Cereblon degradator conjugates and uses thereof

Abstract

Provided herein are cerebron degrading agent antibody conjugates (cDACs) comprising a cerebron degrading agent moiety covalently linked to an antibody. The cDACs target proteins for intracellular degradation and can be used to treat diseases and conditions.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority benefit of U.S. Provisional Application No. 63 / 435,142, filed Dec. 23, 2022, and U.S. Provisional Application No. 63 / 525,282, filed Jul. 6, 2023, the contents of each of these provisional applications being incorporated by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to cereblon - degrading agent antibody conjugates (cDAC) compositions, intermediates for their manufacture, and methods of use thereof. cDACs can be used to promote intracellular degradation of target proteins. Background Art

[0004] Cereblon is a 442 - amino acid multifunctional protein located 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 functions of ion channels, which are important for maintaining cell growth and proliferation. Cereblon is also involved in the occurrence of many diseases, such as 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 Cullins 1 (RoC1) to form a functional E3 ubiquitin ligase complex, which is called the CRL4 / CRBN E3 ubiquitin ligase complex. Cereblon's role as part of this complex includes many target 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 many other proteins. Cereblon is also related to the development of cerebral tissues and, due to its expression in regions such as the hippocampus, is related to memory and learning processes (Higgins, et al., (2004) Neurol. 63(10):1927 - 31).

[0005] Cereblon is a target of immunomodulatory drugs (IMiDs) that are used to regulate the immune response and contain a glutarimide functional group (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, iberdomide, and apremilast. Thalidomide is FDA-approved for the treatment of multiple myeloma. Lenalidomide and pomalidomide are FDA-approved for the treatment of multiple myeloma and other diseases. Cytokine modulation and T-cell co-stimulation by IMiDs result in the production of interleukin-2 in T cells (Schafer et al., (2003) J. Pharmacol. & Exper. Ther. 305:1222-32). IMiDs have pleiotropic effects on multiple immune cells, including natural killer (NK) cell activation and B-cell and monocyte inhibition (Corral et al., (1999) J. Immunol. 163:380-6). The approved drugs thalidomide and the derivatives lenalidomide and pomalidomide have been repurposed as immunomodulatory drugs (IMiDs) for blood 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 in a shallow hydrophobic pocket on the surface of cereblon, and the binding is mediated by the glutarimide ring.

[0006] As the binding protein for IMiDs, cereblon is responsible for the multiple effects of IMiDs like thalidomide and its analogs (P. Ottis, et al. (2017) ACS Chem. Biol. 12(4):892-898; Shi Q, et al. (2017) J ImmunolRes. 2017:9130608; Sperling AS, et al. (2019) Blood134(2):160–170). Cereblon expression can affect cell metabolism and may be a pathogenic effect of the disease even in the absence of IMiDs. Cereblon orthologs are highly conserved from plants to humans, which emphasizes its physiological importance (Zhihua H, et al. (2011) Annu RevPlant Biol. 62(1):299–334).

[0007] The ATP-dependent ubiquitin-proteasome system (UPS) is the major pathway for intracellular protein degradation. The UPS system, including ubiquitin (Ub), proteasome, catalytic enzymes, and specific substrates, plays important roles in various biological processes. Ubiquitination occurs through a series of enzymatic events, particularly through the concerted action of Ub-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Once the substrate protein is polyubiquitinated, it will be recognized and degraded by the proteasome, and the UPS can digest the substrate protein into small peptides. The specific recognition of substrate proteins is the apparent function of E3, and thus E3 plays an important role in determining Ub-mediated protein degradation (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).

[0008] Proteolysis-targeting chimeras (PROTACs) are heterobifunctional degrader constructs that can target and degrade aberrant proteins using the cell's ubiquitin-proteasome machinery. The main mechanism of PROTAC technology is to bring an E3 ubiquitin ligase into proximity with the protein of interest (POI) to be targeted for degradation, leading to the degradation of the POI using the UPS, such as a target protein that is itself a disease mediator (Lu et al., (2015) Cell Cancer 22(6):755-63; Wang, C. et al. (2021) Eur JMed Chem.225:113749). The E3 ligase ligand of a PROTAC can hijack the E3 ligase and label the POI with ubiquitin. During this process, the PROTAC itself is not degraded but is recycled to facilitate 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 mode contrasts with the function of conventional inhibitors, where sequence target binding is required to elicit the desired effect. For typical small molecule drugs driven by standard occupancy, binding affinity is essential for their efficacy. In contrast, PROTACs induce the degradation of the POI via the UPS, an event-driven mode that can be used to overcome the common drawbacks of traditional occupancy-driven small molecule drugs (K.M. 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).

[0009] "Molecular glue" is a degrader construct that mediates proximity-induced protein degradation by inducing or stabilizing protein-protein interactions between an E3 ubiquitin ligase and a POI, interacting more frequently with the ligase or the target POI to form 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 glue can degrade other non-ligandable proteins by orchestrating 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 glue degraders can target the nuclear receptor GSPT1 (Huber, A.D., et al. (2022) ACS Med.Chem.Lett. 13:1311-1320).

[0010] Although both molecular glue and PROTAC are bifunctional protein degraders, they have different mechanisms of action and structural requirements (den Besten, W., et al. (2020) Nat Chem Biol 16:1157–1158). However, cereblon ligands can be components of both PROTAC and molecular glue degraders, which recruit the target POI to the CRL4 / CRBN E3 ubiquitin ligase to degrade the POI (Lu et al., (2015) Cell Cancer 22(6):755-63; Wang, C. et al. (2021) Eur J MedChem. 225:113749). Certain glutarimide compounds such as thalidomide, lenalidomide, and pomalidomide act as molecular glue to enhance or induce interactions between E3 ligases and target proteins, thereby triggering ubiquitination and degradation (Dong G, et al. (2021) J Med Chem. 64(15):10606-10620).

[0011] A protein of interest is bromodomain-containing protein 4 (BRD4). Certain small molecule BRD4 inhibitors interfere with protein-protein interactions and have become the subject of anti-tumor drug development. Limitations include the reversible binding of BRD4 inhibitors (e.g., JQ1, OTX015), requiring high systemic drug concentrations and continuous exposure to ensure sufficient functional inhibition (J. Shi, et al. (2018) Mol.Pharm. 15(9):4139-4147).

[0012] Target protein ligands have been used in PROTACs, where pomalidomide is conjugated with various BRD4 target protein ligands via a PEG linker, and the BRD4 target protein ligand induces significant degradation of BRD4 in BL (Burkitt lymphoma) cells, with a DC50 value below 1 nM (J. Lu, et al. (2015) Chem. Biol. 22(6):755-763). PROTACs and other BRD4 and BET target protein ligands show significant effects on downstream cell proliferation and apoptosis induction in c-MYC, AML (acute myeloid leukemia) cells, and BL cells. (E.W. Georg, et al., (2015) Science 348(6241):1376-1381). These results suggest that cereblon-based PROTACs with BET provide a better and more effective 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). The degradation of BET proteins is related to the linker design in PROTACs (T.A. Bemis, et al (2021), Chem. Commun. 57(8):1026-1029).

[0013] There are limitations or challenges in the design, preparation, and use of antibody compositions covalently linked to drugs, payloads, and other bioactive moieties via linkers. Linkers can be classified as cleavable and non-cleavable based on 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 resistant to proteolytic degradation, such that cleavage occurs only after lysosomal internalization and complete degradation of the antibody. These linkers have higher stability than cleavable linkers but may suffer from lower membrane permeability. In contrast, 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 B-Q, et al., (2012) Nat Biotechnol; Bargh JD, et al., (2019) Chem Soc Rev. 48:4361-74). Thus, some linkers may be unstable in the bloodstream and release unacceptable amounts of drug before internalization into target cells (Khot, A. et al., (2015) Bioanalysis 7(13):1633-1648), while other linkers may provide stability in the bloodstream but intracellular release efficacy may be negatively affected. In addition, linkers that provide the desired intracellular release may have poor stability in the bloodstream. Furthermore, the amount of drug moiety loaded on 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 other parameters that need to be addressed and are often interrelated.

[0014] Accordingly, there is a continuing need to improve the design of antibody conjugates, including linkers and conjugation chemistries, to provide optimized safety and efficacy. In addition, there is a need to enhance and target the delivery of cereblon ligands containing PROTACs and molecular glues to cells containing protein targets. The combination of tumor-associated protein degradation and cereblon immunomodulatory activity can enhance the therapeutic benefit in patients suffering from various hyperproliferative disorders such as cancer. SUMMARY OF THE INVENTION

[0015] The present disclosure generally relates to a conjugate composition, referred to as a cereblon degrader antibody conjugate or "cDAC", wherein the cereblon degrader moiety is covalently linked to an antibody via an antibody linker. In some embodiments, the cereblon degrader moiety comprises a target protein ligand covalently linked via a degrader linker to a cereblon-binding E3 ubiquitin ligase ligand. In other embodiments, the cereblon degrader moiety is a molecular glue. In some embodiments, the disclosed cereblon degrader moiety of the cDAC targets a suitable target cell and is released as a cereblon degrader compound, thereby performing its function of stimulating / inducing ubiquitination of the target protein and achieving its degradation via the ubiquitin-proteasome system (UPS). The cDAC may have enhanced therapeutic benefits for patients suffering from various hyperproliferative disorders such as cancer.

[0016] One aspect of the present disclosure is a cereblon degrader antibody conjugate (cDAC) comprising a cereblon degrader moiety covalently linked to an antibody via a linker (e.g., an antibody linker), wherein the cereblon degrader moiety is covalently linked via a degrader linker or a molecular glue to a cereblon-binding E3 ubiquitin ligase ligand, and the antibody is a thiol-containing antibody.

[0017] Another aspect of the present disclosure is a cDAC having a structure of Formula I:

[0018] Ab-[L1-cD] p I

[0019] or a pharmaceutically acceptable salt thereof,

[0020] wherein:

[0021] Ab is an antibody;

[0022] cD is a cereblon degrader moiety;

[0023] L1 is a linker connecting Ab and cD; and

[0024] p is an integer from 1 to 14.

[0025] Another aspect of the present disclosure is a cereblon degrader-linker intermediate having a structure of Formula II

[0026] X-L3-cD II

[0027] wherein:

[0028] X is a thiol-reactive group;

[0029] L3 is a linker selected from:

[0030] (i) A protease-cleavable non-peptide linker having the following formula:

[0031] -Str-PM-IM-

[0032] where Str is an extender unit covalently linked to X;

[0033] PM is a peptidomimetic unit, and IM is an immolator unit covalently linked to cD;

[0034] (ii) A disulfide linker selected from the following formulas:

[0035] and

[0036] and

[0037] (iii) A linker having the following formula:

[0038]

[0039] where *, indicates the point of attachment to X,

[0040] 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl;

[0041] R 6 is selected from H and C1-C6 alkyl,

[0042] The wavy line indicates the point of attachment to cD,

[0043] R 4a 、R 4b 、R 5a 、R 5a and R 6 The C1-C6 alkyl of are independently and optionally 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; and

[0044] cD is the cereblon degrader moiety, wherein

[0045] (i) cD is a molecular glue (MG); or

[0046] (ii) cD is a cereblon degrader moiety having the formula:

[0047] TPL-L2-E3UL

[0048] wherein:

[0049] TPL is a target protein ligand;

[0050] E3UL is an E3 ubiquitin ligase ligand that binds to cereblon; and

[0051] and L2 is a degrader linker.

[0052] Another aspect of the present disclosure is a cDAC prepared by conjugating an antibody with a cereblon degrader intermediate of formula II.

[0053] Another aspect of the present disclosure is a method for preparing a cDAC, which comprises reacting a thiol-containing antibody with a cereblon degrader intermediate of formula II.

[0054] Another aspect of the present disclosure is a pharmaceutical composition comprising a therapeutically effective amount of a cDAC and one or more pharmaceutically acceptable diluents, vehicles, carriers or excipients.

[0055] Another aspect of the present disclosure is a method for treating cancer, which comprises administering a therapeutically effective amount of a cDAC to a patient in need thereof.

[0056] Another aspect of the present disclosure is the use of a cDAC in the manufacture of a medicament for treating cancer in a mammal.

[0057] Another aspect of the present disclosure is the use of a cDAC for treating cancer in a mammal. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Shows the antiproliferative effect of the BRD4-cereblon degrader on the in vitro potency against KPL-4 and SK-BR-3 cells at 5 days. Cell viability as a percentage of the control was plotted against the concentration (nM) of the cereblon degrader compound cD-5 in a graph.

[0059] Figure 2AThe anti-proliferative effects of the in vitro potency are shown by treating HER2+ KPL-4 cells with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 for 5 days. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0060] Figure 2B The anti-proliferative effects of the in vitro potency are shown by treating HER2+ SK-BR-3 cells with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 for 5 days. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0061] Figure 3A The anti-proliferative effects of the in vitro potency are shown by treating HER2-low / ER+ CAMA1 cells with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 for 5 days. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0062] Figure 3B The anti-proliferative effects of the in vitro potency are shown by treating HER2-low / ER+ EFM19 cells with anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 for 5 days. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0063] Figure 4 The anti-proliferative effects of the in vitro potency are shown by treating various AML cell lines with anti-CD33 BRD4-cereblon degrader antibody conjugate cDAC-3 for 7 days. The AML cell lines are MV-4-11, EOL-1, Molm-13, Nomo-1, HL-60, and OCI-AML-2. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0064] Figure 5AThe anti-proliferative effect of the in vitro potency 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 is shown. Cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0065] Figure 5B The anti-proliferative effect of the in vitro potency 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 is shown. Cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0066] Figure 6A The anti-proliferative effect of the in vitro potency 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 is shown. Cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0067] Figure 6B The anti-proliferative effect of the in vitro potency 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 is shown. Cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0068] Figure 7 The in vivo efficacy of anti-CD33 BRD4-cereblon degrading antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 in reducing tumor volume over time (21 days) in an HL-60 xenograft mouse model at the following doses is shown.

[0069] 1) Vehicle (histidine buffer #8), 100 μL, IV once

[0070] 2) cDAC-4, 3 mg / kg IV once

[0071] 3) cDAC-3, 1 mg / kg IV once

[0072] 4) cDAC-3, 3 mg / kg IV once

[0073] 5) cDAC-3, 10 mg / kg IV once

[0074] 6) cDAC-6, 3 mg / kg IV once

[0075] 7) cDAC-5, 1 mg / kg IV once

[0076] 8) cDAC-5, 3 mg / kg IV once Detailed implementation manners

[0077] Now, certain embodiments of the present disclosure will be described in detail, and examples thereof are shown in the accompanying structures and formulas. Although the present invention will be described in conjunction with the recited embodiments, it should be understood that they are not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalent forms that may be included within the scope provided herein as defined by the claims.

[0078] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. The present invention is in no way limited to the described methods and materials.

[0079] Definitions

[0080] The term "antibody" is used in the broadest sense and specifically encompasses 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 antigen-binding activity. As used herein, "antibody fragment" and all of its grammatical variants are defined as a portion of a full antibody that contains the antigen-binding site or variable region of the full antibody, where the portion does not contain the constant heavy-chain domains of the Fc region of the full 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 an 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) single-chain polypeptides containing only one light-chain variable domain or a fragment thereof containing the three CDRs of the light-chain variable domain, without an associated heavy-chain portion; (3) single-chain polypeptides containing only one heavy-chain variable domain or a fragment thereof containing the three CDRs of the heavy-chain variable domain, without an associated light-chain portion; (4) nanobodies containing 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 an antibody fragment containing one or more heavy chains, the heavy chain may contain any constant domain sequence found in the non-Fc region of the full antibody (e.g., CH1 in the IgG isotype), and / or may contain any hinge region sequence found in the full antibody, and / or may contain a leucine zipper sequence fused to or located within the hinge region sequence or constant domain sequence of the heavy chain.

[0081] "Antibody" refers to a polypeptide containing an antigen-binding region (including complementarity-determining regions (CDRs)) 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 contains a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa) that are linked by disulfide bonds. Each chain is composed of domains, which are called immunoglobulin domains. These domains are classified into different categories by size and function, such as variable domains or regions on the light and heavy chains (V 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 defines a variable region of about 100 to 110 or more amino acids, called the complementarity-determining region, which is mainly responsible for antigen recognition, i.e., the antigen-binding domain. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are large molecules of about 150 kDa composed of four peptide chains. An IgG antibody contains two identical heavy chains of about 50 kDa of the γ class and two identical light chains of about 25 kDa, and thus has a tetrameric quaternary structure. The two heavy chains are linked to each other by disulfide bonds and to the light chains. The resulting tetramer has two identical halves that together form a Y shape. Each end of the fork contains an identical antigen-binding domain. Humans have four IgG subclasses (IgG1, IgG2, IgG3, and IgG4), named in the order of their abundance in serum (i.e., IgG1 is the most abundant). Generally, the antigen-binding domain of an antibody is the most critical in terms of specificity and affinity for binding to cancer cells.

[0082] Antibodies that target specific antigens include bispecific or multispecific antibodies having at least one antigen-binding region that targets a specific antigen. In some embodiments, a targeted monoclonal antibody is a bispecific antibody having at least one antigen-binding region that targets a tumor cell.

[0083] An "antibody construct" refers to an antibody or fusion protein comprising (i) an antigen-binding domain and (ii) an Fc domain.

[0084] 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 the antibody, alone or together with other components that together constitute an antigen-binding construct. Many different types of antibody "fragments" are known in the art, including, for example, (i) Fab fragments, which are monovalent fragments composed of the V L 、V H 、C L and CH1 domains; (ii) F(ab')2 fragments, which are divalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragments, which are composed of the V L and V H domains of a single arm of the antibody; (iv) Fab' fragments, which are generated by disrupting the disulfide bridge of an F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) single-chain Fv (scFv), which are monovalent molecules composed of the two domains of an Fv fragment (i.e., V L and V H ) linked by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain.

[0085] An antibody or antibody fragment can be part of a larger construct, e.g., a conjugate of an antibody fragment with another region or a fusion construct. 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 fusion to a transmembrane domain (optionally using an intervening linker or "stalk" (e.g., a hinge region)) and an optional intracellular signaling domain. For example, an antibody fragment can be fused to the γ and / or δ chains of a T cell receptor to provide a T cell receptor-like construct that binds TROP2. In yet another embodiment, an antibody fragment is part of a bispecific T cell engager (BiTE) that includes a CD1 or CD3 binding domain and a linker.

[0086] "Epitope" means any antigenic determinant or epitope determinant of an antigen to which an antigen-binding domain binds (i.e., at the paratope of the antigen-binding domain). Antigenic determinants generally consist of large groups of chemically reactive surfaces of molecules such as amino acids or sugar side chains, and generally have specific three-dimensional structural features as well as specific charge features.

[0087] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. There are three main classes of Fc receptors: (1) FcγRs that bind IgG, (2) FcαRs that bind IgA, and (3) FcεRs that bind 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 also have different affinities for IgG subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0088] "Amino acid" means any monomer 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-natural (not naturally occurring) amino acids and their stereoisomers. The "stereoisomers" of a given amino acid are isomers that have the same molecular formula and intramolecular bonds but different three-dimensional arrangements of the bonds and atoms (e.g., l-amino acids and the corresponding d-amino acids). Amino acids can be glycosylated (e.g., N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan, or glypication) or deglycosylated. In this document, amino acids may be represented by well-known three-letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0089] Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, 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. Stereoisomers of naturally occurring 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.

[0090] Naturally occurring amino acids include those formed in proteins by post-translational modification, such as citrulline (Cit).

[0091] Non-natural (not naturally occurring) amino acids include, but are not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in L- or D-configurations, which act in a manner similar to naturally occurring amino acids. For example, an "amino acid analog" can be a non-natural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., a carbon bonded to hydrogen, a carboxyl group, and an amino group), but has a modified side chain group or a modified peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. An amino acid mimetic refers to a compound that has a structure different from the general chemical structure of an amino acid but acts in a manner similar to a naturally occurring amino acid.

[0092] "Amino acid side chain" refers to the amino acid group that defines an amino acid and distinguishes one amino acid from other amino acids. For example, the side chains of a representative group of amino acids are: glycine (-H), alanine (-CH3), phenylalanine (-CH2(C6H5)), lysine (-CH2CH2CH2CH2NH2), arginine (-CH2CH2CH2NHC(NH)NH2), leucine -CH2CH(CH3)2, and citrulline (-CH2CH2CH2NHC(O)NH2).

[0093] "Linker" refers to a functional group that covalently links two or more parts in a compound or material. For example, the linking moiety can be used to covalently bind a drug moiety to an antibody construct in the conjugates provided herein or between two or more ligand-binding moieties.

[0094] "Linking moiety" refers to a functional group that covalently bonds two or more parts in a compound. For example, the linking moiety can be used to covalently bind a drug moiety to an antibody in a conjugate. Useful bonds for linking the linking moiety to proteins and other materials include, but are not limited to, amide, amine, ester, carbamate, disulfide, urea, thioether, thiocarbamate, thiocarbonate, and thiourea.

[0095] "Divalent" refers to a chemical moiety containing two attachment points for linking two parts; a multivalent linking moiety can have additional attachment points for linking additional functional groups. A divalent group can be denoted by the suffix "-diyl". For example, divalent linking moieties include divalent polymeric moieties such as divalent poly(ethylene glycol), divalent cycloalkyl, divalent heterocycloalkyl, divalent aryl, and divalent heteroaryl groups. "Divalent cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group" refers to a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl having two attachment points for covalently linking two parts in a molecule or material. The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be substituted or unsubstituted. The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0096] Wavy line Indicates the attachment point of a specified chemical moiety. If a specified chemical moiety has two wavy lines Present, it should be understood that the chemical moiety can be used bilaterally, i.e., read from left to right or from right to left. In some embodiments, a particular moiety having two wavy lines Present is considered to be read from left to right.

[0097] "Alkyl" refers to a straight (linear) or branched chain saturated aliphatic group having the indicated number of carbon atoms. The alkyl group can include any number of carbons, such as from 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, isopropyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, tert-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-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), 1-heptyl, and 1-octyl, and so on. The alkyl group can be substituted or unsubstituted. The substituted alkyl group can be substituted with one or more groups selected from halo, hydroxy, amino, oxo group (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy. The substituted alkyl group can be geminally substituted, where the carbon atoms of the alkyl group form a spiro ring, a cycloalkyl group, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0098] The term "alkylene diyl" refers to a divalent alkyl group. Examples of alkylene diyl include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), etc. Alkylene diyl can also be referred to as an "alkylene" group. Alkylene diyl can be substituted or unsubstituted. Substituted alkylene diyl can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy. Substituted alkylene diyl can be geminally substituted, where the carbon atoms of the alkyl form a spiro ring, cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0099] "Alkenyl" refers to a straight-chain (linear) or branched-chain unsaturated aliphatic group having the indicated number of carbon atoms and at least one carbon-carbon double bond sp2. Alkenyl can contain from 2 to about 12 or more carbon atoms. Alkenyl is a group having "cis" and "trans" orientations or alternatively "E" and "Z" orientations. Examples include, but are not limited to, ethenyl (ethylenyl or vinyl) (-CH=CH2), allyl (-CH2CH=CH2), butenyl, pentenyl, and their isomers. Alkenyl can be substituted or unsubstituted. "Substituted alkenyl" can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0100] The term "alkenylene" or "alkenyl diyl" refers to a straight-chain or branched-chain divalent hydrocarbon group. Examples include, but are not limited to, vinylene (ethylenylene or vinylene) (-CH=CH-), propenylene (-CH2CH=CH-), etc.

[0101] "Alkynyl" refers to a straight-chain (linear) or branched-chain unsaturated aliphatic group 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 their isomers. Alkynyl can be substituted or unsubstituted. "Substituted alkynyl" can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0102] The term "alkynylene" or "alkynyl diyl" refers to a divalent alkynyl group.

[0103] The terms "carbocyclic", "carbocyclic group", "carbocycle" 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 indicated number of atoms. 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, having one or more double or triple bonds in 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.

[0104] The term "cycloalkylene" refers to a divalent cycloalkyl group.

[0105] "Aryl" refers to a monovalent aromatic hydrocarbon group of 6 to 20 carbon atoms (C6-C 20 ) derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. An aryl may be monocyclic, fused to form a bicyclic or tricyclic group, or linked by bonds to form a biaryl. Representative aryls include phenyl, naphthyl and biphenyl. Other aryls include benzyl having a methylene linker group. Some aryls have 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryls have 6 to 10 ring members, such as phenyl or naphthyl.

[0106] The terms "heterocycle", "heterocyclic group" and "heterocyclic ring" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic group having from 3 to about 20 ring atoms, wherein at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur and the remaining ring atoms are C, and wherein one or more of the ring atoms are optionally independently substituted with one or more of the substituents described below. The heterocycle can be a monocyclic ring having from 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P and S) or a bicyclic ring having from 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P and S), such as: a bicyclic [4,5], [5,5], [5,6], or [6,6] system or multiple rings. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A. 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. "Heterocyclic group" also includes groups in which the heterocyclic group is fused to a saturated, partially unsaturated ring or an aromatic carbocyclic or heterocyclic ring. Examples of heterocycles include, but are not limited to: 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, azetidin-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl yl, diazepinyl yl, thiazepinyl groups such as a base, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxolanyl, 1,3-dioxolyl, pyrazolinyl, dithiolanyl, dithiolyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolinylimidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolinyl, and N-pyridylurea. The spiroheterocyclic group is also included within the scope of this definition. Examples of the spiroheterocyclic group include azaspiro[2.5]octanyl and azaspiro[2.4]heptyl. Examples of the heterocyclic group in which two ring atoms are replaced by an oxo(=O) moiety are pyrimidinone and 1,1-dioxothiomorpholinyl. The heterocyclic groups herein may optionally be independently substituted with one or more substituents described herein.

[0107] The term "heteroaryl" refers to a monovalent aromatic group having a 5-, 6- or 7-membered ring and includes a fused ring system having 5 to 20 atoms (wherein at least one is aromatic) containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. Examples of heteroaryl groups are pyridyl (including, for example, 2-hydroxypyridyl), imidazolyl, imidazopyridyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolazinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolinyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furanopyridyl. The heteroaryl groups may optionally be independently substituted with one or more substituents described herein.

[0108] Where possible, the heterocyclic or heteroaryl group may be carbon (carbon-linked) or nitrogen (nitrogen-linked). By way of example but not limitation, the carbon-linked heterocyclic or heteroaryl is bonded at the following positions: the 2, 3, 4, 5 or 6 positions of pyridine, the 3, 4, 5 or 6 positions of pyridazine, the 2, 4, 5 or 6 positions of pyrimidine, the 2, 3, 5 or 6 positions of pyrazine, the 2, 3, 4 or 5 positions of furan, tetrahydrofuran, thiophene, thienyl, pyrrole or tetrahydropyrrole rings, the 2, 4 or 5 positions of oxazole, imidazole or thiazole, the 3, 4 or 5 positions of isoxazole, pyrazole or isothiazole, the 2 or 3 positions of aziridine, the 2, 3 or 4 positions of azetidine, the 2, 3, 4, 5, 6, 7 or 8 positions of quinoline, or the 1, 3, 4, 5, 6, 7 or 8 positions of isoquinoline.

[0109] By way of example and not limitation, the nitrogen-bonded heterocycle or heteroaryl is bonded at the following positions: 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, dihydroindole, 1H-indazole; the 2-position of isoindole or isoindoline; the 4-position of morpholine; and the 9-position of carbazole or β-carboline.

[0110] The term “halo” or “halogen” by itself or as part of another substituent means a fluorine, chlorine, bromine, or iodine atom.

[0111] The term “carbonyl” by itself or as part of another substituent means C(=O) or –C(=O)–, i.e., a carbon atom double-bonded to an oxygen and bonded to two other groups in the moiety having the carbonyl.

[0112] The term “chiral” refers to a molecule having non-superimposability on its mirror image, while the term “achiral” refers to a molecule that is superimposable on its mirror image.

[0113] The term “stereoisomer” refers to compounds having the same chemical composition but different arrangements of atoms or groups in space.

[0114] “Diastereomers” denote stereoisomers having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. A mixture of diastereomers can be separated under high-resolution analytical procedures such as electrophoresis and chromatography.

[0115] “Enantiomers” refer to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0116] The stereochemical definitions and conventions used herein generally follow those described in McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, edited by S.P. Parker; 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 thus exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds described herein are part of the present disclosure, which stereoisomeric forms include, but are not limited to, diastereomers, enantiomers, and atropisomers and mixtures thereof, such as racemic mixtures. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule around its chiral center. The prefixes d and l or (+) and (-) are used to denote the sign of rotation of the compound of plane-polarized light, where (-) or l indicates that the compound is levorotatory. A compound with the prefix (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except for being mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is commonly referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur in the absence of stereoselection or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two optically inactive enantiomeric species. Enantiomers can be separated from a racemic mixture by chiral separation methods such as supercritical fluid chromatography (SFC). Although stereochemistry is explicitly constructed, for example, from X-ray crystallographic data, the assignment of configuration at a chiral center of an isolated enantiomer may be tentative, as described for illustrative purposes in the structure of Table 1.

[0117] The terms “treat”, “treatment” and “treating” refer to any indication of successful treatment or amelioration of an injury, pathology, condition (e.g., cancer) or symptom (e.g., cognitive impairment), including any objective or subjective parameter, such as alleviation; remission; making a symptom or symptoms, injury, pathology or condition more tolerable to the patient; decreasing the rate of progression of a symptom; decreasing the frequency or duration of a symptom or condition or condition; or in some cases, preventing the onset of a symptom. Treatment or amelioration of a symptom can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0118] As used herein, the terms “cancer,” “neoplasm,” and “tumor” are used to refer to cells that exhibit autonomous, unregulated 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 the present disclosure include cancer cells (e.g., cancer cells from an individual having cancer), malignant cancer cells, pre-metastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. Cancers of almost every tissue are known. The phrase “cancer burden” refers to the amount of cancer cells or cancer volume in a subject. Thus, reducing the cancer burden refers to reducing the number or volume of cancer cells in a subject. The term “cancer cell” as used herein refers to any cell that is a cancer cell (e.g., from any cancer treatable in an individual, e.g., isolated from an individual having cancer) or is derived from a cancer cell (e.g., a clone of a cancer cell). For example, a cancer cell can be from an established cancer cell line, can be a primary cell isolated from an individual having cancer, can be a progeny cell of a primary cell isolated from an individual having cancer, etc. In some embodiments, the term can also refer to a portion of a cancer cell, such as a subcellular portion of a cancer cell, a cell membrane portion, or a cell lysate. Many types of cancers are known to those of skill in the art, including solid tumors such as epithelial cancers, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, and circulating cancers such as leukemias.

[0119] As used herein, the term “cancer” includes any form of cancer, including but not limited to solid tumor cancers (e.g., skin cancer, lung cancer, prostate cancer, breast cancer, gastric cancer, bladder cancer, colon cancer, ovarian cancer, pancreatic cancer, kidney cancer, liver cancer, glioblastoma, medulloblastoma, leiomyosarcoma, head and neck squamous cell carcinoma, melanoma, and neuroendocrine cancer) and liquid cancers (e.g., hematological cancers); epithelial cancers; soft tissue tumors; sarcomas; teratomas; melanomas; leukemias; lymphomas; and brain cancers, including minimal residual disease, and including both primary and metastatic tumors.

[0120] The phrases "effective amount" and "therapeutically effective amount" refer to the dose or amount of a therapeutic agent that produces the therapeutic effect for which it is administered. The exact dose will depend on the purpose of the treatment and will be determinable by those 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, 11th ed. (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22nd ed., (Pharmaceutical Press, London, 2012)). In the case of cancer, a therapeutically effective amount of a therapeutic agent can reduce the number of cancer cells; decrease the tumor size; inhibit (i.e., slow down and preferably stop to some extent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more cancer-related symptoms to some extent. To the extent that a therapeutic agent can prevent the growth and / or kill existing cancer cells, it can inhibit cell growth and / or be cytotoxic. For cancer therapy, for example, efficacy can be measured by assessing the time to progression (TTP) and / or determining the response rate (RR).

[0121] "Recipient", "individual", "subject", "host", and "patient" are used interchangeably and refer to any mammalian subject (e.g., human) in need of diagnosis, treatment, or therapy. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo animals, sport animals, or pets such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is a human. A "patient" or "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the patient or individual or subject is a human. In some embodiments, the patient can be a "cancer patient", i.e., a patient suffering from one or more cancer symptoms or at risk of developing one or more cancer symptoms. A "patient population" refers to a group of cancer patients. Such a population can be used to demonstrate statistically significant drug efficacy and / or safety.

[0122] As used herein, the term "administer" 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 slow-release device (e.g., a micro-osmotic pump) to a subject.

[0123] The terms "residue", "moiety", or "group" refer to a component that is covalently bound or linked to another component.

[0124] The term "covalently bound" or "covalently linked" refers to a chemical bond formed by sharing one or more pairs of electrons.

[0125] As used herein, the term "peptidomimetic" or PM refers to a non-peptide chemical moiety that is part of a linker. A peptide is a short chain of (two or more) amino acid monomers linked by peptide (amide) bonds, while a peptidomimetic chemical moiety includes non-amino acid chemical moieties. A peptidomimetic chemical moiety may also contain 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 in any part of its chemical structure.

[0126] CEREBLON Degrader Antibody Conjugate

[0127] The cereblon degrader antibody conjugate (cDAC) provided herein comprises at least one (p)cereblon degrader moiety (cD) covalently linked to an antibody (Ab) via an antibody linker (L1).

[0128] The cereblon degrader antibody conjugate (cDAC) induces target-specific degradation of tumor-associated proteins and confers specificity to minimize off-target toxic effects. In embodiments, the cD forms a cereblon-based ternary complex between the target protein and the E3 ubiquitin ligase cereblon.

[0129] Exemplary embodiments of cDAC have a structure of Formula I:

[0130] Ab-[L1-cD] p I

[0131] or a pharmaceutically acceptable salt thereof,

[0132] wherein:

[0133] Ab is an antibody;

[0134] L1 is an antibody linker;

[0135] cD is the cereblon degrader moiety; and

[0136] p is an integer from 1 to 14.

[0137] In some embodiments, L1 comprises a sacrificial moiety. In embodiments, L1 is L 1a -IM, where IM is the sacrificial moiety and L 1a is any remaining portion of the L1 antibody linker. In embodiments, L1 is the sacrificial moiety IM.

[0138] In some embodiments, cD comprises a cereblon-binding E3 ubiquitin ligase ligand E3UL. In some embodiments, cD is E3UL-cD a , where E3UL is the E3 ubiquitin ligase ligand of the cereblon degrader moiety that binds to cereblon, and cD a is any remaining portion 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.

[0139] Exemplary embodiments of cDAC have a structure of Formula I':

[0140] Ab-[L 1a -IM—E3UL-cD a p

[0141] I'

[0142] ​In some embodiments, the cereblon degrader moiety (cD) of the cDAC is covalently linked to the antibody linker (L1) through an acetalamine group.

[0143] In some embodiments, L1 comprises a sacrificial moiety IM selected from:

[0144]

[0145] wherein

[0146] * indicates the point of attachment to L 1a and ** indicates the point of attachment to cD a and the wavy line indicates the point of attachment to E3UL.

[0147] Exemplary embodiments of the cDAC have a structure of Formula I-A

[0148]

[0149] wherein

[0150] Ab is an antibody;

[0151] L 1a is an antibody linker;

[0152] Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C3-C 20 heterocyclic group, and C3-C 20 heteroaryl;

[0153] The dashed line indicates an optional double bond;

[0154] Z 1 is selected from C(R 1 )2, CR 1 , N, and NR 1a , and

[0155] Z 2 is selected from C(R 2 )2, CR 2 , N, and NR 2a , wherein

[0156] R 1 and R 2 are each independently selected from the group consisting of: H, F, Cl, Br, I, -CN, C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, (C1-C6 alkyl diyl)-(C6-C 20Aryl), -(C1-C6 alkyldiyl)-NR a R b 、-(C1-C6 alkyldiyl)-OR a 、(C1-C6 alkyldiyl)-(C3-C 20 Carbocyclic), (C1-C6 alkyldiyl)-(C2-C 20 Heterocyclic), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclic group, C2-C 20 Heterocyclic group, 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)2NR a and -S(O)3H;

[0157] R 1a and R 2a Each independently selected from the group consisting of: 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 Carbocyclic), (C1-C6 alkyldiyl)-(C2-C 20 Heterocyclic), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20Carbocyclic group, C2-C 20 Heterocyclic group, 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 alkyl diyl)-NR a R b 、-C(=O)OR a 、-NR a R b 、-OR a 、-S(O)R a 、-S(O)2R a 、-S(O)2NR a and -S(O)3H; or

[0158] (i) Two geminal R 1 or two geminal R 2 form a 3- to 6-membered carbocyclic or heterocyclic spiro group, or

[0159] (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, carbocyclic, heterocyclic or heteroaryl group, where

[0160] R a and R b are each independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl;

[0161] Each alkyl, alkylene, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group, and heteroaryl is independent and optionally substituted with one or more groups selected from the following: 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(=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;

[0162] cD a is the remainder of the cereblon degrader moiety; and

[0163] p is an integer from 1 to 14.

[0164] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are each H.

[0165] In some embodiments, ring A is C3-C 20Heteroaryl.

[0166] In some embodiments, ring A is an isoindoline substituted with ═O.

[0167] In some embodiments, the cDAC of formula (I) has the structure of formula I-A’:

[0168]

[0169] wherein X 1 is selected from CH2 and C(═O).

[0170] In some embodiments, the antibody is a thiol-containing antibody.

[0171] In some embodiments, the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

[0172] In some embodiments, the antibody is a cysteine-engineered antibody.

[0173] In some embodiments, the cysteine-engineered antibody comprising one or more cysteine mutations is selected from HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

[0174] In some embodiments, L 1a is a protease-cleavable non-peptide linker.

[0175] 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.

[0176] In some embodiments, L 1a has the structure of formula L1-A

[0177]

[0178] wherein

[0179] * indicates the point of attachment to the cysteine thiol of Ab;

[0180] [[ID=5"3]]R 1 is selected from the group consisting of: C1-C 12 alkylene, C1-C 12 alkylene-C(═O), C1-C 12 alkylene-NH, (CH2CH2O) r C1-C 12Alkylene-NH, (CH2CH2O) r -C(=O), (CH2CH2O) r -C(=O), (CH2CH2O) r -CH2 and C1-C 12 Alkylene-NHC(=O)CH2CH(thiophen-3-yl), wherein

[0181] r is an integer in the range of 1 to 10; and

[0182] C1-C 12 The alkylene is 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;

[0183] 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; and

[0184] 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.

[0185] In some embodiments, AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2 and -CH2CH2CH2NHC(O)NH2.

[0186] In some embodiments, R 1 is a C5 alkylene.

[0187] In some embodiments, R2 and R 3 together form a C4 cycloalkyl ring.

[0188] In some embodiments, AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0189] In some embodiments,

[0190] R 1 is a C5 alkylene;

[0191] R 2 and R 3 together form a C4 cycloalkyl ring; and

[0192] AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0193] In some embodiments, cD a comprises (i) a target protein ligand covalently linked to a degrader linker, or (ii) a molecular glue 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.

[0194] In some embodiments, p is 1, 2, 3, 4, 5, or 6.

[0195] Exemplary embodiments of cDAC have the structure of Formula I-B

[0196]

[0197] wherein

[0198] Ab is an antibody;

[0199] L 1a is an antibody linker;

[0200] Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic, C2-C 20 heterocyclic, and C1-C 20 heteroaryl;

[0201] The dashed line indicates an optional double bond;

[0202] Z 1 is selected from C(R 1 )2, CR 1 , N, and NR 1a , and

[0203] Z2 Selected from C(R 2 )2, CR 2 , N and NR 2a , wherein

[0204] R 1 and R 2 are each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H;

[0205] R 1a and R 2a are independently selected from the group consisting of: H, C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12Alkynyl, (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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or

[0206] (i) Two geminal R 1 or two geminal R 2 form a 3- to 6-membered carbocyclic or heterocyclic spiro group, or

[0207] (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, carbocyclic, heterocyclic or heteroaryl group, where

[0208] R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12alkyl, C2-C 12 alkenyl, and C2-C 12 alkynyl;

[0209] wherein each alkyl, alkylene, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group, and heteroaryl is independent and optionally substituted with one or more groups selected from: 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(=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;

[0210] cD a is the remainder of the cereblon degrader moiety; and

[0211] p is an integer from 1 to 14.

[0212] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R2 Each is H.

[0213] In some embodiments, Ring A is C3-C 20 heteroaryl.

[0214] In some embodiments, Ring A is an isoindoline substituted with =O.

[0215] 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 BRD4.

[0216] In some embodiments, the cDAC of Formula I-B comprises the structure of Formula I-B’

[0217]

[0218] where X 1 is selected from CH₂ and C(=O).

[0219] In some embodiments, the antibody is a thiol-containing antibody.

[0220] In some embodiments, the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

[0221] In some embodiments, the antibody is a cysteine-engineered antibody.

[0222] In some embodiments, the cysteine-engineered antibody has one or more cysteine mutation sites selected from: HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

[0223] In some embodiments, L 1a is a protease-cleavable non-peptide linker.

[0224] In some embodiments, L 1a has the structure of L1-A

[0225]

[0226] where

[0227] * indicates the point of attachment to the cysteine thiol of Ab;

[0228] R 1 is selected from the group consisting of: C1-C 12 alkylene, C1-C 12Alkylene-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)CH2CH(thiophen-3-yl), wherein

[0229] r is an integer in the range of 1 to 10; and

[0230] C1-C 12 The alkylene is 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;

[0231] 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; and

[0232] 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.

[0233] In some embodiments, AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

[0234] In some embodiments, R 1 is a C5 alkylene group.

[0235] In some embodiments, R 2 and R 3 together form a C4 cycloalkyl ring.

[0236] In some embodiments, AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0237] In some embodiments:

[0238] R 1 is a C5 alkylene group;

[0239] 〕R 2 and R 3 together form a C4 cycloalkyl ring; and

[0240] AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0241] In some embodiments, cDa comprises (i) a target protein ligand covalently linked to a degrader linker, or (ii) a molecular glue 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 BRD4.

[0242] In some embodiments, p is 1, 2, 3, 4, 5, or 6.

[0243] In some embodiments, L1 is a sacrificial moiety IM.

[0244] Exemplary embodiments of cDAC have a structure of Formula I":

[0245] Ab-[IM—E3UL-cD a p

[0246] I"

[0247] In some embodiments, IM is:

[0248]

[0249] where

[0250] * indicates the point of attachment to Ab,

[0251] ** indicates the point of attachment to cD a ; and `

[0252] The wavy line indicates the point of attachment to E3UL.

[0253] 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and 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.

[0254] Exemplary embodiments of cDAC have the structure of Formula I-C

[0255]

[0256] wherein

[0257] Ab is an antibody;

[0258] 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and 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;

[0259] Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic, C2-C 20 heterocyclic and C1-C 20 heteroaryl;

[0260] Dashed line Indicates an optional double bond;

[0261] Z 1 is selected from C(R 1 )2, CR 1 , N, and NR 1a , and

[0262] Z 2 is selected from C(R 2 )2, CR 2 , N, and NR 2a , where

[0263] R 1 and R 2 are each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NRa and -S(O)3H;

[0264] R 1a and R 2a are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or

[0265] (i) two geminal R 1 or two geminal R 2 form a 3- to 6-membered carbocyclic or heterocyclic spiro group, or

[0266] (ii) R 1 and R 2 , R 1a and R 2 , R 1 and R 2a , or R 1a and R 2ato form a fused 5- or 6-membered aryl, carbocyclic, heterocyclic or heteroaryl group, wherein

[0267] R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl;

[0268] wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclic, heterocyclic and heteroaryl is independently and optionally substituted with one or more groups selected from: 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(=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;

[0269] cD ais the remainder of the cereblon degrader moiety; and

[0270] p is an integer from 1 to 14.

[0271] In some embodiments, sulfur is conjugated to the cysteine thiol of Ab to form a disulfide bond.

[0272] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are each H.

[0273] In some embodiments, Ring A is a C3-C 20 heteroaryl.

[0274] In some embodiments, Ring A is an isoindoline substituted with =O or an oxo group.

[0275] 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 BRD4.

[0276] In some embodiments, the cDAC of Formula I-C comprises the structure of Formula I-C’

[0277] wherein

[0278] Ab is an antibody;

[0279] 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and 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;

[0280] X1 Selected from CH2 and C(=O);

[0281] Dashed line Indicates an optional double bond;

[0282] Z 1 Selected from C(R 1 )2, CR 1 , N and NR 1a , and

[0283] Z 2 Selected from C(R 2 )2, CR 2 , N and NR 2a , wherein

[0284] R 1 and R 2 are each independently selected from the group consisting of: H, F, Cl, Br, I, -CN, C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, (C1-C6 alkyl diyl)-(C6-C 20 aryl), -(C1-C6 alkyl diyl)-NR a R b , -(C1-C6 alkyl diyl)-OR a , (C1-C6 alkyl diyl)-(C3-C 20 carbocyclic group), (C1-C6 alkyl diyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyl diyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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 alkyl diyl)-NR a R b , -C(=O)OR a , -NR a R b , -NO2, =O, -OR a , -OC(=O)R a , -SR a , -S(O)Ra 、 -S(O)2R a 、 -S(O)2NR a and -S(O)3H;

[0285] R 1a and R 2a are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or

[0286] (i) Two geminal R 1 or two geminal R 2 form a 3- to 6-membered carbocyclic or heterocyclic spiro group, or

[0287] (ii) R 1 and R 2 、 R 1a and R 2 、 R 1 and R 2a 、 or R1a and R 2a form a fused 5- or 6-membered aryl, carbocyclic, heterocyclic or heteroaryl group, wherein

[0288] R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, wherein the phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl;

[0289] wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclic, heterocyclic and heteroaryl is independently and optionally substituted by one or more groups selected from: 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(=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;

[0290] cDa is the remainder of the cereblon degrader moiety; and

[0291] p is an integer from 1 to 14.

[0292] In some embodiments, the antibody is a thiol-containing antibody.

[0293] In some embodiments, the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

[0294] In some embodiments, the antibody is a cysteine-engineered antibody.

[0295] In some embodiments, the cysteine-engineered antibody comprises one or more cysteine mutations selected from the following: HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HCY373C.

[0296] In some embodiments, cD a comprises (i) a target protein ligand covalently linked to a degrader linker, or (ii) a molecular glue 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.

[0297] In some embodiments, p is 1, 2, 3, 4, 5, or 6.

[0298] CEREBLON DEGRADER-LINKER INTERMEDIATE

[0299] A cereblon degrader-linker intermediate (cDLI) is a reagent for a method of preparing 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 linked to the cereblon degrader moiety (cD) via a linker (L3).

[0300] In some embodiments, the cereblon degrader-linker intermediate has a structure of Formula II:

[0301] X-L3-cD II

[0302] Where:

[0303] X is a thiol-reactive group covalently linked to L3;

[0304] L3 is a linker covalently linked to X and cD; and

[0305] cD is a cereblon degrader moiety covalently linked to L3.

[0306] cD can be a hetero-bifunctional divalent cereblon degrader moiety or a molecular glue cereblon degrader moiety.

[0307] In some embodiments, L3 comprises a sacrificial moiety. In an embodiment, L3 is L 3a -IM, where IM is the sacrificial moiety and L 3a is any remaining portion of the L3 linker. In an embodiment, L3 is the sacrificial moiety IM.

[0308] In some embodiments, cD comprises an E3 ubiquitin ligase ligand E3UL that binds to cereblon. In some embodiments, cD is E3UL-cD a where E3UL is the E3 ubiquitin ligase ligand of the cereblon degrader moiety cD that binds to cereblon, and cD a is any remaining portion 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.

[0309] Exemplary embodiments of cDLI have a structure of Formula II’:

[0310] X-L 3a -IM—E3UL-cD a II’

[0311] In some embodiments, the cereblon degrader moiety (cD) of cDLI is linked to the antibody linker (L3) via an acetalamine group.

[0312] In some embodiments, L3 comprises a sacrificial moiety IM selected from:

[0313]

[0314] where

[0315] * indicates the point of attachment to any remaining portion of the L3 linker L 3a ;

[0316] [[ID=5l]]** indicates the point of attachment to cD a ; and

[0317] The wavy line indicates the point of attachment to E3UL.

[0318] Exemplary embodiments of cDLI have a structure of Formula II-A

[0319]

[0320] wherein

[0321] X is a thiol-reactive group;

[0322] L 3a is a linker;

[0323] Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C3-C 20 heterocyclic group, and C3-C 20 heteroaryl;

[0324] The dashed line indicates an optional double bond;

[0325] Z 1 is selected from C(R 1 )2, CR 1 , N, and NR 1a , and

[0326] Z 2 is selected from C(R 2 )2, CR 2 , N, and NR 2a , where

[0327] R 1 and R 2 are each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H;

[0328] R 1a and R 2a Each independently selected from the group consisting of: 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 Carbocyclic), (C1-C6 alkyldiyl)-(C2-C 20 Heterocyclic), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclic group, C2-C 20 Heterocyclic group, 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)2NR a and -S(O)3H; or

[0329] (i) Two geminal Rs 1 or two vicinal Rs 2 form a 3- to 6-membered carbocyclic or heterocyclic spiro group, or

[0330] (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, carbocyclic, heterocyclic or heteroaryl group, where

[0331] R a and R b are each independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where the phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl;

[0332] Each alkyl, alkylenediyl, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group, and heteroaryl is independent and optionally substituted with one or more groups selected from the following: 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(=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;

[0333] cD a is the remainder of the cereblon degrader moiety; and

[0334] p is an integer from 1 to 14.

[0335] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are each H.

[0336] In some embodiments, ring A is C3-C 20Heteroaryl.

[0337] In some embodiments, Ring A is an isoindoline substituted with =0.

[0338] In some embodiments, cD a In some embodiments, TPL comprises a ligand that binds to BRD4.

[0339] In some embodiments, the cDLI of Formula II has the structure of Formula II-A'

[0340]

[0341] in

[0342] X is a thiol-reactive group;

[0343] L 3a is a connector; and

[0344] X 1 Selected from CH2 and C(=O).

[0345] In some embodiments, L 3a It is a non-peptide linker cleavable by protease.

[0346] In some embodiments, XL 3a Having the structure of formula L3-A

[0347]

[0348] in

[0349] R 1 Select from the group consisting of: 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)CH2CH(thiophen-3-yl), wherein

[0350] r is an integer in the range of 1 to 10; and

[0351] C1-C 12The alkylene group is optionally substituted with one or more groups selected from the following: 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;

[0352] R 2 and R 3 together form a C3-C7 cycloalkyl ring optionally substituted with one or more groups selected from the following: 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; and

[0353] 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.

[0354] In some embodiments, AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

[0355] In some embodiments, R 1 is a C5 alkylene group.

[0356] In some embodiments, R 2 and R 3 together form a C4 cycloalkyl ring.

[0357] In some embodiments, AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0358] In some embodiments,

[0359] R 1 is a C5 alkylene group;

[0360] R 2 and R3 Together form a C4 cycloalkyl ring; and

[0361] AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0362] In some embodiments, cDa comprises (i) a target protein ligand covalently linked to a degrader linker, or (ii) a molecular glue 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 BRD4.

[0363] Exemplary embodiments of cDLI have the structure of Formula II-B

[0364]

[0365] wherein

[0366] X is a thiol-reactive group;

[0367] L 3a is a linker;

[0368] Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic, C2-C 20 heterocyclic, and C1-C 20 heteroaryl;

[0369] The dashed line indicates an optional double bond;

[0370] Z 1 is selected from C(R 1 )2, CR 1 , N, and NR 1a , and

[0371] Z 2 is selected from C(R 2 )2, CR 2 , N, and NR 2a , wherein

[0372] R 1 and R 2 are each independently selected from the group consisting of: H, F, Cl, Br, I, -CN, C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, (C1-C6 alkyl diyl)-(C6-C 20 aryl), -(C1-C6 alkyl diyl)-NR a R b, -(C1-C6 alkyldiyl)-OR a , (C1-C6 alkyldiyl)-(C3-C 20 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H;

[0373] R 1a and R 2a are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or

[0374] (i) Two geminal R 1 or two geminal R 2 form a 3- to 6-membered carbocyclic or heterocyclic spiro group, or

[0375] (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, carbocyclic, heterocyclic or heteroaryl group, where

[0376] R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl;

[0377] Each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group, and heteroaryl is independent and optionally substituted with one or more groups selected from the following: 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(=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; and

[0378] cD a is the remainder of the cereblon degrader moiety.

[0379] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are each H.

[0380] In some embodiments, ring A is a C3-C 20 heteroaryl.

[0381] In some embodiments, Ring A is an isatin substituted with =O.

[0382] 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 BRD4.

[0383] In some embodiments, the cDLI of Formula II-B comprises the structure of Formula II-B’

[0384]

[0385] X is a thiol-reactive group;

[0386] L 3a is a linker; and

[0387] X 1 is selected from CH2 and C(=O).

[0388] In some embodiments, L 3a is a protease-cleavable non-peptide linker.

[0389] In some embodiments, X-L 3a has the structure of Formula L3-A

[0390]

[0391] where

[0392] R 1 is selected from the group consisting of: 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)CH2CH(thiophen-3-yl), where

[0393] r is an integer in the range of 1 to 10; and

[0394] C1-C 12The alkylene group is optionally substituted by one or more groups selected from the following: 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;

[0395] R 2 and R 3 together form a C3-C7 cycloalkyl ring optionally substituted by one or more groups selected from the following: 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; and

[0396] 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.

[0397] In some embodiments, AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

[0398] In some embodiments, R 1 is a C5 alkylene group.

[0399] In some embodiments, R 2 and R 3 together form a C4 cycloalkyl ring.

[0400] In some embodiments, AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0401] In some embodiments:

[0402] R 1 is a C5 alkylene group;

[0403] R 2 and R3 together form a C4 cycloalkyl ring; and

[0404] AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

[0405] In some embodiments, cDa comprises (i) a target protein ligand covalently linked to a degrader linker, or (ii) a molecular glue 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 BRD4.

[0406] Exemplary embodiments of cDLI have a structure of Formula II”:

[0407] X-IM—E3UL-cD a II”

[0408] In an embodiment, X-IM comprises where

[0409] ** indicates the point of attachment to cD a and the wavy line indicates the point of attachment to E3UL.

[0410] 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and 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.

[0411] Exemplary embodiments of cDLI have a structure of Formula II-C

[0412]

[0413] where

[0414] R 4a 、R 4b 、R5a and R 5a are each independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and 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;

[0415] Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group and C1-C 20 heteroaryl;

[0416] The dashed line indicates an optional double bond;

[0417] Z 1 is selected from C(R 1 )2, CR 1 , N and NR 1a , and

[0418] Z 2 is selected from C(R 2 )2, CR 2 , N and NR 2a , wherein

[0419] R 1 and R 2 are each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C​​​​20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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 alkylene)-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)2NR a and -S(O)3H;

[0420] R 1a and R 2a are independently selected from the group consisting of: H, C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, (C1-C6 alkylene)-(C6-C 20 aryl), -(C1-C6 alkylene)-NR a R b 、-(C1-C6 alkylene)-OR a 、(C1-C6 alkylene)-(C3-C 20 carbocyclic group), (C1-C6 alkylene)-(C2-C 20 heterocyclic group), (C1-C6 alkylene)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or

[0421] (i) Two geminal R 1 or two vicinal R 2 form a 3- to 6-membered carbocyclic or heterocyclic spiro group, or

[0422] (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, carbocyclic, heterocyclic or heteroaryl group, where

[0423] R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl;

[0424] Each alkyl, alkylene, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group, and heteroaryl is independent and optionally substituted with one or more groups selected from: 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(=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; and

[0425] cD a is the remainder of the cereblon degrader moiety.

[0426] In some embodiments, Z 1 is CR 1 and Z 2 is CR 2 and R 1 and R 2 are each H.

[0427] In some embodiments, ring A is a C3-C 20 heteroaryl.

[0428] In some embodiments, Ring A is an isoindoline substituted with =O or an oxo group.

[0429] 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.

[0430] In some embodiments, the cDLI of Formula II-C comprises the structure of Formula II-C’

[0431]

[0432] where

[0433] 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl, where the C1-C6 alkyl is independently and 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;

[0434] Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group and C1-C 20 heteroaryl;

[0435] The dashed line indicates an optional double bond;

[0436] Z 1 is selected from C(R 1 )2, CR 1 , N and NR 1a , and

[0437] Z 2 is selected from C(R 2 )2, CR 2 , N and NR 2a , where

[0438] R1 and R 2 each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H;

[0439] R 1a and R 2a are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or

[0440] (i) Two geminal R 1 or two geminal R 2 form a 3- to 6-membered carbocyclic or heterocyclic spiro group, or

[0441] (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, carbocyclic, heterocyclic or heteroaryl group, where

[0442] R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl;

[0443] Each alkyl, alkylene, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group, and heteroaryl is independent and optionally substituted with one or more groups selected from: 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(=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; and

[0444] X 1 is selected from CH2 and C(=O); and

[0445] cD a is the remainder of the cereblon degrader moiety.

[0446] In some embodiments, cDa comprises (i) a target protein ligand covalently linked to a degrader linker, or (ii) a molecular glue 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 BRD4.

[0447] Cereblon-binding E3 ubiquitin ligase ligand

[0448] The cereblon-binding E3 ubiquitin ligase ligand (E3UL) is the part that binds to cereblon in the E3 ubiquitin ligase complex.

[0449] In some embodiments, the E3UL contains a glutarimide group.

[0450] In some embodiments, the cereblon degrader moiety (cD) of the antibody conjugate (cDAC) has a structure selected from the following formulae:

[0451]

[0452] where 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;

[0453] Z 1 is selected from C(R 1 )2, CR 1 , N, and NR 1a ;

[0454] Z 2 is selected from C(R 2 )2, CR 2 , N, and NR 2a ;

[0455] R is selected from H and C1-C6 alkyl;

[0456] R 1 and R 2 are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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 、-NO2、=O、-OR a 、-OC(=O)R a 、-SR a 、-S(O)R a 、-S(O)2R a 、-S(O)2NR a and -S(O)3H;

[0457] R 1a and R 2a Independently selected from the group consisting of: 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 Carbocyclic), (C1-C6 alkyldiyl)-(C2-C 20 Heterocyclic), (C1-C6 alkyldiyl)-(C1-C 20 Heteroaryl), C6-C 20 Aryl, C3-C 20 Carbocyclic group, C2-C 20 Heterocyclic group, 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 、-ORa 、 -S(O)R a 、 -S(O)₂R a 、 -S(O)₂NR a and -S(O)₃H;

[0458] or (i) two geminal Rs 1 or two geminal Rs 2 form a 3- to 6-membered carbocyclic or heterocyclic 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, carbocyclic, heterocyclic or heteroaryl group;

[0459] R a and R b are independently selected from H, OH, C₁-C₆ alkyl, phenyl and benzyl, where phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C₁-C 12 alkyl, C₂-C 12 alkenyl and C₂-C 12 alkynyl, and

[0460] A is selected from C₆-C 20 aryl, C₃-C 20 carbocyclic, C₂-C 20 heterocyclic and C₁-C 20 heteroaryl;

[0461] Any of the alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclic, heterocyclic, and heteroaryl groups is independently and optionally substituted by one or more groups independently selected from the following: 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(=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.

[0462] In some embodiments, Z 1 and Z 2 each is CR 1 and R 1 and R 2 each is H.

[0463] In some embodiments, R is H.

[0464] In some embodiments, A is a C6-C 20 aryl.

[0465] In some embodiments,

[0466] Z 1and Z 2 are each CR 1 , where R 1 and R 2 are each H;

[0467] R is H; and

[0468] A is C6-C 20 aryl.

[0469] In some embodiments, the E3 UL of the cereblon degrader moiety (cD) of the antibody conjugate (cDAC) has a structure selected from the following formulae:

[0470] and

[0471] where X 1 is selected from CH2 and C(=O); and the wavy line indicates the point of attachment of the antibody linker L1 of Formula I, the linker L3 of Formula II, and / or the degrader linker L2 of the cereblon degrader moiety cD.

[0472] In some embodiments, X 1 is C(=O).

[0473] Antibody linker

[0474] 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 of the cDAC in the bloodstream and at the same time allow for efficient cleavage upon internalization into target cells. The specific design of the antibody linker affects various aspects of the cDAC pharmacology, including the stability of the drug in circulation, tumor cell permeability, drug-to-antibody ratio (DAR) (i.e., the number of payload molecules carried by each antibody), and the extent of the bystander effect.

[0475] The disclosed antibody linkers can include cleavable non-peptidic peptidomimetic units (PM). The PM can be a substrate for lysosomal proteases, although it does not contain a peptide (WO 2015 / 095227; WO 2015 / 095124; WO 2015 / 095223). For example, a peptidomimetic linker containing cyclobutane-1,1-dicarboxamide is mainly hydrolyzed by cathepsin B, while a valine-citrulline dipeptide linker is not hydrolyzed. Antibody-drug conjugates carrying a PM linker may be as effective and stable in vivo as antibody-drug conjugates with a dipeptide linker (Wei et al., (2018) J. Med. Chem. 61:989-1000).

[0476] In some embodiments, L1 is a protease-cleavable non-peptidic linker having the following formula:

[0477] -Str-PM-IM-

[0478] Wherein Str is an extender unit covalently linked to an antibody; PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to a cereblon degrader moiety.

[0479] In some embodiments, Str has the following formula:

[0480]

[0481] Wherein

[0482] * indicates the point of attachment to the cysteine thiol of the antibody on the succinimide ring, and

[0483] R 1 is selected from the group consisting of: 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)CH2CH(thiophen-3-yl), where r is an integer in the range of 1 to 10, and C1-C 12 alkylene is 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.

[0484] In some embodiments, Str is selected from the following:

[0485]

[0486]

[0487] where * indicates the point of attachment to the cysteine thiol of the antibody.

[0488] In some embodiments of R 1 , C1-C12 The alkylene group is a C1-C5 alkylene group.

[0489] In some embodiments, R 1 is (CH2)5 or a C5 alkylene group.

[0490] In some embodiments, PM has the following formula:

[0491]

[0492] wherein 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, and

[0493] 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.

[0494] In some embodiments, AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

[0495] In some embodiments, R 2 and R 3 together form a C4 cycloalkyl ring; and AA is -CH3.

[0496] In some embodiments, R 2 and R 3 together form a C4 cycloalkyl ring; and AA is -CH2CH2CH2NHC(O)NH2.

[0497] In some embodiments, Formula I includes a sacrificial moiety selected from the following:

[0498]

[0499] where * indicates the connection point to the remainder of L1 and the wavy line indicates the connection point to cD;

[0500] 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl; and

[0501] C1-C6 alkyl is independently and 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.

[0502] In some embodiments, the cereblon degrader moiety (cD) of cDAC is linked to the antibody linker (L1) through an acetalamine group at the nitrogen atom of the succinimide group of cD.

[0503] In some embodiments, the IM-cD of the antibody conjugate (cDAC) comprises a structure selected from:

[0504]

[0505] where the wavy line indicates the connection point to the remainder of linker L1 of the heterobifunctional cD of formula I or the remainder of linker L3 of the molecular glue cD of formula II.

[0506] In some embodiments, Z 1 and Z 2 are each CR 1 and R 1 and R 2 are each H.

[0507] In some embodiments, R is H.

[0508] In some embodiments, A is C6-C 20 aryl.

[0509] In some embodiments,

[0510] Z 1 and Z 2 are each CR1 , wherein R 1 and R 2 are each H;

[0511] R is H; and

[0512] A is C6-C 20 aryl.

[0513] In some embodiments, IM comprises a group selected from 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl)methylcarbamate.

[0514] In some embodiments, L1 forms a disulfide bond with a cysteine thiol of the antibody.

[0515] In some embodiments, Formula I is selected from the following formulas:

[0516]

[0517] In some embodiments, L1 is a linker having the following formula:

[0518]

[0519] wherein

[0520] 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 atom to which they are attached form a three-, four-, or five-membered cycloalkyl or heterocyclic group optionally substituted with F, Cl, and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and optionally 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; and

[0521] * indicates the point of attachment to the cysteine thiol of the antibody, and the wavy line indicates the attachment to the cereblon degrader moiety.

[0522] In some embodiments, L1 is selected from the following formulas:

[0523]

[0524] wherein

[0525] R 4a 、R4b , 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group optionally substituted with F, Cl and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and optionally 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;

[0526] R 6 is selected from H and C1-C6 alkyl; and

[0527] * indicates that sulfur is conjugated to the cysteine thiol of the antibody to form a disulfide bond, and the wavy line indicates the linkage to the cereblon degrader moiety.

[0528] In some embodiments, R 4a and R 4b are each -CH3.

[0529] In some embodiments, R 5a and R 5b are each H.

[0530] In some embodiments, R 6 is H.

[0531] In some embodiments, R 4a and R 4b are each -CH3, R 5a and R 5b are each H, and R 6 is H.

[0532] CEREBLON degrader moiety

[0533] In an embodiment, cD is a bivalent heterobifunctional cD or a molecular glue cD. In some embodiments, the cereblon degrader moiety (cD) has the formula:

[0534] E3UL-cD a

[0535] wherein

[0536] E3UL is a cereblon-binding E3 ubiquitin ligase ligand;

[0537] cD a is the molecular glue moiety; or

[0538] cD a is TPL-L2-, wherein

[0539] TPL is a target protein ligand, and

[0540] L2 is a degrader linker.

[0541] Bivalent cereblon degrader moiety

[0542] In an embodiment, cD comprises a cereblon-binding E3 ubiquitin ligase ligand (E3UL) covalently linked via a degrader linker (L2) to a target protein ligand (TPL) to form a bivalent heterobifunctional cD. In an embodiment, the cereblon degrader moiety (cD) has the formula:

[0543] TPL-L2-E3UL

[0544] wherein:

[0545] TPL is a target protein ligand;

[0546] E3UL is a cereblon-binding E3 ubiquitin ligase ligand;

[0547] L2 is a degrader linker; and

[0548] one of TPL, E3UL, and L2 is linked to L1.

[0549] Target protein ligand

[0550] The target protein ligand (TPL) is the moiety that binds to the protein of interest to be labeled and degraded by the E3 ubiquitin ligase / protease system. TPL is covalently linked via a degrader linker to a cereblon-binding E3 ubiquitin ligase ligand.

[0551] An exemplary target protein of the cereblon degrader antibody conjugate (cDAC) is BRD4. BRD4 is a member of the bromodomain and extra-terminal domain (BET) family and is an attractive target in various pathological conditions, particularly cancers including solid tumors and hematological malignancies. Inhibition of BRD4 in prostate and AML (acute myeloid leukemia) cell lines has shown sensitivity (S.E. Lochrin, et al. (2014) Canc. Biol. Ther. 15(12):1583-1585).

[0552] 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).

[0553] In some embodiments, TPL has a structure of the following formula:

[0554]

[0555] wherein

[0556] R x is selected from F, Cl, and Br; n is 0, 1, 2, or 3;

[0557] R y is selected from H and C1-C6 alkyl; and

[0558] The wavy line indicates the point of attachment to L2.

[0559] In some embodiments, TPL has the following structure:

[0560]

[0561] wherein the wavy line indicates the point of attachment to L2.

[0562] In some embodiments, TPL has the following structure:

[0563]

[0564] wherein the wavy line indicates the point of attachment to L2.

[0565] An exemplary target protein of the cereblon degrader antibody conjugate (cDAC) is GSPT1 (G1 to S phase transition protein 1 homolog), a translation termination factor (Huber, A. et al. (2022) ACS Med. Chem. Lett., 13:1311 - 1320; Powell, C. E. et al. (2020) ACS Chem. Biol. 15:2722 - 2730; Matyskiela, M. E. (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. Thus, GSPT1 is a potential drug target for future chemotherapy (Matyskiela, M. E. et al., (2016) Nature 535(7611), 252 - 7; Surka, C.; et al., (2021) Blood 137(5)661 - 677; Takwale, A. D. et al. (2022) Bioorganic Chemistry 127:105923; Hansen JD, et al. (2021) JMed Chem. 64(4):1835 - 1843).

[0566] Degrader linker

[0567] The degrader linker (L2) is any suitable bifunctional or trifunctional linker unit that is covalently linked to the target protein ligand (TPL) and the cereblon-binding E3 ubiquitin ligase ligand (E3UL). The degrader linker can be covalently linked to the antibody linker L1 to form the cereblon degrader antibody conjugate (cDAC).

[0568] In some embodiments, L2 is selected from:

[0569] -N(R’)-(C1-C 12 alkylene diyl)-N(R’)-、

[0570] -N(R’)-(C2-C 12 alkenylene diyl)-N(R’)-、

[0571] -N(R’)-(C2-C 12 alkynylene diyl)-N(R’)-、

[0572] -N(R’)-(C1-C 12 alkylene diyl)-C(=O)-(N(R’)-、

[0573] -N(R’)-(C1-C 12 alkylene diyl)-(N(R’)-C(=O)CH2O-、

[0574] -N(R’)-(C1-C 12 alkylene)-(N(R’)-C(=O)CH2N(R’)-、

[0575] -N(R’)-(C1-C 12 alkylene)-C(=O)-(N(R’)-(C1-C 12 alkylene)-N(R’)-、

[0576] -N(R’)-(C1-C6 alkylene)-O-(C1-C6 alkylene)-N(R’)-、

[0577] -N(R’)-(CH2CH2O) n -N(R’)-(CH2CH2O) n - where n is an integer from 1 to 4,

[0578] C1-C 12 alkylene,

[0579] C2-C 12 alkenylene and

[0580] C2-C 12 alkynylene,

[0581] where R’ is selected from H, C1-C6 alkylene and the point of attachment to L1; and where

[0582] alkylene, alkenylene and alkynylene are optionally substituted with one or more substituents 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.

[0583] Bivalent cereblon degrader compounds

[0584] Exemplary heterobifunctional cereblon degrader compounds were prepared and characterized and are shown in Table 1.

[0585] Table 1 Bivalent cereblon degrader compounds (cD)

[0586]

[0587]

[0588]

[0589] Molecular glue cereblon degrader moiety

[0590] In embodiments, the cereblon degrader moiety (cD) of the antibody conjugate (cDAC) is a molecular glue cereblon degrader moiety. In such embodiments, the molecular glue cereblon degrader moiety (cD) comprises an E3 UL covalently linked to a molecular glue moiety (cD a ) to form molecular glue cD.

[0591] In some embodiments, molecular glue cD comprises a structure selected from the following formulae:

[0592]

[0593] wherein the wavy line indicates the point of attachment to the antibody linker L1 of formula I or linker L3 of formula II;

[0594] cD a is the molecular glue moiety;

[0595] The dashed line indicates an optional double bond;

[0596] Z 1 is selected from C(R 1 )2, CR 1 , N and NR 1a ;

[0597] Z 2 is selected from C(R 2 )2, CR 2 , N and NR 2a ;

[0598] R 1 and R 2 are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20Aryl, C3-C 20 Carbocyclic group, C2-C 20 Heterocyclic group, 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)2NR a and -S(O)3H;

[0599] R 1a and R 2a independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H;

[0600] or (i) two geminal R 1 or two geminal R 2 form a 3- to 6-membered carbocyclic or heterocyclic 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, carbocyclic, heterocyclic or heteroaryl group;

[0601] R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where the phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl, and

[0602] A is selected from C6-C 20 aryl, C3-C 20 carbocyclic, C2-C 20 heterocyclic and C1-C 20 heteroaryl;

[0603] An alkyl group, an alkylene group, an alkenyl group, an alkynyl group, an aryl group, a carbocyclic group, a heterocyclic group, and a heteroaryl group are each independently and optionally substituted with one or more groups independently selected from the following: 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(=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.

[0604] In some embodiments, Z 1 and Z 2 are each CR 1 , and R 1 and R 2 are each H.

[0605] In some embodiments, R is H.

[0606] In some embodiments, A is a C6-C 20 aryl group.

[0607] In some embodiments,

[0608] Z 1 and Z2 Each is CR 1 , where R 1 and R 2 Each is H;

[0609] R is H; and

[0610] A is C6-C 20 Aryl.

[0611] In some embodiments, the molecular glue cD comprises a structure selected from the following formulas:

[0612]

[0613] in

[0614] The wavy line indicates the point of attachment of the antibody linker L1 of Formula I or the linker L3 of Formula II;

[0615] cD a is a molecular glue portion; and

[0616] X 1 Selected from CH2 and C(=O).

[0617] In some embodiments, X 1 is C(=O).

[0618] CEREBLON Degrader-Linker Intermediate

[0619] The cereblon degrader-linker intermediate (cDLI) is a reagent used in a method for preparing a cereblon degrader-antibody conjugate (cDAC) by conjugating it to a thiol-containing antibody. The cereblon degrader-linker intermediate has a thiol-reactive functional group (X). The thiol-reactive functional group (X) is covalently linked to the cereblon degrader moiety (cD) via a linker (L3).

[0620] In some embodiments, the cereblon degrader-linker intermediate has the structure of Formula II:

[0621] X-L3-cD

[0622] II

[0623] in:

[0624] X is a thiol-reactive group;

[0625] L3 is a linker selected from the following:

[0626] (i) a protease-cleavable non-peptide linker having the formula:

[0627] -Str-PM-Y-

[0628] wherein Str is an extender unit covalently linked to X;

[0629] PM is a peptidomimetic unit, and

[0630] Y is a spacer unit covalently linked to cD;

[0631] (ii) a disulfide linker selected from the following formulae:

[0632]

[0633] and

[0634] (iii) a linker having the following formula:

[0635]

[0636] wherein * indicates the point of attachment to X,

[0637] 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl;

[0638] R 6 is selected from H and C1-C6 alkyl,

[0639] the wavy line indicates the attachment to cD,

[0640] C1-C6 alkyl is independently and optionally 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; and

[0641] cD is a cereblon degrader moiety having the following formula:

[0642] TPL-L2-E3UL

[0643] wherein:

[0644] TPL is a target protein ligand;

[0645] E3UL is an E3 ubiquitin ligase ligand that binds to cereblon;

[0646] L2 is a degrader linker; and

[0647] One of TPL, E3UL, and L2 is linked to L1; or

[0648] cD is a molecular glue.

[0649] In some embodiments, the linkage of L3 to cD comprises a carbamate (-OC(O)NH-) or methylcarbamate (-OC(O)NHCH2-) group.

[0650] In some embodiments, X is selected from the group consisting of maleimide, bromoacetamide, toluenesulfonyl thioether, and 2-pyridyldisulfide, wherein the pyridyl group is optionally substituted with one or two nitro groups.

[0651] In some embodiments, the cereblon degrader-linker intermediate has the following formula:

[0652]

[0653] wherein IM comprises a group selected from 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl)methylcarbamate, and

[0654] 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.

[0655] In some embodiments, AA is selected from H, -CH₃, -CH₂(C₆H₅), -CH₂CH₂CH₂CH₂NH₂, -CH₂CH₂CH₂NHC(NH)NH₂, -CH₂CH(CH₃)₂, and -CH₂CH₂CH₂NHC(O)NH₂.

[0656] In some embodiments, AA is -CH₃ or -CH₂CH₂CH₂NHC(O)NH₂.

[0657] In some embodiments, the cereblon degrader-linker intermediate is selected from the following formulas:

[0658]

[0659] wherein X 1 is selected from CH₂ and C(=O).

[0660] In some embodiments, the cereblon degrader-linker intermediate has the formula:

[0661] wherein L3 is a protease-cleavable non-peptidic linker having the formula:

[0662] -Str-PM-IM-

[0663] wherein Str is an extender unit covalently linked to X;

[0664] PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to cD and has the formula:

[0665] wherein the wavy line is the connection to PM.

[0666] In some embodiments, the cereblon degrader-linker intermediate has the formula:

[0667] wherein L3 is a protease-cleavable non-peptidic linker having the formula:

[0668] -Str-PM-IM-

[0669] wherein Str is an extender unit covalently linked to X;

[0670] PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to L2 of cD and has the formula:

[0671]

[0672] wherein the wavy line is the connection to PM.

[0673] Prepare certain cDLIs in Table 2, which do not have the necessary properties of stability, cleavage efficiency, and conjugation efficiency with the antibody.

[0674] Table 2 Cereblon degrader-linker intermediates (cDLIs) with poor stability and / or low conjugation efficiency

[0675]

[0676]

[0677] The sulfonyl-thio cDLI-1 compound failed to react with the antibody for 3 hours and overnight under the conditions described in Example 102, including pH 8.5 and 3 and 10 equivalents. Analysis by mass spectrometry (LC / MS) showed no expected conjugate product cDAC, ring opening of the succinimide, addition of water (+18 mass units), and addition of Tris buffer to the succinimide ring. Under these conditions, the carbamate functional group formed from the succinimide nitrogen in cDLI-1 was too unstable for conjugation.

[0678] The sulfonamide cDLI-2 and bromo-lenalidomide cDLI-3 compounds did not conjugate to the antibody. The maleimide-sulfonamide compounds cDLI-4 and cDLI-5 failed to conjugate to the cysteine mutant antibody and were unstable in whole blood.

[0679] Various substituted peptide linkers cDLI-6 conjugated to the antibody but failed to cleave in the presence of protease. The (S,S) valine-alanine and (S,S) valine-citrulline versions of cDLI-6, as well as the phenyl and dimethoxyphenyl versions, were tested, and all gave cDAC without cleavage to release the cereblon-degrader moiety or its metabolite, with or without a methyl group near the succinimide nitrogen.

[0680] In a model study, the nitro group of p-nitrobenzyloxymethyl lenalidomide cDLI-7 was reduced to an amine. No cleavage of the aminobenzyloxy group was observed by detecting the appearance of lenalidomide. In a model study, the disulfide group of p-nitropyridyldithio-methyl lenalidomide cDLI-8 was reduced. No cleavage of the disulfide group was observed by detecting the appearance of lenalidomide.

[0681] Sulfonyl-thio cDLI-9 (with or without a methyl group near the succinimide nitrogen) cleaved to release detectable lenalidomide but was unstable, generating hydrolysis products.

[0682] Both sulfonyl-thio cDLI-9 and cDLI-10 failed to conjugate to the antibody under different conditions (pH 8.5 and 3 and 10 equivalents to the antibody) and with a reaction time of 3 hours to overnight. Hydrolysis of the carbonate bond was observed by LC / MS.

[0683] In some embodiments, the cereblon degrader-linker intermediate comprises a TPL selected from the following formula:

[0684] (i)

[0685] wherein R x is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R ySelected from H and C1-C6 alkyl;

[0686] (ii)

[0687] (iii)

[0688] wherein the wavy line indicates the connection point of L2.

[0689] Exemplary cereblon degrader-linker intermediates (cDLIs) are selected from:

[0690]

[0691] and

[0692]

[0693] where 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, and X 1 is selected from CH2 and C(=O).

[0694] In some embodiments, the cereblon degrader-linker intermediate comprises wherein L2 is selected from:

[0695] -N(R)-(C1-C 12 alkyl diyl)-N(R)-,

[0696] -N(R)-(C2-C 12 alkenyl diyl)-N(R)-,

[0697] -N(R)-(C2-C 12 alkynyl diyl)-N(R)-,

[0698] -N(R)-(C1-C 12 alkyl diyl)-C(=O)-(N(R)-,

[0699] -N(R)-(C1-C 12 alkyl diyl)-C(=O)-(N(R)-(C1-C 12 alkyl diyl)-N(R)-,

[0700] -(C1-C6 alkyl diyl)-O-(C1-C6 alkyl diyl)-,

[0701] C1-C 12 alkyl diyl,

[0702] C2-C 12 alkenyl diyl, and

[0703] C2-C 12 alkynylene diyl

[0704] wherein the alkyl diyl, alkenylene diyl and alkynylene diyl 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 H, C1-C6 alkyl diyl and the point of attachment to L3.

[0705] The cereblon degrader-linker intermediates (cDLIs) in Table 3 were prepared, which have the necessary properties of stability, cleavage efficiency, and conjugation efficiency with the antibody. Each cDLI in Table 3 was characterized by NMR and shown by LC / MS to have sufficient purity and the correct mass.

[0706] Examples of cereblon degrader-linker intermediates (cDLIs) in Table 3

[0707]

[0708]

[0709]

[0710] antibody

[0711] The cereblon degrader antibody conjugates (cDACs) provided herein comprise an antibody. Functional variants of the antibody constructs and antigen-binding domains described herein are included within the scope of the antibody embodiments.

[0712] The antibody portion of the cDAC can target cells expressing the antigen, thereby intracellularly delivering the antigen-specific cDAC to the target cells, typically by endocytosis. Although cDACs containing antibodies against antigens not found on the cell surface may result in less specific intracellular delivery of the cereblon degrader portion into the cells, the cDACs can still undergo pinocytosis. The cDACs described herein and methods of using them advantageously utilize antibody recognition on the cell surface and / or endocytosis of the cDAC to deliver the intracellular cereblon degrader portion.

[0713] Trastuzumab, an anti-HER2 antibody

[0714] In certain embodiments, the immunoconjugates described herein (e.g., cDAC) comprise an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody of cDAC 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 US5821337, which is specifically incorporated herein by reference. These antibodies comprise a human framework region and 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 TM from Genentech, Inc.

[0715] Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1κ monoclonal antibody that is a humanized form of the murine anti-HER2 antibody (4D5) and can selectively bind to the extracellular domain of HER2 with high affinity (Kd = 5 nM) in cell-based assays (US5677171; US 5821337; US 6054297; US 6165464; US 6339142; US 6407213; US 6639055; US6719971; US 6800738; US 7074404; Coussens et al., (1985) Science 230:1132-9; Slamon et al., (1989) Science 244:707-12; Slamon et al., (2001) New Engl. J. Med. 344:783-792).

[0716] 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 region of trastuzumab. In some embodiments, the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.

[0717] 7C2, an anti-HER2 antibody

[0718] The anti-HER2 murine antibody 7C2 binds to an epitope in domain I of HER2. See, for example, PCT Publication No. WO 98 / 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 signaling (e.g., PI3K / AKT). In contrast, the binding of pertuzumab to domain II blocks ligand-driven interactions of HER2 with other HER family members (e.g., HER3, HER1, or HER4), and thus also blocks downstream signal transduction. The binding of MAb 7C2 to domain I does not interfere with the binding of trastuzumab or pertuzumab to domains IV and II, respectively, thus providing the potential to combine MAb 7C2 ADC (antibody-drug conjugate) with trastuzumab, trastuzumab emtansine ((T-DM1) and / or pertuzumab. The murine antibodies 7C2, 7C2.B9 are described in WO 1998 / 017797. The anti-HER2 7C2 humanized antibody is disclosed in WO 2016 / 040723.

[0719] In some embodiments, the anti-HER2 antibody of the cDAC described herein comprises a humanized 7C2 anti-HER2 antibody. The humanized 7C2 antibody is an anti-HER2 antibody.

[0720] In some embodiments, the cDACs described herein comprise an anti-HER2 antibody that comprises 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. In some embodiments, the cDACs described herein comprise an anti-HER2 antibody that comprises 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.

[0721] In one aspect, the cDACs described herein comprise an antibody that comprises 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, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8 or 13. In one aspect, the cDACs described herein comprise an antibody that comprises 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, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:6, (b) HVR-H2 comprising the amino acid sequences of SEQ ID NO:7, 11, 12, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8 or 13. In yet another aspect, the antibody comprises (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.

[0722] In another aspect, the cDACs described herein comprise an antibody that comprises 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, (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:5. In one embodiment, the antibody comprises (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.

[0723] In another aspect, the cDAC described herein comprises an antibody that comprises (a) a VH domain that comprises 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 the amino acid sequence selected from SEQ ID NO:8 or 13; and (b) a VL domain that comprises 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. In another aspect, the cDAC described herein comprises: (a) a VH domain that comprises 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 the amino acid sequence selected from SEQ ID NO:8; and (b) a VL domain that comprises 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.

[0724] In another aspect, the cDACs described herein comprise an antibody that 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, (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 comprise an antibody that 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, (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.

[0725] 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 the HVRs of any of the above embodiments and further comprises a human receptor framework, such as a human immunoglobulin framework or a human consensus framework.

[0726] In another aspect, the anti-HER2 antibody of the antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:18. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:2 comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-HER2 antibody comprising the sequence retains the ability to bind to HER2. In certain embodiments, in SEQ ID NO:2, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, in SEQ ID NO:2, a total of 1 to 5 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions or deletions occur in regions outside of the HVRs (i.e., in the FRs). Optionally, the anti-HER2 antibody comprises the VH sequence of SEQ ID NO:2, including post-translational modifications of the sequence. In one specific embodiment, the VH comprises one, two or three HVRs selected from the following: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:6, (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:7, and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:8.

[0727] In another aspect, provided is an anti-HER2 antibody of an antibody-drug conjugate, wherein the antibody comprises: 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, the 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 comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, provided that the anti-HER2 antibody comprising the sequence retains the ability to bind to HER2. In certain embodiments, in SEQ ID NO:1, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, in SEQ ID NO:1, a total of 1 to 5 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the HVRs (i.e., in the FRs). Optionally, the anti-HER2 antibody comprises the VL sequence of SEQ ID NO:1, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two or three HVRs selected from the following: (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.

[0728] In another aspect, provided is an antibody-drug conjugate comprising 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.

[0729] In one embodiment, provided is an antibody-drug conjugate comprising an antibody, wherein the antibody comprises the VH and VL sequences in SEQ ID NO:2 and SEQ ID NO:1, respectively, including post-translational modifications of those sequences.

[0730] In one embodiment, provided is an antibody-drug conjugate comprising an antibody, wherein the antibody comprises the humanized 7C2.v2.2.LA (hu7C2) K149C κ light chain sequence of SEQ ID NO:14

[0731] In one embodiment, provided is an antibody-drug conjugate comprising an antibody, wherein the antibody comprises the Hu7C2 A118C IgG1 heavy chain sequence of SEQ ID NO:15.

[0732] In another aspect, the present disclosure provides antibody-drug conjugates comprising an antibody that binds the same epitope as the anti-HER2 antibodies provided herein. For example, in certain embodiments, immunoconjugates are provided that comprise an antibody that binds 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).

[0733] In some embodiments, the anti-HER2 antibody of the cDAC according to any of the above embodiments is a monoclonal antibody, including human antibodies. In one embodiment, the anti-HER2 antibody of the immunoconjugate is an antibody fragment, such as an Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment. In another embodiment, the immunoconjugate comprises an antibody that is substantially a full-length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody types or isotypes defined herein. In some embodiments, the anti-HER2 antibody is a full-length antibody.

[0734] Humanized 7C2 anti-HER2 antibody sequence listing

[0735]

[0736]

[0737]

[0738] Anti-CD33 antibody

[0739] The anti-CD33 antibody 15G15.33 of the cDAC in Tables 4 and 5 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: 16-21.

[0740] HVR-L1 RSSQSLLHSNGYNYLD (SEQ ID NO:16)

[0741] HVR-L2 LGVNSVS (SEQ ID NO:17)

[0742] HVR-L3 MQALQTPWT (SEQ ID NO:18)

[0743] HVR-H1 NHAIS (SEQ ID NO:19)

[0744] HVR-H2 GIIPIFGTANYAQKFQG (SEQ ID NO:20)

[0745] HVR-H3 EWADVFD (SEQ ID NO:21)

[0746] The anti-CD33 antibody 15G15.33 of cDAC in Tables 4 and 5 contains the light chain variable region of SEQ ID NO:22 and / or the heavy chain variable region of SEQ ID NO:23.

[0747] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGVNSV

[0748] SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK

[0749] (SEQ ID NO:22)

[0750] QVQLVQSGAEVKKPGSSVKVSCKASGGIFSNHAISWVRQAPGQGLEWMGGIIPIFGTANY

[0751] AQKFQGRVTITADESTSTAFMELSSLRSEDTAVYYCAREWADVFDIWGQGTMVTVSS

[0752] (SEQ ID NO:23)

[0753] The anti-CD33 antibody 9C3 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 NO:24 - 29, and the following VL and VH sequences SEQ ID NO:30 - 37.

[0754] 9C3-HVR L1 RASQGIRNDLG(SEQ ID NO:24)

[0755] 9C3-HVR L2 AASSLQS(SEQ ID NO:25)

[0756] 9C3-HVR L3 LQHNSYPWT(SEQ ID NO:26)

[0757] 9C3-HVR H1 GNYMS(SEQ ID NO:27)

[0758] 9C3-HVR H2 LIYSGDSTYYADSVKG (SEQ ID NO:28)

[0759] 9C3-HVR H3 DGYYVSDMVV (SEQ ID NO:29)

[0760] 9C3 V L

[0761] DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF

[0762] SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK

[0763] (SEQ ID NO:30)

[0764] 9C3 V H

[0765] EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS

[0766] VKGRFNISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS

[0767] (SEQ ID NO:31)

[0768] 9C3.2 V L

[0769] DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYA

[0770] ASSLQSGVPSRF

[0771] SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK

[0772] (SEQ ID NO:32)

[0773] 9C3.2 V H

[0774] EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVS

[0775] LIYSGDSTYYADS

[0776] VKGRFTISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGK

[0777] GTTVTVSS

[0778] (SEQ ID NO:33)

[0779] 9C3.3 V L

[0780] DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF

[0781] SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK

[0782] (SEQ ID NO:34)

[0783] 9C3.3 V H

[0784] EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS

[0785] VKGRFSISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS

[0786] (SEQ ID NO:35)

[0787] 9C3.4 V L

[0788] DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF

[0789] SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK

[0790] (SEQ ID NO:36)

[0791] 9C3.4 V H

[0792] EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS

[0793] VKGRFAISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS

[0794] (SEQ ID NO:37)

[0795] In some embodiments, the cDACs described herein comprise an anti-CD33 antibody that comprises 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-L3 comprising the amino acid sequence of SEQ ID NO:26.

[0796] In one aspect, the cDACs described herein comprise an antibody that comprises 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: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. In one embodiment, the antibody comprises HVR-H3, which comprises the amino acid sequence of SEQ ID NO:29. In another embodiment, the antibody comprises: HVR-H3, which comprises the amino acid sequence of SEQ ID NO:29; and HVR-L3, which comprises the amino acid sequence of SEQ ID NO:26. In a further embodiment, the antibody comprises: HVR-H3, which comprises the amino acid sequence of SEQ ID NO:29; HVR-L3, which comprises the amino acid sequence of SEQ ID NO:26; and HVR-H2, which comprises the amino acid sequence of SEQ ID NO:28. In a further embodiment, the antibody comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:27; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:28; and (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:29.

[0797] In another aspect, the cDAC described herein comprises at least one, at least two, or all three VLHVR 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, which comprises the amino acid sequence of SEQ ID NO:24; (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:25; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:26.

[0798] In another aspect, 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, which comprises the amino acid sequence of SEQ ID NO:27, (ii) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:28, and (iii) HVR-H3, which comprises the 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, which comprises the amino acid sequence of SEQ ID NO:24, (ii) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:25; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:26.

[0799] In another aspect, the cDAC described herein comprises: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO:27; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO:28; (c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:29; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:24; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:25; and (f) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:26.

[0800] In any of the above embodiments, the anti-CD33 antibody is humanized. In one embodiment, the anti-CD33 antibody comprises the HVRs of any of the above embodiments and further comprises a human receptor framework, such as a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human receptor framework is a human VLκI consensus (VL KI)Framework and / or VH framework VH1. In certain embodiments, the human receptor framework is the human VLκI consensus (VL KI )framework and / or VH framework VH1, which contains any of the following mutations.

[0801] In another aspect, the anti-CD33 antibody comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequences of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 and / or SEQ ID NO:37. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequences 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, but the anti-CD33 antibody comprising the sequence retains the ability to bind to CD33. In certain embodiments, in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 and / or SEQ ID NO:37, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, in SEQ ID NO:35, SEQ ID NO:33, SEQ ID NO:35 and / or SEQ ID NO:37, a total of 1 to 5 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD33 antibody comprises the VH sequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37, including post-translational modifications of those sequences. In a specific embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence of SEQ ID NO:27, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO:28 and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO:29.

[0802] In another aspect, provided is an anti-CD33 antibody, wherein the antibody comprises 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 sequences of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and / or SEQ ID NO:36. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequences of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and / or SEQ ID NO:36 comprises substitutions (such as conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CD33 antibody comprising the sequence retains the ability to bind to CD33. In certain embodiments, in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and / or SEQ ID NO:36, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain embodiments, in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and / or SEQ ID NO:36, a total of 1 to 5 amino acids are substituted, inserted and / or deleted. In certain embodiments, the substitution, insertion or deletion occurs in a region outside the HVRs (i.e., in the FRs). Optionally, the anti-CD33 antibody comprises the 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 those sequences. In a specific embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:24, (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:25, and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:26.

[0803] In another aspect, provided is an anti-CD33 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.

[0804] 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.

[0805] In another aspect, the present disclosure provides antibodies that bind to the same epitope as the anti-CD33 antibodies provided herein. For example, in certain embodiments, an antibody that binds to the same epitope as the anti-CD33 antibody is provided, which comprises 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.

[0806] In some embodiments, the anti-CD33 antibody is a monoclonal antibody, including human antibodies. In one embodiment, the anti-CD33 antibody is an antibody fragment, such as an Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment. In another embodiment, the antibody is a substantially full-length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody types or isotypes defined herein. In some embodiments, the anti-CD33 antibody is a full-length antibody.

[0807] In a further aspect, the anti-CD33 antibody according to any one of the above embodiments can be incorporated, alone or in combination, into any one of the features described below.

[0808] Antibody variants engineered with cysteine

[0809] In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, e.g., "THIOMAB" TM” or TDC, wherein one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the substituted residues are present at sites of the antibody available for conjugation. As further described herein, by replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties (such as a drug moiety or a linker-drug moiety) to produce an immunoconjugate. In certain embodiments, any one or more of the following residues can be replaced with cysteine: 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. In a specific embodiment, the antibody described herein comprises an HC-A140C (EU numbering) cysteine substitution. In a specific embodiment, the antibody described herein comprises an LC-K149C (Kabat numbering) cysteine substitution. In a specific embodiment, the antibody described herein comprises an HC-A118C (EU numbering) cysteine substitution. Cysteine-engineered antibodies can be generated as described, for example, in US7521541.

[0810] In certain embodiments, the antibody comprises one of the following heavy chain cysteine substitutions:

[0811]

[0812]

[0813] In certain embodiments, the antibody comprises one of the following light chain cysteine substitutions:

[0814]

[0815] Non-limiting exemplary hu7C2.v2.2.LA light chain (LC) K149C THIOMAB TM Having heavy and light chain amino acid sequences of SEQ ID NO:10 and 14, respectively. Non-limiting exemplary hu7C2.v2.2.LA heavy chain (HC) A118C THIOMAB TM Having heavy and light chain amino acid sequences of SEQ ID NO:15 and 9, respectively.

[0816] Antibody target

[0817] In some embodiments, the antibody of the cereblon degrader antibody conjugate (cDAC) is capable of binding to one or more tumor-associated antigens (TAAs), cell surface receptors, and immune-specific antigens to confer targeting specificity to the cereblon degrader antibody conjugate and is capable of delivering the active pharmaceutical moiety safely and systemically.

[0818] Certain tumor-associated antigens are known in the art and antibodies can be generated using methods and information well known in the art. To discover effective cellular targets for cancer diagnosis and treatment, 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 as compared to one or more normal non-cancer cells. Generally, such tumor-associated polypeptides are expressed at higher levels on the surface of cancer cells as compared to the surface of non-cancer cells. The identification of such tumor-associated cell surface antigen polypeptides enables greater specificity for the destruction of cancer cells via antibody-based therapies.

[0819] Examples of TAAs include, but are not limited to, those listed below, including (1)-(55). For convenience, information related to all of these antigens known in the art is listed below and includes the name, alternative name, Genbank accession number, and primary reference, following the nucleic acid and protein sequence identification conventions of the National Center for Biotechnology Information (NCBI). Nucleic acid and protein sequences corresponding to the TAAs listed below (including (1)-(55)) are available in public databases such as GenBank. Antibody-targeted TAAs include all amino acid sequence variants and isoforms having at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the cited references, and / or which exhibit substantially the same biological properties or characteristics as the TAAs having the sequences found in the cited references. For example, TAAs having variant sequences are generally capable of specifically binding to antibodies that specifically bind to the TAAs having the listed corresponding sequences. The sequences and disclosures in the references specifically cited herein are hereby expressly incorporated by reference.

[0820] The sequences and disclosures in the references specifically cited herein are hereby expressly incorporated by reference.

[0821] (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)); WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790 - A1 (pages 38 - 39); WO2002102235 (claim 13; page 296); WO2003055443 (pages 91 - 92); WO200299122 (example 2; pages 528 - 530); WO2003029421 (claim 6); WO2003024392 (claim 2; figure 112); WO200298358 (claim 1; page 183); WO200254940 (pages 100 - 101); WO200259377 (pages 349 - 350); WO200230268 (claim 27; page 376); WO200148204 (example; Figure 4 ) NP_001194 bone morphogenetic protein receptor, type IB / pid = NP_001194.1 - Cross - reference: MIM:603248; NP_001194.1; AY065994.

[0822] (2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486) Biochem. Biophys. Res. Commun. 255(2), 283 - 288(1999), Nature 395(6699):288 - 291(1998), Gaugitsch, H.W., et al. (1992) J. Biol. Chem. 267(16):11267 - 11273); WO2004048938 (Example 2); WO2004032842 (Example IV); WO2003042661 (Claim 12); WO2003016475 (Claim XXXX); WO200278524 (Example 2); WO200299074 (Claim 19; pages 127 - 129); WO200286443 (Claim 27; pages 222, 393); WO2003003906 (Claim 10; page 293); WO200264798 (Claim 33; pages 93 - 95); WO200014228 (Claim 5; pages 133 - 136); US2003224454 (Figure 3); WO2003025138 (Claim 12; page 150); NP_003477 Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 / pid = NP_003477.3 - Homo sapiens Cross - reference: MIM:600182; NP_003477.3; NM_015923; NM_003486_1.

[0823] (3) STEAP1 (prostate six - transmembrane epithelial antigen; Genbank accession number NM_012449) Cancer Res. 61(15), 5857 - 5860(2001), Hubert, R.S., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96(25):14523 - 14528); WO2004065577 (Claim 6); WO2004027049( Figure 1 L); EP1394274 (Example 11); WO2004016225 (Claim 2); WO2003042661 (Claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (Figure 2); WO200289747 (Example 5; pages 618 - 619); WO2003022995 (Example 9; Figure 13A, Example 53; page 173; Example 2; Figure 2A It should be noted that there is an unclear "XXXX" in the translation of Claim in . You may need to check and correct it according to the actual content.);NP_036581 Prostate six transmembrane epithelial antigen Cross-reference: MIM:604415; NP_036581.1; NM_012449_1.

[0824] (4) 0772P (CA125, MUC16, Genbank accession number AF361486) J. Biol. Chem. 276(Number 29): 27371 - 27375 (2001); WO2004045553 (claim 14); WO200292836 (claim 6; Figure 12); WO200283866 (claim 15; pages 116 - 121); US2003124140 (Example 16); US 798959. Cross-reference: GI:34501467; AAK74120.3; AF361486_1.

[0825] (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM_005823) Yamaguchi, N., et al. Biol. Chem. vol. 269(Number 2), 805 - 808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96(Number 20): 11531 - 11536 (1999), Proc. Natl. Acad. Sci. U.S.A. 93(Number 1): 136 - 140 (1996), J. Biol. Chem. 270(Number 37): 21984 - 21990 (1995)); WO2003101283 (claim 14); (WO2002102235 (claim 13; pages 287 - 288); WO2002101075 (claim 4; pages 308 - 309); WO200271928 (pages 320 - 321); WO9410312 (pages 52 - 57); Cross-reference: MIM:601051; NP_005814.2; NM_005823_1.

[0826] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier 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, J. A., et al. (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO2004022778 (claim 2); EP1394274 (example 11); WO2002102235 (claim 13; page 326); EP875569 (claim 1; pages 17-19); WO200157188 (claim 20; page 329); WO2004032842 (example IV); WO200175177 (claim 24; pages 139-140); cross-reference: MIM:604217; NP_006415.1; NM_006424_1.

[0827] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMAB, 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); WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1: page 50); WO200188133 (claim 1; pages 41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11); accession: Q9P283; EMBL; AB040878; BAA95969.1.Genew; HGNC:10737.

[0828] (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; US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (example 5); WO2003105758 (claim 12); US2003206918 (example 5); EP1347046 (claim 1); WO2003025148 (claim 20); Cross-reference: GI:37182378; AAQ88991.1; AY358628_1.

[0829] (9) ETBR (Endothelin B Receptor, Genbank accession number AY275463); Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177, 34 - 39, 1991; Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178, 248 - 255, 1991; Arai H., et al. Jpn. Circ. J. 56, 1303 - 1307, 1992; Arai H., et al. J. Biol. Chem. 268, 3463 - 3470, 1993; Sakamoto A., Yanagisawa M., et al. Biochem. Biophys. Res. Commun. 178, 656 - 663, 1991; Elshourbagy N.A., et al. J. Biol. Chem. 268, 3873 - 3879, 1993; Haendler B., et al. J. Cardiovasc. Pharmacol. 20, s1 - S4, 1992; Tsutsumi M., et al. Gene 228, 43 - 49, 1999; Strausberg R.L., et al. Proc. Natl. Acad. Sci. U.S.A. 99, 16899 - 16903, 2002; Bourgeois C., et al. J. Clin. Endocrinol. Metab. 82, 3116 - 3123, 1997; Okamoto Y., et al. Biol. Chem. 272, 21589 - 21596, 1997; Verheij J.B., et al. Am. J. Med. Genet. 108, 223 - 225, 2002; Hofstra R.M.W., et al. Eur. J. Hum. Genet. 5, 180 - 185, 1997; Puffenberger E.G., et al. Cell 79, 1257 - 1266, 1994; Attie T., et al., Hum. Mol. Genet. 4, 2407 - 2409, 1995; Auricchio A., et al. Hum. Mol. Genet. 5:351 - 354, 1996; Amiel J., et al. Hum. Mol. Genet. 5, 355 - 357, 1996; Hofstra R.M.W., et al., Nat. Genet. 12, 445 - 447, 1996; Svensson P.J., et al. Hum. Genet. 103, 145 - 148, 1998; Fuchs S., et al. Mol. Med. 7, 115 - 124, 2001; Pingault V., et al. (2002) Hum. Genet. 111, 198-206; WO2004045516 (Claim 1); WO2004048938 (Example 2); WO2004040000 (Claim 151); WO2003087768 (Claim 1); WO2003016475 (Claim 1); WO2003016475 (Claim 1); WO200261087(. Figure 1 ); WO2003016494 (Figure 6); WO2003025138 (Claim 12; Page 144); WO200198351 (Claim 1; Pages 124-125); EP522868 (Claim 8; Figure 2); WO200177172 (Claim 1; Pages 297-299); US2003109676; US6518404 (Figure 3); US5773223 (Claim 1a; Col 31-34); WO2004001004.

[0830] (10) MSG783 (RNF124, Hypothetical protein FLJ20315, Genbank accession number NM_017763); WO2003104275 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page 61); WO2003018621 (Claim 1); WO2003024392 (Claim 2; Figure 93); WO200166689 (Example 6); Cross-reference: LocusID: 54894; NP_060233.2; NM_017763_1.

[0831] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, Prostate cancer-associated gene 1, Prostate cancer-associated protein 1, Prostate hexamembrane epithelial antigen 2, Hexamembrane prostate protein, Genbank accession number AF455138) Lab. Invest. 82(11):1573-1582 (2002)); WO2003087306; US2003064397 (Claim 1; Figure 1 ); WO200272596 (Claim 13; Pages 54-55); WO200172962 (Claim 1; Figure 4B); WO2003104270 (Claim 11); WO2003104270 (Claim 16); US2004005598 (Claim 22); WO2003042661 (Claim 12); US2003060612 (Claim 12; Figure 10); WO200226822 (Claim 23; Figure 2); WO200216429 (Claim 12; Figure 10); Cross References: GI:22655488; AAN04080.1; AF455138_1.

[0832] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, Transient Receptor Potential Cation Channel, Subfamily M Member 4, Genbank Accession No. NM_017636) Xu, X.Z., et al. Proc. Natl. Acad. Sci. U.S.A. 98(19):10692 - 10697(2001), Cell 109(3):397 - 407(2002), J. Biol. Chem. 278(33):30813 - 30820(2003)); US2003143557 (Claim 4); WO200040614 (Claim 14; Pages 100 - 103); WO200210382 (Claim 1; Figure 9A); WO2003042661 (Claim 12); WO200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794 (Claim 14; Figure 1 A - D); Cross References: MIM:606936; NP_060106.2; NM_017636_1.

[0833] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, Teratocarcinoma - Derived Growth Factor, Genbank Accession No. 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); US2003224411 (Claim 1); WO2003083041 (Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; Pages 52 - 53); WO2003024392 (Claim 2; Figure 58); WO200216413 (Claim 1; Pages 94 - 95, 105); WO200222808 (Claim 2; Figure 1); US5854399 (Example 2; Col 17-18); US5792616 (Figure 2); Cross References: MIM:187395; NP_003203.1; NM_003212_1.

[0834] (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 J.J., et al. J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol. 35, 1025-1031, 1998; Weis J.J., et al. Proc. Natl. Acad. Sci. U.S.A. 83, 5639-5643, 1986; Sinha S.K., et al. (1993) J. Immunol. 150, 5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim 9); WO2004045520 (Example 4); WO9102536 (Figures 9.1-9.9); WO2004020595 (Claim 1); Accessions: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

[0835] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin - associated β), B29, Genbank accession number NM_000626 or 11038674); Proc. Natl. Acad. Sci. U.S.A. (2003) 100(7):4126 - 4131, Blood (2002) 100(9):3068 - 3076, Muller et al. (1992) Eur. J. Immunol. 22(6):1621 - 1625); WO2004016225 (claim 2, figure 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (example 2); US2002150573 (claim 5, page 15); US5644033; WO2003048202 (claim 1, pages 306 and 309); WO 99 / 558658, US6534482 (claim 13, figure 17A / B); WO200055351 (claim 11, pages 1145 - 1146); cross - reference: MIM:147245; NP_000617.1; NM_000626_1.

[0836] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 - domain - containing phosphatase - anchored protein 1a), SPAP1B, SPAP1C, Genbank accession number 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. U.S.A. 98(17):9772 - 9777 (2001), Xu, M.J., et al. (2001) Biochem. Biophys. Res. Commun. 280(3):768 - 775; WO2004016225 (claim 2); WO2003077836; WO200138490 (claim 5; figure 18D - 1 - 18D - 2); WO2003097803 (claim 12); WO2003089624 (claim 25); cross - reference: MIM:606509; NP_110391.2; NM_030764_1.

[0837] (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. U.S.A. 82, 6497-6501, 1985; Swiercz J.M., et al. J. Cell Biol. 165, 869-880, 2004; Kuhns J.J., 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; WO2004048938 (Example 2); WO2004027049( Figure 1 I); WO2004009622; WO2003081210; WO2003089904 (Claim 9); WO2003016475 (Claim 1); US2003118592; WO2003008537 (Claim 1); WO2003055439 (Claim 29; Figure 1 A-B); WO2003025228 (Claim 37; Figure 5C); WO200222636 (Example 13; Pages 95-107); WO200212341 (Claim 68; Figure 7 ); WO200213847 (Pages 71-74); WO200214503 (Pages 114-117); WO200153463 (Claim 2; Pages 41-46); WO200141787 (Page 15); WO200044899 (Claim 52; Figure 7 ); WO200020579 (Claim 3; Figure 2); US5869445 (Claim 3; Col 31-38); WO9630514 (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.

[0838] (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 R.L., et al. Proc. Natl. Acad. Sci. U.S.A. 99:16899 - 16903, 2002; WO2004063709; EP1439393 (claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (claim 12); WO200278524 (Example 2); WO200286443 (claim 27; page 427); WO200260317 (claim 2); accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728.

[0839] (19) MDP (DPEP1, Genbank accession number BC017023) Proc. Natl. Acad. Sci. U.S.A. 99(26):16899 - 16903 (2002)); WO2003016475 (claim 1); WO200264798 (claim 33; pages 85 - 87); JP05003790 (Figures 6 - 8); WO9946284 (Figure 9); cross - reference: MIM:179780; AAH17023.1; BC017023_1.

[0840] (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession number AF184971); Clark H.F., et al. Genome Res. 13, 2265-2270, 2003; Mungall A.J., 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; EP1394274 (Example 11); US2004005320 (Example 5); WO2003029262 (pages 74-75); WO2003002717 (Claim 2; page 63); WO200222153 (pages 45-47); US2002042366 (pages 20-21); WO200146261 (pages 57-59); WO200146232 (pages 63-65); WO9837193 (Claim 1; pages 55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

[0841] (21) Brevican (BCAN, BEHAB, Genbank accession number AF229053); Gary S.C., et al., Gene 256, 139 - 147, 2000; Clark H.F., et al., Genome Res. 13, 2265 - 2270, 2003; Strausberg R.L., et al., Proc. Natl. Acad. Sci. U.S.A. 99, 16899 - 16903, 2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; Figure 52); US2003119122 (claim 1; Figure 52); US2003119126 (claim 1); US2003119121 (claim 1; Figure 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; Figure 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1).

[0842] (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)); WO2003042661 (claim 12); WO200053216 (claim 1; page 41); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (pages 128 - 132); WO200053216 (claim 1; page 42); Cross - reference: MIM:600997; NP_004433.2; NM_004442_1.

[0843] (23) ASLG659 (B7h, Genbank accession number AX092328); US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (Figure 3); US2003165504 (claim 1); US2003124140 (Example 2); US2003065143 (Figure 60); WO2002102235 (claim 13; page 299); US2003091580 (Example 2); WO200210187 (claim 6; Figure 10); WO200194641 (claim 12; Figure 7 b); WO200202624 (claim 13; Figure 1 A-1B); US2002034749 (claim 54; pages 45 - 46); WO200206317 (Example 2; pages 320 - 321, claim 34; pages 321 - 322); WO200271928 (pages 468 - 469); WO200202587 (Example 1; Figure 1 ); WO200140269 (Example 3; pages 190 - 192); WO200036107 (Example 2; pages 205 - 207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (pages 233 - 234, 452 - 453); WO 0116318.

[0844] (24) PSCA (Prostate Stem Cell Antigen Precursor, Genbank accession number AJ297436) Reiter R.E., et al. Proc. Natl. Acad. Sci. U.S.A. 95, 1735 - 1740, 1998; Gu Z., et al. Oncogene 19, 1288 - 1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3): 783 - 788; WO2004022709; EP1394274 (Example 11); US2004018553 (claim 17); WO2003008537 (claim 1); WO200281646 (claim 1; page 164); WO 2003003906 (claim 10; page 288); WO 200140309 (Example 1; Figure 17); US2001055751 (Example 1; Figure 1 b); 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 1 B); Accession: O43653; EMBL; AF043498; AAC39607.1.

[0845] (25) GEDA (Genbank Accession No. 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 - reference: GI:30102449; AAP14954.1; AY260763_1.

[0846] (26) BAFF - R (B - cell Activation Factor Receptor, BLyS Receptor 3, BR3, Genbank Accession No. AF116456); BAFF Receptor / pid = NP_443177.1 - Homo sapiens Thompson, J.S., et al. Science 293(5537), 2108 - 2111 (2001); WO2004058309; WO2004011611; WO2003045422 (Example; pages 32 - 33); WO2003014294 (Claim 35; Figure 6B );WO2003035846 (Claim 70; pages 615 - 616); WO200294852 (Col 136 - 137); WO200238766 (Claim 3; page 133); WO200224909 (Example 3; Figure 3); Cross - reference: MIM:606269; NP_443177.1; NM_052945_1; AF132600.

[0847] (27) CD22 (B - cell Receptor CD22 - B Isoform, BL - CAM, Lyb - 8, Lyb8, SIGLEC - 2, FLJ22814, Genbank Accession No. AK026467); Wilson et al. (1991) J.Exp.Med. 173:137 - 146; WO2003072036 (Claim 1; Figure 1 );Cross - reference: MIM:107266; NP_001762.1; NM_001771_1.

[0848] (28) CD79a (CD79A, CD79α, immunoglobulin-associated α, a B cell-specific protein that covalently interacts with Igβ (CD79B) and forms a complex with IgM molecules on the surface, transducing signals involved in B cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2, Genbank accession number NP_001774.10) WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13 - 14); WO9958658 (claim 13, Figure 16); WO9207574( Figure 1 ); US5644033; 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.

[0849] (29) CXCR5 (Burkitt lymphoma receptor 1, a G protein-coupled receptor activated by the CXCL13 chemokine, which plays a role in lymphocyte migration and humoral defense and is involved in the possible development of HIV-2 infection as well as AIDS, lymphoma, myeloma, and leukemia); 372 aa, pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession number NP_001707.1) WO 2004040000; WO2004 / 015426; US2003105292 (Example 2); US6555339 (Example 2); WO 2002 / 61087( Figure 1);WO200157188 (Claim 20, page 269); WO200172830 (pages 12 - 13); WO 2000 / 22129 (Example 1, pages 152 - 153, Example 2, pages 254 - 256); WO 199928468 (Claim 1, page 38); US 5440021 (Example 2, col 49 - 52); WO9428931 (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.

[0850] (30) HLA - DOB (β subunit of MHC class II molecule (Ia antigen), which 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; WO9958658 (Claim 13, Figure 15); US6153408 (Col 35 - 38); US5976551 (col 168 - 170); US6011146 (col 145 - 146); Kasahara et al. (1989) Immunogenetics 30(1):66 - 68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111 - 14119.

[0851] (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an extracellular ATP-gated ion channel that may be involved in synaptic transmission and neurogenesis, and whose deficiency 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; WO2004047749; WO2003072035 (claim 10); Touchman et al. (2000) Genome Res. 10:165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82).

[0852] (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_001773.1) WO2004042346 (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.

[0853] (33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family that regulates B-cell activation and apoptosis, and whose 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) US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al. (1996) Genomics 38(3):299-304; Miura et al. (1998) Blood 92:2815-2822; WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages 24-26).

[0854] (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain containing C2-type Ig-like and ITAM domains, which may play 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) WO2003077836; WO200138490 (claim 6, Figures 18E-1-18-E-2); Davis et al. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7).

[0855] (35) IRTA2 (immunoglobulin superfamily receptor translocation-associated 2, a putative immune receptor that may play a role in B cell development and lymphoma genesis; gene dysregulation caused by translocation in some B cell malignancies); 977 aa, 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, AY506558; NP_112571.1. WO2003024392 (claim 2, Figure 97); Nakayama et al. (2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (claim 3, Figures 18B-1-18B-2).

[0856] (36) TENB2 (TMEFF2, brain tumor suppressor protein, TPEF, HPP1, TR, a putative transmembrane proteoglycan that is related to EGF / heterodimeric regulatory protein family growth factors and follistatin); 374 aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession numbers AF179274; AY358907, CAF85723, CQ782436WO 2004074320; JP2004113151; WO 2003042661; WO2003009814; EP1295944 (pages 69 - 70); WO 200230268 (page 329); WO 200190304; US2004249130; US2004022727; WO 2004063355; US 2004197325; US2003232350; US2004005563; US2003124579; 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.

[0857] (37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R.P. et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106(33), 13731 - 13736; Kummer, M.P. et al. (2009) J. Biol. Chem. 284(4), 2296 - 2306.

[0858] (38) TMEFF1 (Transmembrane protein with EGF-like and two follistatin-like domains 1; Tumor suppressor in brain-1); H7365; C9orf2; C9ORF2; U19878; X83961; NM_080655; NM_003692; Harms, P.W. (2003) Genes Dev. 17(21), 2624-2629; Gery, S. et al. (2003) Oncogene 22(18):2723-2727.

[0859] (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, M.H. et al. (2009) Mol. Cell. Biol. 29(8), 2264-2277; Treanor, J.J. et al. (1996) Nature 382(6586):80-83.

[0860] (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, A.G. et al. (2003) Int. J. Cancer 103(6), 768-774; Zammit, D.J. et al. (2002) Mol. Cell. Biol. 22(3):946-952.

[0861] (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.

[0862] (42) Ly6G6D (Lymphocyte antigen 6 complex, 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.

[0863] (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.

[0864] (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.

[0865] (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.

[0866] (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, B.F. et al. (1996) FEBS Lett. 394(3):325-329.

[0867] (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.

[0868] (48) ASPHD1 (Aspartate beta-hydroxylase domain-containing protein 1, LOC253982); NP_859069.2; NM_181718.3; Gerhard, D.S. et al. (2004) Genome Res. 14(10B):2121-2127.

[0869] (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.

[0870] (50) TMEM118 (Ring finger protein, transmembrane 2; RNFT2; FLJ14627); NP_001103373.1; NM_001109903.1; Clark, H.F. et al. (2003) Genome Res. 13(10):2265-2270; Scherer, S.E. et al. (2006) Nature 440(7082):346-351.

[0871] (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. U.S.A. 100(11):6759-6764; Takeda, S. et al. (2002) FEBS Lett. 520(1-3):97-101.

[0872] (52) CD33, a member of the sialic acid-binding immunoglobulin-like lectin family, is a 67 kDa glycosylated transmembrane protein. In addition to committed bone marrow monocytes and erythroid progenitors, CD33 can also be expressed on most myeloid and monocytic leukemia cells. It is not seen on the earliest pluripotent stem cells, mature granulocytes, lymphoid cells, or non-hematopoietic 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 hydrophobic residues, similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in many inhibitory receptors.

[0873] (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 such as cell adhesion, intercellular signaling, glycoprotein turnover, and roles in inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined. This gene is closely linked to other CTL / CTLD superfamily members in 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 that consists of a single C-type lectin-like domain (not expected to bind calcium or sugars), a stalk region, a transmembrane domain, and a short cytoplasmic tail region that contains an ITIM motif.

[0874] (54) TROP2 (Tumor-associated calcium signal transducer 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 transducer that is differentially expressed in many cancers. It signals cells to self-renew, proliferate, invade, and survive. It has stem cell-like qualities. TROP2 is expressed in many normal tissues, although 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). The overexpression of TROP2 has prognostic significance. Several ligands that interact with TROP2 have been proposed. TROP2 signals cells via different pathways and is transcriptionally regulated through a complex network of several transcription factors.

[0875] Human TROP2 (TACSTD2: Tumor-associated calcium signal transducer 2, GA733-1, EGP-1, M1S1; hereinafter referred to as hTROP2) is a type I single-pass transmembrane cell membrane protein composed of 323 amino acid residues. The presence of cell membrane proteins is involved in immune resistance, which is common in human trophoblasts and cancer cells (Faulk W P, et al. (1978), Proc. Natl. Acad. Sci. 75(4):1947-1951). It has been previously proposed that an antigen molecule recognized by a monoclonal antibody against a cell membrane protein in a human choriocarcinoma cell line was identified and designated as TROP2 as one of the molecules expressed in human trophoblast cells (Lipinski M, et al. (1981), Proc. Natl. Acad. Sci. 78(8), 5147-5150). This molecule was also designated as the tumor antigen GA733-1 recognized by the mouse monoclonal antibody GA733 (Linnenbach A J, et al., (1989) Proc. Natl. Acad. Sci. 86(1), 27-31), which was recognized by the mouse monoclonal antibody RS7-3G11 obtained by immunizing with a non-small cell lung cancer cell line or an epithelial glycoprotein (EGP-1; Basu A, et al., Int. J. Cancer, 62(4), 472-479 (1995)). However, in 1995, the TROP2 gene was cloned, and it was confirmed that all these molecules are the same molecule (Fornaro M, et al., (1995) Int. J. Cancer, 62(5), 610-618). The DNA sequence and amino acid sequence of hTROP2 are available in public databases and can be cited, for example, under accession numbers NM_002353 and NP_002344 (NCBI).

[0876] In response to this information indicating its association with cancer, various anti-hTROP2 antibodies have been established to date, and their anti-tumor effects have been studied. Among these antibodies, unconjugated antibodies that exhibit anti-tumor activity by themselves in a nude mouse xenograft model have been disclosed (WO 2008 / 144891; WO 2011 / 145744; WO 2011 / 155579; WO 2013 / 077458), as well as antibodies that exhibit anti-tumor activity as ADCs with cytotoxic drugs (WO 2003 / 074566; WO2011 / 068845; WO 2013 / 068946; US 7999083). However, the intensity or coverage of their activity is still insufficient, and the medical need for hTROP2 as a therapeutic target has not been met.

[0877] TROP2 expression in cancer cells is associated with drug resistance. Several strategies target TROP2 in cancer cells, including antibodies, antibody fusion proteins, chemical inhibitors, nanoparticles, etc. In vitro and preclinical studies using these various therapies have led to significant inhibition of tumor cell growth in vitro and in vivo in mice. Clinical studies have explored the potential applications of Trop2 as a prognostic biomarker and as a therapeutic target for reversing drug resistance.

[0878] (55) CD123 (IL-4, IL3RA, IL3ry, IL3RAY, interleukin-3 receptor) is a protein found on cells that helps transmit the signal of interleukin-3, which is an important soluble cytokine in the immune system. The gene encoding the receptor is located in the pseudoautosomal region of the X and Y chromosomes. This receptor belongs to the type I cytokine receptor family and is a heterodimer with a unique α chain paired with a common β (βc or CD131) subunit. The gene of the α subunit is 40 kilobases long and has 12 exons. CD123 is the 70kD transmembrane α chain of the IL-3 receptor. Alone, CD123 binds IL-3 with low affinity; when CD123 binds to CDw131 (common β chain), it binds IL-3 with high affinity. CD123 does not transduce intracellular signals when binding IL-3 and requires the β chain to achieve this function. CD123 serves as a diagnostic, prognostic, and therapeutic biomarker in some hematological malignancies, especially the co-expression of CD123 and TCF4 in acute leukemia by immunohistochemistry is highly specific and sensitive for blastic plasmacytoid dendritic cell neoplasm (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) Cardiovasc Hematol Disord Drug Targets 19(3):195 - 204).

[0879] Exemplary embodiments of antibody targets are HER2 and CD33.

[0880] In an embodiment, the antibody has a free cysteine thiol group that can be used to conjugate with the electrophilic group of a cereblon degrader-linker intermediate (cDLI).

[0881] The thiol-containing antibody can be a native cysteine thiol or a reduced intrachain or interchain disulfide amino acid residue.

[0882] The thiol-containing antibody can be a cysteine-engineered antibody in which one or more cysteine residues have been introduced by mutagenesis according to known techniques, and site-specific conjugation of the cereblon degrader-linker intermediate is provided by cysteine substitution at sites where the engineered cysteine is available for conjugation, without disturbing 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 US7521541; US 7723485; US2012 / 0121615; WO 2009 / 052249). One, two, three or more cysteine amino acids can be introduced into the cysteine-engineered antibody.

[0883] The cDAC 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 its symmetric structure, the cysteine-engineered IgG antibody can allow up to two cDLIs to conjugate to each mutant cysteine site. For example, a cysteine-engineered antibody having one mutant cysteine site allows up to two cDLIs to conjugate to give a theoretical maximum DAR of 2. A cysteine-engineered antibody having two mutant cysteine sites allows up to four cDLIs to conjugate to give a theoretical maximum DAR of 4. A cysteine-engineered antibody having three mutant cysteine sites allows up to six cDLIs to conjugate to give a theoretical maximum DAR of 6.

[0884] Cysteine thiols are reactive nucleophiles at neutral pH. Unlike most amines, they are protonated and less nucleophilic near pH 7. Since the free thiol (RSH, mercapto) group is relatively reactive, proteins with cysteine residues usually exist in an oxidized form as disulfide-linked oligomers or have internal bridging disulfide groups. Antibody cysteine thiol groups are generally more reactive, i.e., more nucleophilic, towards electrophilic conjugation reagents than antibody amines or hydroxyl groups. Engineering cysteine thiol groups by mutating various amino acid residues of a protein to cysteine amino acids can be problematic, especially in the case of unpaired (free Cys) residues or residues that are relatively prone to reaction or oxidation. In concentrated solutions of proteins, whether in the periplasm of Escherichia coli (E. coli), in culture supernatants, or in partially or fully purified proteins, unpaired Cys residues on the protein surface can pair and oxidize to form intermolecular disulfides and thus form protein dimers or multimers. Disulfide dimer formation renders new Cys pairs non-reactive towards conjugation to drugs, ligands, or other labels. In addition, if the protein oxidizes to form an intramolecular disulfide bond between a newly engineered Cys and an existing Cys residue, both Cys groups are unavailable for active site participation and interaction. In addition, the protein can become inactive or non-specific through misfolding or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311:1-9).

[0885] In some embodiments, a cysteine-engineered antibody can have a reactive cysteine thiol residue introduced at a site on the light chain (such as the 149-lysine site (LC K149C)) or a site on the heavy chain (such as the 122-serine site (HC S122C)), as numbered by Kabat numbering. In other embodiments, the cysteine-engineered antibody has a cysteine residue introduced at the 118-alanine site (EU numbering) of the heavy chain (HC A118C). This mutation site is numbered 121 by sequential numbering and alternatively numbered 114 by Kabat numbering. In other embodiments, the cysteine-engineered antibody has a mutant cysteine residue introduced into one of the following: (i) the light chain at G64C, R142C, K188C, L201C, T129C, S114C, V205C, or E105C according to Kabat numbering; (ii) the heavy chain at D101C, A140C, L177C, V184C, T205C, or S122C according to Kabat numbering; or (iii) other cysteine mutant antibodies, such as those 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, pp. 189-203; WO 2011 / 156328; US 9000130. In other embodiments, the cysteine-engineered antibody comprises one or more cysteine mutations selected from the following: HCA118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, HC Y373C.

[0886] In certain embodiments, the antibodies provided herein can be further modified to include additional non-protein moieties known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propanediol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, poly(propylene oxide / ethylene oxide) copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, and the like.

[0887] In some embodiments, the cDAC is a mixture of cereblon degrader antibody conjugate compounds, wherein the average drug load of each antibody in the mixture of cereblon degrader antibody conjugate compounds is from about 2 to about 6.

[0888] The present disclosure includes all reasonable combinations and permutations of the features of the embodiments of Formulae I-II.

[0889] Let p represent the drug - loading amount, that is, the number of cereblon - degrader moieties (CD) of each antibody (Ab) in the cereblon - degrader antibody conjugate (cDAC) of Formula I. The loading amount (p) can range from 1 to about 8 CD moieties per antibody. The cDAC of Formula I includes a mixture or collection of antibodies conjugated to CD moieties in the range of 1 to 8. In some embodiments, the number of CD moieties that can be conjugated to the antibody is limited by the number of reactive or available amino - acid side - chain residues such as lysine and cysteine. In some embodiments, free cysteine residues are introduced into the antibody amino - acid sequence by the methods described herein. In these aspects, p can be 1, 2, 3, 4, 5, 6, 7, or 8 and ranges therebetween, such as 1 to 8 or 2 to 6. Exemplary cDACs of Formula I include, but are not limited to, antibodies having 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123 - 138). In some embodiments, without engineering, one or more free cysteine residues are already present in the antibody that form intra - and inter - chain disulfide bonds (native disulfide groups), in which case the existing free reduced cysteine residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to antibody conjugation in order to generate one or more free native cysteine residues.

[0890] For some cDACs, p may be limited by the number of attachment sites on the antibody. For example, in the case where the attachment 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 thiol groups with sufficiently high reactivity through which the drug can be attached. In other embodiments, one or more lysine amines in the antibody may be available and reactive towards conjugation with the cereblon - degrader - linker intermediate of Formula II. In certain embodiments, a higher drug - loading amount, e.g., p>5, may result in aggregation, insolubility, toxicity, or decreased cell permeability of certain antibody - drug conjugates. In certain embodiments, the average CD loading amount of the 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 denatured to expose reactive nucleophilic groups such as lysine or cysteine.

[0891] The drug - loading amount (drug / antibody ratio) of the cDAC can be controlled in different ways, such as: (i) limiting the molar excess of the cereblon - degrader - linker intermediate compound relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) targeting optimized antibody reactive moieties or limiting the reducing denaturation conditions.

[0892] It should be understood that when more than one nucleophilic group of the antibody reacts with the drug, the resulting product is a mixture of cDAC compounds that has a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody (DAR) can be calculated from the mixture by a dual ELISA antibody assay that is specific for the antibody and specific for the drug. Individual cDAC molecules in the mixture can be identified by mass spectrometry and separated by HPLC such as 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 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, Vol. 45, March 2004; Alley 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, Vol. 45, March 2004). In certain embodiments, homogeneous cDAC with a single drug - loading can be separated from the conjugation mixture by electrophoresis or chromatography.

[0893] In some embodiments, the cereblon degrader moiety of the cDAC has the formula:

[0894] TPL - L2 - E3UL

[0895] Wherein:

[0896] TPL is a target protein ligand;

[0897] E3UL is an E3 ubiquitin ligase ligand that binds to cereblon;

[0898] L2 is a degrader linker; and

[0899] One of TPL, E3UL, and L2 is linked to L1; or

[0900] The cereblon degrader moiety is a molecular glue.

[0901] In some embodiments, TPL has the following formula:

[0902]

[0903] Wherein

[0904] R x is selected from F, Cl, and Br, and n is 0, 1, 2, or 3;

[0905] R y is selected from H and C1-C6 alkyl; and

[0906] The wavy line indicates the connection point of L2.

[0907] In some embodiments, TPL has the following formula:

[0908]

[0909] Wherein the wavy line indicates the connection point of L2.

[0910] In some embodiments, TPL has the following formula:

[0911]

[0912] Where the wavy line indicates the connection point of L2.

[0913] In some embodiments, TPL targets BRD4, GSPT1, BET, BRM (SMARCA2), KRAS, and SHP2.

[0914] In some embodiments, E3UL contains a succinimide group.

[0915] In some embodiments, E3UL is selected from:

[0916]

[0917] Where X 1 is selected from CH2 and C(=O); and the wavy line indicates the connection point of L1 or L2.

[0918] In some embodiments, the cereblon degrader antibody conjugate has the following formula:

[0919]

[0920] Wherein L1 is a protease-cleavable non-peptide linker having the formula:

[0921] -Str-PM-IM-

[0922] Wherein Str is an extender unit covalently linked to X;

[0923] PM is a peptidomimetic unit, and

[0924] IM is a sacrificial unit covalently linked to the glutarimide group of E3UL and has the formula:

[0925] Wherein the wavy line is the connection to PM.

[0926] In some embodiments, the cereblon degrader antibody conjugate has the formula:

[0927]

[0928] Wherein L1 is a protease-cleavable non-peptide linker having the formula:

[0929] -Str-PM-IM-

[0930] Wherein Str is an extender unit covalently linked to X;

[0931] PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to L2 of cD and has the formula:

[0932]

[0933] Wherein the wavy line is the connection to PM.

[0934] In some embodiments, L2 is selected from:

[0935] -N(R)-(C1-C 12 alkanediyl)-N(R)-,

[0936] -N(R)-(C2-C 12 alkenediyl)-N(R)-,

[0937] -N(R)-(C2-C 12 alkynediyl)-N(R)-,

[0938] -N(R)-(C1-C 12 alkanediyl)-C(=O)-(N(R)-,

[0939] -N(R)-(C1-C 12 alkylene diyl)-(N(R)-C(=O)CH2O-,

[0940] -N(R)-(C1-C 12 alkylene diyl)-(N(R)-C(=O)CH2N(R)-,

[0941] -N(R)-(C1-C 12 alkylene diyl)-C(=O)-(N(R)-(C1-C 12 alkylene diyl)-N(R)-,

[0942] -N(R)-(C1-C6 alkylene diyl)-O-(C1-C6 alkylene diyl)-N(R)-,

[0943] -N(R)-(CH2CH2O) n -N(R)-(CH2CH2O) n -, where n is an integer from 1 to 4,

[0944] C1-C 12 alkylene diyl,

[0945] C2-C 12 alkenylene diyl, and

[0946] C2-C 12 alkynylene diyl,

[0947] where R is selected from H, C1-C6 alkylene diyl and the point of attachment to L1; and

[0948] alkylene diyl, alkenylene diyl and alkynylene diyl are optionally substituted by 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.

[0949] In some embodiments, the cDAC of formula I is selected from:

[0950]

[0951]

[0952] where R x is selected from F, Cl, Br, n is 0, 1, 2 or 3; R ySelected from H and C1-C6 alkyl, and X 1 Selected from CH2 and C(=O).

[0953] In some embodiments, Formula I is selected from the following formulas:

[0954]

[0955] And

[0956]

[0957] Wherein R x Is selected from F, Cl, and Br, and n is 0, 1, 2, or 3; R y Selected from H and C1-C6 alkyl, and X 1 Selected from CH2 and C(=O).

[0958] In some embodiments, the cDAC of Formula I is selected from:

[0959]

[0960]

[0961] And

[0962]

[0963] In some embodiments, p of cDAC is 1, 2, 3, 4, 5, or 6.

[0964] Table 4 shows exemplary cDACs prepared with cereblon degrader-linker intermediates from Table 2 and their assay data.

[0965] Table 4 Examples of cereblon-degrading antibody conjugates (cDACs)

[0966]

[0967]

[0968] Biological activity of cDAC

[0969] Generally, the cytotoxic or cytostatic activity of the cereblon degrader antibody conjugate (cDAC) is measured by exposing mammalian cells having a receptor protein (e.g., HER2) to the cDAC antibody in cell culture medium; culturing the cells for a period of about 6 hours to about 5 days and measuring cell viability. Cell-based in vitro assays are used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the cDACs described herein.

[0970] The in vitro potency of the cDACs described herein was measured by a cell proliferation assay as described in Example 103. The cDACs described herein showed surprising and unexpected potency in inhibiting tumor cell proliferation. The potency of the cDACs is related to the target antigen expression of the cells. The conjugates tested were able to bind to specific antigens expressed on the cell surface and cause the death of these cells in vitro.

[0971] The luminescent cell viability assay is a commercially available (Promega Corp., Madison, WI) homogeneous assay based on the recombinant expression of coleopteran luciferase (US 5583024; US 5674713; US 5700670). This cell proliferation assay determines the number of viable cells in a culture based on the quantification of ATP present, which is an indicator of metabolically active cells (Crouch et al. (1993) J. Immunol. Meth. 160:81 - 88; US 6602677). The assay was performed in 96 - well format, making it suitable for automated high - throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs 6:398 - 404). The homogeneous assay procedure involves adding a single reagent ( reagent) directly to the cells cultured in serum - supplemented medium. No cell washing, medium removal, and multiple pipetting steps are required. After adding the reagent and mixing, the system can detect as few as 15 cells / well in 10 minutes in 384 - well format. Cells can be treated continuously with cDAC, or they can be treated and separated from cDAC. Typically, cells treated briefly (i.e., 3 hours) exhibit the same potency effect as continuously treated cells. The assay can be performed in 96 - well or 384 - well format, making it 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 ( reagent) directly to the cultured cells. This causes cell lysis and generates a luminescence signal produced by the luciferase reaction. The luminescence signal is proportional to the amount of ATP present, which in turn is proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or a CCD camera imaging device. The luminescence output is expressed in relative light units (RLU).

[0972] The homogeneous "add - mix - measure" format causes cell lysis and generates a luminescence signal proportional to the amount of ATP present. The amount of ATP is proportional to the number of cells present in the culture. The assay generates a "luminescent" luminescence signal produced by the luciferase reaction, which typically has a half-life greater than five hours, depending on the cell type and culture medium used. Live cells are reflected in relative light units (RLU). The substrate beetle luciferin is oxidized and decarboxylated by recombinant firefly luciferase, converting ATP to AMP and generating photons simultaneously.

[0973] Cell-based in vitro assays are used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of cDAC as described herein. Generally, the cytotoxic or cytostatic activity of cDAC is measured by the following steps: exposing mammalian cells expressing an antigen such as HER2, ER (estrogen receptor), or CD33 polypeptide to cDAC in cell culture medium; culturing the cells for about 6 hours to about 5 days; and measuring cell viability.

[0974] Figure 1 The anti-proliferative effect of the BRD4-cereblon degrader on the in vitro potency of KPL-4 and SK-BR-3 cells at 5 days is shown. Cell viability as a percentage of the control was plotted against the concentration (nM) of the cereblon degrader compound cD-5 from Table 1 in a graph. The IC 50 for KPL-4 was 0.65 nM. The IC 50 for SK-BR03 was 0.41 nM. These results demonstrate the significant potency of the cereblon degrader compound.

[0975] Figure 2A The anti-proliferative effect of the in vitro potency of HER2+ KPL-4 cells after 5 days of treatment with the anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 in Table 4 is shown. Cell viability as a percentage of the control was plotted against the concentration (μg / mL) of cDAC in a graph. Figure 2B The anti-proliferative effect of the in vitro potency of HER2+ SK-BR-3 cells after 5 days of treatment with the anti-HER27C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 in Table 3 is shown. Cell viability as a percentage of the control was plotted against the concentration (μg / mL) of cDAC in a graph.

[0976] Figure 3AThe anti-proliferative effects of the in vitro potency are shown by treating HER2-low / ER+CAMA1 cells with the anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 in Table 4 for 5 days. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph. Figure 3B The anti-proliferative effects of the in vitro potency are shown by treating HER2-low / ER+EFM19 cells with the anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4 for 5 days. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph. Table 5 shows that anti-HER2 cDAC is active in both HER2+ and HER2-low breast cancer cell lines, while the off-target anti-CD33 cDAC is not.

[0977] Table 5 In vitro potency of BRD4-cereblon degrader antibody conjugates (cDAC)

[0978]

[0979] Figure 4 The anti-proliferative effects of the in vitro potency are shown by treating AML cell lines with the anti-CD33 BRD4-cereblon degrader antibody conjugate cDAC-3 for 7 days. The AML cell lines are MV-4-11, EOL-1, Molm-13, Nomo-1, HL-60, and OCI-AML-2. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph. Table 6 shows that cDAC-3 is active in inhibiting various AML cell lines.

[0980] Table 6 In vitro potency of anti-CD33 BRD4-cereblon degrader antibody conjugate cDAC-3 in various AML cell lines

[0981] AML cell line EC50 MV-4-11 0.1058 EOL-1 0.001122 Molm-13 2.590 Nomo-1 0.008795 HL-60 0.0003994 OCI-AML-2 0.0001918

[0982] Figure 5A The anti-proliferative effects of the in vitro potency are shown by treating EOL-1 AML cells with the anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4 for 5 days. The cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph. Figure 5BThe anti-proliferative effects of the in vitro potency are shown by treating HL-60 AML cells with the anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4 for 5 days. Cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph. Table 6 shows Figure 5A and 5B the EC50 values of the cDACs.

[0983] Figure 6A The anti-proliferative effects of the in vitro potency are shown by treating Molm-13 AML cells with the anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4 for 3 days. Cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph. Figure 6B The anti-proliferative effects of the in vitro potency are shown by treating MV-4-11 AML cells with the anti-HER2 7C2 and anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 from Table 4 for 3 days. Cell viability as a percentage of the control was plotted against the concentration of cDAC (μg / mL) in a graph.

[0984] Table 7 shows the EC50 values of the BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-5, and cDAC-6 from Figure 5A , 5B , 6A, and 6B and the in vitro potency in AML cell lines. cDAC-3 and cDAC-5 contain a thiolated human anti-CD33 antibody, while cDAC-4 and cDAC-6 contain a thiolated human anti-7C2 (HER2) antibody. It can be seen that the targeted cDAC3 and cDAC-5 have a concentration-dependent effect in inhibiting AML cells with the CD33 receptor, while the non-targeted cDAC-4 and cDAC-6 show the expected lower potency.

[0985] Table 7 from Figure 5A , 5B , 6A, 6B of the in vitro potency (EC 50 nM) of the BRD4-cereblon degrader antibody conjugates cDAC3-6 in AML cell lines

[0986]

[0987] Cell killing assays of 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 gave the IC50 values (ng / ml) in Table 8. It can be seen that the targeted cDAC3 and cDAC-5 have a concentration-dependent effect on AML cells with CD33 receptor, while the non-targeted cDAC-4 and cDAC-6 have the expected lower potency.

[0988] Table 8 Cell killing assays of BRD4-cereblon degrader antibody conjugates cDAC3-6 in AML cell lines measured by IC50 (ng / ml)

[0989]

[0990] In vitro anti-proliferative effects of exemplary cDACs indicate that the cDACs described herein are biologically active and consist of a variety of antibodies, including those that bind to tumor-associated antigens and cell surface receptor proteins described herein. The exemplary cDACs of Table 3 contain antibodies that bind to the tumor-associated antigens HER2 and CD33. HER2 is highly expressed at levels of millions of copies per cell in certain solid tumors such as breast cancer and gastric cancer. CD33 is expressed at low copy numbers of approximately 10,000 per cell in hematological malignancies such as leukemia and lymphoma. The recycling and internalization mechanisms are different between the HER2 and CD33 cell surface proteins. Thus, demonstration of the significant in vitro potency of exemplary cDACs containing HER2 and CD33 reasonably suggests that cDACs described herein containing other antibodies in addition to anti-HER2 and anti-CD33 will have similar biological activity.

[0991] The in vivo efficacy of cDACs was measured in mouse tumor xenograft studies (Examples 104-105). The cDACs described herein showed surprising and unexpected target-dependent and dose-dependent potency in inhibiting tumor growth. The efficacy of cDACs may be related to the target antigen expression of tumor cells.

[0992] The efficacy of the cDACs provided herein was measured in vivo by implanting allografts or xenografts of cancer cells in rodents and treating the tumors with cDAC. Variable results are expected depending on the cell line, the specificity of the antibody binding of the cDAC to receptors present on the cancer cells, the dosing regimen, and other factors. The in vivo efficacy of cDAC can be measured using transgenic explant mouse models that express moderate to high levels of tumor-associated antigens, including KPL4 that expresses HER2 and BJAB that expresses CD22. Subjects can be treated once with cDAC and monitored for 3 to 6 weeks to measure the time to tumor doubling, log cell kill, and tumor shrinkage. Follow-up dose response and multi-dose experiments can be conducted.

[0993] For example, the in vivo efficacy of the anti-HER2 cDAC described herein can be measured using a HER2-overexpressing transgenic explant mouse model (Phillips et al. (2008) Cancer Res. 68:9280-90). Allografts were propagated from Fo5 mmtv transgenic mice that are (Genentech, Inc.) therapy non-responsive or poorly responsive. According to Examples 104-105, subjects were treated one or more times with a cDAC at a certain dose level (mg / kg) and a placebo buffer control (vehicle), and monitored for two weeks or longer to measure the time to tumor doubling, log cell kill, and tumor shrinkage.

[0994] Figure 7 The in vivo efficacy of anti-CD33 BRD4-cereblon degrading antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6 to reduce tumor volume over time (21 days) at the following doses in an HL-60 xenograft mouse model is shown.

[0995] 1) Vehicle (histidine buffer #8), 100 μL, IV once

[0996] 2) cDAC-4, 3 mg / kg IV once

[0997] 3) cDAC-3, 1 mg / kg IV once

[0998] 4) cDAC-3, 3 mg / kg IV once

[0999] 5) cDAC-3, 10 mg / kg IV once

[1000] 6) cDAC-6, 3 mg / kg IV once

[1001] 7) cDAC-5, 1 mg / kg IV once

[1002] 8) cDAC-5, 3 mg / kg IV once

[1003] As Figure 7 shown, the anti-CD33 BRD4-cereblon degrader antibody conjugates cDAC-3 and cDAC-5 from Table 4 performed according to Example 105 showed significant dose-dependent activity in inhibiting tumor growth in the murine HL-60 human leukemia cell line. At the 1 mg / kg dose, cDAC-5 showed higher 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 was linked to the antibody linker via an acetalamine structure (Table 3, cDLI-5), while the indolinone group of the cereblon degrader moiety of cDAC-3 was linked to the antibody linker via a carbamate group (Table 3, cDLI-1). At the 3 mg / kg dose, both cDAC-3 and cDAC-5 (lines 4 and 8 respectively in Figure 7 ) resulted in tumor volumes below the limit of quantification. In contrast, Figure 7 also illustrates that the non-targeting HER2 controls cDAC-4 and cDAC-6 had an initial response in tumors in the matched 3 mg / kg groups (lines 2 and 6 respectively), but ultimately grew at the end of the study (>21 days). This comparison demonstrates the targeted specific efficacy of the BRD4-cereblon degrader antibody conjugates provided herein (e.g., cDAC-3 and cDAC-5). The LALA-PG mutation within the Fc domain of the anti-CD33 antibody ablates Fc-FcR-mediated effector functions without affecting the required affinity (Schlothauer, T. et al. (2016) Protein Engineering, Design & Selection, 29(10):457–466). In addition, the cereblon degrader antibody conjugates provided herein (e.g., cDAC-3 and cDAC-5) were shown to be tolerated in mice at 3 mg / kg or lower doses.

[1004] The in vivo and whole blood stability of cDAC can be measured and evaluated according to standard assays, including Example 104.

[1005] For the whole blood assays 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 the 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 profiles allowed determination of the average DAR (drug-to-antibody ratio). Table 9 shows that in all media, both cDAC-4 and cDAC-6 were stable at room temperature for 24 hours, with cDAC-4 being slightly more stable than cDAC-6.

[1006] Table 9 Buffer and whole blood (WB) stability of various BRD4-cereblon degrader antibody conjugates cDAC-4 and cDAC-6

[1007]

[1008]

[1009] Drug Compositions

[1010] In another aspect, the present disclosure provides compositions, such as pharmaceutical or pharmacological compositions or formulations, comprising a cereblon degrader antibody conjugate (cDAC) or multiple cDACs as described herein and a pharmaceutical or pharmacological carrier.

[1011] The cDAC can be formulated for parenteral administration, such as by intradermal, subcutaneous (subQ), intramuscular (IM), or intravenous (IV) injection, infusion, or administration into the body cavity or lumen of an organ. Alternatively, the cDACs described herein can be injected into a specific site of the body that has been otherwise placed, such as into a tumor. Compositions for injection will generally comprise a solution of the cDAC dissolved in a pharmaceutical carrier. Acceptable vehicles and solvents that can be used include water and isotonic solutions of one or more salts (such as sodium chloride), e.g., Ringer's solution. Additionally, sterile, non-volatile oils can conventionally be used as a solvent or suspending medium. For this purpose, any mild, non-volatile oil can be used, including synthetic mono- or di-glycerides of fatty acids. Additionally, fatty acids such as oleic acid can also be used in the preparation of injectables. These compositions are desirably sterile and generally do not contain undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutical adjuvants to approximate physiological conditions as required, such as pH regulators and buffers, toxicity regulators, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[1012] The composition can contain any suitable cDAC concentration. The concentration of cDAC in the composition can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the patient. In certain embodiments, the concentration of cDAC in an injectable solution formulation will be in the range of about 0.1% (w / w) to about 10% (w / w).

[1013] Method of treating cancer with a cereblon degrader antibody conjugate

[1014] By inducing target-specific degradation of tumor-associated proteins and conferring specificity to minimize off-target toxic effects, the cereblon degrader antibody conjugates (cDACs) provided herein can be used to treat diseases and disorders such as cancer. The cDAC directs a tumor-associated antigen-binding antibody to cells that express the antigen and deliver the cereblon degrading (cD) moiety to the target cells. The target protein is ubiquitinated and subsequently degraded.

[1015] Provided herein is a method of treating cancer with a pharmaceutical composition of a cereblon degrader antibody conjugate (cDAC) provided herein. The method comprises administering to a subject in need thereof a therapeutically effective amount of a cDAC as described herein, such as a patient having cancer and in need of treating cancer. The method comprises administering a therapeutically effective amount of a cDAC selected from Table 3.

[1016] In certain embodiments, the disclosed cDACs include those having anti-cancer activity. The cDAC selectively delivers an effective dose of the active form of the cereblon degrader moiety to tumor tissue, whereby greater selectivity (i.e., lower effective dose) can be achieved while increasing the therapeutic index ("therapeutic window") relative to the unconjugated cereblon degrader compound.

[1017] It is contemplated that the disclosed cDACs can be used to treat, for example, various hyperproliferative diseases or disorders characterized by overexpression of tumor antigens. Exemplary hyperproliferative conditions include benign or malignant solid tumors and hematological conditions such as leukemia and lymphoid malignancies.

[1018] Examples of cancers to be treated herein include, but are not limited to, epithelial cancer, lymphoma, embryonal tumor, sarcoma, leukemia or lymphocytic malignancies (including acute myeloid leukemia), squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer 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.

[1019] In one aspect, cDAC is provided for use as a medicament. In certain embodiments, cDAC is also provided herein for use in the methods described herein for treating an individual, the method comprising administering to the individual an effective amount of cDAC. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as a therapeutic agent as described herein.

[1020] In a further aspect, the use of cDAC as described herein in the manufacture or preparation of a medicament is also provided. In one aspect, the medicament is for the treatment of cancer, the method comprising administering to an individual suffering from cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as a therapeutic agent as described herein.

[1021] Administration can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. A variety of dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsed infusion. The cDAC dose can be in the range of 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 cDAC dose can be about 100, 200, 300, 400 or 500 μg / kg. The cDAC dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. The cDAC dose can also be outside of these ranges, depending on the particular conjugate and the type and severity of the cancer or disorder being treated. The frequency of administration can be in the range of single dose per week to multiple doses, or more frequently. In some embodiments, cDAC is administered about once a month to about five times a week. In some embodiments, cDAC is administered once a week.

[1022] The disclosed cDAC can be used alone or in combination with other therapeutic agents in a treatment regimen. The cDAC can be administered in the same treatment cycle, on the same treatment day as one or more other drugs, and optionally simultaneously with one or more other drugs in the regimen. For example, for cancer treatment administered every 3 weeks, each co-administered drug is administered on day 1 of the 3-week cycle. For example, cDAC can be co-administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. Such combination therapies encompass co-administration (where two or more therapeutic agents are included in the same composition or separate formulations), as well as separate administration, in which case the administration of cDAC can occur before, simultaneously with, and / or after the administration of the additional therapeutic agent. cDAC can also be used in combination with radiation therapy.

[1023] The disclosed cDAC can be used to treat HER2-positive (HER2+) cancers comprising cancer cells having a higher than normal level of HER2. Examples of HER2-positive cancers include HER2-positive breast cancer and HER2-positive gastric cancer. Optionally, the HER2-positive cancer has an immunohistochemical (IHC) score of 2+ or 3+ by in situ hybridization (ISH) amplification ratio. The term "HER2-positive cell" refers to a cell that expresses HER2 on its surface. cDAC can also be used to treat HER2-low tumor types.

[1024] Examples

[1025] Example 1 Synthesis of 1-(5-aminopentyl)-1H-pyrrole-2,5-dione hydrochloride 1

[1026]

[1027] According to the procedure of WO 2017 / 214024 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). After the mixture was stirred at room temperature for 2 hours, it was heated under reflux for 8 hours. The organic solvent was removed under reduced pressure and the residue was extracted with EtOAc (500 mL × 3) and washed with H2O. The combined organic layers were dried over Na2SO4 and concentrated to obtain the crude product. It was washed with petroleum ether to obtain 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (250 g, 77.4%) as a white solid. 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 under reflux under N2 for 8 hours. The mixture was concentrated and the residue was purified by silica gel column chromatography (PE:EtOAc = 3:1) to obtain tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamate (13 g, 10%). HCl / EtOAc (50 mL) was added dropwise 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). After the mixture was stirred at room temperature for 5 hours, it was filtered and the solid was dried to obtain 1-(5-aminopentyl)-1H-pyrrole-2,5-dione hydrochloride, 1 (16 g, 73.7%). 1 H NMR (400 MHz, 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).

[1028] Synthesis of Example 2 (S)-1-(1-(4-(hydroxymethyl)phenylamino)-1-oxo-5-ureidopentan-2-yl)carbamoyl)cyclobutanecarboxylic acid 2

[1029]

[1030] To a mixture of (S)-2-amino-5-ureidovaleric acid 2a (17.50 g, 0.10 mol) in a mixture of dioxane and H2O (50 mL / 75 mL) was added K2CO3 (34.55 g, 0.25 mol). Fmoc-Cl (30.96 g, 0.12 mol) was added slowly at 0 °C. The reaction mixture was warmed 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 × 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-ureidovaleric acid 2b (38.0 g, 95.6%). 2b was commercially available.

[1031] 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 eq) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline EEDQ, Sigma-Aldrich CAS Reg. No. 16357-59-8 (3.2 g, 13 mmol, 1.3 eq). The mixture was stirred at room temperature under N2 for 16 h and then concentrated to give a brown solid. MTBE (200 mL) was added and the mixture 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 (4.2 g, 84%) as an orange solid. LCMS (ESI): m / z 503.0 [M+1].

[1032] To a stirred solution of 2c (4.2 g, 8.3 mmol) in dry DMF (20 ml) at room temperature was added dropwise piperidine (1.65 mL, 17 mmol, 2 eq). The mixture was stirred at room temperature for 30 min to form 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 and LCMS showed consumption of 2c. It was concentrated to dryness under reduced pressure (ensuring no residual piperidine) and the residue was partitioned between EtOAc and H2O (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-ureidovaleramide 2d (2.2 g, 94%) as an oily residue (containing a small amount of DMF).

[1033] Commercially available 1,1-diethyl cyclobutane-1,1-dicarboxylate (CAS Reg. No. 3779-29-1) is converted to the semi-acid / ester 1-ethyl cyclobutane-1,1-dicarboxylate (CAS Reg No. 54450-84-9) by limited saponification with aqueous base and activated to the NHS ester 1-(2,5-dioxopyrrolidin-1-yl) 1-ethyl cyclobutane-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 No. 125700-67-6, Sigma-Aldrich B-2903)) and N-hydroxysuccinimide.

[1034] To a solution of 1-(2,5-dioxopyrrolidin-1-yl) 1-ethyl cyclobutane-1,1-dicarboxylate (8 g, 29.7 mmol) in DME (50 mL) was added a solution of 2d (6.0 g, 21.4 mmol) and NaHCO3 (7.48 g, 89.0 mmol) in water (30 mL). After the mixture was stirred 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)cyclobutane-1-carboxylate 2e (6.4 g, 68.7%). LCMS (ESI): m / z 435.0 [M+1]

[1035] To a stirred solution of 2e (6.4 g, 14.7 mmol) in a mixture of THF and MeOH (20 mL / 10 mL) at room temperature was added a solution of LiOH·H2O (1.2 g, 28.6 mmol) in H2O (20 mL). After the reaction mixture was stirred 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-yl)carbamoyl)cyclobutane-1-carboxylic acid 2 (3.5 g, yield: 58.5%). LCMS (ESI): m / z 406.9 [M+1]. 11H NMR (400 MHz, methanol-d4) δ 8.86 (d, J = 8.4 Hz, 2H), 8.51 (d, J = 8.4 Hz, 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).

[1036] 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

[1037]

[1038] At 0 °C, diisopropylethylamine, DIPEA (1.59 g, 12.3 mmol) and bis(2-oxo-3-oxazolidinyl)phosphinic chloride, BOP-Cl (CAS Reg. No. 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)cyclobutane carboxylic acid 2 (1 g, 2.46 mmol) in DMF (10 mL), 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 a 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 silica gel column chromatography (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. 11H NMR (400 MHz, DMSO-d6): δ 10.00 (s, 1H), 7.82 - 7.77 (m, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 6.96 (s, 2H), 5.95 (t, J = 6.4 Hz, 1H), 5.39 (s, 2H), 5.08 (t, J = 5.6 Hz, 1H), 4.40 - 4.35 (m, 3H), 4.09 (d, J = 4.8 Hz, 1H), 3.01 (d, J = 3.2 Hz, 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).

[1039] Synthesis of Example 4 (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

[1040]

[1041] At 0 °C, a solution of 3 (2.0 g, 3.5 mmol) in N,N-dimethylformamide DMF or N-methylpyrrolidone NMP (50 mL) was treated dropwise in portions with thionyl chloride SOCl2 (1.25 g, 10.5 mmol). The reaction remained yellow. The reaction was monitored by LC / MS, indicating a conversion > 90%. After the reaction mixture was stirred at 20 °C for 30 minutes or several hours, it was diluted with water (50 mL) and extracted with EtOAc (50 mL x 3). The organic layer was dried, concentrated and purified by flash column (DCM:MeOH = 20:1) to form 4 as a grey solid, also known as MC-CBDK-cit-PAB-Cl. LCMS: (5 - 95, AB, 1.5 minutes), 0.696 minutes, m / z = 589.0 [M+1] + .

[1042] Synthesis of Example 5 (S)-4-(2-(1-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamoyl)cyclobutanecarbonylamino)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate 5

[1043]

[1044] To a solution of 3 in anhydrous DMF was added diisopropylethylamine (DIEA), followed by addition of PNP carbonate (bis(4-nitrophenyl) carbonate). The reaction solution was stirred at room temperature (r.t.) for 4 h and the mixture was purified by preparative HPLC to give 5. LCMS (ESI): M+H + = 736.29.

[1045] Example 6 Synthesis of 4-nitrophenyl 2-(pyridin-2-yldisulfanyl)ethyl carbonate 6

[1046]

[1047] According to the protocol of WO 2016 / 040825 incorporated herein, 1,2-bis(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-nitrophenyl chloroformate in triethylamine and acetonitrile gave 4-nitrophenyl 2-(pyridin-2-yldisulfanyl)ethyl carbonate 6.

[1048] Example 7 Synthesis of 2-((5-nitropyridin-2-yl)disulfanyl)ethylamine hydrochloride 7

[1049]

[1050] 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 addition of 2-aminoethanethiol hydrochloride (183 mg, 1.61 mmol). The reaction mixture was stirred at room temperature overnight, concentrated in vacuo to remove the solvent, and the residue was washed with DCM (30 mL×4) to give 7 (300 mg, 69.6%) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.28 (d, J = 2.4 Hz, 1H), 8.56 (dd, J = 8.8, 2.4 Hz, 1H), 8.24 (s, 4H), 8.03 (d, J = 8.8 Hz, 1H), 3.15 - 3.13 (m, 2H), 3.08 - 3.06 (m, 2H)

[1051] Example 8 Synthesis of 4-nitrophenyl 2-((5-nitropyridin-2-yl)disulfanyl)ethyl carbonate 8

[1052]

[1053] Under N2, 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 / CH3OH (250 mL / 250 mL) was stirred at room temperature for 24 h. After the mixture was concentrated under vacuum, the residue was diluted with DCM (300 mL). Manganese dioxide MnO2 (10 g) was added, and the mixture was stirred at room temperature for another 0.5 h. The mixture was purified by silica gel column chromatography (DCM / MeOH = 100 / 1 to 100 / 1) to afford 2-((5-nitropyridin-2-yl)disulfanyl)ethanol as a brown oil (2.2 g, 61.1%). 1 H NMR (400 MHz, CDCl3) δ 9.33 (d, J = 2.8 Hz, 1H), 8.38 - 8.35 (dd, J = 9.2, 2.8 Hz, 1H), 7.67 (d, J = 9.2 Hz, 1H), 4.10 (t, J = 7.2 Hz, 1H), 3.81 - 3.76 (q, 2H), 3.01 (t, J = 5.2 Hz, 2H).

[1054] 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 afford 8 as a light brown oil (270 mg, 33.1%). 1 H NMR (400 MHz, CDCl3) δ 9.30 (d, J = 2.4 Hz, 1H), 8.43 - 8.40 (dd, J = 8.8, 2.4 Hz, 1H), 8.30 - 8.28 (m, 2H), 7.87 (d, J = 8.8 Hz, 1H), 7.39 - 7.37 (m, 2H), 4.56 (t, J = 6.4 Hz, 2H), 3.21 (t, J = 6.4 Hz, 2H).

[1055] Example 9 Synthesis of 2-((5-nitropyridin-2-yl)disulfanyl)propan-1-amine 9

[1056]

[1057] To a stirred solution of 1-aminopropan-2-ol (10 g, 133 mmol) in MeOH (360 mL) and H2O (40 mL) was added Boc2O (37 g, 169 mmol). The reaction mixture was stirred at room temperature for 5 h, concentrated and purified by chromatography (EtOAc / PE = 10%-50%) to afford tert-butyl 2-hydroxypropylcarbamate (19.8 g, yield: 85%) as a colorless oil.

[1058] To a stirred solution of tert-butyl 2-hydroxypropylcarbamate (10 g, 57 mmol) and Et3N (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 h, washed with ice water (200 mL x 3) and brine (200 mL). The organic layer was concentrated to afford 1-(tert-butoxycarbonylamino)propan-2-yl methanesulfonate (12 g, yield: 83%) as a red oil.

[1059] 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 ethanethiolate, 5.4 g, 47.3 mmol) in H2O (100 mL). The reaction mixture was stirred at 60 °C for 12 h. The mixture was concentrated and extracted with DCM (200 ml x 2). The combined organic layers were concentrated and purified by chromatography to afford S-1-(tert-butoxycarbonylamino)propan-2-yl thioacetate (1.1 g, yield: 20%) as a red solid. 1 H NMR (400 MHz, CDCl3-d) 1.30 (d, J = 7.09 Hz, 3H) 1.44 (s, 9H) 2.33 (s, 3H) 3.16 - 3.42 (m, 2H) 3.58 - 3.71 (m, 1H)

[1060] To a stirred solution of S-1-(tert-butoxycarbonylamino)propan-2-yl thioacetate (500 mg, 2.15 mmol) in MeOH (5 mL) was added HCl / MeOH (10 mL) dropwise. The reaction mixture was stirred at room temperature for 3 h, concentrated to afford 1-aminopropane-2-thiol hydrochloride, which was used directly in the next step.

[1061] 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. MnO2 (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 x 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.

[1062] Synthesis of ((2,6-dioxo-3-(1-oxoisoindolin-2-yl)piperidin-1-yl)methyl)carbamic acid 4-nitrobenzyl ester 10 in Example 10

[1063]

[1064] Synthesis of ((2,6-dioxo-3-(1-oxoisoindolin-2-yl)piperidin-1-yl)methyl)carbamic acid 2-((5-nitropyridin-2-yl)disulfanyl)ethyl ester 11 in Example 11

[1065]

[1066] 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 in Example cD-1

[1067]

[1068] 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 in Example cD-2

[1069]

[1070] Synthesis of Example cD-3 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

[1071]

[1072] Synthesis of Example cD-4 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

[1073]

[1074] Synthesis of Example cD-5 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

[1075]

[1076] Synthesis of Example cD-6 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

[1077]

[1078] Synthesis of Example cDLI-1 (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]azulen-6-carboxamido)heptyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamic acid 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)propanamido)benzyl ester cDLI-1

[1079]

[1080] Preparation of tert-butyl (7-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)heptyl)carbamate cDLI-1c

[1081] 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. Then NaBH(OAc)3 (1226.2 mg, 5.79 mmol) was 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 completion of the reaction. The reaction mixture was washed with water (30 mL x 2), the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude, which was purified by silica gel column chromatography (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] + .

[1082] Preparation of tert-butyl 3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidine-1-carboxylate cDLI-1d

[1083] To a solution of cDLI-1c (300.00 mg, 0.63 mmol) and di-tert-butyl dicarbonate Boc2O (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) indicated completion of the reaction. The mixture was diluted with dichloromethane (45 mL) and washed with aqueous citric acid solution (15 mL), water (15 mL), and saturated brine (15 mL). The organic layer was concentrated and purified by flash column (eluting with 0 - 60% EtOAc in petroleum ether) to afford cDLI-1d (300 mg, 83%) as a pale yellow solid.

[1084] 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

[1085] 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 uL, 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 h. TLC (5% MeOH in DCM, R f = 0.6) indicated completion of the reaction. The mixture was concentrated and used directly for the next step. To the crude product (277.27 mg, 0.44 mmol, theoretical) was added (S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl) allylcarbamate cDLI-1e (243 mg, 0.87 mmol) and 4A molecular sieves in dichloromethane (5 mL), and together with N,N-dimethylformamide DMF (1 mL) 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 h. TLC (10% MeOH in DCM, R f= 0.5) indicates the completion of the reaction. The mixture was filtered and 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 as a pale yellow solid (75 mg, 19.6%). LCMS (10 - 80, AB, 7.0 min): RT = 4.491 min, m / z = 877.5 [M+H] + .

[1086] 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

[1087] At 25 °C, Pd(PPh3)4 (19.8 mg, 0.02 mmol) was added to a solution of cDLI-1f (75.00 mg, 0.09 mmol) and 1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (also known as 1,3-dimethylbarbituric acid (66.8 mg, 0.43 mmol)) in dichloromethane (3 mL) and methanol (3 mL). The reaction mixture was stirred at 25 °C for 3 h under a nitrogen atmosphere. TLC (10% MeOH in DCM, R f = 0.3) indicates the completion of the reaction. 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 as a white solid (30 mg, 44.2%). LCMS (5 - 95, AB, 1.5 min): RT = 0.883 min, m / z = 793.4 [M+H] + .

[1088] 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

[1089] To a solution of cDLI-1g (30.00 mg, 0.04 mmol) and 1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxylic acid 2,5-dioxopyrrolidin-1-yl ester 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 completion of the reaction. The mixture was concentrated and purified by preparative TLC (10% MeOH in DCM) to afford 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] + .

[1090] (7-Aminoheptyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamic acid 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)propionamido)benzyl ester cDLI-1j Preparation

[1091] 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 indicated completion of the reaction. The mixture was concentrated to afford cDLI-1j (9.20 mg, 100%) as a pale yellow solid.

[1092] Preparation of cDLI-1

[1093] 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]azepine-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 benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate HATU, CAS Reg. No. 148893-10-1 (8.00 mg, 0.02 mmol) in N,N-dimethylformamide (1 mL) was added N,N-diisopropylethylamine (0.01 mL, 0.05 mmol). The mixture was stirred at 25 °C for 5 minutes. Then cDLI-1j (9.20 mg, 0.01 mmol) was added. Then the mixture was stirred at 25 °C for 1 hour. LCMS (10-80 AB / 7.0 minutes)): RT = 3.840 minutes, [M+H]+ 1365.3 showed 16% of the desired product. Then the mixture was filtered and the filtrate was sent to preparative HPLC (acetonitrile in water 30-60 / 0.225% FA) to give cDLI-1 (1.90 mg, 13.1%) as a pale yellow solid. LCMS (5-95, AB, 1.5 minutes)): RT (220 / 254 nm) = 0.88 minutes, m / z = 1365.4 [M+H] + 。

[1094] Synthesis of Example cDLI-5 ((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]azepine-9-carboxamido)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamic acid 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl ester cDLI-5

[1095]

[1096]

[1097] Preparation of ((3-(4-((7-((tert-Butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamic acid 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl ester cDLI-5c

[1098] To a solution of diphenylphosphoryl azide DPPA (0.05 mL, 0.22 mmol) and 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 and then (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 minutes): R T = 3.735 minutes, m / z = 1098.3 [M+H] + showed 18% of the desired product. The mixture was then filtered and the filtrate was sent to preparative HPLC (acetonitrile in water 30 - 60 / 0.225% FA) to afford cDLI-5c (20 mg, 19.3%) as a pale yellow solid. LCMS (5 - 95, AB, 1.5 minutes): R T = 0.909 minutes, m / z = 1098.7 [M+H] + .

[1099] Preparation of ((3-(4-((7-Aminoheptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamic acid 4-((S)-2-(1-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)benzyl ester cDLI-5d

[1100] cDLI-5c (15.0 mg, 0.01 mmol) was stirred in a mixture of 5% trifluoroacetic acid in hexafluoroisopropanol HFIP (1 mL) at 25 °C for 1 h. The mixture was concentrated to afford cDLI-5d as the TFA salt as a white solid (15 mg, 99%). LCMS (5-95, AB, 1.5 min): R T = 0.756 min, m / z = 998.7 [M+H] + .

[1101] Preparation of cDLI-5

[1102] 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). The mixture was stirred at 25 °C for 5 min. Then 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]azepine-6-carboxylic acid cDLI-5e (15.0 mg, 0.01 mmol) was added. The mixture was stirred at 25 °C for 1 h. LCMS (5-95 AB / 1.5 min): RT = 0.870 min, m / z = 741.3 [M / 2+H]+ showing 18% of the desired product. The mixture was then filtered and the filtrate was sent to preparative HPLC (acetonitrile in water 30-60 / 0.2,25% FA) to afford cDLI-5 as a white solid (6.4 mg, 31.4%). LCMS (5-95, AB, 1.5 min): RT (220 / 254 nm) = 0.863 min, m / z = 1480.9 [M+H] + .

[1103] Example cDLI-6 S-methylthiosulfonate 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]azepine-9-carboxamido)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamoyl)oxy)-2-methylpropan-2-yl) ester cDLI-6 synthesis

[1104]

[1105] Preparation of tert-butyl (7-(methoxy(methyl)amino)-7-oxoheptyl)carbamate cDLI-6a

[1106] 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 the addition of 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 completion of the reaction. The reaction mixture was poured into water (50 mL) and extracted with DCM (50 mL x 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 as a colorless oil (2 g, 85%). LCMS (5 - 95, AB, 1.5 min): R T = 0.772 min, m / z = 189.1 [M - 100 + H] + . 1 H NMR (400 MHz, chloroform-d): δ = 4.52 (br s, 1H), 3.68 (s, 3H), 3.18 (s, 3H), 3.11 (d, J = 6.0 Hz, 2H), 2.41 (t, J = 7.2 Hz, 2H), 1.70 - 1.59 (m, 2H), 1.52 - 1.42 (m, 11H), 1.39 - 1.32 (m, 4H)

[1107] Preparation of tert-butyl (7-oxoheptyl)carbamate cDLI-6b

[1108] At -78 °C, to a solution of cDLI-6a (500.00 mg, 1.73 mmol) in tetrahydrofuran (12 mL) was added lithium aluminum hydride LiAlH4 (98.70 mg, 2.60 mmol). 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) indicated completion of the reaction. Saturated aqueous NH4Cl was slowly added dropwise with stirring at 0 °C and the mixture was filtered and extracted with ethyl acetate (30 mL * 3). The organic layer was washed with H2O (20 mL) and brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give cDLI-6b as a colorless oil (390.00 mg, 98%), which was used directly in the next step.

[1109] (7-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)heptyl)carbamic acid tert-butyl ester Preparation of cDLI-6d

[1110] To a solution of 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione, lenalidomide (CAS Reg. No. 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 h. Then sodium triacetoxyborohydride STAB, NaBH(OAc)3 (735.73 mg, 3.47 mmol) was added to the mixture and stirred at 25 °C for 12 h. LCMS (5-95 AB / 1.5 min): R T = 0.889 min, m / z = 373.1 [M-100+H] + showed 28% of the desired product. The reaction mixture was washed with water (30 mL x 2), 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] + .

[1111] 2-(3-(4-((7-((tert-Butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)acetic acid tert-butyl ester Preparation of cDLI-6e

[1112] To a mixture of cDLI-6d (370.00 mg, 0.78 mmol) in N,N-dimethylformamide (20 mL) was added K2CO3 (173.2 mg, 1.25 mmol) and tert-butyl bromoacetate (0.15 mL, 1.02 mmol). The mixture was stirred at 25 °C for 2 h. TLC (10% MeOH in DCM, R f= 0.8) indicates the completion of the reaction. The mixture was filtered, EtOAc (60 mL) was added and washed with water (20 mL). The organic layer was dried over Na2SO4, filtered and concentrated to obtain the crude product, which was purified by silica chromatography eluting with 0 - 1.5% MeOH in DCM to give cDLI-6e as a white solid (320.00 mg, 70%). LCMS (5 - 95, AB, 1.5 min): RT = 0.990 min, m / z = 587.4 [M + H] + 。

[1113] Preparation of tert-butyl 2-(3-(4-((7-aminoheptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)acetate cDLI-6f

[1114] 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 - 95 AB / 1.5 min): RT = 0.705 min, m / z = 431.2 [M + H]+ showing 70% of the desired product. The mixture was concentrated to give cDLI-6f as a yellow oil (296.00 mg, 99.7%).

[1115] Preparation of 2-(3-(4-((7-((tert-butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)acetic acid cDLI-6g

[1116] At 25 °C, to a solution of cDLI-6f (296.00 mg, 0.54 mmol) in methanol (5 mL) was added Boc2O (0.19 mL, 0.82 mmol) and triethylamine Et3N (0.23 mL, 1.63 mmol), 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]+ showing 70% of the desired product. The mixture was concentrated and purified by reverse phase chromatography (Xtimate (Welch Materials, Inc.) 150*25 mm*5 um, acetonitrile in water 60 - 80.6 / 0.225% FA) to give cDLI-6g as a white solid (110 mg, 38%). LCMS (5 - 95, AB, 1.5 min): RT = 0.895 min, m / z = 553.3 [M + Na] + 。

[1117] Preparation of S-(1-((((3-(4-((7-((tert-Butoxycarbonyl)amino)heptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamoyloxy)-2-methylpropan-2-yl) methanesulfonate cDLI-6h

[1118] 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) methanesulfonate (48.6 mg, 0.26 mmol), followed by triethylamine (0.03 mL, 0.20 mmol). The mixture was stirred at 25 °C for 10 minutes and heated to 90 °C under N2 atmosphere for 3 hours. TLC (10% MeOH in DCM, R f = 0.6) indicated completion of the reaction. 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), the organic layer was dried over Na2SO4, concentrated and purified by preparative TLC (10% MeOH in DCM, R f = 0.6) to afford cDLI-6h (40 mg, 43%) as a white solid. LCMS (5-95, AB, 1.5 minutes): RT = 0.958 minutes, m / z = 734.4 [M+H] + .

[1119] Preparation of S-(1-((((3-(4-((7-Aminoheptyl)amino)-1-oxoisoindolin-2-yl)-2,6-dioxopiperidin-1-yl)methyl)carbamoyloxy)-2-methylpropan-2-yl) methanesulfonate cDLI-6i

[1120] 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 hour. The mixture was concentrated to afford the TFA salt of cDLI-6i (32 mg, 98.1%) as a yellow solid. LCMS (5-95, AB, 1.5 minutes): RT = 0.758 minutes, m / z = 612.3 [M+H] + .

[1121] 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]azulen-6-carboxylic acid cDLI-6j

[1122] 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 h. TLC (10% methanol in dichloromethane, R f = 0.3) indicated completion of the reaction. Water (20 mL) was added to the mixture, the aqueous layer was acidified to pH = 3 with 2 M HCl, then extracted with EtOAc (30 mL * 4), and the organic layer was dried over Na2SO4, filtered and concentrated to give cDLI-6j as a yellow solid (95 mg, 98%). LCMS (5-95, AB, 1.5 min): RT = 0.770 min, m / z = 501.2 [M+H] + .

[1123] Preparation of cDLI-6

[1124] 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. Then the mixture was filtered and the filtrate was subjected to preparative HPLC (acetonitrile 30-60 in water / 0.225% FA) to give cDLI-6 as a pale yellow solid (6.1 mg, 12%). LCMS (5-95, AB, 1.5 min): RT (220 / ​254 nm) = 0.893 min, m / z = 1094.5 [M+H] + .

[1125] Preparation of the cysteine-engineered antibody of Example 101

[1126] For large-scale antibody production, the antibody was produced in CHO cells. The vectors encoding VL and VH were transfected into CHO cells, and the IgG was purified from the cell culture medium by protein affinity chromatography.

[1127] As initially isolated, the engineered cysteine residues in the antibody exist as mixed disulfides with cellular thiols (e.g., glutathione) and are thus not available for conjugation. Partial reduction (e.g., with DTT) of these antibodies, purification, and reoxidation with dehydroascorbic acid (DHAA) yield antibodies with free cysteine thiols available for conjugation, as described previously (Junutula et al. (2008) Nat. Biotechnol. 26:925-932; US2011 / 0301334). Briefly, the antibody is combined with the cereblon degrader-linker intermediate to allow for optimal conjugation to the free cysteine residues of the antibody. After several hours, the cereblon degrader-antibody conjugate is purified.

[1128] Under certain conditions, the cysteine-engineered antibody is made reactive for conjugation to the cereblon degrader-linker intermediate by treatment with a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride) in 50 mM Tris pH 7.5 and 2 mM EDTA at 37 °C for 3 hours or overnight at room temperature (Getz et al., (1999) Anal. Biochem. 273:73-80; Soltec Ventures, Beverly, MA). The full-length cysteine-engineered monoclonal antibody (THIOMAB TM )(Gomez et al., (2010) Biotechnology and Bioeng. 105(4):748-760; Gomez et al., (2010) Biotechnol. Prog. 26:1438-1445) expressed in CHO cells is reduced, e.g., with approximately 50-fold excess DTT at room temperature to reduce disulfide bonds that may form between the newly introduced cysteine residues and cysteines present in the culture medium. The reduced THIOMAB TM is 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 is acidified by addition of 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 is eluted with 50 mM Tris (pH 7.5), 2 mM EDTA.

[1129] The 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 Service, National Institutes of Health, Bethesda, MD). The 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) unless noted as the Kabat system. Single letter amino acid abbreviations are used.

[1130] The full-length cysteine-engineered monoclonal antibody (THIOMAB TM ) expressed in CHO cells bears cysteine adducts (cystine), or is glutathionylated on the engineered cysteine due to the cell culture conditions. To release the reactive thiol group of the engineered cysteine, the THIOMAB TM is dissolved in 500 mM sodium borate and 500 mM sodium chloride at approximately pH 8.0 and reduced with approximately 50 to 100-fold excess of 1 mM TCEP at 37 °C for approximately 1 to 2 hours. Alternatively, DTT is used as the reducing agent. The formation of interchain disulfide bonds is monitored by non-reducing SDS-PAGE or by denaturing reverse phase HPLC PLRP column chromatography. The reduced THIOMAB TM is 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.

[1131] By performing reoxidation, disulfide bonds are re-established between the cysteine residues present in the parental Mab. The eluted reduced THIOMAB TMTreatment is carried out for about 3 hours or about 3 hours with 15X or 2 mM dehydroascorbic acid (dhAA) in 50 mM Tris-Cl (pH 7.5) at pH 7, or overnight with 200 nM to 2 mM aqueous copper sulfate solution (CuSO4) at room temperature. Other oxidants (i.e., oxidation reagents) and oxidation conditions known in the art can be used. Ambient air oxidation may also be effective. This mild partial reoxidation step effectively forms intrachain disulfide bonds with high fidelity. The buffer is exchanged by elution through Sephadex G25 resin and eluted with PBS containing 1 mM DTPA. The reduced antibody concentration and thiol concentration from the absorbance at 280 nm of the solution are determined by reaction with DTNB (Aldrich, Milwaukee, WI) and measuring the absorbance at 412 nm, thereby checking the thiol / antibody value.

[1132] On a TSQ Quantum Triple quadrupole TM mass spectrometer (ThermoElectron, San Jose California) with an extended mass range. The sample is chromatographically separated on a 1000A micropore column (50 mm × 2.1 mm, Polymer Laboratories, Shropshire, UK) heated to 75 °C. 30%-40% B can be used (solvent A: 0.05% TFA in water, solvent B: acetonitrile containing 0.04% TFA), and the eluate is directly ionized using an electrospray source. The data is collected by a data system and deconvoluted using (Novatia, LLC, New Jersey). Before LC / MS analysis, the antibody or conjugate (50 micrograms) is treated with PNGase F (2 units / ml; PROzyme, San Leandro, CA) at 37 °C for 2 hours to remove N-linked carbohydrates.

[1133] The hydrophobic interaction chromatography (HIC) sample is injected onto a butyl HIC NPR column (2.5 micron particle size, 4.6 mm × 3.5 cm) (Tosoh Bioscience) and eluted with a linear gradient of 0 to 70% at 0.8 ml / minute (A: 1.5 M ammonium sulfate solution in 50 mM potassium phosphate, pH 7, B: 50 mM potassium phosphate pH 7, 20% isopropanol). An Agilent 1100 series HPLC system equipped with a multi-wavelength detector and Chemstation software is used to resolve and quantify each antibody species with different drug ratios.

[1134] Example 102 Conjugation of Cereblon Degrader-Linker Intermediate (cDLI) and Antibody

[1135] After the reduction and re-oxidation procedures of Example 101, the cysteine-engineered antibody (THIOMAB TM ) was dissolved in PBS (phosphate-buffered saline) buffer and frozen on ice. An excess (about 1.5 moles to 20 equivalents) of the cereblon degrader-linker intermediate activated with a thiol-reactive group such as pyridyldisulfide, maleimide, or bromoacetamide was dissolved in DMSO, diluted in acetonitrile and water, and added to the frozen, reduced, and re-oxidized antibody in PBS. Typically, the cereblon degrader-linker intermediate from a DMSO stock in 50 mM Tris at a concentration of about 20 mM and pH 8 was added to the antibody, and monitored for about 1 to about 24 hours until the reaction was complete, as determined by LC-MS analysis of the reaction mixture. When the reaction was complete, an excess of a capping reagent such as ethyl maleimide was added to quench the reaction and cap any unreacted antibody thiol groups. The conjugate mixture could be 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 a G25 resin in PBS, filtered through a 0.2 μm filter under sterile conditions, and stored frozen.

[1136] For example, after dilution with 20 mM sodium succinate at pH 5, the crude cDAC was 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. The cDAC was formulated into 20 mM His / acetate (pH 5) with 240 mM sucrose using a gel filtration column. The cDAC was characterized by UV spectroscopy to determine the protein concentration, analytical SEC (size exclusion chromatography) for aggregation analysis, and LC-MS before and after treatment with Lysine C endopeptidase.

[1137] Size exclusion chromatography analysis was performed using a Shodex KW802.5 column with 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 / minute. The aggregation state of the cDAC was determined by integrating the peak area absorbance eluting at 280 nm.

[1138] LC-MS analysis can be performed using an Agilent QTOF 6520 ESI instrument. For example, cDAC was treated with Tris (pH 7.5) containing 1:500 w / w intracellular protease Lys C (Promega) at 37 °C for 30 minutes. The resulting cleavage fragments were loaded onto a heated to 80 °C (Angstrom), 8 μm (micrometer) PLRP-S (highly cross-linked polystyrene) chromatographic column and eluted with a gradient of 30% B to 40% B in 5 minutes. Mobile phase A was H2O containing 0.05% TFA, and mobile phase B was acetonitrile containing 0.04% TFA. The flow rate was 0.5 ml / minute. Before electrospray ionization and MS analysis, protein elution was monitored by UV absorbance detection at 280 nm. Chromatographic resolution of the unconjugated Fc fragment, residual unconjugated Fab, and drug-conjugated Fab could generally be achieved. The obtained m / z spectra were deconvoluted using Mass Hunter TM software (Agilent Technologies) to calculate the mass of the antibody fragments.

[1139] Example 103 In Vitro Cell Proliferation Assay

[1140] The efficacy of cDAC was measured by a cell proliferation assay using the following protocol (CELLTITER GLO TM Luminescent Cell Viability Assay, Promega Corp. Technical Bulletin TB288; Mendoza et al. (2002) Cancer Res. 62:5485-5488):

[1141] 1. An aliquot of 40 μl of a cell culture containing approximately 4000 cells (SK-BR-3, KPL-4, CAMA1, EFM19, MV-4-11, EOL-1, Molm-13, Nomo-1, HL-60, and OCI-AML-2 expressing HER) was deposited into each well of a 384-well opaque-wall plate.

[1142] 2. Control wells containing medium and no cells were prepared.

[1143] 3. cDAC (n = 3) was added to the experimental wells and incubated for 3 to 5 days.

[1144] 4. The plate was equilibrated to room temperature for approximately 30 minutes.

[1145] 5. An equal volume of CELLTITER GLO TM reagent as the volume of cell culture medium present in each well was added.

[1146] 6. Mix the contents on an orbital shaker for 15 minutes to induce cell lysis.

[1147] 7. Incubate the plate at room temperature for 5 minutes to stabilize the luminescence signal.

[1148] 8. Record the luminescence and report it as % activity in a graph, where the RLU (Relative Luminescence Units) is normalized to the control (no antibody control minus no cell control).

[1149] At Figure 1 、 Figure 2A - 2B 、 Figure 3A - 3B 、 Figure 4 、 Figure 5A - 5B and Figure 6A - 6B the data is plotted and presented as individual points for each replicate (n = 3) of each antibody. This protocol is a modification of CELLTITER GLO TM luminescent cells. The cell line can be grown in a medium containing RPMI - 1640, 20% HI - FBS, 2 mM L - glutamine.

[1150] Example 104 Whole Blood Stability Assay

[1151] Whole Blood Incubation: Matrix collected in lithium heparin tubes (shipped by the vendor (BioIVT, Westbury NY)). Unfrozen plasma and whole blood were collected in the afternoon, cold shipped (2 - 8 °C) overnight to arrive within 18 hours after collection, while frozen plasma was collected and shipped frozen under normal delivery conditions. The cDAC source material was formulated into a buffer (1X PBS [pH 7.4], 0.5% bovine serum albumin, 15 ppm Proclin TM ) and then further diluted to a final concentration of 100 μg / mL. After mixing, 150 μL of the whole blood / buffer stability sample was aliquoted into two separate tubes at two different time points. Then the 0 - hour time point was placed at - 80 °C. Whole blood samples were generated, with two 150 - μL aliquots for the 0 and 24 - hour whole blood time points. The 0 - hour sample was immediately placed in an - 80 °C freezer and simultaneously incubated in a 37 °C incubator with shaking for 24 hours (approx. 700 rpm). Aliquots were collected after storage in an - 80 °C freezer for 24 hours until affinity capture LC - MS was performed. The matrices used to generate the samples were mouse (CB17 SCID), rat (Sprague - Dawley), monkey (cynomolgus), and human.

[1152] In vitro stability sample analysis: Wash the streptavidin (SA)-coated magnetic beads (Thermo Fisher Scientific, catalog number 60210) twice with HBS-EP buffer (GE Healthcare Life Sciences, catalog number BR-1001-88), then mix with the biotinylated extracellular domain of the target (e.g., human HER2), or an anti-idiotypic antibody for specific capture, or biotinylated human IgG for general capture using KingFisher Flex (Thermo Fisher Scientific), and incubate for 2 hours at room temperature with gentle stirring. Then wash the SA-bead / biotin capture probe complex twice with HBS-EP buffer, mix with cDAC or precursor stability samples pre-diluted 1:16 with HBS-EP buffer, and incubate for 2 hours at room temperature with gentle stirring. After 2 hours, wash the SA-bead / biotin capture probe / sample complex twice with HBS-EP buffer, and then deglycosylate by incubating overnight with PNGase F (New England Biolabs, catalog number P0704B). Then wash the SA-bead / biotin capture probe / sample complex twice with HBS-EP buffer, followed by two washes with water (Optima TM LC / MS Grade, Fisher Chemical, catalog #W6-1), and finally wash once with 10% acetonitrile. Place the beads in 30% acetonitrile / 0.1% formic acid and elute for 30 minutes at room temperature with gentle stirring, then collect the beads. Then load the eluted sample onto an LC-MS (Thermo Scientific Q-Exactive Plus) for analysis. Inject and load 10 μL of the cDAC sample onto a Waters C4 column (1000 μm × 10 cm) maintained at 65 °C. Separate cDAC on-column at a flow rate of 20 μL / min using a Waters Acquity UPLC system, with the following gradient: 20% B (100% acetonitrile + 0.1% formic acid) at 0 - 2 minutes; 35% B at 2.5 minutes; 65% B at 5 minutes; 95% B at 5.5 minutes; 5% B at 6 minutes. The column is directly coupled to a Thermo Scientific Q-Exactive Plus mass spectrometer for on-line detection, which operates in positive electrospray ionization mode and acquires a mass range of m / z 500 to 4000 Da.

[1153] Example 105 Tumor Growth Inhibition In Vivo Efficacy in HL-60 Mice Expressing CD33

[1154] Prior to a single treatment on Day 0, tumors were established and allowed to grow in CD33-expressing HL-60 mice to a volume of 150 - 200 mm 3 (as measured using calipers). Tumor volume was measured using calipers according to the following formula: V (mm 3 ) = 0.5 A X B 2 , where A and B are the long and short diameters, respectively. Mice were euthanized before the tumor volume reached 3000 mm 3 or when the tumor showed signs of impending ulceration. Data collected from each experimental group (10 mice per group) are represented as mean + SE.

[1155] Alternatively, the Fo5 mouse mammary tumor model was used to evaluate the in vivo efficacy of an anti-HER2 cereblon-degrading agent antibody conjugate (cDAC) after a single intravenous injection, and 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), which is incorporated herein by reference. The anti-HER2 cDAC was tested using the Fo5 model, which is a transgenic mouse model in which the human HER2 gene is overexpressed in the mammary epithelium under the transcriptional regulation of the mouse mammary tumor virus promoter (MMTV-HER2). HER2 overexpression leads to the spontaneous development of mammary tumors. Mammary tumors from one of these founder animals (founder #5 [Fo5]) were propagated in subsequent generations of FVB mice by serial transplantation of tumor fragments (sized approximately 2 × 2 mm). All studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. At the start of the study and 14 days after transplantation, each cDAC (single dose) was administered intravenously to nine animals. The initial tumor size was approximately 200 mm 3 volume.

[1156] Other mammary fat pad transplantation efficacy models can be used as described (Chen et al. (2007) Cancer Res. 67:4924 - 4932) to evaluate tumor volume after a single intravenous administration, and tumors excised from mice carrying intraperitoneal tumors were then serially implanted into the mammary fat pads of recipient mice.

[1157] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if each reference were specifically and individually indicated to be incorporated by reference and are set forth in their entirety herein.

Claims

1. A cereblon degrader antibody conjugate having a structure of Formula I-A, wherein Ab is an antibody; L 1a is an antibody linker; Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C3-C 20 heterocyclic group and C3-C 20 heteroaryl; Dashed line Indicates an optional double bond; Z 1 selected from C(R 1 )2, CR 1 , N and NR 1a , and Z 2 selected from C(R 2 )2, CR 2 , N and NR 2a , wherein R 1 and R 2 each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; R 1a and R 2a each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or (i) Two geminal Rs 1 or two geminal Rs 2 form a 3- to 6-membered carbocyclic or heterocyclic 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 Forming a fused 5-membered or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group, wherein R a and R b are each independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, where the phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl, and -C2-C 12 alkynyl; wherein each alkyl, alkylene, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group and heteroaryl is independently and optionally substituted with one or more groups selected from: 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(=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; cD a is the remainder of the cereblon degrader moiety; and p is an integer from 1 to 14.

2. The cereblon degrader antibody conjugate according to claim 1, wherein Z 1 is CR 1 , and Z 2 is CR 2 , and wherein R 1 and R 2 are each H.

3. The cereblon degrading agent antibody conjugate according to any one of claims 1 and 2, wherein ring A is a C3-C 20 heteroaryl.

4. The cereblon degrader antibody conjugate according to claim 3, wherein Ring A is isoindoline substituted with =O.

5. The cereblon degrader antibody conjugate according to any one of claims 1 to 4, having a structure of Formula I-A': wherein X 1 is selected from CH2 and C(=O).

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

7. The cereblon degrader antibody conjugate according to 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 degrader antibody conjugate according to any one of claims 1 to 7, wherein the antibody is a cysteine-engineered antibody.

9. The cereblon degrader antibody conjugate according to claim 8, wherein the 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.

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

11. The cereblon degrader antibody conjugate according to any one of claims 1 to 10, wherein L 1a has a structure of formula L1-A: wherein * indicates the point of attachment to the cysteine thiol of the Ab; R 1 is selected from the group consisting of: 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)CH2CH(thiophen-3-yl), wherein r is an integer in the range of 1 to 10; and C1-C 12 The alkylene group is optionally substituted by one or more groups selected from the following: 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; and 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.

12. The cereblon degrader antibody conjugate according to claim 11, wherein AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

13. The cereblon degrading agent antibody conjugate according to claim 11, wherein R 1 is a C5 alkylene group.

14. The cereblon degrading agent antibody conjugate according to any one of claims 11 to 13, wherein R 2 and R 3 together form a C4 cycloalkyl ring.

15. The cereblon degrader antibody conjugate according to any one of claims 11 to 14, wherein AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

16. The cereblon degrader antibody conjugate according to claim 11, wherein R 1 is a C5 alkylene group; R 2 and R 3 together form a C4 cycloalkyl ring; and AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

17. The cereblon degrader antibody conjugate according to any one of claims 1 to 16, wherein cDa comprises (i) a target protein ligand covalently linked to a degrader linker, or (ii) a molecular glue moiety.

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

19. A cereblon degrader antibody conjugate having the structure of Formula I-B, wherein Ab is an antibody; L 1a is an antibody linker; Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group and C1-C 20 heteroaryl; Dashed line Indicates an optional double bond; Z 1 selected from C(R 1 )2, CR 1 , N and NR 1a , and Z 2 selected from C(R 2 )2, CR 2 , N and NR 2a , wherein R 1 and R 2 each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; R 1a and R 2a are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or (i) Two geminal Rs 1 or two geminal Rs 2 form a 3- to 6-membered carbocyclic or heterocyclic 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, carbocyclic, heterocyclic or heteroaryl group, where R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl; Each alkyl, alkylene, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group, and heteroaryl is independent and optionally substituted with one or more groups selected from: 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(=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; cD a the remainder of the cereblon degrader moiety; and p is an integer from 1 to 14.

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

21. The cereblon degrading agent antibody conjugate according to any one of claims 19 and 20, wherein ring A is a C3-C 20 heteroaryl.

22. The cereblon degrader antibody conjugate according to claim 21, wherein Ring A is an isoindoline substituted with =O.

23. The cereblon degrader antibody conjugate according to any one of claims 19 to 22, which has the structure of Formula I-B’: wherein X 1 is selected from CH2 and C(=O).

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

25. The cereblon degrader antibody conjugate according to 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 degrader antibody conjugate according to any one of claims 19 to 25, wherein the antibody is a cysteine-engineered antibody.

27. The cereblon degrader antibody conjugate according to claim 26, wherein the cysteine-engineered antibody comprises a cysteine mutation selected from the following: HC A118C, LC K149C, HC A140C, LC V205C, LCS121C, HC L174C, HC L177C, and HC Y373C.

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

29. The cereblon degrading agent antibody conjugate according to any one of claims 19 to 28, wherein L 1a has the structure of formula L1-A: wherein * indicates the attachment point to the cysteine thiol of the Ab; R 1 selected from the group consisting of: 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)CH2CH(thiophen-3-yl), wherein r is an integer in the range of 1 to 10; and C1-C 12 The alkylene group is optionally substituted with one or more groups selected from the following: 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; and 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.

30. The cereblon degrader antibody conjugate according to claim 29, wherein AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

31. The cereblon degrading agent antibody conjugate according to claim 29, wherein R 1 is a C5 alkylene group.

32. The cereblon degrading agent antibody conjugate according to any one of claims 29 to 31, wherein R 2 and R 3 together form a C4 cycloalkyl ring.

33. The cereblon degrader antibody conjugate according to any one of claims 29 to 32, wherein AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

34. The cereblon degrader antibody conjugate according to claim 29, wherein R 1 is a C5 alkylene group; R 2 and R 3 together form a C4 cycloalkyl ring; and AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

35. The cereblon degrader antibody conjugate according to any one of claims 19 to 34, wherein cDa comprises (i) a target protein ligand covalently linked to the degrader linker, or (ii) a molecular glue moiety.

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

37. A cereblon degrader antibody conjugate having the structure of Formula I-C, wherein Ab is an 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and 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; Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group and C1-C 20 heteroaryl; Dashed line Indicates an optional double bond; Z 1 selected from C(R 1 )2, CR 1 , N and NR 1a , and Z 2 selected from C(R 2 )2, CR 2 , N and NR 2a , wherein R 1 and R 2 each independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; R 1a and R 2a are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or (i) Two geminal Rs 1 or two geminal Rs 2 form a 3- to 6-membered carbocyclic or heterocyclic 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, carbocyclic, heterocyclic or heteroaryl group, wherein R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, where the phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl; Each alkyl, alkylene, alkenyl, alkynyl, aryl, carbocyclic group, heterocyclic group, and heteroaryl is independent and optionally substituted with one or more groups selected from the following: 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(=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; cD a is the remainder of the cereblon degrader moiety; and p is an integer from 1 to 14.

38. The cereblon degrader antibody conjugate according to claim 37, wherein sulfur is conjugated to the cysteine thiol of the Ab to form a disulfide bond.

39. The cereblon degrading agent antibody conjugate according to claim 37, wherein Z 1 is CR 1 , and Z 2 is CR 2 , and wherein R 1 and R 2 are each H.

40. The cereblon degrading agent antibody conjugate according to any one of claims 37 to 39, wherein Ring A is a C3-C 20 heteroaryl.

41. The cereblon degrader antibody conjugate according to claim 40, wherein ring A is an isoindoline substituted with =O.

42. The cereblon degrader antibody conjugate according to any one of claims 37 to 41, which has a structure of formula I-C’: wherein X 1 is selected from CH2 and C(=O).

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

44. The cereblon degrader antibody conjugate according to 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 degrader antibody conjugate according to any one of claims 37 to 44, wherein the antibody is a cysteine-engineered antibody.

46. The cereblon degrader antibody conjugate according to claim 45, wherein the cysteine-engineered antibody comprises a cysteine mutation selected from: HC A118C, LC K149C, HC A140C, LC V205C, LCS121C, HC L174C, HC L177C, and HC Y373C.

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

48. The cereblon degrading agent antibody conjugate according to any one of claims 37 to 47, wherein L 1a has a structure of formula L1-A: wherein * indicates the point of attachment to the cysteine thiol of the Ab; R 1 selected from the group consisting of: 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)CH2CH(thiophen-3-yl), where r is an integer in the range of 1 to 10; C1-C 12 The alkylene group is optionally substituted with one or more groups selected from the following: 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; and 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.

49. The cereblon degrader antibody conjugate according to claim 48, wherein AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

50. The cereblon degrading agent antibody conjugate according to claim 48, wherein R 1 is a C5 alkylene group.

51. The cereblon degrading agent antibody conjugate according to any one of claims 48 to 50, wherein R 2 and R 3 together form a C4 cycloalkyl ring.

52. The cereblon degrader antibody conjugate according to any one of claims 48 to 51, wherein AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

53. The cereblon degrader antibody conjugate according to claim 48, wherein R 1 is a C5 alkylene group; R 2 and R 3 together form a C4 cycloalkyl ring; and AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

54. The cereblon degrader antibody conjugate according to any one of claims 37 to 53, wherein cD comprises (i) a target protein ligand covalently linked to a degrader linker, or (ii) a molecular glue moiety.

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

56. A cereblon degrader antibody conjugate comprising a cereblon degrader moiety covalently linked to an antibody via an antibody linker, wherein the cereblon degrader moiety is: (a) a target protein ligand covalently linked to a cereblon-binding E3 ubiquitin ligase ligand via a degrader linker, or (b) a molecular glue, and the antibody is a thiol-containing antibody.

57. The cereblon degrader antibody conjugate according to claim 56, wherein the thiol-containing antibody binds to a tumor-associated antigen or a cell surface receptor.

58. The cereblon degrader antibody conjugate according to claim 57, wherein the tumor-associated antigen or cell surface receptor is selected from the group consisting of: (1) BMPR1B (bone morphogenetic protein type IB receptor); (2)E16 (LAT1, SLC7A5); (3)STEAP1 (prostate six-transmembrane epithelial antigen); (4)MUC16 (0772P, CA125); (5)MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin); (6)Napi2b (NAPI-3B, NPTIIb, SLC34A2, solute carrier 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 cDNA2700050C12 gene); (9)ETBR (endothelin 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, prostate six-transmembrane epithelial antigen 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 Hs 73792); (15)CD79b (CD79B, CD79β, IGb (immunoglobulin-related β), B29); (16)FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase-anchoring protein 1a), SPAP1B, SPAP1C); (17)HER2; (18)NCA; (19)MDP; (20)IL20Rα; (21)biglycan; (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 α); (29)CXCR5 (Burkitt lymphoma receptor 1); (30) HLA-DOB (β 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), 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 with EGF-like and two follistatin-like domains 1; Brain tumor suppressor protein-1); (39) GDNF-Ra1 (GDNF family receptor alpha1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; 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 locus G6D; Ly6-D, MEGT1); (43) LGR5 (G protein-coupled receptor 5 containing leucine-rich repeats; 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) CD123.

59. The cereblon degrader antibody conjugate according to claim 58, wherein the tumor-associated antigen or cell surface receptor is HER2 or CD33.

60. The cereblon degrader antibody conjugate according to any one of claims 56 to 59, which has the formula I: Ab-[L1-cD] p I or a pharmaceutically acceptable salt thereof, wherein: Ab is the antibody; L1 is the antibody linker; cD is the cereblon degrader moiety; and p is an integer from 1 to 14.

61. The cereblon degrader antibody conjugate according to claim 60, wherein the antibody is a cysteine-engineered antibody.

62. The cereblon degrader antibody conjugate according to claim 61, wherein the cysteine-engineered antibody comprises a cysteine mutation selected from the following: A118C, LCK149C, HC A140C, LC V205C, LC S121C, HCL174C, HC L177C, HC Y373C.

63. The cereblon degrader antibody conjugate according to claim 60, wherein the linkage between L1 and cD comprises an acetalamine group.

64. The cereblon degrader antibody conjugate according to claim 60, wherein the linkage between L1 and cD comprises a carbamate (-OC(O)NH-) or methylcarbamate (-OC(O)NHCH2-) group.

65. The cereblon degrader antibody conjugate according to claim 60, wherein L1 is a protease-cleavable non-peptide linker having the following formula: -Str-PM-IM- where Str is an extension unit covalently linked to the antibody; PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to the cereblon degrader moiety.

66. The cereblon degrader antibody conjugate according to claim 65, wherein Str has the following formula: where * indicates the point of attachment of the succinimidyl ring to the cysteine thiol of the antibody, and R 1 selected from the group consisting of: 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)CH2CH(thiophen-3-yl), where r is an integer in the range of 1 to 10, and C1-C 12 alkylene is 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.

67. The cereblon degrading agent antibody conjugate according to claim 66, wherein R 1 is (CH2)5.

68. The cereblon degrader antibody conjugate according to any one of claims 65 to 67, wherein PM has the following formula: wherein 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; and 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.

69. The cereblon degrader antibody conjugate according to claim 68, wherein AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

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

71. The cereblon degrader antibody conjugate according to claim 65, which has the following formula: wherein cD is the cereblon degrader moiety.

72. The cereblon degrader antibody conjugate according to claim 71, which is selected from:

73. The cereblon degrader antibody conjugate according to claim 71, which has the following formula:

74. The cereblon degrader antibody conjugate according to claim 73, which is selected from:

75. The cereblon degrader antibody conjugate according to claim 74, which is selected from:

76. The cereblon degrader antibody conjugate according to claim 60, wherein L1 forms a disulfide bond with a cysteine thiol of the antibody.

77. The cereblon degrader antibody conjugate according to claim 60, wherein L1 is selected from: where * indicates that sulfur is conjugated to the cysteine thiol of the antibody to form a disulfide bond, and 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted by F, Cl and C1-C6 alkyl, where the C1-C6 alkyl is independently and optionally substituted by 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 6 selected from H and C1-C6 alkyl; and The wavy line indicates the connection to the cereblon degrader moiety.

78. The cereblon degrading agent antibody conjugate according to claim 77, wherein R 4a and R 4b is -CH3, R 5a and R 5b is H, and R 6 is H.

79. The cereblon degrader antibody conjugate according to claim 60, wherein L1 is a linker having the following formula: where * indicates the connection point to the cysteine thiol of the antibody, R 4a and R 4b are independently selected from H and C1-C6 alkyl, or R 4a and R 4b together with the carbon atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl, wherein the C1-C6 alkyl is independently and 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; The wavy line indicates the connection to the cereblon degrader moiety.

80. The cereblon degrader antibody conjugate according to claim 60, wherein the cereblon degrader moiety has the following formula: TPL-L2-E3UL wherein: TPL is a target protein ligand; E3UL is a cereblon-binding E3 ubiquitin ligase ligand; L2 is a degrader linker; and one of TPL, E3UL, and L2 is connected to L1; or the cereblon degrader moiety is a molecular glue.

81. The cereblon degrader antibody conjugate according to claim 80, wherein TPL has the following formula: where R x selected from F, Cl and Br, and n is 0, 1, 2 or 3; R y selected from H and C1-C6 alkyl; and The wavy line indicates the connection point of L2.

82. The cereblon degrader antibody conjugate according to claim 80, wherein TPL has the following formula: where the wavy line indicates the connection point of L2.

83. The cereblon degrader antibody conjugate according to claim 80, wherein TPL has the following formula: where the wavy line indicates the connection point of L2.

84. The cereblon degrader antibody conjugate according to claim 80, wherein TPL targets BRD4, GSPT1, BET, BRM (SMARCA2), KRAS, and SHP2.

85. The cereblon degrader antibody conjugate according to claim 80, wherein E3UL comprises a succinimide group.

86. The cereblon degrader antibody conjugate according to claim 80, wherein E3UL is selected from: wherein X 1 is selected from CH2 and C(=O); and the wavy line indicates the point of attachment of L1 or L2.

87. The cereblon degrader antibody conjugate according to claim 80, which has the following formula: Wherein L1 is a protease-cleavable non-peptide linker having the following formula: -Str-PM-IM- Where Str is an extender unit covalently linked to X; PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to the succinimide group of E3UL and has the following formula: Where the wavy line is the connection to PM.

88. The cereblon degrader antibody conjugate according to claim 80, which has the following formula: Wherein L1 is a protease-cleavable non-peptide linker having the following formula: -Str-PM-IM- Where Str is an extender unit covalently linked to X; PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to L2 of cD and has the following formula: Where the wavy line is the connection to PM.

89. The cereblon degrader antibody conjugate according to claim 80, wherein L2 is selected from: -N(R)-(C1-C 12 alkylene diyl)-N(R)-, -N(R)-(C2-C 12 alkenediyl)-N(R)-, -N(R)-(C2-C 12 alkynediyl)-N(R)-, -N(R)-(C1-C 12 alkanediyl)-C(=O)-(N(R)-, -N(R)-(C1-C 12 alkanediyl)-(N(R)-C(=O)CH2O-, -N(R)-(C1-C 12 alkylene diyl)-(N(R)-C(=O)CH2N(R)-, -N(R)-(C1-C 12 alkylene)-C(=O)-(N(R)-(C1-C 12 alkylene)-N(R)-, -N(R)-(C1-C6 alkylene)-O-(C1-C6 alkylene)-N(R)-, -N(R)-(CH2CH2O) n -N(R)-(CH2CH2O) n -, where n is an integer from 1 to 4, C1-C 12 alkyl diyl, C2-C 12 alkenediyl, and C2-C 12 alkynediyl, Where R is selected from H, C1-C6 alkylene and the connection point to L1; and The alkylene, alkenylene and alkynylene are optionally substituted with one or more groups selected from the following: 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.

90. The cereblon degrader antibody conjugate according to claim 60, which is selected from: 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, and X 1 is selected from CH2 and C(=O).

91. The cereblon degrader antibody conjugate according to claim 60, which is selected from: 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, and X 1 is selected from CH2 and C(=O).

92. The cereblon degrader antibody conjugate according to claim 60, which is selected from:

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

94. The cereblon degrader antibody conjugate according to any one of claims 60 to 92, which comprises a mixture of cereblon degrader antibody conjugate compounds, wherein the average drug loading of each antibody in the mixture of cereblon degrader antibody conjugate compounds is about 2 to about 6.

95. The cereblon degrader antibody conjugate according to claim 60, wherein the cereblon degrader (cD) moiety is a molecular glue selected from: wherein the wavy line indicates the connection point of L1, and the dashed line indicates an optional double bond; Z 1 selected from C(R 1 )2, CR 1 , N and NR 1a ; Z 2 Selected from C(R 2 )2, CR 2 , N and NR 2a ; R is selected from H and C1-C6 alkyl; R 1 and R 2 are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR[[ID=6③]] a and -S(O)3H; R 1a and R 2a are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or (i) two geminal Rs 1 or two geminal Rs 2 form a 3- to 6-membered carbocyclic or heterocyclic 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, carbocyclic, heterocyclic or heteroaryl group, wherein R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl and benzyl, wherein phenyl and benzyl are optionally substituted by one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl and C2-C 12 alkynyl; Ring A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group and C1-C 20 heteroaryl; and The alkyl, alkylene, alkenyl, alkynyl, aryl, carbocyclic, heterocyclic and heteroaryl are independently and optionally substituted with one or more independently selected from the following groups: 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(=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.

96. The cereblon degrader antibody conjugate according to claim 95, which comprises a cD structure selected from the following: wherein the wavy line indicates the point of attachment to the remainder of L1.

97. The cereblon degrader antibody conjugate according to claim 96, which comprises a sacrificial moiety selected from the following: wherein * indicates the point of attachment to the remainder of L1, and the wavy line indicates the point of attachment to the 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl; and C1-C6 alkyl is independently and 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.

98. The cereblon degrader antibody conjugate according to claim 95, wherein the cereblon degrader moiety is selected from: wherein X 1 is selected from CH2 and C(=O); and the wavy line indicates the point of attachment of L1.

99. A cereblon degrader-linker intermediate having Formula II: X-L3-cD II Wherein: X is a thiol-reactive group; L3 is a linker selected from: (i) A protease-cleavable non-peptide linker having the following formula: -Str-PM-IM- Where Str is an extender unit covalently linked to X; PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to cD; (ii) A disulfide bond linker selected from: And (iii) A linker having the following formula: Wherein, * indicates the point of attachment to X, 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, Optionally substituted with F, Cl and C1-C6 alkyl, R 6 selected from H and C1-C6 alkyl The wavy line indicates the connection to cD, C1-C6 alkyl is independently and 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; and cD is a cereblon degrader moiety having the following formula: TPL-L2-E3UL Wherein: TPL is a target protein ligand; E3UL is a cereblon-binding E3 ubiquitin ligase ligand; and And L2 is a degrader linker; or cD is a molecular glue.

100. The cereblon degrader-linker intermediate according to claim 99, wherein the connection between L3 and cD comprises a carbamate (-OC(O)NH-) or methylcarbamate (-OC(O)NHCH2-) group.

101. The cereblon degrader-linker intermediate according to claim 99, wherein the cereblon degrader moiety is a molecular glue selected from: Where the wavy line indicates the point of attachment of L3, and the dashed line indicates an optional double bond; Z 1 selected from C(R 1 )2, CR 1 , N, and NR 1a ; Z 2 selected from C(R 2 )2, CR 2 , N and NR 2a ; R is selected from H and C1-C6 alkyl; R 1 and R 2 are independently selected from the group consisting of: H, F, Cl, Br, I, -CN, C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, (C1-C6 alkyl diyl)-(C6-C 20 aryl), -(C1-C6 alkyl diyl)-NR a R b , -(C1-C6 alkyl diyl)-OR a , (C1-C6 alkyl diyl)-(C3-C 20 carbocyclic group), (C1-C6 alkyl diyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyl diyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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 alkyl diyl)-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)2NR a and -S(O)3H; R 1a and R 2a are independently selected from the group consisting of: 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 carbocyclic group), (C1-C6 alkyldiyl)-(C2-C 20 heterocyclic group), (C1-C6 alkyldiyl)-(C1-C 20 heteroaryl), C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group, 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)2NR a and -S(O)3H; or (i) two geminal Rs 1 or two geminal Rs 2 form a 3- to 6-membered carbocyclic or heterocyclic 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, carbocyclic, heterocyclic or heteroaryl group; R a and R b are independently selected from H, OH, C1-C6 alkyl, phenyl, and benzyl, where the phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of: F, Cl, -CN, C1-C 12 alkyl, C2-C 12 alkenyl, and C2-C 12 alkynyl, and A is selected from C6-C 20 aryl, C3-C 20 carbocyclic group, C2-C 20 heterocyclic group and C1-C 20 heteroaryl; An alkyl group, an alkylene group, an alkenyl group, an alkynyl group, an aryl group, a carbocyclic group, a heterocyclic group, and a heteroaryl group are each independently and optionally substituted with one or more groups independently selected from: 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(═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.

102. The cereblon degrader-linker intermediate according to claim 101, which comprises a structure selected from the following: wherein the wavy line indicates the point of attachment to the remainder of L3.

103. The cereblon degrader-linker intermediate according to claim 101, which comprises a sacrificial moiety selected from the following: wherein * indicates the point of attachment to the remainder of L3, and the wavy line indicates the point of attachment to the remainder of L3; 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 atom to which they are attached form a three-, four- or five-membered cycloalkyl or heterocyclic group, optionally substituted with F, Cl and C1-C6 alkyl; R 6 selected from H and C1-C6 alkyl; the wavy line indicates the connection to cD; and The C1-C6 alkyl groups are independent and optionally substituted by 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.

104. The cereblon degrader-linker intermediate according to claim 99, wherein the cereblon degrader moiety is selected from: wherein X 1 is selected from CH2 and C(=O); and the wavy line indicates the point of attachment of L3.

105. The cereblon degrader-linker intermediate according to claim 99, wherein X is selected from the group consisting of maleimide, bromoacetamide, tosylthioether, and 2-pyridyldisulfide, wherein the pyridyl group is optionally substituted by one or two nitro groups.

106. The cereblon degrader-linker intermediate according to claim 99, which has the following formula: wherein IM contains a group selected from 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl)methylcarbamate, and 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.

107. The cereblon degrader-linker intermediate according to claim 106, wherein AA is selected from H, -CH3, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, and -CH2CH2CH2NHC(O)NH2.

108. The cereblon degrader-linker intermediate according to claim 106 or 107, wherein AA is -CH3 or -CH2CH2CH2NHC(O)NH2.

109. The cereblon degrader-linker intermediate according to claim 99, which is selected from: where X 1 is selected from CH2 and C(=O).

110. The cereblon degrader-linker intermediate according to claim 109, which has the following formula: wherein L3 is a protease-cleavable non-peptide linker having the following formula: -Str-PM-IM- wherein Str is an extender unit covalently linked to X; PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to cD and has the following formula: wherein the wavy line is the connection to PM.

111. The cereblon degrader-linker intermediate according to claim 109, which has the following formula: wherein L3 is a protease-cleavable non-peptide linker having the following formula: -Str-PM-IM- wherein Str is an extender unit covalently linked to X; PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to L2 of cD and has the following formula: where the wavy line is the connection to PM.

112. The cereblon degrader-linker intermediate according to claim 99, wherein TPL is selected from: (i) 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; (ii) (iii) where the wavy line indicates the connection point of L2.

113. The cereblon degrader-linker intermediate according to claim 99, wherein L2 is selected from: -N(R)-(C1-C 12 alkanediyl)-N(R)-, -N(R)-(C2-C 12 alkenylene)-N(R)-, -N(R)-(C2-C 12 alkynediyl)-N(R)-, -N(R)-(C1-C 12 alkylene)-C(=O)-(N(R)-, -N(R)-(C1-C 12 alkylene)-C(=O)-(N(R)-(C1-C 12 alkylene)-N(R)-, -(C1-C6 alkanediyl)-O-(C1-C6 alkanediyl)-, C1-C 12 Alkylene diyl, C2-C 12 alkenyl diyl, and C2-C 12 alkynediyl, wherein the alkanediyl, alkenediyl, and alkynediyl 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 H, C1-C6 alkanediyl, and the connection point to L3.

114. The cereblon degrader intermediate according to claim 99, wherein cD is selected from: 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; and X 1 is selected from CH2 and C(=O).

115. The cereblon degrader-linker intermediate according to claim 99, which is selected from: 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, and X 1 is selected from CH2 and C(=O).

116. The cereblon degrader-linker intermediate according to claim 99, which is selected from:

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

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

119. A method for preparing the cereblon degrader-linker intermediate of formula II according to claim 99, the method comprising reacting the X-L3 moiety with the cereblon degrader moiety.

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

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

122. The method according to claim 121, wherein the cancer is selected from epithelial cancer; lymphoma; germ cell tumor; sarcoma; leukemia or lymphocytic malignancy, including acute myeloid leukemia; squamous cell carcinoma; epithelial squamous cell carcinoma; lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma; peritoneal cancer; hepatocellular carcinoma; gastric or stomach cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; hepatoma; 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.

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

124. The method according to claim 121, wherein the cancer is BRD4-dependent.

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

126. A cereblon degrader antibody conjugate according to any one of claims 1 to 98 or a pharmaceutical composition according to claim 120, which is used in a method for treating cancer.

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