 and BET target protein ligands showed significant effects on downstream cell proliferation and apoptosis induction in c-MYC, AML (acute myeloid leukemia) cells, and BL cells (EW Georg, et al. (2015) Science 348(6241):1376-1381). These results demonstrate that cereblon-based PROTACs with BET offer a better and more efficient strategy for targeting BRD4 than traditional small molecule inhibitors (L. Bai, et al., (2017) Canc. Res. 77(9):2476-2487; C. Qin, et al., (2018) J. Med. Chem. 61(15):6685-6704; J. Zhang, et al. (2020) Bioorg. Chem. 99:103817). BET protein degradation correlated with the linker design of the PROTAC (T.A. Bemis, et al. (2021) Chem. Commun. 57(8):1026-1029).

[0012] There are limitations and challenges in the design, preparation, and use of antibody compositions covalently linked to drugs, payloads, or other biologically active moieties via linkers. Linkers can be classified into cleavable and non-cleavable linkers according to their chemical properties (Beck A, et al., (2017) Nat Rev Drug Discov. 16(6):315-37; Tsuchikama K, et al., (2018) Protein Cell. 9:33-46). Non-cleavable linkers consist of stable bonds that are resistant to proteolysis, and as a result, cleavage occurs only after lysosomal internalization and complete antibody degradation. These linkers have higher stability than cleavable ones but may suffer from lower membrane permeability. Conversely, cleavage of cleavable linkers can depend on external pH (acid-labile linkers), specific lysosomal proteases (protease-cleavable linkers), or glutathione reduction of disulfide linkers (Shen BQ, ra, (2012) Nat Biotechnol; Bargh JD, et al., (2019) Chem Soc Rev. 48:4361-74). Thus, some linkers may be unstable in the bloodstream, releasing unacceptable amounts of drug before internalization in target cells (Khot, A. et al., (2015) Bioanalysis 7(13):1633-1648), while other linkers may provide stability in the bloodstream, but the effectiveness of intracellular release may be adversely affected. Furthermore, linkers that provide the desired intracellular release may have poor stability in the bloodstream. Furthermore, the amount of drug moiety carried by the antibody, quantified as the drug-to-antibody ratio (DAR), the amount of aggregates formed in the conjugation reaction, and the yield of the final purified conjugate that can be obtained are parameters that need to be addressed and are often interrelated.

[0013] Therefore, there is a continuing need for improved antibody conjugate design, including linker and conjugation chemistry, to provide optimized safety and efficacy. Furthermore, there is a need for enhanced targeted delivery of PROTACs and cereblon ligands containing molecular adhesives to cells containing protein targets. The combination of tumor-associated protein degradation and cereblon immunomodulatory activity may enhance therapeutic benefits in patients suffering from various hyperproliferative disorders, such as cancer. Summary of the Invention

[0014] The present disclosure generally relates to conjugate compositions, referred to as cereblon degrader-antibody conjugates or "cDACs," in which a cereblon degrader moiety is covalently attached to an antibody via an antibody linker. In some embodiments, the cereblon degrader moiety comprises a target protein ligand covalently linked to a cereblon-binding E3 ubiquitin ligase ligand via a degrader linker. In other embodiments, the cereblon degrader moiety is a molecular adhesive. In some embodiments, the cereblon degrader moiety of the disclosed cDACs is targeted to appropriate target cells and released as a cereblon degrader compound, thereby functioning to stimulate / induce ubiquitination of the target protein, leading to its degradation by the ubiquitin-proteasome system (UPS). cDACs may provide improved therapeutic benefit in patients suffering from various hyperproliferative disorders, such as cancer.

[0015] One aspect of the present disclosure is a cereblon degrader antibody conjugate (cDAC) comprising a cereblon degrader moiety covalently attached to an antibody by a linker (e.g., an antibody linker), wherein the cereblon degrader moiety is a target protein ligand, or molecular glue, covalently attached to a cereblon-binding E3 ubiquitin ligase ligand by the degrader linker, and the antibody is a thiol-containing antibody.

[0016] Another aspect of the present disclosure is a compound of formula I: Ab-[L1-cD] p I or a pharmaceutically acceptable salt thereof, During the ceremony, Ab is antibody; cD is the cereblon degradation product portion; L1 is a linker attached to Ab and cD; p is an integer of 1 to 14.

[0017] Another aspect of the present disclosure is a compound of formula II: X-L3-cD II a cereblon degrader linker intermediate having the structure During the ceremony, X is a thiol-reactive group; L3 is (i) Formula: -Str-PM-IM- wherein Str is a stretcher unit covalently attached to X; PM is a peptidomimetic unit, IM is an immolator unit covalently attached to cD and has the formula: (ii) Formula: [ka] [ka] and [ka] a disulfide linker selected from: and Formula (iii): [ka] (where * indicates the point of attachment to X; R 4a , R 4b , R 5a , and R 5a are each independently selected from H and C1-C6 alkyl, or R 4aand R 4b together with the carbon atom to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl, optionally substituted with F, Cl, and C1-C6 alkyl; R 6 is selected from H and C1-C6 alkyl; The wavy line indicates the attachment point to cD. R 4a , R 4b , R 5a , R 5a and R 6 wherein the C1-C6 alkyl is independently selected from a linker having F, Cl, -CN, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; cD is the cereblon degrader moiety, (i) CD is a molecular glue (MG), or (ii) cD has the formula: TPL-L2-E3UL (In the formula, TPL is a target protein ligand; E3UL is the cereblon-binding E3 ubiquitin ligase ligand; L2 is a degradant linker).

[0018] Another aspect of the present disclosure is a cDAC prepared by conjugation of an antibody with a cereblon degradation intermediate of formula II.

[0019] Another aspect of the present disclosure is a method of preparing cDAC comprising reacting a thiol-containing antibody with a cereblon degrader intermediate of formula II.

[0020] Another aspect of the disclosure is a pharmaceutical composition comprising a therapeutically effective amount of cDAC and one or more pharmaceutically acceptable diluents, vehicles, carriers, or excipients.

[0021] Another aspect of the disclosure is a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of cDAC.

[0022] Another aspect of the disclosure is the use of cDAC in the manufacture of a medicament for treating cancer in a mammal.

[0023] Another aspect of the present disclosure is the use of cDACs for the treatment of cancer in a mammal. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows the in vitro potency of the anti-proliferative effects of BRD4-cereblon degrader on KPL-4 and SK-BR-3 cells at day 5. Cell viability as a percent of control is plotted against the concentration (nM) of cereblon degrader compound cD-5. [Figure 2A] Figure 2A shows the in vitro anti-proliferative effect of HER2+ KPL-4 cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 3. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 2B] Figure 2B shows the in vitro antiproliferative effects of HER2+ SK-BR-3 cells after treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 for 5 days. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 3A]Figure 3A shows the in vitro antiproliferative effect of HER2-low / ER+ CAMA1 cells after treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 for 5 days. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 3B] Figure 3B shows the in vitro antiproliferative effects of HER2-low / ER+ EFM19 cells after treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 for 5 days. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 4] Figure 4 shows the in vitro antiproliferative effect of the anti-CD33 BRD4-cereblon degrader antibody conjugate cDAC-3 on various AML cell lines after 7 days of treatment. The AML cell lines were MV-4-11, EOL-1, Molm-13, Nomo-1, HL-60, and OCI-AML-2. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 5A] Figure 5A shows the in vitro antiproliferative effects of EOL-1 AML cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 5B] Figure 5B shows the in vitro antiproliferative effects of HL-60 AML cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 6A]Figure 6A shows the in vitro antiproliferative effects of Molm-13 AML cells after 3 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 6B] Figure 6B shows the in vitro antiproliferative effects of MV-4-11 AML cells after 3 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). [Figure 7] Figure 7 shows the in vivo efficacy of anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 at the following doses in reducing tumor volume over time (21 days) in an HL-60 xenograft mouse model: 1) Vehicle (Histidine Buffer #8), 100 μL, 1 IV; 2) cDAC-4, 3 mg / kg, 1 IV; 3) cDAC-3, 1 mg / kg, 1 IV; 4) cDAC-3, 3 mg / kg, 1 IV; 5) cDAC-3, 10 mg / kg, 1 IV; 6) cDAC-6, 3 mg / kg, 1 IV; 7) cDAC-5, 1 mg / kg, 1 IV; and 8) cDAC-5, 3 mg / kg, 1 IV. DETAILED DESCRIPTION OF THE INVENTION

[0025] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope provided herein as defined by the claims.

[0026] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0027] definition The term "antibody" is used in the broadest sense and encompasses, among other things, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. As used herein, an "antibody fragment" and all grammatical variations thereof are defined as a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, which portion does not include the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including, but not limited to, (1) single-chain Fv (scFv) molecules; (2) a single-chain polypeptide comprising only one light-chain variable domain, or a fragment thereof comprising the three CDRs of the light-chain variable domain and no associated heavy-chain portion; (3) a single-chain polypeptide comprising only one heavy-chain variable region, or a fragment thereof comprising the three CDRs of the heavy-chain variable region and no associated light-chain portion; (4) nanobodies comprising a single Ig domain or other specific single-domain binding module from a non-human species; and (5) multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain may contain any of the constant domain sequences found in the non-Fc region of an intact antibody (e.g., CH1 in IgG isotypes), and / or may contain any of the hinge region sequences found in an intact antibody, and / or may contain a leucine zipper sequence fused to or located within the hinge region or constant domain sequence of the heavy chain.

[0028] An "antibody" refers to a polypeptide comprising an antigen-binding region (including complementarity-determining regions (CDRs)) derived from an immunoglobulin gene or a fragment thereof. The term "antibody" specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa) linked by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains are divided into various categories based on size and function, e.g., variable domains or regions on the light and heavy chains (V and V, respectively). L and V H ) and constant domains or regions on the light and heavy chains (C L and C H ). The N-terminus of each chain, called the paratope, defines a variable region of approximately 100–110 or more amino acids primarily responsible for antigen recognition, i.e., the antigen-binding domain. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which subsequently define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are large molecules of approximately 150 kDa composed of four peptide chains. IgG antibodies contain two identical class gamma heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thus forming a tetrameric quaternary structure. The two heavy chains are linked to each other and to the light chains by disulfide bonds. The resulting tetramer has two identical halves, which together form a Y-like shape. Each end of the fork contains an identical antigen-binding domain. There are four IgG subclasses in humans (IgG1, IgG2, IgG3, and IgG4), named in order of their abundance in serum (i.e., IgG1 is the most abundant). The antigen-binding domain of an antibody is usually most important for the specificity and affinity of binding to cancer cells.

[0029] Antibodies that target a specific antigen include bispecific or multispecific antibodies that have at least one antigen-binding region that targets a specific antigen. In some embodiments, the targeting monoclonal antibody is a bispecific antibody that has at least one antigen-binding region that targets tumor cells.

[0030] "Antibody construct" refers to an antibody or fusion protein comprising (i) an antigen-binding domain and (ii) an Fc domain.

[0031] In some embodiments, the binding agent is an antigen-binding antibody "fragment," which is a construct that comprises at least the antigen-binding region of an antibody, either alone or together with other components that together form an antigen-binding construct. For example, (i) V L , V H , C L (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V(ab')2 fragment, which is a monovalent fragment consisting of the V domain of a single arm of an antibody; and (iv) a Fab fragment, which is a monovalent fragment consisting of the CH1 domain of an antibody. L and V H These include many different types of antibody "fragments," including Fv fragments consisting of two domains, (iv) Fab' fragments, which are obtained by cleaving the disulfide bridges of the F(ab')2 fragment using mild reducing conditions, (v) disulfide-stabilized Fv fragments (dsFv), and (vi) single-chain Fv (scFv), which are Fv fragments (i.e., V) linked by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain. L and V H ) is a monovalent molecule consisting of two domains.

[0032] An antibody or antibody fragment can be part of a larger construct, e.g., a conjugate or fusion construct of the antibody fragment to additional domains. For example, in some embodiments, an antibody fragment can be fused to an Fc region as described herein. In other embodiments, an antibody fragment (e.g., Fab or scFv) can be part of a chimeric antigen receptor or chimeric T cell receptor, e.g., by fusing to a transmembrane domain (optionally with an intervening linker or "stalk" (e.g., hinge region)) and optional intercellular signaling domains. For example, an antibody fragment can be fused to the gamma and / or delta chains of a T cell receptor to provide a T cell receptor-like construct that binds to TROP2. In yet another embodiment, the antibody fragment is part of a bispecific T cell engager (BiTE) comprising a CD1- or CD3-binding domain and a linker.

[0033] "Epitope" (i.e., in the paratope of an antigen-binding domain) means any antigenic or epitopic determinant of the antigen to which the antigen-binding domain binds. Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0034] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. There are three major classes of Fc receptors: (1) FcγR, which binds IgG; (2) FcαR, which binds IgA; and (3) FcεR, which binds IgE. The FcγR family includes several members, such as FcγI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), and FcγRIIIB (CD16B). Fcγ receptors have different affinities for IgG and for IgG subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0035] "Amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally occurring α-amino acids and their stereoisomers, as well as non-naturally occurring (non-naturally occurring) amino acids and their stereoisomers. A "stereoisomer" of a given amino acid refers to an isomer that has the same molecular formula and intramolecular bond but a different three-dimensional arrangement of bonds and atoms (e.g., an l-amino acid and the corresponding d-amino acid). Amino acids can be glycosylated (e.g., N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan, or glycosylation) or deglycosylated. Amino acids can be referred to herein by either the commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0036] Naturally occurring amino acids are those encoded by genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0037] Naturally occurring amino acids include those formed in proteins by post-translational modifications, such as citrulline (Cit).

[0038] Non-natural (non-naturally occurring) amino acids include, but are not limited to, L- or D-configuration amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids that function in a similar manner to naturally occurring amino acids. For example, "amino acid analogs" can be non-natural amino acids that have the same basic chemical structure as naturally occurring amino acids (i.e., carbons bonded to hydrogen, carboxyl groups, and amino groups), but have modified side groups or modified peptide backbones, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. "Amino acid mimetics" refer to compounds that have a structure different from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids.

[0039] "Amino acid side chain" refers to the group of an amino acid that defines the amino acid and distinguishes one amino acid from another. For example, side chains of representative amino acids are glycine (-H), alanine (-CH), phenylalanine (-CH(CH)), lysine (-CHCHCHCHNH), arginine (-CHCHCHNHC(NH)NH), leucine -CHCH(CH) and citrulline (-CHCHCHNHC(O)NH).

[0040] "Linker" refers to a functional group that covalently links two or more moieties in a compound or substance. For example, a linking moiety can be used to covalently link a drug moiety to an antibody construct in a conjugate provided herein or between two or more ligand-binding moieties.

[0041] "Linking moiety" refers to a functional group that covalently links two or more moieties in a compound. For example, a linking moiety can be used to covalently link a drug moiety to an antibody in a conjugate. Useful bonds for linking a linking moiety to proteins and other substances include, but are not limited to, amide, amine, ester, carbamate, disulfide, urea, thioether, thiocarbamate, thiocarbonate, and thiourea.

[0042] "Divalent" refers to a chemical moiety that contains two points of attachment for linking two moieties; a polyvalent linking moiety may have additional points of attachment for linking additional functional groups. A divalent radical may be indicated by the suffix "diyl." For example, divalent linking moieties include divalent polymer moieties, such as divalent poly(ethylene glycol), divalent cycloalkyl, divalent heterocycloalkyl, divalent aryl, and divalent heteroaryl groups. A "divalent cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group" refers to a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group that has two points of attachment for covalently linking two moieties in a molecule or substance. The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may be substituted or unsubstituted. The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0043] Wavy line [ka] represents the point of attachment of a particular chemical moiety. [ka] It will be understood that when present, the chemical moiety may be used on both sides, i.e., reading from left to right or right to left. In some embodiments, the two wavy lines present [ka] Certain parts with a . are considered to be used when read from left to right.

[0044] "Alkyl" refers to a straight-chain (straight-chain) or branched saturated aliphatic group having the indicated number of carbon atoms. Alkyl can contain any number of carbons, for example, 1 to 12. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et-CH2CH3), 1-propyl (n-PR, n-propyl, -CH2CH2CH3), 2-propyl (i-PR, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2- Propyl (t-Bu, t-butyl-C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2 and the like. The alkyl groups can be substituted or unsubstituted. Substituted alkyl groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.Substituted alkyl groups may also be geminally substituted when the carbon atoms of the alkyl form a spiro, cycloalkyl group, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0045] The term "alkyldiyl" refers to a divalent alkyl group. Examples of alkyldiyl groups include, but are not limited to, methylene (-CH-), ethylene (-CHCH-), propylene (-CHCHCH-), and the like. Alkyldiyl groups are sometimes referred to as "alkylene" groups. Alkyldiyl groups can be substituted or unsubstituted. Substituted alkyldiyl groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy. Substituted alkyldiyl groups can also be geminally substituted when the carbon atoms of the alkyl form a spiro, cycloalkyl group, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0046] "Alkenyl" refers to a straight-chain (straight-chain) or branched unsaturated aliphatic group having the indicated number of carbon atoms and at least one sp2 carbon-carbon double bond. Alkenyls can contain from 2 to about 12 or more carbon atoms. Alkenyl groups are groups having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. Examples include, but are not limited to, ethylenyl or vinyl (-CH=CH2), allyl (-CH2CH=CH2), butenyl, pentenyl, and their isomers. Alkenyl groups can be substituted or unsubstituted. "Substituted alkenyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0047] The terms "alkenylene" or "alkenyldiyl" refer to a straight-chain or branched divalent hydrocarbon radical. Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), and allyl (-CHCH=CH-).

[0048] "Alkynyl" refers to a straight-chain (straight-chain) or branched unsaturated aliphatic radical having the indicated number of carbon atoms and at least one carbon-carbon triple bond (sp). Alkynyl can contain from 2 to about 12 or more carbon atoms. For example, C2-C6 alkynyl includes, but is not limited to, ethynyl (-C≡CH), propynyl (propargyl, -CH2C≡CH), butynyl, pentynyl, hexynyl, and isomers thereof. Alkynyl groups can be substituted or unsubstituted. "Substituted alkynyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0049] The terms "alkynylene" or "alkynyldiyl" refer to a divalent alkynyl group.

[0050] The terms "carbocycle," "carbocyclyl," "carbocyclic ring," and "cycloalkyl" refer to saturated or partially unsaturated, monocyclic, fused bicyclic, spiro, or bridged polycyclic ring assemblies containing 3 to 12 ring atoms or the number of atoms indicated. Saturated monocyclic carbocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocycles include, for example, norbornane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. Carbocyclic groups may also be partially unsaturated and have one or more double or triple bonds within the ring. Representative partially unsaturated carbocyclic groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene.

[0051] The term "cycloalkyldiyl" refers to a divalent cycloalkyl group.

[0052] "Aryl" refers to a monovalent aromatic hydrocarbon radical of 6 to 20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system (C6-C 20 ) Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or joined by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl, which has a methylene linking group. Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl.

[0053] The terms "heterocycle," "heterocyclic ring," and "heterocyclic ring" are used interchangeably herein and refer to saturated or partially unsaturated (i.e., having one or more double and / or triple bonds in the ring) carbocyclic radicals of 3 to about 20 ring atoms, where at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, and the remaining ring atoms are C, and one or more ring atoms are optionally substituted independently with one or more substituents described below. A heterocycle can be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), e.g., a bicyclo[4,5], [5,5], [5,6], or [6,6] system, or more rings. Heterocycles are described in Paquette, Leo A; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. "Heterocycle" also includes radicals in which a heterocyclic radical is fused with a saturated, partially unsaturated ring, or an aromatic carbocyclic or heterocyclic ring.Examples of heterocyclic rings include morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, and azocanyl. Examples of heterocyclic ... Examples of spiroheterocyclyl moieties include azaspiro[2.5]octanyl and azaspiro[2.4]heptanyl. Examples of heterocyclic groups in which two ring atoms are substituted with oxo (=O) moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.

[0054] The term "heteroaryl" refers to a monovalent aromatic radical of a 5-, 6-, or 7-membered ring, including fused ring systems of 5 to 20 atoms (at least one of which is aromatic) containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, and the like. benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiaphenyl, benzothiazolyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

[0055] The heterocycle or heteroaryl group may be carbon (carbon-linked) or nitrogen (nitrogen-linked) linked, where possible. By way of example and not limitation, a carbon-linked heterocycle or heteroaryl may be bonded at the 2-, 3-, 4-, 5-, or 6-position of a pyridine, the 3-, 4-, 5-, or 6-position of a pyridazine, the 2-, 4-, 5-, or 6-position of a pyrimidine, the 2-, 3-, 5-, or 6-position of a pyrazine, the 2-, 3-, 4-, or 5-position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2-, 4-, or 5-position of an oxazole, imidazole, or thiazole, the 3-, 4-, or 5-position of an isoxazole, pyrazole, or isothiazole, the 2-, or 3-position of an aziridine, the 2-, 3-, or 4-position of an azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of a quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of an isoquinoline.

[0056] By way of example, and without limitation, a nitrogen-linked heterocycle or heteroaryl is bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or β-carboline.

[0057] The terms "halo" and "halogen," by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0058] The term "carbonyl," by itself or as part of another substituent, refers to C(=O) or -C(=O)-, i.e., a carbon atom double-bonded to oxygen and bonded to two other groups in the carbonyl-containing moiety.

[0059] The term "chiral" refers to molecules that have the property of not being superimposable on their mirror image partners, while the term "achiral" refers to molecules that are superimposable on their mirror image partners.

[0060] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0061] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high-resolution analytical procedures such as electrophoresis and chromatography.

[0062] "Enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0063] Stereochemical definitions and conventions used herein generally follow S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds described herein may contain asymmetric or chiral centers and therefore may exist in different stereoisomeric forms. All stereoisomers of the compounds described herein, including, but not limited to, diastereomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemic mixtures, are intended to form part of this disclosure. Many organic compounds exist in optically active forms, i.e., they can rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to indicate the sign of rotation of plane-polarized light by a compound, with (-) or 1 meaning the compound is levorotatory. Compounds with the prefix (+) or d meaning dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers are also sometimes called enantiomers, and mixtures of such isomers are often called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species that lacks optical activity. Enantiomers may be separated from a racemic mixture by chiral separation methods, such as supercritical fluid chromatography (SFC). While assignment of configuration at chiral centers in separated enantiomers may be tentative, and is shown in the structures in Table 1 for illustrative purposes, stereochemistry has been definitively assigned from X-ray crystallographic data, etc.

[0064] The terms "treat," "treatment," and "treating" refer to any indication of success in treating or ameliorating an injury, pathology, condition (e.g., cancer) or symptom (e.g., cognitive impairment), including any objective or subjective parameter such as reduction; remission; reducing symptoms or making the symptom, injury, pathology, or condition more tolerable to the patient; slowing the rate of progression of a symptom; reducing the frequency or duration of a symptom or condition; or, in some circumstances, preventing the onset of a symptom. Treating or ameliorating a symptom can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0065] The terms "cancer," "neoplasm," and "tumor" are used herein to refer to cells that exhibit autonomous, uncontrolled growth, such that the cells exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, and / or treatment in the context of this disclosure include cancer cells (e.g., cancer cells derived from an individual with cancer), malignant cancer cells, premetastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. Cancers of virtually all tissues are known. The phrase "cancer burden" refers to the amount of cancer cells or the volume of cancer in a subject. Thus, reducing cancer burden refers to reducing the number of cancer cells or the volume of cancer cells in a subject. As used herein, the term "cancer cell" refers to a cancer cell (e.g., isolated from any cancer that can treat an individual, e.g., from an individual with cancer) or any cell derived from a cancer cell, e.g., a clone of a cancer cell. For example, a cancer cell can be derived from an established cancer cell line, a primary cell isolated from an individual with cancer, a progeny cell from a primary cell isolated from an individual with cancer, etc. In some embodiments, the term may also refer to a part of a cancer cell, such as an intracellular portion of the cancer cell, a portion of the cell membrane, or a cell lysate. There are many types of cancer known to those skilled in the art, including solid tumors such as carcinoma, sarcoma, glioblastoma, melanoma, lymphoma, and myeloma, and circulating cancers such as leukemia.

[0066] As used herein, the term "cancer" includes any form of cancer, including, but not limited to, solid tumor cancers (e.g., skin, lung, prostate, breast, stomach, bladder, colon, ovarian, pancreatic, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head and neck squamous cell carcinoma, melanoma, and neuroendocrine) and liquid cancers (e.g., blood cancers); carcinoma; soft tissue tumor; sarcoma; teratoma; melanoma; leukemia; lymphoma; and brain cancer, including minimal residual disease, and including both primary and metastatic tumors.

[0067] The phrases "effective amount" and "therapeutically effective amount" refer to the dose or amount of a therapeutic agent administered that produces a therapeutic effect. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11 th Edition(McGraw-Hill,2006);andRemington:The Science and Practice of Pharmacy,22 nd Edition, (see Pharmaceutical Press, London, 2012). In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent a therapeutic agent may prevent growth and / or kill existing cancer cells, the therapeutic agent may be cytostatic and / or cytotoxic. With respect to cancer treatment, efficacy may be measured, for example, by assessing the time to progression (TTP) and / or determining the response rate (RR).

[0068] "Recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., a human) for whom diagnosis, treatment, or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, and the like. In certain embodiments, the mammal is a human. A "patient" or "individual" or "subject" is a mammal. Mammals include, but are not limited to, farm animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the patient, individual, or subject is a human. In some embodiments, the patient may be a "cancer patient," i.e., a patient suffering from or at risk of suffering from one or more symptoms of cancer. A "patient population" refers to a group of cancer patients. Populations such as these can be used to demonstrate statistically significant efficacy and / or safety of a drug.

[0069] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or implantation of a sustained-release device, e.g., a mini-osmotic pump, into a subject.

[0070] The terms "residue," "moiety," or "group" refer to a moiety that is covalently bonded or linked to another moiety.

[0071] The terms "covalently bound" or "covalently linked" refer to a chemical bond formed by the sharing of one or more electron pairs.

[0072] As used herein, the term "peptidomimetic" or PM refers to a non-peptide chemical moiety as part of a linker. A peptide is a short chain (two or more) of amino acid monomers linked by peptide (amide) bonds, whereas a peptidomimetic chemical moiety includes non-amino acid chemical moieties. A peptidomimetic chemical moiety may also include one or more amino acids separated by one or more non-amino acid chemical units. A peptidomimetic chemical moiety does not contain two or more adjacent amino acids linked by peptide bonds anywhere in its chemical structure.

[0073] Cereblon Degrader Antibody Conjugate S The cereblon degrading agent antibody conjugates (cDACs) provided herein comprise at least one (p) cereblon degrading agent moiety (cD) covalently attached to an antibody (Ab) by an antibody linker (L1).

[0074] Cereblon degrader antibody conjugates (cDACs) induce targeted degradation of tumor-associated proteins, providing specificity that minimizes off-target toxic effects. In some embodiments, cDACs form a cereblon-based ternary complex between the target protein and the E3 ubiquitin ligase cereblon.

[0075] An exemplary embodiment of a cDAC has Formula I: Ab-[L1-cD] p I or a pharmaceutically acceptable salt thereof, (In the formula, Ab is antibody; L1 is the antibody linker; cD is the cereblon degradation product portion; p is an integer of 1 to 14.

[0076] In some embodiments, L1 comprises an immolator moiety. 1a -IM, where IM is the immolator part, and L 1ais any remainder of the L1 antibody linker. In some embodiments, L1 is an immolator moiety, IM.

[0077] In some embodiments, the cD comprises the cereblon-binding E3 ubiquitin ligase ligand E3UL. In some embodiments, the cD comprises E3UL-cD a and E3UL is the cereblon-binding, E3 ubiquitin ligase ligand of the cereblon degrader moiety, cD a is any remainder of cD. In some embodiments, cD a is TPL-L2-, where TPL is a target protein ligand and L2 is a degrader linker. In some embodiments, TPL comprises a ligand that binds to BRD4.

[0078] An exemplary embodiment of a cDAC is represented by Formula I Ab-[L 1a -IM-E3UL-cD a ] p I' It has.

[0079] In some embodiments, the cereblon degrader portion of cDAC (cD) is covalently attached to the antibody linker (L1) via an amino group.

[0080] In some embodiments, L1 is [ka] and [ka] and an immolator moiety IM selected from *L 1a ** indicates the bond point with cD a The wavy line indicates the point of attachment to E3UL.

[0081] An exemplary embodiment of a cDAC has the structure of formula IA: [ka] During the ceremony, Ab is antibody; L 1a is the antibody linker; Ring A is C6-C 20 Aryl, C3-C 20 Carbocyclyl, C3-C 20 Heterocyclyl, and C3-C 20 heteroaryl; Dashed line [ka] indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a is selected from Z 2 is C(R 2 )2, CR 2 , N, and NR 2a is selected from R 1 and R 2 are each independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NRa R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are each independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR aR b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; or (i) Two geminal R 1 Or two geminal R 2 forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii)R 1 and R 2 , R 1a and R 2 , R 1 and R 2a , or R 1a and R 2a form a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R a and R b are each independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are each independently selected from F, Cl, —CN, C1-C6 alkyl, 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl; Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -C H(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -C H(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N optionally substituted with one or more groups selected from HC(=NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H; CD a is the remainder of the cereblon degrader moiety; p is an integer of 1 to 14.

[0082] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are H, respectively.

[0083] In some embodiments, ring A is C3-C 20 It is heteroaryl.

[0084] In some embodiments, ring A is an isoindoline substituted with ═O.

[0085] In some embodiments, the cDAC of formula I has the structure of formula I-A': [ka] In the formula, X 1 is selected from CH2 and C(=O).

[0086] In some embodiments, the antibody is a thiol-containing antibody.

[0087] In some embodiments, the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

[0088] In some embodiments, the antibody is a cysteine ​​engineered antibody.

[0089] In some embodiments, the cysteine ​​engineered antibody comprising one or more cysteine ​​mutations is selected from HCA118C, LCK149C, HCA140C, LCV205C, LCS121C, HCL174C, HCL177C, and HCY373C.

[0090] In some embodiments, L 1a is a protease-cleavable non-peptide linker.

[0091] In some embodiments, cD a is TPL-L2-, where TPL is a target protein ligand and L2 is a degrader linker. In some embodiments, TPL comprises a ligand that binds to BRD4.

[0092] In some embodiments, L 1ahas the structure of formula L1-A, [ka] During the ceremony, * indicates the point of attachment of Ab to the cysteine ​​thiol; R 1 is C1-C 12 Alkylene, C1-C 12 Alkylene-C(=O), C1-C 12 Alkylene-NH, (CH2CH2O) r , C1-C 12 Alkylene-NH, (CH2CH2O) r -C(=O), (CH2CH2O) r -C(=O), (CH2CH2O) r -CH2, and C1-C 12 alkylene-NHC(=O)CHCH(thiophen-3-yl), r is an integer ranging from 1 to 10; C1-C 12 The alkylene may be substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; R 2 and R 3 together form a C3-C7 cycloalkyl ring optionally substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; AA is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

[0093] In some embodiments, AA is selected from H, —CH3, —CH2(C6H5), —CH2CH2CH2CH2NH2, —CH2CH2CH2NHC(NH)NH2, —CH2CH(CH3)2, and —CH2CH2CH2NHC(O)NH2.

[0094] In some embodiments, R 1 is a C5 alkylene.

[0095] In some embodiments, R 2 and R 3 taken together form a C4 cycloalkyl ring.

[0096] In some embodiments, AA is —CH 3 or —CH 2 CH 2 CH 2 NHC(O)NH 2 .

[0097] In some embodiments, R 1 is a C5 alkylene; R 2 and R 3 together form a C4 cycloalkyl ring; AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0098] In some embodiments, cD a comprises (i) a target protein ligand covalently attached to a degrader linker, or (ii) a molecular adhesive moiety. In some embodiments, cD a is TPL-L2-, where TPL is a target protein ligand and L2 is a degrader linker. In some embodiments, TPL comprises a ligand that binds to BRD4.

[0099] In some embodiments, p is 1, 2, 3, 4, 5, or 6.

[0100] An exemplary embodiment of a cDAC has the structure of formula IB: [ka] During the ceremony, Ab is antibody; L 1a is the antibody linker; Ring A is C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, and C1-C 20 is heteroaryl; Dashed line [ka] indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a is selected from Z 2 is C(R 2 )2, CR 2 , N, and NR 2a is selected from R 1 and R 2 are each independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a or selected from the group consisting of -S(O)H; (i) Two geminal R 1 Or two geminal R 2 forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii)R 1 and R 2 , R 1a and R 2 , R 1 and R 2a Or R 1a and R 2a forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are independently selected from F, Cl, —CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl, Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -C H(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -C H(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N optionally substituted with one or more groups selected from HC(=NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H; CD a is the remainder of the cereblon degrader moiety; p is an integer of 1 to 14.

[0101] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are H, respectively.

[0102] In some embodiments, ring A is C3-C 20 It is heteroaryl.

[0103] In some embodiments, ring A is an isoindoline substituted with ═O.

[0104] In some embodiments, cD a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0105] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0106] In some embodiments, the cDAC of formula IB has the structure of formula I-B': [ka] In the formula, X 1 is selected from CH2 and C(=O).

[0107] In some embodiments, the antibody is a thiol-containing antibody.

[0108] In some embodiments, the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

[0109] In some embodiments, the antibody is a cysteine ​​engineered antibody.

[0110] In some embodiments, the cysteine ​​engineered antibody has a cysteine ​​mutation site selected from one or more of HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

[0111] In some embodiments, L 1a is a protease-cleavable non-peptide linker.

[0112] In some embodiments, L 1a has the structure of formula L1-A, [ka] During the ceremony, * indicates the point of attachment of Ab to the cysteine ​​thiol; R 1 is C1-C 12 Alkylene, C1-C 12 Alkylene-C(=O), C1-C 12 Alkylene-NH, (CH2CH2O) r , C1-C 12 Alkylene-NH, (CH2CH2O) r -C(=O), (CH2CH2O) r -C(=O), (CH2CH2O) r -CH2, and C1-C 12 alkylene-NHC(=O)CHCH(thiophen-3-yl), r is an integer ranging from 1 to 10; C1-C 12 The alkylene may be substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; R 2 and R 3 together form a C3-C7 cycloalkyl ring optionally substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; AA is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

[0113] In some embodiments, AA is selected from H, —CH3, —CH2(C6H5), —CH2CH2CH2CH2NH2, —CH2CH2CH2NHC(NH)NH2, —CH2CH(CH3)2, and —CH2CH2CH2NHC(O)NH2.

[0114] In some embodiments, R 1 is a C5 alkylene.

[0115] In some embodiments, R 2 and R 3 taken together form a C4 cycloalkyl ring.

[0116] In some embodiments, AA is —CH 3 or —CH 2 CH 2 CH 2 NHC(O)NH 2 .

[0117] In some embodiments, R 1 is a C5 alkylene; R 2 and R 3 together form a C4 cycloalkyl ring; AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0118] In some embodiments, the cDa comprises (i) a target protein ligand covalently attached to a degrader linker, or (ii) a molecular adhesive moiety. a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0119] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0120] In some embodiments, p is 1, 2, 3, 4, 5, or 6.

[0121] In some embodiments, L1 is an immolator moiety, IM.

[0122] An exemplary embodiment of a cDAC is represented by Formula I Ab-[IM-E3UL-cD a ] p I” It has the following structure.

[0123] In some embodiments, the IM is [ka] and During the ceremony, * indicates the point of attachment to AB ** indicates CD a indicates the point of attachment to The wavy line indicates the point of attachment to E3UL.

[0124] In such embodiments, R 4a , R 4b , R 5a , and R 5a are independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C1-C6 alkyl, where C1-C6 alkyl is optionally substituted independently with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0125] An exemplary embodiment of a cDAC has the structure of formula IC: [ka] During the ceremony, Ab is antibody; R 4a , R 4b , R 5a , and R 5a are each independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C1-C6 alkyl, wherein C1-C6 alkyl is optionally substituted independently with one or more groups selected from F, Cl, —CN, —NH2, —CH2NH2, —OH, —OCH3, —OCH2CH3, —OCH2CH2OCH3, —OCH2CH2OH, —OCH2CH2N(CH3)2, —OCH2F, —OCHF2, —OCF3, —OP(O)(OH)2, —S(O)2N(CH3)2, —SCH3, —S(O)2CH3, and —S(O)3H; Ring A is C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, and C1-C 20 is heteroaryl; Dashed line [ka] indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a is selected from Z 2 is C(R 2 )2, CR 2 , N, and NR 2a is selected from R 1and R 2 are each independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NRa R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a or selected from the group consisting of -S(O)H; (i) Two geminal R 1 Or two geminal R 2 forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii)R 1 and R 2 , R 1a and R 2 , R 1 and R 2a Or R 1a and R 2a forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R a and R bare independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are independently selected from F, Cl, —CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl, Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -C H(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -C H(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N optionally substituted with one or more groups selected from HC(=NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H; CD a is the remainder of the cereblon degrader moiety; p is an integer of 1 to 14.

[0126] In some embodiments, sulfur is attached to a cysteine ​​thiol of the Ab to form a disulfide bond.

[0127] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are H, respectively.

[0128] In some embodiments, ring A is C3-C 20 It is heteroaryl.

[0129] In some embodiments, ring A is isoindoline substituted with ═O or oxo.

[0130] In some embodiments, cD a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0131] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0132] In some embodiments, the cDAC of formula IC has the structure of formula IC': [ka] During the ceremony, Ab is antibody; R 4a , R 4b , R 5a , and R 5a are each independently selected from H and C1-C6 alkyl, or R 4a and R 4btogether with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C1-C6 alkyl, wherein C1-C6 alkyl is optionally substituted independently with one or more groups selected from F, Cl, —CN, —NH2, —CH2NH2, —OH, —OCH3, —OCH2CH3, —OCH2CH2OCH3, —OCH2CH2OH, —OCH2CH2N(CH3)2, —OCH2F, —OCHF2, —OCF3, —OP(O)(OH)2, —S(O)2N(CH3)2, —SCH3, —S(O)2CH3, and —S(O)3H; X 1 is selected from CH2 and C(=O); Dashed line [ka] indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a is selected from Z 2 is C(R 2 )2, CR 2 , N, and NR 2a is selected from R 1 and R 2 are each independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NRa R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a or selected from the group consisting of -S(O)H; (i) Two geminal R 1 Or two geminal R 2 forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii)R 1 and R 2 , R 1a and R 2 , R 1 and R 2a Or R 1a and R 2a forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are independently selected from F, Cl, —CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl, Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -C H(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -C H(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N optionally substituted with one or more groups selected from HC(=NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H; CD a is the remainder of the cereblon degrader moiety; p is an integer of 1 to 14.

[0133] In some embodiments, the antibody is a thiol-containing antibody.

[0134] In some embodiments, the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

[0135] In some embodiments, the antibody is a cysteine ​​engineered antibody.

[0136] In some embodiments, the cysteine ​​engineered antibody comprises one or more cysteine ​​mutations selected from HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

[0137] In some embodiments, cD a comprises (i) a target protein ligand covalently attached to a degrader linker, or (ii) a molecular adhesive moiety. In some embodiments, cD a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0138] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0139] In some embodiments, p is 1, 2, 3, 4, 5, or 6.

[0140] Cereblon degrader linker intermediate The cereblon degrader-linker intermediate (cDLI) is a reagent for a method of producing a cereblon degrader-antibody conjugate (cDAC) by conjugation with a thiol-containing antibody. The cereblon degrader-linker intermediate has a thiol-reactive functional group (X). The thiol-reactive functional group (X) is covalently attached to the cereblon degrader moiety (cD) by a linker (L3).

[0141] In some embodiments, the cereblon degrading agent antibody conjugate has Formula II X-L3-cD II having the structure wherein X is a thiol-reactive group covalently attached to L3; L3 is a linker covalently linking X and cD; cD is the cereblon degrader moiety covalently bound to L3; cD can be a heterobifunctional bivalent cereblon degrader moiety or a molecular adhesive cereblon degrader moiety.

[0142] In some embodiments, L3 comprises an immolator moiety. 3a -IM, where IM is the immolator part, and L 3a is an optional remainder of the L3 linker.

[0143] In some embodiments, L3 is an immolator moiety, IM.

[0144] In some embodiments, the cD comprises the cereblon-binding E3 ubiquitin ligase ligand E3UL. In some embodiments, the cD comprises E3UL-cD a E3UL is the cereblon-binding, E3 ubiquitin ligase ligand and the cD of the cereblon degrader moiety. a is any remainder of cD. In some embodiments, cD a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0145] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0146] An exemplary embodiment of a cDLI is represented by formula II' XL 3a -IM-E3UL-cD a II' It has the following structure.

[0147] In some embodiments, the cereblon degrader portion of the cDLI (cD) is linked to the antibody linker (L3) via an amino group.

[0148] In some embodiments, L3 is: [ka] and [ka] and an immolator moiety IM selected from * indicates L3 linker, L 3a represents the bond to any remainder of ** indicates CD a indicates the point of attachment to The wavy line indicates the point of attachment to E3UL.

[0149] An exemplary embodiment of a cDLI has the structure of Formula II-A: [ka] During the ceremony, X is a thiol-reactive group; L 3a is the linker; Ring A is C6-C 20 Aryl, C3-C 20 Carbocyclyl, C3-C 20 Heterocyclyl, and C3-C 20 heteroaryl; Dashed line [ka] indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a is selected from Z 2 is C(R 2 )2, CR 2 , N, and NR 2a is selected from R 1 and R 2 are each independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NRa R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are each independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; or (i) Two geminal R 1 Or two geminal R 2 forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii)R 1 and R 2 , R 1a and R 2 , R 1 and R 2a , or R 1a and R 2a form a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R a and R b are each independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are each independently selected from F, Cl, —CN, C1-C6 alkyl, 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl; Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -C H(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -C H(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N optionally substituted with one or more groups selected from HC(=NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H; CD a is the remainder of the cereblon degrader moiety; p is an integer of 1 to 14.

[0150] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are H, respectively.

[0151] In some embodiments, ring A is C3-C 20 It is heteroaryl.

[0152] In some embodiments, ring A is an isoindoline substituted with ═O.

[0153] In some embodiments, cD a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0154] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0155] In some embodiments, the cDLI of formula II has the structure of formula II-A': [ka] During the ceremony, X is a thiol-reactive group; L 3a is the linker; X 1 is selected from CH2 and C(=O).

[0156] In some embodiments, L 3a is a protease-cleavable non-peptide linker.

[0157] In some embodiments, XL 3a has the structure of formula L3-A, [ka] During the ceremony, R 1 is C1-C 12 Alkylene, C1-C 12 Alkylene-C(=O), C1-C 12 Alkylene-NH, (CH2CH2O) r , C1-C 12 Alkylene-NH, (CH2CH2O) r-C(=O), (CH2CH2O) r -C(=O), (CH2CH2O) r -CH2, and C1-C 12 alkylene-NHC(=O)CHCH(thiophen-3-yl), r is an integer ranging from 1 to 10; C1-C 12 The alkylene may be substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; R 2 and R 3 together form a C3-C7 cycloalkyl ring optionally substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; AA is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

[0158] In some embodiments, AA is selected from H, —CH3, —CH2(C6H5), —CH2CH2CH2CH2NH2, —CH2CH2CH2NHC(NH)NH2, —CH2CH(CH3)2, and —CH2CH2CH2NHC(O)NH2.

[0159] In some embodiments, R 1is a C5 alkylene.

[0160] In some embodiments, R 2 and R 3 taken together form a C4 cycloalkyl ring.

[0161] In some embodiments, AA is —CH 3 or —CH 2 CH 2 CH 2 NHC(O)NH 2 .

[0162] In some embodiments, R 1 is a C5 alkylene; R 2 and R 3 together form a C4 cycloalkyl ring; AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0163] In some embodiments, the cDa comprises (i) a target protein ligand covalently attached to a degrader linker, or (ii) a molecular adhesive moiety. a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0164] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0165] An exemplary embodiment of a cDLI has the structure of formula II-B: [ka] During the ceremony, X is a thiol-reactive group; L 3a is the linker; Ring A is C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, and C1-C 20 is heteroaryl; Dashed line [ka] indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a is selected from Z 2 is C(R 2 )2, CR 2 , N, and NR 2a is selected from R 1 and R 2 are each independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)Ra , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a or selected from the group consisting of -S(O)H; (i) Two geminal R 1 Or two geminal R 2forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii)R 1 and R 2 , R 1a and R 2 , R 1 and R 2a Or R 1a and R 2a forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are independently selected from F, Cl, —CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl, Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -C H(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -C H(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N optionally substituted with one or more groups selected from HC(=NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H; CD a is the remainder of the cereblon degrader moiety.

[0166] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are H, respectively.

[0167] In some embodiments, ring A is C3-C 20 It is heteroaryl.

[0168] In some embodiments, ring A is an isoindoline substituted with ═O.

[0169] In some embodiments, cD a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0170] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0171] In some embodiments, the cDLI of formula II-B has formula II-B': [ka] The structure of X is a thiol-reactive group; L 3a is the linker; X 1 is selected from CH2 and C(=O).

[0172] In some embodiments, L 3a is a protease-cleavable non-peptide linker.

[0173] In some embodiments, XL 3a is the formula L3-A: [ka] having the structure During the ceremony, R 1 is C1-C 12 Alkylene, C1-C 12 Alkylene-C(=O), C1-C 12 Alkylene-NH, (CH2CH2O) r , C1-C 12 Alkylene-NH, (CH2CH2O) r-C(=O), (CH2CH2O) r -C(=O), (CH2CH2O) r -CH2, and C1-C 12 alkylene-NHC(=O)CHCH(thiophen-3-yl), r is an integer ranging from 1 to 10; C1-C 12 The alkylene may be substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; R 2 and R 3 together form a C3-C7 cycloalkyl ring optionally substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; AA is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

[0174] In some embodiments, AA is selected from H, —CH3, —CH2(C6H5), —CH2CH2CH2CH2NH2, —CH2CH2CH2NHC(NH)NH2, —CH2CH(CH3)2, and —CH2CH2CH2NHC(O)NH2.

[0175] In some embodiments, R 1is a C5 alkylene.

[0176] In some embodiments, R 2 and R 3 taken together form a C4 cycloalkyl ring.

[0177] In some embodiments, AA is —CH 3 or —CH 2 CH 2 CH 2 NHC(O)NH 2 .

[0178] In some embodiments, R 1 is a C5 alkylene; R 2 and R 3 together form a C4 cycloalkyl ring; AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0179] In some embodiments, the cDa comprises (i) a target protein ligand covalently attached to a degrader linker, or (ii) a molecular adhesive moiety. a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0180] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0181] An exemplary embodiment of a cDLI has Formula II″: X-IM-E3UL-cD a II” It has the following structure.

[0182] In some embodiments, the X-IM [ka] Including, ** is CD a The wavy line indicates the point of attachment to E3UL.

[0183] In such embodiments, R 4a , R 4b , R 5a , and R 5a are independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C-C alkyl, wherein the C-C alkyl is optionally substituted independently with one or more groups selected from F, Cl, —CN, —NH, —CHNH, —OH, —OCH, —OCHCH, —OCHCHOCH, —OCHCHOH, —OCHCHN(CH), —OCHF, —OCHF, —OCF, —OP(O)(OH), —S(O)N(CH), —SCH, —S(O)CH, and —S(O)H.

[0184] An exemplary embodiment of a cDLI has formula II-C: [ka] having the structure During the ceremony, R 4a , R 4b , R 5a , and R 5a are each independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C1-C6 alkyl, wherein C1-C6 alkyl is optionally substituted independently with one or more groups selected from F, Cl, —CN, —NH2, —CH2NH2, —OH, —OCH3, —OCH2CH3, —OCH2CH2OCH3, —OCH2CH2OH, —OCH2CH2N(CH3)2, —OCH2F, —OCHF2, —OCF3, —OP(O)(OH)2, —S(O)2N(CH3)2, —SCH3, —S(O)2CH3, and —S(O)3H; Ring A is C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, and C1-C 20 is heteroaryl; Dashed line [ka] indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a is selected from Z 2 is C(R 2 )2, CR 2 , N, and NR 2a is selected from R 1 and R 2 are each independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b, -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a, -S(O)NR a or selected from the group consisting of -S(O)H; (i) Two geminal R 1 Or two geminal R 2 forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii)R 1 and R 2 , R 1a and R 2 , R 1 and R 2a Or R 1a and R 2a forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are independently selected from F, Cl, —CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl, Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -C H(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -C H(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N optionally substituted with one or more groups selected from HC(=NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H; CD a is the remainder of the cereblon degrader moiety.

[0185] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are H, respectively.

[0186] In some embodiments, ring A is C3-C 20 It is heteroaryl.

[0187] In some embodiments, ring A is isoindoline substituted with ═O or oxo.

[0188] In some embodiments, cD a is TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0189] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0190] In some embodiments, the cDLI of formula II-C has formula II-C': [ka] The structure of During the ceremony, R 4a , R 4b , R 5a , and R 5a are each independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C1-C6 alkyl, wherein C1-C6 alkyl is optionally substituted independently with one or more groups selected from F, Cl, —CN, —NH2, —CH2NH2, —OH, —OCH3, —OCH2CH3, —OCH2CH2OCH3, —OCH2CH2OH, —OCH2CH2N(CH3)2, —OCH2F, —OCHF2, —OCF3, —OP(O)(OH)2, —S(O)2N(CH3)2, —SCH3, —S(O)2CH3, and —S(O)3H; Ring A is C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, and C1-C 20is heteroaryl; Dashed line [ka] indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a is selected from Z 2 is C(R 2 )2, CR 2 , N, and NR 2a is selected from R 1 and R 2 are each independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -ORa , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a or selected from the group consisting of -S(O)H; (i) Two geminal R 1 Or two geminal R 2forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii)R 1 and R 2 , R 1a and R 2 , R 1 and R 2a Or R 1a and R 2a forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are independently selected from F, Cl, —CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl, Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -C H(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -C H(CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N optionally substituted with one or more groups selected from HC(=NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H; X 1 is selected from CH2 and C(=O); CD a is the remainder of the cereblon degrader moiety.

[0191] In some embodiments, the cDa comprises (i) a target protein ligand covalently attached to a degrader linker, or (ii) a molecular adhesive moiety. ais TPL-L2-, where TPL is the target protein ligand and L2 is the degrader linker.

[0192] In some embodiments, the TPL comprises a ligand that binds to BRD4.

[0193] Celeron binding, E3 ubiquitin ligase ligand Cereblon-binding E3 ubiquitin ligase ligand (E3UL) is the moiety of the E3 ubiquitin ligase complex that binds to cereblon.

[0194] In some embodiments, E3UL comprises a glutarimide group.

[0195] In some embodiments, the cereblon degrader portion (cD) of the antibody conjugate (cDAC) has a structure selected from the following formulas: [ka] and [ka] wherein the wavy line indicates the point of attachment to the antibody linker L1 of Formula I or the linker L3 of Formula II, and the dashed line indicates an optional double bond; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a Selected from; Z 2 is C(R 2 )2, CR 2 , N, and NR 2a Selected from; R is selected from H and C1-C6 alkyl; R 1 and R 2 are independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl, C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; or (i) two geminal R 1 Or two geminal R 2 forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii) R 1 and R 2 , R 1a and R 2 , R 1 and R 2a , or R 1a and R 2a forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl, or heteroaryl group; R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are independently selected from F, Cl, —CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl, A is C6-C 20Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, and C1-C 20 heteroaryl; Alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl are independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH (OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH (CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO 2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -N and optionally substituted with one or more groups selected from HC(=NH)NH2, -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0196] In some embodiments, Z 1 and Z 2 are CR 1 and R 1 and R 2 are H respectively.

[0197] In some embodiments, R is H.

[0198] In some embodiments, A is C6-C 20 It is aryl.

[0199] In some embodiments, Z 1 and Z 2 are CR 1 and R 1 and R 2 are H, respectively; R is H; A is C6-C 20 It is aryl.

[0200] In some embodiments, the E3UL of the cereblon degrader portion (cD) of the antibody conjugate (cDAC) has the following formula: [ka] and [ka] having a structure selected from In the formula, X 1 is selected from CH2 and C(=O); the wavy lines indicate the points of attachment of the antibody linker L1 of Formula I, the linker L3 of Formula II and / or the degradation linker L2 of the cereblon degradation moiety cD.

[0201] In some embodiments, X 1 is C(=O).

[0202] antibody linker The antibody linker (L1) is a bifunctional linker that covalently attaches the antibody (Ab) to the cereblon degrader moiety (cD). The disclosed antibody linkers provide stability for cDAC in the bloodstream and enable efficient cleavage upon internalization into target cells. The specific design of the antibody linker influences aspects of cDAC pharmacology, including drug stability in the circulation, tumor cell permeability, drug-to-antibody ratio (DAR), i.e., the number of payload molecules carried by each antibody, and the extent of bystander effects.

[0203] The disclosed antibody linkers may contain cleavable non-peptidic peptidomimetic units (PMs). PMs do not contain peptides but can be substrates for lysosomal proteases (WO 2015 / 095227; WO 2015 / 095124; WO 2015 / 095223). For example, cyclobutane-1,1-dicarboxamide-containing peptidomimetic linkers are primarily hydrolyzed by cathepsin B, while valine-citrulline dipeptide linkers are not. Antibody-drug conjugates with PM linkers may be as effective and stable in vivo as those with dipeptide linkers (Wei et al., (2018) J. Med. Chem. 61:989-1000).

[0204] In some embodiments, L1 is a protease-cleavable non-peptide linker having the formula: -Str-PM-IM- where Str is a stretcher unit covalently attached to the antibody; PM is a peptidomimetic unit, and IM is an immolator unit covalently attached to the cereblon degrader moiety.

[0205] In some embodiments, Str has the formula: [ka] have During the ceremony, * indicates the point of attachment on the succinimidyl ring to the cysteine ​​thiol of the antibody; R 1 is C1-C12 Alkylene, C1-C 12 Alkylene-C(=O), C1-C 12 Alkylene-NH, (CH2CH2O) r , C1-C 12 Alkylene-NH, (CH2CH2O) r -C(=O), (CH2CH2O) r -C(=O), (CH2CH2O) r -CH2, and C1-C 12 alkylene-NHC(=O)CHCH(thiophen-3-yl), r is an integer ranging from 1 to 10, C1-C 12 The alkylene may be substituted with one or more groups selected from F, Cl, -CN, -NH, -CHNH, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H.

[0206] In some embodiments, Str is: [ka] [ka] [ka] [ka] is selected from where * indicates the point of attachment to the cysteine ​​thiol of the antibody.

[0207] R 1 In some embodiments, C1-C 12 Alkylene is C1-C5 alkylene.

[0208] In some embodiments, R 1is (CH2)5 or C5 alkylene.

[0209] In one embodiment, PM has the formula: [ka] and In the formula, R 2 and R 3 are taken together to form a C3-C7 cycloalkyl ring optionally substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H; AA is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

[0210] In some embodiments, AA is selected from H, —CH3, —CH2(C6H5), —CH2CH2CH2CH2NH2, —CH2CH2CH2NHC(NH)NH2, —CH2CH(CH3)2, and —CH2CH2CH2NHC(O)NH2.

[0211] In some embodiments, R 2 and R 3 taken together is a C4 cycloalkyl ring; AA is -CH3.

[0212] In some embodiments, R 2 and 3 taken together form a C4 cycloalkyl ring; AA is -CH2CH2CH2NHC(O)NH2.

[0213] In some embodiments, Formula I [ka] and [ka] and an immolator portion selected from where * indicates the point of attachment to the rest of L1 and the wavy line indicates the point of attachment to cD; R 4a , R 4b , R 5a , and R 5a are independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl, optionally substituted with F, Cl, and C1-C6 alkyl; The C1-C6 alkyl may be substituted with one or more groups independently selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0214] In some embodiments, the cereblon degrader portion of cDAC (cD) is linked to the antibody linker (L1) via the amino group of the nitrogen atom of the glutarimide group of cD.

[0215] In some embodiments, the IM-cD of the antibody conjugate (cDAC) is: [ka] and [ka] and wherein the wavy line indicates the point of attachment to the remainder L1 of the linker of the heterobifunctional cD of Formula I or the remainder L3 of the linker of the molecular adhesive cD of Formula II.

[0216] In some embodiments, Z 1 and Z 2 are CR 1 and R 1 and R 2 are H respectively.

[0217] In some embodiments, R is H.

[0218] In some embodiments, A is C6-C 20 It is aryl.

[0219] In some embodiments, Z 1 and Z 2 are CR 1 and R 1 and R 2 are H, respectively; R is H; A is C6-C 20 It is aryl.

[0220] In some embodiments, IM comprises a group selected from 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl)methylcarbamate.

[0221] In some embodiments, L1 forms a disulfide bond with a cysteine ​​thiol of the antibody.

[0222] In some embodiments, Formula I has the formula: [ka] , [ka] , and [ka] is selected from.

[0223] In some embodiments, L1 is a linker having the formula: [ka] During the ceremony, R 4a and R 4b are each independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C1-C6 alkyl, wherein C1-C6 alkyl is optionally substituted independently with one or more groups selected from F, Cl, —CN, —OH, —OCH3, —OCH2CH3, —OCH2CH2OCH3, —OCH2CH2OH, —OCH2CH2N(CH3)2, —OCH2F, —OCHF2, —OCF3, —OP(O)(OH)2, —S(O)2N(CH3)2, —SCH3, —S(O)2CH3, and —S(O)3H; * indicates the point of attachment to the antibody cysteine ​​thiol, and the wavy line indicates the attachment to the cereblon degrader moiety.

[0224] In some embodiments, L1 is a group of the formula: [ka] [ka] and [ka] is selected from During the ceremony, R 4a , R 4b , R 5a , and R 5aare each independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C1-C6 alkyl, wherein C1-C6 alkyl is optionally substituted independently with one or more groups selected from F, Cl, —CN, —OH, —OCH3, —OCH2CH3, —OCH2CH2OCH3, —OCH2CH2OH, —OCH2CH2N(CH3)2, —OCH2F, —OCHF2, —OCF3, —OP(O)(OH)2, —S(O)2N(CH3)2, —SCH3, —S(O)2CH3, and —S(O)3H; R 6 is selected from H and C1-C6 alkyl; The * indicates that the sulfur is attached to the cysteine ​​thiol of the antibody to form a disulfide bond, and the wavy line indicates attachment to the cereblon degrader moiety.

[0225] In some embodiments, R 4a and R 4b are -CH3, respectively.

[0226] In some embodiments, R 5a and R 5b are H, respectively.

[0227] In some embodiments, R 6 is H.

[0228] In some embodiments, R 4a and R 4b are -CH3 and R 5a and R 5b are H and R, respectively. 6 is H.

[0229] Cereblon decomposition agent part In some embodiments, the cD is a bivalent heterobifunctional cD or a molecular adhesive cD. In some embodiments, the cereblon degrader moiety (cD) has the formula: E3UL-cD a and During the ceremony, E3UL is the cereblon-binding E3 ubiquitin ligase ligand; CD a is a molecular adhesive moiety; or CD a is TPL-L2-, TPL is a target protein ligand, L2 is a degradant linker.

[0230] Bivalent cereblon cleavage moiety In some embodiments, the cD comprises a cereblon-binding E3 ubiquitin ligase ligand (E3UL) covalently linked to a target protein ligand (TPL) by a degrader linker (L2) to form a bivalent heterobifunctional cD. In some embodiments, the cereblon degrader moiety (cD) has the formula: TPL-L2-E3UL and (In the formula, TPL is a target protein ligand; E3UL is the cereblon-binding E3 ubiquitin ligase ligand; L2 is a degradant linker; TPL, E3UL, and one of L2 are bound to L1.

[0231] Target protein ligand S Target protein ligands (TPLs) are moieties that bind to proteins of interest for tagging and degradation by the E3 ubiquitin ligase / proteasome system. TPLs are covalently linked to cereblon-bound E3 ubiquitin ligase ligands by a degrader linker.

[0232] An exemplary target protein of cereblon degrader antibody conjugate (cDAC) is BRD4. BRD4 is a member of the bromodomain and extraterminal domain (BET) family and is an attractive target in various pathological conditions, especially cancer, including solid tumors and hematological malignancies. Prostate and AML (acute myeloid leukemia) cell lines show sensitivity to BRD4 inhibition (SELochrin, et al. (2014) Canc. Biol. Ther. 15(12):1583-1585).

[0233] Additional exemplary target proteins for cereblon degrader antibody conjugates (cDACs) include, but are not limited to, GSPT1, BET, BRM (SMARCA2), KRAS, and SHP2 (Wang, C. et al. (2021) Eur J Med Chem. 225:113749).

[0234] In some embodiments, TPL has the following formula: [ka] having the structure During the ceremony, R x is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R y is selected from H and C1-C6 alkyl; The wavy line indicates the point of attachment to L2.

[0235] In some embodiments, the TPL has the structure: [ka] where the wavy line indicates the point of attachment to L2.

[0236] In some embodiments, the TPL has the structure: [ka] where the wavy line indicates the point of attachment to L2.

[0237] An exemplary target protein for cereblon degrader antibody conjugates (cDACs) is the translation termination factor GSPT1 (G1 to S phase transition protein 1 homolog) (Huber, A. et al. (2022) ACS Med. Chem. Lett., 13:1311-1320; Powell, CE et al. (2020) ACS Chem. Biol. 15:2722-2730; Matyskiela, ME (2016) Nature 535(7611):252-257). GSPT1 is upregulated in many cancers, particularly hematopoietic malignancies, and acute leukemia cells have been shown to be highly sensitive to GSPT1 degradation. Therefore, GSPT1 is a potential drug target for future chemotherapy (Matyskiela, ME et al., (2016) Nature 535(7611), 252-7; Surka, C.; et al., (2021) Blood 137(5) 661-677; Takwale, AD et al. (2022) Bioorganic Chemistry 127:105923; Hansen JD, et al. (2021) J Med Chem. 64(4):1835-1843).

[0238] Degradant Linker The degrader linker (L2) is any suitable bifunctional or trifunctional linker unit that covalently binds the target protein ligand (TPL) and the cereblon-associated E3 ubiquitin ligase ligand (E3UL). The degrader linker may be covalently attached to the antibody linker L1 to form a cereblon degrader antibody conjugate (cDAC).

[0239] In some embodiments, L2 is selected from: -N(R')-(C1-C 12 alkyldiyl)-N(R')-, -N(R')-(C2-C 12 alkenyldiyl)-N(R')-, -N(R')-(C2-C 12 alkynyldiyl)-N(R')-, -N(R')-(C1-C12 alkyldiyl)-C(=O)-(N(R')-, -N(R')-(C1-C 12 alkyldiyl)-(N(R')-C(=O)CHO-, -N(R')-(C1-C 12 alkyldiyl)-(N(R')-C(=O)CHN(R)-, -N(R')-(C1-C 12 alkyldiyl)-C(=O)-(N(R)-(C1-C 12 alkyldiyl)-N(R')-, —N(R′)—(C1-C6 alkyldiyl)-O—(C1-C6 alkyldiyl)-N(R′)—, -N(R')-(CH2CH2O) n -N(R')-(CH2CH2O) n wherein n is an integer from 1 to 4; C1-C 12 Alkyldiyl, C2-C 12 alkenyldiyl, and C2-C 12 alkynyldiyl; In the formula, R' is H, C1-C6 alkyldiyl, and L 1への結合点から選択され、 The alkyldiyl, alkenyldiyl, and alkynyldiyl may be substituted with one or more groups selected from F, Cl, -CN, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0240] Bivalent cereblon degrader compounds Exemplary heterobifunctional cereblon degrader compounds were prepared and characterized and are shown in Table 1. [Table 1] TIFF2026502860000064.tif215170

[0241] Molecular adhesive Cereblon decomposer part In some embodiments, the cereblon degrader moiety (cD) of the antibody conjugate (cDAC) is a molecular adhesive cereblon degrader moiety. In such embodiments, the molecular adhesive cereblon degrader moiety (cD) is a molecular adhesive moiety (cD a ) to form molecular adhesive cD.

[0242] In some embodiments, the molecular adhesive cD is [ka] and [ka] and wherein the wavy line indicates the point of attachment to the antibody linker L1 of Formula I or the linker L3 of Formula II; CD a is the molecular adhesive moiety; Dashed lines indicate optional double bonds; Z 1 is C(R 1 )2, CR 1 , N, and NR 1a Selected from; Z 2 is C(R 2 )2, CR 2 , N, and NR 2a Selected from; R 1 and R 2 are independently H, F, Cl, Br, I, -CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl, C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; R 1a and R 2a are independently H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, (C1-C6 alkyldiyl)-(C6-C 20 aryl), -(C1-C6 alkyldiyl)-NR a R b , -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclyl), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclyl), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20Heterocyclyl, C1-C 20 Heteroaryl, -C(=NH)NH(OH), -C(=NH)NH2, -C(=O)NR a R b , -C(=O)NR a -NR a R b , -C(=O)NH(C1-C6 alkyldiyl)-NR a R b , -C(=O)OR a , -NR a R b , -OR a , -S(O)R a , -S(O)2R a , -S(O)NR a and —S(O)H; or (i) two geminal R 1 Or two geminal R 2 forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii) R 1 and R 2 , R 1a and R 2 , R 1 and R 2a , or R 1a and R 2a forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl, or heteroaryl group; R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, and phenyl and benzyl are independently selected from F, Cl, —CN, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C2-C 12 alkynyl, A is C6-C 20 Aryl, C3-C 20 Carbocyclyl, C2-C 20 Heterocyclyl, and C1-C 20 heteroaryl; Alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl are independently F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH( OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH( CH3)CN, -C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C (CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N (CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC( and optionally substituted with one or more groups independently selected from: -NH)NH, -NHC(=O)NH, -NO, =O, -OH, -OCH, -OCHCH, -OCHCHOCH, -OCHCHOH, -OCHCHN(CH), -OCHF, -OCHF, -OCF, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H.

[0243] In some embodiments, Z 1 and Z 2 are CR 1 and R 1 and R 2 are H respectively.

[0244] In some embodiments, R is H.

[0245] In some embodiments, A is C6-C 20 It is aryl.

[0246] In some embodiments, Z 1 and Z 2 are CR 1 and R 1 and R 2 are H, respectively; R is H; A is C6-C 20 It is aryl.

[0247] In some embodiments, the molecular adhesive cD is [ka] and [ka] and During the ceremony, The wavy line indicates the point of attachment to the antibody linker L1 of Formula I or linker L3 of Formula II; CD a is the molecular adhesive moiety; X 1 is selected from CH2 and C(=O).

[0248] In some embodiments, X 1 is C.

[0249] Cereblon degrader linker intermediate The cereblon degrader-linker intermediate (cDLI) is a reagent for a method of producing a cereblon degrader-antibody conjugate (cDAC) by conjugation with a thiol-containing antibody. The cereblon degrader-linker intermediate has a thiol-reactive functional group (X). The thiol-reactive functional group (X) is covalently attached to the cereblon degrader moiety (cD) by a linker (L3).

[0250] In some embodiments, the cereblon degrading agent antibody conjugate has Formula II X-L3-cD II having the structure (In the formula, X is a thiol-reactive group; L3 is: (i) Formula: -Str-PM-Y- wherein Str is a stretcher unit covalently attached to X; PM is a peptidomimetic unit, Y is a spacer unit covalently linked to cD; (ii) Formula: [ka] [ka] and [ka] and Formula (iii): [ka] (where * indicates the point of attachment to X; R 4a , R 4b , R 5a , and R 5a is independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atoms to which they are attached form a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with F, Cl, and C1-C6 alkyl; R 6 is selected from H and C1-C6 alkyl; The wavy line indicates binding to cD; C1-C6 alkyl is a linker selected from, independently, F, Cl, -CN, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H); cD is a compound of the formula: TPL-L2-E3UL 1. A cereblon degrading agent antibody conjugate comprising: (In the formula, TPL is a target protein ligand; E3UL is a cereblon-binding, E3 ubiquitin ligase ligand; L2 is a degradant linker; one of TPL, E3UL, or L2 is bound to L1, or cD is a molecular glue.

[0251] In some embodiments, the bond of L3 to cD comprises a carbamate (-OC(O)NH-) or methylcarbamate (-OC(O)NHCH2-) group.

[0252] In some embodiments, X is selected from the group consisting of maleimide, bromoacetamide, toluenesulfonyl sulfide, and 2-pyridyl disulfide, wherein the pyridyl is optionally substituted with one or two nitro groups.

[0253] In some embodiments, the cereblon degrader-linker intermediate has the formula: [ka] and wherein IM comprises a group selected from 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl)methylcarbamate; AA is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

[0254] In some embodiments, AA is selected from H, —CH3, —CH2(C6H5), —CH2CH2CH2CH2NH2, —CH2CH2CH2NHC(NH)NH2, —CH2CH(CH3)2, and —CH2CH2CH2NHC(O)NH2.

[0255] In some embodiments, AA is —CH 3 or —CH 2 CH 2 CH 2 NHC(O)NH 2 .

[0256] In some embodiments, the cereblon degrader-linker intermediate has the formula: [ka] and [ka] is selected from In the formula, X 1 is selected from CH2 and C(=O).

[0257] In some embodiments, the cereblon degrader-linker intermediate has the formula: [ka] and wherein L3 is a protease-cleavable non-peptide linker having the formula: -Str-PM-IM- Str is a stretcher unit covalently linked to X; PM is a peptidomimetic unit, IM is an immolator unit covalently attached to cD and has the formula: [ka] and the wavy line is the bond to the PM.

[0258] In some embodiments, the cereblon degrader-linker intermediate has the formula: [ka] and wherein L3 is a protease-cleavable non-peptide linker having the formula: -Str-PM-IM- Str is a stretcher unit covalently linked to X; PM is a peptidomimetic unit, IM is an immolator unit covalently attached to L2 of cD and has the following formula: [ka] and the wavy line is the bond to the PM.

[0259] Certain cDLIs in Table 2 were prepared that did not have the required properties of stability, cleavage efficiency, and antibody binding efficiency. [Table 2] TIFF2026502860000081.tif58170

[0260] The sulfonylthio cDLI-1 compound did not react with the antibody under the conditions described in Example 102: 3 and 10 equivalents at pH 8.5 for 3 hours and overnight. Analysis by mass spectrometry (LC / MS) showed neither the expected conjugated product cDAC, nor the opening of the glutarimide ring, nor the addition of water (+18 mass units) nor Tris buffer to the glutarimide ring. The carbamate functional group formed by the glutarimide nitrogen in cDLI-1 is too unstable under these conditions for conjugation.

[0261] The sulfenamide cDLI-2 and bromo-lenalidomide cDLI-3 compounds did not bind to the antibody. The maleimide sulfenamide compounds cDLI-4 and cDLI-5 failed to bind to the cysteine-mutated antibody and were not stable in whole blood.

[0262] Various substituted peptide linkers of cDLI-6 were conjugated to the antibody but were not cleavable in the presence of proteases. The (S,S)valine-alanine and (S,S)valine-citrulline forms of cDLI-6 were tested, as were the phenyl and dimethoxyphenyl forms, and both with and without a methyl group adjacent to the glutarimide nitrogen. All resulted in cDAC that did not cleave to release cereblon degradation moieties or their metabolites.

[0263] In a model study, the nitro group of para-nitrobenzyloxymethyl lenalidomide (cDLI-7) was reduced to an amine. No cleavage of the para-aminobenzyloxy group was observed upon the addition of lenalidomide. In a model study, the disulfide group of para-nitropyridyl disulfide methyl lenalidomide (cDLI-8) was reduced. No cleavage of the disulfide group was observed upon the addition of lenalidomide.

[0264] Sulfonyl-thio cDLI-9, with or without a methyl adjacent to the glutarimide nitrogen, cleaved to release detectable lenalidomide, but was not stable and produced hydrolysis products.

[0265] Both sulfonyl-thio cDLI-9 and cDLI-10 failed to bind to the antibody under different conditions: pH 8.5, 3 and 10 equivalents relative to the antibody, and reaction times ranging from 3 hours to overnight. Hydrolysis of the carbonate bond was monitored by LC / MS.

[0266] In some embodiments, the cereblon degrader-linker intermediate has the following formula: (i) [ka] (In the formula, Rx is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R y is selected from H and C1-C6 alkyl; (ii) [ka] (iii) [ka] (wherein the wavy line indicates the point of attachment to L2) The TPL is selected from:

[0267] Exemplary cereblon degrader-linker intermediates (cDLIs) include: [ka] [ka] and [ka] Selected from R x is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R y is selected from H and C1-C6 alkyl; X 1 is selected from CH2 and C(=O).

[0268] In some embodiments, the cereblon degrader-linker intermediate, L2, is: -N(R)-(C1-C 12 alkyldiyl)-N(R)-, -N(R)-(C2-C 12 alkenyldiyl)-N(R)-, -N(R)-(C2-C 12 alkynyldiyl)-N(R)-, -N(R)-(C1-C 12 alkyldiyl)-C(=O)-(N(R)-, -N(R)-(C1-C 12alkyldiyl)-C(=O)-(N(R)-(C1-C 12 alkyldiyl)-N(R)-, -(C1-C6 alkyldiyl)-O-(C1-C6 alkyldiyl-, C1-C 12 Alkyldiyl, C2-C 12 alkenyldiyl, and C2-C 12 is selected from wherein the alkyldiyl, alkenyldiyl, and alkyldiyl are optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, and R is selected from one or more of H, C1-C6 alkyldiyl, and a point of attachment to L3.

[0269] The cereblon degrader-linker intermediates (cDLIs) in Table 3 were prepared with the required properties of stability, cleavage efficiency, and antibody binding efficiency. Each cDLI in Table 3 was characterized by NMR and shown to have sufficient purity and the correct mass by LC / MS. [Table 3] TIFF2026502860000089.tif203170

[0270] antibody The cereblon degrading agent antibody conjugates (cDACs) provided herein comprise antibodies. Included within the scope of antibody embodiments are functional variants of the antibody constructs and antigen-binding domains described herein.

[0271] The antibody portion of the cDAC can target cells that express the antigen, thereby delivering the antigen-specific cDAC intracellularly to the target cell, typically via endocytosis. cDACs containing antibodies against antigens not found on the cell surface can reduce the specificity of intracellular delivery of the cereblon degrader portion, but the cDACs can still be pinocytosed. The cDACs and methods of use described herein advantageously utilize antibody recognition on the cell surface and / or endocytosis of the cDAC to deliver the cereblon degrader portion intracellularly.

[0272] Trastuzumab, anti-HER2 antibody In certain embodiments, the immunoconjugates (e.g., cDACs) described herein comprise an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody of the cDACs comprises a humanized anti-HER2 antibody, such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8, as described in Table 3 of U.S. Patent No. 5,821,337, which is specifically incorporated herein by reference. These antibodies comprise human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also known as trastuzumab and is commercially available under the trade name HERCEPTIN™ (Genentech, Inc.).

[0273] Trastuzumab (CAS 180288-69-1, HERCEPTIN™, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1 kappa monoclonal antibody that selectively binds to the extracellular domain of HER2 with high affinity (Kd=5 nM) in cell-based assays. It is a humanized form of the murine anti-HER2 antibody (4D5) (U.S. Pat. Nos. 5,677,171, 5,821,337, 6,054,297, 6,165,464, 6,339,142, 6,407,213, 6,639,055, 6,719,971, 6,800,738, 7,074,404; Coussens et al. (1985) Science 230:1132-9; Slamon et al. (1985) Science 230:1132-9). al(1989)Science 244:707-12, Slamon et al(2001)New Engl.J.Med.344:783-792).

[0274] In some embodiments, the antibody construct or antigen-binding domain comprises the CDR regions of trastuzumab. In some embodiments, the anti-HER2 antibody further comprises the framework regions of trastuzumab. In some embodiments, the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.

[0275] 7C2, anti-HER2 antibody Anti-HER2 murine antibody 7C2 binds to an epitope in domain I of HER2. See, for example, PCT Publication No. WO98 / 17797. This epitope is different from the epitope bound by trastuzumab, which binds to domain IV of HER2, and the epitope bound by pertuzumab, which binds to domain II of HER2. By binding to domain IV, trastuzumab disrupts the ligand-independent HER2-HER3 complex, thereby inhibiting downstream signal transduction (e.g., PI3K / AKT). In contrast, binding of pertuzumab to domain II disrupts ligand-driven HER2 interaction with other HER family members (e.g., HER3, HER1, or HER4), thereby also inhibiting downstream signal transduction. The binding of MAb7C2 to domain I does not interfere with the binding of trastuzumab or pertuzumab to domains IV and II, respectively, thereby allowing the possibility of combining MAb7C2 ADCs (antibody-drug conjugates) with trastuzumab, trastuzumab emtansine (T-DM1), and / or pertuzumab. Mouse antibodies 7C2 and 7C2.B9 are described in WO 1998 / 017797. Anti-HER2 7C2 humanized antibodies are disclosed in WO 2016 / 040723.

[0276] In some embodiments, the anti-HER2 antibody of the cDACs described herein comprises the humanized 7C2 anti-HER2 antibody. The humanized 7C2 antibody is an anti-HER2 antibody.

[0277] In some embodiments, the cDACs described herein comprise an anti-HER2 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, 11, or 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8 or 13; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 3; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 4; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

[0278] In some embodiments, the cDACs described herein comprise an anti-HER2 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 3, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 4, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

[0279] In one aspect, the cDACs described herein include antibodies comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, 11, or 12, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8 or 13. In one aspect, the cDACs described herein include antibodies comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

[0280] In a further embodiment, the antibody comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, 11, or 12, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8 or 13. In a further embodiment, the antibody comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

[0281] In another aspect, the cDACs described herein include antibodies comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 3, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 4, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

[0282] In one embodiment, the antibody comprises (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 3, (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 4, and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

[0283] In another aspect, a cDAC described herein comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, 11, or 12, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 8 or 13, and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 3, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 4, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

[0284] In another aspect, a cDAC described herein comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:6, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:7, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:8, and (b) at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:3, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:4, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:5.

[0285] In another aspect, the cDACs described herein include antibodies comprising (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, 11, or 12, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8 or 13, (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 3, (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 4, and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5. In another aspect, the cDACs described herein include antibodies comprising (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8, (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 3, (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 4, and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

[0286] In any of the above embodiments, the anti-HER2 antibody of the antibody-drug conjugate is humanized. In one embodiment, the anti-HER2 antibody of the antibody-drug conjugate comprises an HVR of any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0287] In another aspect, the anti-HER2 antibody of the antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-HER2 antibody comprising that sequence retains the ability to bind to HER2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 2. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 2. In certain embodiments, the substitutions, insertions, or deletions occur within regions outside the HVRs (i.e., within the FRs). Optionally, the anti-HER2 antibody comprises the VH sequence of SEQ ID NO: 2, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

[0288] In another aspect, an anti-HER2 antibody of an antibody-drug conjugate is provided, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 1 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-HER2 antibody comprising that sequence retains the ability to bind to HER2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 1. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 1. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-HER2 antibody comprises the VL sequence of SEQ ID NO: 1, including post-translational modifications of the sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 3, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 4, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

[0289] In another aspect, an antibody-drug conjugate is provided that comprises an anti-HER2 antibody, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above.

[0290] In one embodiment, an antibody-drug conjugate is provided comprising an antibody, wherein the antibody comprises the VH and VL sequences of SEQ ID NO: 2 and SEQ ID NO: 1, respectively, including post-translational modifications of these sequences.

[0291] In one embodiment, an antibody-drug conjugate is provided that comprises an antibody comprising the humanized 7C2.v2.2.LA (hu7C2)K149C kappa light chain sequence of SEQ ID NO: 14.

[0292] In one embodiment, an antibody-drug conjugate is provided comprising an antibody comprising the Hu7C2 A118C IgG1 heavy chain sequence of SEQ ID NO: 15.

[0293] In a further aspect, provided herein is an antibody-drug conjugate comprising an antibody that binds to the same epitope as the anti-HER2 antibodies provided herein. For example, in certain embodiments, provided is an immunoconjugate comprising an antibody that binds to the same epitope as an anti-HER2 antibody comprising the VH sequence of SEQ ID NO: 2 and the VL sequence of SEQ ID NO: 1, respectively.

[0294] In some embodiments, the anti-HER2 antibody of the cDAC according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-HER2 antibody of the immunoconjugate is an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the immunoconjugate comprises a substantially full-length antibody, e.g., an IgG1 antibody, an IgG2a antibody, or an antibody of another antibody class or isotype as defined herein. In some embodiments, the anti-HER2 antibody is a full-length antibody. [table] TIFF2026502860000090.tif255170TIFF2026502860000091.tif39170

[0295] Anti-CD33 antibody The cDAC anti-CD33 antibody 15G15.33 in Tables 4 and 5 contains three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3), SEQ ID NOs: 16-21. HVR-L1 RSSQSLLHSNGYNYLD (SEQ ID NO: 16) HVR-L2 LGVNSVS (SEQ ID NO: 17) HVR-L3 MQALQTPWT (SEQ ID NO: 18) HVR-H1 NHAIS (SEQ ID NO: 19) HVR-H2 GIIPIFGTANYAQKFQG (SEQ ID NO: 20) HVR-H3 EWADVFD (SEQ ID NO: 21)

[0296] The cDAC anti-CD33 antibody 15G15.33 in Tables 4 and 5 comprises the light chain variable region of SEQ ID NO:22 and / or the heavy chain variable region of SEQ ID NO:23. EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGVNSV SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK (SEQ ID NO: 22) QVQLVQSGAEVKKPGSSVKVSCKASGGIFSNHAISWVRQAPGQGLEWMGGIIPIFGTANY AQKFQGRVTITADESTSTAFMELSSLRSEDTAVYYCAREWADVFDIWGQGTMVTVSS (SEQ ID NO: 23)

[0297] The anti-CD33 antibody 9C3 comprises three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3) and three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3), SEQ ID NOs: 24-29, and the following VL and VH sequences, SEQ ID NOs: 30-37. 9C3-HVR L1 RASQGIRNDLG (SEQ ID NO: 24) 9C3-HVR L2 AASSLQS (SEQ ID NO: 25) 9C3-HVR L3 LQHNSYPWT (SEQ ID NO: 26) 9C3-HVR H1 GNYMS (SEQ ID NO: 27) 9C3-HVR H2 LIYSGDSTYYADSVKG (SEQ ID NO: 28) 9C3-HVR H3 DGYYVSDMVV (SEQ ID NO: 29) 9C3 V L DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK (SEQ ID NO: 30) 9C3 V H EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS VKGRFNISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS (SEQ ID NO: 31) 9C3.2V L DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK (SEQ ID NO: 32) 9C3.2V H EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS VKGRFTISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS (SEQ ID NO: 33) 9C3.3V L DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK (SEQ ID NO: 34) 9C3.3V H EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS VKGRFSISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS (SEQ ID NO: 35) 9C3.4V L DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK (SEQ ID NO: 36) 9C3.4V H EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS VKGRFAISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS (SEQ ID NO: 37)

[0298] In some embodiments, the cDACs described herein comprise an anti-CD33 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L13 comprising the amino acid sequence of SEQ ID NO: 26.

[0299] In one aspect, the cDACs described herein comprise an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29. In one embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29. In another embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29 and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In a further embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28. In a further embodiment, the antibody comprises (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29.

[0300] In another aspect, the cDACs described herein comprise at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

[0301] In another embodiment, the anti-CD33 antibody comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 29; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

[0302] In another aspect, the cDACs described herein comprise (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29, (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

[0303] In any of the above embodiments, the anti-CD33 antibody is humanized. In one embodiment, the anti-CD33 antibody comprises an HVR as in any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework. In a specific embodiment, the human acceptor framework comprises a human VLkappa I consensus (VL KI ) framework and / or VH framework VH1. In certain embodiments, the human acceptor framework is a human VLkappaI consensus (VL) framework comprising any one of the following mutations: KI ) framework and / or VH framework VH1.

[0304] In another aspect, an anti-CD33 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, although an anti-CD33 antibody comprising that sequence retains the ability to bind to CD33. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 35, SEQ ID NO: 33, SEQ ID NO: 35, and / or SEQ ID NO: 37. In certain embodiments, the substitutions, insertions, or deletions occur within regions outside the HVRs (i.e., within the FRs). Optionally, the anti-CD33 antibody comprises a VH sequence of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and / or SEQ ID NO: 37, including post-translational modifications of the sequence. In certain embodiments, the VH is selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29.

[0305] In another aspect, anti-CD33 antibodies are provided that comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and / or SEQ ID NO:36. In certain embodiments, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and / or SEQ ID NO:36 contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, although anti-CD33 antibodies comprising such sequences retain the ability to bind to CD33. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and / or SEQ ID NO:36. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and / or SEQ ID NO:36. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-CD33 antibody comprises a VL sequence of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and / or SEQ ID NO:36, including post-translational modifications of the sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:24, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:25, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.

[0306] In another aspect, there is provided an anti-CD33 antibody comprising a VH of any of the embodiments provided above, and a VL of any of the embodiments provided above.

[0307] In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:31 and SEQ ID NO:30, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:33 and SEQ ID NO:32, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:35 and SEQ ID NO:34, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:37 and SEQ ID NO:36, respectively, including post-translational modifications of those sequences.

[0308] In a further aspect, provided herein are antibodies that bind to the same epitope as the anti-CD33 antibodies provided herein. For example, in certain embodiments, provided are antibodies that bind to the same epitope as the anti-CD33 antibodies comprising the VH sequences of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and / or SEQ ID NO:37, and the VL sequences of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and / or SEQ ID NO:36, respectively.

[0309] In some embodiments, the anti-CD33 antibody is a monoclonal antibody, including a human antibody. In one embodiment, the anti-CD33 antibody is an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a substantially full-length antibody, e.g., an IgG1 antibody, an IgG2a antibody, or other antibody class or isotype as defined herein. In some embodiments, the anti-CD33 antibody is a full-length antibody.

[0310] In a further aspect, an anti-CD33 antibody according to any of the above embodiments may incorporate any of the features, alone or in combination, as described below.

[0311] Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "THIOMAB™," in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at sites on the antibody that are available for conjugation. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: Any one or more of K149 (Kabat numbering) of the light chain, V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, A140 (EU numbering) of the heavy chain, L174 (EU numbering) of the heavy chain, Y373 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region may be substituted with cysteine. In certain embodiments, the antibodies described herein comprise an HC-A140C (EU numbering) cysteine ​​substitution. In certain embodiments, the antibodies described herein comprise an LC-K149C (Kabat numbering) cysteine ​​substitution. In certain embodiments, the antibodies described herein comprise an HC-A118C (EU numbering) cysteine ​​substitution. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0312] In certain embodiments, the antibody comprises one of the following heavy chain cysteine ​​substitutions: [table] TIFF2026502860000092.tif86170

[0313] In certain embodiments, the antibody comprises one of the following light chain cysteine ​​substitutions: [table] TIFF2026502860000093.tif41170

[0314] A non-limiting exemplary hu7C2.v2.2.LA light chain (LC)K149CTHIOMAB™ has the heavy and light chain amino acid sequences of SEQ ID NOs: 10 and 14, respectively. A non-limiting exemplary hu7C2.v2.2.LA heavy chain (HC)A118CTHIOMAB™ has the heavy and light chain amino acid sequences of SEQ ID NOs: 15 and 9, respectively.

[0315] antibody target In some embodiments, the antibodies of the cereblon degrading agent antibody conjugates (cDACs) may bind to one or more tumor-associated antigens (TAA), cell surface receptors, and immunospecific antigens, conferring specificity to the targeting of the cereblon degrading agent antibody conjugates and enabling safe systemic delivery of the active drug moiety.

[0316] Certain tumor-associated antigens are known in the art and can be prepared for use in generating antibodies using methods and information well known in the art.In an attempt to discover effective cellular targets for cancer diagnosis and therapy, researchers have attempted to identify transmembrane or other tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal, non-cancerous cells.In many cases, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells.The identification of such tumor-associated cell surface antigen polypeptides allows for greater specificity in targeting cancer cells for destruction by antibody-based therapy.

[0317] Examples of TAAs include, but are not limited to, those listed below, including (1) through (55). For convenience, information related to these antigens (all known in the art) is listed below, including the name, synonyms, GenBank accession number, and primary reference(s), followed by the conventions for identifying the nucleic acid and protein sequences from the National Center for Biotechnology Information (NCBI). Nucleic acid and protein sequences corresponding to the TAAs listed below, including (1) through (55), are available in public databases such as GenBank. TAAs targeted by antibodies include all amino acid sequence variants and isoforms that have at least about 70%, 80%, 85%, 90%, or 95% sequence identity to the sequences identified in the cited references and / or exhibit substantially the same biological properties or characteristics as the TAA having the sequence found in the cited reference. For example, TAA having a variant sequence can generally specifically bind to an antibody that specifically binds to the TAA having the corresponding sequence listed. The sequences and disclosures in the references specifically cited herein are expressly incorporated by reference.

[0318] The sequences and disclosures in the references specifically cited herein are expressly incorporated by reference.

[0319] (1) BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession number NM_001203) ten Dijke, P. et al. Science 264(5155):101-104(1994), Oncogene 14(11):1377-1382(1997)); WO 2004063362 (Claim 2); WO 2003042661 (Claim 12); U.S. Patent Application Publication No. 2003134790 (pages 38-39); WO 2002102235 (Claim 13; page 296); WO 2003055443 (pages 91-92); WO 200299122 (Example 2; pages 528-530); Publication No. 2003029421 (Claim 6); WO 2003024392 (Claim 2; Figure 112); WO 200298358 (Claim 1; page 183); WO 200254940 (pages 100-101); WO 200259377 (pages 349-350); WO 200230268 (Claim 27; page 376); WO 200148204 (Example; Figure 4) NP_001194 Bone morphogenetic protein receptor type B / pid=NP_001194.1 - Cross reference: MIM:603248; NP_001194.1; AY065994.

[0320] (2) E16 (LAT1, SLC7A5, Genbank Accession No. NM_003486) Biochem. Biophys. Res. Commun. 255(2), 283-288(1999), Nature 395(6699):288-291(1998), Gaugitsch, HW et al. (1992) J. Biol. Chem. 267(16):11267-11273; WO 2004048938 (Example 2); WO 2004032842 (Example IV); WO 2003042661 (Claim 12); WO 2003016475 (Claim 1); WO 200278524 (Example 2); WO 2002990 No. 74 (Claim 19; pages 127-129); WO 200286443 (Claim 27; pages 222, 393); WO 2003003906 (Claim 10; page 293); WO 200264798 (Claim 33; pages 93-95); WO 200014228 (Claim 5; pages 133-136); U.S. Patent Application Publication No. 2003224454 (Figure 3); WO 2003025138 (Claim 12; page 150); NP_003477 Solute transporter family 7 (cationic amino acid transporters, y+ system), member 5 / pid=NP_003477.3 - Homo sapiens cross references: MIM:600182;NP_003477.3;NM_015923;NM_003486_1.

[0321] (3) STEAP1 (six-transmembrane epithelial antigen of the prostate, Genbank accession number NM_012449) Cancer Res. 61(15), 5857-5860 (2001), Hubert, RS et al. (1999) Proc. Natl. Acad. Sci. USA 96(25): 14523-14528; WO 2004065577 (claim 6); WO 2004027049 (Figure 1L); EP 1394274 (Example 11); WO 2004016225 (claim 2); WO 2003 042661 (Claim 12); U.S. Patent Application Publication No. 2003157089 (Example 5); U.S. Patent Application Publication No. 2003185830 (Example 5); U.S. Patent Application Publication No. 2003064397 (Figure 2); WO 200289747 (Example 5; pages 618-619); WO 2003022995 (Example 9; Figure 13A, Example 53; page 173, Example 2; Figure 2A); NP_036581 Six-transmembrane epithelial antigen of the prostate Cross references: MIM:604415; NP_036581.1; NM_012449_1.

[0322] (4) 0772P (CA125, MUC16, Genbank Accession No. AF361486) J. Biol. Chem. 276(29):27371-27375 (2001)); WO 2004045553 (Claim 14); WO 200292836 (Claim 6; Figure 12); WO 200283866 (Claim 15; 116-121); U.S. Patent Application Publication No. 2003124140 (Example 16); U.S. Patent No. 798959 Cross-reference: GI:34501467; AAK74120.3; AF361486_1.

[0323] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, GenBank accession number NM_005823) Yamaguchi, N. et al. Biol. Chem. 269(2), 805-808 (1994), Proc. Natl. Acad. Sci. USA 96(20):11531-11536 (1999), Proc. Natl. Acad. Sci. USA 93(1):136-140 (1996), J. Biol. Chem. 27 0(37):21984-21990 (1995)); WO 2003101283 (Claim 14); (WO 2002102235 (Claim 13; pages 287-288); WO 2002101075 (Claim 4; pages 308-309); WO 200271928 (pages 320-321); WO 9410312 (pages 52-57); Cross-references: MIM:601051; NP_005814.2; NM_005823_1.

[0324] (6) Napi2b (Napi3b, NAPI-3B, NPTIIb, SLC34A2, solute transporter family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM_006424) J. Biol. Chem. 277(22):19665-19672(2002), Genomics 62(2):281-284 (1999), Field, JA et al. (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO 2004022778 (Claim 2); EP 1394274 (Example 11); WO 2002102235 (Claim 13; page 326); EP 875569 (Claim 1; pages 17-19); WO 200157188 ​​(Claim 20; page 329); WO 2004032842 (Example IV); WO 200175177 (Claim 24; pages 139-140); Cross-references: MIM:604217; NP_006415.1; NM_006424_1.

[0325] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (Semaphorin) 5B, GenBank accession number AB040878) Nagase T et al. (2000) DNA Res.7(2):143-150); WO 2004000997 (Claim 1); WO 2003003984 (Claim 1); WO 200206339 (Claim 1; page 50); WO 200188133 (Claim 1; pages 41-43, 48-58); WO 2003054152 (Claim 20); WO 2003101400 (Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1.Genew; HGNC: 10737.

[0326] (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, GenBank accession number AY358628); Ross et al. (2002) Cancer Res.62:2546-2553; U.S. Patent Application Publication No. 2003129192 (Claim 2); U.S. Patent Application Publication No. 2004044180 (Claim 12); U.S. Patent Application Publication No. 2004044179 (Claim 11); U.S. Patent Application Publication No. 2003096961 (Claim 11); U.S. Patent Application Publication No. 2003232056 (Example 5); International Publication No. 2003105758 (Claim 12); U.S. Patent Application Publication No. 2003206918 (Example 5); EP1347046 (Claim 1); International Publication No. 2003025148 (Claim 20); Cross-references: GI:37182378; AAQ88991.1; AY358628_1.

[0327] (9)ETBR(Enzyme B strain and Genbank accession number AY275463);Nakamuta M.ら.Biochem.Biophys.Res.Commun.177,34-39,1991;Ogawa Y. Biochem.Biophys.Res.Commun.178,248-255,1991;CloudH.Jpn.Circ.J.56,1303-1307,1992;Cloud H.J.Biol.Chem.268,3463-3470,1993;Sakamoto A.,Yanagisawa M.ら.Biochem.Biophys.Res.Commun.178,656-663,1991;Elshourbagy NAら.J.Biol.Chem.268,3873-3879,1993;Haendler B.ら.J.Cardiovasc.Pharmacol.20,s1-S4,1992;Tsutsumi M.ら.Gene 228,43-49,1999;Strausberg RL.Proc.Natl.Acad.Sci.USA99,16899-16903,2002;Bourgeois C. J. Clin.Endocrinol.Metab.82,3116–3123,1997;Okamoto Y.Biol.Chem.272,21589–21596,1997;Verheij JBら.Am.J.Med.Genet.108,223-225,2002;Hofstra RMWら.Eur.J.Hum.Genet.5,180-185,1997;Puffenberger EGら.Cell 79,1257-1266,1994;Attie T.ら,Hum.Mol.Genet.4,2407-2409,1995;Auricchio A.ら.Hum.Mol.Genet.5:351-354,1996;Amiel J.ら.Hum.Mol.Genet.5,355-357,1996;Hofstra RMWら.Nat.Genet.12,445-447,1996;Svensson PJら.Hum.Genet.103,145-148,1998;Fuchs S.ら.Mol.Med.7,115-124,2001;Pingault V.ら.(2002)Hum.Genet.111,198-206; WO 2004045516 (Claim 1); WO 2004048938 (Example 2); WO 2004040000 (Claim 151); WO 2003087768 (Claim 1); WO 2003016475 (Claim 1); WO 2003016475 (Claim 1); WO 200261087 (Figure 1); WO 2003016494 (Figure 6); WO 2003025138 (Claim 12; page 144); WO 200198351 (Claim 1; pages 124-125); EP 522868 (Claim 8; Figure 2); WO 200177172 (Claim 1; pages 297-299); U.S. Patent Application Publication No. 2003109676; U.S. Patent No. 6,518,404 (Figure 3); U.S. Patent No. 5,773,223 (Claim 1a; columns 31-34); WO 2004001004.

[0328] (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763); WO 2003104275 (Claim 1); WO 2004046342 (Example 2); WO 2003042661 (Claim 12); WO 2003083074 (Claim 14; page 61); WO 2003018621 (Claim 1); WO 2003024392 (Claim 2; Figure 93); WO 200166689 (Example 6); Cross-references: LocusID: 54894; NP_60233.2; NM_017763_1.

[0329] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, GenBank accession number AF455138) Lab. Invest. 82(11):1573-1582 (2002)); WO 2003087306; U.S. Patent Application Publication No. 2003064397 (Claim 1; Figure 1); WO 200272596 (Claim 13; pages 54-55); WO No. 200172962 (Claim 1; Figure 4B); WO 2003104270 (Claim 11); WO 2003104270 (Claim 16); U.S. Patent Application Publication No. 2004005598 (Claim 22); WO 2003042661 (Claim 12); U.S. Patent Application Publication No. 2003060612 (Claim 12; Figure 10); WO 200226822 (Claim 23; Figure 2); WO 200216429 (Claim 12; Figure 10); Cross-references: GI:22655488; AAN04080.1; AF455138_1.

[0330] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636) Xu, XZ et al. Proc. Natl. Acad. Sci. USA 98(19):10692-10697(2001), Cell 109(3):397-407 (2002), J. Biol. Chem. 278(33):30813-30820 (2003)); U.S. Patent Application Publication No. 2003143557 (Claim 4); WO 200040614 (Claim 14; pages 100-103); WO 200210382 (Claim 1; Figure 9A); WO 2003042661 (Claim 12); WO 200230268 (Claim 27; page 391); U.S. Patent Application Publication No. 2003219806 (Claim 4); WO 200162794 (Claim 14; Figures 1A-1D); Cross-references: MIM:606936; NP_060106.2; NM_017636_1.

[0331] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession number NP_003203 or NM_003212) Ciccodicola, A. et al. EMBO J. 8(7):1987-1991 (1989), Am. J. Hum. Genet. 49(3):555-565 (1991)); U.S. Patent Application Publication No. 2003224411 (Claim 1); WO 2003083041 (Example 1); WO 2003034984 (Claim 12); WO 200288170 (Claim 2; pages 52-53); WO No. 2003024392 (Claim 2; Figure 58); WO 200216413 (Claim 1; pages 94-95, 105); WO 200222808 (Claim 2; Figure 1); U.S. Pat. No. 5,854,399 (Example 2; columns 17-18); U.S. Pat. No. 5,792,616 (Figure 2); Cross-references: MIM:187395; NP_003203.1; NM_003212_1.

[0332] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792 Genbank accession number M26004) Fujisaku et al. (1989) J. Biol. Chem. 264(4):2118-2125; Weis JJ et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M. et al. Proc. Natl. Acad. Sci. USA 84, 9194-9198, 1987; Barel M. et al. Mol. Immunol. 35, 1025-1031, 1998; Weis JJ et al. Proc. Natl. Acad. Sci. USA 83, 5639-5643, 1986; Sinha SK et al. (1993) J. Immunol. 150, 5311-5320; WO 2004045520 (Example 4); U.S. Patent Application Publication No. 2004005538 (Example 1); WO 2003062401 (Claim 9); WO 2004045520 (Example 4); WO 9102536 (Figures 9.1 to 9.9); WO 2004020595 (Claim 1); Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

[0333] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related beta), B29, GenBank Accession No. NM_000626 or 11038674) Proc. Natl. Acad. Sci. USA (2003) 100(7):4126-4131, Blood (2002) 100(9):3068-3076, Muller et al. (1992) Eur. J. Immunol. 22(6):1621-1625; International Publication No. 2004016225 (claim 2, figure 140); International Publication No. 2003087768, U.S. Patent Application Publication No. 20041018 No. 74 (Claim 1, page 102); WO 2003062401 (Claim 9); WO 200278524 (Example 2); U.S. Patent Application Publication No. 2002150573 (Claim 5, page 15); U.S. Patent No. 5,644,033; WO 2003048202 (Claim 1, pages 306 and 309); WO 99 / 558658, U.S. Patent No. 6,534,482 (Claim 13, Figure 17A / B); WO 200055351 (Claim 11, pages 1145-1146); Cross-references: MIM:147245; NP_000617.1; NM_000626_1.

[0334] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchor protein 1a SH2 domain containing), SPAP1B, SPAP1C, Genbank accession numbers NM_030764, AY358130) Genome Res. 13(10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99(8):2662-2669(2002), Proc. Natl. Acad. Sci. USA 98(17):9772-9777(2001), Xu, MJ et al. (2001) Biochem. Biophys. Res. Commun. 280(3):768-775; WO 2004016225 (Claim 2); WO 2003077836; WO 200138490 (Claim 5; Figure 18D-1 to Figure 18D-2); WO 2003097803 (Claim 12); WO 2003089624 (Claim 25); Cross-references: MIM:606509; NP_110391.2; NM_030764_1.

[0335] (17) HER2 (ErbB2, GenBank accession number M11730) Coussens L. et al. Science (1985) 230(4730):1132-1139; Yamamoto T. et al. Nature 319, 230-234, 1986; Semba K. et al. Proc. Natl. Acad. Sci. USA 82, 6497-6501, 1985; Swiercz JM et al. J. Cell Biol. 165, 869-880, 2004; Kuhns JJ et al. J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S. et al. Nature 421, 756-760, 2003; Ehsani A. et al. (1993) Genomics 15,426-429; WO 2004048938 (Example 2); WO 2004027049 (Figure 1I); WO 2004009622; WO 2003081210; WO 2003089904 (Claim 9); WO 2003016475 (Claim 1); U.S. Patent Application Publication No. 2003118592; Publication No. 2003008537 (Claim 1); International Publication No. 2003055439 (Claim 29; Figures 1A-B); International Publication No. 2003025228 (Claim 37; Figure 5C); International Publication No. 200222636 (Example 13; pages 95-107); International Publication No. 200212341 (Claim 68; Figure 7); International Publication No. 200213847 (pages 71-74) WO 200214503 (pp. 114-117); WO 200153463 (Claim 2; pp. 41-46); WO 200141787 (p. 15); WO 200044899 (Claim 52; Figure 7); WO 200020579 (Claim 3; Figure 2); U.S. Pat. No. 5,869,445 (Claim 3; columns 31-38); WO 9630514 (Claim 2; pages 56-61); EP1439393 (Claim 7); WO2004043361 (Claim 7); WO2004022709; WO200100244 (Example 3; Figure 4); Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.

[0336] (18) NCA (CEACAM6, Genbank accession number M18728); Barnett T. et al. Genomics 3, 59-66, 1988; Tawaragi Y. et al. Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg RL et al. Proc. Natl. Acad. Sci. USA 99:16899-16903, 2002; WO 2004063709; EP 1439393 (claim 7); WO 2004044178 (example 4); WO 2004031238; WO 2003042661 (claim 12); WO 200278524 (example 2); WO 200286443 (claim 27; page 427); WO 200260317 (claim 2); Accessions: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728.

[0337] (19) MDP (DPEP1, Genbank Accession No. BC017023) Proc. Natl. Acad. Sci. USA 99(26):16899-16903 (2002)); WO 2003016475 (Claim 1); WO 200264798 (Claim 33; pp. 85-87); JP 05003790 (Figures 6-8); WO 9946284 (Figure 9); Cross References: MIM:179780; AAH17023.1; BC017023_1.

[0338] (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971); Clark HF et al. Genome Res. 13, 2265-2270, 2003; Mungall AJ et al. Nature 425, 805-811, 2003; Blumberg H. et al. Cell 104, 9-19, 2001; Dumoutier L. et al. J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J. et al. J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S. et al. (2003) Biochemistry 42:12617-12624; Sheikh F. et al. (2004) J. Immunol. 172, 2006-2010; EP 1394274 (Example 11); U.S. Patent Application Publication No. 2004005320 (Example 5); WO 2003029262 (pages 74-75); WO 2003002717 (claim 2; page 63); WO 200222153 (pages 45-47) ;U.S. Patent Application Publication No. 2002042366 (pages 20-21); WO 200146261 (pages 57-59); WO 200146232 (pages 63-65); WO 9837193 (claim 1; pages 55-59); Deposits: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

[0339] (21) Brevican (BCAN, BEHAB, Genbank Accession No. AF229053) Gary SC et al. Gene 256, 139-147, 2000; Clark HF et al. Genome Res. 13, 2265-2270, 2003; Strausberg RL et al. Proc. Natl. Acad. Sci. USA 99, 16899-16903, 2002; U.S. Patent Application Publication No. 2003186372 (Claim 11); U.S. Patent Application Publication No. 2003186373 (Claim 11); U.S. Patent Application Publication No. 2003119131 (Claim 1; Figure 52); U.S. Patent Application Publication No. 2003119122 (Claim 1; Figure 52); U.S. Patent Application Publication No. 2003119126 (Claim 1); U.S. Patent Application Publication No. 2003119121 (Claim 1; Figure 52); U.S. Patent Application Publication No. 2003119129 (Claim 1); U.S. Patent Application Publication No. 2003119130 (Claim 1); U.S. Patent Application Publication No. 2003119128 (Claim 1; Figure 52); U.S. Patent Application Publication No. 2003119125 (Claim 1); International Publication No. 2003016475 (Claim 1); International Publication No. 200202634 (Claim 1).

[0340] (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442) Chan, J. and Watt, V.M., Oncogene 6(6), 1057-1061 (1991) Oncogene 10(5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000)); WO 2003042661 (Claim 12); WO 200053216 (Claim 1; page 41); WO 2004065576 (Claim 1); WO 2004020583 (Claim 9); WO 2003004529 (pages 128-132); WO 200053216 (Claim 1; page 42); Cross-references: MIM:600997; NP_004433.2; NM_004442_1.

[0341] (23) ASLG659 (B7h, Genbank Accession No. AX092328) U.S. Patent Application Publication No. 20040101899 (Claim 2); WO 2003104399 (Claim 11); WO 2004000221 (Figure 3); U.S. Patent Application Publication No. 2003165504 (Claim 1); U.S. Patent Application Publication No. 2003124140 (Example 2); U.S. Patent Application Publication No. 2003065143 (Figure 60); WO 2002102235 (Claim 13; 299); U.S. Patent Application Publication No. 2003091580 (Example 2); WO 200210187 (Claim 6; Figure 10); WO 200194641 (Claim 12; Figure 7b); WO 200202 No. 624 (claim 13; Figures 1A-1B); U.S. Patent Application Publication No. 2002034749 (claim 54; pages 45-46); WO 200206317 (Example 2; pages 320-321, claim 34; pages 321-322); WO 200271928 (pages 468-469); WO 200202587 (Example 1; Figure 1); WO WO 200140269 (Example 3; pages 190-192); WO 200036107 (Example 2; pages 205-207); WO 2004053079 (Claim 12); WO 2003004989 (Claim 1); WO 200271928 (pages 233-234, 452-453); WO 0116318.

[0342] (24) PSCA (Prostate Stem Cell Antigen Precursor, Genbank Accession Number AJ297436) Reiter RE et al. Proc. Natl. Acad. Sci. USA 95, 1735-1740, 1998; Gu Z. et al. Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; WO 2004022709; EP 1394274 (Example 11); U.S. Patent Application Publication No. 2004018553 (Claim 17); WO 2003008537 (Claim 1); WO 200281646 (Claim 1; page 164); WO 2003003906 (Claim 10; page 288 p.); WO 200140309 (Example 1; Figure 17); U.S. Patent Application Publication No. 2001055751 (Example 1; Figure 1b); WO 200032752 (Claim 18; Figure 1); WO 1998 / 51805 (Claim 17; page 97); WO 1998 / 51824 (Claim 10; page 94); WO 1998 / 40403 (Claim 2; Figure 1B); Accession: O43653; EMBL; AF043498; AAC39607.1.

[0343] (25) GEDA (Genbank accession number AY260763), AAP14954 lipoma HMGIC fusion partner-like protein / pid=AAP14954.1—Homo sapiens species: Homo sapiens (human) WO2003054152 (claim 20), WO2003000842 (claim 1), WO2003023013 (Example 3, claim 20), US2003194704 (claim 45), cross-references: GI:30102449; AAP14954.1; AY260763_1. WO 2003000842 (Claim 1), WO 2003023013 (Example 3, Claim 20), U.S. Patent Application Publication No. 2003194704 (Claim 45), Cross References: GI:30102449; AAP14954.1; AY260763_1.

[0344] (26) BAFF-R (cell-activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456), BAFF receptor / pid=NP_443177.1-Homo sapiens, Thompson, J.S. et al. Science 293(5537), 2108-2111(2001), International Publication No. 2004058309, International Publication No. 2004011611, International Publication No. 2003045422 (Examples, 32-33) 33), WO2003014294 (Claim 35, Figure 6B), WO2003035846 (Claim 70, pages 615-616), WO200294852 (Cols. 136-137), WO200238766 (Claim 3, page 133), WO200224909 (Example 3, Figure 3), Cross References: MIM:606269, NP_443177.1, NM_052945_1, AF132600.

[0345] (27) CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number AK026467); Wilson et al. (1991) J. Exp. Med. 173:137-146; WO 2003072036 (Claim 1; Figure 1); Cross-references: MIM:107266; ​​NP_001762.1; NM_001771_1.

[0346] (28) CD79a (CD79A, CD79α, immunoglobulin-related alpha, B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with Ig M molecules on the surface, transducing signals involved in B cell differentiation), pI: 4.84, molecular weight: 25028 TM: 2 [P] gene chromosome: 19q13.2, GenBank accession number NP_001774.10) WO 2003088808, U.S. Patent Application Publication No. 20030228319; WO 2003062401 (claim 9); U.S. Patent Application Publication No. 2002 No. 150573 (claim 4, pages 13-14); WO 9958658 (claim 13, Figure 16); WO 9207574 (Figure 1); U.S. Pat. No. 5,644,033; Ha et al. (1992) J. Immunol. 148(5):1526-1531; Mueller et al. (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al. (1994) Immunogenetics 40(4):287-295; Preud'homme et al. (1992) Clin. Exp. Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol. 148(2)633-637; Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464.

[0347] (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense and plays a role in HIV-2 infection and possibly the development of AIDS, lymphoma, myeloma, and leukemia); 372aa, pI: 8.54 MW: 41959 TM: 7[P]Gene Chromosome: 11q23.3, Genbank accession number NP_001707.1) WO 2004040000; WO 2004 / 015426; U.S. Patent Application Publication No. 2003105292 (Example 2); U.S. Patent No. 6,555,339 (Example 2); WO 2002 / 61087 (Figure 1); WO 200157188 ​​(claims 20, 269 pages); WO 200172830 (pages 12-13); WO 2000 / 2212 No. 9 (Example 1, pages 152-153, Example 2, pages 254-256); WO 199928468 (Claim 1, page 38); U.S. Pat. No. 5,440,021 (Example 2, columns 49-52); WO 9428931 (pages 56-58); WO 1992 / 17497 (Claim 7, Figure 5); Dobner et al. (1992) Eur. J. Immunol. 22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779.

[0348] (30) HLA-DOB (beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes); 273 aa, pI: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, GenBank accession number NP_002111.1) Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson et al. (1989) Immunogenetics 29(6):411-413; Beck et al. (1992) J. Mol. Biol. 228:433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci. USA 99:16899-16903; Servenius et al. (1987) J. Biol. Chem. 262:8759-8766; Beck et al. (1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) Tissue Antigens 59:512-519; WO 9958658 (claim 13, Figure 15); U.S. Patent No. 6,153,408 (columns 35-38); U.S. Patent No. 5,976,551 (columns 168-170); U.S. Patent No. 6,011,146 (columns 145-146); Kasahara et al. (1989) Immunogenetics 30(1):66-68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111-14119.

[0349] (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and defects may contribute to the pathophysiology of idiopathic detrusor instability; 422 aa), pI: 7.63, MW: 47206 TM: 1 [P] gene chromosome: 17p13.3, GenBank accession number NP_002552.2) Le et al. (1997) FEBS Lett. 418(1-2): 195-199; WO 2004047749; WO 2003072035 (claim 10); Touchman et al. (2000) Genome Res.10:165-173; WO 200222660 (Claim 20); WO 2003093444 (Claim 1); WO 2003087768 (Claim 1); WO 2003029277 (page 82).

[0350] (32) CD72 (B cell differentiation antigen CD72, Lyb-2), pI: 8.66, MW: 40225 TM: 1 [P] gene chromosome: 9p13.3, GenBank accession number NP_1773.1) WO 2004042346 (claim 65); WO 2003 / 026493 (pages 51-52, 57-58); WO 2000 / 75655 (pages 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12): 4870-4877; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99: 16899-16903.

[0351] (33) LY64 (Lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus; 661 aa, pI: 6.20, MW: 74147 TM: 1 [P] gene chromosome: 5q12, GenBank accession number NP_005573.1) U.S. Patent Application Publication No. 2002193567; WO 9707198 (claim 11, pp. 39-42); Miura et al. (1996) Genomics 38(3):299-304; Miura et al. (1998) Blood 92:2815-2822; WO 2003083047; WO 9744452 (claim 8, pages 57-61); WO 200012130 (pages 24-26).

[0352] (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for immunoglobulin Fc domains containing a C2 Ig-type domain and an ITAM domain, may have a role in B lymphocyte differentiation; 429 aa, pI: 5.28, MW: 46925 TM: 1 [P] gene chromosome: 1q21-1q22, GenBank accession number NP_443170.1) WO 2003077836; WO 200138490 (claim 6, Figures 18E-1 to 18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO 2003089624 (claim 8); EP 1347046 (claim 1); WO 2003089624 (claim 7).

[0353] (35) IRTA2 (Immunoglobulin superfamily receptor translocation-associated 2, a putative immune receptor with a potential role in B cell development and lymphopoiesis; deregulation of the gene by translocation occurs in several B cell malignancies); 977aa, pI: 6.88 MW: 106468 TM: 1[P] gene chromosome: 1q21, GenBank accession numbers: Human: AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse: AK089756, AY158090, AY506 558; NP_112571.1. WO 2003024392 (Claim 2, Figure 97); Nakayama et al. (2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO 2003077836; WO 200138490 (Claim 3, Figures 18B-1 to 18B-2).

[0354] (36) TENB2 (related to the EGF / Heregulin family of growth factors and follistatin; TMEFF2, Tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan); 374 aa, NCBI accession numbers: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession numbers AF179274; AY358907, CAF85723, CQ782436 International Publication No. 2004074320; Japanese Patent Application Publication No. 2004113151; International Publication No. 2003042661; International Publication No. 2003009814; EP1295944 (pages 69-70); International Publication No. 200230268 (page 329); International Publication No. 200190304; U.S. Patent Application Publication No. 2004249130; U.S. Patent Application Publication No. 2004022727; International Publication No. 2004063355; U.S. Patent Application Publication No. 2004197325; U.S. Patent Application Publication No. 2003232350; U.S. Patent Application Publication No. 2004005563; U.S. Patent Application Publication No. 2003124579; Horie et al. (2000) Genomics 67:146-152; Uchida et al. (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et al. (2000) Cancer Res. 60:4907-12; Glynne-Jones et al. (2001) Int J Cancer. Oct 15;94(2):178-84.

[0355] (37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, RP et al. (2009) Proc. Natl. Acad. Sci. USA 106(33), 13731-13736; Kummer, MP et al. (2009) J. Biol. Chem. 284(4), 2296-2306.

[0356] (38) TMEFF1 (transmembrane protein 1 with EGF-like domain and two follistatin-like domains; tomoregulin-1); H7365; C9orf2; C9ORF2; U19878; X83961; NM_080655; NM_003692; Harms, PW (2003) Genes Dev. 17(21), 2624-2629; Gery, S. et al. (2003) Oncogene 22(18):2723-2727.

[0357] (39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); U95847; BC014962; NM_145793NM_005264; Kim, MH et al. (2009) Mol. Cell. Biol. 29(8), 2264-2277; Treanor, JJ et al. (1996) Nature 382(6586):80-83.

[0358] (40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen, AG et al. (2003) Int. J. Cancer 103(6), 768-774; Zammit, DJ et al. (2002) Mol. Cell. Biol. 22(3):946-952.

[0359] (41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); NP_001007539.1; NM_001007538.1; Furushima, K. et al. (2007) Dev. Biol. 306(2), 480-492; Clark, H. F. et al. (2003) Genome Res. 13(10):2265-2270.

[0360] (42) Ly6G6D (lymphocyte antigen 6 complex, gene locus G6D; Ly6-D, MEGT1); NP_067079.2; NM_021246.2; Mallya, M. et al. (2002) Genomics 80(1):113-123; Ribas, G. et al. (1999) J. Immunol. 163(1):278-287.

[0361] (43) LGR5 (Leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et al. (2009) Am. J. Epidemiol. 170(5):537-545; Yamamoto, Y. et al. (2003) Hepatology 37(3):528-533.

[0362] (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); NP_066124.1; NM_020975.4; Tsukamoto, H. et al. (2009) Cancer Sci. 100(10):1895-1901; Narita, N. et al. (2009) Oncogene 28(34):3058-3068.

[0363] (45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226); NP_059997.3; NM_017527.3; Ishikawa, N. et al. (2007) Cancer Res. 67(24):11601-11611; de Nooij-van Dalen, A.G. et al. (2003) Int. J. Cancer 103(6):768-774;

[0364] (46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_006134.1; NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105(1-2):162-164; O'Dowd, BF et al. (1996) FEBS Lett. 394(3):325-329;

[0365] (47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12); NP_115940.2; NM_032551.4; Navenot, J.M. et al. (2009) Mol. Pharmacol. 75(6):1300-1306; Hata, K. et al. (2009) Anticancer Res. 29(2):617-623.

[0366] (48) ASPHD1 (Aspartate beta-hydroxylase domain containing 1; LOC253982); NP_859069.2; NM_181718.3; Gerhard, DS et al. (2004) Genome Res. 14(10B):2121-2127;

[0367] (49) Tyrosinase (TYR; OCAIA; OCA1A; Tyrosinase; SHEP3); NP_000363.1; NM_000372.4; Bishop, D.T. et al. (2009) Nat. Genet. 41(8):920-925; Nan, H. et al. (2009) Int. J. Cancer 125(4):909-917;

[0368] (50) TMEM118 (RING finger protein, transmembrane 2, RNFT2, FLJ14627); NP_001103373.1; NM_001109903.1; Clark, HF et al. (2003) Genome Res. 13(10):2265-2270; Scherer, SE et al. (2006) Nature 440(7082):346-351.

[0369] (51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, T.A. et al. (2003) Proc. Natl. Acad. Sci. USA 100(11):6759-6764; Takeda, S. et al. (2002) FEBS Lett. 520(1-3):97-101.

[0370] (52) CD33, a member of the sialic acid-binding immunoglobulin-like lectin family, is a 67-kDa glycosylated transmembrane protein. It is expressed on most myeloid and monocytic leukemia cells, as well as on myelomonocytic and erythroid progenitor cells. It is not found on early pluripotent stem cells, mature granulocytes, lymphocytes, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin. Invest. 75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33 contains two tyrosine residues in its cytoplasmic tail, each followed by a hydrophobic residue similar to the immunoreceptor tyrosine-based inhibition motif (ITIM) found in many inhibitory receptors.

[0371] (53) CLL-1 (CLEC12A, MICL, and DCAL2) encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, including roles in cell adhesion, cell-cell signaling, glycoprotein turnover, and inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternative splice transcript variants of this gene have been described, although the full-length nature of some of these variants has not been determined. This gene is closely linked to other CTL / CTLD superfamily members within the natural killer gene complex region on chromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9(5):585-90; van Rhenen A et al. (2007) Blood 110(7):2659-66; Chen CH et al. (2006) Blood 107(4):1459-67; Marshall AS et al. (2006) Eur. J. Immunol. 36(8):2159-69; Bakker AB et al. (2005) Cancer Res. 64(22):8443-50; Marshall AS et al. (2004) J. Biol. Chem. 279(15):14792-802). CLL-1 has been shown to be a type II transmembrane receptor containing a single C-type lectin-like domain (not predicted to bind either calcium or sugars), a stalk region, a transmembrane domain, and a short cytoplasmic tail containing ITIM motifs.

[0372] (54) TROP2 (tumor-associated calcium signal transduction 2) is a transmembrane glycoprotein encoded by the TACSTD2 gene (Linnenbach AJ, et al. (1993) Mol Cell Biol. 13(3):1507-15; Calabrese G, et al. (2001) Cytogenet Cell Genet. 92(1-2):164-5). TROP2 is an intracellular calcium signal transduction protein differentially expressed in many cancers. Trop2 signals cells for self-renewal, proliferation, invasion, and survival. It has stem cell-like properties. Although TROP2 is expressed in many normal tissues, in contrast, it is overexpressed in many cancers (Ohmachi T, et al., (2006) Clin. Cancer Res., 12(10), 3057-3063; Muhlmann G, et al., (2009) J. Clin. Pathol., 62(2), 152-158; Fong D, et al., (2008) Br. J. Cancer, 99(8), 1290-1295; Fong D, et al., (2008) Mod. Pathol., 21(2), 186-191; Ning S, et al., (2013) Neurol. Sci., 34(10), 1745-1750). TROP2 overexpression is prognostically significant. Several ligands that interact with TROP2 have been proposed. TROP2 signals cells through various pathways and is transcriptionally regulated by a complex network of several transcription factors.

[0373] Human TROP2 (TACSTD2: tumor-associated calcium signaling substance 2, GA733-1, EGP-1, M1S1; hereafter referred to as hTROP2) is a 323-amino acid single-pass transmembrane type 1 plasma membrane protein. The existence of a plasma membrane protein involved in immune resistance common to human trophoblasts and cancer cells has long been suggested (Faulk WP, et al. (1978), Proc. Natl. Acad. Sci. 75(4):1947-1951). Furthermore, an antigenic molecule recognized by a monoclonal antibody targeting a plasma membrane protein of a human choriocarcinoma cell line was identified and named TROP2 as one of the molecules expressed in human trophoblasts (Lipinski M, et al. (1981), Proc. Natl. Acad. Sci. 78(8), 5147-5150). This molecule was identified as tumor antigen GA733-1, recognized by the mouse monoclonal antibody GA733 obtained by immunization with gastric cancer cell lines or non-small cell lung cancer cells (Linnenbach AJ, et al., (1989) Proc. Natl. Acad. Sci. 86(1), 27-31), and as epithelial glycoprotein EGP-1, recognized by the mouse monoclonal antibody RS7-3G11 obtained by immunization with non-small cell lung cancer cells (Basu A, et al., Int. J. Cancer, 62(4), 472-479(1995)). However, in 1995, the cloning of the TROP2 gene confirmed that these molecules were all identical (Fornaro M, et al., (1995) Int. J. Cancer, 62(5), 610-618). The DNA and amino acid sequences of hTROP2 are available in public databases and may be referenced, for example, under accession numbers NM_002353 and NP_002344 (NCBI).

[0374] In response to this information suggesting a relationship with cancer, several anti-hTROP2 antibodies have been established and their anti-tumor effects have been studied.Among these antibodies, for example, in nude mouse xenograft models, non-conjugated antibodies that show anti-tumor activity by themselves (WO2008 / 144891; WO2011 / 145744; WO2011 / 155579; WO2013 / 077458) and antibodies that show anti-tumor activity as antibody-drug conjugates (ADCs) combined with cytotoxic drugs (WO2003 / 074566; WO2011 / 068845; WO2013 / 068946; US Patent No. 7999083) have been disclosed.However, the strength or coverage of their activity is still insufficient, and the medical needs for hTROP2 as a therapeutic target are unmet.

[0375] Expression of TROP2 in cancer cells correlates with drug resistance. Several strategies target TROP2 on cancer cells, including antibodies, antibody fusion proteins, chemical inhibitors, and nanoparticles. In vitro and preclinical studies using these various therapeutic treatments have resulted in significant inhibition of tumor cell growth both in vitro and in vivo in mice. Clinical trials are investigating the potential application of Trop2 both as a prognostic biomarker and as a therapeutic target for reversing resistance.

[0376] (55) CD123 (IL-4, IL3RA, IL3ry, IL3RAY, interleukin-3 receptor) is a protein found on cells that helps transduce signals from interleukin-3, a soluble cytokine important in the immune system. The gene encoding the receptor is located in the pseudoautosomal region of the X and Y chromosomes. The receptor belongs to the type I cytokine receptor family and is a heterodimer with a unique alpha chain paired with a common beta (beta c or CD131) subunit. The gene for the alpha subunit is 40 kilobases long and contains 12 exons. CD123 is the 70 kD transmembrane alpha chain of the IL-3 receptor. Alone, CD123 binds IL-3 with low affinity; when CD123 associates with CDw131 (the common beta chain), it binds IL-3 with high affinity. CD123 does not transduce intracellular signals upon binding to IL-3; it requires the beta chain for this function. CD123 serves as a diagnostic, prognostic, and therapeutic marker in several hematological malignancies. In particular, co-expression of CD123 and TCF4 by immunohistochemistry in acute leukemia is highly specific and sensitive for blastic plasmacytoid dendritic cell neoplasms (BPDCN) (Sun Q, et al. (1996) Blood 87:83; Herling M, et al. (2003) Blood 101:5007; Charles N, et al. (2010) Nat. Med. 16:701; Martin-Gayo E, et al. (2010) Blood 115:5366; Testa U, et al. (2019) Cancers 11(9):1358-1388; Shi M, et al. (2019) Cardiovascular Hematol Disord Drug Targets 19(3):195-204).

[0377] Exemplary embodiments of antibody targets are HER2 and CD33.

[0378] In some embodiments, the antibody has a free cysteine ​​thiol group available for conjugation with an electrophilic group of a cereblon degrader-linker intermediate (cDLI).

[0379] Thiol-containing antibodies can have naturally occurring cysteine ​​thiols or reduced intrachain or interchain disulfide amino acid residues.

[0380] The thiol-containing antibody may be a cysteine-engineered antibody in which one or more cysteine ​​residues have been introduced by mutagenesis according to known techniques, where the engineered cysteines provide for site-specific attachment of a cereblon degrader linker intermediate via cysteine ​​substitution at available sites for attachment without perturbing immunoglobulin folding and assembly or altering antigen binding and effector function (Junutula, et al. (2008) Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; Shen, B. et al. (2012) Nat. Biotechnol. 30(2):184-189; Sukumaran et al. (2015) Pharm. Res 32:1884-1893, U.S. Patent No. 7,521,541; U.S. Patent No. 7,723,485; U.S. Patent Application Publication No. 2012 / 0121615; WO 2009 / 052249). Cysteine ​​engineered antibodies can have one, two, three, or more cysteine ​​amino acids introduced into them.

[0381] cDACs can be formed by conjugating one or more antibody cysteine ​​thiol groups to a molar excess of the cereblon degrader-linker intermediate (cDLI) of Formula II. Due to their symmetrical structure, cysteine-engineered IgG antibodies can conjugate up to two cDLIs with each mutated cysteine ​​site. For example, a cysteine-engineered antibody with one mutated cysteine ​​site can conjugate up to two cDLIs, giving a theoretical maximum DAR of 2. A cysteine-engineered antibody with two mutated cysteine ​​sites can conjugate up to four cDLIs, giving a theoretical maximum DAR of 4. A cysteine-engineered antibody with three mutated cysteine ​​sites can conjugate up to six cDLIs, giving a theoretical maximum DAR of 6.

[0382] Unlike most amines, which become protonated near pH 7 and have low nucleophilicity, cysteine ​​thiols are reactive nucleophiles at neutral pH. Because free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine ​​residues often exist in their oxidized form as disulfide-linked oligomers or internally crosslinked disulfide groups. Antibody cysteine ​​thiol groups are generally more reactive toward electrophilic conjugation reagents, i.e., more nucleophilic, than antibody amine or hydroxyl groups. Manipulating cysteine ​​thiol groups by mutating various amino acid residues in proteins to cysteine ​​amino acids can be potentially problematic, especially for unpaired (free Cys) residues or residues that are more prone to reaction or oxidation. In concentrated solutions of proteins, whether in the periplasm of E. coli, in culture supernatant, or in partially or fully purified proteins, unpaired Cys residues on the surface of proteins can pair and oxidize to form intermolecular disulfides, thus forming protein dimers or multimers. The formation of disulfide dimers renders the new Cys unreactive for conjugation to drugs, ligands, or other labels. Furthermore, if a protein forms an intermolecular disulfide bond between a newly engineered Cys residue and an existing Cys residue through oxidation, neither Cys group will be available for active site participation and interaction. Furthermore, proteins can become inactive or nonspecific due to misfolding or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem 311:1-9).

[0383] In some embodiments, a cysteine ​​engineered antibody may have a reactive cysteine ​​thiol residue introduced at a site on the light chain such as lysine 149 (LCK149C) or at a site on the heavy chain such as serine 122 (HCS122C), as numbered by Kabat numbering. In other embodiments, a cysteine ​​engineered antibody has a cysteine ​​residue introduced at alanine 118 (EU numbering) of the heavy chain (HCA118C). This mutation site is alternatively numbered 121 in sequential numbering, or 114 in Kabat numbering. In other embodiments, a cysteine ​​engineered antibody has a mutated cysteine ​​residue introduced at: (i) a light chain of G64C, R142C, K188C, L201C, T129C, S114C, V205C, or E105C according to Kabat numbering; (ii) a heavy chain of D101C, A140C, L177C, V184C, T205C, or S122C according to Kabat numbering; or (iii) other cysteine-mutated antibodies as described in Bhakta, S. et al., (2013) "Engineering THIOMABs for Site-Specific Conjugation of Thiol-Reactive Linkers", Laurent Ducry (ed.), Antibody-Drug Conjugates, Methods in Molecular Biology, vol. 1045, pages 189-203; WO 2011 / 156328; U.S. Patent Application Publication No. 9000130.

[0384] In other embodiments, the cysteine ​​engineered antibody comprises one or more cysteine ​​mutations selected from HCA118C, LCK149C, HCA140C, LCV205C, LCS121C, HCL174C, HCL177C, HCY373C.

[0385] In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if multiple polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used therapeutically under defined conditions.

[0386] In some embodiments, the cDAC is a mixture of cereblon degrading agent antibody conjugate compounds, and the average drug loading per antibody in the mixture of cereblon degrading agent antibody conjugate compounds is from about 2 to about 6.

[0387] The present disclosure includes all reasonable combinations and permutations of features of the embodiments of Formulas I-II.

[0388] Drug loading, represented by p, is the number of cereblon degrader moieties (CD) per antibody (Ab) in the cereblon degrader-antibody conjugate (cDAC) of Formula I. Loading (p) can range from 1 to about 8 CD moieties per antibody. cDACs of Formula I include mixtures or populations of antibodies conjugated with 1 to about 8 CD moieties. In some embodiments, the number of CD moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues, such as lysine and cysteine. In some embodiments, a free cysteine ​​residue is introduced into the antibody amino acid sequence by the methods described herein. In such embodiments, p can be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges thereof (e.g., 1 to 8 or 2 to 6). Exemplary cDACs of Formula I include, but are not limited to, antibodies with one, two, three, or four engineered cysteine ​​amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine ​​residues already exist in the antibody that form intrachain and interchain disulfide bonds (native disulfide groups) without engineering, in which case the existing free, reduced cysteine ​​residues can be used to conjugate the antibody to a drug. In some embodiments, the antibody is exposed to reducing conditions to generate one or more free cysteine ​​residues prior to conjugation of the antibody.

[0389] For some cDACs, p may be limited by the number of binding sites on the antibody.

[0390] For example, when the linkage is a cysteine ​​thiol, as in certain exemplary embodiments described herein, the antibody may have only one or a limited number of cysteine ​​thiol groups, or may have only one or a limited number of sufficiently reactive thiol groups to which a drug may be attached. In other embodiments, one or more lysine amino groups in the antibody may be available and reactive for conjugation with a cereblon degrader-linker intermediate of Formula II. In certain embodiments, higher drug loading, e.g., p greater than 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the average cD loading relative to cDAC ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

[0391] The loading of cDAC (drug / antibody ratio) can be controlled in various ways, for example, by: (i) limiting the molar excess of cereblon degrader-linker intermediate compound relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing denaturation conditions for optimized antibody reactivity.

[0392] It should be understood that when more than one nucleophilic group on an antibody reacts with a drug, the resulting product is a mixture of cDAC compounds with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody (DAR) can be calculated from this mixture by antibody-specific and drug-specific duplicate ELISA antibody assays. Individual cDAC molecules can be identified in a mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography (see, e.g., McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, KJ, et al. "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate", Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, SC, et al. "Controlling the location of drug attachment in antibody-drug conjugates", Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogeneous cDACs having a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0393] In some embodiments, the cereblon degrader portion of cDAC has the formula: TPL-L2-E3UL and (In the formula, TPL is a target protein ligand; E3UL is the cereblon-binding E3 ubiquitin ligase ligand; L2 is a degradant linker; one of TPL, E3UL, or L2 is bound to L1, or The cereblon degraded portion is a molecular glue.

[0394] In one embodiment, TPL has the formula: [ka] wherein R x is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R y is selected from H and C1-C6 alkyl; The wavy line indicates the attachment point of L2.

[0395] In one embodiment, TPL has the formula: [ka] where the wavy line indicates the point of attachment to L2.

[0396] In one embodiment, TPL has the formula: [ka] where the wavy line indicates the point of attachment to L2.

[0397] In some embodiments, TPL targets BRD4, GSPT1, BET, BRM (SMARCA2), KRAS, and SHP2.

[0398] In some embodiments, E3UL comprises a glutarimide group.

[0399] In some embodiments, E3UL is [ka] and [ka] is selected from In the formula, X 1 is selected from CH2 and C(=O); the wavy line indicates the point of attachment of L1 or L2.

[0400] In some embodiments, the cereblon degrading agent antibody conjugate has the formula: [ka] and wherein L1 is a group represented by the formula: -Str-PM-IM- Str is a stretcher unit covalently linked to X; PM is a peptidomimetic unit, IM is an immolator unit covalently attached to the glutarimide group of E3UL and has the following formula: [ka] and the wavy line is the bond to the PM.

[0401] In some embodiments, the cereblon degrading agent antibody conjugate has the formula: [ka] and wherein L1 is a group represented by the formula: -Str-PM-IM- a protease-cleavable non-peptide linker having the formula: Str is a stretcher unit covalently linked to X; PM is a peptidomimetic unit, IM is an immolator unit covalently attached to L2 of cD and has the following formula: [ka] and the wavy line is the bond to the PM.

[0402] In some embodiments, L2 is: -N(R)-(C1-C 12 alkyldiyl)-N(R)-, -N(R)-(C2-C 12 alkenyldiyl)-N(R)-, -N(R)-(C2-C 12 alkynyldiyl)-N(R)-, -N(R)-(C1-C 12 alkyldiyl)-C(=O)-(N(R)-, -N(R)-(C1-C 12 alkyldiyl)-(N(R)-C(=O)CHO-, -N(R)-(C1-C 12 alkyldiyl)-(N(R)-C(=O)CHN(R)-, -N(R)-(C1-C 12 alkyldiyl)-C(=O)-(N(R)-(C1-C 12 alkyldiyl)-N(R)-, -N(R)-(C1-C6 alkyldiyl)-O-(C1-C6 alkyldiyl)-N(R)-, -N(R)-(CH2CH2O) n -N(R)-(CH2CH2O) n wherein n is an integer from 1 to 4; C1-C 12 Alkyldiyl, C2-C 12 alkenyldiyl, and C2-C 12 alkynyldiyl; R is selected from H, C1-C6 alkyldiyl, and a point of attachment to L1; Alkyldiyl, alkenyldiyl, and alkynyldiyl may be substituted with one or more groups selected from F, Cl, -CN, -NH2, -CH2NH2, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0403] In some embodiments, the cDAC of formula I is [ka] [ka] and [ka] is selected from In the formula, R x is selected from F, Cl, Br, and n is 0, 1, 2, or 3; R y is selected from H and C1-C6 alkyl; X 1 is selected from CH2 and C(=O).

[0404] In some embodiments, Formula I has the formula: [ka] [ka] and [ka] Selected from R x is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R y is selected from H and C1-C6 alkyl; X 1 is selected from CH2 and C(=O).

[0405] In some embodiments, the cDAC of formula I is [ka] [ka] [ka] [ka] and [ka] is selected from.

[0406] In some embodiments, p of the cDAC is 1, 2, 3, 4, 5 or 6.

[0407] Table 4 shows exemplary cDACs prepared using the cereblon degrader-linker intermediates in Table 2 and their assay data. [Table 4]

[0408] Biological activity of cDAC Generally, the cytotoxic or cytostatic activity of cereblon degrader-antibody conjugates (cDACs) is measured by exposing mammalian cells bearing a receptor protein, such as HER2, to cDAC antibodies in cell culture medium; the cells are cultured for approximately 6 hours to approximately 5 days, and cell viability is measured. Using cell-based in vitro assays, we measured viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of cDACs, as described herein.

[0409] The in vitro efficacy of cDAC described herein was measured by cell proliferation assay as described in Example 103. The cDAC described herein showed surprising and unexpected efficacy in inhibiting tumor cell proliferation. The efficacy of cDAC correlated with the target antigen expression of cells. The conjugates tested can bind to specific antigens expressed on the surface of cells and cause the death of those cells in vitro.

[0410] The CellTiter-Glo® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, WI) homogeneous assay based on recombinant expression of Coleoptera luciferase (U.S. Pat. Nos. 5,583,024, 5,674,713, and 5,700,670). This cell proliferation assay determines the number of viable cells in culture based on quantification of ATP present, an indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth. 160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay is performed in a 96-well format, making it amenable to automated high-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6:398-404). The homogeneous assay procedure involves adding a single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. Washing of cells, removal of medium, and multiple pipetting steps are not required. This system detects as few as 15 cells / well in 10 minutes after adding and mixing reagents in a 384-well format. Cells can be continuously treated with cDACs, or treated and then separated from cDACs. Generally, cells treated for a short period (i.e., 3 hours) exhibited the same potency as cells treated continuously. The assay can be performed in a 96-well or 384-well format, suitable for automated high-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedure involves adding a single reagent (CellTiter-Glo® Reagent) directly to cultured cells. This results in cell lysis and the generation of a luminescent signal generated by a luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or a CCD camera imaging device. Luminous output is expressed as relative light units (RLU).

[0411] This homogeneous "add-mix-measure" format results in cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. The CellTiter-Glo® Assay generates a "glow-type" luminescent signal produced by a luciferase reaction, which typically has a half-life of greater than 5 hours, depending on the cell type and medium used. Viable cells are reflected in relative luminescence units (RLU). Beetle luciferin, a substrate, is oxidatively decarboxylated by recombinant firefly luciferase, concomitantly converting ATP to AMP and generating photons.

[0412] Cell-based in vitro assays are used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the cADCs of the present invention. Generally, the cytotoxic or cytostatic activity of cADCs is measured by exposing mammalian cells expressing an antigen, such as HER2, ER (estrogen receptor), or CD33 polypeptide, to the cADCs in cell culture medium, culturing the cells for about 6 hours to about 5 days, and measuring cell viability.

[0413] Figure 1 shows the in vitro potency of the anti-proliferative effects of BRD4-cereblon degrader on KPL-4 and SK-BR-3 cells at day 5. Cell viability as a percentage of control is plotted against the concentration (nM) of cereblon degrader compound cD-5 from Table 1. IC for KPL-4 50 The IC against SK-BR03 was 0.65 nM. 50 The α-amyloid activity of the cereblon degrader compounds was 0.41 nM. These results demonstrate significant potency of the cereblon degrader compounds.

[0414] Figure 2A shows the in vitro anti-proliferative effect of HER2+ KPL-4 cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). Figure 2B shows the in vitro anti-proliferative effect of HER2+ SK-BR-3 cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 3. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL).

[0415] Figure 3A shows the in vitro anti-proliferative effect of HER2 low / ER+ CAMA1 cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). Figure 3A shows the in vitro anti-proliferative effect of HER2 low / ER+ EFM19 cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). Table 5 shows that anti-HER2 cDAC is active in both HER2+ and HER2-low breast cancer cell lines, whereas the off-target anti-CD33 cDAC is not. [Table 5]

[0416] Figure 4 shows the in vitro antiproliferative effect of anti-CD33 BRD4-cereblon degrader antibody conjugate cDAC-3 after 7 days of treatment of AML cell lines. The AML cell lines were MV-4-11, EOL-1, Molm-13, Nomo-1, HL-60, and OCI-AML-2. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). Table 6 shows that cDAC-3 is active in inhibiting various AML cell lines. [Table 6]

[0417] Figure 5A shows the in vitro anti-proliferative effects of EOL-1 AML cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). Figure 5B shows the in vitro anti-proliferative effects of HL-60 AML cells after 5 days of treatment with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). Table 6 shows the EC50 values ​​of the cDACs in Figures 5A and 5B.

[0418] Figure 6A shows the in vitro anti-proliferative effect of Molm-13 AML cells after treatment for 3 days with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL). Figure 6B shows the in vitro anti-proliferative effect of MV-4-11 AML cells after treatment for 3 days with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4. Cell viability as a percentage of control is plotted against the concentration of cDAC (μg / mL).

[0419] Table 7 shows EC50 values ​​for the potency of BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-5, and cDAC-6 and their in vitro efficacy in AML cell lines from Figures 5A, 5B, 6A, and 6B. cDAC-3 and cDAC-5 contain a thiohuman anti-CD33 antibody, whereas cDAC-4 and cDAC-6 contain a thiohuman anti-7C2 (HER2) antibody. It can be seen that the targeted cDAC3 and cDAC-5 have a concentration-dependent effect on inhibiting AML cells through the CD33 receptor, while the non-targeted cDAC-4 and cDAC-6 showed lower potency than expected. [Table 7]

[0420] Cell killing assays on AML cells treated with CD33 BRD4-cereblon degrader antibody conjugates cDAC3 and cDAC-5, and 7C2 BRD4-cereblon degrader antibody conjugates cDAC4 and cDAC-6 yielded the IC50 values ​​(ng / ml) shown in Table 8. It can be seen that the targeted cDAC3 and cDAC-5 have a concentration-dependent effect on AML cells bearing the CD33 receptor, while the non-targeted cDAC-4 and cDAC-6 have lower potency than expected. [Table 8]

[0421] The in vitro antiproliferative effects of exemplary cDACs demonstrate that the cDACs described herein are biologically active, comprising a wide variety of antibodies, including those that bind to the tumor-associated antigens and cell surface receptor proteins described herein. The exemplary cDACs in Table 3 include antibodies that bind to the tumor-associated antigens HER2 and CD33. HER2 is highly expressed at levels of several million copies per cell in certain solid tumors, such as breast cancer and gastric cancer. CD33 is expressed at a much lower copy number of approximately 10,000 copies per cell in hematological malignancies, such as leukemia and lymphoma. The recycling and internalization mechanisms differ between HER2 and CD33 cell surface proteins. Therefore, the demonstration of significant in vitro efficacy of exemplary cDACs comprising HER2 and CD33 reasonably suggests that the cDACs described herein comprising antibodies other than anti-HER2 and anti-CD33 are similarly biologically active.

[0422] The in vivo efficacy of cDAC was measured in tumor xenograft studies in mice (Examples 104-105). The cDACs described herein showed surprising and unexpected target- and dose-dependent efficacy in inhibiting tumor growth. The efficacy of cDACs can be correlated with the target antigen expression of tumor cells.

[0423] The efficacy of the cADCs provided herein is measured in vivo by implanting cancer cell allografts or xenografts in rodents and treating the tumors with cADCs. Variable results are expected depending on the cell line, the specificity of the cADC's antibody binding to the receptors present on the cancer cells, the dosing regimen, and other factors. The in vivo efficacy of cADCs was measured using a transgenic explant mouse model expressing moderate to high levels of tumor-associated antigens, including Her2-expressing KPL4 and CD22-expressing BJAB. Subjects may be treated once with cADCs and monitored for 3-6 weeks to measure tumor doubling time, log cell kill, and tumor shrinkage. Follow-up dose-response and multi-dose studies may be performed.

[0424] For example, the in vivo efficacy of the anti-HER2 cDAC described herein can be measured using a high-expressing HER2 transgenic explant mouse model (Phillips et al. (2008) Cancer Res. 68:9280-90). Allografts are grown from Fo5 mmtv transgenic mice that are unresponsive or poorly responsive to HERCEPTIN® (Genentech, Inc.) therapy. Subjects are treated once or more with specific dose levels (mg / kg) of cDAC and a placebo buffer control (vehicle) and monitored for two or more weeks to measure tumor doubling, log cell kill, and time to tumor shrinkage according to Examples 104-105.

[0425] Figure 7 shows the in vivo efficacy of anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 at the following doses in reducing tumor volume over time (21 days) in an HL-60 xenograft mouse model. 1) Vehicle (Histidine Buffer #8), 100 μL, 1 IV 2)cDAC-4, 3mg / kg, IV once 3) cDAC-3, 1 mg / kg IV once 4)cDAC-3, 3mg / kg, IV once 5)cDAC-3, 10mg / kg, IV once 6)cDAC-6, 3mg / kg, IV once 7) cDAC-5, 1 mg / kg IV once 8)cDAC-5, 3mg / kg, once IV

[0426] As shown in Figure 7, the anti-CD33BRD4-cereblon degrader antibody conjugates cDAC-3 and cDAC-5 from Table 4 demonstrated clear dose-dependent activity in inhibiting tumor growth in the HL-60 human leukemia cell line in mice, as performed according to Example 105. At a 1 mg / kg dose, cDAC-5 demonstrated greater efficacy than cDAC-3 (line 7 vs. line 3 in the figure). The amine of the glutarimide group of the cereblon degrader moiety of cDAC-5 is linked to the antibody linker via an amino structure (Table 3, cDLI-5), while the indolinone group of the cereblon degrader moiety of cDAC-3 is linked to the antibody linker via a carbamate group (Table 3, cDLI-1). At a 3 mg / kg dose, tumor volumes were below the limit of quantitation for both cDAC-3 and cDAC-5 (lines 4 and 8, respectively, in Figure 7). In contrast, Figure 7 also shows that tumors from the non-targeted HER2 control cDAC-4 and cDAC-6 in the corresponding 3 mg / kg groups (line 2 and line 6, respectively) had an initial response but ultimately grew by the end of the study (>21 days). This contrast demonstrated the target-specific efficacy of the BRD4-cereblon degrader antibody conjugates provided herein (e.g., cDAC-3 and cDAC-5). The LALA-PG mutation in the Fc domain of anti-CD33 antibodies abolishes Fc-FcR-mediated effector function without affecting the desired affinity (Schlothauer, T. et al. (2016) Protein Engineering, Design & Selection, 29(10):457-466). Furthermore, doses of 3 mg / kg or less of the cereblon degrader antibody conjugates provided herein (e.g., cDAC-3 and cDAC-5) were shown to be tolerable in mice.

[0427] The in vivo and whole blood stability of cDAC can be measured and evaluated according to standard assays, including those in Example 104.

[0428] According to the whole blood assay of Example 104, the stability of cDAC-4 and cDAC-6 was measured in buffer, cynomolgus monkey whole blood, human whole blood, mouse whole blood and rat whole blood.At certain time points, samples were subjected to capture by biotinylated extracellular domain (ECD) of HER2 antigen immobilized on streptavidin magnetic beads.After washing the beads, the samples were eluted and analyzed by LC / MS (liquid chromatography / mass spectrometry).Identification and characterization by mass and LC elution profile allowed the determination of average DAR (drug-to-antibody ratio).Table 9 shows that both cDAC-4 and cDAC-6 were stable in all media at room temperature for 24 hours, and cDAC-4 was slightly more stable than cDAC-6. [Table 9]

[0429] Pharmaceutical Composition In another aspect, provided herein is a composition, e.g., a pharmaceutically or pharmacologically acceptable composition or formulation, comprising a cereblon degrading agent antibody conjugate (cDAC) or multiple cDACs described herein and a pharmaceutically or pharmacologically acceptable carrier.

[0430] cDACs can be formulated for parenteral administration, such as intradermal, subcutaneous (subcutaneous), intramuscular (IM), or intravenous (IV) injection, infusion, or administration into a cavity or lumen of an organ. Alternatively, the cDACs described herein can be injected into specific body sites, such as tumors. Injectable compositions generally contain a solution of cDAC dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be used include water and isotonic solutions of one or more salts, such as sodium chloride, such as Ringer's solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland, fixed oil, including synthetic mono- or diglycerides, can be used. Additionally, fatty acids, such as oleic acid, can also be used in the preparation of injectables. These compositions are desirably sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0431] The composition may contain any suitable concentration of cDAC. The concentration of cDAC in the composition may vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., in accordance with the particular mode of administration selected and the patient's needs. In certain embodiments, the concentration of cDAC in the injectable solution formulation ranges from about 0.1% (w / w) to about 10% (w / w).

[0432] Methods of treating cancer using cereblon degrader-antibody conjugates By providing specificity that induces targeted degradation of tumor-associated proteins and minimizes off-target toxic effects, the cereblon degrader antibody conjugates (cDACs) provided herein may be useful in treating diseases and disorders such as cancer. cDACs transport tumor-associated antigen-binding antibodies to cells that express the antigen and deliver the cereblon degrader (cD) moiety to the target cells. The target protein is ubiquitinated and subsequently degraded.

[0433] Provided herein are methods for treating cancer using pharmaceutical compositions of cereblon-degrading agent antibody conjugates (cDACs) provided herein. The method comprises administering a therapeutically effective amount of an cDAC described herein to a subject in need of treatment, such as a patient with cancer and in need of cancer treatment. The method comprises administering a therapeutically effective amount of a cDAC selected from Table 3.

[0434] In certain embodiments, the disclosed cDACs include those with anti-cancer activity. The cDACs selectively deliver an effective amount of the active form of the cereblon degrader moiety to tumor tissue, thereby achieving greater selectivity (i.e., lower effective doses) while increasing the therapeutic index ("therapeutic window") compared to unconjugated cereblon degrader compounds.

[0435] It is contemplated that the disclosed cDAC can be used to treat various hyperproliferative diseases or disorders, such as those characterized by the overexpression of tumor antigens.Exemplary hyperproliferative disorders include benign or malignant solid tumors and blood diseases such as leukemia and lymphatic tumors.

[0436] Examples of cancers to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia or lymphoid malignancies (including acute myeloid leukemia), squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0437] In another aspect, cDACs are provided for use as pharmaceuticals.In certain embodiments, the cDACs described herein are also provided for use in a method for treating an individual, comprising administering an effective amount of cDACs to the individual.In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, for example, as described herein, to the individual.

[0438] In a further aspect, the present invention also provides a use of a cDAC as described herein in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer, and the method comprises administering an effective amount of the medicament to an individual with cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0439] Administration can be by any suitable route, for example, intravenous or subcutaneous injection, depending on whether administration is brief or chronic. Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time points, bolus administration, and pulse infusion. The dose of cDAC can range from about 5 mg / kg (body weight) to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The dose of cDAC can be about 100, 200, 300, 400, or 500 μg / kg. The dose of cDAC can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The dose of cDAC can also be outside these ranges, depending on the specific conjugate and the type and severity of the cancer or disorder being treated. The dosing frequency can range from a single administration to multiple administrations per week, or more frequently. In some embodiments, cDACs are administered about once a month to about five times every five weeks, hi some embodiments, cDACs are administered once a week.

[0440] The disclosed cDAC can be used alone or in combination with other therapeutic agents in a treatment regimen.cDAC can be administered in a regimen with one or more other drugs, during the same treatment cycle, on the same treatment day as one or more other drugs, and optionally simultaneously with one or more other drugs.For example, in the case of cancer therapy administered every three weeks, each simultaneously administered drug is administered on the first day of each three-week cycle.For example, cDAC can be administered simultaneously with at least one additional therapeutic agent, such as a chemotherapy drug.Such combination therapy includes combined administration (when two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case cDAC can be administered before, simultaneously, and / or after the administration of the additional therapeutic agent.cDAC can also be used in combination with radiation therapy.

[0441] The disclosed cDACs can be useful for treating HER2-positive (HER2+) cancers, including cancer cells with higher than normal levels of HER2. Examples of HER2-positive cancers include HER2-positive breast cancer and HER2-positive gastric cancer. In some cases, HER2-positive cancers have an immunohistochemistry (IHC) score of 2+ or 3+ by in situ hybridization (ISH) amplification ratio. The term "HER2-positive cells" refers to cells that express HER2 on their surface. cDACs can also be useful in treating HER2-low tumor types. [Example]

[0442] Example 1 Synthesis of 1-(5-aminopentyl)-1H-pyrrole-2,5-dione hydrochloride, 1 [ka] Following the procedure of WO 2017 / 214024, which is incorporated herein by reference, maleic anhydride furan-2,5-dione (150 g, 1.53 mol) was added to a stirred solution of 6-aminohexanoic acid (201 g, 1.53 mol) in HOAc (1000 mL). The mixture was stirred at room temperature for 2 hours and then heated to reflux for 8 hours. The organic solvent was removed under reduced pressure, and the residue was extracted with EtOAc (500 mL x 3) and washed with HO. The combined organic layers were dried over NaSO and concentrated to give the crude product, which was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid as a white solid (250 g, 77.4%). DPPA (130 g, 473 mmol) and TEA (47.9 g, 473 mmol) were added to a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (100 g, 473 mmol) in t-BuOH (200 mL). The mixture was heated at reflux under N for 8 hours. The mixture was concentrated, and the residue was purified by column chromatography on silica gel (PE: EtOAc = 3:1) to give tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) pentylcarbamate (13 g, 10%). To a solution of tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamate (28 g, 992 mmol) in anhydrous EtOAc (30 mL) was added dropwise HCl / EtOAc (50 mL). After stirring the mixture at room temperature for 5 h, it was filtered and the solid was dried to give 1-(5-aminopentyl)-1H-pyrrole-2,5-dione hydrochloride 1 (16 g, 73.7%). 1 H NMR (400MHz, DMSO-d6): δ 8.02(s,2H),6.99(s,2H),3.37-3.34(m,2H),2.71-2.64(m,2H),1.56-1.43(m,4H),1.23-1.20(m,2H).

[0443] Example 2 Synthesis of (S)-1-(1-(4-(hydroxymethyl)phenylamino)-1-oxo-5-ureidopentan-2-ylcarbamoyl)cyclobutanecarboxylic acid, 2 [ka] [ka] To a mixture of (S)-2-amino-5-ureidopentanoic acid 2a (17.50 g, 0.10 mol) in a mixture of dioxane and HO (50 mL / 75 mL) was added KCO (34.55 g, 0.25 mol). Fmoc-Cl (30.96 g, 0.12 mol) was added to 0 The mixture was slowly added at °C. The reaction mixture was allowed to warm to room temperature over 2 h. The organic solvent was removed under reduced pressure, and the aqueous slurry was adjusted to pH = 3 with 6 M HCl solution and extracted with EtOAc (100 mL x 3). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoic acid 2b (38.0 g, 95.6%). 2b is commercially available.

[0444] To a solution of 2b (4 g, 10 mmol) in a mixture of DCM and MeOH (100 mL / 50 mL) was added (4-aminophenyl)methanol (1.6 g, 13 mmol, 1.3 equiv.) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, EEDQ, described in Sigma-Aldrich CAS Registry Number 16357-59-8 (3.2 g, 13 mmol, 1.3 equiv.). The mixture was stirred under N2 at room temperature for 16 h, after which it was concentrated to give a brown solid. MTBE (200 mL) was added, and it was stirred at 15 °C for 2 h. The solid was collected by filtration and washed with MTBE (50 mL × 2) to give (S)-(9H-fluoren-9-yl)methyl (1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate 2c as an orange solid (4.2 g, 84%). LCMS (ESI): m / z 503.0 [M+1].

[0445] To a stirred solution of 2c (4.2 g, 8.3 mmol) in dry DMF (20 mL) was added piperidine (1.65 mL, 17 mmol, 2 equiv.) dropwise at room temperature. The mixture was stirred at room temperature for 30 min, forming a solid precipitate. Dry DCM (50 mL) was added, and the mixture immediately became clear. The mixture was stirred at room temperature for an additional 30 min, after which LCMS showed that 10e had been consumed. It was concentrated to dryness under reduced pressure (ensuring no piperazine remained), and the residue was partitioned between EtOAc and HO (50 mL / 20 mL). The aqueous phase was washed with EtOAc (50 mL × 2) and concentrated to give (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide 2d (2.2 g, 94%) as an oily residue (containing a small amount of DMF).

[0446] Commercially available 1,1-cyclobutanedicarboxylic acid, 1,1-diethyl ester (CAS Registry Number 3779-29-1) is converted to the half-acid / ester 1,1-cyclobutanedicarboxylic acid, 1-ethyl ester (CAS Registry Number 54450-84-9) by limited saponification with aqueous base, followed by activation to the NHS ester, 1-(2,5-dioxopyrrolidin-1-yl)1-ethylcyclobutane-1,1-dicarboxylate, using a coupling reagent such as TBTU (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, also known as N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate, CAS Registry Number 125700-67-6, Sigma-Aldrich B-2903) and N-hydroxysuccinimide.

[0447] To a solution of 1-(2,5-dioxopyrrolidin-1-yl)1-ethylcyclobutane-1,1-dicarboxylate (8 g, 29.7 mmol) in DME (50 mL) was added 2d (6.0 g, 21.4 mmol) and NaHCO3 (7.48 g, 89.0 mmol) in water (30 mL). After stirring the mixture at room temperature for 16 h, it was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (DCM:MeOH = 10:1) to give (S)-ethyl 1-((1-(4-(hydroxymethyl)phenyl)-2-oxo-6-ureidohexan-3-yl)carbamoyl)cyclobutanecarboxylate 2e (6.4 g, 68.7%) as a white solid. LCMS (ESI): m / z 435.0 [M+1]

[0448] To a stirred solution of 2e (6.4 g, 14.7 mmol) in a mixture of THF and MeOH (20 mL / 10 mL) was added a solution of LiOH·HO (1.2 g, 28.6 mmol) in HO (20 mL) at room temperature. After stirring the reaction mixture at room temperature for 16 h, the solvent was removed under reduced pressure, and the resulting residue was purified by preparative HPLC to give (S)-1-(1-(4-(hydroxymethyl)phenylamino)-1-oxo-5-ureidopentan-2-ylcarbamoyl)cyclobutanecarboxylic acid 2 (3.5 g, 58.5% yield). LCMS (ESI): m / z 406.9 [M+1]. 1 H NMR(400MHz, methanol-d4)δ 8.86(d,J=8.4Hz,2H),8.51(d,J=8.4Hz,2H),5.88-5.85(m,1H),5.78(s,2H),4.54-4.49(m,3H),4.38-4.32(m,1H) ,3.86-3.75(m,1H),3.84-3.80(m,2H),3.28-3.21(m,1H),3.30-3.24(m,1H),3.00-2.80(m,1H),2.37-2.28(m,2H).

[0449] Example 3 Synthesis of S)—N-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)-N-(1-(4-(hydroxymethyl)phenylamino)-1-oxo-5-ureidopentan-2-yl)cyclobutane-1,1-dicarboxamide, 3 [ka] Diisopropylethylamine, DIPEA (1.59 g, 12.3 mmol), and bis(2-oxo-3-oxazolidinyl)phosphinic chloride, BOP-Cl (CAS Registry Number 68641-49-6, Sigma-Aldrich, 692 mg, 2.71 mmol) were added to a solution of (S)-1-(1-(4-(hydroxymethyl)phenylamino)-1-oxo-5-ureidopentan-2-ylcarbamoyl)cyclobutanecarboxylic acid 2 (1 g, 2.46 mmol) in DMF (10 mL) at 0 °C, followed by the addition of 1-(5-aminopentyl)-1H-pyrrole-2,5-dione hydrochloride 1 (592 mg, 2.71 mmol). The mixture was stirred at 0 °C for 0.5 h. The reaction mixture was quenched with citric acid solution (10 mL) and extracted with DCM / MeOH (10:1). The organic layer was dried and concentrated, and the residue was purified by column chromatography on silica gel (DCM:MeOH=10:1) to give 3 (1.0 g, 71%), also known as MC-CBDK-cit-PAB-OH. LCMS (ESI): M+H + =571.28. 1H NMR(400MHz,DMSO-d6):δ 10.00(s,1H),7.82-7.77(m,2H),7.53(d,J=8.4Hz,2H),7.19(d,J=8.4Hz,2H),6.96(s, 2H),5.95(t,J=6.4Hz,1H),5.39(s,2H),5.08(t,J=5.6Hz,1H),4.40-4.35(m,3H),4.09 (d,J=4.8Hz,1H),3.01(d,J=3.2Hz,2H),3.05-2.72(m,4H),2.68-2.58(m,3H),2.40-2. 36(m,4H),1.72-1.70(m,3H),1.44-1.42(m,1H),1.40-1.23(m,6H),1.21-1.16(m,4H).

[0450] Example 4 Synthesis of (S)—N-(1-(4-(chloromethyl)phenylamino)-1-oxo-5-ureidopentan-2-yl)-N-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)cyclobutane-1,1-dicarboxamide, 4 [ka] A solution of 3 (2.0 g, 3.5 mmol) in N,N-dimethylformamide, DMF or N-methylpyrrolidone, NMP (50 mL) was treated dropwise with thionyl chloride, SOCl (1.25 g, 10.5 mmol) at 0 °C in several portions. The reaction remained yellow. The reaction was monitored by LC / MS, which showed >90% conversion. After the reaction mixture was stirred at 20 °C for 30 min or for several hours, it was diluted with water (50 mL) and extracted with EtOAc (50 mL × 3). The organic layer was dried, concentrated, and purified by flash column (DCM:MeOH = 20:1) to form 4, also known as MC-CBDK-cit-PAB-Cl, as a gray solid. LCMS: (5-95, AB, 1.5 min), 0.696 min, m / z = 589.0 [M+1] + .

[0451] Example 5 Synthesis of (S)-4-(2-(1-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamoyl)cyclobutanecarboxamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate, 5 [ka] To a solution of 3 in anhydrous DMF, diisopropylethylamine (DIEA) was added, followed by PNP carbonate (bis(4-nitrophenyl)carbonate). The reaction solution was stirred at room temperature (rt) for 4 hours, and the mixture was purified by preparative HPLC to give 5. LCMS (ESI): M+H + =736.29.

[0452] Example 6 Synthesis of 4-nitrophenyl 2-(pyridin-2-yldisulfanyl)ethyl carbonate, 6 [ka] Following the procedure of WO 2016 / 040825, which is incorporated herein, 1,2-di(pyridin-2-yl)disulfane and 2-mercaptoethanol were reacted in pyridine and methanol at room temperature to give 2-(pyridin-2-yldisulfanyl)ethanol. Acylation with 4-nitrophenylcarbonochloridate in triethylamine and acetonitrile gave 4-nitrophenyl 2-(pyridin-2-yldisulfanyl)ethyl carbonate 6.

[0453] Example 7 Synthesis of 2-((5-nitropyridin-2-yl)disulfanyl)ethanamine hydrochloride, 7 [ka] To a mixture of 1,2-bis(5-nitropyridin-2-yl)disulfane (1.0 g, 3.22 mmol) in anhydrous DMF / MeOH (25 mL / 25 mL) was added HOAc (0.1 mL), followed by 2-aminoethanethiol hydrochloride (183 mg, 1.61 mmol). After stirring the reaction mixture at room temperature overnight, it was concentrated in vacuo to remove the solvent, and the residue was washed with DCM (30 mL × 4) to give 7 as a pale yellow solid (300 mg, 69.6%). 1 H NMR(400MHz,DMSO-d6)δ 9.28(d,J=2.4Hz,1H),8.56(dd,J=8.8,2.4Hz,1H),8.24(s,4H),8.03(d,J=8.8Hz,1H),3.15-3.13(m,2H),3.08-3.06(m,2H)

[0454] Example 8 Synthesis of 4-nitrophenyl 2-((5-nitropyridin-2-yl)disulfanyl)ethyl carbonate, 8 [ka] A solution of 1,2-bis(5-nitropyridin-2-yl)disulfane (9.6 g, 30.97 mmol) and 2-mercaptoethanol (1.21 g, 15.49 mmol) in anhydrous DCM / CHOH (250 mL / 250 mL) was stirred at room temperature under N for 24 h. The mixture was concentrated in vacuo, and the residue was diluted with DCM (300 mL). Manganese oxide MnO (10 g) was added, and the mixture was stirred at room temperature for an additional 0.5 h. The mixture was purified by column chromatography on silica gel (DCM / MeOH = 100 / 1 to 100 / 1) to give 2-((5-nitropyridin-2-yl)disulfanyl)ethanol (2.2 g, 61.1%) as a brown oil. 1 H NMR(400MHz,CDCl3)δ 9.33(d,J=2.8Hz,1H),8.38-8.35(dd,J=9.2,2.8Hz,1H),7.67(d,J=9.2Hz,1H),4.10(t,J=7.2Hz,1H),3.81-3.76(q,2H),3.01(t,J=5.2Hz,2H).

[0455] To a solution of 2-((5-nitropyridin-2-yl)disulfanyl)ethanol (500 mg, 2.15 mmol) in anhydrous DMF (10 mL) was added DIEA (834 mg, 6.45 mmol), followed by PNP carbonate (bis(4-nitrophenyl)carbonate, 1.31 g, 4.31 mmol). The reaction solution was stirred at room temperature for 4 h, and the mixture was purified by preparative HPLC (FA) to give 8 (270 mg, 33.1%) as a light brown oil. 1 H NMR(400MHz,CDCl3)δ 9.30(d,J=2.4Hz,1H),8.43-8.40(dd,J=8.8,2.4Hz,1H),8.30-8.28(m,2H),7.87 (d,J=8.8Hz,1H),7.39-7.37(m,2H),4.56(t,J=6.4Hz,2H),3.21(t,J=6.4Hz,2H).

[0456] Example 9 Synthesis of 2-((5-nitropyridin-2-yl)disulfanyl)propan-1-amine, 9 [ka] To a stirred solution of 1-aminopropan-2-ol (10 g, 133 mmol) in MeOH (360 mL) and HO (40 mL) was added BocO (37 g, 169 mmol). After the reaction mixture was stirred at room temperature for 5 h, it was concentrated and purified by chromatography (EtOAc / PE = 10%-50%) to give tert-butyl 2-hydroxypropylcarbamate as a colorless oil (19.8 g, yield: 85%).

[0457] To a stirred solution of tert-butyl 2-hydroxypropylcarbamate (10 g, 57 mmol) and EtN (17 g, 171 mmol) in DCM (130 mL) was added a solution of MsCl (methanesulfonyl chloride, 13 g, 114 mmol). The reaction mixture was stirred at room temperature for 4 hours and then washed with ice water (200 mL x 3) and brine (200 mL). The organic layer was concentrated to give 1-(tert-butoxycarbonylamino)propan-2-yl methanesulfonate as a red oil (12 g, 83% yield).

[0458] To a stirred solution of 1-(tert-butoxycarbonylamino)propan-2-yl methanesulfonate (6 g, 23.7 mmol) in acetone (70 mL) was added a solution of potassium thioacetate (potassium ethanethioate, 5.4 g, 47.3 mmol) in HO (100 mL). The reaction mixture was stirred at 60° C. for 12 hours. The mixture was concentrated and extracted with DCM (200 ml×2). The combined organic layer was concentrated and purified by chromatography to give S-1-(tert-butoxycarbonylamino)propan-2-yl ethanethioate as a red solid (1.1 g, yield: 20%). 1 H NMR(400MHz, CDCl3-d)1.30(d,J=7.09Hz,3H)1.44(s,9H)2.33(s,3H)3.16-3.42(m,2H)3.58-3.71(m,1H).

[0459] To a stirred solution of S-1-(tert-butoxycarbonylamino)propan-2-ylethanethioate (500 mg, 2.15 mmol) in MeOH (5 mL) was added dropwise HCl / MeOH (10 mL). The reaction mixture was stirred at room temperature for 3 h and then concentrated to give 1-aminopropane-2-thiol hydrochloride, which was used directly in the next step.

[0460] To a solution of 1,2-bis(5-nitropyridin-2-yl)disulfane (1.33 g, 4.3 mmol) in DCM (35 mL) was added a solution of 1-aminopropane-2-thiol hydrochloride (273 mg, 2.15 mmol). The mixture was stirred at 15 °C for 12 h. MnO (374.1 mg, 4.3 mmol) was added to the mixture and stirred at 15 °C for 10 min. The solid was washed with DCM (100 mL) and MeOH (30 mL × 3). The solution was concentrated to give 9 as a yellow solid (300 mg, 57%). LCMS (ESI): RT = 0.546 min, M+H + =245.7.

[0461] Example 10 Synthesis of 4-nitrobenzyl ((2,6-dioxo-3-(1-oxoisoindolin-2-yl)piperidin-1-yl)methyl)carbamate, 10 [ka]

[0462] Example 11 Synthesis of 2-((5-nitropyridin-2-yl)disulfanyl)ethyl ((2,6-dioxo-3-(1-oxoisoindolin-2-yl)piperidin-1-yl)methyl)carbamate, 11 [ka]

[0463] Example cD-1 Synthesis of 4-(3,5-difluoropyridin-2-yl)-N-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)propyl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10 triazadibenzo[cd,f]azulene-6-carboxamide, cD-1 [ka] [ka]

[0464] Example cD-2 Synthesis of 4-(3,5-difluoropyridin-2-yl)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10-triazadibenzo[cd,f]azulene-6-carboxamide, cD-2 [ka] [ka]

[0465] Example cD-3 Synthesis of 4-(3,5-difluoropyridin-2-yl)-N-(5-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentyl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10-triazadibenzo[cd,f]azulene-6-carboxamide, cD-3 [ka]

[0466] Example cD-4 Synthesis of 4-(3,5-difluoropyridin-2-yl)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)hexyl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10-triazadibenzo[cd,f]azulene-6-carboxamide, cD-4 [ka] [ka]

[0467] Example cD-5 Synthesis of 4-(3,5-difluoropyridin-2-yl)-N-(7-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)heptyl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10-triazadibenzo[cd,f]azulene-6-carboxamide, cD-5 [ka] [ka]

[0468] Example cD-6 Synthesis of 4-((3-cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-dimethylisoxazol-4-yl)-N-(5-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentyl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxamide, cD-6 [ka] [ka]

[0469] Example cDLI-1 Synthesis of 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)propanamido)benzyl (7-(4-(3,5-difluoropyridin-2-yl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10-triazadibenzo[cd,f]azulene-6-carboxamido)heptyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate, cDLI-1 [ka] [ka] [ka]

[0470] Preparation of tert-butyl (7-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)heptyl)carbamate, cDLI-1c To a solution of tert-butyl (7-oxoheptyl)carbamate, cDLI-1b (636.9 mg, 2.78 mmol) and 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione, cDLI-1a (600.00 mg, 2.31 mmol) in anhydrous dichloromethane (50 mL) was added acetic acid (0.02 mL, 0.26 mmol). The mixture was stirred at 25 °C for 2 h. NaBH(OAc) (1226.2 mg, 5.79 mmol) was then added to the mixture, and the mixture was stirred at 25 °C for 12 h. TLC (60% EtOAc in petroleum ether, R f =0.5) indicated the reaction was complete. The reaction mixture was washed with water (30 mL × 2), and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by column chromatography on silica gel (solvent gradient: 0-6% methanol in dichloromethane) to give cDLI-1c (0.70 g, 64%) as a pale yellow oil. LCMS (5-95, AB, 1.5 min): RT = 0.892 min, m / z = 495.2 [M+Na] + .

[0471] Preparation of tert-butyl 3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidine-1-carboxylate, cDLI-1d To a solution of cDLI-1c (300.00 mg, 0.63 mmol) and di-tert-butyl decarbonate, BocO (207.8 mg, 0.95 mmol) in dichloromethane (20 mL) was added 4-dimethylaminopyridine (116.3 mg, 0.95 mmol) and triethylamine (0.13 mL, 0.95 mmol). The mixture was stirred at 25 °C for 2 h. TLC (60% EtOAc in petroleum ether, R f =0.6), indicating the reaction was complete. The mixture was diluted with dichloromethane (45 mL) and washed with aqueous citric acid (15 mL), water (15 mL), and saturated brine (15 mL). The organic layer was concentrated and purified by flash column chromatography (eluted with 0–60% EtOAc in petroleum ether) to give cDLI-1d (300 mg, 83%) as a pale yellow solid.

[0472] Preparation of tert-butyl 3-(4-((((4-((S)-2-(((allyloxy)carbonyl)amino)propanamido)benzyl)oxy)carbonyl)(7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidine-1-carboxylate, cDLI-1f To a mixture of triphosgene (150.00 mg, 0.51 mmol) and 4A molecular sieves in dichloromethane (10 mL) was added a solution of N,N-diisopropylethylamine (228.10 μL, 1.3 mmol) and cDLI-1d (250.00 mg, 0.44 mmol) in dichloromethane (10 mL). The mixture was stirred at 25° C. for 1 hour. TLC (5% MeOH in DCM, R f=0.6), which indicated the reaction was complete. The mixture was concentrated and used directly in the next step. To the crude product (277.27 mg, 0.44 mmol, theoretical), allyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)carbamate, cDLI-1e (243 mg, 0.87 mmol) and 4A molecular sieves in dichloromethane (5 mL) and N,N-dimethylformamide, DMF (1 mL) were added, along with triethylamine (0.18 mL, 1.31 mmol) and 4-dimethylaminopyridine, DMAP (160.00 mg, 1.31 mmol). The mixture was stirred at 35° C. for 12 hours. TLC (10% MeOH in DCM, R f =0.5) indicated the reaction was complete. The mixture was filtered, diluted with DCM (50 mL), and washed with saturated citric acid (10 mL) and saturated brine (10 mL). The organic layer was concentrated in vacuo and purified by flash column (eluting with 0-10% MeOH in DCM) to give cDLI-1f (75 mg, 19.6%) as a pale yellow solid. LCMS (10-80, AB, 7.0 min): RT = 4.491 min, m / z = 877.5 [M+H] + .

[0473] Preparation of tert-butyl 3-(4-((((4-((S)-2-aminopropanamido)benzyl)oxy)carbonyl)(7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidine-1-carboxylate, cDLI-1g To a solution of cDLI-1f (75.00 mg, 0.09 mmol) and 1,3-dimethylbarbituric acid and 1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (66.8 mg, 0.43 mmol) in dichloromethane (3 mL) and methanol (3 mL) was added Pd(PPh3)4 (19.8 mg, 0.02 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 3 h under a nitrogen atmosphere. TLC (10% MeOH in DCM, R f=0.3) indicated the reaction was complete. The mixture was filtered, and the filtrate was concentrated to give the crude product, which was purified by preparative TLC (10% MeOH in DCM) to give cDLI-1g (30 mg, 44.2%) as a white solid. LCMS (5-95, AB, 1.5 min): RT = 0.883 min, m / z = 793.4 [M+H] + .

[0474] Preparation of tert-butyl 3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)(((4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)propanamido)benzyl)oxy)carbonyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidine-1-carboxylate, cDLI-1i To a solution of cDLI-1g (30.00 mg, 0.04 mmol) and 2,5-dioxopyrrolidin-1-yl 1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxylate, cDLI-1h (46.0 mg, 0.11 mmol) in N,N-dimethylformamide (1 mL) was added N,N-diisopropylethylamine (14.7 mg, 0.11 mmol). The reaction mixture was stirred at 20 °C for 2 h. TLC (10% MeOH in DCM, R f =0.5) indicated the reaction was complete. The mixture was concentrated and purified by preparative TLC (10% MeOH in DCM) to give cDLI-1i (10 mg, 24.4%) as a white solid. LCMS (10-80, AB, 7.0 min): RT = 4.728 min, m / z = 1083.7 [M+H] + .

[0475] Preparation of 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)propanamido)benzyl(7-aminoheptyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate, cDLI-1j To a solution of cDLI-1i (10.00 mg, 0.01 mmol) in dichloromethane, DCM (1 mL) was added trifluoroacetic acid, TFA (0.2 mL, 0.13 mmol). The mixture was stirred at 25 °C for 1 h. TLC showed that the reaction was complete. The mixture was concentrated to give cDLI-1j (9.20 mg, 100%) as a pale yellow solid.

[0476] Preparation of cDLI-1 To a solution of 4-(3,5-difluoropyridin-2-yl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10-triazadibenzo[cd,f]azulene-6-carboxylic acid, cDLI-1k (10.0 mg, 0.02 mmol), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; hexafluorophosphate azabenzotriazole tetramethyluronium, HATU, CAS registration number 148893-10-1 (8.00 mg, 0.02 mmol) in N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (0.01 mL, 0.05 mmol) was added. The mixture was stirred at 25°C for 5 minutes. Then, cDLI-1j (9.20 mg, 0.01 mmol) was added. The mixture was then stirred at 25°C for 1 hour. LCMS (10-80 AB / 7.0 min): RT = 3.840 min, [M+H] = 1365.3, indicating 16% of the desired product. The mixture was then filtered, and the filtrate was sent to prep-HPLC (acetonitrile 30-60 / 0.225% FA in water) to give cDLI-1 (1.90 mg, 13.1%) as a pale yellow solid. LCMS (5-95, AB, 1.5 min): RT (220 / 254 nm) = 0.88 min, m / z = 1365.4 [M+H] + .

[0477] Example cDLI-5 Synthesis of 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl((3-(4-((7-(7-(3,5-difluoropyridin-2-yl)-2-methyl-10-((methylsulfonyl)methyl)-3-oxo-3,4,6,7-tetrahydro-2H-2,4,7-triazadibenzo[cd,f]azulene-9-carboxamido)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamate, cDLI-5 [ka] [ka] [ka]

[0478] Preparation of 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl((3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamate, cDLI-5c To a solution of diphenylphosphoryl azide, DPPA (0.05 mL, 0.22 mmol), 2-(3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)acetic acid, cDLI-5a (50.0 mg, 0.09 mmol) in N,N-dimethylformamide, DMF (2 mL) was added N,N-diisopropylethylamine, DIEA (0.08 mL, 0.47 mmol). The mixture was stirred at 25° C. for 5 minutes, after which (S)—N-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)-N-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)cyclobutane-1,1-dicarboxamide, cDLI5b (107.6 mg, 0.19 mmol) was added. The mixture was stirred at 90° C. for 1 hour. LCMS (10-80, AB / 7.0 min): R T =3.735 min, m / z=1098.3[M+H] + showed 18% of the desired product. The mixture was then filtered, and the filtrate was sent for preparative HPLC (acetonitrile 30-60 / 0.225% FA in water) to give cDLI-5c (20 mg, 19.3%) as a pale yellow solid. LCMS (5-95, AB, 1.5 min): R T=0.909min, m / z=1098.7[M+H] + .

[0479] Preparation of 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl((3-(4-((7-aminoheptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamate, cDLI-5d A mixture of DLI-5c (15.0 mg, 0.01 mmol) in 5% trifluoroacetic acid in hexafluoroisopropanol, HFIP (1 mL) was stirred at 25° C. for 1 h. The mixture was concentrated to give cDLI-5d (15 mg, 99%) as a TFA salt as a white solid. LCMS (5-95, AB, 1.5 min): R T =0.756 min, m / z=998.7[M+H] + .

[0480] Preparation of cDLI-5 To a solution of cDLI-5d (13.5 mg, 0.03 mmol) in N,N-dimethylformamide (1 mL) was added HATU (11.8 mg, 0.03 mmol) and N,N-diisopropylethylamine (0.01 mL, 0.07 mmol). After stirring the mixture at 25 °C for 5 min, 4-(3,5-difluoropyridin-2-yl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10-triazadibenzo[cd,f]azulene-6-carboxylic acid, cDLI-5e (15.0 mg, 0.01 mmol), was added. The mixture was stirred at 25 °C for 1 h. LCMS (5-95AB / 1.5 min): RT = 0.870 min, m / z = 741.3 [M / 2+H] + showed 18% of the desired product. The mixture was then filtered, and the filtrate was sent to preparative HPLC (acetonitrile 30-60 / 0.225% FA in water) to give cDLI-5 (6.4 mg, 31.4%) as a white solid. LCMS (5-95, AB, 1.5 min): RT (220 / 254 nm) = 0.863 min, m / z = 1480.9 [M+H] + .

[0481] Example cDLI-6 Synthesis of S-(1-((((3-(4-((7-(7-(3,5-difluoropyridin-2-yl)-2-methyl-10-((methylsulfonyl)methyl)-3-oxo-3,4,6,7-tetrahydro-2H-2,4,7-triazadibenzo[cd,f]azulene-9-carboxamide)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamoyl)oxy)-2-methylpropan-2-yl)methanesulfonothioate, cDLI-6 [ka] [ka]

[0482] Preparation of tert-butyl (7-(methoxy(methyl)amino)-7-oxoheptyl)carbamate, cDLI-6a To a solution of 7-((tert-butoxycarbonyl)amino)heptanoic acid (5.0 g, 20.38 mmol) in dichloromethane (20 mL) was added EDCI (5.86 g, 30.57 mmol) and triethylamine (7.91 mL, 61.14 mmol), followed by N,O-dimethylhydroxylamine hydrochloride (0.87 g, 8.97 mmol) and 4-dimethylaminopyridine (0.10 g, 0.82 mmol). The mixture was stirred at 25 °C for 16 h. TLC (50% ethyl acetate in petroleum ether, R f =0.8) indicated the reaction was complete. The reaction mixture was poured into water (50 mL) and extracted with DCM (50 mL × 3). The combined organic layers were washed with saturated ammonium chloride (100 mL), dried over Na2SO4, filtered, and concentrated to give the crude product, which was purified by column chromatography (0-30% ethyl acetate in petroleum ether, R f =0.8) to give cDLI-6a (2 g, 85%) as a colorless oil. LCMS (5-95, AB, 1.5 min): T =0.772min, m / z=189.1[M-100+H] + . 1 H NMR (400MHz, chloroform-d): δ=4.52(br s,1H),3.68(s,3H),3.18(s,3H),3.11(d,J=6.0Hz,2H),2.41(t,J=7.2Hz,2H),1.70-1.59(m,2H),1.52-1.42(m,11H),1.39-1.32(m,4H)

[0483] Preparation of tert-butyl(7-oxoheptyl)carbamate, cDLI-6b To a solution of cDLI-6a (500.00 mg, 1.73 mmol) in tetrahydrofuran (12 mL) was added lithium aluminum hydride LiAlH (98.70 mg, 2.60 mmol) at −78° C. The mixture was warmed to 0° C. and stirred at this temperature for 30 min. TLC (30% EtOAc in petroleum ether, R f=0.5), which indicated the reaction was complete. With stirring at 0 °C, saturated aqueous NH4Cl was slowly added, and the mixture was filtered and extracted with ethyl acetate (30 mL × 3). The organic layer was washed with HO (20 mL) and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give cDLI-6b (390.00 mg, 98%) as a colorless oil, which was used directly for further purification.

[0484] Preparation of tert-butyl (7-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)heptyl)carbamate, cDLI-6d To a solution of 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione, lenalidomide (CAS registration 191732-72-6), cDLI-6c (360.00 mg, 1.39 mmol), and cDLI-6b (382.11 mg, 1.67 mmol) in N,N-dimethylformamide (3 mL) was added acetic acid (0.01 mL, 0.16 mmol). The mixture was stirred at 25 °C for 4 hours. Sodium triacetoxyborohydride, STAB, NaBH(OAc) (735.73 mg, 3.47 mmol) was then added to the mixture and stirred at 25 °C for 12 hours. LCMS (5-95 AB / 1.5 min): R T =0.889min, m / z=373.1[M-100+H] + The elution yielded the desired product in 28 fractions. The reaction mixture was washed with water (30 mL × 2), and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (solvent gradient: 0-6% methanol in dichloromethane) to give cDLI-6d (360 mg, 55%) as a pale yellow solid. LCMS (5-95, AB, 1.5 min): RT = 0.887 min, m / z = 495.3 [M + Na] + .

[0485] Preparation of tert-butyl 2-(3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)acetate, cDLI-6e To a mixture of cDLI-6d (370.00 mg, 0.78 mmol) in N,N-dimethylformamide (20 mL) was added KCO (173.2 mg, 1.25 mmol) and tert-butyl-bromoacetate (0.15 mL, 1.02 mmol), and the mixture was stirred at 25 °C for 2 h. TLC (10% MeOH in DCM, R f =0.8) indicated the reaction was complete. The mixture was filtered, EtOAc (60 mL) was added, and washed with water (20 mL). The organic layer was dried over Na2SO4, filtered, and purified by silica chromatography eluting with 0-1.5% MeOH in DCM to give cDLI-6e (320.00 mg, 70%) as a white solid. LCMS (5-95, AB, 1.5 min): RT = 0.990 min, m / z = 587.4 [M+H] + .

[0486] Preparation of tert-butyl 2-(3-(4-((7-aminoheptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)acetate, cDLI-6f To a mixture of cDLI-6e (320 mg, 0.55 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (3.0 mL, 38.94 mmol), and the mixture was stirred at 25 °C for 2 h. LCMS (5-95AB / 1.5 min): RT = 0.705 min, m / z = 431.2 [M + H] + showed 70% of the desired product. The mixture was concentrated to give cDLI-6f (296.00 mg, 99.7%) as a yellow oil.

[0487] Preparation of 2-(3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)acetic acid, cDLI-6g To a solution of cDLI-6f (296.00 mg, 0.54 mmol) in methanol (5 mL) was added BocO (0.19 mL, 0.82 mmol) and triethylamine, EtN (0.23 mL, 1.63 mmol) at 25 °C, and the mixture was stirred at 25 °C for 12 h. LCMS (5-95 AB / 1.5 min): RT = 0.903 min, m / z = 531.3 [M+H] + indicated 70% of the desired product. The mixture was concentrated and purified by reverse-phase chromatography (Xtimate C18® (Welch Materials) 150 × 25 mm × 5 μm, acetonitrile 60–80.6 / 0.225% FA in water) to give cDLI-6g (110 mg, 38%) as a white solid. LCMS(5-95,AB,1.5min):RT=0.895min,m / z=553.3[M+Na] + .

[0488] Preparation of S-(1-((((3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamoyl)oxy)-2-methylpropan-2-yl)methanesulfonothioate, cDLI-6h To a solution of cDLI-6h (70.00 mg, 0.13 mmol) and diphenylphosphoryl azide, DPPA (0.03 mL, 0.13 mmol) in toluene (5 mL) was added S-(1-hydroxy-2-methylpropan-2-yl)methanesulfonothioate (48.6 mg, 0.26 mmol), followed by triethylamine (0.03 mL, 0.20 mmol). The mixture was stirred at 25 °C for 10 min and heated at 90 °C under a N atmosphere for 3 h. TLC (10% MeOH in DCM, R f =0.6) indicated the reaction was complete. The mixture was filtered and the organic layer was concentrated in vacuo. The resulting mixture was diluted with EtOAc (40 mL) and washed with water (20 mL), and the organic layer was dried over Na2SO4, concentrated, and analyzed by preparative TLC (10% MeOH in DCM, R f=0.6) to give cDLI-6h (40 mg, 43%) as a white solid. LCMS (5-95, AB, 1.5 min): RT = 0.958 min, m / z = 734.4 [M+H] + .

[0489] Preparation of S-(1-((((3-(4-((7-aminoheptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamoyl)oxy)-2-methylpropan-2-yl)methanesulfonothioate, cDLI-6i To a mixture of cDLI-6h (32.00 mg, 0.04 mmol) in dichloromethane (0.50 mL) was added trifluoroacetic acid (0.5 mL, 6.49 mmol), and the mixture was stirred at 25 °C for 1 h. The mixture was concentrated to give the TFA salt of cDLI-6i (32 mg, 98.1%) as a yellow solid. LCMS (5-95, AB, 1.5 min): RT = 0.758 min, m / z = 612.3 [M+H] + .

[0490] Preparation of 4-(3,5-difluoropyridin-2-yl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11 tetrahydro-1H-1,4,10 triazadibenzo[cd,f]azulene-6-carboxylic acid, cDLI-6j To a solution of methyl 4-(3,5-difluoropyridin-2-yl)-10-methyl-7-((methylsulfonyl)methyl)-11-oxo-3,4,10,11-tetrahydro-1H-1,4,10-triazadibenzo[cd,f]azulene-6-carboxylate (100.0 mg, 0.19 mmol) in tetrahydrofuran (10 mL), methanol (10 mL), and water (2.5 mL) was added lithium hydroxide monohydrate (118.2 mg, 1.94 mmol). The mixture was stirred at 40° C. for 16 hours. TLC (10% methanol in dichloromethane, R f= 0.3) indicated the reaction was complete. Water (20 mL) was added to the mixture, and the aqueous layer was acidified to pH = 3 with 2 M HCl, then extracted with EtOAc (30 mL × 4). The organic layer was dried over Na2SO4, filtered, and concentrated to give cDLI-6j (95 mg, 98%) as a yellow solid. LCMS (5-95, AB, 1.5 min): RT = 0.770 min, m / z = 501.2 [M+H] + .

[0491] Preparation of cDLI-6 To a solution of cDLI-6j (44.1 mg, 0.09 mmol) in N,N-dimethylformamide (1 mL) was added HATU (38.6 mg, 0.10 mmol) and N,N-diisopropylethylamine (0.04 mL, 0.22 mmol). The mixture was stirred at 25 °C for 5 min. Then, cDLI-6i 3 (32.0 mg, 0.04 mmol) was added. The mixture was stirred at 25 °C for 1 h. The mixture was then filtered, and the filtrate was subjected to preparative e-HPLC (acetonitrile 30-60 / 0.225% FA in water) to give cDLI-6 (6.1 mg, 12%) as a pale yellow solid. LCMS (5-95, AB, 1.5 min): RT (220 / 254 nm) = 0.893 min, m / z = 1094.5 [M+H]. + .

[0492] Example 101 Preparation of Cysteine ​​Engineered Antibodies For large-scale production of antibodies, the antibodies were produced in CHO cells. Vectors encoding VL and VH were transfected into CHO cells, and IgG was purified from the cell culture medium by protein affinity chromatography.

[0493] As initially isolated, the engineered cysteine ​​residues in the antibodies are unavailable for conjugation because they exist as mixed disulfides with cellular thiols (e.g., glutathione). Partial reduction (e.g., with DTT), purification, and reoxidation with dehydroascorbic acid (DHAA) of these antibodies yields antibodies with free cysteine ​​sulfhydryl groups available for conjugation, as previously described (Junutula et al. (2008) Nat. Biotechnol. 26:925-932; U.S. Patent Application Publication No. 2011 / 0301334). Briefly, the antibodies were combined with a cereblon-degrading agent-linker intermediate to allow conjugation to the free cysteine ​​residues of the antibody. After several hours, the cereblon-degrading agent-antibody conjugate was purified.

[0494] Under specific conditions, cysteine ​​engineered antibodies were made reactive for conjugation with cereblon degrader-linker intermediates by treatment with reducing agents such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride (Getz et al., (1999) Anal. Biochem. 273:73-80; Soltec Ventures, Beverly, MA) in 50 mM Tris pH 7.5 containing 2 mM EDTA for 3 hours at 37°C or overnight at room temperature. Cysteine ​​engineered antibodies were made reactive for conjugation with cereblon degrader-linker intermediates in CHO cells (Gomez et al., (2010) Biotechnology and A full-length cysteine-engineered monoclonal antibody (THIOMAB™) expressed in a cysteine-engineered antibody (Bioeng. 105(4):748-760; Gomez et al., (2010) Biotechnol. Prog. 26:1438-1445) was reduced with approximately 50-fold excess DTT, e.g., overnight at room temperature, to reduce disulfide bonds that may have formed between the newly introduced cysteine ​​residue and cysteines present in the culture medium. The reduced THIOMAB™ was diluted and loaded onto a HiTrap S column in 10 mM sodium acetate (pH 5) and eluted with PBS containing 0.3 M sodium chloride. Alternatively, the antibody was acidified by adding 1 / 20 volume of 10% acetic acid, diluted with 10 mM succinate pH 5, loaded onto the column, and then washed with 10 column volumes of succinate buffer. The column was eluted with 50 mM Tris pH 7.5, 2 mM EDTA.

[0495] Light chain amino acids are numbered according to Kabat (Kabat et al., "Sequences of proteins of immunological interest," (1991), 5th ed., US Dept of Health and Human Services, National Institutes of Health, Bethesda, MD). Heavy chain amino acids are numbered according to the EU numbering system (Edelman et al., (1969) Proc. Natl. Acad. of Sci. 63(1):78-85), except where noted as the Kabat system. Single-letter amino acid abbreviations are used.

[0496] Full-length cysteine-engineered monoclonal antibody (THIOMAB™) expressed in CHO cells either has a cysteine ​​adduct (cystine) or, due to cell culture conditions, is glutathionylated on the engineered cysteine. To release the reactive thiol group of the engineered cysteine, THIOMAB™ was dissolved in 500 mM sodium borate and 500 mM sodium chloride at approximately pH 8.0 and reduced with approximately a 50- to 100-fold excess of 1 mM TCEP at 37°C for approximately 1 to 2 hours. Alternatively, DTT was used as the reducing agent. Interchain disulfide bond formation was monitored by either non-reducing SDS-PAGE or denaturing reverse-phase HPLC PLRP column chromatography. The reduced THIOMAB™ was diluted and loaded onto a HiTrap SP FF column in 10 mM sodium acetate (pH 5) and eluted with PBS containing 0.3 M sodium chloride or 50 mM Tris-Cl (pH 7.5) containing 150 mM sodium chloride.

[0497] Reoxidation was performed to reestablish disulfide bonds between cysteine ​​residues present in the parent Mab. The eluted reduced THIOMAB™ was treated with 15x or 2 mM dehydroascorbic acid (dhAA) (pH 7) for approximately 3 hours, or 50 mM Tris-Cl (pH 7.5) for approximately 3 hours, or with 200 nM to 2 mM aqueous copper sulfate (CuSO4) at room temperature overnight. Other oxidants (i.e., oxidizing agents) and oxidation conditions known in the art can be used. Ambient air oxidation can also be effective. This mild, partial reoxidation step efficiently forms intrachain disulfides with high fidelity. The buffer was exchanged by elution with Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol / antibody value was confirmed by determining the reduced antibody concentration from the solution's absorbance at 280 nm and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) and by determining the absorbance at 412 nm.

[0498] Liquid chromatography / mass spectrometry was performed on a TSQ Quantum Triple quadrupole™ mass spectrometer with extended mass range (Thermo Electron, San Jose, California). Samples were chromatographed on a PRLP-S®, 1000A, microbore column (50 mm x 2.1 mm, Polymer Laboratories, Shropshire, UK) heated to 75°C. A linear gradient of 30-40% B (solvent A: 0.05% TFA in water, solvent B: 0.04% TFA in acetonitrile) was used, with direct ionization of the eluent using an electrospray source. Data were collected by an Xcalibur® data system, and deconvolution was performed using ProMass® (Novatia, LLC, New Jersey). Prior to LC / MS analysis, antibodies or conjugates (50 micrograms) were treated with PN Gase F (2 units / ml; PROzyme, San Leandro, CA) for 2 hours at 37°C to remove N-linked carbohydrates.

[0499] Hydrophobic interaction chromatography (HIC) samples were injected onto a butyl HIC NPR column (2.5 micron particle size, 4.6 mm x 3.5 cm) (Tosoh Bioscience) and eluted with a 0-70% linear gradient at 0.8 ml / min (A: 1.5 M ammonium sulfate in 50 mM ammonium phosphate (pH 7); B: 50 mM potassium phosphate (pH 7), 20% isopropanol). Antibody species with different ratios of drug per antibody were resolved and quantified using an Agilent 1100 series HPLC system equipped with a multi-wavelength detector and Chemstation software.

[0500] Example 102 Conjugation of Cereblon Degrader-Linker Intermediate (cDLI) to Antibody Following the reduction and reoxidation procedure of Example 101, the cysteine-engineered antibody (THIOMAB™) was dissolved in PBS (phosphate-buffered saline) buffer and chilled on ice. Approximately 1.5 mol to 20 equivalents of an excess of cereblon degrader-linker intermediate activated with a thiol-reactive group, such as pyridyl disulfide, maleimide, or bromoacetamide, was dissolved in DMSO, diluted with acetonitrile and water, chilled, reduced, and added to the reoxidized antibody in PBS. Typically, the cereblon degrader-linker intermediate was added to the antibody from a DMSO stock at a concentration of approximately 20 mM in 50 mM Tris (pH 8). The reaction mixture was analyzed by LC-MS analysis and monitored for approximately 1 to 24 hours until completion. Once the reaction was complete, an excess of capping reagent, such as ethylmaleimide, was added to quench the reaction and cap any unreacted antibody thiol groups. The conjugation mixture was loaded and eluted through a HiTrap SP FF column to remove excess drug and other impurities. The reaction mixture was concentrated by centrifugal ultrafiltration, and the resulting cysteine ​​engineered cereblon degrader-antibody conjugate (cDAC) was purified and desalted by elution through G25 resin in PBS, filtered through a 0.2 μm filter under sterile conditions, and frozen for storage.

[0501] For example, crude cDAC was diluted with 20 mM sodium succinate, pH 5, and then applied to a cation exchange column. The column was washed with at least 10 column volumes of 20 mM sodium succinate, pH 5, and the antibody was eluted with PBS. Using a gel filtration column, cDAC was formulated in 20 mM His / acetate (pH 5) containing 240 mM sucrose. AAC was characterized before and after treatment with lysine C-endopeptidase by UV spectroscopy to measure protein concentration, analytical size exclusion chromatography (SEC) for aggregation analysis, and LC-MS.

[0502] Size-exclusion chromatography was performed using a Shodex KW802.5 column in 0.2 M potassium phosphate (pH 6.2) with 0.25 mM potassium chloride and 15% IPA at a flow rate of 0.75 ml / min. The aggregation state of cDAC was determined by integration of the elution peak area absorbance at 280 nm.

[0503] LC-MS analysis may be performed using an Agilent QTOF 6520 ESI instrument. For example, cDAC was treated with endoproteinase LysC (Promega) at 1:500 w / w in Tris (pH 7.5) for 30 min at 37 °C. The resulting cleaved fragments were loaded onto a 1000 Å, 8 μm PLRP-S (high-crosslinked polystyrene) column heated to 80 °C and eluted with a gradient of 30% B to 40% B over 5 min. Mobile phase A was HO with 0.05% TFA, and mobile phase B was acetonitrile with 0.04% TFA. The flow rate was 0.5 ml / min. Protein elution was monitored by UV absorbance detection at 280 nm prior to electrospray ionization and MS analysis. Chromatographic resolution of unconjugated Fc fragments, residual unconjugated Fab, and drug-labeled Fab was routinely achieved. The resulting m / z spectra were deconvoluted using Mass Hunter™ software (Agilent Technologies) to calculate the masses of the antibody fragments.

[0504] Example 103 In vitro cell proliferation assay The efficacy of cDAC was measured by a cell proliferation assay using the following protocol (CELLITER GLO™ Luminescent Cell Viability Assay, Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62:5485-5488): 1. A 40 μl aliquot of cell culture containing approximately 4000 cells (HER-expressing SK-BR-3, KPL-4, CAMA1, EFM19, MV-4-11, EOL-1, Molm-13, Nomo-1, HL-60, and OCI-AML-2) in medium was deposited into each well of a 384-well opaque-walled plate. 2. Control wells containing medium but no cells were prepared. 3. cDAC (n=3) was added to experimental wells and incubated for 3-5 days. 4. The plate was equilibrated at room temperature for approximately 30 minutes. 5. A volume of CELLTITER GLO™ Reagent equal to the volume of cell culture medium present in each well was added. 6. The contents were mixed on an orbital shaker for 15 minutes to induce cell lysis. 7. The plate was incubated at room temperature for 5 minutes to stabilize the luminescent signal. 8. Luminescence was recorded and reported graphically as % activity, and RLU (relative luminescence units) normalized to controls (no antibody control minus no cell control).

[0505] Data were plotted as individual points for each replicate (n=3) for each antibody and are shown in Figures 1, 2A-2B, 3A-3B, 4, 5A-5B, and 6A-6B. The protocol is a modified version of the CELLITER GLO™ Luminescent Cell. Cell lines can be grown in medium containing RPMI-1640, 20% HI-FBS, and 2 mM L-glutamine.

[0506] Example 104 Whole Blood Stability Assay Whole blood incubation: Matrix (shipped by the vendor (BioIVT, Westbury, NY)) was collected in lithium heparin-containing tubes. Unfrozen plasma and whole blood were collected in the afternoon and shipped refrigerated (2–8°C) overnight to arrive within 18 hours of collection, while frozen plasma was collected and shipped frozen under normal delivery conditions. cDAC source material was incubated in a buffer solution (1x PBS [pH 7.4], 0.5% bovine serum albumin, 15 ppm Proclin HCl).(商標) The whole blood / buffer solution was formulated at 1 mg / mL in PBS and then further diluted to a final concentration of 100 μg / mL. After mixing, 150 μL of whole blood / buffer stability samples were aliquoted into two separate tube sets at two different time points and then placed at -80°C for the 0-hour time point. Whole blood samples were generated, with two 150 μL aliquots for the 0-hour and 24-hour time points for whole blood. The 0-hour sample was immediately placed in a -80°C freezer and shaken (approximately 700 rpm) in a 37°C incubator for 24 hours. The aliquots stored in the -80°C freezer were collected at 24 hours until affinity capture LC-MS analysis was performed. The matrices used to generate the samples were mouse (CB17 SCID), rat (Sprague-Dawley), monkey (cynomolgus monkey), and human.

[0507] In vitro stability sample analysis: Streptavidin (SA)-coated magnetic beads (Thermo Fisher Scientific, catalog no. 60210) were washed twice with HBS-EP buffer (GE Healthcare Life Sciences, catalog no. BR-1001-88) and then mixed with either biotinylated extracellular domain of target (e.g., human HER2) or anti-idiotypic antibody for specific capture or biotinylated human IgG for general capture using a KingFisher Flex (Thermo Fisher Scientific) and incubated for 2 hours at room temperature with gentle agitation. The SA-bead / biotin capture probe complex was then washed twice with HBS-EP buffer and mixed with cDAC or precursor stability sample prediluted 1:16 in HBS-EP buffer and incubated for 2 hours at room temperature with gentle agitation. After 2 hours, the SA-bead / biotin capture probe / sample complexes were washed twice with HBS-EP buffer and then deglycosylated by overnight incubation with PNGase F (New England Biolabs, catalog no. P0704B). The SA-bead / biotin capture probe / sample complexes were then washed twice with HBS-EP buffer, followed by two washes with water (Optima™ LC / MS grade, Fisher Chemical, catalog no. W6-1), and finally one wash with 10% acetonitrile. Before collecting the beads, they were placed in 30% acetonitrile / 0.1% formic acid for 30 minutes at room temperature with gentle agitation. The eluted sample was then loaded onto an LC-MS (Thermo Scientific Q-Exactive Plus) for analysis. Ten μL of the cDAC sample was injected and loaded onto a Waters C4 column (1000 μm x 10 cm) maintained at 65°C. cDAC was separated on a Waters Acquity UPLC system at a flow rate of 20 μL / min with the following gradient: 20% B (100% acetonitrile + 0.1% formic acid) from 0 to 2 min, 35% B at 2.5 min, 65% B at 5 min, 95% B at 5.5 min, and 5% B at 6 min.The column was directly coupled for online detection to a Thermo Scientific Q-Exactive Plus mass spectrometer operated in positive electrospray ionization mode with an acquisition mass range of m / z 500–4000 Da.

[0508] Example 105 Tumor Growth Inhibition, In Vivo Efficacy in CD33-Expressing HL-60 Mice Prior to the single treatment on day 0, tumors were established and grown to 150–200 mm in CD33-expressing HL-60 mice. 3 Tumor volume was calculated using the formula: V (mm 3 )=0.5A×B 2 (where A and B are the short and long diameters, respectively). 3 Mice were sacrificed before reaching 0.05 or when tumors showed signs of impending ulceration. Data collected from each experimental group (10 mice per group) are expressed as mean + standard error.

[0509] Alternatively, the in vivo efficacy of anti-HER2 cereblon degrader antibody conjugate (cDAC) after a single intravenous dose was evaluated using the Fo5 mouse mammary tumor model, as previously described (Phillips GDL, Li GM, Dugger DL, et al. Targeting HER2-Positive Breast Cancer with Trastuzumab-DM1, an Antibody-Cytotoxic Drug Conjugate. (2008) Cancer Res. 68:9280-90, incorporated herein by reference). Anti-HER2 cDAC was evaluated using the Fo5 model, a transgenic mouse model in which the human HER2 gene is overexpressed in mammary epithelial cells under the transcriptional control of the mouse mammary tumor virus promoter (MMTV-HER2). HER2 overexpression causes spontaneous mammary tumor development. A mammary tumor from one of these founder animals (Founder Number 5 [Fo5]) is propagated in subsequent generations of FVB mice by stepwise transplantation of tumor fragments (approximately 2'2 mm in size). All studies are performed in accordance with the Guide for the Care and Use of Laboratory Animals. Each cDAC (single dose) is administered intravenously to nine animals at the start of the study and 14 days after transplantation. Initial tumor size is approximately 200 mm. 3 is the volume of.

[0510] Tumor volume after a single intravenous administration was assessed, and other mammary fat pad transplant efficacy models may be used as described (Chen et al. (2007) Cancer Res. 67:4924-4932), using tumors excised from mice bearing intraperitoneal tumors and then serially passaged into the mammary fat pads of recipient mice.

[0511] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

Claims

1. Formula IA 【Chemistry 1】 cereblon degrading agent antibody conjugate having the structure: During the ceremony, Ab is an antibody; L １ａ is the antibody linker; Ring A is C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ３ -C ２０ heterocyclyl, and C ３ -C ２０ heteroaryl; Dashed line 【Chemistry 2】 represents an optional double bond; Z １ is C(R １ ) ２ , C.R. １ , N, and NR １ａ is selected from Z ２ is C(R ２ ) ２ , C.R. ２ , N, and NR ２ａ is selected from R １ and R ２ are each independently H, F, Cl, Br, I, —CN, or C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -NO ２ , =O, -OR ａ , -OC(=O)R ａ , -SR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H; R １ａ and R ２ａ are each independently H, C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -OR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H; or (i) Two geminal R １ Or two geminal R ２ forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii) R １ and R ２ , R １ａ and R ２ , R １ and R ２ａ , or R １ａ and R ２ａ form a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R ａ and R ｂ are each independently H, OH, or C １ -C ６ alkyl, phenyl, and benzyl, where phenyl and benzyl are selected from F, Cl, —CN, C １ -C １２ Alkyl, C ２ -C １２ alkenyl, and C ２ -C １２ alkynyl; Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N (CH ３ ) ２ , -CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-CC(CH ３ ) ２ CONG ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ 、-NHCOCH ３ 、-NN(CH ３ COCH ３ 、-NHS(O) ２ CH ３ 、-N(CH ３ )C(CH ３ ) ２ CONG ２ 、-N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-OCH ２ F, -OCHF ２ 、-OCF ３ 、-OP(O)(OH) ２ 、-S(O) ２ N(CH) ３ ) ２ 、-SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H, CD ａ is the remainder of the cereblon degrader moiety; and A cereblon degrading agent-antibody conjugate wherein p is an integer from 1 to 14.

2. Z １ is CR １ and Z ２ is CR ２ and R １ and R ２ The cereblon degrading agent antibody conjugate of claim 1, wherein each of

3. Ring A is C ３ -C ２０ The cereblon degrading agent antibody conjugate of claim 1 or 2, which is heteroaryl.

4. 4. The cereblon degrading agent-antibody conjugate of claim 3, wherein ring A is an isoindoline substituted with =O.

5. 5. The cereblon degrading agent-antibody conjugate of any one of claims 1 to 4, having formula IA': 【Transformation 3】 having the structure In the formula, X １ is CH ２ and C(=O).

6. The cereblon degrading agent-antibody conjugate of any one of claims 1 to 5, wherein the antibody is a thiol-containing antibody.

7. The cereblon degrading agent-antibody conjugate of any one of claims 1 to 6, wherein the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

8. The cereblon degrading agent-antibody conjugate of any one of claims 1 to 7, wherein the antibody is a cysteine ​​engineered antibody.

9. 9. The cereblon degrading agent-antibody conjugate of claim 8, wherein the cysteine ​​engineered antibody comprises cysteine ​​mutations selected from HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

10. L １ａ The cereblon degrading agent-antibody conjugate of any one of claims 1 to 9, wherein is a protease-cleavable non-peptide linker.

11. The cereblon degrading agent-antibody conjugate of any one of claims 1 to 10, wherein L １ａ But, formula L １ -A 【Chemistry 4】 wherein: * indicates the point of attachment to the cysteine ​​thiol of the Ab; R １ is C １ -C １２ Alkylene, C １ -C １２ Alkylene-C(=O), C １ -C １２ Alkylene-NH, (CH ２ CH ２ O) ｒ , C １ -C １２ Alkylene-NH, (CH ２ CH ２ O) ｒ -C(=O), (CH ２ CH ２ O) ｒ -C(=O), (CH ２ CH ２ O) ｒ -CH ２ , and C １ -C １２ Alkylene -NHC(=O)CH ２ CH(thiophen-3-yl), r is an integer ranging from 1 to 10; C １ -C １２ Alkylene is F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H; R ２ and R ３ together, F, Cl, -CN, -NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ C optionally substituted with one or more groups selected from H ３ -C ７ forming a cycloalkyl ring; cereblon degrading agent antibody conjugate, wherein AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

12. AA is H, —CH ３ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ NH ２ , -CH ２ CH ２ CH ２ NHC (NH) NH ２ , -CH ２ CH (CH ３ ) ２ , and -CH ２ CH ２ CH ２ NHC(O)NH ２ The cereblon degrading agent-antibody conjugate of claim 11, selected from:

13. R １ is C ５ The cereblon degrading agent-antibody conjugate of claim 11, which is alkylene.

14. R ２ and R ３ Together they form C ４ The cereblon degrading agent-antibody conjugate of any one of claims 11 to 13, which forms a cycloalkyl ring.

15. AA is -CH ３ or -CH ２ CH ２ CH ２ NHC(O)NH ２ The cereblon degrading agent-antibody conjugate of any one of claims 11 to 14, wherein

16. R １ is C ５ alkylene; R ２ and R ３ But together, C ４ forming a cycloalkyl ring; AA is -CH ３ or -CH ２ CH ２ CH ２ NHC(O)NH ２ The cereblon degrading agent antibody conjugate of claim 11, wherein

17. 17. The cereblon degrading agent antibody conjugate of any one of claims 1 to 16, wherein cDa comprises (i) a target protein ligand covalently attached to a degrading agent linker, or (ii) a molecular adhesive moiety.

18. The cereblon degrading agent antibody conjugate of any one of claims 1 to 17, wherein p is 1, 2, 3, 4, 5 or 6.

19. Formula I-B 【Transformation 5】 cereblon degrading agent antibody conjugate having the structure: During the ceremony, Ab is an antibody; L １ａ is the antibody linker; Ring A is C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ heterocyclyl, and C １ -C ２０ is heteroaryl; Dashed line 【Transformation 6】 represents an optional double bond; Z １ is C(R １ ) ２ , C.R. １ , N, and NR １ａ is selected from Z ２ is C(R ２ ) ２ , C.R. ２ , N, and NR ２ａ is selected from R １ and R ２ are each independently H, F, Cl, Br, I, —CN, or C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -NO ２ , =O, -OR ａ , -OC(=O)R ａ , -SR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H; R １ａ and R ２ａ are independently H, C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -OR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H, or (i) Two geminal R １ Or two geminal R ２ forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii) R １ and R ２ , R １ａ and R ２ , R １ and R ２ａ Or R １ａ and R ２ａ forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R ａ and R ｂ are independently H, OH, C １ -C ６ alkyl, phenyl, and benzyl, where phenyl and benzyl are selected from F, Cl, —CN, C １ -C １２ Alkyl, C ２ -C １２ alkenyl, and C ２ -C １２ alkynyl, Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N (CH ３ ) ２ , -CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-CC(CH ３ ) ２ CONG ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ 、-NHCOCH ３ 、-N(CH ３ COCH ３ 、-NHS(O) ２ CH ３ 、-N(CH ３ )C(CH ３ ) ２ CONG ２ 、-N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-OCH ２ F, -OCHF ２ 、-OCF ３ 、-OP(O)(OH) ２ 、-S(O) ２ N(CH) ３ ) ２ 、-SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H, CD ａ is the remainder of the cereblon degrader moiety; A cereblon degrading agent-antibody conjugate wherein p is an integer from 1 to 14.

20. Z １ is CR １ and Z ２ is CR ２ and R １ and R ２ The cereblon degrading agent antibody conjugate of claim 19, wherein each is H.

21. Ring A is C ３ -C ２０ The cereblon degrading agent antibody conjugate of claim 19 or 20, which is heteroaryl.

22. 22. The cereblon degrading agent antibody conjugate of claim 21, wherein ring A is an isoindoline substituted with =0.

23. 23. The cereblon degrading agent-antibody conjugate of any one of claims 19 to 22, having formula I-B' 【Transformation 7】 having the structure In the formula, X １ is CH ２ and C(=O).

24. The cereblon degrading agent-antibody conjugate of any one of claims 19 to 23, wherein the antibody is a thiol-containing antibody.

25. The cereblon degrading agent-antibody conjugate of any one of claims 19 to 24, wherein the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

26. The cereblon degrading agent-antibody conjugate of any one of claims 19 to 25, wherein the antibody is a cysteine ​​engineered antibody.

27. 27. The cereblon degrading agent-antibody conjugate of claim 26, wherein said cysteine ​​engineered antibody comprises a cysteine ​​mutation selected from HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

28. L １ａ The cereblon degrading agent-antibody conjugate of any one of claims 19 to 27, wherein is a protease-cleavable non-peptide linker.

29. The cereblon degrading agent-antibody conjugate of any one of claims 19 to 28, wherein L １ａ But, formula L １ -A 【Transformation 8】 wherein: * indicates the point of attachment to the cysteine ​​thiol of the Ab; R １ is C １ -C １２ Alkylene, C １ -C １２ Alkylene-C(=O), C １ -C １２ Alkylene-NH, (CH ２ CH ２ O) ｒ , C １ -C １２ Alkylene-NH, (CH ２ CH ２ O) ｒ -C(=O), (CH ２ CH ２ O) ｒ -C(=O), (CH ２ CH ２ O) ｒ -CH ２ , and C １ -C １２ Alkylene -NHC(=O)CH ２ CH(thiophen-3-yl), r is an integer ranging from 1 to 10; C １ -C １２ Alkylene is F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H; R ２ and R ３ together, F, Cl, -CN, -NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ C optionally substituted with one or more groups selected from H ３ -C ７ forming a cycloalkyl ring; cereblon degrading agent antibody conjugate, wherein AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

30. AA is H, —CH ３ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ NH ２ , -CH ２ CH ２ CH ２ NHC (NH) NH ２ , -CH ２ CH (CH ３ ) ２ , and -CH ２ CH ２ CH ２ NHC(O)NH ２ 30. The cereblon degrading agent antibody conjugate of claim 29, selected from:

31. R １ is C ５ 30. The cereblon degrading agent-antibody conjugate of claim 29, which is alkylene.

32. R ２ and R ３ Together they form C ４ The cereblon degrading agent-antibody conjugate of any one of claims 29 to 31, which forms a cycloalkyl ring.

33. AA is -CH ３ or -CH ２ CH ２ CH ２ NHC(O)NH ２ The cereblon degrading agent antibody conjugate of any one of claims 29 to 32, wherein

34. R １ is C ５ alkylene; R ２ and R ３ But together, C ４ forming a cycloalkyl ring; AA is -CH ３ or -CH ２ CH ２ CH ２ NHC(O)NH ２ 30. The cereblon degrading agent antibody conjugate of claim 29, wherein

35. The cereblon degrading agent antibody conjugate of any one of claims 19 to 34, wherein the cDa comprises (i) a target protein ligand covalently attached to a degrading agent linker, or (ii) a molecular adhesive moiety.

36. The cereblon degrading agent antibody conjugate of any one of claims 19 to 35, wherein p is 1, 2, 3, 4, 5 or 6.

37. Formula IC 【Chemistry 9】 cereblon degrading agent antibody conjugate having the structure: During the ceremony, Ab is an antibody; R ４ａ , R ４ｂ , R ５ａ , and R ５ａ are each independently H and C １ -C ６ alkyl, or R ４ａ and R ４ｂ together with the carbon atoms to which they are attached, form F, Cl, and C １ -C ６ forming a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted by alkyl, C １ -C ６ Alkyl is independently F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H; Ring A is C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ heterocyclyl, and C １ -C ２０ is heteroaryl; Dashed line 【Chemistry 10】 represents an optional double bond; Z １ is C(R １ ) ２ , C.R. １ , N, and NR １ａ is selected from Z ２ is C(R ２ ) ２ , C.R. ２ , N, and NR ２ａ is selected from R １ and R ２ are each independently H, F, Cl, Br, I, —CN, or C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -NO ２ , =O, -OR ａ , -OC(=O)R ａ , -SR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H; R １ａ and R ２ａ are independently H, C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -OR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H, or (i) Two geminal R １ Or two geminal R ２ forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii) R １ and R ２ , R １ａ and R ２ , R １ and R ２ａ Or R １ａ and R ２ａ forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R ａ and R ｂ are independently H, OH, C １ -C ６ alkyl, phenyl, and benzyl, where phenyl and benzyl are selected from F, Cl, —CN, C １ -C １２ Alkyl, C ２ -C １２ alkenyl, and C ２ -C １２ alkynyl, Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is independently F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N (CH ３ ) ２ , -CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-CC(CH ３ ) ２ CONG ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ 、-NHCOCH ３ 、-N(CH ３ COCH ３ 、-NHS(O) ２ CH ３ 、-N(CH ３ )C(CH ３ ) ２ CONG ２ 、-N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-OCH ２ F, -OCHF ２ 、-OCF ３ 、-OP(O)(OH) ２ 、-S(O) ２ N(CH) ３ ) ２ 、-SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H, CD ａ is the remainder of the cereblon degrader moiety; A cereblon degrading agent-antibody conjugate wherein p is an integer from 1 to 14.

38. 38. The cereblon degrading agent antibody conjugate of claim 37, wherein said sulfur is conjugated to a cysteine ​​thiol of said Ab to form a disulfide bond.

39. Z １ is CR １ and Z ２ is CR ２ and R １ and R ２ and each is H.

40. Ring A is C ３ -C ２０ The cereblon degrading agent antibody conjugate of any one of claims 37 to 39, which is heteroaryl.

41. 41. The cereblon degrading agent antibody conjugate of claim 40, wherein ring A is an isoindoline substituted with =0.

42. 42. The cereblon degrading agent-antibody conjugate of any one of claims 37 to 41, having formula IC' 【Chemistry 11】 wherein X １ is CH ２ and C(=O).

43. The cereblon degrading agent-antibody conjugate of any one of claims 37 to 42, wherein the antibody is a thiol-containing antibody.

44. The cereblon degrading agent-antibody conjugate of any one of claims 37 to 43, wherein the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

45. The cereblon degrading agent-antibody conjugate of any one of claims 37 to 44, wherein the antibody is a cysteine ​​engineered antibody.

46. 46. ​​The cereblon degrading agent-antibody conjugate of claim 45, wherein said cysteine ​​engineered antibody comprises a cysteine ​​mutation selected from HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

47. L １ａ The cereblon degrading agent-antibody conjugate of any one of claims 37 to 46, wherein is a protease-cleavable non-peptide linker.

48. 48. The cereblon degrading agent-antibody conjugate of any one of claims 37 to 47, comprising: L １ａ But, formula L １ -A 【Chemistry 12】 having the structure During the ceremony, * indicates the point of attachment to the cysteine ​​thiol of the Ab; R １ is C １ -C １２ Alkylene, C １ -C １２ Alkylene-C(=O), C １ -C １２ Alkylene-NH, (CH ２ CH ２ O) ｒ , C １ -C １２ Alkylene-NH, (CH ２ CH ２ O) ｒ -C(=O), (CH ２ CH ２ O) ｒ -C(=O), (CH ２ CH ２ O) ｒ -CH ２ , and C １ -C １２ Alkylene -NHC(=O)CH ２ CH(thiophen-3-yl), where r is an integer ranging from 1 to 10; C １ -C １２ Alkylene is F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H; R ２ and R ３ together, F, Cl, -CN, -NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ C optionally substituted with one or more groups selected from H ３ -C ７ forming a cycloalkyl ring; cereblon degrading agent antibody conjugate, wherein AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

49. AA is H, —CH ３ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ NH ２ , -CH ２ CH ２ CH ２ NHC (NH) NH ２ , -CH ２ CH (CH ３ ) ２ , and -CH ２ CH ２ CH ２ NHC(O)NH ２ 49. The cereblon degrading agent antibody conjugate of claim 48, selected from:

50. R １ is C ５ The cereblon degrading agent-antibody conjugate of claim 48, which is alkylene.

51. R ２ and R ３ Together they form C ４ The cereblon degrading agent-antibody conjugate of any one of claims 48 to 50, which forms a cycloalkyl ring.

52. AA is -CH ３ or -CH ２ CH ２ CH ２ NHC(O)NH ２ The cereblon degrading agent antibody conjugate of any one of claims 48 to 51, wherein

53. R １ is C ５ alkylene; R ２ and R ３ But together, C ４ forming a cycloalkyl ring; AA is -CH ３ or -CH ２ CH ２ CH ２ NHC(O)NH ２ 49. The cereblon degrading agent antibody conjugate of claim 48, wherein

54. 54. The cereblon degrading agent antibody conjugate of any one of claims 37 to 53, wherein cD comprises (i) a target protein ligand covalently attached to a degrading agent linker, or (ii) a molecular adhesive moiety.

55. The cereblon degrading agent antibody conjugate of any one of claims 37 to 54, wherein p is 1, 2, 3, 4, 5 or 6.

56. 1. A cereblon degrading agent antibody conjugate comprising a cereblon degrading agent moiety covalently attached to an antibody by an antibody linker, the cereblon degrader moiety is (a) a target protein ligand covalently linked to a cereblon-bound E3 ubiquitin ligase ligand by a degrader linker, or (b) a molecular adhesive; A cereblon degrader-antibody conjugate, wherein the antibody is a thiol-containing antibody.

57. 57. The cereblon degrading agent-antibody conjugate of claim 56, wherein said thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

58. the tumor-associated antigen or cell surface receptor is (1) BMPR1B (bone morphogenetic protein receptor type IB); (2) E16 (LAT1, SLC7A5); (3) STEAP1 (six-transmembrane epithelial antigen of the prostate); (4) MUC16 (0772P, CA125); (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin); (6) Napi2b (NAPI-3B, NPTIIb, SLC34A2, solute transporter family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b); (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM), and short cytoplasmic domain, (semaphorin) 5B); (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene); (9) ETBR (endothelin type B receptor); (10) MSG783 (RNF124, hypothetical protein FLJ20315); (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane epithelial antigen of the prostate 2, six-transmembrane prostate protein); (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor); (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs73792); (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related beta), B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C); (17) HER2; (18) NCA; (19) MDP; (20) IL20Rα; (21) Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3); (27) CD22 (B cell receptor CD22-B isoform); (28) CD79a (CD79A, CD79α, immunoglobulin-related alpha); (29) CXCR5 (Burkitt's lymphoma receptor 1); (30) HLA-DOB (beta subunit of MHC class II molecule (Ia antigen)); (31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5); (32) CD72 (B cell differentiation antigen CD72, Lyb-2); (33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family); (34) FcRH1 (Fc receptor-like protein 1); (35) FcRH5 (IRTA2, immunoglobulin superfamily receptor translocation-associated 2); (36) TENB2 (putative transmembrane proteoglycan); (37) PMEL17 (silver homolog; SILV, D12S53E, PMEL17, SI, SIL); (38) TMEFF1 (transmembrane protein 1 with an EGF-like domain and two follistatin-like domains; tomoregulin-1); (39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha 1; GFR-ALPHA-1); (40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1); (41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); (42) Ly6G6D (lymphocyte antigen 6 complex, gene locus G6D; Ly6-D, MEGT1); (43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67); (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); (45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226); (46) GPR19 (G protein-coupled receptor 19; Mm.4787); (47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12); (48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982); (49) Tyrosinase (TYR; OCAIA, OCA1A, tyrosinase, SHEP3); (50) TMEM118 (RING finger protein, transmembrane 2; RNFT2; FLJ14627); (51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); (52) CD33; (53) CLL-1; (54) TROP2; and (55) The cereblon degrading agent antibody conjugate of claim 57, wherein the antibody conjugate is selected from the group consisting of CD123.

59. 59. The cereblon degrading agent antibody conjugate of claim 58, wherein said tumor-associated antigen or cell surface receptor is HER2 or CD33.

60. Formula I: Ab-[L] １ -cD] ｐ I 60. The cereblon degrading agent-antibody conjugate of any one of claims 56 to 59, or a pharmaceutically acceptable salt thereof, comprising: During the ceremony, Ab is an antibody; L １ is the antibody linker; cD is the cereblon degrader moiety; A cereblon degrading agent-antibody conjugate or a pharmaceutically acceptable salt thereof, wherein p is an integer from 1 to 14.

61. 61. The cereblon degrading agent antibody conjugate of claim 60, wherein the antibody is a cysteine ​​engineered antibody.

62. 62. The cereblon degrading agent-antibody conjugate of claim 61, wherein said cysteine ​​engineered antibody comprises a cysteine ​​mutation selected from A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, HC Y373C.

63. L １ The cereblon degrading agent antibody conjugate of claim 60, wherein the bond to cD comprises an aminal group.

64. L １ The bond to cD is carbamate (—OC(O)NH—) or methylcarbamate (—OC(O)NHCH ２ The cereblon degrading agent-antibody conjugate of claim 60, comprising a -) group.

65. 61. The cereblon degrading agent antibody conjugate of claim 60, wherein L １ But the formula: -Str-PM-IM- wherein Str is a stretcher unit covalently attached to said antibody; PM is a peptidomimetic unit and IM is an immolator unit covalently attached to said cereblon degrader moiety.

66. 66. The cereblon degrading agent-antibody conjugate of claim 65, wherein Str is of the following formula: 【Chemistry 13】 where * indicates the point of attachment on the succinimidyl ring to the cysteine ​​thiol of the antibody; R １ is C １ -C １２ Alkylene, C １ -C １２ Alkylene-C(=O), C １ -C １２ Alkylene-NH, (CH ２ CH ２ O) ｒ , C １ -C １２ Alkylene-NH, (CH ２ CH ２ O) ｒ -C(=O), (CH ２ CH ２ O) ｒ -C(=O), (CH ２ CH ２ O) ｒ -CH ２ , and C １ -C １２ Alkylene -NHC(=O)CH ２ CH(thiophen-3-yl), r is an integer ranging from 1 to 10, and C １ -C １２ Alkylene is F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H. A cereblon degrading agent antibody conjugate optionally substituted with one or more groups selected from:

67. R １ But (CH ２ ) ５ 67. The cereblon degrading agent antibody conjugate of claim 66, wherein

68. 68. The cereblon degrading agent antibody conjugate of any one of claims 65 to 67, wherein PM is represented by the following formula: 【Chemistry 14】 wherein R ２ and R ３ together, F, Cl, -CN, -NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H, C ３ -C ７ forming a cycloalkyl ring, cereblon degrading agent antibody conjugate, wherein AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

69. AA is H, —CH ３ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ NH ２ , -CH ２ CH ２ CH ２ NHC (NH) NH ２ , -CH ２ CH (CH ３ ) ２ , and -CH ２ CH ２ CH ２ NHC(O)NH ２ 69. The cereblon degrading agent antibody conjugate of claim 68, selected from:

70. The cereblon degrading agent-antibody conjugate of any one of claims 65 to 69, wherein IM comprises a group selected from 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl)methylcarbamate.

71. 66. The cereblon degrading agent-antibody conjugate of claim 65, having the formula: 【Chemistry 15】 wherein cD is a cereblon degrader moiety. 【Request Item 72】 【Chemistry 16】 and 【Chemistry 17】 72. The cereblon degrading agent antibody conjugate of claim 71, selected from:

73. formula: [Chemistry 18] 72. The cereblon degrading agent-antibody conjugate of claim 71, comprising: 【Request Item 74】 【Chemistry 19】 and 【Chemistry 20】 74. The cereblon degrading agent antibody conjugate of claim 73, selected from: 【Request Item 75】 【Chemistry 21】 【Chemistry 22】 and 【Chemistry 23】 75. The cereblon degrading agent antibody conjugate of claim 74, selected from:

76. L １ 61. The cereblon degrading agent antibody conjugate of claim 60, wherein forms a disulfide bond with a cysteine ​​thiol of the antibody.

77. 61. The cereblon degrading agent antibody conjugate of claim 60, wherein L １ but the following: 【Chemistry 24】 【Chemistry 25】 and 【Chemistry 26】 is selected from wherein * indicates that sulfur is conjugated to a cysteine ​​thiol of the antibody to form a disulfide bond; R ４ａ , R ４ｂ , R ５ａ , and R ５ａ are independently H and C １ -C ６ alkyl, or R ４ａ and R ４ｂ together with the carbon atoms to which they are attached, form F, Cl, and C １ -C ６ forming a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with alkyl, C １ -C ６ Alkyl is independently F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H; R ６ is H and C １ -C ６ alkyl; Cereblon degrader antibody conjugate, with wavy lines indicating binding to the cereblon degrader moiety.

78. R ４ａ and R ４ｂ Ga-CH ３ and R ５ａ and R ５ｂ is H and R ６ The cereblon degrading agent antibody conjugate of claim 77, wherein is H.

79. 61. The cereblon degrading agent antibody conjugate of claim 60, wherein L １ But the formula: 【Chemistry 27】 wherein * indicates the point of attachment to the cysteine ​​thiol of the antibody; R ４ａ and R ４ｂ are independently H and C １ -C ６ alkyl, or R ４ａ and R ４ｂ together with the carbon atoms to which they are attached, form F, Cl, and C １ -C ６ forming a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted by alkyl, C １ -C ６ Alkyl is independently F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H; Cereblon degrader antibody conjugate, wavy line indicates binding to the cereblon degrader moiety.

80. 61. The cereblon degrading agent antibody conjugate of claim 60, wherein the cereblon degrading agent moiety has the formula: TPL-L ２ -E3UL wherein TPL is the target protein ligand; E3UL is cereblon-binding E3 ubiquitin ligase ligand; L ２ is a degrader linker; TPL, E3UL, L ２ One of them is L １ is bound to, or A cereblon degrader antibody conjugate, wherein said cereblon degrader moiety is a molecular adhesive.

81. 81. The cereblon degrading agent antibody conjugate of claim 80, wherein TPL has the formula: 【Chemistry 28】 wherein R ｘ is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R ｙ is H and C １ -C ６ alkyl; The wavy line is L ２ Cereblon degrader antibody conjugate showing the attachment points.

82. 81. The cereblon degrading agent antibody conjugate of claim 80, wherein TPL has the formula: 【Chemistry 29】 where the wavy line represents L ２ Cereblon degrader antibody conjugate showing the attachment point to

83. 81. The cereblon degrading agent antibody conjugate of claim 80, wherein TPL has the formula: 【Transformation 30】 In the formula, the wavy line represents L ２ Cereblon degrader antibody conjugate showing the attachment point to

84. 81. The cereblon degrading agent antibody conjugate of claim 80, wherein TPL targets BRD4, GSPT1, BET, BRM (SMARCA2), KRAS, and SHP2.

85. The cereblon degrading agent antibody conjugate of claim 80, wherein E3UL comprises a glutarimide group.

86. E3UL, 【Chemistry 31】 and 【Chemistry 32】 is selected from In the formula, X １ is CH ２ and C(=O); the wavy line is L １ or L ２ 81. The cereblon degrading agent antibody conjugate of claim 80, exhibiting the following attachment points:

87. 81. The cereblon degrading agent antibody conjugate of claim 80, having the formula: 【Transformation 33】 and wherein L1 is a group represented by the formula: -Str-PM-IM- is a protease-cleavable non-peptide linker having the formula: where Str is a stretcher unit covalently linked to X; PM is a peptidomimetic unit; IM is an immolator unit covalently attached to the glutarimide group of E3UL and has the formula: 【Transformation 34】 and where the wavy line is the bond to PM, cereblon degrader antibody conjugate.

88. 81. The cereblon degrading agent antibody conjugate of claim 80, having the formula: 【Chemistry 35】 and In the ceremony, L １ is the formula: -Str-PM-IM- is a protease-cleavable non-peptide linker having the formula: where Str is a stretcher unit covalently linked to X; PM is a peptidomimetic unit; IM is CD L ２ and an immolator unit covalently bonded to the 【Transformation 36】 and where the wavy line is the bond to PM, cereblon degrader antibody conjugate.

89. L ２ but, -N(R)-(C １ -C １２ alkyldiyl)-N(R)-, -N(R)-(C ２ -C １２ alkenyldiyl)-N(R)-, -N(R)-(C ２ -C １２ alkynyldiyl)-N(R)-, -N(R)-(C １ -C １２ alkyldiyl)-C(═O)-(N(R)-, -N(R)-(C １ -C １２ alkyldiyl)-(N(R)-C(=O)CH ２ O-, -N(R)-(C １ -C １２ alkyldiyl)-(N(R)-C(=O)CH ２ N(R)-, -N(R)-(C １ -C １２ alkyldiyl)-C(=O)-(N(R)-(C １ -C １２ alkyldiyl)-N(R)-, -N(R)-(C １ -C ６ alkyldiyl)-O-(C １ -C ６ alkyldiyl)-N(R)-, -N(R)-(CH ２ CH ２ O) ｎ -N(R)-(CH ２ CH ２ O) ｎ - (n is an integer from 1 to 4), C １ -C １２ Alkyldiyl, C ２ -C １２ alkenyldiyl, and C ２ -C １２ alkynyldiyl; R is H, C １ -C ６ Alkyldiyl, and L １ selected from the points of attachment to Alkyldiyl, alkenyldiyl, and alkynyldiyl are substituted with F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ 81. The cereblon degrading agent antibody conjugate of claim 80, optionally substituted with one or more groups selected from: H. 【Request Item 90】 【Chemistry 37】 【Transformation 38】 and 【Chemistry 39】 and R ｘ is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R ｙ However, H and C １ -C ６ alkyl; X １ But CH ２ 61. The cereblon degrading agent antibody conjugate of claim 60, wherein said conjugate is selected from: and C(=O). 【Request Item 91】 【Chemistry 40】 【Chemistry 41】 and 【Chemistry 42】 and R ｘ is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R ｙ However, H and C １ -C ６ alkyl; X １ But CH ２ 61. The cereblon degrading agent antibody conjugate of claim 60, wherein said conjugate is selected from: and C(=O). 【Request Item 92】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 and 【Chemistry 47】 61. The cereblon degrading agent antibody conjugate of claim 60, selected from:

93. The cereblon degrading agent antibody conjugate of any one of claims 60 to 92, wherein p is 1, 2, 3, 4, 5 or 6.

94. The cereblon degrading agent antibody conjugate of any one of claims 60 to 92, comprising a mixture of cereblon degrading agent antibody conjugate compounds, wherein the average drug loading per antibody in said mixture of cereblon degrading agent antibody conjugate compounds is from about 2 to about 6.

95. 61. The cereblon degrading agent antibody conjugate of claim 60, wherein the cereblon degrading agent (cD) moiety is: 【Chemistry 48】 and 【Chemistry 49】 a molecular adhesive selected from In the formula, the wavy line represents L １ The dashed line indicates the connection point of [Transformation 50] represents an optional double bond; Z １ is C(R １ ) ２ , C.R. １ , N, and NR １ａ Selected from: Z ２ is C(R ２ ) ２ , C.R. ２ , N, and NR ２ａ Selected from: R is H and C １ -C ６ alkyl; R １ and R ２ are independently H, F, Cl, Br, I, —CN, C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -NO ２ , =O, -OR ａ , —OC(═O)R ａ , -SR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H; R １ａ and R ２ａ are independently H, C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -OR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H; or (i) two geminal R １ Or two geminal R ２ forms a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii) R １ and R ２ , R １ａ and R ２ , R １ and R ２ａ Or R １ａ and R ２ａ forms a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, R ａ and R ｂ are independently H, OH, C １ -C ６ alkyl, phenyl, and benzyl, wherein phenyl and benzyl are selected from F, Cl, —CN, C １ -C １２ Alkyl, C ２ -C １２ alkenyl, and C ２ -C １２ alkynyl, Ring A is C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ heterocyclyl, and C １ -C ２０ heteroaryl; and Alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl are independently F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N (CH ３ ) ２ , -CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-CC(CH ３ ) ２ CONG ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ 、-NHCOCH ３ 、-N(CH ３ COCH ３ 、-NHS(O) ２ CH ３ 、-NN(CH ３ )C(CH ３ ) ２ CONG ２ 、-NN(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-OCH ２ F, -OCHF ２ 、-OCF ３ 、-OP(O)(OH) ２ 、-S(O) ２ N(CH) ３ ) ２ 、-SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H. A cereblon degrading agent antibody conjugate optionally substituted with one or more groups selected from: 【Request Item 96】 【Chemistry 51】 and 【Chemistry 52】 96. The cereblon degrading agent antibody conjugate of claim 95, comprising a cD structure selected from: In the formula, the wavy line represents L １ cereblon degrader antibody conjugate showing the point of attachment to the remainder of 【Request Item 97】 【Chemistry 53】 and 【Chemistry 54】 97. The cereblon degrading agent-antibody conjugate of claim 96, comprising an immolator moiety selected from: In the formula, * represents L １ indicates the point of attachment to the rest of cD, and the wavy line indicates the point of attachment to cD; R ４ａ , R ４ｂ , R ５ａ , and R ５ａ are independently H and C １ -C ６ alkyl, or R ４ａ and R ４ｂ together with the carbon atoms to which they are attached, form F, Cl, and C １ -C ６ forming a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with alkyl; C １ -C ６ Alkyl is independently F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H. A cereblon degrading agent antibody conjugate optionally substituted with one or more groups selected from:

98. the cereblon degrader moiety: 【Transformation 55】 and 【Transformation 56】 96. The cereblon degrading agent antibody conjugate of claim 95, selected from: In the formula, X １ is CH ２ and C(=O); the wavy line is L １ cereblon degrader antibody conjugate showing the binding moiety.

99. Formula II 8-8 ３ -cD II 1. A cereblon degrader-linker intermediate comprising: (In the formula, X is a thiol-reactive group; L ３ teeth, (i) Formula: -Str-PM-IM- a protease-cleavable non-peptide linker having where Str is a stretcher unit covalently attached to X; PM is a peptidomimetic unit; IM is an immolator unit covalently bound to cD (ii) 【Chemistry 57】 【Chemistry 58】 and 【Chemistry 59】 a disulfide linker selected from and (iii) Formula: 【Transformation 60】 where * indicates the point of attachment to X; R ４ａ , R ４ｂ , R ５ａ , and R ５ａ are independently H and C １ -C ６ alkyl, or R ４ａ and R ４ｂ together with the carbon atoms to which they are attached, form F, Cl, and C １ -C ６ forming a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with alkyl; R ６ is H and C １ -C ６ alkyl, The wavy line indicates binding to cD; C １ -C ６ Alkyl is independently F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H) is a linker selected from cD has the formula: TPL-L ２ -E3UL (In the formula, TPL is the target protein ligand; E3UL is cereblon-binding E3 ubiquitin ligase ligand; L ２ is a degradant linker; or cD is a molecular glue).

100. L ３ The bond to cD is carbamate (—OC(O)NH—) or methylcarbamate (—OC(—O)NHCH ２ 100. The cereblon degrading agent-linker intermediate of claim 99, comprising a -) group.

101. 100. The cereblon degrader-linker intermediate of claim 99, wherein the cereblon degrader moiety is: 【Chemistry 61】 and 【Transformation 62】 a molecular adhesive selected from In the formula, the wavy line represents L ３ indicates the point of attachment of the group, and the dashed line indicates an optional double bond; Z １ is C(R １ ) ２ , C.R. １ , N, and NR １ａ Selected from: Z ２ is C(R ２ ) ２ , C.R. ２ , N, and NR ２ａ Selected from: R is H and C １ -C ６ alkyl; R １ and R ２ are independently H, F, Cl, Br, I, —CN, C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -NO ２ , =O, -OR ａ , —OC(═O)R ａ , -SR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H; R １ａ and R ２ａ are independently H, C １ -C １２ Alkyl, C ２ -C １２ Alkenyl, C ２ -C １２ Alkynyl, (C １ -C ６ alkyldiyl)-(C ６ -C ２０ aryl), -(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -(C １ -C ６ alkyldiyl)-OR ａ , (C １ -C ６ alkyldiyl)-(C ３ -C ２０ carbocyclyl), (C １ -C ６ alkyldiyl)-(C ２ -C ２０ heterocyclyl), (C １ -C ６ alkyldiyl)-(C １ -C ２０ Heteroaryl), C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ Heterocyclyl, C １ -C ２０ Heteroaryl, —C(═NH)NH(OH), —C(═NH)NH ２ , —C(═O)NR ａ R ｂ , —C(═O)NR ａ -NR ａ R ｂ , -C(=O)NH(C １ -C ６ alkyldiyl)-NR ａ R ｂ , -C(=O)OR ａ , -NR ａ R ｂ , -OR ａ , -S(O)R ａ , -S(O) ２ R ａ , -S(O) ２ NR ａ , and -S(O) ３ H; or (i) two geminal R １ Or two geminal R ２ form a 3- to 6-membered carbocyclyl or heterocyclyl spiro group, or (ii) R １ and R ２ , R １ａ and R ２ , R １ and R ２ａ Or R １ａ and R ２ａ form a fused 5- or 6-membered aryl, carbocyclyl, heterocyclyl, or heteroaryl group; R ａ and R ｂ are independently H, OH, C １ -C ６ alkyl, phenyl, and benzyl, wherein phenyl and benzyl are selected from F, Cl, —CN, C １ -C １２ Alkyl, C ２ -C １２ alkenyl, and C ２ -C １２ alkynyl, A is C ６ -C ２０ Aryl, C ３ -C ２０ Carbocyclyl, C ２ -C ２０ heterocyclyl, and C １ -C ２０ heteroaryl; Alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl are independently F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N (CH ３ ) ２ , -CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-CC(CH ３ ) ２ CONG ２ 、-NH ２ 、-NHCH ３ 、-NN(CH ３ ) ２ 、-NHCOCH ３ 、-NN(CH ３ COCH ３ 、-NHS(O) ２ CH ３ 、-NN(CH ３ )C(CH ３ ) ２ CONG ２ 、-NN(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-OCH ２ F, -OCHF ２ 、-OCF ３ 、-OP(O)(OH) ２ 、-S(O) ２ N(CH) ３ ) ２ 、-SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ A cereblon degrader-linker intermediate optionally substituted with one or more groups independently selected from H. 【Request Item 102】 【Chemistry 63】 and 【Chemistry 64】 102. The Revlon degrader-linker intermediate of claim 101, comprising a structure selected from: In the formula, the wavy line represents L ３ cereblon degrader-linker intermediate showing the point of attachment to the remainder of 【Request Item 103】 【Chemistry 65】 and ​ 102. The cereblon degrader-linker intermediate of claim 101, comprising an immolator moiety selected from: In the formula, * represents L ３ The wavy line indicates the point of attachment to the rest of L ３ indicates the point of attachment to the remainder of R ４ａ , R ４ｂ , R ５ａ , and R ５ａ are independently H and C １ -C ６ alkyl, or R ４ａ and R ４ｂ together with the carbon atoms to which they are attached, form F, Cl, and C １ -C ６ forming a 3-, 4-, or 5-membered cycloalkyl or heterocyclyl optionally substituted with alkyl; R ６ is H and C １ -C ６ alkyl; The wavy line indicates the point of attachment to cD; C １ -C ６ Alkyl is independently F, Cl, —CN, —NH ２ , -CH ２ NH ２ , —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ , -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H.

104. the cereblon degrader moiety: 【Transformation 67】 and 【Transformation 68】 100. The cereblon degrading agent-linker intermediate of claim 99, selected from: In the formula, X １ is CH ２ and C(=O); the wavy line is L ３ cereblon degrader-linker intermediate showing the binding site.

105. 100. The cereblon degrader-linker intermediate of claim 99, wherein X is selected from the group consisting of maleimide, bromoacetamide, toluenesulfonyl sulfide, and 2-pyridyl disulfide, wherein the pyridyl is optionally substituted with one or two nitro groups.

106. 100. The Revlon degrader-linker intermediate of claim 99, having the formula: 【Transformation 69】 and wherein IM comprises a group selected from 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl)methylcarbamate; cereblon degrader-linker intermediate, wherein AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

107. AA is H, —CH ３ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ NH ２ , -CH ２ CH ２ CH ２ NHC (NH) NH ２ , -CH ２ CH (CH ３ ) ２ , and -CH ２ CH ２ CH ２ NHC(O)NH ２ The cereblon degrading agent-linker intermediate of claim 106, selected from:

108. AA is -CH ３ or -CH ２ CH ２ CH ２ NHC(O)NH ２ The cereblon degrading agent-linker intermediate of claim 106 or 107, 【Request Item 109】 【Chemistry 70】 and 【Chemistry 71】 100. The Revlon degrader-linker intermediate of claim 99, selected from: In the formula, X １ is CH ２ and C(═O).

110. 110. The cereblon degrader-linker intermediate of claim 109, having the formula: 【Chemistry 72】 and In the ceremony, L ３ is the formula: -Str-PM-IM- is a protease-cleavable non-peptide linker having the formula: where Str is a stretcher unit covalently linked to X; PM is a peptidomimetic unit; IM is an immolator unit covalently attached to cD and has the formula: 【Transformation 73】 and The wavy line indicates the attachment of the cereblon degrader-linker intermediate to the PM.

111. 110. The cereblon degrader linker intermediate of claim 109, having the formula: 【Chemistry 74】 and In the ceremony, L ３ is the formula: -Str-PM-IM- is a protease-cleavable non-peptide linker having the formula: where Str is a stretcher unit covalently linked to X; PM is a peptidomimetic unit; IM is CD's L ２ and an immolator unit covalently bonded to the 【Chemistry 75】 The cereblon degrader-linker intermediate has the following structure:

112. 100. The cereblon degrader-linker intermediate of claim 99, wherein TPL is: 【Transformation 76】 (i) (In the formula, R ｘ is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R ｙ However, H and C １ -C ６ alkyl); (ii) 【Chemical Formula 77】 and (iii) 【Transformation 78】 (Wherein, the wavy line represents L ２ (indicating the point of attachment to A cereblon degrader-linker intermediate selected from:

113. L ２ but, -N(R)-(C １ -C １２ alkyldiyl)-N(R)-, -N(R)-(C ２ -C １２ alkenyldiyl)-N(R)-, -N(R)-(C ２ -C １２ alkynyldiyl)-N(R)-, -N(R)-(C １ -C １２ alkyldiyl)-C(═O)-(N(R)-, -N(R)-(C １ -C １２ alkyldiyl)-C(=O)-(N(R)-(C １ -C １２ alkyldiyl)-N(R)-, -(C １ -C ６ alkyldiyl)-O-(C １ -C ６ Alkyldiyl-, C １ -C １２ Alkyldiyl, C ２ -C １２ alkenyldiyl, and C ２ -C １２ alkynyldiyl; Alkyldiyl, alkenyldiyl, and alkynyldiyl are substituted with F, Cl, —OH, —OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N (CH ３ ) ２ and R is optionally substituted with one or more groups selected from H, C １ -C ６ Alkyldiyl, and L ３ 100. The cereblon degrading agent-linker intermediate of claim 99, wherein the point of attachment to

114. CD is, 【Transformation 79】 【Chemistry 80】 and 【Chemistry 81】 is selected from R ｘ is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R ｙ However, H and C １ -C ６ alkyl; X １ But CH ２ and C(=O). 【Request Item 115】 【Chemistry 82】 【Chemistry 83】 and 【Chemical 84】 is selected from In the formula, R ｘ is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R ｙ However, H and C １ -C ６ alkyl; X １ But CH ２ and C(=O). 【Request Item 116】 【Chemistry 85】 【Chemical 86】 【Transformation 87】 【Chemical 88】 【Chemistry 89】 and 【Chemistry 90】 100. The cereblon degrading agent-linker intermediate of claim 99, selected from:

117. A cereblon degrader-antibody conjugate prepared by conjugation of a thiol-containing antibody with a cereblon degrader intermediate selected from Table 3.

118. 100. A method for preparing a cereblon degrading agent-antibody conjugate, comprising reacting a thiol-containing antibody with the cereblon degrading agent-linker intermediate of formula II of claim 99.

119. 100. A method for preparing a cereblon degrader-linker intermediate of formula II of claim 99, comprising reacting an X-L3 moiety with a cereblon degrader moiety.

120. 99. A pharmaceutical composition comprising the cereblon degrading agent-antibody conjugate of any one of claims 1 to 98 and one or more pharmaceutically acceptable diluents, vehicles, carriers or excipients.

121. 120. A method for treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the cereblon degrading agent-antibody conjugate of any one of claims 1 to 98 or the pharmaceutical composition of claim 120.

122. 122. The method of claim 121, wherein the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia or lymphoid malignancies including acute myeloid leukemia, squamous cell carcinoma, epithelial squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung cancer including adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic cancer, anal cancer, penile cancer, and head and neck cancer.

123. 122. The method of claim 121, wherein the cancer is breast cancer or acute myeloid leukemia.

124. 122. The method of claim 121, wherein the cancer is BRD4 dependent.

125. Use of the cereblon degrading agent-antibody conjugate of any one of claims 1 to 98 or the pharmaceutical composition of claim 120 in the manufacture of a medicament for treating cancer in a mammal.

126. A cereblon degrading agent-antibody conjugate according to any one of claims 1 to 98 or a pharmaceutical composition according to claim 120 for use in a method for treating cancer.

Images