CN120417934A - CEREBLON degrader conjugates and uses thereof - Google Patents

Abstract

Translated fromChinese

本文提供了包含共价连接至抗体的cereblon降解剂部分的cereblon降解剂抗体缀合物(cDAC)。所述cDAC靶向蛋白质以进行细胞内降解，并且可以用于治疗疾病和疾患。

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

Description

CEREBLON degrading agent conjugate and use thereof

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application Ser. No. 63/435,142, filed on Ser. No. 2022, 12, 23, and U.S. provisional application Ser. No. 63/525,282, filed on Ser. No. 7, 2023, each of which is incorporated by reference in its entirety.

Technical Field

The present disclosure relates generally to cereblon degradant antibody conjugate (cDAC) compositions, intermediates for their manufacture, and methods of use thereof. cDAC can be used to promote intracellular degradation of a target protein.

Background

Cereblon is a 442-amino acid multifunctional protein located in the cytoplasm, nucleus and peripheral membrane of the human brain and other tissues (Wada et al Biochem. Biophys. Res. Comm.477:388-94 (2016)). Cereblon ensure normal metabolic and physiological functions of the ion channel, which is important for maintaining cell growth and proliferation. Cereblon are also involved in the occurrence of many diseases, such as cancer (Shi et al, (2017) j.immunol.res. Article No. 9130608). Cereblon interact with DNA damage binding protein-1 (DDB 1), cullin 4 (Cul 4A and Cul 4B), and the regulator factor (RoC 1) of Cullins 1 to form a functional E3 ubiquitin ligase complex, which is referred to as CRL4/CRBN E3 ubiquitin ligase complex. Cereblon the role of this complex as part of it includes a number of target proteins that undergo proteolysis (degradation) via the ubiquitin-proteasome pathway (Chang et al, (2011) int. J. Biochem. Mol. Biol.2 (3): 287-94). The complex ubiquitinates many other proteins. Cereblon also relates to the development of cerebral tissue and, due to its expression in regions such as the hippocampus, to the memory and learning processes (Higgins, et al, (2004) neurol.63 (10): 1927-31).

Cereblon are targets for immunomodulatory drugs (IMiD) that modulate immune responses and contain glutarimide functionality (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 class of IMiD includes thalidomide and analogs of lenalidomide, pomalidomide, iberdomide, and apremilast. Thalidomide is approved by the FDA for the treatment of multiple myeloma. LenalidomideAnd pomalidomideAre approved by the FDA for the treatment of multiple myeloma and other diseases. Cytokine modulation by IMiD and T cell co-stimulation results in the production of interleukin-2 in T cells (Schafer et al, (2003) J.Pharmacol. & Exper. Ther. 305:1222-32). IMiD has pleiotropic effects on a variety of 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 reused as immunomodulating drugs (IMiD) 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 shallow hydrophobic pockets of the cereblon surface and that binding is mediated by glutarimide rings.

As binding proteins for IMiD, cereblon is responsible for various effects of IMiD like thalidomide and its analogs (P.ottis, et al (2017) ACS chem. Biol.12 (4): 892-898; shi Q, et al (2017) J Immunol Res.2017:9130608;Sperling AS, et al (2019) Blood134 (2): 160-170). Cereblon expression can affect cellular metabolism and can be a pathogenic effect of disease even in the absence of IMiD. Cereblon orthologs are highly conserved from Plant to human, which underscores their physiological importance (Zhihua H, et al (2011) Annu Rev Plant biol.62 (1): 299-334).

The ATP-dependent ubiquitin-proteinase system (UPS) is the main pathway for intracellular protein degradation. UPS systems (including ubiquitin (Ub), proteasomes, catalytic enzymes, and specific substrates) play an important role in various biological processes. Ubiquitination occurs through a series of enzymatic events, particularly under the synergistic action of Ub activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3). Once the substrate protein is polyubiquitinated, it is recognized and degraded by the proteasome, and the UPS can digest the substrate protein into small peptides. The specific recognition of the substrate protein is an apparent function of E3, so 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.Scheestra, et al, (2019), comput.Structure.Biotechnol.J.17:160-176; P.Ottis, et al, (2017) ACS chem.biol.12 (4): 892-898).

The proteolytically targeted chimera (PROTAC) is a heterobifunctional degradant construct capable of targeting degradation of aberrantly acting proteins using the ubiquitin-proteasome mechanism of cells. The main mechanism of PROTAC technology is to use UPS to degrade proteins of interest (POIs), such as target proteins that are themselves disease mediators (Lu et al, (2015) CELL CANCER (6): 755-63; wang, C. Et al (2021) Eur J Med chem.225: 113749), by bringing E3 ubiquitin ligase into proximity with the POIs that are to be targeted to degrade leading to degradation of the target protein. PROTAC E3 ligase ligand can hijack E3 ligase and label POI with ubiquitin. In this process PROTAC is not itself degraded, but is recovered to promote 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 pattern is in contrast to the function of conventional inhibitors, where sequence target binding is necessary to stimulate the desired effect. For a typical small molecule drug driven by standard occupancy, binding affinity is essential for its efficacy. In contrast, PROTAC induces degradation of POIs by UPS, an event-driven mode that can be used to overcome the common disadvantages of traditional placeholder-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. Edit Engl.55 (6): 1966-1973).

A "molecular gel" is a degradant construct that mediates near-induced protein degradation of a ligase (more frequently) or target POI interaction by inducing or stabilizing protein-protein interactions between the E3 ubiquitin ligase and the 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 gums can degrade other non-ligand proteins by coordinating the direct interaction 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 gel degradation agents can target nuclear receptor GSPT (Huber, A.D., et al (2022) ACS Med. Chem. Lett. 13:1311-1320).

Although both molecular gums and PROTAC are bifunctional protein degrading agents, they have different mechanisms of action and structural requirements (den Besten, w., et al (2020) Nat Chem Biol 16:1157-1158). However, the cereblon ligand may be a component of both PROTAC and a molecular gum degrading agent that recruits targeted POIs to the CRL4/CRBN E3 ubiquitin ligase to degrade POIs (Lu et al, (2015) CELL CANCER (6): 755-63; wang, C. Et al (2021) Eur J Med chem.225: 113749). Certain glutarimide compounds such as thalidomide, lenalidomide, and pomalidomide act as molecular gums to enhance or induce interactions between the E3 ligase and the target protein, triggering ubiquitination and degradation (Dong G, et al (2021) J Med chem.64 (15): 10606-10620).

One protein of interest is bromodomain-containing protein 4 (BRD 4). Certain small molecule BRD4 inhibitors interfere with protein-protein interactions and have been the subject of anti-tumor drug development. Limitations include reversible binding of BRD4 inhibitors (e.g., JQ1, OTX 015), requiring large systemic drug concentrations and sustained exposure to ensure adequate functional inhibition (j. Shi, et al (2018) mol.pharm.15 (9): 4139-4147).

Target protein ligands have been used in PROTAC, in which pomalidomide binds to various BRD4 target protein ligands through PEG linkers, which BRD4 target protein ligands induce significant degradation of BRD4 in BL (burkitt lymphoma) cells, with DC50 values below 1nM (j.lu, et al (2015) chem. Biol.22 (6): 755-763). PROTAC and other BRD4 and BET target protein ligands showed significant effects on downstream cell proliferation and apoptosis induction of c-MYC, AML (acute myeloid leukemia) cells, BL cells. (E.W. Georg et al, (2015) Science 348 (6241): 1376-1381). These results indicate that CEREBlon-based PROTAC with BET provides a better and more efficient strategy for targeting BRD4 than traditional small molecule inhibitors (L.Bai, et al, (2017) Canc.Res.77 (9): 2476-2487; C.Qin, et al, (2018) J.Med.chem.61 (15): 6685-6704; J.zhang, et al (2020) bioorg.chem.99: 103817). Degradation of BET proteins is associated with linker design in PROTAC (T.A. Bemis, et al (2021), chem. Commun.57 (8): 1026-1029).

Limitations or challenges exist in the design, manufacture, and use of antibody compositions that are covalently linked to drugs, payloads, and other biologically active moieties through linkers. The linkers can be divided into cleavable and non-cleavable linkers according to their chemical nature (Beck A, et al, (2017) Nat Rev Drug discovery.16 (6): 315-37; tsuhikama K, et al, (2018) Protein cell.9:33-46). The non-cleavable linker consists of a stable bond that is 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 the cleavable linker may depend on the external pH (acid labile linker), specific lysosomal proteases (protease cleavable linker) 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 blood stream, releasing unacceptable amounts of drug prior to internalization in the target cells (Khot, a. Et al, (2015) Bioanalysis 7 (13): 1633-1648), while other linkers may provide stability in the blood stream, but the effectiveness of intracellular release may be negatively impacted. In addition, the stability of the linker in the blood stream to provide the desired intracellular release may be poor. Furthermore, the amount of drug moiety loaded on the antibody (quantified as drug to antibody ratio (DAR)), the amount of aggregates formed in the conjugation reaction, and the yield of final purified conjugate available are other parameters that need to be addressed and are typically interrelated.

Accordingly, there is a continuing need to improve the design of antibody conjugates, including linker and linker chemistry, to provide optimized safety and efficacy. In addition, there is a need to enhance and target delivery of cereblon ligands containing PROTAC and molecular gums to cells containing protein targets. The combination of tumor-associated protein degradation and cereblon immunomodulatory activity may enhance the therapeutic benefit of patients suffering from various hyperproliferative disorders such as cancer.

Disclosure of Invention

The present disclosure generally relates to a conjugate composition, referred to as a cereblon degrading agent antibody conjugate or "cDAC," in which the cereblon degrading agent moiety is covalently linked to the antibody through an antibody linker. In some embodiments, the cereblon degrading agent portion comprises a target protein ligand covalently linked to a cereblon-bound E3 ubiquitin ligase ligand through a degrading agent linker. In other embodiments, the cereblon degrading agent moiety is a molecular gel. In some embodiments, the cereblon degradant portion of the disclosed cDAC targets the appropriate target cells and is released as a cereblon degradant compound, thereby performing its function of stimulating/inducing ubiquitination of the target protein and achieving its degradation by the ubiquitin-proteinase system (UPS). cDAC may have enhanced therapeutic benefits for patients suffering from various hyperproliferative disorders such as cancer.

One aspect of the disclosure is a cereblon-degrading agent antibody conjugate (cDAC) comprising a cereblon-degrading agent moiety covalently attached to an antibody through a linker (e.g., an antibody linker), wherein the cereblon-degrading agent moiety is covalently attached to a cereblon-bound E3 ubiquitin ligase ligand through a degrading agent linker or molecular gel, and the antibody is a thiol-containing antibody.

Another aspect of the present disclosure is a cDAC having the structure of formula I:

Ab-[L₁-cD]_p I

Or a pharmaceutically acceptable salt thereof,

Wherein:

ab is an antibody;

cD is the cereblon degradant moiety;

L₁ is a linker to Ab and cD, and

P is an integer of 1 to 14.

Another aspect of the present disclosure is a cereblon degradation agent-linker intermediate having the structure of 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 formula:

-Str-PM-IM-

Wherein Str is an extender unit covalently linked to X;

PM is a peptidomimetic unit, and IM is a sacrificial (immolator) unit covalently linked to cD;

(ii) A disulfide linker selected from the formula:

And

And

(Iii) A linker having the formula:

wherein X indicates the point of connection to X,

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl;

R⁶ is selected from H and C₁-C₆ alkyl,

The wavy line indicates the point of connection to the cD,

C₁-C₆ alkyl of R^4a、R^4b、R^5a、R^5a and R⁶ 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)₃ H, and

CD is the cereblon degradant moiety, wherein

(I) cD is Molecular Gel (MG), or

(Ii) cD is the cereblon degradant moiety having the formula:

TPL-L₂-E3UL

Wherein:

TPL is a target protein ligand;

e3UL is a cereblon-bound E3 ubiquitin ligase ligand, and

And L₂ is a degradant linker.

Another aspect of the disclosure is a cDAC prepared by conjugating an antibody to a cereblon degradant intermediate of formula II.

Another aspect of the disclosure is a method for preparing a cDAC comprising reacting a thiol-containing antibody with a cereblon degrading agent intermediate of formula II.

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

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

Another aspect of the disclosure is the use of a cDAC in the manufacture of a medicament for the treatment of cancer in a mammal.

Another aspect of the disclosure is the use of a cDAC for treating cancer in a mammal.

Drawings

FIG. 1 shows the antiproliferative effect of BRD4-cereblon degrading agents on the in vitro efficacy of KPL-4 and SK-BR-3 cells at 5 days. Cell viability is plotted as a percentage of control against the concentration (nM) of the cereblon degradant compound cD-5.

FIG. 2A shows the anti-proliferative effect of in vitro efficacy after 5 days by treating HER2+KPL-4 cells with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 in Table 3. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 2B shows the antiproliferative effect of in vitro efficacy after 5 days by treating HER2+SK-BR-3 cells with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degradant antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 3A shows the antiproliferative effect of the in vitro potency of HER 2-low/ER+CAMA 1 cells after 5 days of treatment with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 3B shows the antiproliferative effect of the in vitro potency of HER 2-low/ER+EFM19 cells after 5 days of treatment with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 4 shows the antiproliferative effect of in vitro potency after 7 days of treatment of various AML cell lines with anti-CD 33 BRD4-cereblon degrading agent antibody conjugate cDAC-3. AML cell lines are MV-4-11, EOL-1, molm-13, nomo-1, HL-60 and OCI-AML-2. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 5A shows the antiproliferative effect of the in vitro potency of EOL-1AML cells after 5 days of treatment with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degradant antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 5B shows the antiproliferative effect of the in vitro potency of HL-60AML cells after 5 days of treatment with anti-HER 2 7C2 and anti-CD 33 cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 6A shows the antiproliferative effect of the in vitro potency of Molm-13AML cells after 3 days of treatment with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degradant antibody conjugates cDAC-3, cDAC-4, cDAC-5, and cDAC-6. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 6B shows the antiproliferative effect of the in vitro potency of MV-4-11AML cells after 3 days of treatment with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 7 shows the in vivo efficacy of anti-CD 33 BRD4-cereblon degrading antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 in HL-60 xenograft mouse models to reduce tumor volume over time (21 days) at the following doses.

1) Vehicle (histidine buffer # 8), 100 μl, IV one time

2) CDAC-4,3mg/kg IV once

3) CDAC-3,1mg/kg IV once

4) CDAC-3,3mg/kg IV once

5) CDAC-3,10mg/kg IV once

6) CDAC-6,3mg/kg IV once

7) CDAC-5,1mg/kg IV once

8) CDAC-5,3mg/kg IV once

Detailed Description

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

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 methods and materials described.

Definition of the definition

The term "antibody" is used in its broadest sense and specifically covers 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. An "antibody fragment" as used herein, and all grammatical variants thereof, is defined as a portion of an intact antibody that comprises the antigen binding site or variable region of the intact antibody, wherein the portion does not contain the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, fab '-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 one contiguous amino acid residue (referred to herein as a "single chain antibody fragment" or "single chain polypeptide"), including but not limited to (1) a single chain Fv (scFv) molecule, (2) a single chain polypeptide comprising only one light chain variable domain or a fragment thereof comprising three CDRs of a light chain variable domain, without an associated heavy chain portion, (3) a single chain polypeptide comprising only one heavy chain variable domain or a fragment thereof comprising three CDRs of a heavy chain variable domain, without an associated light chain portion, (4) a nanobody comprising a single Ig domain or other specific single domain binding module from a non-human species, and (5) a multi-specific or multivalent structure formed from an antibody fragment. In an antibody fragment comprising one or more heavy chains, the heavy chains may comprise any constant domain sequence found in the non-Fc region of the whole antibody (e.g., CH1 in the IgG isotype), and/or may comprise any hinge region sequence found in the whole antibody, and/or may comprise a leucine zipper sequence fused to or located in the hinge region sequence or constant domain sequence of the heavy chain.

An "antibody" refers to a polypeptide comprising antigen binding regions (including Complementarity Determining Regions (CDRs)) from an immunoglobulin gene or 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. Exemplary immunoglobulin (antibody) structural units comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" (about 50-70 kDa) chain linked by disulfide bonds. Each chain consists of a domain, which is called an immunoglobulin domain. These domains fall into different classes by size and function, such as variable domains or regions on the light and heavy chains (V_L and V_H, respectively) and constant domains or regions on the light and heavy chains (C_L and C_H, respectively). The N-terminal end of each strand defines a variable region of about 100 to 110 or more amino acids, termed paratope, primarily responsible for antigen recognition, i.e., the antigen binding domain. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes IgG, igM, igA, igD and IgE, respectively. IgG antibodies are large molecules of about 150kDa consisting of four peptide chains. IgG antibodies contain two identical gamma-class heavy chains of about 50kDa and two identical light chains of about 25kDa, and thus have a tetrameric quaternary structure. The two heavy chains are linked to each other and to the light chain by disulfide bonds. The resulting tetramer has two identical halves which together form a Y-shape. Each end of the fork contains the same antigen binding domain. Humans have four subclasses of IgG (IgG 1, igG2, igG3, and IgG 4), named in the order of their abundance in serum (i.e., igG1 is the most abundant). In general, the antigen binding domain of an antibody is most critical in terms of specificity and affinity for binding to cancer cells.

Antibodies targeting a particular antigen include bispecific or multispecific antibodies having at least one antigen-binding region that targets a particular antigen. In some embodiments, the targeting monoclonal antibody is a bispecific antibody having at least one antigen binding region that targets tumor cells.

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

In some embodiments, the binding agent is an antigen-binding antibody "fragment" that is a construct that comprises at least the antigen-binding region of the antibody, alone or together with other components that make up the antigen-binding construct. Many different types of antibody "fragments" are known in the art, including, for example, (i) Fab fragments, which are monovalent fragments consisting of V_L、V_H、C_L and CH₁ domains, (ii) F (ab ')₂ fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) Fv fragments, which consist of the V_L and V_H domains of a single arm of an antibody, (iv) Fab ' fragments, which are produced by disrupting the disulfide bridge of the F (ab ')₂ fragment using mild reducing conditions, (V) disulfide stabilized Fv fragments (dsFv), and (vi) single chain Fv (scFv), which are monovalent molecules consisting of the two domains of the Fv fragments (i.e., V_L and V_H) connected by a synthetic linker that enables the two domains to be synthesized as a single polypeptide chain.

The antibody or antibody fragment may be part of a larger construct, for example, a conjugate or fusion construct of the antibody fragment with additional regions. For example, in some embodiments, an antibody fragment may be fused to an Fc region as described herein. In other embodiments, the antibody fragment (e.g., fab or scFv) may be part of a chimeric antigen receptor or chimeric T cell receptor, for example by fusion to a transmembrane domain (optionally using an intermediate linker or "handle" (e.g., hinge region)) and optionally an intercellular signaling domain. For example, the antibody fragment can be fused to the gamma and/or delta chain of a T cell receptor to provide a T cell receptor-like construct that binds TROP 2. In yet another embodiment, the antibody fragment is part of a bispecific T cell adapter (BiTE) comprising a CD1 or CD3 binding domain and a linker.

"Epitope" means any epitope or epitope determinant of an antigen to which an antigen binding domain binds (i.e., at the paratope of an antigen binding domain). An epitope is typically composed of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and typically has specific three-dimensional structural features as well as specific charge features.

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) FcγR which binds IgG, (2) FcαR which binds IgA, and (3) FcεR which binds IgE. The fcγr family includes several members such as fcγi (CD 64), fcγriia (CD 32A), fcγriib (CD 32B), fcγriiia (CD 16A) and fcγriiib (CD 16B). Fcγ receptors differ in affinity for IgG and also have different affinities for IgG subclasses (e.g., igG1, igG2, igG3, and IgG 4).

"Amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally occurring α -amino acids and stereoisomers thereof, as well as non-natural (non-naturally occurring) amino acids and stereoisomers thereof. "stereoisomers" of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but differing in the three-dimensional arrangement of bonds and atoms (e.g., l-amino acid and corresponding d-amino acid). The amino acid may be glycosylated (e.g., N-linked glycans, O-linked glycans, phosphoglycans, C-linked glycans, or glycosyl phosphatidylinositol (glypication)) or deglycosylated. In this context, amino acids may be represented by the well-known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission.

Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that have been modified later, such as hydroxyproline, gamma-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 (gin), 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 alpha-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.

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

Non-natural (non-naturally occurring) amino acids include, but are not limited to, amino acid analogs, amino acid mimics, synthetic amino acids, N-substituted glycine, and N-methyl amino acids of the L-or D-configuration, which function in a manner similar to naturally occurring amino acids. For example, an "amino acid analog" may be an unnatural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., carbon bonded to hydrogen, carboxyl, amino), but has a modified side chain group or modified peptide backbone, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid but that function in a similar manner as a naturally occurring amino acid.

An "amino acid side chain" is an 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(C₆ H₅), lysine (-CH₂CH₂CH₂CH₂NH₂), arginine (-CH₂CH₂CH₂NHC(NH)NH₂), leucine-CH₂CH(CH₃)₂, and citrulline (-CH₂CH₂CH₂NHC(O)NH₂).

"Linker" refers to a functional group that covalently links two or more moieties in a compound or material. For example, a linking moiety may be used to covalently bind a drug moiety to an antibody construct in a conjugate provided herein or between two or more ligand binding moieties.

"Linking moiety" refers to a functional group that covalently bonds two or more moieties in a compound. For example, the linking moiety may be used to covalently bind the drug moiety to the antibody in the conjugate. Useful linkages for linking the linking moiety to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, disulfides, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

"Divalent" refers to a chemical moiety containing two points of attachment for linking two moieties, and a multivalent linking moiety may have additional points of attachment for linking additional functional groups. The divalent group may be represented 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 group having two points of attachment for covalently linking two moieties in a molecule or material. Cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups may be substituted or unsubstituted. The cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano and alkoxy.

Wave lineRepresenting the point of attachment of the designated chemical moiety. If the chemical moiety is specified to have two wavy linesIf present, it should be understood that the chemical moiety may be used bilaterally, i.e., read from left to right or right to left. In some embodiments, reading from left to right is considered to have two waveform linesThe specific portion that exists.

"Alkyl" refers to a straight (linear) or branched saturated aliphatic group having the indicated number of carbon atoms. The alkyl group may include any number of carbons, for example 1 to 12. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH₃), ethyl (Et, -CH₂CH₃) 1-propyl (n-Pr, n-propyl, -CH₂CH₂CH₃), 2-propyl (i-Pr, isopropyl, -CH (CH₃)₂), a, 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)), a catalyst, 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)), a catalyst, 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)), a catalyst for the preparation of a pharmaceutical composition, 2, 3-dimethyl-2-butyl (-C (CH₃)₂CH(CH₃)₂), 3-dimethyl-2-butyl (-CH (CH₃)C(CH₃)₃), 1-heptyl, 1-octyl, and the like). Alkyl groups may be substituted or unsubstituted. The substituted alkyl group may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=o), alkylamino, amido, acyl, nitro, cyano and alkoxy. The substituted alkyl group may be geminal substituted wherein the carbon atom of the alkyl group forms a spiro ring, cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

The term "alkyldiyl" refers to a divalent alkyl group. Examples of alkyldiyls include, but are not limited to, methylene (-CH₂ -), ethylene (-CH₂CH₂ -), propylene (-CH₂CH₂CH₂ -), and the like. Alkyldiyl may also be referred to as an "alkylene" group. Alkyldiyl groups may be substituted or unsubstituted. The substituted alkyldiyl group may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=o), alkylamino, amido, acyl, nitro, cyano and alkoxy. The substituted alkyldiyl may be geminal substituted wherein the carbon atom of the alkyl group forms a spiro ring, cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

"Alkenyl" refers to a straight-chain (straight-chain) or branched unsaturated aliphatic group having the indicated number of carbon atoms and at least one carbon-carbon double bond sp 2. Alkenyl groups may contain 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, vinyl (ethylenyl or vinyl) (-ch=ch₂), allyl (-CH₂CH＝CH₂), butenyl, pentenyl, and isomers thereof. Alkenyl groups may be substituted or unsubstituted. "substituted alkenyl" may be substituted with one or more alkenyl groups selected from halo, hydroxy, amino, oxo (=o), alkylamino, amido, acyl, nitro, cyano and alkoxy.

The term "alkenylene" or "alkenyldiyl" refers to a straight or branched chain divalent hydrocarbon group. Examples include, but are not limited to, vinylidene (ETHYLENYLENE or vinyl) (-ch=ch-), propenyl (-CH₂ ch=ch-), and the like.

"Alkynyl" refers to a straight-chain (straight-chain) or branched unsaturated aliphatic group having the indicated number of carbon atoms and at least one carbon-carbon triple bond sp. Alkynyl groups can contain 2 to about 12 or more carbon atoms. For example, C₂-C₆ alkynyl includes, but is not limited to, ethynyl (-c≡ch), propynyl (propargyl, -CH₂ c≡ch), butynyl, pentynyl, hexynyl, and isomers thereof. Alkynyl groups may be substituted or unsubstituted. "substituted alkynyl" may be substituted with one or more alkenyl groups selected from halo, hydroxy, amino, oxo (=o), alkylamino, amido, acyl, nitro, cyano and alkoxy.

The term "alkynylene" or "alkynediyl" refers to a divalent alkynyl group.

The terms "carbocycle", "carbocyclyl", "carbocycle" and "cycloalkyl" refer to a saturated or partially unsaturated, monocyclic, fused bicyclic, spiro or bridged polycyclic ring assembly containing from 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, decalin, and adamantane. The carbocyclic group may also be partially unsaturated, having one or more double or triple bonds in the ring. Representative carbocyclic groups that are partially unsaturated 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.

The term "cycloalkyldiyl" refers to a divalent cycloalkyl group.

"Aryl" refers to a monovalent aromatic hydrocarbon radical of 6 to 20 carbon atoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Aryl groups may be monocyclic, fused to form a bicyclic or tricyclic group, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl groups having methylene linker groups. Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl.

The terms "heterocycle (heterocycle)", "heterocyclyl" and "heterocycle (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, the remaining ring atoms being C, wherein one or more ring atoms are optionally independently substituted with one or more substituents described below. The heterocycle may be a single ring having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P and S) or having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P and S), for example a bicyclo [4,5], [5,6], or [6,6] system or 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,1950to present),, especially volumes 13, 14, 16, 19 and 28, and J.Am.chem.Soc. (1960) 82:5566. "heterocyclyl" also includes groups in which the heterocyclyl is fused to a saturated, partially unsaturated ring or 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, azo-1-yl, azetidin-1-yl, octahydropyrido [1,2-a ] pyrazin-2-yl, [1,4] diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholino, thiomorpholino, thianyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxacycloheptyl, thietanyl, oxazazinBasic, diazaRadical, thiazasA group, a 2-pyrrolinyl group, a 3-pyrrolinyl group, an indolinyl group, a 2H-pyranyl group, a 4H-pyranyl group, a dioxanyl group, a 1, 3-dioxapentyl group, a pyrazolinyl group, a dithianyl group, a dithiocyclopentyl group, a dihydropyranyl group, a dihydrothienyl group, a dihydrofuryl group, a pyrazolinyl imidazolinyl group, an imidazolidinyl group, a 3-azabicyclo [3.1.0] hexanyl group, a 3-azabicyclo [4.1.0] heptanyl group, an azabicyclo [2.2.2] hexanyl group, a 3H-indolylquinolinyl group and an N-pyridylurea group. Spiro heterocyclyl moieties are also included within the scope of this definition. Examples of heterocyclyl moieties of the spiro ring include azaspiro [2.5] octanyl and azaspiro [2.4] heptyl. Examples of heterocyclyl groups in which 2 ring atoms are substituted with oxo (=o) moieties are pyrimidinonyl and 1, 1-dioxo-thiomorpholinyl. The heterocyclic groups herein may be optionally substituted independently with one or more substituents described herein.

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

The heterocyclic or heteroaryl group may be carbon (carbon linked) or nitrogen (nitrogen linked) bonded where possible. For example, but not by way of limitation, the carbon-bonded heterocycle or heteroaryl is bonded at the 2,3, 4, 5 or 6 position of pyridine, the 3, 4, 5 or 6 position of pyridazine, the 2, 4, 5 or 6 position of pyrimidine, the 2,3, 5 or 6 position of pyrazine, the 2,3, 4 or 5 position of furan, tetrahydrofuran, thiafuran, thiophene, pyrrole or tetrahydropyrrole ring, the 2, 4 or 5 position of oxazole, imidazole or thiazole, the 3, 4 or 5 position of isoxazole, pyrazole or isothiazole, the 2 or 3 position of aziridine, the 2,3 or 4 position of azetidine, the 2,3, 4, 5, 6, 7 or 8 position of quinoline, or the 1,3, 4, 5, 6, 7 or 8 position of isoquinoline.

For example, but not by way of limitation, the nitrogen-bonded heterocycle or heteroaryl is bonded at the 1-position of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of an isoindole or isoindoline, the 4-position of morpholine, and the 9-position or β -carboline of carbazole.

The term "halo" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom.

The term "carbonyl" by itself or as part of another substituent means C (=o) or-C (=o) -, i.e. a carbon atom that is double bonded to oxygen and bonded to two other groups in the moiety having a carbonyl group.

The term "chiral" refers to a molecule that has no overlap with a mirror partner, while the term "achiral" refers to a molecule that can overlap with its mirror partner.

The term "stereoisomers" refers to compounds having the same chemical composition but different arrangements of atoms or groups in space.

"Diastereoisomers" means stereoisomers which have 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. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography.

"Enantiomer" refers to two stereoisomers of a compound that are mirror images of each other that are non-superimposable.

The stereochemical definitions and conventions used herein generally follow the editions of S.P.Parker, mcGraw-Hill Dictionary of CHEMICAL TERMS (1984) McGraw-Hill Book Company, new York, and Eliel, E. And Wilen, S., "Stereochemistry of Organic Compounds", john Wiley & Sons, inc., new York,1994. The compounds described herein may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds described herein, including but not limited to diastereomers, enantiomers and atropisomers and mixtures thereof, such as racemic mixtures, form part of the present disclosure. Many organic compounds exist in optically active form, i.e. they have the ability to rotate plane-polarized light planes. In describing optically active compounds, the prefixes D and L or R and S are used to represent the absolute configuration of the molecule about its chiral center. The prefixes d and l or (+) and (-) are used to denote the sign of the rotation of the compound to plane polarized light, where (-) or 1 indicates that the compound is left-handed. Compounds with (+) or d prefix are dextrorotatory. These stereoisomers are identical for a given chemical structure, except that they are mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. The 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur without stereoselectivity 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 may be separated from the racemic mixture by chiral separation methods such as Supercritical Fluid Chromatography (SFC). While stereochemistry such as from x-ray crystallography data is explicitly constructed, the configuration assignment at the chiral centers of the isolated enantiomers may be tentative, described in the structure of table 1 for illustrative purposes.

The terms "treat" (teat, treatment and treating) "refer to any sign of successful treatment or amelioration of a injury, pathology, condition (e.g., cancer), or symptom (e.g., cognitive disorder), including any objective or subjective parameter, such as alleviation, alleviation of symptoms or making the symptom, injury, pathology, or condition more tolerable to the patient, reduction of the rate of symptom progression, reduction of the frequency or duration of the symptom or condition, or, in some cases, prevention of the onset of the symptom. Treatment or amelioration of symptoms can be based on any objective or subjective parameter, including, for example, the results of physical examination.

The terms "cancer," "neoplasm," and "tumor" are used herein to refer to cells that exhibit self-regulated, unregulated growth such that the cells exhibit an abnormal growth phenotype characterized by a significant loss of control of 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 with cancer), malignant cancer cells, pre-metastatic cancer cells, and non-metastatic cancer cells. Almost every tissue cancer is known. The phrase "cancer burden" refers to the amount of cancer cells or the volume of cancer in a subject. Thus, reducing the burden of cancer refers to reducing the number of cancer cells or the 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 that an individual may be treated for, e.g., isolated from an individual with cancer) or derived from a cancer cell (e.g., a clone of a cancer cell). For example, the cancer cells may be from established cancer cell lines, may be primary cells isolated from individuals with cancer, may be progeny cells of primary cells isolated from individuals with cancer, and the like. In some embodiments, the term may also refer to a portion of a cancer cell, such as a subcellular portion, cell membrane portion, or cell lysate of a cancer cell. 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, as well as circulating cancers such as leukemia.

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, stomach 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.

The phrases "effective amount" and "therapeutically effective amount" refer to the dose or amount of the therapeutic agent that produces the therapeutic effect to which it is administered. The exact dosage will depend on the purpose of the treatment and will be determinable by one of skill in the art using known techniques (see, e.g., lieberman, pharmaceutical Dosage Forms (volume 1-3, ,1992);Lloyd,The Art,Science and Technology of Pharmaceutical Compounding(1999);Pickar,Dosage Calculations(1999);Goodman&Gilman's The Pharmacological Basis of Therapeutics,, 11 th edition (McGraw-Hill, 2006), and Remington: THE SCIENCE AND PRACTICE of Pharmacy, 22 nd edition (Pharmaceutical Press, london, 2012)). In the case of cancer, a therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells, reduce the size of the tumor, inhibit (i.e., slow and preferably stop to some extent) the infiltration of cancer cells into surrounding organs, inhibit (i.e., slow and preferably stop to some extent) the metastasis of the tumor, inhibit to some extent the growth of the tumor, and/or alleviate to some extent one or more symptoms associated with the cancer.

"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, sports or pets, such as dogs, horses, cats, cattle, sheep, goats, pigs, camels, and the like. 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., cattle, 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 may be a "cancer patient," i.e., a patient suffering from or at risk of suffering from one or more symptoms of cancer. By "patient population" is meant a group of cancer patients. Such populations may be used to demonstrate statistically significant drug efficacy and/or safety.

As used herein, the term "administration" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration or implantation of a sustained-release device (e.g., a micro-osmotic pump) in a subject.

The term "residue", "moiety" or "group" refers to a component that is covalently bound or linked to another component.

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

As used herein, the term "peptidomimetic" or PM means a non-peptide chemical moiety that is part of a linker. The peptide is a short chain of (two or more) amino acid monomers linked by peptide (amide) bonds, while the peptidomimetic chemical moiety comprises a non-amino acid chemical moiety. The peptidomimetic chemical moiety can also comprise one or more amino acids separated by one or more non-amino acid-containing units. The peptidomimetic chemical moiety does not comprise two or more adjacent amino acids linked by peptide bonds in any portion of its chemical structure.

CEREBLON degrading agent antibody conjugates

The cereblon degradation agent antibody conjugates (cDAC) provided herein comprise at least one (p) cereblon degradation agent moiety (cD) covalently linked to an antibody (Ab) through an antibody linker (L₁).

The cereblon degradant antibody conjugate (cDAC) induces target-specific degradation of tumor-associated proteins and brings 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.

An exemplary embodiment of a cDAC has the structure of formula I:

Ab-[L₁-cD]_p I

Or a pharmaceutically acceptable salt thereof,

Wherein:

ab is an antibody;

l₁ is an antibody linker;

cD is the cereblon degradation agent part, and

P is an integer of 1 to 14.

In some embodiments, L₁ comprises a sacrificial moiety. In embodiments, L₁ is L_1a -IM, wherein IM is the sacrificial moiety and L_1a is any remaining portion of the L₁ antibody linker. In an embodiment, L₁ is a sacrificial moiety IM.

In some embodiments, the cD comprises a cereblon-bound E3 ubiquitin ligase ligand E3UL. In some embodiments, cD is E3UL-cD_a, wherein E3UL is the E3 ubiquitin ligase ligand of the cereblon-bound cereblon-degrading moiety, and cD_a is any remaining portion of cD. In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

An exemplary embodiment of the cDAC has the structure of formula I':

Ab-[L_1a-IM—E3UL-cD_a]_p

I’

In some embodiments, the cereblon-degrading agent portion (cD) of the cDAC is covalently linked to the antibody linker (L₁) through an acetal amine group.

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

Wherein the method comprises the steps of

* Indicates the connection point with L_1a, × indicates the connection point with cD_a, and the waveform line indicates the connection point with E3 UL.

Exemplary embodiments of cDAC have the structure of formula I-A

Wherein the method comprises the steps of

Ab is an antibody;

l_1a is an antibody linker;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₃-C₂₀ heterocyclyl, and C₃-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are each independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-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, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

Wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, 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)₃ H;

cD_a is the remainder of the cereblon degradant portion, and

P is an integer of 1 to 14.

In some embodiments, Z¹ is CR¹ and Z² is CR², and R¹ and R² are each H.

In some embodiments, ring a is C₃-C₂₀ heteroaryl.

In some embodiments, ring a is an isoindoline substituted with =o.

In some embodiments, the cDAC of formula (I) has the structure of formula I-A':

Wherein X¹ is selected from CH₂ and C (=o).

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

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

In some embodiments, the antibody is a cysteine engineered antibody.

In some embodiments, the cysteine engineered antibodies comprising one or more cysteine mutations are selected from the group consisting of HC a118C, LC K149C, HC a140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

In some embodiments, L_1a is a protease cleavable non-peptide linker.

In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

In some embodiments, L_1a has the structure of formula L₁ -A

Wherein the method comprises the steps of

* Indicating the point of attachment to the cysteine thiol of Ab;

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=O), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂ and C₁-C₁₂ alkylene-NHC (=O) CH₂ CH (thiophen-3-yl), wherein

R is an integer in the range of 1 to 10, and

C₁-C₁₂ 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)₃ H;

R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

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

In some embodiments, R¹ is C₅ alkylene.

In some embodiments, R² and R³ together form a C₄ cycloalkyl ring.

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

In some embodiments of the present invention, in some embodiments,

R¹ is C₅ alkylene;

R² and R³ together form a C₄ cycloalkyl ring, and

AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

In some embodiments, cD_a comprises (i) a target protein ligand covalently linked to a degradant linker, or (ii) a molecular gelatin moiety. In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

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

Exemplary embodiments of cDAC have the structure of formula I-B

Wherein the method comprises the steps of

Ab is an antibody;

l_1a is an antibody linker;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

Wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, 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)₃ H;

cD_a is the remainder of the cereblon degradant portion, and

P is an integer of 1 to 14.

In some embodiments, Z¹ is CR¹ and Z² is CR², and R¹ and R² are each H.

In some embodiments, ring a is C₃-C₂₀ heteroaryl.

In some embodiments, ring a is an isoindoline substituted with =o.

In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

In some embodiments, the cDAC of formula I-B comprises a structure of formula I-B

Wherein X¹ is selected from CH₂ and C (=o).

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

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

In some embodiments, the antibody is a cysteine engineered antibody.

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

In some embodiments, L_1a is a protease cleavable non-peptide linker.

In some embodiments, L_1a has the structure of formula L₁ -A

Wherein the method comprises the steps of

* Indicating the point of attachment to the cysteine thiol of Ab;

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=O), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂ and C₁-C₁₂ alkylene-NHC (=O) CH₂ CH (thiophen-3-yl), wherein

R is an integer in the range of 1 to 10, and

C₁-C₁₂ 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)₃ H;

R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

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

In some embodiments, R¹ is C₅ alkylene.

In some embodiments, R² and R³ together form a C₄ cycloalkyl ring.

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

In some embodiments:

r¹ is C₅ alkylene;

R² and R³ together form a C₄ cycloalkyl ring, and

AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

In some embodiments cDa comprises (i) a target protein ligand covalently linked to a degradant linker, or (ii) a molecular gelatin moiety. In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

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

In some embodiments, L₁ is a sacrificial moiety IM.

An exemplary embodiment of a cDAC has the structure of formula I ":

Ab-[IM—E3UL-cD_a]_p

I”

in some embodiments, IM is:

Wherein the method comprises the steps of

* Indicating the point of attachment to Ab,

* Indicating the point of attachment to cD_a, and

The wavy line indicates the point of connection with E3 UL.

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, 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)₃ H.

Exemplary embodiments of cDAC have the structure of formula I-C

Wherein the method comprises the steps of

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

Wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, 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)₃ H;

cD_a is the remainder of the cereblon degradant portion, and

P is an integer of 1 to 14.

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

In some embodiments, Z¹ is CR¹ and Z² is CR², and R¹ and R² are each H.

In some embodiments, ring a is C₃-C₂₀ heteroaryl.

In some embodiments, ring a is an isoindoline substituted with =o or oxo.

In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

In some embodiments, the cDAC of formula I-C comprises a structure of formula I-C

Wherein the method comprises the steps of

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H;

X¹ is selected from CH₂ and C (=o);

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

Wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, 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)₃ H;

cD_a is the remainder of the cereblon degradant portion, and

P is an integer of 1 to 14.

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

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

In some embodiments, the antibody is a cysteine engineered antibody.

In some embodiments, the cysteine engineered antibody comprises one or more cysteine mutations selected from the group consisting of HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

In some embodiments, cD_a comprises (i) a target protein ligand covalently linked to a degradant linker, or (ii) a molecular gelatin moiety. In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

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

CEREBLON degrading agent-linker intermediate

The cereblon degradation agent-linker intermediate (cDLI) is an agent for use in a method of preparing a cereblon degradation agent antibody conjugate (cDAC) by conjugation with thiol-containing antibodies. The cereblon degrading agent-linker intermediate has a thiol reactive functional group (X). The thiol-reactive functional group (X) is covalently linked to the cereblon degradant moiety (cD) through a linker (L₃).

In some embodiments, the cereblon degrading agent-linker intermediate has the structure of formula II:

X-L₃-cD II

Wherein:

x is a thiol-reactive group covalently linked to L₃;

L₃ is a linker covalently linked to X and cD, and

CD is the cereblon degrading agent moiety covalently linked to L₃.

The cD may be a heterobifunctional divalent cereblon degradant moiety or a molecular gel cereblon degradant moiety.

In some embodiments, L₃ comprises a sacrificial moiety. In an embodiment, L₃ is L_3a -IM, wherein IM is the sacrificial moiety and L_3a is any remaining portion of the L₃ linker. In an embodiment, L₃ is a sacrificial moiety IM.

In some embodiments, the cD comprises a cereblon-bound E3 ubiquitin ligase ligand E3UL. In some embodiments, cD is E3UL-cD_a, wherein E3UL is the E3 ubiquitin ligase ligand of the cereblon-bound cereblon degradant moiety cD, and cD_a is any remaining portion of cD. In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

The exemplary embodiment of cDLI has the structure of formula II':

X-L_3a-IM—E3UL-cD_a II’

In some embodiments, the cereblon-degrading agent portion (cD) of cDLI is linked to the antibody linker (L₃) through an aminal group.

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

Wherein the method comprises the steps of

* Indicating the point of connection to any remaining portion of L₃ connector L_3a,

* Indicating the point of attachment to cD_a, and

The wavy line indicates the point of connection with E3 UL.

CDLI an exemplary embodiment has the structure of formula II-A

Wherein the method comprises the steps of

X is a thiol-reactive group;

l_3a is a linker;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₃-C₂₀ heterocyclyl, and C₃-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are each independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-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, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

Wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, 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)₃ H;

cD_a is the remainder of the cereblon degradant portion, and

P is an integer of 1 to 14.

In some embodiments, Z¹ is CR¹ and Z² is CR², and R¹ and R² are each H.

In some embodiments, ring a is C₃-C₂₀ heteroaryl.

In some embodiments, ring a is an isoindoline substituted with =o.

In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

In some embodiments, cDLI of formula II has the structure of formula II-A

Wherein the method comprises the steps of

X is a thiol-reactive group;

l_3a is a linker, and

X¹ is selected from CH₂ and C (=o).

In some embodiments, L_3a is a protease cleavable non-peptide linker.

In some embodiments, X-L_3a has the structure of formula L₃ -A

Wherein the method comprises the steps of

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=O), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂ and C₁-C₁₂ alkylene-NHC (=O) CH₂ CH (thiophen-3-yl), wherein

R is an integer in the range of 1 to 10, and

C₁-C₁₂ 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)₃ H;

R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

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

In some embodiments, R¹ is C₅ alkylene.

In some embodiments, R² and R³ together form a C₄ cycloalkyl ring.

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

In some embodiments of the present invention, in some embodiments,

R¹ is C₅ alkylene;

R² and R³ together form a C₄ cycloalkyl ring, and

AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

In some embodiments cDa comprises (i) a target protein ligand covalently linked to a degradant linker, or (ii) a molecular gelatin moiety. In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

CDLI an exemplary embodiment has the structure of formula II-B

Wherein the method comprises the steps of

X is a thiol-reactive group;

l_3a is a linker;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl 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)₃ H, and

CD_a is the remainder of the cereblon degradant portion.

In some embodiments, Z¹ is CR¹ and Z² is CR², and R¹ and R² are each H.

In some embodiments, ring a is C₃-C₂₀ heteroaryl.

In some embodiments, ring a is an isoindoline substituted with =o.

In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

In some embodiments, cDLI of formula II-B comprises a structure of formula II-B

X is a thiol-reactive group;

l_3a is a linker, and

X¹ is selected from CH₂ and C (=o).

In some embodiments, L_3a is a protease cleavable non-peptide linker.

In some embodiments, X-L_3a has the structure of formula L₃ -A

Wherein the method comprises the steps of

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=O), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂ and C₁-C₁₂ alkylene-NHC (=O) CH₂ CH (thiophen-3-yl), wherein

R is an integer in the range of 1 to 10, and

C₁-C₁₂ 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)₃ H;

R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

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

In some embodiments, R¹ is C₅ alkylene.

In some embodiments, R² and R³ together form a C₄ cycloalkyl ring.

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

In some embodiments:

r¹ is C₅ alkylene;

R² and R³ together form a C₄ cycloalkyl ring, and

AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

In some embodiments cDa comprises (i) a target protein ligand covalently linked to a degradant linker, or (ii) a molecular gelatin moiety. In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

The exemplary embodiment of cDLI has the structure of formula II ":

X-IM—E3UL-cD_a II”

in an embodiment, an X-IM comprisesWherein the method comprises the steps of

* Indicates the connection point with cD_a, and the waveform line indicates the connection point with E3 UL.

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, 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)₃ H.

CDLI an exemplary embodiment has the structure of formula II-C

Wherein the method comprises the steps of

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl 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)₃ H, and

CD_a is the remainder of the cereblon degradant portion.

In some embodiments, Z¹ is CR¹ and Z² is CR², and R¹ and R² are each H.

In some embodiments, ring a is C₃-C₂₀ heteroaryl.

In some embodiments, ring a is an isoindoline substituted with =o or oxo.

In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

In some embodiments cDLI of formula II-C comprises a structure of formula II-C

Wherein the method comprises the steps of

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl 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)₃ H, and

X¹ is selected from CH₂ and C (=O), and

CD_a is the remainder of the cereblon degradant portion.

In some embodiments cDa comprises (i) a target protein ligand covalently linked to a degradant linker, or (ii) a molecular gelatin moiety. In some embodiments, cD_a is TPL-L₂ -, where TPL is the target protein ligand and L₂ is the degradant linker. In some embodiments, the TPL comprises a ligand that binds BRD 4.

CEREBLON-bound E3 ubiquitin ligase ligand

The cereblon-bound E3 ubiquitin ligase ligand (E3 UL) is the moiety in the E3 ubiquitin ligase complex that binds cereblon.

In some embodiments, E3UL comprises a glutarimide group.

In some embodiments, the cereblon degradant portion (cD) of the antibody conjugate (cDAC) has a structure selected from the group consisting of:

Wherein the wavy line indicates the point of attachment to antibody linker L₁ of formula I or linker L₃ of formula II, and the dashed line indicates an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N, and NR^1a;

Z² is selected from C (R²)₂、CR², N, and NR^2a;

R is selected from H and C₁-C₆ alkyl;

R¹ and R² are independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H;

Or (i) two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or (ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group;

R^a and R^b are independently 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-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl, and

A is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

Wherein any alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, and heteroaryl is 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)₃ H.

In some embodiments, Z¹ and Z² are each CR¹ and R¹ and R² are each H.

In some embodiments, R is H.

In some embodiments, a is C₆-C₂₀ aryl.

In some embodiments of the present invention, in some embodiments,

Z¹ and Z² are each CR¹, wherein R¹ and R² are each H;

R is H, and

A is C₆-C₂₀ aryl.

In some embodiments, the E3UL of the cereblon degradant portion (cD) of the antibody conjugate (cDAC) has a structure selected from the group consisting of:

And

Wherein X¹ 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 degradant linker L₂ of the cereblon degradant moiety cD.

In some embodiments, X¹ is C (=o).

Antibody linkers

The antibody linker (L₁) is a bifunctional linker covalently linking the antibody (Ab) to the cereblon degradant moiety (cD). The disclosed antibody linkers provide stability of the cDAC in the blood stream and at the same time allow for efficient cleavage upon internalization into the target cell. The specific design of antibody linkers affects various aspects of cDAC pharmacology, including the stability of the drug in the circulation, tumor cell permeability, drug to antibody ratio (DAR) (i.e., the number of payload molecules carried by each antibody), and the extent of bystander effects.

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

In some embodiments, L₁ is a protease-cleavable non-peptide linker having the formula:

-Str-PM-IM-

Wherein Str is an extender unit covalently linked to the antibody, PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to the cereblon degradant moiety.

In some embodiments, str has the formula:

Wherein the method comprises the steps of

* Indicating the point of attachment of the succinimidyl ring to the cysteine thiol of the antibody, and

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=o), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂ and C₁-C₁₂ alkylene-NHC (=o) CH₂ CH (thiophen-3-yl), wherein R is an integer ranging from 1 to 10, and C₁-C₁₂ alkylene is optionally substituted ：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)₃ H with one or more groups selected from.

In some embodiments, str is selected from the following:

wherein the point of attachment to the cysteine thiol of the antibody is indicated.

In some embodiments of R¹, the C₁-C₁₂ alkylene is C₁-C₅ alkylene.

In some embodiments, R¹ is (CH₂)₅ or C₅ alkylene.

In some embodiments, PM has the formula:

Wherein R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

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

In some embodiments, R² and R³ together form a C₄ cycloalkyl ring, and AA is-CH₃.

In some embodiments, R² and R³ together form a C₄ cycloalkyl ring, and AA is-CH₂CH₂CH₂NHC(O)NH₂.

In some embodiments, formula I comprises a sacrificial moiety selected from the group consisting of:

Wherein the points of connection to the rest of L₁ are indicated and the wavy lines indicate points of connection to cD;

R^4a、R^4b、R^5a and R^5a are independently selected from H and C₁-C₆ alkyl, or R^4a and R^4b together with the carbon atom to which they are bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, 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)₃ H.

In some embodiments, the cereblon degradant portion (cD) of the cDAC is linked to the antibody linker (L₁) through an aminal group of the nitrogen atom of the glutarimide group of the cD.

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

Wherein the wavy line indicates the point of attachment to the remainder of linker L₁ of heterobifunctional cD of formula I or the remainder of linker L₃ of molecular gel cD of formula II.

In some embodiments, Z¹ and Z² are each CR¹ and R¹ and R² are each H.

In some embodiments, R is H.

In some embodiments, a is C₆-C₂₀ aryl.

In some embodiments of the present invention, in some embodiments,

Z¹ and Z² are each CR¹, wherein R¹ and R² are each H;

R is H, and

A is C₆-C₂₀ aryl.

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

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

In some embodiments, formula I is selected from the following formulas:

In some embodiments, L₁ is a linker having the formula:

Wherein the method comprises the steps of

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H, and

* Indicates the point of attachment to the cysteine thiol of the antibody, and the wavy line indicates the attachment to the cereblon degradant moiety.

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

Wherein the method comprises the steps of

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H;

R⁶ is selected from H and C₁-C₆ alkyl, and

* Indicates that sulfur is conjugated to the cysteine thiol of the antibody to form disulfide bonds, and the wavy line indicates the linkage to the cereblon degradant moiety.

In some embodiments, R^4a and R^4b are each-CH₃.

In some embodiments, R^5a and R^5b are each H.

In some embodiments, R⁶ is H.

In some embodiments, R^4a and R^4b are each-CH₃,R^5a and R^5b are each H, and R⁶ is H.

CEREBLON degrading agent fraction

In embodiments, the cD is a divalent heterobifunctional cD or a molecular gel cD. In some embodiments, the cereblon degradant portion (cD) has the formula:

E3UL-cD_a

Wherein the method comprises the steps of

E3UL is a cereblon-bound E3 ubiquitin ligase ligand;

cD_a is a molecular gel moiety, or

CD_a is TPL-L₂ -, wherein

TPL is a target protein ligand, and

L₂ is a degradant linker.

Divalent CEREBLON degrading agent fraction

In embodiments, the cD comprises a cereblon-bound E3 ubiquitin ligase ligand (E3 UL) covalently linked to a Target Protein Ligand (TPL) through a degradant linker (L2) to form a bivalent heterobifunctional cD. In embodiments, the cereblon degradant portion (cD) has the formula:

TPL-L₂-E3UL

Wherein:

TPL is a target protein ligand;

e3UL is a cereblon-bound E3 ubiquitin ligase ligand;

l₂ is a degrading agent linker, and

One of TPL, E3UL, and L₂ is connected to L₁.

Target protein ligands

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 to the cereblon-bound E3 ubiquitin ligase ligand through a degradant linker.

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

Additional exemplary target proteins for the cereblon degradant antibody conjugate (cDAC) include, but are not limited to GSPT1, BET, BRM (SMARCA 2), KRAS, and SHP2 (Wang, C.et al (2021) Eur J Med chem.225: 113749).

In some embodiments, the TPL has the structure of the formula:

Wherein the method comprises the steps of

R^x is selected from F, cl and Br, n is 0,1, 2 or 3;

r^y is selected from H and C₁-C₆ alkyl, and

The wavy line indicates the point of connection to L₂.

In some embodiments, the TPL has the following structure:

Wherein the wavy line indicates the point of connection with L₂.

In some embodiments, the TPL has the following structure:

Wherein the wavy line indicates the point of connection with L₂.

Exemplary target proteins for the cereblon degradant antibody conjugate (cDAC) are GSPT (G1 to S phase change protein 1 homolog), 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 susceptible to GSPT1 degradation. Thus GSPT 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) J Med chem.64 (4): 1835-1843).

Degradation agent joint

The degradation linker (L₂) is any suitable bi-or tri-functional linker unit that is covalently linked to the Target Protein Ligand (TPL) and the cereblon-bound E3 ubiquitin ligase ligand (E3 UL). The degradant linker may be covalently linked to the antibody linker L₁ to form a cereblon degradant antibody conjugate (cDAC).

In some embodiments, L₂ is selected from:

-N (R ') - (C₁-C₁₂ Alkyldiyl) -N (R') -

-N (R ') - (C₂-C₁₂ alkenyldiyl) -N (R') -

-N (R ') - (C₂-C₁₂ alkynyldiyl) -N (R') -

-N (R ') - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R') -),

-N (R ') - (C₁-C₁₂ alkyldiyl) - (N (R') -C (=o) CH₂ O-

-N (R ') - (C₁-C₁₂ alkyldiyl) - (N (R ') -C (=o) CH₂ N (R ') -,

-N (R ') - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R ') - (C₁-C₁₂ alkyldiyl) -N (R ') -,

-N (R ') - (C₁-C₆ Alkyldiyl) -O- (C₁-C₆ Alkyldiyl) -N (R') -

N (R') - (CH₂CH₂O)_n-N(R')-(CH₂CH₂O)_n -, where N is an integer from 1 to 4,

A C₁-C₁₂ alkyldiyl group,

C₂-C₁₂ Alkenyldiyl

C₂-C₁₂ alkynyl diradical (C₂-C₁₂) and (C) is used for preparing the catalyst,

Wherein R' is selected from H, C₁-C₆ alkyl diradicals and the point of attachment to L₁, and wherein

Alkyldiyl, alkenyldiyl and alkynyldiyl are optionally substituted ：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)₃ H with one or more substituents selected from the group consisting of.

Divalent cereblon degrading agent compounds

Exemplary heterobifunctional cereblon degradant compounds were prepared and characterized and are shown in table 1.

TABLE 1 bivalent cereblon degradation agent compound (cD)

Molecular gel CEREBLON degrading agent part

In embodiments, the cereblon degradant portion (cD) of the antibody conjugate (cDAC) is a molecular gel cereblon degradant portion. In such embodiments, the molecular gel cereblon degradant portion (cD) comprises an E3UL covalently linked to the molecular gel portion (cD_a) to form the molecular gel cD.

In some embodiments, the molecular gel cD comprises a structure selected from the following formulas:

Wherein the wavy line indicates the point of attachment to antibody linker L₁ of formula I or linker L₃ of formula II;

cD_a is a molecular gel moiety;

the dashed line indicates an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N, and NR^1a;

Z² is selected from C (R²)₂、CR², N, and NR^2a;

R¹ and R² are independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H;

Or (i) two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or (ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group;

R^a and R^b are independently 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-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl, and

A is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl and heteroaryl are 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)₃ H.

In some embodiments, Z¹ and Z² are each CR¹ and R¹ and R² are each H.

In some embodiments, R is H.

In some embodiments, a is C₆-C₂₀ aryl.

In some embodiments of the present invention, in some embodiments,

Z¹ and Z² are each CR¹, wherein R¹ and R² are each H;

R is H, and

A is C₆-C₂₀ aryl.

In some embodiments, the molecular gel cD comprises a structure selected from the following formulas:

Wherein the method comprises the steps of

The wavy line indicates the point of attachment of antibody linker L₁ of formula I or linker L₃ of formula II;

cD_a is a molecular gel moiety, and

X¹ is selected from CH₂ and C (=o).

In some embodiments, X¹ is C (=o).

CEREBLON degrading agent-linker intermediate

The cereblon degradation agent-linker intermediate (cDLI) is an agent for use in a method of preparing a cereblon degradation agent antibody conjugate (cDAC) by conjugation with thiol-containing antibodies. The cereblon degrading agent-linker intermediate has a thiol reactive functional group (X). The thiol-reactive functional group (X) is covalently linked to the cereblon degradant moiety (cD) through a linker (L₃).

In some embodiments, the cereblon degrading agent-linker intermediate has the structure of 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 formula:

-Str-PM-Y-

Wherein Str is an extender unit covalently linked to X;

PM is a peptidomimetic unit, and

Y is a spacer unit covalently linked to cD;

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

And

(Iii) A linker having the formula:

wherein X indicates the point of connection 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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl;

R⁶ is selected from H and C₁-C₆ alkyl,

The wavy line indicates the connection to the cD,

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)₃ H, and

CD is the cereblon degradant moiety having the formula:

TPL-L₂-E3UL

Wherein:

TPL is a target protein ligand;

e3UL is a cereblon-bound E3 ubiquitin ligase ligand;

l₂ is a degrading agent linker, and

One of TPL, E3UL and L₂ is connected to L₁, or

CD is molecular gel.

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

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

In some embodiments, the cereblon degradant-linker intermediate has the formula:

Wherein IM comprises a group selected from the group consisting of 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl) methylcarbamate, and

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

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

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

In some embodiments, the cereblon degrading agent-linker intermediate is selected from the following formulas:

Wherein X¹ is selected from CH₂ and C (=o).

In some embodiments, the cereblon degrading agent-linker intermediate comprises the formula:

wherein L₃ is a protease-cleavable non-peptide linker having the 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 formula:

Wherein the wavy line is a connection to the PM.

In some embodiments, the cereblon degrading agent-linker intermediate comprises the formula:

wherein L₃ is a protease-cleavable non-peptide linker having the 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 formula:

Wherein the wavy line is a connection to the PM.

Some cDLI in table 2 were prepared that did not have the necessary properties for stability, cleavage efficiency, and conjugation efficiency with the antibodies.

TABLE 2 cereblon degrading agent-linker intermediate (cDLI) with poor stability and/or poor conjugation efficiency

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 that there was no expected ring opening of the conjugation product cDAC, glutarimide, addition of water (+18 mass units) and addition of Tris buffer to the glutarimide ring. Under these conditions, the carbamate functionality formed from the glutarimide nitrogen in cDLI-1 is too labile for conjugation.

The sulfenamide cDLI-2 and bromo-lenalidomide cDLI-3 compounds were not conjugated to antibodies. Maleimide-sulfenamide compounds cDLI-4 and cDLI-5 failed to conjugate with cysteine mutant antibodies and were unstable in whole blood.

Various substituted peptide linkers cDLI-6 were conjugated to the antibodies, but failed to cleave in the presence of proteases. The (S, S) valine-alanine and (S, S) valine-citrulline versions as well as the phenyl and dimethoxyphenyl versions of cDLI-6 were tested and all given cDAC without cleavage to release the cereblon-degradant moiety or its metabolites with or without methyl groups near the glutarimide nitrogen.

In one model study, the nitro group of p-nitrobenzyloxymethyl lenalidomide cDLI-7 was reduced to an amine. By detecting the presence of lenalidomide, no cleavage of p-aminobenzyloxy was observed. The disulfide group of p-nitropyridyldithiomethyllenalidomide cDLI-8 was reduced in one model study. By detecting the presence of lenalidomide, no cleavage of disulfide groups was observed.

Sulfonyl-thio cDLI-9 (with or without methyl groups near the glutarimide nitrogen) cleaves to release detectable lenalidomide, but is unstable, yielding a hydrolysate.

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

In some embodiments, the cereblon degradant-linker intermediate comprises a TPL selected from the following formulas:

(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)

wherein the wavy line indicates the point of connection of L₂.

An exemplary cereblon degradant-linker intermediate (cDLI) is selected from:

And

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 X¹ is selected from CH₂ and C (=o).

In some embodiments, the cereblon degradant-linker intermediate comprises wherein L₂ is selected from the group consisting of:

-N (R) - (C₁-C₁₂ Alkyldiyl) -N (R) -,

-N (R) - (C₂-C₁₂ alkenyldiyl) -N (R) -,

-N (R) - (C₂-C₁₂ alkynyldiyl) -N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R) - (C₁-C₁₂ alkyldiyl) -N (R) -,

- (C₁-C₆ Alkyldiyl) -O- (C₁-C₆ Alkyldiyl) -,

A C₁-C₁₂ alkyldiyl group,

C₂-C₁₂ alkenyldiyl, and

C₂-C₁₂ alkynyl diradical (C₂-C₁₂) and (C) is used for preparing the catalyst,

Wherein the alkyl, alkenyl and alkynyl groups are optionally substituted ：F、Cl、-OH、-OCH₃、-OCH₂CH₃、-OCH₂CH₂OCH₃、-OCH₂CH₂OH、-OCH₂CH₂N(CH₃)₂, with one or more groups selected from the group consisting of and R is selected from H, C₁-C₆ alkyl groups and the point of attachment to L₃.

The cereblon-linker intermediate (cDLI) in table 3 was prepared, which had the necessary properties of stability to the antibody, cleavage efficiency and conjugation efficiency. Each cDLI in table 3 was characterized by NMR and was shown to be of sufficient purity and correct mass by LC/MS.

TABLE 3 examples of cereblon degradant-linker intermediates (cDLI)

Antibodies to

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

The antibody portion of the cDAC can target antigen-expressing cells, thereby delivering antigen-specific cDAC to the target cells, typically by endocytosis. While cDAC comprising antibodies to antigens not found on the cell surface may result in less specific intracellular delivery of the cereblon degrading agent moiety into the cell, cDAC may still undergo pinocytosis. The cDAC and methods of use thereof described herein advantageously utilize antibody recognition on the cell surface and/or endocytosis of the cDAC to deliver the cereblon degradant moiety within the cell.

Trastuzumab, anti-HER 2 antibodies

In certain embodiments, an immunoconjugate (e.g., cDAC) described herein comprises an anti-HER 2 antibody. In some embodiments, the anti-HER 2 antibody of the cDAC comprises a humanized anti-HER 2 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 US 5821337, which is specifically incorporated herein by reference. These antibodies comprise a human framework region and the complementarity determining region of the murine antibody (4D 5) that binds HER 2. Humanized antibody huMAb4D5-8, also known as trastuzumab, is commercially available under the trade name hercetin^TM (Genentech, inc.).

Trastuzumab (CAS 180288-69-1,HuMAb4D5-8,rhuMAb HER2,Genentech) is a recombinant DNA derived IgG1 kappa monoclonal antibody which is a humanized form of murine anti-HER 2 antibody (4D 5) which can selectively bind to the extracellular domain (US 5677171;US 5821337;US 6054297;US 6165464;US 6339142;US 6407213;US 6639055;US 6719971;US 6800738;US 7074404;Coussens of HER2 et al, (1985) Science230:1132-9; slamon et al, (1989) Science 244:707-12; slamon et al, (2001) New Engl. J. Med. 344:783-792) with high affinity (Kd=5 nM) in a cell-based assay.

In some embodiments, the antibody construct or antigen binding domain comprises CDR regions of trastuzumab. In some embodiments, the anti-HER 2 antibody further comprises a trastuzumab framework region. In some embodiments, the anti-HER 2 antibody further comprises one or two variable regions of trastuzumab.

7C2, anti-HER 2 antibodies

The anti-HER 2 murine antibody 7C2 binds to an epitope in domain I of HER 2. See, for example, PCT publication No. WO 98/17797. This epitope is different from the epitope bound by trastuzumab to domain IV of HER2 and the epitope bound by pertuzumab to domain II of HER 2. Trastuzumab disrupts the ligand-independent HER2-HER3 complex by binding domain IV, thereby inhibiting downstream signaling (e.g., PI 3K/AKT). In contrast, binding of pertuzumab to domain II prevents ligand-driven HER2 from interacting with other HER family members (e.g., HER3, HER1, or HER 4), and thus also prevents downstream signal transduction. Binding of MAb 7C2 to domain I does not interfere with binding of trastuzumab or pertuzumab to domains IV and II, respectively, thereby providing the potential to combine MAb 7C2 ADC (antibody drug conjugate) with trastuzumab-maytansinoid conjugate ((T-DM 1) and/or pertuzumab.

In some embodiments, the anti-HER 2 antibodies to cDAC described herein comprise humanized 7C2 anti-HER 2 antibodies. The humanized 7C2 antibody is an anti-HER 2 antibody.

In some embodiments, a cDAC described herein comprises an anti-HER 2 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 6, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7, 11, or 12, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8 or 13, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 3, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 4, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 5. In some embodiments, a cDAC described herein comprises an anti-HER 2 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:6, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:7, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:8, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:3, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:4, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

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

In another aspect, a cDAC described herein comprises an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 3, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 4, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 5. 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.

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

In another aspect, a cDAC described herein comprises an antibody comprising (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 another aspect, a cDAC described herein comprises an antibody comprising (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.

In any of the above embodiments, the anti-HER 2 antibody of the antibody-drug conjugate is humanized. In one embodiment, the anti-HER 2 antibody of the antibody-drug conjugate comprises the HVR of any one of the embodiments described above, and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-HER 2 antibody of the antibody-drug conjugate comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID No. 2 comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-HER 2 antibody comprising the sequence retains the ability to bind to HER 2. 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 substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-HER 2 antibody comprises the VH sequence of SEQ ID NO. 2, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 6, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8.

In another aspect, an anti-HER 2 antibody of an antibody-drug conjugate is provided, 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, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID No. 1 comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an anti-HER 2 antibody comprising the sequence retains the ability to bind to HER 2. 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 HVR (i.e., in the FR). Optionally, the anti-HER 2 antibody comprises the VL sequence of SEQ ID NO. 1, including post-translational modifications of that sequence. In a specific embodiment, the VL comprises one, two or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:3, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:4, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 5.

In another aspect, there is provided an antibody-drug conjugate comprising an anti-HER 2 antibody, wherein the antibody comprises a VH in any embodiment as provided above and a VL in any embodiment as provided above.

In one embodiment, an antibody-drug conjugate comprising an antibody is provided, 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.

In one embodiment, an antibody-drug conjugate comprising an antibody is provided, wherein the antibody comprises the humanized 7c2.v2.2.La (hu 7C 2) K149 ck light chain sequence of SEQ ID No. 14

In one embodiment, an antibody-drug conjugate comprising an antibody is provided, wherein the antibody comprises the Hu7C 2A 118C IgG1 heavy chain sequence of SEQ ID NO. 15.

In another aspect, provided herein are antibody-drug conjugates comprising an antibody that binds to the same epitope as an anti-HER 2 antibody provided herein. For example, in certain embodiments, immunoconjugates comprising antibodies that bind the same epitope as an anti-HER 2 antibody (comprising the VH sequence of SEQ ID NO:2 and the VL sequence of SEQ ID NO:1, respectively) are provided.

In some embodiments, the anti-HER 2 antibody of the cDAC according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-HER 2 antibody of the immunoconjugate is an antibody fragment, e.g., 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, e.g., an IgG1 antibody, an IgG2a antibody, or other antibody types or isotypes as defined herein. In some embodiments, the anti-HER 2 antibody is a full length antibody.

Humanized 7C2 anti-HER 2 antibody sequence table

Anti-CD 33 antibodies

Anti-CD 33 antibody 15G15.33 to 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.

HVR-L1 RSSQSLLHSNGYNYLD (SEQ ID NO:16)

HVR-L2 LGVNSVS (SEQ ID NO:17)

HVR-L3 MQALQTPWT (SEQ ID NO:18)

HVR-H1 NHAIS (SEQ ID NO:19)

HVR-H2 GIIPIFGTANYAQKFQG (SEQ ID NO:20)

HVR-H3 EWADVFD (SEQ ID NO:21)

Anti-CD 33 antibodies 15G15.33 to cDAC in tables 4 and 5 comprise the light chain variable region of SEQ ID NO. 22 and/or the heavy chain variable region of SEQ ID NO. 23.

EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGVNSV

SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK

(SEQ ID NO:22)

QVQLVQSGAEVKKPGSSVKVSCKASGGIFSNHAISWVRQAPGQGLEWMGGIIPIFGTANY

AQKFQGRVTITADESTSTAFMELSSLRSEDTAVYYCAREWADVFDIWGQGTMVTVSS

(SEQ ID NO:23)

The anti-CD 33 antibody 9C3 comprises three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3), SEQ ID NOS: 24-29 and the following VL and VH sequences SEQ ID NOS: 30-37.

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

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

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

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

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

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

9C3 V_L

DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF

SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK

(SEQ ID NO:30)

9C3 V_H

EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS

VKGRFNISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS

(SEQ ID NO:31)

9C3.2 V_L

DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYA

ASSLQSGVPSRF

SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK

(SEQ ID NO:32)

9C3.2 V_H

EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVS

LIYSGDSTYYADS

VKGRFTISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGK

GTTVTVSS

(SEQ ID NO:33)

9C3.3 V_L

DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF

SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK

(SEQ ID NO:34)

9C3.3 V_H

EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS

VKGRFSISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS

(SEQ ID NO:35)

9C3.4 V_L

DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRF

SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK

(SEQ ID NO:36)

9C3.4 V_H

EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWVRQAPGKGLEWVSLIYSGDSTYYADS

VKGRFAISRDISKNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKGTTVTVSS

(SEQ ID NO:37)

In some embodiments, a cDAC described herein comprises an anti-CD 33 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:27, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:28, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:29, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:24, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:25, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

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

In another aspect, a cDAC described herein 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. 24, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 25, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 26. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 24, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 25, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 26.

In another aspect, an anti-CD 33 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:27, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:28 and (iii) HVR-H3 comprising 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 comprising the amino acid sequence of SEQ ID NO:24, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:25, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

In another aspect, a cDAC described herein comprises (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.

In any of the above embodiments, the anti-CD 33 antibody is humanized. In one embodiment, the anti-CD 33 antibody comprises the HVR of any one of the embodiments above, and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework is a human VL kappa I consensus (VL_KI) framework and/or a VH framework VH₁. In certain embodiments, the human acceptor framework is a human VL kappa I consensus (VL_KI) framework and/or a VH framework VH₁ comprising any one of the following mutations.

In another aspect, an anti-CD 33 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35 and/or SEQ ID NO. 37. In certain embodiments, a VH sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35 and/or SEQ ID NO. 37 comprises a substitution (e.g. a conservative substitution), insertion or deletion relative to a reference sequence, but an anti-CD 33 antibody comprising the sequence retains the ability to bind CD 33. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35 and/or SEQ ID NO. 37. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted and/or deleted in SEQ ID NO:35, SEQ ID NO:33, SEQ ID NO:35 and/or SEQ ID NO: 37. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-CD 33 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 comprising the amino acid sequence of SEQ ID NO. 27, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 28 and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 29.

In another aspect, an anti-CD 33 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and/or SEQ ID NO: 36. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34 and/or SEQ ID NO. 36 comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to a reference sequence, but an anti-CD 33 antibody comprising the sequence retains the ability to bind CD 33. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and/or SEQ ID NO: 36. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and/or deleted in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and/or SEQ ID NO: 36. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the anti-CD 33 antibody comprises the VL sequences 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 specific embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 24, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 25, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 26.

In another aspect, an anti-CD 33 antibody is provided, wherein the antibody comprises a VH as in any one of the embodiments provided above and a VL as in any one of the embodiments provided above.

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.

In another aspect, provided herein are antibodies that bind to the same epitope as the anti-CD 33 antibodies provided herein. For example, in certain embodiments, an antibody that binds to the same epitope as an anti-CD 33 antibody is provided that comprises the VH sequence of SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35 and/or SEQ ID NO. 37 and the VL sequence of SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34 and/or SEQ ID NO. 36, respectively.

In some embodiments, the anti-CD 33 antibody is a monoclonal antibody, including a human antibody. In one embodiment, the anti-CD 33 antibody is an antibody fragment, such as 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 as defined herein. In some embodiments, the anti-CD 33 antibody is a full length antibody.

In a further aspect, the anti-CD 33 antibody according to any of the above embodiments may incorporate any of the features described below, alone or in combination.

Through cysteine engineering engineered antibody variants

In certain embodiments, it may be desirable to produce a cysteine engineered antibody, e.g., "THIOMAB^TM" or TDC, in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the substituted residue is present at a site available for conjugation to an antibody. As further described herein, by substituting those residues with cysteines, reactive thiol groups are thereby located at accessible sites of the antibody, and can be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates. In certain embodiments, any one or more of the following residues, 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 Fc region of the heavy chain, may be substituted with cysteine. In specific embodiments, the antibodies described herein comprise HC-A140C (EU numbering) cysteine substitutions. In specific embodiments, the antibodies described herein comprise LC-K149C (Kabat numbering) cysteine substitutions. In specific embodiments, the antibodies described herein comprise HC-A118C (EU numbering) cysteine substitutions. Cysteine engineered antibodies may be generated as described, for example, in US 7521541.

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

in certain embodiments, the antibody comprises one of the following light chain cysteine substitutions:

A non-limiting exemplary hu7C2.v2.2.LA Light Chain (LC) K149C THIOMAB^TM has the heavy and light chain amino acid sequences of SEQ ID NOS 10 and 14, respectively. A non-limiting exemplary hu7C2.v2.2.LA Heavy Chain (HC) A118C THIOMAB^TM has the heavy and light chain amino acid sequences of SEQ ID NOs 15 and 9, respectively.

Antibody targets

In some embodiments, antibodies to the cereblon degradant antibody conjugate (cDAC) are capable of binding to one or more Tumor Associated Antigens (TAA), cell surface receptors, and immunospecific antigens to confer targeted specificity to the cereblon degradant antibody conjugate, and are capable of safe and systemic delivery of active drug moieties.

Certain tumor-associated antigens are known in the art and can be prepared for antibody production using methods and information well known in the art. To find effective cellular targets for cancer diagnosis and treatment, researchers have attempted to identify transmembrane or additional 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. In general, such tumor-associated polypeptides are expressed in greater amounts on the surface of cancer cells than on the surface of non-cancer cells. The recognition of such tumor-associated cell surface antigen polypeptides allows for higher specificity for destruction of cancer cells via antibody-based therapies.

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 names, alternative names, genbank accession numbers, and major references, following the National Center for Biotechnology Information (NCBI) nucleic acid and protein sequence recognition convention. Nucleic acid and protein sequences corresponding to 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 TAAs having the sequences found in the cited references. For example, TAAs having variant sequences are typically capable of specifically binding to antibodies that specifically bind to TAAs having the corresponding sequences listed. The sequences and disclosures of the references specifically cited herein are expressly incorporated by reference.

The sequences and disclosures of the references specifically cited herein are expressly incorporated by reference.

(1) BMPR1B (bone morphogenic protein receptor type IB, genbank accession No. NM-001203) ten Dijke, P., et al Science 264 (5155): 101-104 (1994), oncogene14 (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; FIG. 112), WO200298358 (claim 1; page 183), WO200254940 (pages 100-101), WO200259377 (pages 349-350), WO200230268 (claim 27; page 376), WO200148204 (example; FIG. 4) NP-001194 bone morphogenic protein receptor, type IB/d=NP-001194.1-cross-referenced: 603248, MIM 194, A0694; A_06591).

(2) E16 (LAT 1, 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 1), 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 (FIG. 3), WO2003025138 (claim 12; page 150), NP-003477 solute carrier family 7 (cationic amino acid transporter, y+ system), member 5/pid=NP-003477.3-person cross-reference: 182, NP-003477.3; NM-013-015923 NM 486).

(3) STEAP1 (prostate six transmembrane epithelial antigen; genbank accession No. NM-014449) 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 (FIG. 1L), EP1394274 (example 11), WO2004016225 (claim 2), WO2003042661 (claim 12), US2003157089 (example 5), US2003185830 (example 5), US2003064397 (FIG. 2), WO200289747 (example 5; pages 618-619), WO2003022995 (example 9; FIG. 13A, example 53; page 173; example 2; FIG. 2A), NP-036581 prostate six transmembrane epithelial antigen cross-reference: MIM:604415, NP-0381.1; NM-01449_1.

(4) 0772P (CA 125, MUC16, genbank accession No. AF 361486) J.biol.chem.276 (29): 27371-27375 (2001), WO2004045553 (claim 14), WO200292836 (claim 6; FIG. 12), WO200283866 (claim 15; pages 116-121), US2003124140 (example 16), US 798959. Cross-references GI:34501467; AAK 741120.3; AF361486_1.

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiator, mesothelin, genbank accession No. NM-005823) Yamaguchi, N.et al Biol.Chem.269(2),805-808(1994),Proc.Natl.Acad.Sci.U.S.A.96(20):11531-11536(1999),Proc.Natl.Acad.Sci.U.S.A.93(1):136-140(1996),J.Biol.Chem.270(37):21984-21990(1995));WO2003101283( claim 14, (WO 2002102235 (claim 13; pages 287-288), WO2002101075 (claim 4; pages 308-309), WO200271928 (pages 320-321), WO9410312 (pages 52-57), cross-references MIM: 60051; NP-005814.2; NM-005823_1).

(6) Napi3b (NAPI-3B, NPTIIb, SLC A2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3b, genbank accession No. 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): -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-references: MIM:604217; NP-415.1; NM 0061).

(7) Sema5b (FLJ 10372, KIAA1445, mm.42015, sema5B, SEMAG, sema5b Hlog, sema domain, heptathrombospondin repeat (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (Sema 5b, genbank accession No. AB 040878) 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 numbers Q9P283, EMBL, AB040878, baa95969.1.genew, HGNC 10737.

(8) PSCA hlg (2700050C 12Rik, C530008O16Rik, RIKEN CDNA 2700050C12, RIKEN CDNA 2700050C12 genes, genbank accession number AY 358628), 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-references: GI:37182378; AAQ88991.1, AY358628_1.

(9) ETBR (endothelin B receptor, genbank accession AY); M. biochem.Biophys.Res.Commun.177,34-39,1991; ogawa Y.178, 248-255,1991; arai H, J.circ.56, 1303-1307,1992; arai H, J.biol.chem.268,3463-, yanagisawa M, biochem.Biophys.Res.Commun.178,656-, J.biol.chem.268,3873-, J.Cardiovic.Phacol.20, S1-S. Tsutsumi M., et al Gene-, et al, proc.Natl.Acad.Sci.U.S. A.99,16899-, et al J.Clin.Endocrinol.Metab.82,3116-, et al biol.chem.272,21589-, et al am.J.Med.Genet.108,223-, et al Eur.J.hum.Genet.5,180-, et al Cell-, et al, hum.mol.Genet.4,2407-, et al hum.mol.5:351-354, 1996; amiel J., etal hum.Mol.Genet.5,355-, et al Nat.Genet.12,445-, et al hum.Genet.103,145-148,1998; fuchs S., -et al, et.Mol.115, et.Genet.No. 20040, WO 1, no. 2002; WO (example 2), WO (claim 151), WO (claim 1), WO (FIG. 6), WO (claim 12, page 144), WO (claim 1, pages 124-125), EP (claim 8; FIG. 2), WO (claim 1, pages 297-299), US (FIG. 3), US (claim 1a; col 31-34), WO.

(10) MSG783 (RNF 124, hypothetical protein FLJ20315, genbank accession No. NM-017763), WO2003104275 (claim 1), WO2004046342 (example 2), WO2003042661 (claim 12), WO2003083074 (claim 14; page 61), WO2003018621 (claim 1), WO2003024392 (claim 2; FIG. 93), WO200166689 (example 6), cross-references LocusID:54894, NP-060233.2, and NM-017763_1.

(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, genbank accession No. AF 455138) Lab. Invest.82 (11): 1573-1582 (2002));WO 2003087306; US2003064397 (claim 1; FIG. 1), WO200272596 (claim 13; pages 54-55); WO200172962 (claim 1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; FIG. 10); WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12; FIG. 10); cross-reference: GI 225488; AAN 6580.1; AF455138 1).

(12) TrpM4 (BR 22450, FLJ20041, 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; fig. 9A), WO2003042661 (claim 12), WO200230268 (claim 27; page 391), US2003219806 (claim 4), WO200162794 (claim 14; fig. 1A-D), cross references MIM 606936; np_060106.2, NM 017636_1.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma 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; FIG. 58), WO200216413 (Claim 1; pages 94-95, 105), WO200222808 (Claim 2; FIG. 1), US5854399 (example 2; col 17-18), US5792616 (FIG. 2), cross-references: 187395; NP-003203.1; NM 003212_1).

(14) CD21 (CR 2 (complement receptor 2) or C3DR (C3 d/Epstein Barr virus receptor) or Hs.73792Genbank 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), U.S. 2004005538 (example 1), WO2003062401 (claim 9), WO2004045520 (example 4), WO9102536 (FIGS. 9.1-9.9), WO2004020595 (claim 1), accession numbers P20023, Q13866, Q14212, EMBL 26004, AAA35786.1.

(15) CD79B (CD 79B, CD79 9β, IGb (immunoglobulin related β), B29, genbank accession No. NM-000626 or 11038674) Proc.Natl.Acad.Sci.U.S. (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, FIG. 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, US 1474482 (claim 13, FIG. 17A/B), WO200055351 (claim 11, pages 5-6), cross-referenced: 000626, NM 000626.5, NP 1; and NP 617.000626.

(16) FcRH2 (IFGP, IRTA4, SPAP1A (SH 2 domain-containing phosphatase-anchored protein 1A), SPAP1B, SPAP C, 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; FIGS. 18D-1-18D-2), WO2003097803 (claim 12), WO2003089624 (claim 25), cross-references: MIM:606509; NP 110391.2; NM_030764_1.

(17) HER2 (ErbB 2, genbank accession No. M11730) Coussens l, et al Science (1985) 230 (4730): 1132-1139); yamamoto T., et al Nature Coussens-234, 1986; semba K., et al Proc. Natl. Acad. Sci. U.S. A.82, 6497-Coussens, et al J.cell biol.165, 869-Coussens, et al J.biol.chem.274,36422-36427,1999; cho H.—S., et al Nature Coussens-Coussens, et al (1993) Genomics Coussens-429; WO2004048938 (example 2); WO Coussens (FIG. 1I), WO Coussens (claim 9), WO Coussens (claim 1), US Coussens, WO Coussens (claim 1), WO Coussens (claim 29; FIGS. 1A-B), WO Coussens (claim 37; FIG. 5C), WO Coussens (example 13; pages 95-107), WO Coussens (claim 68; FIG. 7), WO Coussens (pages 71-74), WO Coussens (pages 114-117), WO Coussens (claim 2; pages 41-46), WO Coussens (page 15), WO Coussens (claim 52; FIG. 7), WO Coussens (claim 3; FIG. 2), US Coussens (claim 3; col 31-38), WO Coussens (claim 2; pages 56-61), EP Coussens (claim 7), WO Coussens (example 3; FIG. 4), login: P04BL; EMM 2; A35808; A3932.BL Coussens; EM1.BL Coussens).

(18) NCA (CEACAM 6, 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 numbers P40199, Q1499; EMBL; M29541; AAA59915.1.EMBL; M18728).

(19) MDP (DPEP 1, genbank accession BC 017023) Proc.Natl.Acad.Sci.U.S.A.99 (26): 168903 (2002)), WO2003016475 (claim 1), WO200264798 (claim 33; pages 85-87), JP05003790 (FIGS. 6-8), WO9946284 (FIG. 9), cross-references MIM 179780, AAH17023.1, BC017023_1.

(20) IL20Rα (IL 20Ra, ZCYTOR7, genbank accession number AF 184971); clark H.F., et al Genome Res.13,2265-2270,2003;Mungall A.J, et al Nature425,805-811,2003;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), U.S. 2004005320 (example 5), WO2003029262 (pages 74-75), WO 3995 (claim 2; page 63), WO200222153 (pages 45-47), U.S. Pat. No. 20-21), WO200146261 (pages 57-59), WO200146232 (pages 63-65), WO9837193 (page 55-55), UHF, Q.349, Q.Kq.Kq.Kq.74, Q.Kq.Q.Kq.8, Q.Kq.9.

(21) Brevican (BCAN, BEHAB, genbank accession No. AF 229053) 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; fig. 52); US2003119122 (claim 1; fig. 52); US2003119126 (claim 1); US2003119121 (claim 1; fig. 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; fig. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1).

(22) EphB2R (DRT, ERK, hek, EPHT, tyro5, genbank accession No. 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-references MIM:600997, NP-004433.2, NM-004442_1.

(23) ASLG659 (B7 h, genbank accession number AX 092328) U.S. Pat. No. 20040101899 (claim 2), WO2003104399 (claim 11), WO2004000221 (FIG. 3), U.S. Pat. No. 2003165504 (claim 1), U.S. Pat. No. 2003124140 (example 2), U.S. Pat. No. 2003065143 (FIG. 60), WO2002102235 (claim 13, page 299), U.S. Pat. No. 2003091580 (example 2), WO200210187 (claim 6, FIG. 10), WO200194641 (claim 12, FIG. 7B), WO200202624 (claim 13, FIGS. 1A-1B), U.S. Pat. No. 2002034749 (claim 54, pages 45-46), WO200206317 (pages 320-321, page 34; pages 321-322), WO200271928 (pages 468-469), WO200202587 (example 1, FIG. 1), WO200140269 (example 3; pages 190-192), WO200036107 (pages 205-207), WO 5342 (claim 12), WO2003004989 (claim 1), WO200271928 (pages 45-234, pages 3563-453).

(24) PSCA (prostate Stem cell antigen precursor, genbank accession number AJ 297436) 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; FIG. 17); US2001055751 (example 1; FIG. 1B); WO 200032752 (claim 18; FIG. 1); WO 1998/51805 (claim 17); page 97); WO 1998/51824 (claim 10; page 94); WO 1998/40539; WO 4384; AF 4325; FIGS. 653).

(25) GEDA (Genbank accession number AY 260763), AAP14954 lipoma HMGIC fusion partner-like protein/pid=AAP 14954.1-Chirenin species: chirenin (human) WO2003054152 (claim 20), WO2003000842 (claim 1), WO2003023013 (example 3, claim 20), US2003194704 (claim 45), cross-references: GI: 3010249, AAP14954.1, AY260763_1.

(26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3, genbank accession No. AF 116456), BAFF receptor/pid=NP-443177.1-Chinesian Thompson, J.S., et al Science 293 (5537), 2108-2111 (2001), WO2004058309, WO2004011611, WO2003045422 (example; pages 32-33), WO2003014294 (claim 35; FIG. 6B), WO2003035846 (claim 70; pages 615-616), WO200294852 (Col 136-137), WO200238766 (claim 3; page 133), WO200224909 (example 3; FIG. 3), cross-references: 606269, NP-443177.1, NM-052945_1, AF132600).

(27) CD22 (B cell receptor CD22-B isoforms, BL-CAM, lyb-8, lyb8, SIGLEC-2, FLJ22814, genbank accession number AK 026467); wilson et al. (1991) Exp. Med.173:137-146; WO2003072036 (claim 1; FIG. 1), cross-references MIM 107266; NP_001762.1; NM_001771_1.

(28) CD79a (CD 79A, CD. Alpha., immunoglobulin-related. Alpha., and B cell-specific proteins that interact covalently with Ig beta (CD 79B) and form complexes with IgM molecules at the surface, transduce 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, FIG. 16), WO9207574 (FIG. 1), US5644033, ha et al (1992) J.Immunol.148 (FIG. 1), mueller et al (1992) Eur.J.22, 1621-1625, hashimoto et al (1994) Immunogenetics (4) and Preud' house et al (1992) in 146-14, WO9958658 (see FIG. 146-14), WO9958658 (1992) J.Immunol.148 (1992) J.1526-1531, mueller et al (1992) Eur.J.22, hashimoto et al (1994) 49) N.141-figure 146, sagu.1988).

(29) CXCR5 (Burkitt lymphoma receptor 1, a G protein-coupled receptor activated by CXCL13 chemokines, which plays a role in lymphocyte migration and humoral defense, in HIV-2 infection and in the possible development of AIDS, lymphoma, myeloma and leukemia), 372aa, pI:8.54MW:41959TM:7[ P ] gene chromosome 11q23.3, genbank accession No. NP-001707.1) WO 2004040000, WO2004/015426; U.S. 2003105292 (example 2), U.S. Pat. No. 2, 6555339 (example 2), WO 2002/61087 (FIG. 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), U.S. Pat. No. 49-52), WO9428931 (pages 56-58), WO 2/97 (1997), U.S. Pat. No. 2/6107 (example 2), WO 2002/61087 (see FIGS. 12-269, etc., pages 309-269 (see FIGS. 12-309, 2799, etc.), WO 4992 (see FIGS. 1992, 2799).

(30) HLA-DOB (beta subunit of MHC class II molecule (Ia antigen), which binds to peptide and presents it to CD4+ T lymphocytes); 273aa, pI 6.56MW 30630 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 (6): 411-413; beck et al (1992) J.mol.228:433-441; strausberg et al (2002) Proc.Natl.Aca.Sci 99:1699; servernius 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:512-519; WO 9958585858 (claim 13, FIG. 15); US6153408 (Col 35-38); US 34168-170); strausberg et al (2002) Proc.Acad.99:16903; servernius et al (1987) J.biol.262: 8759-8766; beck et al (1996) J.mol.255:1-13; naruse et al (1989) J.14168).

(31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an extracellular ATP-gated ion channel, may be involved in synaptic transmission and neurogenesis, the lack of which may lead to the pathophysiology of idiopathic detrusor instability); 422 aa), pI 7.63, MW:47206TM:1[ P ] gene chromosome 17P13.3, genbank accession No. 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).

(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 99:168916-903.

(33) LY64 (lymphocyte antigen 64 (RP 105), a type I membrane protein of the Leucine Rich Repeat (LRR) family, which regulates B cell activation and apoptosis, whose loss of function is associated with increased disease activity in patients with systemic lupus erythematosus), 661aa, pI:6.20, MW: 741147TM: 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).

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for an immunoglobulin Fc domain comprising C2-type Ig-like and ITAM domains, which may play a role in B lymphocyte differentiation), 429aa, pI:5.28, MW: 463205. TM.: 1[ P ] gene chromosome 1q21-1q22, genbank accession number NP-443170.1) WO2003077836, WO200138490 (claim 6, FIGS. 18E-1-18E-2), davis et al (2001) Proc.Natl. Acad. Sci USA98 (17): 9772-9777; WO2003089624 (claim 8), EP1347046 (claim 1), WO2003089624 (claim 7).

(35) IRTA2 (immunoglobulin superfamily receptor translocation related 2, putative immunoreceptor that may play a role in B cell development and lymphogenesis; gene dysregulation by translocation in some B cell malignancies), 977aa, pI:6.88MW: 1064638 TM:1[ P ] gene chromosome 1q21, genbank accession numbers: human AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; mice: AK089756, AY158090, AY506558; NP-112571.1. WO2003024392 (claim 2, FIG. 97), nakayama et al (2000) biochem. Biophys. Res. Commun.277 (1): 124-127, WO2003077836, WO200138490 (claim 3, FIGS. 18B-1-18B-2).

(36) TENB2 (TMEFF 2, brain tumor-inhibiting protein, TPEF, HPP1, TR, a putative transmembrane proteoglycan, which is related to EGF/catabolin family growth factors and follistatin), 374aa, NCBI accession numbers AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP-057276; NCBI gene: 23671;OMIM:605734;SwissProt Q9UIK5;Genbank accession number AF179274;AY358907,CAF85723,CQ782436WO 2004074320;JP 2004113151;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. Res. Commun.266:593-602; liang et al (2000) Cancer Res.60:4907-12; glynne-Jones et al (2001) Int J caner. Oct 15;94 (2): 178-84).

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

(38) TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; brain tumor oncostatin -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).

(39) GDNF-Ra1 (GDNF family receptor α1;GFRA1;GDNFR;GDNFRA;RETL1;TRNR1;RET1L;GDNFR-α1;GFR-α-1);U95847;BC014962;NM_145793NM_005264;Kim,M.H. et al (2009) mol. Cell. Biol.29 (8), 2264-2277; trenor, J.et al (1996) Nature 382 (6586): 80-83.

(40) Ly6E (lymphocyte antigen 6 complex gene 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.

(41) TMEM46 (SHISA homolog 2 (Xenopus); SHISA 2); 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.

(42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D, ly6-D, MEGT 1), 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.

(43) LGR5 (leucine-rich repeat-rich G-protein coupled receptor 5; GPR49, GPR 67); NP-003658.1; NM-003667.2; salanti, G. Et al (2009) am. J. Epidemic mol.170 (5): 537-545; yamamoto, Y. Et al (2003) Hepatology 37 (3): 528-533).

(44) RET (RET proto-Oncogene, MEN2A, HSCR1, MEN2B, MTC1, PTC, CDHF12, hs.168414, RET51, RET-ELE 1), 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.

(45) LY6K (lymphocyte antigen 6 complex gene locus K, LY, K, HSJ001348, FLJ 35226), 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.cancer103 (6): 768-774).

(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).

(47) GPR54 (KISS 1 receptor, KISS1R, GPR54 4, HOT7T175, AXOR 12), 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.

(48) ASPHD1 (containing aspartic acid β -hydroxylase domain 1, LOC 253982), NP-859069.2, NM-181718.3, gerhard, D.S. et al (2004) Genome Res.14 (10B): 2121-2127.

(49) Tyrosinase (TYR, OCAIA, OCA A, tyrosinase, SHEP 3), 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).

(50) TMEM118 (cycloproteins, transmembrane 2; RNFT2; FLJ 14627); 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).

(51) GPR172A (G protein coupled receptor 172A, GPCR, FLJ11856, D15Ertd747 e), NP-078807.1, NM-024131.3, 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.

(52) CD33, a member of the sialic acid binding immunoglobulin-like lectin family, is a 67kDa glycosylated transmembrane protein. In addition to committed bone marrow mononuclear cells and erythroid progenitors, CD33 can be expressed on most myeloid and monocytic leukemia cells. It was not seen on the earliest multipotent 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 on its cytoplasmic tail, each followed by a hydrophobic residue, similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in many inhibitory receptors.

(53) CLL-1 (CLEC 12A, MICL and DCAL) 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 multiple 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 transcriptional variants of this gene have been described, but the full length nature of some of these variants has not been established. This gene is tightly 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 includes a single C-type lectin-like domain (which is expected to not bind calcium or sugar), a region, a transmembrane domain, and a short stem comprising an itc motif.

(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 signaling transducer that is differentially expressed in many cancers. It signals cells to self-renew, proliferate, invade and survive. It has a quality similar to stem cells. 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). Overexpression of TROP2 has prognostic significance. Several ligands have been proposed that interact with TROP 2. TROP2 signals cells via different pathways and is regulated by transcription via a complex network of several transcription factors.

Human TROP2 (TACSTD 2: tumor-associated calcium signal transducer 2, GA733-1, EGP-1, M1S1; hereinafter abbreviated as hTROP) is a single-pass transmembrane type 1 cell membrane protein composed of 323 amino acid residues. Whereas the presence of cell membrane proteins is involved in immunological resistance, which is common for 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 antigen molecules recognized by monoclonal antibodies directed against cell membrane proteins in the human choriocarcinoma cell line be identified and designated TROP2 as one of the molecules expressed in human trophoblasts (LIPINSKI M, et al (1981), proc. Natl. Acad. Sci.78 (8), 5147-5150). This molecule is also designated 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 obtained by immunization with a gastric cancer cell line or an epithelial glycoprotein (EGP-1; basu A, et al, int. J. Cancer,62 (4), 472-479 (1995)) was recognized by the mouse monoclonal antibody RS7-3G11 obtained by immunization with a non-small cell lung cancer cell. However, in 1995, the TROP2 gene was cloned and it was confirmed that all of these molecules were identical (Fornaro M, et al, (1995) int. J. Cancer,62 (5), 610-618). The DNA sequence and amino acid sequence of hTROP2 are available on public databases and can be referenced, for example, under accession numbers nm_002353 and np_002344 (NCBI).

For such information indicating association with cancer, various anti-hTROP 2 antibodies have been established so far and their anti-tumor effects have been studied. Among these antibodies, non-conjugated antibodies which exhibit anti-tumor activity by themselves, for example in a nude mouse xenograft model (WO 2008/144891; WO 2011/145744; WO 2011/155579; WO 2013/077458) are disclosed as antibodies which exhibit anti-tumor activity as ADCs with cytotoxic drugs (WO 2003/074566; WO 2011/068845; WO 2013/068946;US 7999083). However, the intensity or coverage of its activity is still insufficient and the medical needs of hTROP2 as a therapeutic target are not met.

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

(55) CD123 (IL-4, IL3RA, IL3ry, IL3RAY, interleukin-3 receptor) is a protein found on cells that helps to transmit the signal of interleukin-3, an important soluble cytokine in the immune system. Genes encoding receptors are located in the pseudo-autosomal regions of the X and Y chromosomes. The receptor belongs to the family of type I cytokine receptors and is a heterodimer with a unique alpha chain paired with a common beta (βc or CD 131) subunit. The gene for the alpha subunit is 40 kilobases long and has 12 exons. CD123 is the 70kD transmembrane alpha chain of the IL-3 receptor. Separately, CD123 binds IL-3 with low affinity, while CD123 binds to CDw131 (common beta chain) with high affinity. CD123 does not transduce intracellular signals upon binding to IL-3 and requires a β chain to achieve this function. CD123 acts as a diagnostic, prognostic and therapeutic marker in some hematological malignancies, particularly acute leukemia CD123 and TCF4 co-expression by immunohistochemistry, is highly specific and sensitive to blast plasmacytoid dendritic cell tumors (BPDCN) (Sun Q, et al (1996) Blood 87:83; herling M, et al (2003) Blood101:5007; charles N, et al (2010) Nat. Med.16:701; martin-Gayo E, et al (2010) Blood 115:5366; testa U, et al (2019) Cancers (9): 1358-1388; shi M, et al (2019) Cardiovasc Hematol Disord Drug Targets (3): 195-204).

Exemplary embodiments of antibody targets are HER2 and CD33.

In embodiments, the antibodies have free cysteine thiol groups that can be used to conjugate with electrophilic groups of the cereblon degradant-linker intermediate (cDLI).

The thiol-containing antibody may be a natural cysteine thiol or reduced intra-or inter-chain disulfide amino acid residues.

Thiol-containing antibodies may be cysteine engineered antibodies in which one or more cysteine residues have been introduced by mutagenesis according to known techniques and site-specific conjugation of the cereblon-degrading agent-linker intermediate is provided by cysteine substitution at the site where the engineered cysteines are available for conjugation, while not interfering with immunoglobulin folding and assembly or altering antigen binding and effector functions (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 32:1884-1893US 7521541;US 7723485;US2012/011615; wo 2009/052249). One, two, three or more cysteine amino acids may be introduced into the cysteine engineered antibody.

CDAC may be formed by conjugating one or more antibody cysteine thiol groups to a molar excess of the cereblon degradant-linker intermediate (cDLI) of formula II. Due to its symmetrical structure, cysteine engineered IgG antibodies can allow up to two cDLI to be conjugated to each mutant cysteine site. For example, a cysteine engineered antibody with one mutated cysteine site allows conjugation of up to two cDLI to give a theoretical maximum DAR of 2. Cysteine engineered antibodies with two mutated cysteine sites allowed conjugation of up to four cDLI to give a theoretical maximum DAR of 4. Cysteine engineered antibodies with three mutated cysteine sites allowed conjugation of up to six cDLI to give a theoretical maximum DAR of 6.

Cysteine thiols are reactive nucleophiles at neutral pH, unlike most amines, they are protonated and less nucleophilic near pH 7. Because the free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues are typically present in the oxidized form of disulfide-linked oligomers or have internally bridged disulfide groups. The antibody cysteine thiol group is typically more reactive, i.e., more nucleophilic, to the electrophilic conjugation reagent than the antibody amine or hydroxyl group. Engineering cysteine thiol groups by mutating various amino acid residues of a protein into cysteine amino acids can be problematic, particularly in the case of unpaired (free Cys) residues or residues that are relatively easy to react or to oxidize. In concentrated solutions of proteins, whether in the periplasm of E.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 the new Cys unreactive for conjugation to drugs, ligands, or other labels. Furthermore, if the protein oxidizes between a newly engineered Cys and an existing Cys residue to form an intramolecular disulfide bond, neither Cys group is available for active site participation and interaction. In addition, proteins can become inactive or non-specific by misfolding or loss of tertiary structure (Zhang et al (2002) Anal. Biochem. 311:1-9).

In some embodiments, the cysteine engineered antibody may have reactive cysteine thiol residues introduced at a site on the light chain, such as the 149-lysine site (LC K149C), or at 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 position (EU numbering) of the heavy chain (HC a 118C). The mutation site is numbered 121 by sequence number, alternatively numbered 114 by Kabat number. In other embodiments, the cysteine engineered Antibody has a mutant cysteine residue introduced into (i) the light chain at G64C, R142C, K188C, L201C, T129C, S114C, V C or E105C according to Kabat numbering, (ii) the heavy chain at D101C, A140C, L177C, V184C, T C or S122C according to Kabat numbering, or (iii) other cysteine mutant antibodies, such as those edited by Bhakta, S.et al ,(2013)"Engineering THIOMABs for Site-Specific Conjugation of Thiol-Reactive Linkers",Laurent Ducry(), anti-bodies-Drug Conjugates, methods in Molecular Biology, volume 1045, pages 189-203, as described in WO 2011/156328;US 9000130. In other embodiments, the cysteine engineered antibody comprises one or more cysteine mutations selected from the group consisting of HC A118C, LC K149C, HC A140C, LC V205C, LC S121C, HC L174C, HC L177C, HC Y373C.

In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Moieties suitable for derivatization of antibodies 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), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers) and dextran or poly (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer may have any molecular weight and may or may not have branching. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular characteristics or functions of the antibody to be improved, whether the antibody derivative will be used in a defined-condition therapy, and the like.

In some embodiments, the cDAC is a mixture of cereblon degradant antibody conjugate compounds, wherein the average drug loading of each antibody in the mixture of cereblon degradant antibody conjugate compounds is between about 2 and about 6.

The present disclosure includes all reasonable combinations and permutations of features of embodiments of formulas I-II.

The drug loading, i.e., the number of cereblon degradant moieties (CDs) per antibody (Ab) in the cereblon degradant antibody conjugate (cDAC) of formula I, is indicated by p. The loading (p) may range from 1 to about 8 CD portions per antibody. The cDAC of formula I includes a mixture or collection of antibodies conjugated to a cD moiety in the range of 1 to 8. In some embodiments, the number of cD moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues (such as lysine and cysteine). In some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. In these aspects, p may be 1,2, 3, 4, 5, 6, 7 or 8 and ranges thereof, such as 1 to 8 or 2 to 6. Exemplary cDACs of formula I include, but are not limited to, antibodies with 1,2, 3, or 4 engineered cysteine amino acids (Lyon, R.et al (2012) Methods in enzyme.502:123-138). In some embodiments, without the use of engineering, one or more free cysteine residues are already present in the antibody forming intra-and inter-chain disulfide bonds (natural disulfide groups), in which case the existing free reduced cysteine residues can be used to conjugate the antibody to a drug. In some embodiments, prior to conjugation of the antibody, the antibody is exposed to reducing conditions so as to produce one or more free native cysteine residues.

For some cDACs, p may be limited by the number of attachment sites on the antibody. For example, where the linkage is a cysteine thiol, as in certain exemplary embodiments described herein, the antibody may have only one or a limited number of cysteine thiol groups, or may have only one or a limited number of thiol groups with sufficiently high reactivity through which the drug may be linked. In other embodiments, one or more lysinylamino groups in the antibody may be available and reactive for conjugation with the cereblon degrading agent-linker intermediate of formula II. In certain embodiments, higher drug loading, 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 drug loading 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 exhibit reactive nucleophilic groups such as lysine or cysteine.

The drug loading (drug/antibody ratio) of the cDAC can be controlled in different ways, for example, (i) to limit the molar excess of cereblon degradant-linker intermediate compound relative to antibody, (ii) to limit conjugation reaction time or temperature, and (iii) to limit reductive denaturation conditions for the optimized antibody reactive moiety.

It will be appreciated that when more than one nucleophilic group of an antibody is reacted with a drug, then the resulting product is a mixture of cDAC compounds having a distribution of one or more drug moieties attached to the antibody. The average amount of Drug (DAR) in each antibody can be calculated from the mixture by a double ELISA antibody assay, which 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, e.g., hydrophobic interaction chromatography (see, e.g., mcDonagh et al, (2006) prot.engr.design & Selection 19 (7): 299-307; hamburg et al, (2004) clin.cancer res.10:7063-7070; hamburg 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,2004Annual Meeting,2004, 27 to 31, proceedings of the AACR, 45, 3 months 2004; alley et al, "Controlling the location of drug ATTACHMENT IN anti-drug conjugates," abstract No. 627,American Association for Cancer Research,2004Annual Meeting,2004, 27 to 31, proceedings of the AACR, 45, 2004). In certain embodiments, a homogeneous cDAC having a single drug loading may be separated from the coupling mixture by electrophoresis or chromatography.

In some embodiments, the cereblon degrading agent portion of the cDAC has the formula:

TPL-L₂-E3UL

Wherein:

TPL is a target protein ligand;

e3UL is a cereblon-bound E3 ubiquitin ligase ligand;

l₂ is a degrading agent linker, and

One of TPL, E3UL and L₂ is connected to L₁, or

The cereblon degrading agent part is molecular gel.

In some embodiments, the TPL has the formula:

Wherein the method comprises the steps of

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

The wavy line indicates the connection point of L₂.

In some embodiments, the TPL has the formula:

Wherein the wavy line indicates the connection point of L₂.

In some embodiments, the TPL has the formula:

wherein the wavy line indicates the point of connection of L₂.

In some embodiments, TPL targets BRD4, GSPT1, BET, BRM (SMARCA 2), KRAS, and SHP2.

In some embodiments, E3UL comprises a glutarimide group.

In some embodiments, E3UL is selected from:

Wherein X¹ is selected from CH₂ and C (=O), and the wavy line indicates the point of attachment of L₁ or L₂.

In some embodiments, the cereblon degradant antibody conjugate has the formula:

wherein L₁ is a protease-cleavable non-peptide linker having the 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 the glutarimide group of E3UL and has the formula:

Wherein the wavy line is a connection to the PM.

In some embodiments, the cereblon degradant antibody conjugate has the formula:

wherein L₁ is a protease-cleavable non-peptide linker having the 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 formula:

Wherein the wavy line is a connection to the PM.

In some embodiments, L₂ is selected from:

-N (R) - (C₁-C₁₂ Alkyldiyl) -N (R) -,

-N (R) - (C₂-C₁₂ alkenyldiyl) -N (R) -,

-N (R) - (C₂-C₁₂ alkynyldiyl) -N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) - (N (R) -C (=o) CH₂ O-),

-N (R) - (C₁-C₁₂ alkyldiyl) - (N (R) -C (=o) CH₂ N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R) - (C₁-C₁₂ alkyldiyl) -N (R) -,

-N (R) - (C₁-C₆ Alkyldiyl) -O- (C₁-C₆ Alkyldiyl) -N (R) -,

N (R) - (CH₂CH₂O)_n-N(R)-(CH₂CH₂O)_n -, where N is an integer from 1 to 4,

A C₁-C₁₂ alkyldiyl group,

C₂-C₁₂ alkenyldiyl, and

C₂-C₁₂ alkynyl diradical (C₂-C₁₂) and (C) is used for preparing the catalyst,

Wherein R is selected from H, C₁-C₆ alkyl diradicals and the point of attachment to L₁, and

Alkyldiyl, alkenyldiyl and alkynyldiyl are optionally substituted by one or more groups selected from the group consisting of ：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)₃ H.

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

Wherein R^x is selected from F, cl, br, n is 0, 1,2 or 3;R^y is selected from H and C₁-C₆ alkyl, and X¹ is selected from CH₂ and C (=o).

In some embodiments, formula I is selected from the following formulas:

And

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 X¹ is selected from CH₂ and C (=o).

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

And

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

Table 4 shows exemplary cDAC and assay data for preparation with the cereblon degradant-linker intermediates from table 2.

TABLE 4 examples of cereblon degrading antibody conjugates (cDAC)

Biological Activity of cDAC

Typically, the cytotoxic or cytostatic activity of a cereblon degradant antibody conjugate (cDAC) is measured by exposing mammalian cells having a receptor protein (e.g., HER 2) to a cDAC antibody in a 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 were used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the cDAC described herein.

The in vitro potency of the cDAC described herein was measured by a cell proliferation assay as described in example 103. The cDAC described herein show surprising and unexpected efficacy in inhibiting tumor cell proliferation. Efficacy of cDAC correlates with target antigen expression by the cell. The conjugates tested were able to bind to specific antigens expressed on the cell surface and resulted in the death of these cells in vitro.

The luminous cell viability assay is a commercially available (Promega corp., madison, WI) homogeneous assay method based on recombinant expression of coleopteran luciferases (US 5583024; US5674713; US 5700670). This cell proliferation assay determines the number of living cells in culture based on the quantification of ATP present, an indicator of metabolically active cells (Crouch et al (1993) J.Immunol. Meth.160:81-88; U.S. Pat. No. 6,66,677).The assay was performed in a 96-well format, making it suitable for automated High Throughput Screening (HTS) (Cree et al (1995) ANTICANCER DRUGS 6:398-404). Homogeneous assay procedures involve the addition of a single reagentReagents) are added directly to cells cultured in serum-supplemented medium. Cell washing, removal of the medium and multiple pipetting steps are not required. After the reagents were added and mixed, the system detected as few as 15 cells/well in 384 well format within 10 minutes. Cells may be treated continuously with the cDAC, or they may be treated and separated from the cDAC. In general, the transiently treated cells (i.e., 3 hours) exhibited the same potent effect as the continuously treated cells. The assay can be performed in 96-well or 384-well formats, making it suitable for automated High Throughput Screening (HTS). See Cree et al, (1995) ANTICANCER DRUGS, 6:398-404. The measurement procedure includes the steps of mixing the single reagentReagents) are added directly to the cultured cells. This causes the cells to lyse and generate a luminescent signal generated by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which in turn is proportional to the number of living cells present in the culture. The data may be recorded by a photometer or a CCD camera imaging device. The luminous output is expressed in Relative Light Units (RLU).

The homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent 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 signal produced by the luciferase reaction, which typically has a half-life of greater than five hours, depending on the cell type and medium used. Living cells are reflected in Relative Luminescence Units (RLU). The substrate beetle luciferin is oxidative decarboxylated by the recombinant firefly luciferase while converting ATP to AMP and generating photons.

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

FIG. 1 shows the antiproliferative effect of BRD4-cereblon degrading agents on the in vitro efficacy of KPL-4 and SK-BR-3 cells at 5 days. Cell viability is plotted as a percentage of control against the concentration (nM) of the cereblon degradant compound cD-5 from Table 1. IC₅₀ for KPL-4 was 0.65nM. IC₅₀ for SK-BR03 was 0.41nM. These results demonstrate the remarkable efficacy of the cereblon degradant compound.

FIG. 2A shows the anti-proliferative effect of in vitro efficacy after 5 days by treating HER2+KPL-4 cells with anti-HER 27C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 in Table 4. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph. FIG. 2B shows the anti-proliferative effect of in vitro efficacy after 5 days by treating HER2+SK-BR-3 cells with anti-HER 27C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 in Table 3. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

FIG. 3A shows the anti-proliferative effect of in vitro efficacy after 5 days by treating HER 2-low/ER+CAMA 1 cells with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 in Table 4. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph. FIG. 3B shows the antiproliferative effect of the in vitro potency of HER 2-low/ER+EFM19 cells after 5 days of treatment with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 from Table 4. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph. Table 5 shows that anti-HER 2 cDAC was active in both HER2+ and HER 2-low breast cancer cell lines, while off-target anti-CD 33 cDAC was not active.

TABLE 5 in vitro efficacy of BRD4-cereblon degrading agent antibody conjugate (cDAC)

FIG. 4 shows the antiproliferative effect of in vitro potency after 7 days of AML cell lines by treatment with anti-CD 33 BRD4-cereblon degrading agent antibody conjugate cDAC-3. AML cell lines are MV-4-11, EOL-1, molm-13, nomo-1, HL-60 and OCI-AML-2. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph. Table 6 shows that cDAC-3 is active in inhibiting various AML cell lines.

TABLE 6 in vitro potency of anti-CD 33 BRD4-cereblon degrading agent antibody conjugate cDAC-3 in various AML cell lines

AML cell lines	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

FIG. 5A shows the anti-proliferative effect of in vitro efficacy after 5 days by treating EOL-1AML cells with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 from Table 4. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph. FIG. 5B shows the antiproliferative effect of in vitro potency after 5 days of treatment of HL-60AML cells with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degradant antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 from Table 4. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph. Table 6 shows EC50 values for the cDAC of fig. 5A and 5B.

FIG. 6A shows the anti-proliferative effect of in vitro efficacy after 3 days by treating Molm-13 AML cells with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 from Table 4. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph. FIG. 6B shows the antiproliferative effect of in vitro potency after 3 days of MV-4-11AML cells treated with anti-HER 2 7C2 and anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 from Table 4. Cell viability is plotted as a percentage of control versus concentration of cDAC (μg/mL) in a graph.

Table 7 shows the EC50 values for in vitro potency in BRD4-cereblon degrader antibody conjugates cDAC-3, cDAC-5 and cDAC-6 from FIGS. 5A, 5B, 6A and 6B, and AML cell lines. cDAC-3 and cDAC-5 comprise a thio-human anti-CD 33 antibody, and cDAC-4 and cDAC-6 comprise a thio-human anti-7C 2 (HER 2) antibody. It can be seen that targeting cDAC3 and cDAC-5 has a concentration-dependent effect in inhibiting AML cells with CD33 receptor, whereas non-targeting cDAC-4 and cDAC-6 shows the expected lower efficacy.

TABLE 7 in vitro potency (EC₅₀ nM) of BRD4-cereblon degrader antibody conjugate cDAC3-6 in AML cell lines from FIGS. 5A, 5B, 6A, 6B

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 targeting cDAC3 and cDAC-5 has a concentration-dependent effect on AML cells with CD33 receptor, rather than targeting cDAC-4 and cDAC-6, has the expected lower efficacy.

TABLE 8 cell killing assay of BRD4-cereblon degrader antibody conjugate cDAC3-6 in AML cell lines measured as IC50 (ng/ml)

The in vitro antiproliferative effects of exemplary cDAC indicate that the cDAC described herein are biologically active, consisting of a variety of antibodies, including those that bind to the tumor-associated antigens and cell surface receptor proteins described herein. The exemplary cDAC of table 3 comprises antibodies that bind to the tumor associated antigens HER2 and CD 33. HER2 is highly expressed in certain solid tumors (such as breast and gastric cancers) at millions of copies per cell. CD33 is expressed in hematological malignancies (such as leukemia and lymphoma) at a low copy number of about 10,00 per cell. The mechanisms of circulation and internalization differ between HER2 and CD33 cell surface proteins. Thus, proof of significant in vitro efficacy of exemplary cDAC comprising HER2 and CD33 reasonably suggests that cDAC comprising antibodies other than anti-HER 2 and anti-CD 33 described herein will have similar biological activity.

In vivo efficacy of cDAC was measured in mouse tumor xenograft studies (examples 104-105). The cDAC described herein show surprising and unexpected efficacy in inhibiting tumor growth, both targeting-dependent and dose-dependent. Efficacy of cDAC may be related to target antigen expression by tumor cells.

Efficacy of the cDAC provided herein was measured in vivo by implantation of an allograft or xenograft of cancer cells in rodents and treatment of the tumor with the cDAC. Variable results are expected depending on the cell line, the specificity of antibody binding of the cDAC to the receptor present on the cancer cell, the dosing regimen, and other factors. In vivo efficacy of cDAC can be measured using transgenic explant mouse models expressing moderate to high levels of tumor-associated antigens, including KPL4 expressing HER2 and BJAB expressing CD 22. Subjects can be treated once with cDAC and monitored over 3 to 6 weeks to measure the time to tumor doubling, log cell killing and tumor shrinkage. Subsequent dose responses and multi-dose experiments can be performed.

For example, in vivo efficacy of the anti-HER 2 cDAC described herein can be measured by a mouse model that highly expresses a HER2 transgenic explant (Phillips et al (2008) Cancer Res.68:9280-90). Allograft propagation from Fo5 mmtv transgenic mice, the mice pair(Genentech, inc.) therapy did not respond or did not respond poorly. According to examples 104-105, subjects were treated one or more times with a dose level (mg/kg) of cDAC and placebo buffer control (vehicle) and monitored over two weeks or more to measure the time to tumor doubling, log cell killing and tumor shrinkage.

FIG. 7 shows the in vivo efficacy of anti-CD 33 BRD4-cereblon degrading antibody conjugates cDAC-3, cDAC-4, cDAC-5 and cDAC-6 in HL-60 xenograft mouse models to reduce tumor volume over time (21 days) at the following doses.

1) Vehicle (histidine buffer # 8), 100 μl, IV one time

2) CDAC-4,3mg/kg IV once

3) CDAC-3,1mg/kg IV once

4) CDAC-3,3mg/kg IV once

5) CDAC-3,10mg/kg IV once

6) CDAC-6,3mg/kg IV once

7) CDAC-5,1mg/kg IV once

8) CDAC-5,3mg/kg IV once

As shown in FIG. 7, anti-CD 33 BRD4-cereblon degrading agent antibody conjugates cDAC-3 and cDAC-5 from Table 4 performed according to example 105 showed significant dose-dependent activity in the mouse HL-60 human leukemia cell line to inhibit tumor growth. At a dose of 1mg/kg, cDAC-5 showed a higher therapeutic effect than cDAC-3 (line 7 vs line 3 in the figure). The amine of the glutarimide group of the cereblon degradant portion of cDAC-5 was linked to the antibody linker through an aminal structure (Table 3, cDLI-5), while the indolinone group of the cereblon degradant portion of cDAC-3 was linked to the antibody linker through a carbamate group (Table 3, cDLI-1). At a dose of 3mg/kg, both cDAC-3 and cDAC-5 (row 4 and row 8, respectively, in FIG. 7) resulted in tumor volumes below the quantification limit. In contrast, figure 7 also illustrates that non-targeted HER2 controls cDAC-4 and cDAC-6 had an initial response in tumors of the matched 3mg/kg group (row 2 and row 6, respectively), but eventually grew (> 21 days) at the end of the study. This comparison demonstrates the targeting-specific efficacy of BRD4-cereblon degradant antibody conjugates provided herein (e.g., cDAC-3 and cDAC-5). The LALA-PG mutation within the Fc domain of anti-CD 33 antibodies ablates Fc-FcR mediated effector functions without affecting the desired affinity (Schlothauer, T.et al (2016) Protein Engineering, design & Selection,29 (10): 457-466). In addition, the cereblon degradant antibody conjugates provided herein (e.g., cDAC-3 and cDAC-5) were shown to be tolerated in mice at doses of 3mg/kg or less.

In vivo and whole blood stability of the cDAC can be measured and evaluated according to standard assays, including example 104.

According to the whole blood assay of example 104, 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, the samples were subjected to capture of the biotinylated extracellular domain (ECD) of HER2 antigen immobilized on streptavidin magnetic beads. After washing the microbeads, the sample is eluted and analyzed by LC/MS (liquid chromatography/mass spectrometry). Identification and characterization by mass and LC elution curves allows determination of average DAR (drug to antibody ratio). Table 9 shows that in all media, cDAC-4 and cDAC-6 were both stable at room temperature for 24 hours, with cDAC-4 being slightly more stable than cDAC-6.

TABLE 9 buffer and Whole Blood (WB) stability of various BRD4-cereblon degrading agent antibody conjugates cDAC-4 and cDAC-6

Pharmaceutical composition

In another aspect, provided herein are compositions, e.g., pharmaceutical or pharmacological compositions or formulations, comprising a cereblon degradation agent antibody conjugate (cDAC) or a plurality of cdacs as described herein and a pharmaceutical or pharmacological carrier.

The cDAC may be formulated for parenteral administration, such as intradermal, subcutaneous (subcutaneous), intramuscular (IM) or Intravenous (IV) injection, infusion or administration into a body cavity or lumen of an organ. Alternatively, the cDAC described herein may be injected into a specific site that is otherwise placed into the body, such as a tumor. The composition for injection will typically comprise a solution of cDAC dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that may be used include isotonic solutions of water and one or more salts, such as sodium chloride, for example ringer's solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. These compositions are desirably sterile and generally free of undesirable materials. These compositions may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances in the desired approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g. sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

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

Methods of treating cancer with cereblon degradant antibody conjugates

The cereblon degradant antibody conjugates (cDAC) provided herein can be used to treat diseases and disorders, such as cancer, by inducing target-specific degradation of tumor-associated proteins and imparting specificity to minimize off-target toxic effects. cDAC directs tumor-associated antigen-binding antibodies to cells expressing the antigen and delivers the cereblon degradation (cD) moiety to target cells. The target protein is ubiquitinated and then degraded.

Provided herein are methods of treating cancer with the pharmaceutical compositions of the cereblon degradant antibody conjugates (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 suffering from cancer and in need of treatment for cancer. The method comprises administering a therapeutically effective amount of a cDAC selected from table 3.

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 degradant moiety to the tumor tissue, thereby allowing greater selectivity (i.e., lower effective dose) while increasing the therapeutic index ("therapeutic window") relative to the unconjugated cereblon degradant compound.

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

Examples of cancers to be treated herein include, but are not limited to, epithelial cancers, lymphomas, blastomas, sarcomas, leukemias, or lymphocytic malignancies (including acute myeloid leukemia), squamous cell cancers, epithelial squamous cell cancers, lung cancers (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cancers), peritoneal cancers, hepatocellular cancers, gastric or stomach cancers (including gastrointestinal cancers), pancreatic cancers, glioblastomas, cervical cancers, ovarian cancers, liver cancers, bladder cancers, hepatomas, breast cancers, colon cancers, rectal cancers, colorectal cancers, endometrial or uterine cancers, salivary gland cancers, kidney or renal cancers, prostate cancers, vulval cancers, thyroid cancers, liver cancers, anal cancers, penile cancers, and head and neck cancers.

In one aspect, a cDAC is provided for use as a medicament. In certain embodiments, provided herein are also cDAC in a method for treating an individual described herein, 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, e.g., a therapeutic agent as described herein.

In a further aspect, also provided herein is the use of a cDAC as described herein in the manufacture or preparation of a medicament. In one aspect, the medicament is for treating cancer, the method comprising administering to an individual having 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, e.g., a therapeutic agent as described herein.

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

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

The disclosed cDAC can be used to treat HER2 positive (her2+) cancers that contain cancer cells with higher than normal levels of HER 2. Examples of HER2 positive cancers include HER2 positive breast cancer and HER2 positive stomach cancer. Optionally, the HER2 positive cancer has an Immunohistochemical (IHC) score of 2+ or 3+ by an In Situ Hybridization (ISH) amplification ratio. The term "HER2 positive cells" refers to cells that express HER2 on their surface. cDAC can also be used to treat HER 2-low tumor types.

Examples

Example 11 Synthesis of- (5-aminopentyl) -1H-pyrrole-2, 5-dione hydrochloride 1

Maleic anhydride, furan-2, 5-dione (150 g,1.53 mol) was added to a stirred solution of 6-aminocaproic acid (201 g,1.53 mol) in HOAc (1000 mL) following the procedure of WO 2017/214024, incorporated herein by reference. 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 H₂ O. The combined organic layers were dried over Na₂SO₄ and concentrated to give the crude product. It was washed with petroleum ether to give 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) are 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 for 8 hours under N₂. The mixture was concentrated and the residue was purified by silica gel column chromatography (PE: etoac=3:1) to give tert-butyl 5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentylcarbamate (13 g, 10%). To a solution of tert-butyl 5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentylcarbamate (28 g,992 mmol) in anhydrous EtOAc (30 mL) was added HCl/EtOAc (50 mL) dropwise. After stirring the mixture at room temperature for 5 hours, it is filtered and the solid is dried to give 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione hydrochloride ,1(16g,73.7％).¹H NMR(400MHz,DMSO-d₆):δ8.02(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).

Example 2 Synthesis of (S) -1- (1- (4- (hydroxymethyl) phenylamino) -1-oxo-5-ureidopentan-2-ylcarbamoyl) cyclobutanecarboxylic acid 2

To a mixture of (S) -2-amino-5-ureidovaleric acid 2a (17.50 g,0.10 mol) in a mixture of dioxane and H₂ O (50 mL/75 mL) was added K₂CO₃ (34.55 g,0.25 mol). Fmoc-Cl (30.96 g,0.12 mol) was slowly added at 0 ℃. The reaction mixture was warmed to room temperature over 2 hours. The organic solvent was removed under reduced pressure and the aqueous slurry was adjusted to ph=3 with 6M 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-ureidopentanoic acid 2b (38.0 g, 95.6%). 2b are commercially available.

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,13mmol,1.3 eq) and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline EEDQ, sigma-ALDRICH CAS Reg. No.16357-59-8 (3.2 g,13mmol,1.3 eq). After the mixture was stirred at room temperature for 16 hours under N₂, it was concentrated to give a brown solid. MTBE (200 mL) was added and stirred at 15℃for 2 hours. The solid was collected by filtration and washed with MTBE (50 ml×2) to give (1- ((4- (hydroxymethyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) carbamic acid (S) - (9H-fluoren-9-yl) methyl ester 2c (4.2 g, 84%) as an orange solid. LCMS (ESI) m/z 503.0[ M+1].

To a stirred solution of 2c (4.2 g,8.3 mmol) in dry DMF (20 ml) was added piperidine (1.65 mL,17mmol,2 eq) dropwise at room temperature. The mixture was stirred at room temperature for 30 minutes, and a solid precipitate formed. Dry DCM (50 mL) was added and the mixture immediately became clear. The mixture was stirred at room temperature for an additional 30 minutes and LCMS showed 10e consumption. It was concentrated to dryness under reduced pressure (to ensure no residual piperidine) and the residue was partitioned between EtOAc and H₂ O (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).

Commercially available 1, 1-diethyl 1, 1-cyclobutanedicarboxylate (CAS Reg. No. 3779-29-1) is converted to the 1-ethyl half-acid/ester (CAS Reg. No. 54450-84-9) by limited saponification with aqueous base and activation of the N-hydroxysuccinimide to the NHS ester cyclobutane-1, 1-dicarboxylic acid 1- (2, 5-dioxopyrrolidin-1-yl) 1-ethyl ester with coupling reagents 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).

To a solution of 1- (2, 5-dioxopyrrolidin-1-yl) 1-ethyl cyclobutane-1, 1-dicarboxylic acid ester (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 stirring the mixture at room temperature for 16 hours, 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-ureidohexane-3-yl) carbamoyl) cyclobutanecarboxylate 2e (6.4 g, 68.7%) as a white solid. LCMS (ESI): M/z435.0[ M+1]

To a stirred solution of 2e (6.4 g,14.7 mmol) in a mixture of THF and MeOH (20 mL/10 mL) was added a solution of LiOH H₂ O (1.2 g,28.6 mmol) in H₂ O (20 mL) at room temperature. After the reaction mixture was stirred at room temperature for 16 hours, the solvent was removed under reduced pressure, and the obtained residue was purified by preparative HPLC to give (S) -1- (1- (4- (hydroxymethyl) phenylamino) -1-oxo-5-ureidopentan-2-ylcarbamoyl) cyclobutanecarboxylic acid 2 (3.5 g, yield: 58.5%). LCMS (ESI) m/z 406.9[ M+1].¹ H NMR (400 MHz, methanol -d₄)δ8.86(d,J＝8.4Hz,2H),8.51(d,J＝8.4Hz,2H),5.88-5.85(m,1H),5.78(s,2H),4.54-4.49(m,3H),4.38-4.32(m,1H),3.86-3.75(m,1H),3.84-3.80(m,2H),3.28-3.21(m,1H),3.30-3.24(m,1H),3.00-2.80(m,1H),2.37-2.28(m,2H).)

Example 3 Synthesis of S) -N- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) -N- (1- (4- (hydroxymethyl) phenylamino) -1-oxo-5-ureidopentan-2-yl) cyclobutane-1, 1-dicarboxamide 3

Diisopropylethylamine, DIPEA (1.59 g,12.3 mmol) and bis (2-oxo-3-oxazolidinyl) phosphinoyl chloride, BOP-Cl (CAS reg. No.68641-49-6, sigma-Aldrich,692mg,2.71 mmol) were added to a solution of (S) -1- (1- (4- (hydroxymethyl) phenylamino) -1-oxo-5-ureidopentan-2-ylcarbamoyl) cyclobutanecarboxylic acid 2 (1 g,2.46 mmol) in DMF (10 mL), followed by 1- (5-aminopentyl) -1H-pyrrole-2, 5-dione hydrochloride 1 (592 mg,2.71 mmol) at 0 ℃. The mixture was stirred at 0 ℃ for 0.5 hours. The reaction mixture was quenched with citric acid solution (10 mL) and extracted with DCM/MeOH (10:1). The organic layer was dried and concentrated, and the residue was purified by silica gel column chromatography (DCM: meoh=10:1) to give 3 (1.0 g, 71%), also referred to as MC-CBDK-cit-PAB-OH.LCMS(ESI)):M+H⁺＝571.28.¹H NMR(400MHz,DMSO-d₆):δ10.00(s,1H),7.82-7.77(m,2H),7.53(d,J＝8.4Hz,2H),7.19(d,J＝8.4Hz,2H),6.96(s,2H),5.95(t,J＝6.4Hz,1H),5.39(s,2H),5.08(t,J＝5.6Hz,1H),4.40-4.35(m,3H),4.09(d,J＝4.8Hz,1H),3.01(d,J＝3.2Hz,2H),3.05-2.72(m,4H),2.68-2.58(m,3H),2.40-2.36(m,4H),1.72-1.70(m,3H),1.44-1.42(m,1H),1.40-1.23(m,6H),1.21-1.16(m,4H).

Example 4 Synthesis of (S) -N- (1- (4- (chloromethyl) phenylamino) -1-oxo-5-ureidopentan-2-yl) -N- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) cyclobutane-1, 1-dicarboxamide 4

A solution of 3 (2.0 g,3.5 mmol) in N, N-dimethylformamide DMF or N-methylpyrrolidone NMP (50 mL) was treated batchwise dropwise with thionyl chloride SOCl₂ (1.25 g,10.5 mmol) at 0 ℃. The reaction was still yellow. The reaction was monitored by LC/MS, indicating a conversion of >90%. After stirring the reaction mixture at 20 ℃ for 30 minutes or 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 min), 0.696 min, m/z=589.0 [ m+1]⁺.

EXAMPLE 54 Synthesis of (S) -4- (2- (1- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentylcarbamoyl) cyclobutanecarboxamido) -5-ureidovalerylamino) benzyl ester 5

Diisopropylethylamine (DIEA) was added to a solution of 3 in anhydrous DMF followed by PNP carbonate (bis (4-nitrophenyl) carbonate). The reaction solution was stirred at room temperature (r.t.) for 4 hours, and the mixture was purified by preparative HPLC to give 5.LCMS (ESI)) m+h⁺ = 736.29.

EXAMPLE 6 Synthesis of 4-nitrophenyl 2- (pyridin-2-yldisulfanyl) ethyl carbonate 6

According to the protocol of WO 2016/040825 incorporated herein, 1, 2-bis (pyridin-2-yl) disulfane and 2-mercaptoethanol are reacted in pyridine and methanol at room temperature to give 2- (pyridin-2-yldisulfanyl) ethanol. Acylation with 4-nitrophenyl chloroformate in triethylamine and acetonitrile gives 4-nitrophenyl 2- (pyridin-2-yldisulfanyl) ethylcarbonate 6.

Example 7 2 Synthesis of- ((5-nitropyridin-2-yl) disulfanyl) ethanamine hydrochloride 7

To a mixture of 1, 2-bis (5-nitropyridin-2-yl) dithioalkane (1.0 g,3.22 mmol) in anhydrous DMF/MeOH (25 mL/25 mL) was added HOAc (0.1 mL) followed by 2-aminoethylthiol hydrochloride (183 mg,1.61 mmol). The reaction mixture was stirred at room temperature overnight, concentrated in vacuo to remove solvent, and the residue was washed with DCM (30 ml×4) to give a pale yellow solid 7(300mg,69.6％).¹H NMR(400MHz,DMSO-d₆)δ9.28(d,J＝2.4Hz,1H),8.56(dd,J＝8.8,2.4Hz,1H),8.24(s,4H),8.03(d,J＝8.8Hz,1H),3.15-3.13(m,2H),3.08-3.06(m,2H)

EXAMPLE 8 Synthesis of 4-nitrophenyl 2- ((5-nitropyridin-2-yl) disulfanyl) ethyl carbonate 8

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/CH₃ OH (250 mL/250 mL) was stirred at room temperature under N₂ for 24 hours. After concentrating the mixture in vacuo, the residue was diluted with DCM (300 mL). Manganese oxide MnO₂ (10 g) was added and the mixture was stirred at room temperature for an additional 0.5 hours. The mixture was purified by silica gel column chromatography (DCM/meoh=100/1 to 100/1) to give 2- ((5-nitropyridin-2-yl) disulfanyl) ethanol as a brown oil (2.2g,61.1％).¹H NMR(400MHz,CDCl₃)δ9.33(d,J＝2.8Hz,1H),8.38-8.35(dd,J＝9.2,2.8Hz,1H),7.67(d,J＝9.2Hz,1H),4.10(t,J＝7.2Hz,1H),3.81-3.76(q,2H),3.01(t,J＝5.2Hz,2H).

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 1.31g,4.31 mmol). The reaction solution was stirred at room temperature for 4 hours, and the mixture was purified by preparative HPLC (FA) to give a light brown oil 8(270mg,33.1％).¹H NMR(400MHz,CDCl₃)δ9.30(d,J＝2.4Hz,1H),8.43-8.40(dd,J＝8.8,2.4Hz,1H),8.30-8.28(m,2H),7.87(d,J＝8.8Hz,1H),7.39-7.37(m,2H),4.56(t,J＝6.4Hz,2H),3.21(t,J＝6.4Hz,2H).

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

To a stirred solution of 1-aminopropan-2-ol (10 g,133 mmol) in MeOH (360 mL) and H₂ O (40 mL) was added Boc₂ O (37 g,169 mmol). The reaction mixture was stirred at room temperature for 5 hours, concentrated and purified by chromatography (EtOAc/pe=10% -50%) to give tert-butyl 2-hydroxypropyl carbamate (19.8 g, yield: 85%) as a colorless oil.

To a stirred solution of tert-butyl 2-hydroxypropyl carbamate (10 g,57 mmol) and Et₃ N (17 g,171 mmol) in DCM (130 mL) was added a solution of MsCl (methanesulfonyl chloride, 13g,114 mmol). The reaction mixture was stirred at room temperature for 4 hours, which was washed with ice water (200 mL x 3) and brine (200 mL). The organic layer was concentrated to give 1- (t-butoxycarbonylamino) propan-2-yl methanesulfonate as a red oil (12 g, yield: 83%).

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, 5.4g,47.3 mmol) in H₂ O (100 mL). The reaction mixture was stirred at 60 ℃ for 12 hours. The mixture was concentrated and extracted with DCM (200 ml x 2). The combined organic layers were concentrated and purified by chromatography to give S-1- (tert-butoxycarbonylamino) propan-2-yl thioacetate as a red solid (1.1 g, yield) ：20％).¹H NMR(400MHz,CDCl3-d)1.30(d,J＝7.09Hz,3H)1.44(s,9H)2.33(s,3H)3.16-3.42(m,2H)3.58-3.71(m,1H)

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 hours, and concentrated to give 1-aminopropane-2-thiolate, which was used directly in the next step.

To a solution of 1, 2-bis (5-nitropyridin-2-yl) dithioalkane (1.33 g,4.3 mmol) in DCM (35 mL) was added a solution of 1-aminopropane-2-thiolate (279 mg,2.15 mmol). The mixture was stirred at 15 ℃ for 12 hours. MnO₂ (374.1 mg,4.3 mmol) was added to the mixture and stirred at 15℃for 10 min. The solid was washed with DCM (100 mL) and MeOH (30 mL. Times.3). The solution was concentrated to give 9 (300 mg, 57%) as a yellow solid. LCMS (ESI)) rt=0.546 minutes, m+h⁺ =245.7.

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

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

Example cD-1 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 Synthesis

Example cD-2 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 Synthesis

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 Synthesis

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 Synthesis

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 Synthesis

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 Synthesis

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 ] azulene-6-carboxamido) heptyl) (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) carbamic acid 4S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutane-1-carboxamide) propionylamino) benzyl ester cDLI-1

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

To a solution of tert-butyl (7-oxoheptyl) carbamate cDLI-1b (636.9 mg,2.78 mmol) and 3- (4-amino-1-oxoisoindolin-2-yl) piperidine-2, 6-dione cDLI-1a (600.00 mg,2.31 mmol) in dry dichloromethane (50 mL) was added acetic acid (0.02 mL,0.26 mmol). The mixture was stirred at 25 ℃ for 2 hours. NaBH (OAc)₃ (1226.2 mg,5.79 mmol) was then added to the mixture and the mixture was stirred at 25℃for 12 hours. TLC (60% etoac in petroleum ether, R_f =0.5) indicated the reaction was complete. 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 a crude product, 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 ]⁺.

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

To a solution of cDLI-1c (300.00 mg,0.63 mmol) and di-tert-butyl dicarbonate Boc₂ O (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 ℃ for 2 hours. TLC (60% etoac in petroleum ether, R_f =0.6) indicated the reaction was complete. The mixture was diluted with dichloromethane (45 mL), and washed with aqueous citric acid (15 mL), water (15 mL), saturated brine (15 mL). The organic layer was concentrated and purified by flash column (eluting with 0-60% etoac in petroleum ether) to give cDLI-1d (300 mg, 83%) as a pale yellow solid.

Preparation of tert-butyl 3- (4- ((((4- ((S) -2- (((allyloxy) carbonyl) amino) propionylamino) benzyl) oxy) carbonyl) (7- ((tert-butoxycarbonyl) amino) heptyl) amino) -1-oxoisoindolin-2-yl) -2, 6-dioxopiperidine-1-carboxylate cDLI-1f

To a mixture of triphosgene (150.00 mg,0.51 mmol) and 4A molecular sieve 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 ℃ for 1 hour. TLC (5% meoh in DCM, r_f =0.6) indicated the reaction was complete. The mixture was concentrated and used directly in the next step. To the crude product (277.27 mg,0.44mmol, theory) was added allyl (S) - (1- ((4- (hydroxymethyl) phenyl) amino) -1-oxopropan-2-yl) carbamate cDLI-1e (243 mg,0.87 mmol) and 4A molecular sieve in dichloromethane (5 mL), and N, N-dimethylformamide DMF (1 mL) was added together with triethylamine (0.18 mL,1.31 mmol) and 4-dimethylaminopyridine DMAP (160.00 mg,1.31 mmol). The mixture was stirred at 35 ℃ for 12 hours. TLC (10% meoh in DCM, r_f =0.5) indicated the reaction was complete. The mixture was filtered and diluted with DCM (50 mL), washed with saturated citric acid (10 mL), saturated brine (10 mL). The organic layer was concentrated in vacuo and purified by flash column (eluting with 0-10% meoh in DCM) to give cDLI-1f (75 mg, 19.6%) as a pale yellow solid. LCMS (10-80, ab,7.0 min): rt=4.491 min, m/z=877.5 [ m+h ]⁺.

Preparation of tert-butyl 3- (4- ((((4- ((S) -2-aminopropionylamino) benzyl) oxy) carbonyl) (7- ((tert-butoxycarbonyl) amino) heptyl) amino) -1-oxoisoindolin-2-yl) -2, 6-dioxopiperidine-1-carboxylate cDLI-1g

To a solution of cDLI-1f (75.00 mg,0.09 mmol) and 1, 3-dimethylpyrimidine-2, 4,6 (1 h,3h,5 h) -trione (also known as1, 3-dimethylbarbituric acid (66.8 mg,0.43 mmol)) in dichloromethane (3 mL) and methanol (3 mL) at 25 ℃ was added Pd (PPh₃)₄ (19.8 mg,0.02 mmol). The reaction mixture was stirred under nitrogen atmosphere at 25 ℃ for 3 hours.tlc (10% meoh in DCM, r_f =0.3) indicated that the reaction was complete the mixture was filtered and the filtrate concentrated to give the crude product which was purified by preparative TLC (10% meoh in DCM) to give cDLI-1g (30 mg, 44.2%) LCMS (5-95, ab,1.5 min): rt=0.883 min [ m/z=793.⁺ ] as a white solid.

Preparation of 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) propionylamino) benzyl) oxy) carbonyl) amino) -1-oxoisoindolin-2-yl) -2, 6-dioxopiperidine-1-carboxylic acid tert-butyl ester cDLI-1i

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 ℃ for 2 hours. TLC (10% meoh in DCM, r_f =0.5) indicated the reaction was complete. The mixture was concentrated and purified by preparative TLC (10% meoh in DCM) to give cDLI-1i (10 mg, 24.4%) as a white solid. LCMS (10-80, ab,7.0 min): rt= 4.728 min, m/z=1083.7 [ m+h ]⁺.

Preparation of 4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutane-1-carboxamido) propionylamino) benzyl (7-aminoheptyl) (2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) carbamate cDLI-1j

To a solution of cDLI-1i (10.00 mg,0.01 mmol) in dichloromethane DCM (1 mL) was added trifluoroacetic acid TFA (0.2 mL,0.13 mmol). The mixture was stirred at 25 ℃ for 1 hour. TLC indicated the reaction was complete. The mixture was concentrated to give cDLI-1j (9.20 mg, 100%) as a pale yellow solid.

CDLI-1 preparation

To a solution of 4- (3, 5-difluoropyridin-2-yl) -10-methyl-7- ((methylsulfonyl) methyl) -11-oxo-3, 4,10, 11-tetrahydro-1H-1, 4, 10-triazadibenzo [ cd, f ] azulene-6-carboxylic acid cDLI-1k (10.0 mg,0.02 mmol) and 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyluronium HATU, CAS 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 ℃ for 5 minutes. cDLI-1j (9.20 mg,0.01 mmol) was then added. The mixture was then stirred at 25 ℃ for 1 hour. LCMS (10-80 AB/7.0 min)): rt=3.840 min, [ m+h ] +1365.3 shows 16% 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 give cDLI-1 (1.90 mg, 13.1%) as a pale yellow solid. LCMS (5-95, ab,1.5 min)): RT (220/254 nm) =0.88 min, m/z=1365.4 [ m+h ]⁺.

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

Preparation of 4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutane-1-carboxamido) -5-ureidopentanoylamino) benzyl ((3- (4- ((7- ((tert-butoxycarbonyl) amino) hept-1-oxoisoindolin-2-yl) -2, 6-dioxopiperidin-1-yl) methyl) carbamate cDLI c

To a solution of diphenyl azide phosphate DPPA (0.05 mL,0.22 mmol), 2- (3- (4- ((7- ((t-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 ℃ for 5 minutes, 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 cDLI b (107.6 mg,0.19 mmol) was added. The mixture was stirred at 90 ℃ for 1 hour. LCMS (10-80, ab/7.0 min): R_T = 3.735 min, m/z = 1098.3[ m+h ]⁺ showed 18% of the desired product. The mixture was then filtered and the filtrate was sent to preparative HPLC (acetonitrile in water 30-60/0.225% fa) to give cDLI c (20 mg, 19.3%) as a pale yellow solid. LCMS (5-95, ab,1.5 min): R_T = 0.909 min, m/z = 1098.7[ m+h ]⁺.

Preparation of 4- ((S) -2- (1- ((5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentyl) carbamoyl) cyclobutane-1-carboxamide) -5-ureidovalerylamino) benzyl ((3- (4- ((7-aminoheptyl) amino) -1-oxoisoindolin-2-yl) -2, 6-dioxopiperidin-1-yl) methyl) carbamate cDLI d

A mixture of cDLI-5C (15.0 mg,0.01 mmol) in 5% trifluoroacetic acid in hexafluoroisopropanol HFIP (1 mL) was stirred at 25℃for 1 hour. The mixture was concentrated to give cDLI-5d (15 mg, 99%) as TFA salt as a white solid. LCMS (5-95, ab,1.5 min): R_T = 0.756 min, m/z = 998.7[ m+h ]⁺.

CDLI-5 preparation

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 ℃ for 5 minutes. 4- (3, 5-difluoropyridin-2-yl) -10-methyl-7- ((methanesulfonyl) methyl) -11-oxo-3, 4,10, 11-tetrahydro-1H-1, 4, 10-triazadibenzo [ cd, f ] azulene-6-carboxylic acid cDLI e (15.0 mg,0.01 mmol) was then added. The mixture was stirred at 25 ℃ for 1 hour. LCMS (5-95 AB/1.5 min): rt=0.870 min, m/z=741.3 [ m/2+H ] + showed 18% of the desired product. The mixture was then filtered and the filtrate was sent to preparative HPLC (acetonitrile in water 30-60/0.225% fa) to give cDLI-5 (6.4 mg, 31.4%) as a white solid. LCMS (5-95, ab,1.5 min): RT (220/254 nm) =0.863 min, m/z=1480.9 [ m+h ]⁺.

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

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

To a solution of 7- ((t-butoxycarbonyl) amino) heptanoic acid (5.0 g,20.38 mmol) in dichloromethane (20 mL) were added EDCI (5.86 g,30.57 mmol) and triethylamine (7.91 mL,61.14 mmol), followed by N, O-dimethylhydroxylamine hydrochloride (0.87 g,8.97 mmol), 4-dimethylaminopyridine (0.10 g,0.82 mmol). The mixture was stirred at 25 ℃ for 16 hours. TLC (50% ethyl acetate in petroleum ether, R_f =0.8) indicated the reaction was complete. The reaction mixture was poured into water (50 mL) and extracted with DCM (50 mL 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 (2 g, 85%) as a colorless oil. 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＝6.0Hz,2H),2.41(t,J＝7.2Hz,2H),1.70-1.59(m,2H),1.52-1.42(m,11H),1.39-1.32(m,4H)

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

To a solution of cDLI-6a (500.00 mg,1.73 mmol) in tetrahydrofuran (12 mL) was added lithium aluminum hydride LiAlH₄ (98.70 mg,2.60 mmol) at-78 ℃. The mixture was warmed to 0 ℃ and stirred at that temperature for 30 minutes. TLC (30% etoac in petroleum ether, r_f =0.5) indicated the reaction was complete. Saturated aqueous NH₄ Cl was slowly added with stirring at 0 ℃ and the mixture was filtered and extracted with ethyl acetate (30 ml x 3). The organic layer was washed with H₂ O (20 mL) and brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give cDLI-6b (390.00 mg, 98%) as a colorless oil, which was used directly in the next step.

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

To a solution of 3- (4-amino-1-oxoisoindolin-2-yl) piperidine-2, 6-dione, lenalidomide (CAS 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 ℃ for 4 hours. Sodium triacetoxyborohydride STAB, naBH (OAc)₃ (735.73 mg,3.47 mmol) was then added to the mixture and stirred at 25℃for 12 hours. 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, and 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 ]⁺.

Preparation of tert-butyl 2- (3- (4- ((7- ((tert-butoxycarbonyl) amino) heptyl) amino) -1-oxoisoindolin-2-yl) -2, 6-dioxopiperidin-1-yl) acetate cDLI-6e

To a mixture of cDLI-6d (370.00 mg,0.78 mmol) in N, N-dimethylformamide (20 mL) was added K₂CO₃ (173.2 mg,1.25 mmol) and tert-butyl-bromoacetate (0.15 mL,1.02 mmol), and the mixture was stirred at 25℃for 2 h. TLC (10% meoh in DCM, r_f =0.8) indicated the reaction was complete. The mixture was filtered, etOAc (60 mL) was added and washed with water (20 mL). The organic layer was dried over Na₂SO₄, filtered and concentrated to give the crude product, which was purified by silica chromatography eluting with 0-1.5% meoh in DCM to give cDLI-6e (320.00 mg, 70%) as a white solid. LCMS (5-95, ab,1.5 min): rt=0.990 min, m/z=587.4 [ m+h ]⁺.

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

To a mixture of cDLI-6e (320 mg,0.55 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (3.0 mL,38.94 mmol) and the mixture was stirred at 25℃for 2 h. LCMS (5-95 AB/1.5 min): rt=0.705 min, m/z=431.2 [ m+h ] + showed 70% of the desired product. The mixture was concentrated to give cDLI-6f (296.00 mg, 99.7%) as a yellow oil.

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

To a solution of cDLI-6f (296.00 mg,0.54 mmol) in methanol (5 mL) at 25℃were added Boc₂ O (0.19 mL,0.82 mmol) and triethylamine Et₃ N (0.23 mL,1.63 mmol) and the mixture was stirred at 25℃for 12 h. LCMS (5-95 AB/1.5 min): rt=0.903 min, m/z=531.3 [ m+h ] + showed 70% of the desired product. The mixture was concentrated and purified by reverse phase chromatography (Xtimate(WELCH MATERIALS, inc.) 150 x 25mm x 5um, acetonitrile 60-80.6/0.225% fa in water) to yield cDLI-6g (110 mg, 38%) as a white solid. LCMS (5-95, ab,1.5 min): rt=0.895 min, m/z=553.3 [ m+na ]⁺.

Preparation of S- (1- ((((3- (4- ((7- ((tert-butoxycarbonyl) amino) heptyl) amino) -1-oxoisoindolin-2-yl) -2, 6-dioxopiperidin-1-yl) methyl) carbamoyl) oxy) -2-methylpropan-2-yl) methylthiosulfonate cDLI-6h

To a solution of cDLI-6h (70.00 mg,0.13 mmol) and diphenyl azide phosphate DPPA (0.03 mL,0.13 mmol) in toluene (5 mL) was added S- (1-hydroxy-2-methylpropan-2-yl) methylthiosulfonate (48.6 mg,0.26 mmol) followed by triethylamine (0.03 mL,0.20 mmol). The mixture was stirred at 25 ℃ for 10 minutes and heated to 90 ℃ under an atmosphere of N₂ for 3 hours. TLC (10% meoh in DCM, r_f =0.6) indicated the reaction was complete. The mixture was filtered and the organic layer was concentrated in vacuo. The resulting mixture was diluted with EtOAc (40 mL) and washed with water (20 mL), the organic layer was dried over Na₂SO₄, concentrated and purified by preparative TLC (10% meoh in DCM, r_f =0.6) to give cDLI-6h (40 mg, 43%) as a white solid. LCMS (5-95, ab,1.5 min): rt=0.958 min, m/z=734.4 [ m+h ]⁺.

Preparation of S- (1- ((((3- (4- ((7-aminoheptyl) amino) -1-oxoisoindolin-2-yl) -2, 6-dioxopiperidin-1-yl) methyl) carbamoyl) oxy) -2-methylpropan-2-yl) methylthio sulfonate cDLI-6i

To a mixture of cDLI-6h (32.00 mg,0.04 mmol) in dichloromethane (0.50 mL) was added trifluoroacetic acid (0.5 mL,6.49 mmol) and the mixture was stirred at 25℃for 1 h. The mixture was concentrated to give cDLI-6i TFA salt as a yellow solid (32 mg, 98.1%). LCMS (5-95, ab,1.5 min): rt=0.758 min, m/z=612.3 [ m+h ]⁺.

Preparation of 4- (3, 5-difluoropyridin-2-yl) -10-methyl-7- ((methylsulfonyl) methyl) -11-oxo-3, 4,10, 11-tetrahydro-1H-1, 4, 10-triazadibenzo [ cd, f ] azulene-6-carboxylic acid cDLI-6j

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

CDLI-6 preparation

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 ℃ for 5 minutes. cDLI-6i 3 (32.0 mg,0.04 mmol) was then added. The mixture was stirred at 25 ℃ for 1 hour. The mixture was then filtered and the filtrate was sent to preparative HPLC (acetonitrile in water 30-60/0.225% fa) to give cDLI-6 (6.1 mg, 12%) as a pale yellow solid. LCMS (5-95, ab,1.5 min): RT (220/254 nm) =0.893 min, m/z=1094.5 [ m+h ]⁺.

Example 101 preparation of cysteine engineered antibodies

For large scale antibody production, antibodies were produced in CHO cells. Vectors encoding VL and VH were transfected into CHO cells and IgG was purified from the cell culture medium by protein affinity chromatography.

As originally isolated, the engineered cysteine residues in antibodies exist as mixed disulfides with cellular thiols (e.g., glutathione) and are therefore not available for conjugation. Partial reduction (e.g., with DTT), purification and reoxidation of these antibodies with dehydroascorbic acid (DHAA) yields antibodies with free cysteine sulfhydryl groups available for conjugation as previously described (Junutula et al (2008) Nat. Biotechnol.26:925-932; U.S. Pat. No. 1/0301334). Briefly, antibodies were combined with the cereblon degradant-linker intermediate to allow for the most conjugation to the free cysteine residues of the antibody. After a few hours, the cereblon degradant antibody conjugate was purified.

Under certain conditions, 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) was reduced with a 50-fold excess of DTT at room temperature to reduce disulfide bonds that may form between newly introduced cysteine residues and cysteine present in the medium (Getz et al, (1999) Anal. Biochem.273:73-80;Soltec Ventures,Beverly,MA) to conjugation with the cereblon degradation agent-linker intermediate, the full length cysteine engineered monoclonal antibody expressed in CHO cells (THIOMAB^TM) (Gomez et al, (2010) Biotechnology and bioeng.105 (4): 748-760; gomez et al, (2010) Biotechnol. Prog.26: 1438-1445) was reduced with a 50-fold excess of DTT at room temperature, and the resulting eluate was loaded onto a column of 10mM sodium acetate, pH7.5, pH 10mM buffer (pH 7.5) was eluted with 10mM buffer, 10mM buffer (10.10 mM buffer pH 10, pH 10) and the volume eluted with the buffer.

The light chain amino acids are numbered according to Kabat (Kabat et al Sequences of proteins of immunological interest, (1991) 5th edition, USDept of HEALTH AND Human Service, national Institutes of Health, bethesda, md.). Heavy chain amino acids are numbered according to the EU numbering system (Edelman et al (1969) Proc. Natl. Acad. Of Sci.63 (1): 78-85), unless noted as the Kabat system. Single letter amino acid abbreviations are used.

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

By performing reoxidation, disulfide bonds are reestablished between cysteine residues present in the parent Mab. Eluted reduced THIOMAB^TM was treated with 15X or 2mM dehydroascorbic acid (dhAA) in 50mM Tris-Cl (pH 7.5) for about 3 hours or about 3 hours at pH 7 or 200nM to 2mM copper sulfate in water (CuSO₄) overnight at room temperature. Other oxidizing agents (i.e., oxidizing agents) and oxidizing conditions known in the art may be used. Ambient air oxidation may also be effective. This mild partial reoxidation step effectively forms intrachain disulfide bonds with high fidelity. The buffer was exchanged by elution through Sephadex G25 resin and eluted with 1mM DTPA in PBS. Thiol/antibody values were checked by measuring the reduced antibody concentration and thiol concentration from absorbance at 280nm of the solution by reaction with DTNB (Aldrich, milwaukee, WI) and measuring absorbance at 412 nm.

Liquid chromatography/mass spectrometry was performed on a TSQ Quantum Triple quadrupole^TM mass spectrometer (Thermo Electron, san Jose California) with an extended mass range. The sample was heated to 75 °cChromatographic separation was performed on 1000A microwell columns (50 mm. Times. 2.1mm,Polymer Laboratories,Shropshire,UK). 30% -40% B (solvent A: 0.05% TFA in water, solvent B: acetonitrile containing 0.04% TFA) can be used to directly ionize the eluate using an electrospray source. Data byData system collection and use(Novatia, LLC, new Jersey) deconvolution was performed. Prior to LC/MS analysis, the antibody or conjugate (50 micrograms) was treated with PNGase F (2 units/ml; PROzyme, san Leandro, calif.) for 2 hours at 37℃to remove the N-linked carbohydrate.

A Hydrophobic Interaction Chromatography (HIC) sample was injected onto a butyl HIC NPR column (2.5 micron particle size, 4.6 mM. Times.3.5 cm) (Tosoh Bioscience) and dissolved with a linear gradient of 0 to 70% at 0.8 ml/min (A: 1.5M ammonium sulfate in 50mM potassium phosphate, pH 7, B:50mM potassium phosphate pH 7,20% isopropyl alcohol). An Agilent 1100 series HPLC system equipped with a multi-wavelength detector and Chemstation software was used to resolve and quantify the antibody species with different drug ratios for each antibody.

EXAMPLE 102 conjugation of cereblon degrading agent-linker intermediate (cDLI) and antibody

Following the reduction and reoxidation procedure of example 101, cysteine engineered antibodies (THIOMAB^TM) were dissolved in PBS (phosphate buffered saline) buffer and frozen on ice. An excess (about 1.5 molar to 20 equivalents) of the cereblon degradation agent-linker intermediate activated with thiol-reactive groups (such as pyridyl disulfide, maleimide or bromoacetamide) was dissolved in DMSO, diluted in acetonitrile and water, and added to the frozen, reduced and reoxidized antibodies in PBS. Typically, the cereblon degradant-linker intermediate in DMSO stock in 50mM Tris at a concentration of about 20mM, pH 8, is added to the antibody and monitored until the reaction is complete for about 1 to about 24 hours, as determined by LC-MS analysis of the reaction mixture. When the reaction is complete, an excess of capping reagent such as ethylmaleimide is added to quench the reaction and cap any unreacted antibody thiol groups. The conjugate mixture may be loaded and eluted through HITRAP SP FF columns to remove excess drug and other impurities. The reaction mixture was concentrated by centrifugal ultrafiltration and the resulting cysteine engineered cereblon degradant antibody conjugate (cDAC) was purified and desalted by elution with G25 resin in PBS, filtered through 0.2 μm filter under sterile conditions, and stored frozen.

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

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

LC-MS analysis can be performed using an Agilent QTOF 6520ESI instrument. For example, cDAC was treated with Tris (pH 7.5) containing 1:500w/w intracellular protease Lys C (Promega) at 37℃for 30 min. Loading the resulting cleaved fragments to a temperature of 80℃C(Angstrom), 8 μm (micrometer) PLRP-S (highly crosslinked polystyrene) column and eluted with a gradient of 30% B to 40% B in 5 minutes. Mobile phase a was H₂ O with 0.05% tfa and mobile phase B was acetonitrile with 0.04% tfa. The flow rate was 0.5 ml/min. Protein elution was monitored by UV absorbance detection at 280nm prior to electrospray ionization and MS analysis. Chromatographic resolution of unconjugated Fc fragments, residual unconjugated Fab, and pharmaceutical Fab can generally be achieved. The obtained m/z spectra were deconvolved with Mass Hunter^TM software (Agilent Technologies) to calculate the Mass of the antibody fragments.

Example 103 in vitro cell proliferation assay

Efficacy of the cDAC was measured by a cell proliferation assay using the following protocol (CELLTITER GLO^TMLuminescent Cell Viability Assay,Promega Corp.Technical Bulletin TB288;Mendoza et al (2002) Cancer Res.62:5485-5488:

1. An aliquot of 40. Mu.l of cell culture containing about 4000 cells (HER-expressing SK-BR-3, KPL-4, CAMA1, EFM19, MV-4-11, EOL-1, molm-13, nomo-1, HL-60 and OCI-AML-2) was deposited in each well of a 384 well opaque wall plate.

2. Control wells containing medium and no cells were prepared.

3. CDAC (n=3) was added to the experimental wells and incubated for 3 to 5 days.

4. The plates were equilibrated to room temperature for about 30 minutes.

5. CELLTITER GLO^TM reagent volumes were added equal to the volume of cell culture medium present in each well.

6. The contents were mixed on an orbital shaker for 15 minutes to induce cell lysis.

7. Plates were incubated for 5 minutes at room temperature to stabilize the luminescence signal.

8. Luminescence was recorded and reported graphically as% activity, with RLU (relative luminescence units) normalized to control (no antibody control minus no cell control).

The data are plotted and illustrated in fig. 1, 2A-2B, 3A-3B, 4, 5A-5B, and 6A-6B as separate points for each repeat (n=3) of each antibody. The protocol is a modification of CELLTITER GLO^TM luminous cells. Cell lines can be grown in medium containing RPMI-1640, 20% HI-FBS, 2mM L-glutamine.

EXAMPLE 104 Whole blood stability determination

Whole blood incubation the matrix (carried by the supplier (BioIVT, westbury N.Y.) collected in a tube containing heparin lithium. Unfrozen plasma and whole blood were collected in the afternoon, cold transported (2-8 ℃) overnight to arrive within 18 hours after collection, while frozen plasma was collected and frozen transported under normal delivery conditions. The cDAC source material was formulated as 1mg/mL buffer (1 XPBS [ pH 7.4],0.5% bovine serum albumin, 15ppm Proclin^TM) and then further diluted to a final concentration of 100 μg/mL. After mixing, 150 μl of whole blood/buffer stable samples were aliquoted into two separate sets of tubes at two different time points. The 0 hour time point was then placed at-80 ℃. 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 a-80 ℃ refrigerator and simultaneously shaken in a 37 ℃ incubator for 24 hours (about 700 rpm). Aliquots were collected after 24 hours storage in a-80 ℃ refrigerator until affinity capture LC-MS was performed. Matrices used to generate samples were mice (CB 17 SCID), rats (Sprague-Dawley), monkeys (cynomolgus monkey) and humans.

In vitro stability sample analysis magnetic beads coated with Streptomycin (SA) (Thermo FISHER SCIENTIFIC, cat# 60210) were washed 2 times with HBS-EP buffer (GE HEALTHCARE LIFE SCIENCES, cat# BR-1001-88) and then mixed with biotinylated extracellular domain of the target (e.g. human HER 2) or anti-idiotype antibody for specific capture or biotinylated human IgG for universal capture using KINGFISHER FLEX (Thermo FISHER SCIENTIFIC) and incubated for 2 hours at room temperature with gentle agitation. The SA-bead/biotin capture probe complexes were then washed 2 times with HBS-EP buffer, mixed with cDAC or precursor stable samples pre-diluted 1:16 with HBS-EP buffer, and incubated for 2 hours at room temperature with gentle agitation. After 2 hours, the SA-bead/biotin capture probe/sample complex was washed 2 times with HBS-EP buffer and then deglycosylated via incubation with PNGase F (NEW ENGLAND Biolabs, cat# P0704B) overnight. The SA-bead/biotin capture probe/sample complex was then washed 2 times with HBS-EP buffer, followed by 2 water washes (Optima^TM LC/MS Grade, FISHER CHEMICAL, catalog #W6-1), and finally 1 time with 10% acetonitrile. The beads were placed in 30% acetonitrile/0.1% formic acid to elute for 30 minutes at room temperature with gentle stirring, and then the beads were collected. The eluted samples were then loaded onto LC-MS (Thermo SCIENTIFIC Q-Exactive Plus) for analysis. 10 μl of cDAC sample was injected and loaded onto a Waters C4 column (1000 μm 10 cm) maintained at 65deg.C. cDAC was isolated on the column at a flow rate of 20. Mu.L/min using Waters Acquity UPLC system, with a gradient of 20% B (100% acetonitrile+0.1% formic acid) at 0-2 min, 35% B at 2.5 min, 65% B at 5min, 95% B at 5.5 min, 5% B at 6 min. The column was used directly for on-line detection with a Thermo SCIENTIFIC Q-Exactive Plus mass spectrometer operating in positive electrospray ionization mode with a collection mass in the range of m/z 500 to 4000Da.

EXAMPLE 105 in vivo efficacy of tumor growth inhibition in CD 33-expressing HL-60 mice

Tumors were established and allowed to grow to a volume of 150-200mm³ (as measured using calipers) in CD33 expressing HL-60 mice prior to a single treatment on day 0. Tumor volumes were measured using calipers according to the following formula V (mm³)＝0.5A X B², where A and B are the long and short diameters, respectively.) mice were euthanized before tumor volumes reached 3000mm³ or when tumors showed evidence of impending ulcers.

Alternatively, fo5 mouse mammary tumor models were used to assess in vivo efficacy of anti-HER 2 cereblon degradant antibody conjugates (cDACs) following single dose 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 human HER2 gene was overexpressed in the mammary epithelium HER2 overexpression resulted in spontaneous development of mammary tumors of one of these founder animals (founder #5[ Fo5 ]) were propagated in FVB mice of the next few generations by successive transplantation of tumor fragments (about 2X 2mm in size).

Other breast fat pad implantation efficacy models can be used as described (Chen et al (2007) Cancer res.67:4924-4932), tumor volumes following a single intravenous administration can be assessed, and tumor resected from intraperitoneal tumor-bearing mice can be used, followed by continuous transfer into the recipient mouse's breast fat pad.

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

Claims (126)

1.A cereblon degrading agent antibody conjugate having the structure of formula I-A,

Wherein the method comprises the steps of

Ab is an antibody;

l_1a is an antibody linker;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₃-C₂₀ heterocyclyl, and C₃-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are each independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-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, wherein phenyl and benzyl are optionally substituted with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and-C₂-C₁₂ alkynyl;

Wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, 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)₃ H;

cD_a is the remainder of the cereblon degradant portion, and

P is an integer of 1 to 14.

2. The cereblon degradant antibody conjugate of claim 1, wherein Z¹ is CR¹ and Z² is CR², and wherein R¹ and R² are each H.

3. The cereblon degradant antibody conjugate of any one of claims 1 and 2, wherein ring a is C₃-C₂₀ heteroaryl.

4. The cereblon degradant antibody conjugate of claim 3, wherein ring a is an isoindoline substituted with =o.

5. The cereblon degradant antibody conjugate of any one of claims 1 to 4, having the structure of formula I-a':

Wherein X¹ is selected from CH₂ and C (=o).

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

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

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

9. The cereblon degradant antibody conjugate of claim 8, wherein the cysteine engineered antibody comprises a cysteine mutation selected from the group consisting of HC a118C, LC K149C, HC a140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

10. The cereblon degradant antibody conjugate of any one of claims 1 to 9, L_1a is a protease-cleavable non-peptide linker.

11. The cereblon degradant antibody conjugate of any one of claims 1 to 10, wherein L_1a has the structure of formula L₁ -a:

Wherein the method comprises the steps of

* Indicating the point of attachment to the cysteine thiol of the Ab;

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=O), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂ and C₁-C₁₂ alkylene-NHC (=O) CH₂ CH (thiophen-3-yl), wherein

R is an integer in the range of 1 to 10, and

C₁-C₁₂ 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)₃ H;

R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

12. The cereblon degradant antibody conjugate of 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 degradant antibody conjugate of claim 11, wherein R¹ is C₅ alkylene.

14. The cereblon degradant antibody conjugate of any one of claims 11 to 13, wherein R² and R³ together form a C₄ cycloalkyl ring.

15. The cereblon degradant antibody conjugate of any one of claims 11 to 14, wherein AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

16. The cereblon degradant antibody conjugate of claim 11, wherein

R¹ is C₅ alkylene;

R² and R³ together form a C₄ cycloalkyl ring, and

AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

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

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

19. A cereblon degrading agent antibody conjugate having the structure of formula I-B,

Wherein the method comprises the steps of

Ab is an antibody;

l_1a is an antibody linker;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl, 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, and C₂-C₁₂ alkynyl;

Wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, 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)₃ H;

cD_a is the remainder of the cereblon degradant portion, and

P is an integer of 1 to 14.

20. The cereblon degradant antibody conjugate of claim 19, wherein Z¹ is CR¹ and Z² is CR², and wherein R¹ and R² are each H.

21. The cereblon degradant antibody conjugate of any one of claims 19 and 20, wherein ring a is C₃-C₂₀ heteroaryl.

22. The cereblon degradant antibody conjugate of claim 21, wherein ring a is an isoindoline substituted with =o.

23. The cereblon degradant antibody conjugate of any one of claims 19 to 22, having the structure of formula I-B':

Wherein X¹ is selected from CH₂ and C (=o).

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

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

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

27. The cereblon degradant antibody conjugate of claim 26, wherein the cysteine engineered antibody comprises a cysteine mutation selected from the group consisting of HC a118C, LC K149C, HC a140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

28. The cereblon degradant antibody conjugate of any one of claims 19 to 27, L_1a being a protease-cleavable non-peptide linker.

29. The cereblon degradant antibody conjugate of any one of claims 19 to 28, wherein L_1a has the structure of formula L₁ -a:

Wherein the method comprises the steps of

* Indicating the point of attachment to the cysteine thiol of the Ab;

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=O), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂ and C₁-C₁₂ alkylene-NHC (=O) CH₂ CH (thiophen-3-yl), wherein

R is an integer in the range of 1 to 10, and

C₁-C₁₂ 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)₃ H;

R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

30. The cereblon degradant antibody conjugate of 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 degradant antibody conjugate of claim 29, wherein R¹ is C₅ alkylene.

32. The cereblon degradant antibody conjugate of any one of claims 29 to 31, wherein R² and R³ together form a C₄ cycloalkyl ring.

33. The cereblon degradant antibody conjugate of any one of claims 29 to 32, wherein AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

34. The cereblon degradant antibody conjugate of claim 29, wherein

R¹ is C₅ alkylene;

R² and R³ together form a C₄ cycloalkyl ring, and

AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

35. The cereblon degradant antibody conjugate of any one of claims 19 to 34, wherein cDa comprises (i) a target protein ligand covalently linked to a degradant linker, or (ii) a molecular gel moiety.

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

37. A cereblon degrading agent antibody conjugate having the structure of formula I-C,

Wherein the method comprises the steps of

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H;

Ring a is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

Dotted lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N and NR^1a, and

Z² is selected from C (R²)₂、CR², N and NR^2a, wherein

R¹ and R² are each independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H, or

(I) Two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl;

Wherein each alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl, 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)₃ H;

cD_a is the remainder of the cereblon degradant portion, and

P is an integer of 1 to 14.

38. The cereblon degradant antibody conjugate of claim 37, wherein sulfur is conjugated to the cysteine thiol of the Ab to form a disulfide bond.

39. The cereblon degradant antibody conjugate of claim 37, wherein Z¹ is CR¹ and Z² is CR², and wherein R¹ and R² are each H.

40. The cereblon degradant antibody conjugate of any one of claims 37 to 39, wherein ring a is C₃-C₂₀ heteroaryl.

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

42. The cereblon degradant antibody conjugate of any one of claims 37 to 41, which has the structure of formula I-C:

Wherein X¹ is selected from CH₂ and C (=o).

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

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

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

46. The cereblon degradant antibody conjugate of claim 45, wherein the cysteine-engineered antibody comprises a cysteine mutation selected from the group consisting of HC a118C, LC K149C, HC a140C, LC V205C, LC S121C, HC L174C, HC L177C, and HC Y373C.

47. The cereblon degradant antibody conjugate of any one of claims 37 to 46, L_1a being a protease-cleavable non-peptide linker.

48. The cereblon degradant antibody conjugate of any one of claims 37 to 47, wherein L_1a has the structure of formula L₁ -a:

Wherein the method comprises the steps of

* Indicating the point of attachment to the cysteine thiol of the Ab;

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=O), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂, and C₁-C₁₂ alkylene-NHC (=O) CH₂ CH (thiophen-3-yl), wherein R is an integer ranging from 1 to 10;

C₁-C₁₂ 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)₃ H;

R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

49. The cereblon degradant antibody conjugate of 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 degradant antibody conjugate of claim 48, wherein R¹ is C₅ alkylene.

51. The cereblon degradant antibody conjugate of any one of claims 48 to 50, wherein R² and R³ together form a C₄ cycloalkyl ring.

52. The cereblon degradant antibody conjugate of any one of claims 48 to 51, wherein AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

53. The cereblon degradant antibody conjugate of claim 48, wherein

R¹ is C₅ alkylene;

R² and R³ together form a C₄ cycloalkyl ring, and

AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

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

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

56. A cereblon degrading agent antibody conjugate comprising a cereblon degrading agent moiety covalently linked to an antibody through an antibody linker,

Wherein the cereblon degrading agent moiety is (a) a target protein ligand covalently linked to a cereblon-bound E3 ubiquitin ligase ligand through a degrading agent linker, or (b) a molecular gel, and

The antibody is a thiol-containing antibody.

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

58. The cereblon degradant antibody conjugate of claim 57, wherein the tumor associated antigen or cell surface receptor is selected from the group consisting of:

(1) BMPR1B (bone morphogenic protein IB type receptor);

(2)E16(LAT1、SLC7A5);

(3) STEAP1 (prostate six transmembrane epithelial antigen);

(4)MUC16(0772P、CA125);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiator, mesothelin);

(6) Napi2b (NAPI-3B, NPTIIb, SLC A2, solute carrier family 34 (sodium phosphate) member 2, sodium-dependent phosphate transporter type II 3 b);

(7) Sema5B (FLJ 10372, KIAA1445, mm.42015, sema5B, SEMAG, sema5B Hlog, sema domain, heptathrombospondin repeat (type 1 and type 1), transmembrane domain (TM) and short cytoplasmic domain, (Sema) 5B);

(8) PSCA hlg (2700050C 12Rik, C530008O16Rik, RIKEN CDNA 2700050C12, RIKEN CDNA 2700050C12 genes);

(9) ETBR (endothelin B receptor);

(10) MSG783 (RNF 124, hypothetical protein FLJ 20315);

(11) STEAP2 (hgnc_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer related gene 1, prostate cancer related protein 1, prostate six transmembrane epithelial antigen 2, six transmembrane prostate protein);

(12) TrpM4 (BR 22450, FLJ20041, trpM4B, transient receptor potential cation channel, subfamily M member 4);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF, teratoma-derived growth factor);

(14) CD21 (CR 2 (complement receptor 2) or C3DR (C3 d/Epstein Barr virus receptor) or Hs 73792);

(15) CD79B (CD 79B, CD79 9β, IGb (immunoglobulin related β), B29);

(16) FcRH2 (IFGP 4, IRTA4, SPAP1A (SH 2 domain-containing phosphatase anchor 1A), SPAP1B, SPAP C);

(17)HER2;

(18)NCA;

(19)MDP;

(20)IL20Rα;

(21) Short proteoglycans;

(22)EphB2R;

(23)ASLG659;

(24)PSCA;

(25)GEDA;

(26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR 3);

(27) CD22 (B cell receptor CD22-B isoform);

(28) CD79a (CD 79A, CD79a, immunoglobulin-related a);

(29) CXCR5 (Burkitt lymphoma receptor 1);

(30) HLA-DOB (beta subunit of MHC class II molecule (Ia antigen));

(31) P2X5 (purinergic receptor P2X ligand-gated ion channel 5);

(32) CD72 (B cell differentiation antigen CD72, lyb-2);

(33) LY64 (lymphocyte antigen 64 (RP 105), leucine-rich repeat (LRR) family of type I membrane proteins);

(34) FcRH1 (Fc receptor-like protein 1);

(35) FcRH5 (IRTA 2, immunoglobulin superfamily receptor translocation related 2);

(36) TENB2 (assuming transmembrane proteoglycans);

(37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL);

(38) TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; brain tumor oncostatin-1);

(39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha 1; GFR-alpha 1);

(40) Ly6E (lymphocyte antigen 6 complex gene locus E; ly67, RIG-E, SCA-2, TSA-1);

(41) TMEM46 (SHISA homolog 2 (Xenopus); SHISA 2);

(42) Ly6G6D (lymphocyte antigen 6 complex gene locus G6D; ly6-D, MEGT 1);

(43) LGR5 (G-protein coupled receptor 5 containing leucine-rich repeats; GPR49, GPR 67);

(44) RET (RET protooncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF; hs.168714; RET51; RET-ELE 1);

(45) LY6K (lymphocyte antigen 6 complex gene locus K; LY6K; HSJ001348; FLJ 35226);

(46) GPR19 (G protein coupled receptor 19; mm.4787);

(47) GPR54 (KISS 1 receptor; KISS1R; GPR54; HOT7T175; AXOR 12);

(48) ASPHD1 (aspartic acid containing β -hydroxylase domain 1; loc253982);

(49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP 3);

(50) TMEM118 (cyclophilin, transmembrane 2; RNFT2; FLJ 14627);

(51) GPR172A (G protein coupled receptor 172A; GPCR41; FLJ11856;

D15Ertd747e);

(52)CD33;

(53)CLL-1;

(54) TROP2, and

(55)CD123。

59. The cereblon degradant antibody conjugate of claim 58, wherein the tumor associated antigen or cell surface receptor is HER2 or CD33.

60. The cereblon degradant antibody conjugate of any one of claims 56 to 59, having 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 degradation agent part, and

P is an integer of 1 to 14.

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

62. The cereblon degradant antibody conjugate of claim 61, wherein the cysteine-engineered antibody comprises a cysteine mutation selected from the group consisting of a118C, LCK149C, HC a140C, LC V205C, LC S121C, HC L174C, HC L177C, HC Y373C.

63. The cereblon degradant antibody conjugate of claim 60, wherein the linkage of L₁ to cD comprises an aminal group.

64. The cereblon degradant antibody conjugate of claim 60, wherein the linkage of L₁ to cD comprises a carbamate (-OC (O) NH-) or methylcarbamate (-OC (O) NHCH₂ -) group.

65. The cereblon degradant antibody conjugate of claim 60, wherein L₁ is a protease-cleavable non-peptide linker having the formula:

-Str-PM-IM-

wherein Str is an extender unit covalently linked to the antibody, PM is a peptidomimetic unit, and IM is a sacrificial unit covalently linked to the cereblon degrading agent moiety.

66. The cereblon degradant antibody conjugate of claim 65, wherein Str has the formula:

wherein indicates the point of attachment of the succinimidyl ring to the cysteine thiol of the antibody, and

R¹ is selected from the group consisting of C₁-C₁₂ alkylene, C₁-C₁₂ alkylene-C (=o), C₁-C₁₂ alkylene-NH, (CH₂CH₂O)_r、C₁-C₁₂ alkylene -NH、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-C(＝O)、(CH₂CH₂O)_r-CH₂ and C₁-C₁₂ alkylene-NHC (=o) CH₂ CH (thiophen-3-yl), wherein R is an integer ranging from 1 to 10, and C₁-C₁₂ alkylene is optionally substituted ：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)₃ H with one or more groups selected from.

67. The cereblon degradant antibody conjugate of claim 66, wherein R¹ is (CH₂)₅.

68. The cereblon degradant antibody conjugate of any one of claims 65 to 67, wherein PM has the formula:

Wherein R² and R³ together form a C₃-C₇ cycloalkyl ring ：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)₃ H optionally substituted with one or more groups selected from

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

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

71. The cereblon degradant antibody conjugate of claim 65, having the formula:

wherein cD is the cereblon degradant moiety.

72. The cereblon degradant antibody conjugate of claim 71, which is selected from the group consisting of:

73. the cereblon degradant antibody conjugate of claim 71, having the formula:

74. The cereblon degradant antibody conjugate of claim 73, which is selected from the group consisting of:

75. the cereblon degradant antibody conjugate of claim 74, which is selected from the group consisting of:

76. The cereblon degradant antibody conjugate of claim 60, wherein L₁ forms a disulfide bond with a cysteine thiol of the antibody.

77. The cereblon degradant antibody conjugate of claim 60, wherein L₁ is selected from the group consisting of:

Wherein indicates that sulphur is conjugated to a 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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H;

R⁶ is selected from H and C₁-C₆ alkyl, and

The wavy line indicates the attachment to the cereblon degradant moiety.

78. The cereblon degradant antibody conjugate of claim 77, wherein R^4a and R^4b are-CH₃,R^5a and R^5b are H, and R⁶ is H.

79. The cereblon degradant antibody conjugate of claim 60, wherein L₁ is a linker having the formula:

wherein the point of attachment to the cysteine thiol of the antibody is indicated,

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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl, wherein 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)₃ H;

the wavy line indicates the attachment to the cereblon degradant moiety.

80. The cereblon degradant antibody conjugate of claim 60, wherein the cereblon degradant moiety has the formula:

TPL-L₂-E3UL

Wherein:

TPL is a target protein ligand;

e3UL is a cereblon-bound E3 ubiquitin ligase ligand;

l₂ is a degrading agent linker, and

One of TPL, E3UL and L₂ is connected to L₁, or

The cereblon degrading agent part is molecular gel.

81. The cereblon degradant antibody conjugate of claim 80, wherein TPL has the formula:

Wherein the method comprises the steps of

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

The wavy line indicates the connection point of L₂.

82. The cereblon degradant antibody conjugate of claim 80, wherein TPL has the formula:

wherein the wavy line indicates the point of connection of L₂.

83. The cereblon degradant antibody conjugate of claim 80, wherein TPL has the formula:

wherein the wavy line indicates the point of connection of L₂.

84. The cereblon degradant antibody conjugate of claim 80, wherein TPL targets BRD4, GSPT1, BET, BRM (SMARCA 2), KRAS, and SHP2.

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

86. The cereblon degradant antibody conjugate of claim 80, wherein E3UL is selected from the group consisting of:

Wherein X¹ is selected from CH₂ and C (=O), and the wavy line indicates the point of attachment of L₁ or L₂.

87. The cereblon degradant antibody conjugate of claim 80, having the formula:

wherein L₁ is a protease-cleavable non-peptide linker having the 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 the glutarimide group of E3UL and has the formula:

Wherein the wavy line is a connection to the PM.

88. The cereblon degradant antibody conjugate of claim 80, having the formula:

wherein L₁ is a protease-cleavable non-peptide linker having the 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 formula:

Wherein the wavy line is a connection to the PM.

89. The cereblon degradant antibody conjugate of claim 80, wherein L₂ is selected from the group consisting of:

-N (R) - (C₁-C₁₂ Alkyldiyl) -N (R) -,

-N (R) - (C₂-C₁₂ alkenyldiyl) -N (R) -,

-N (R) - (C₂-C₁₂ alkynyldiyl) -N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) - (N (R) -C (=o) CH₂ O-,

-N (R) - (C₁-C₁₂ Alkyldiyl) - (N (R) -C (=O) CH₂ N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R) - (C₁-C₁₂ alkyldiyl) -N (R) -,

-N (R) - (C₁-C₆ Alkyldiyl) -O- (C₁-C₆ Alkyldiyl) -N (R) -,

N (R) - (CH₂CH₂O)_n-N(R)-(CH₂CH₂O)_n -, where N is an integer from 1 to 4,

A C₁-C₁₂ alkyldiyl group,

C₂-C₁₂ alkenyldiyl, and

C₂-C₁₂ alkynyl diradical (C₂-C₁₂) and (C) is used for preparing the catalyst,

Wherein R is selected from H, C₁-C₆ alkyl diradicals and the point of attachment to L₁, and

Alkyldiyl, alkenyldiyl and alkynyldiyl are optionally substituted by one or more groups selected from the group consisting of ：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)₃ H.

90. The cereblon degradant antibody conjugate of claim 60, which is selected from the group consisting of:

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 X¹ is selected from CH₂ and C (=o).

91. The cereblon degradant antibody conjugate of claim 60, which is selected from the group consisting of:

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 X¹ is selected from CH₂ and C (=o).

92. The cereblon degradant antibody conjugate of claim 60, which is selected from the group consisting of:

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

94. The cereblon degradant antibody conjugate of any one of claims 60 to 92, comprising a mixture of cereblon degradant antibody conjugate compounds, wherein the average drug loading of each antibody in the mixture of cereblon degradant antibody conjugate compounds is between about 2 and about 6.

95. The cereblon degradant antibody conjugate of claim 60, wherein the cereblon degradant (cD) moiety is a molecular gum selected from the group consisting of:

Wherein the wavy line indicates the point of connection of L₁, and the dashed lineIndicating an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N, and NR^1a;

Z² is selected from C (R²)₂、CR², N, and NR^2a;

R is selected from H and C₁-C₆ alkyl;

R¹ and R² are independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H;

Or (i) two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or

(Ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl, 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 with one or more groups independently selected from the group consisting of F, cl, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, and C₂-C₁₂ alkynyl;

Ring A is selected from the group consisting of C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl, and

Alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl and heteroaryl are 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)₃ H.

96. The cereblon degradant antibody conjugate of claim 95, comprising a cD structure selected from the group consisting of:

wherein the wavy line indicates the point of connection with the rest of L₁.

97. The cereblon degradant antibody conjugate of claim 96, comprising a sacrificial moiety selected from the group consisting of:

Wherein a connection point to the remainder of L₁ is indicated and a waveform line indicates a connection point 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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, 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)₃ H.

98. The cereblon degradant antibody conjugate of claim 95, wherein the cereblon degradant moiety is selected from the group consisting of:

Wherein X¹ is selected from CH₂ and C (=O), and the wavy line indicates the point of attachment of L₁.

99. A cereblon degrading agent-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 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;

(ii) A disulfide bond linker selected from the group consisting of:

And

(Iii) A linker having the formula:

wherein X indicates the point of connection 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 ternary, quaternary or five membered cycloalkyl or heterocyclyl,

Optionally substituted with F, cl and C₁-C₆ alkyl,

R⁶ is selected from H and C₁-C₆ alkyl,

The wavy line indicates the connection to the 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)₃ H, and

CD is the cereblon degradant moiety having the formula:

TPL-L₂-E3UL

Wherein:

TPL is a target protein ligand;

e3UL is a cereblon-bound E3 ubiquitin ligase ligand, and

And L₂ is a degradant linker, or

CD is molecular gel.

100. The cereblon-linker intermediate of claim 99, wherein the linkage of L₃ to cD comprises a carbamate (-OC (O) NH-) or methylcarbamate (-OC (O) NHCH₂ -) group.

101. The cereblon degrading agent-linker intermediate of claim 99, wherein the cereblon degrading agent portion is a molecular gum selected from the group consisting of:

Wherein the wavy line indicates the point of attachment of L₃ and the dotted line indicates an optional double bond;

Z¹ is selected from C (R¹)₂、CR¹, N, and NR^1a;

Z² is selected from C (R²)₂、CR², N, and NR^2a;

R is selected from H and C₁-C₆ alkyl;

R¹ and R² are independently selected from the group consisting of H, F, cl, br, I, -CN, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^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)₃ H;

R^1a and R^2a are independently selected from the group consisting of H, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, (C₁-C₆ Alkyldiyl) - (C₆-C₂₀ aryl), - (C₁-C₆ Alkyldiyl) -NR^aR^b、-(C₁-C₆ Alkyldiyl) -OR^a、(C₁-C₆ Alkyldiyl) - (C₃-C₂₀ carbocyclyl), (C₁-C₆ Alkyldiyl) - (C₂-C₂₀ heterocyclyl), (C₁-C₆ Alkyldiyl) - (C₁-C₂₀ heteroaryl), C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, C₁-C₂₀ heteroaryl 、-C(＝NH)NH(OH)、-C(＝NH)NH₂、-C(＝O)NR^aR^b、-C(＝O)NR^a-NR^aR^b、-C(＝O)NH(C₁-C₆ alkyldiyl )-NR^aR^b、-C(＝O)OR^a、-NR^aR^b、-OR^a、-S(O)R^a、-S(O)₂R^a、-S(O)₂NR^a and-S (O)₃ H;

Or (i) two gem R¹ or two gem R² form a 3-to 6-membered carbocyclyl or heterocyclyl screw group, or (ii) R¹ and R²、R^1a and R²、R¹ and R^2a, or R^1a and R^2a form a fused 5-or 6-membered aryl, carbocyclyl, heterocyclyl or heteroaryl group;

R^a and R^b are independently 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-C₁₂ alkyl, C₂-C₁₂ alkenyl and C₂-C₁₂ alkynyl, and

A is selected from C₆-C₂₀ aryl, C₃-C₂₀ carbocyclyl, C₂-C₂₀ heterocyclyl, and C₁-C₂₀ heteroaryl;

alkyl, alkyldiyl, alkenyl, alkynyl, aryl, carbocyclyl, heterocyclyl and heteroaryl are 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)₃ H.

102. The cereblon degradation agent-linker intermediate of claim 101, comprising a structure selected from the group consisting of:

wherein the wavy line indicates the point of connection with the rest of L₃.

103. The cereblon degradation agent-linker intermediate of claim 101, comprising a sacrificial moiety selected from the group consisting of:

wherein points of connection to the rest of L₃ are indicated, and the wavy lines indicate points of connection to the rest 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 bound form a ternary, quaternary or five membered cycloalkyl or heterocyclyl, optionally substituted with F, cl and C₁-C₆ alkyl;

R⁶ is selected from H and C₁-C₆ alkyl;

the waveform line indicates the connection to cD, 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)₃ H.

104. The cereblon degrading agent-linker intermediate of claim 99, wherein the cereblon degrading agent moiety is selected from the group consisting of:

Wherein X¹ is selected from CH₂ and C (=O), and the wavy line indicates the point of attachment of L₃.

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

106. The cereblon-linker intermediate of claim 99, having the formula:

Wherein IM comprises a group selected from the group consisting of 4-aminobenzyl, 4-aminobenzyloxycarbonyl, and (4-aminobenzyl) methylcarbamate, and

AA is a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and citrulline.

107. The cereblon degrading agent-linker intermediate of 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 degrading agent-linker intermediate of claim 106 or 107, wherein AA is-CH₃ or-CH₂CH₂CH₂NHC(O)NH₂.

109. The cereblon-linker intermediate of claim 99, which is selected from the group consisting of:

Wherein X¹ is selected from CH₂ and C (=o).

110. The cereblon degradation agent-linker intermediate of claim 109, having the formula:

wherein L₃ is a protease-cleavable non-peptide linker having the 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 formula:

Wherein the wavy line is a connection to the PM.

111. The cereblon degradation agent-linker intermediate of claim 109, having the formula:

wherein L₃ is a protease-cleavable non-peptide linker having the 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 formula:

Wherein the wavy line is a connection to the PM.

112. The cereblon-linker intermediate of claim 99, wherein TPL is selected from the group consisting of:

(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)

wherein the wavy line indicates the point of connection of L₂.

113. The cereblon-linker intermediate of claim 99, wherein L₂ is selected from:

-N (R) - (C₁-C₁₂ Alkyldiyl) -N (R) -,

-N (R) - (C₂-C₁₂ alkenyldiyl) -N (R) -,

-N (R) - (C₂-C₁₂ alkynyldiyl) -N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R) -,

-N (R) - (C₁-C₁₂ alkyldiyl) -C (=o) - (N (R) - (C₁-C₁₂ alkyldiyl) -N (R) -,

- (C₁-C₆ Alkyldiyl) -O- (C₁-C₆ Alkyldiyl) -,

A C₁-C₁₂ alkyldiyl group,

C₂-C₁₂ alkenyldiyl, and

C₂-C₁₂ alkynyl diradical (C₂-C₁₂) and (C) is used for preparing the catalyst,

Wherein the alkyl, alkenyl and alkynyl groups are optionally substituted ：F、Cl、-OH、-OCH₃、-OCH₂CH₃、-OCH₂CH₂OCH₃、-OCH₂CH₂OH、-OCH₂CH₂N(CH₃)₂, with one or more groups selected from the group consisting of and R is selected from H, C₁-C₆ alkyl groups and the point of attachment to L₃.

114. The cereblon degradant intermediate of claim 99, wherein cD is selected from the group consisting of:

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 X¹ is selected from CH₂ and C (=O).

115. The cereblon-linker intermediate of claim 99, which is selected from the group consisting of:

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 X¹ is selected from CH₂ and C (=o).

116. The cereblon-linker intermediate of claim 99, which is selected from the group consisting of:

117. a cereblon degradant antibody conjugate prepared by conjugation of a thiol-containing antibody to a cereblon degradant intermediate selected from table 3.

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

119. A process for preparing a cereblon degrading agent-linker intermediate of formula II according to claim 99, the process comprising reacting an X-L3 moiety with a cereblon degrading agent moiety.

120. A pharmaceutical composition comprising the cereblon degradation agent antibody conjugate of any one of claims 1-98 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 degradation agent antibody conjugate of any one of claims 1 to 98 or the pharmaceutical composition of claim 120.

122. The method of claim 121, wherein the cancer is selected from the group consisting of epithelial cancers, lymphomas, blastomas, sarcomas, leukemias or lymphoblastic malignancies, including acute myelogenous 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 gastric 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, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

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

124. The method of claim 121, wherein the cancer has BRD4 dependence.

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

126. The cereblon degradant antibody conjugate of any one of claims 1 to 98 or the pharmaceutical composition of claim 120, for use in a method for treating cancer.