Neodegrader Conjugate

Abstract

The present invention provides neodegraders and neodegraders conjugated to binding moieties. Also provided are compositions comprising the conjugates. The compounds and compositions are useful for treating a disease or condition, such as cancer, in a subject in need of treatment.

Description

[Technical field]

[0001] Reference to Electronically Provided Sequence Listings The contents of the Sequence Listing, provided electronically with this application in an ASCII text file (Name: 4547_016PC02_Seqlisting_ST25; Size: 24,777 bytes; and Created: May 31, 2022), are hereby incorporated by reference in their entirety.

[0002] Field The present invention provides a neodegrader conjugate, in which the neodegrader is conjugated to a binding moiety. Also provided is a composition comprising the conjugate. The conjugate and composition are useful for treating cancer in a subject in need of treatment. [Background technology]

[0003] background Proteolysis has been demonstrated as a therapeutic strategy by the efficacy of immunomodulatory imide drugs. These compounds bind to cereblon (CRBN) and inhibit CRL4. CRBN They have the ability to promote the recruitment and ubiquitination of substrate proteins mediated by E3 ubiquitin ligases. Immunomodulatory imides are thought to act as "molecular glue" and fill the binding interface as a hydrophobic patch that reprograms the protein interaction between the ligase and the neosubstrate.

[0004] Despite the excitement these compounds have generated as novel cancer treatments, to date their use has been limited to hematological malignancies such as multiple myeloma and myelodysplastic syndromes (MDS). The expanding library of compounds that can function by degrading other cancer proteins, many of which are considered "undruggable," is an active area of ​​drug development. Thus, there is a continuing need for novel compounds that can target these alternative cancer proteins and treat a wide range of cancers. Summary of the Invention

[0005] overview Treatment of cancer patients with small molecule GSPT1 degraders has been shown to drive clinical responses, but is associated with serious adverse events (AEs). Cancers often express antigens on their surface that are not expressed or are expressed at much lower levels on healthy cells. The present invention is based on the discovery that the combination of a GSPT1 degrading payload molecule and an antibody that binds to a cell surface antigen on cancer cells can improve both the clinical efficacy and tolerability of GSPT1 degraders.

[0006] In one embodiment, the present invention provides a method for producing a compound of formula (I): [ka] [During the ceremony, a is 1 to 10; L is [ka] is a linker selected from where [ka] is the point of attachment to the nitrogen atom; and [ka] is the point of attachment to Bm; and Bm is a binding moiety capable of specifically binding to a protein, e.g., a protein that is a cell surface antigen. or a pharma- ceutically acceptable salt thereof.

[0007] In some embodiments, the binding moiety is an antibody, antibody fragment or antigen-binding fragment. In some embodiments, a is 2-8.

[0008] In some embodiments, L is [ka] It is.

[0009] In some embodiments, L is [ka] It is.

[0010] In some embodiments, Bm is an antibody or an antigen-binding portion thereof. In some embodiments, the protein to which the binding moiety binds is a surface antigen. In some embodiments, the surface antigen is 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, C D15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31 , CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62 P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Lypto-1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin 3, cadherin 6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, ephrin A4, ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24. HMWMAA, HPV E6, hTERT, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (すなわち, CD25), IL-2Rβ (すなわち, CD 122), IL-2Rγ (すなわち, CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22 Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), インターフェロン receptor, インテグリン (α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbincluding β3 integrin), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumain, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / Galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostaglandin E, phosphodiesterase, phosphodiesterase in, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras, Ras mutants, Rh factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin-C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1 or combinations thereof.

[0011] In some embodiments, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or a combination thereof. In some embodiments, the surface antigen comprises CD33.

[0012] In certain embodiments, the antibody is selected from the group consisting of rituximab, trastuzumab, gemtuzumab, CD33AB, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMy9-6-IgG4-S228P, belantamab, indatuximab, cetuximab, dinutuximab, anti-CD38A2 antibody, HuAT13 / 5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, and veltuzumab. In some embodiments, the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMy9-6-IgG4-S228P, CD33AB, lintuzumab, or gemtuzumab. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1 (VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain variable region (VL) CDR1 (VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 5, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. In one embodiment, the antibody is CD33AB.

[0013] In one embodiment, the present invention provides a compound of formula (II): [ka] or a pharma- ceutically acceptable salt thereof.

[0014] In one embodiment, the present invention provides a compound of formula (III): [ka] or a pharma- ceutically acceptable salt thereof.

[0015] In one embodiment, the present invention provides a compound of formula (IV): [ka] where Bm is a binding moiety that specifically binds to a protein, e.g., a protein that is a cell surface antigen. or a pharma- ceutically acceptable salt thereof.

[0016] In one embodiment, the present invention provides a method for producing a compound according to formula (V): [ka] where Bm is a binding moiety that specifically binds to a protein, e.g., a protein that is a cell surface antigen. or a pharma- ceutically acceptable salt thereof.

[0017] In some embodiments, the invention provides a compound of formula (IV) or (V), wherein Bm is an antibody or an antigen-binding portion thereof. In some embodiments, the protein to which the binding moiety specifically binds is a surface antigen. In some embodiments, the surface antigen is 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, C D28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, C D72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto-1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin 3, cadherin 6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, ephrin A4, ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e., CD25), IL-2Rβ (i.e., CD122), IL-2Rγ (i.e., CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R), IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbincluding β3 integrin), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumain, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / Galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, protease Stain, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, Rh factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin-C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1 or combinations thereof.

[0018] In some embodiments, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR, GD2, PDGFR, TEM1 / CD248, TROP-2, or a combination thereof.

[0019] In some embodiments, the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMy9-6-IgG4-S228P, belantamab, indatuximab, cetuximab, dinutuximab, anti-CD38A2 antibody, HuAT13 / 5 antibody, CD33AB, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, or veltuzumab.

[0020] In some embodiments, the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMy9-6-IgG4-S228P, CD33AB, lintuzumab or gemtuzumab. In some embodiments, the antibody or antigen-binding portion thereof comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6 and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:4 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the antibody is CD33AB.

[0021] In certain embodiments, the present invention provides a pharmaceutical composition comprising the above conjugate or compound, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers.

[0022] In some embodiments, the present invention provides a method for treating cancer or myelodysplastic syndrome (MDS) in a subject in need of treatment, comprising administering to the subject a pharma- ceutically acceptable amount of the conjugate, compound, or composition described above, or a pharma- ceutically acceptable salt thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological / blood cancer. In some embodiments, the cancer is breast cancer, gastric cancer, lymphoma, acute myeloid leukemia, multiple myeloma, head and neck cancer, squamous cell carcinoma, and / or hepatocellular carcinoma. In some embodiments, the cancer is refractory or resistant to Mylotarg.

[0023] In some embodiments, the method further comprises administering to the subject a pharma- ceutically acceptable amount of an additional agent before, after, or simultaneously with the conjugate or compound or a pharma- ceutically acceptable salt thereof. In some embodiments, the additional agent is a cytotoxic agent or an immune response modifier. In some embodiments, the immune response modifier is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, and / or a LAG-3 inhibitor.

[0024] In one embodiment, the present invention provides a method for preparing a conjugate of formula (I) or a pharma- ceutically acceptable salt thereof, comprising combining a binding moiety with a conjugate of formula (I-1): [ka] [During the ceremony, L' is [ka] Selected from; where [ka] is the point of attachment to the nitrogen atom. or a pharma- ceutically acceptable salt thereof.

[0025] In certain embodiments, the method further comprises reducing the linking moiety prior to reaction with the compound of formula (I-1).

[0026] In some embodiments, L' is [ka] It is.

[0027] In some embodiments, L' is [ka] It is.

[0028] In certain embodiments, the compound of formula (I-1) is reacted with a binding moiety comprising an antibody or an antigen-binding portion thereof.

[0029] In some embodiments, the antibody or antigen-binding portion thereof binds to a surface antigen. In some embodiments, the surface antigen is 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, C D15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31 , CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62 P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Lypto-1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin 3, cadherin 6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, ephrin A4, ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24. HMWMAA, HPV E6, hTERT, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα(すなわち, CD25), IL-2Rβ(すなわち, CD122 ), IL-2Rγ (すなわち, CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22 Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), インターフェロン receptor, インテグリン (α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbincluding β3 integrin), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumain, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / Galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, protease Stain, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, Rh factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin-C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1 or combinations thereof.

[0030] In some embodiments, the surface antigens include HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, Trop-2, or a combination thereof.

[0031] In some embodiments, the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMy9-6-IgG4-S228P, belantamab, indatuximab, cetuximab, dinutuximab, anti-CD38A2 antibody, CD33AB, HuAT13 / 5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, or veltuzumab.

[0032] In some embodiments, the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMy9-6-IgG4-S228P, CD33AB, lintuzumab or gemtuzumab. In some embodiments, the antibody or antigen binding portion thereof comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6 and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 4 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. [Brief description of the drawings]

[0033] [Figure 1A]FIG. 1A shows the in vivo activity of representative neodegrader conjugates against MV411 (CD33+) tumors. The X-axis shows days after dosing. The Y-axis shows tumor volume (mm3) after dosing with vehicle, 3.02 mg / kg CD33AB-compound (Ia), 2.94 mg / kg CD33AB-compound (Ib), 0.1 mg / kg Mylotarg, 50 mg / kg×21 venetoclax, or 5 mg / kg bid×10 CC-90009.

[0034] [Figure 1B] FIG. 1B shows the in vivo activity of representative neodegrader conjugates against MV411 (CD33+) tumors. The X-axis shows days after dosing. The Y-axis shows tumor volume (mm3) after dosing with vehicle, 3 mg / kg CD33AB-compound (Ic), 2.83 mg / kg CD33AB-compound (Ie), 3.02 mg / kg CD33AB-compound (Ia), 2.99 mg / kg CD33AB-compound (Ih), 2.94 mg / kg CD33AB-compound (Ib), 0.1 mg / kg Mylotarg, 50 mg / kg×21 venetoclax, or 5 mg / kg bid×10 CC-90009.

[0035] [Diagram 2] FIG. 2 shows the in vitro activity of CD33AB-Compound (Ia), CC-90009 and Mylotarg against AML (CD33+) and non-AML (CD33-) cells.

[0036] [Diagram 3] FIG. 3 shows the in vitro activity of CD33AB-Compound (Ia) conjugates against MV4-11 (CD33+) cells. The X-axis shows concentration. The Y-axis shows % cell viability after administration of non-CD33 binding antibody-Neodegrader conjugates, CD33AB, Neodegrader P1, Venetoclax, CC-885, CC-90009, Mylotarg and CD33AB-Compound (Ia).

[0037] [Figure 4]FIG. 4 shows the in vitro activity of CD33AB-Compound (Ia) conjugates, Mylotarg and CC-90009 against patient-derived primary relapsed / refractory AML cells.

[0038] [Diagram 5] FIG. 5 shows the in vitro activity of CD33AB-Compound (Ia) conjugates, CC-90009 and Mylotarg in normal erythroid, myeloid and megakaryocytic progenitor cells as measured by colony forming cell (CFC) assay.

[0039] [Figure 6] FIG. 6 shows the in vivo activity of CD33AB-Compound (Ia) against MV4-11 (CD33+) tumors. In the left graph, the X-axis shows the number of days after administration, and the Y-axis shows the tumor volume (mm3) after administration of vehicle, 5 mg / kg bid CD-90009, 0.1 mg / kg Mylotarg, 50 mg / kg qd venetoclax, and 3 mg / kg CD33AB-Compound (Ia). In the right graph, the X-axis shows the number of days after administration, and the Y-axis shows the tumor volume (mm3) after administration of vehicle, 3 mg / kg CD33 non-binding antibody neodegrader conjugate, 8 mg / kg azacytidine, and 50 mg / kg venetoclax, 1 mg / kg CD33AB-Compound (Ia), and 3 mg / kg CD33AB-Compound (Ia).

[0040] [Figure 7] FIG. 7 shows the in vivo activity of CD33AB-Compound (Ia) in MV4-11 and OCI-AML2 cells.

[0041] [Figure 8] 8 is a Western blot showing the degradation of GSPT1 after in vitro treatment of MV4-11 cells with CD33AB-compound (Ia) conjugate. GSPT1 is completely degraded in 12 hours when treated with CD33AB-compound (Ia) conjugate.

[0042] [Figure 9]FIG. 9 shows the sustained in vitro effect of CD33AB-compound (Ia) on GSPT1 (top) and the pharmacokinetics of the CD33AB-compound (Ia) conjugate (QDx1 IV) (bottom).

[0043] [Figure 10] Figures 10A and 10B show the in vitro activity of CD33AB-Compound (Ia) conjugate against Mylotarg-insensitive AML cells (AML-193 (Figure 10A) and Kasumi-6 (Figure 10B). The X-axis shows the concentration and the Y-axis shows the percent viability of the cell lines after treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Detailed Description The present invention relates to a compound of formula (I): [ka] [During the ceremony, a is 1 to 10; L is [ka] is a linker selected from where [ka] is the point of attachment to the nitrogen atom; and [ka] is the point of attachment to Bm; and Bm is a binding moiety capable of specifically binding to a protein. or a pharma- ceutically acceptable salt thereof. In certain embodiments, the binding moiety is an antibody, an antibody fragment, or an antigen-binding fragment.

[0045] The invention also provides the above compounds fused to a binding moiety, compositions comprising said compounds or said conjugates or methods of using or making said compounds or said conjugates.

[0046] I. Definition In order that this description may be more readily understood, certain terms are first defined. Further definitions are given throughout the detailed description.

[0047] It is understood that a singular reference to an item refers to one or more of that item; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. That is, the terms "a," "one or more," and "at least one" may be used interchangeably herein. It is further noted that the claims may be drafted to exclude any optional element. That is, this description is intended to serve as a predicate for the use of exclusive terms such as "solely," "only," and the like, or the use of a negative limitation in connection with the limitation of a claim element.

[0048] Furthermore, "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used herein in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" used in a phrase such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0049] When embodiments are described herein using the term "comprising," it is understood that other similar embodiments described using "consisting of" and / or "consisting essentially of" are also provided.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000 provide those skilled in the art with a general dictionary of many of the terms used herein.

[0051] Units, prefixes, and symbols are shown in the form recognized by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. When a range of values ​​is described, it is understood that each integer value and each fraction between the stated upper and lower limits of the range is also specifically disclosed, along with each subrange of such values. The upper and lower limits of any range can be independently included or excluded within the range, and each range in which either, neither, or both of the limits are included is also included in the invention. Thus, ranges described herein are understood to include all omissions of values ​​within the range, including the stated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subranges in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0052] When values ​​are specified, it is understood that values ​​that are approximately the same quality or quantity as the stated value are also within the scope of the present invention. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the present invention. Conversely, when different elements or groups of elements are individually disclosed, their combinations are also disclosed. When any element of the present invention is disclosed as having multiple options, the disclosed examples in which each option is excluded, either alone or in any combination with other options, are also disclosed herein; more than one element of the present invention may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0053] As used herein, the term "DAR" refers to the drug-antibody ratio of a conjugate, which is the average number of neodegrader-linker complexes linked to each antibody. In some embodiments, the DAR of the conjugates described herein is 1-10. In some embodiments, the DAR of the conjugates described herein is 1-8. In some embodiments, the DAR of the conjugates described herein is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 1 .2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.

[0054] As used herein, the term "antibody" refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulins can be from any species. However, in certain embodiments, the immunoglobulins are of human, murine, or rabbit origin.

[0055] The term "single domain antibody", also known as nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain with a molecular weight of about 12 kDa to about 15 kDa. Single antibodies can be based on heavy chain variable domains or on light chains. An example of a single domain antibody is the V H H fragment and V NAR Including, but not limited to, fragments.

[0056] An "antibody fragment" comprises a portion of an intact antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab').sub.2 and Fv fragments; bispecific antibodies; linear antibodies; fragments produced by a Fab expression library which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions) and epitope-binding fragments of any of the above, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0057] An "intact antibody" is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0058] The term "monoclonal antibody" as used herein refers to a substantially homogeneous antibody, i.e., an antibody obtained from a population in which the individual antibodies constituting the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to polyclonal antibody preparations which include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not intended to require production of the antibody by any particular method. For example, monoclonal antibodies for use in the present invention may be produced by hybridoma methods or by recombinant DNA methods. "Monoclonal antibodies" may also be isolated from phage antibody libraries.

[0059] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belong to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belong to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include "primatized" antibodies which contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0060] Various methods are used to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses cells from various species, including mice (murine), hamsters, rats, and humans. Other methods used to produce MAbs use genetic engineering, including recombinant DNA technology. Monoclonal antibodies produced by these techniques include chimeric and humanized antibodies, among others. Chimeric antibodies combine DNA coding regions from more than one type of species. For example, chimeric antibodies can be derived from variable regions from mouse and constant regions from human. Humanized antibodies are primarily human, even if they contain non-human portions. Like chimeric antibodies, humanized antibodies can contain fully human constant regions. However, unlike chimeric antibodies, the variable regions can be partially human. The non-human, synthetic portions of humanized antibodies are often derived from the CDRs of mouse antibodies. Either way, these regions are important for the antibody to be able to recognize and bind to a specific antigen. Although useful for diagnosis and short-term therapy, mouse antibodies cannot be administered long-term to humans without increasing the risk of adverse immunogenic responses. This response, called human anti-mouse antibody (HAMA), occurs when the human immune system recognizes the mouse antibody as foreign and attacks it. The HAMA response can lead to toxic shock or even death.

[0061] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portion of the administered antibody, and may have the added benefit of activating a secondary human immune response, such as antibody-dependent cellular cytotoxicity.

[0062] An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like.

[0063] Depending on the amino acid sequence of the constant domain of the heavy chain, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0064] The term "about" is used herein to mean approximately, roughly, in the region or area. When the term "about" is used in conjunction with a numerical value, it modifies the range by extending the boundaries above and below the numerical value set forth. In general, the term "about" can modify the numerical value by, for example, a variance of 10 percent above or below (higher or lower) the stated value.

[0065] The terms "administration", "administer" and grammatical variants thereof refer to the introduction of a composition, such as the EVs (e.g., exosomes) of the present invention, into a subject via a pharma- ceutically acceptable route. The introduction of a composition, such as the EVs (e.g., exosomes) of the present invention, into a subject can be by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal or subcutaneous), rectal, intralymphatic, intrathecal, periocular or topical. Administration includes self-administration and administration by another. A suitable route of administration allows the composition or agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0066] The term "antibody" as used herein includes immunoglobulins and fragments thereof, whether natural or partially or wholly synthetically produced. The term also encompasses any protein having a binding domain that is homologous to an immunoglobulin binding domain. "Antibody" further includes polypeptides comprising framework regions from immunoglobulin genes or fragments thereof that specifically bind and recognize an antigen. The use of the term antibody is meant to include complete antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as, for example, scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, bispecific antibodies and antibody-related polypeptides. Antibodies include bispecific and multispecific antibodies, so long as they exhibit the desired biological activity or function. In certain embodiments of the present invention, the biologically active molecule is a molecule comprising an antibody or an antigen-binding fragment thereof.

[0067] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to an antibody attached, e.g., covalently, to a therapeutic agent (sometimes referred to herein as a drug, agent or active pharmaceutical ingredient) or agent. In certain embodiments of the invention, the biologically active molecule is an antibody-drug conjugate.

[0068] As used herein, the term "approximately" when applied to one or more values ​​of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" refers to a range of values ​​that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise specified or clear from the context (except when such value exceeds 100% of possible values).

[0069] "Conservative amino acid substitution" refers to an amino acid residue that is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when an amino acid in a polypeptide is replaced with another amino acid of the same side chain family, the substitution is considered conservative. In other embodiments, strings of amino acids can be conservatively replaced with structurally similar strings that differ in the order and / or composition of side chain family members.

[0070] As used herein, the term "conserved" refers to nucleotides or amino acid residues, respectively, of a polynucleotide or polypeptide sequence that do not vary at the same position in two or more sequences being compared. A relatively conserved nucleotide or amino acid is one that is more conserved in related sequences than a nucleotide or amino acid that appears elsewhere in the sequence.

[0071] In some embodiments, two or more sequences are considered "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are considered "highly conserved" if they are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. In some embodiments, two or more sequences are considered "conserved" if they are at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to portions, parts, regions, or features thereof.

[0072] As used herein, the terms "linked" and "conjugated" are used interchangeably and refer to the covalent or non-covalent attachment of two or more moieties, including a neodegrader and a binding moiety, respectively. In some embodiments, the linked or conjugated may include a linker.

[0073] The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Usually, an amino acid sequence variant has at least about 70% sequence identity with at least one receptor binding domain of a native antibody or at least one ligand binding domain of a native receptor, and typically has at least about 80% and more typically at least about 90% sequence identity with such receptor or ligand binding domain. Amino acid sequence variants have substitutions, deletions and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by trivial names, one-letter and three-letter codes.

[0074] "Sequence identity" is defined as the percentage of residues in amino acid sequence variants that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignment are well known in the art. One computer program is "Align 2" by Genentech, Inc., filed with the United States Copyright Office, Washington, DC 20559, on December 10, 1991, together with the manual.

[0075] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. An example of an FcR is a native sequence human FcR. Additionally, an FcR can be one that binds IgG antibodies (gamma receptors), and includes receptors of the Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA ("activating receptor") and Fc.gamma.RIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0076] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that is complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed.

[0077] A "native antibody" is a heterotetrameric glycoprotein of about 150,000 daltons, usually composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0078] The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Each of the variable domains of naturally occurring heavy and light chains contains four FRs that adopt a predominantly beta-sheet configuration, connected by three hypervariable regions that connect, and in some cases form part of, a beta-sheet structure. The hypervariable regions of each chain are held in close proximity to each other by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0079] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al supra) and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0080] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0081] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions contribute to the antigen binding specificity of the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, but with a lower affinity than the complete binding site.

[0082] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0083] The "light chains" of antibodies from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0084] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0085] The term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites, which comprises a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen-binding sites.

[0086] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. Humanization is a method of transferring mouse antigen-binding information to a non-immunogenic human antibody acceptor, resulting in many therapeutically useful drugs. The humanization process generally begins with the transfer of all six mouse complementarity determining regions (CDRs) into a human antibody framework. These CDR-grafted antibodies generally do not retain the original antigen-binding affinity, and in fact, affinity is often severely impaired. Besides the CDRs, select non-human antibody framework residues must also be incorporated to maintain the proper CDR conformation. Transfer of mouse framework residues critical for supporting the conformational structure of the grafted CDRs into the human acceptor has been shown to restore antigen binding and affinity. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable regions have been replaced with residues from the hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate with the desired specificity, affinity and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0087] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody is purified (1) to the extent of greater than 95% or greater than 99% by weight of the antibody as determined by the Lowry method, (2) to the extent sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a gas phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions by use of Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0088] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Immature cell division and proliferation leads to the formation of malignant tumors that can invade nearby tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary, metastatic and recurrent cancers.

[0089] As used herein, the term "immune response" refers to a biological response of a vertebrate to foreign agents, which response protects the organism against these agents and the diseases caused by them. Immune responses are mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or by the liver, which result in the selective targeting, binding, damaging, destroying, and / or eliminating from the vertebrate body, invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses can be mediated, for example, by T cells, e.g., effector T cells or CD4 T cells, or by the action of soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or by the liver. + or CD8 +The term "T cell" and "T lymphocyte" as used herein are interchangeable and refer to any lymphocyte produced or processed by the thymus. In one embodiment, a T cell is a CD4 + In one embodiment, the T cells are CD8 + In some embodiments, the T cell is a NKT cell.

[0090] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0091] The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of an agent (e.g., a neodegrader or neodegrader conjugate disclosed herein) that provides a desired biological, therapeutic and / or prophylactic result. The result may be a decrease, amelioration, alleviation, reduction, delay and / or alleviation of one or more of the signs, symptoms or causes of a disease or any other desired alteration of a biological system. For solid tumors, an effective amount includes an amount that shrinks the tumor and / or reduces the tumor growth rate (e.g., to tumor growth inhibition) or prevents or delays other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered one or more times. An effective amount of the composition can, for example, (i) reduce the number of cancer cells; (ii) reduce the size of a tumor; (iii) inhibit, delay, slow or stop to some extent cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent) or stop tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0092] In some embodiments, a "therapeutically effective amount" is an amount of a neodegrader or neodegrader conjugate that has a clinically proven effect in significantly reducing cancer or slowing the progression (regression) of cancer, such as advanced solid tumors. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those of skill in the art, such as assaying the activity of the agent in human subjects in clinical trials, animal model systems predictive of human efficacy, or in vitro assays.

[0093] As used herein, the term "standard of care" refers to a procedure that is accepted by medical professionals as the appropriate treatment for a certain type of illness and is widely used by medical practitioners. This term may be used interchangeably with any of the following terms: "best practice," "standard of medical care," and "standard of care."

[0094] As an example, an "anti-cancer drug" promotes cancer regression or prevents further tumor growth in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer.

[0095] The terms "effective" and "effectiveness" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cell, organ and / or organism level resulting from drug administration.

[0096] The term "immune checkpoint inhibitors" as used herein refers to molecules that fully or partially reduce, inhibit, prevent or modulate one or more checkpoint proteins. Checkpoint proteins control T cell activation or function. A number of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2. Pardoll, DM, Nat Rev Cancer 12(4): 252-64 (2012). These proteins are responsible for costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins control and maintain self-tolerance as well as the duration and strength of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies.

[0097] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent or slow down (reduce) undesired physiological changes or disorders, such as the progression or spread of cancer. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., non-worsening) of the disease state, delay or slow down of disease progression, improvement or palliative and remission (whether partial or complete) of the disease state, whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to life expectancy without treatment. Those in need of treatment include those already with the condition or disorder as well as those predisposed to the condition or disorder or those in whom the condition or disorder is to be prevented.

[0098] II. Neodegrader The present invention relates to a compound represented by formula (P1): [ka] We offer Neo Degrader.

[0099] In certain embodiments, a neodegrader is a molecule that forms a ternary complex with an E3 ubiquitin ligase that can target proteins for degradation.

[0100] III. Neodegrader Conjugates The present invention provides conjugates of one or more of the neodegraders and binding moieties disclosed herein. These conjugates bind to cereblon (CRBN) and inhibit CRL4. CRBN Proteins can be degraded by promoting the recruitment and ubiquitination of substrate proteins mediated by E3 ubiquitin ligases. These agents act as "molecular glue" and fill the binding interface as a hydrophobic patch that reprograms the ligase and neo-substrate protein interactions.

[0101] In one embodiment, the present invention relates to a compound represented by formula (I): [ka] [During the ceremony, a is 1 to 10; L is [ka] is a linker selected from where [ka] is the point of attachment to the nitrogen atom; and [ka] is the point of attachment to Bm. or a pharma- ceutically acceptable salt thereof.

[0102] In some embodiments, the neodegrader conjugates described herein have in vitro antiproliferative activity against tumor cell lines. In some embodiments, the neodegrader conjugates comprising a neodegrader and a binding moiety have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% greater in vitro antiproliferative activity than the neodegrader alone or the binding moiety alone. In some embodiments, the neodegrader conjugates comprising a neodegrader and a binding moiety have at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater in vitro antiproliferative activity than the neodegrader alone or the binding moiety alone.

[0103] In some embodiments, the neodegrader conjugates described herein have in vitro antiproliferative activity against a BT-474 breast cancer cell line, e.g., antiproliferative activity against the BT-474 breast cancer cell line, as compared to the neodegrader alone or the binding moiety alone. In some embodiments, the neodegrader conjugates described herein have in vitro antiproliferative activity against a SK-BR-3 breast cancer cell line, e.g., antiproliferative activity against the SK-BR-3 breast cancer cell line, as compared to the neodegrader alone or the binding moiety alone. In some embodiments, the neodegrader conjugates described herein have in vitro antiproliferative activity against a NCI-N87 gastric cancer cell line, e.g., antiproliferative activity against the NCI-N87 gastric cancer cell line, as compared to the neodegrader alone or the binding moiety alone. In some embodiments, the neodegrader conjugates described herein have in vitro antiproliferative activity against Daudi lymphoma cell lines, e.g., antiproliferative activity against Daudi lymphoma cell lines as compared to the neodegrader alone or the binding moiety alone. In some embodiments, the neodegrader conjugates described herein have in vitro antiproliferative activity against HL-60 acute myeloid leukemia cell lines, e.g., antiproliferative activity against HL-60 acute myeloid leukemia cell lines as compared to the neodegrader alone or the binding moiety alone. In some embodiments, the neodegrader conjugates described herein have in vitro antiproliferative activity against Ramos non-Hodgkin's lymphoma cell lines, e.g., antiproliferative activity against Ramos non-Hodgkin's lymphoma cell lines as compared to the neodegrader alone or the binding moiety alone. In some embodiments, the neodegrader conjugates described herein have in vitro antiproliferative activity against the MV411 AML cell line, e.g., antiproliferative activity against the MV411 AML cell line that is greater than that of the neodegrader alone or the binding moiety alone. In some embodiments, the neodegrader conjugates described herein can maintain antiproliferative activity in the presence of human serum. The neodegrader conjugates described herein can be used to treat cancer.

[0104] In one embodiment, an antibody-neodegrader conjugate (AnDC) is a conjugate of one or more neodegraders disclosed herein and an antibody or antigen-binding portion thereof disclosed herein.

[0105] III.A. Linkers The neodegraders of the present invention are linked to a binding moiety via a linker. As used herein, the term "linker" refers to any chemical moiety capable of connecting a binding moiety (Bm) to a neodegrader P1.

[0106] In some embodiments, the linker may comprise a heterobifunctional group. In the present invention, the term "heterobifunctional group" refers to a chemical moiety that connects the linker of which it is a part to the binding moiety. Heterobifunctional groups are characterized as having different reactive groups at either end of the chemical moiety. Conjugation to "Bm" can be achieved via chemical or enzymatic conjugation or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the binding moiety with the reactive handles of the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via engineered unnatural amino acids, where unnatural amino acids with the desired reactive handles are inserted on "Bm". In enzymatic conjugation, an enzyme mediates the coupling of the linker with accessible amino residues on the binding moiety. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical and enzymatic conjugation can also be used sequentially, for example, enzymatic conjugation can be used to introduce unique reactive handles onto "Bm" for use in subsequent chemical conjugation.

[0107] In some embodiments, the heterobifunctional group is [ka] [During the ceremony, [ka] is the point of attachment to the remainder of the linker; and [ka] is the point of attachment to Bm. It is.

[0108] In some embodiments, L is a beta-glucuronidase cleavable linker. [ka] [During the ceremony, [ka] is the attachment point to neodegrader P1; and [ka] is the point of attachment to the binding moiety. is a beta-glucuronidase cleavable linker selected from:

[0109] III.B. Joining part The present invention provides a neodegrader conjugated to a binding moiety. As used herein, the term "binding moiety" refers to any molecule that recognizes and binds to a cell surface marker or receptor. In some embodiments, the binding moiety is not limited to a polypeptide moiety, but is attached to a protein. In addition to targeting the neodegrader to a specific cell, tissue, or location, the binding moiety may also have certain therapeutic effects, such as antiproliferative (cytostatic and / or cytotoxic) activity on the target cell or pathway. In some embodiments, the binding moiety may contain or be engineered to contain at least one chemically reactive group, such as a carboxylic acid, amine, thiol, or chemically reactive amino acid moiety or side chain. In some embodiments, the binding moiety may include a targeting moiety that binds to or complexes with a cell surface molecule, such as a cell surface receptor or antigen of a target cell population. After specific binding or complexing with the receptor, the cell is capable of taking up the targeting moiety or neodegrader conjugate, which is then internalized into the cell.

[0110] In some embodiments, the group "Bm" can be a moiety capable of specifically binding to a cell surface molecule. In some embodiments, the group "Bm" can be a peptide or protein that binds to a cell surface receptor or antigen.

[0111] In some embodiments, the group "Bm" can be an antibody, an antibody fragment, or an antigen-binding fragment. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. A target antigen generally has multiple binding sites, also called epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen can have more than one corresponding antibody. The term "antibody" herein is used in the broadest sense and specifically includes monoclonal antibodies, single domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric, or derived from other species.

[0112] Monoclonal antibodies that can be conjugated to neodegraders are homogeneous populations of antibodies against a particular antigenic determinant (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids or fragments thereof). Monoclonal antibodies (mAbs) against an antigen of interest can be produced using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, hybridoma techniques, human B-cell hybridoma techniques and EBV-hybridoma techniques. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA and IgD, and any subclass thereof. Hybridomas producing mAbs for use in the present invention can be cultured in vitro or in vivo.

[0113] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be produced by any of a number of techniques known in the art.

[0114] The antibody may be a bispecific antibody. Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities. Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed using affinity chromatography steps, is rather cumbersome and the product yield is low.

[0115] A different approach fuses antibody variable domains with the desired binding specificity (antibody-antigen binding site) to immunoglobulin constant domain sequences. The fusion can be with an immunoglobulin heavy chain constant domain, including at least a portion of the hinge, C.sub.H2 and C.sub.H3 regions. The first heavy chain constant region (C.sub.H1) can include the site necessary for light chain binding, present in at least one of the fusions. Nucleic acids with sequences encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected with a suitable host organism. In embodiments when three polypeptide chains are used in an unequal ratio in the construction to obtain optimal yields, this allows for great flexibility in adjusting the mutual proportions of the three polypeptide fragments. However, it is possible to insert the coding sequences of two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in equal ratios results in high yields or when the ratio is not particularly important.

[0116] A bispecific antibody may have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates separation of the desired bispecific compound from undesired immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule facilitates the separation process. Using such techniques, bispecific antibodies may be prepared for conjugation to neodegraders in the treatment or prevention of diseases as defined herein.

[0117] Hybrid or bifunctional antibodies may be derived biologically, ie, using cell fusion techniques, or chemically, particularly using cross-linking or disulfide bridge forming agents, and may comprise whole antibodies or fragments thereof.

[0118] The antibody may be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to a cancer cell antigen, a viral antigen, or a microbial antigen, or other antibody that binds to a tumor cell or matrix. In this context, "functional activity" means that the fragment, derivative, or analog can elicit anti-anti-idiotypic antibodies that recognize the same antigen as the antibody from which the fragment, derivative, or analog is derived. Specifically, in an exemplary embodiment, the antigenicity of the idiotype of an immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences C-terminal to the CDR sequences that specifically recognize the antigen. To determine which CDR sequences bind to an antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art.

[0119] Other useful antibodies include fragments of antibodies that contain the variable region, the light chain constant region and the CH1 domain of the heavy chain, such as, but not limited to, F(ab')2 fragments that can be produced by pepsin digestion of the antibody molecule and Fab fragments produced by reduction of the disulfide bridges of the F(ab')2 fragments. Other useful antibodies are antibody heavy and light chain dimers or minimal fragments thereof such as Fv or single chain antibodies (SCAs) or any other molecule with the same specificity as an antibody.

[0120] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, containing both human and non-human portions, which can be produced using standard recombinant DNA techniques, are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a mouse monoclonal and a human immunoglobulin constant region. Humanized antibodies are antibody molecules from non-human species that have one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0121] Fully human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which express human heavy and light chain genes. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93). Other human antibodies can be obtained commercially, e.g., from Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0122] Fully human antibodies that recognize a selected epitope can be produced using a technique called "guided selection". In this approach, a selected non-human monoclonal antibody, e.g., a murine antibody, is used as a guide to select a fully human antibody that recognizes the same epitope. Human antibodies can also be produced using a variety of techniques known in the art, including phage display libraries.

[0123] An antibody can be, for example, a fusion protein of an antibody or a functionally active fragment thereof in which the antibody is covalently (e.g., a peptide bond) fused at the N-terminus or C-terminus to the amino acid sequence of another protein that is not an antibody (or a portion thereof, such as at least a 10, 20 or 50 amino acid portion of the protein). The antibody or fragment thereof can be covalently linked to the other protein at the N-terminus of the constant domain.

[0124] Antibodies include analogs and derivatives that are modified, i.e., have any type of molecule covalently attached, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. By way of example, but not by way of limitation, derivatives and analogs of antibodies include those that are further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular antibody units or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like. Additionally, analogs or derivatives can contain one or more unnatural amino acids.

[0125] The antibody in the neodegrader conjugate may include an antibody having a modification (e.g., substitution, deletion, or addition) at an amino acid residue that interacts with an Fc receptor. In particular, the antibody includes an antibody having a modification at an amino acid residue that has been identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor. Antibodies immunospecific for cancer cell antigens can be obtained commercially, for example, from Genentech (San Francisco, Calif.), or can be produced by any method known to those skilled in the art, for example, by chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a similar database, publications, or by routine cloning and sequencing.

[0126] In some embodiments, the antibody of the neodegrader conjugate can be a monoclonal antibody, such as a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the antibody can be an antibody fragment, such as a Fab fragment.

[0127] Antibodies known for the treatment or prevention of cancer can be conjugated to the neodegraders described herein. Antibodies immunospecific for cancer cell antigens can be obtained commercially or produced by any method known to those skilled in the art, such as, for example, recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include the humanized anti-HER2 monoclonal antibody Herceptin® (trastuzumab) for the treatment of patients with metastatic breast cancer; Rituxan® (rituximab; Genentech), a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (oregovomab; AltaRex Corporation, MA), a murine antibody for the treatment of ovarian cancer; Panorex (edrecolomab; Glaxo Wellcome, NC), a murine IgG2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (cetuximab; Imclone Systems Inc., NY), an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers such as head and neck cancer; Vitaxin (etaracizumab; MedImmune, Inc., NY), a humanized antibody for the treatment of sarcoma. MD); Campus I / H (alemtuzumab, Leukosite, MA), a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA), a humanized anti-CD33 IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (epratuzumab, Immunomedics, Inc., NJ), a humanized anti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA), a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc.), a radiolabeled murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma., CA); Allomune (BioTransplant, CA), a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; Avastin (bevacizumab, Genentech, Inc., CA), a humanized anti-VEGF antibody for the treatment of lung and colorectal cancer; epratuzumab (Immunomedics, Inc., NJ and Amgen, CA), an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0128] Other antibodies useful in neodegrader conjugates include, but are not limited to, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, daratumumab, STI-6129, lintuzumab, huMy9-6, belantamab, indatuximab, dinutuximab, anti-CD38A2 antibodies, HuAT13 / 5 H3s antibodies, ibritumomab, tositumomab, panitumumab, tremelimumab, ticilimumab, catumaxomab, and veltuzumab. In some embodiments, the antibody is selected from the group consisting of rituximab, trastuzumab, pertuzumab, huMy9-6-IgG4-S228P, lintuzumab, and gemtuzumab.

[0129] Other antibodies useful in neodegrader conjugates are directed to the following antigens: CA125 (ovarian), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha fetoprotein (carcinoma), CA 242 (colorectal), placental alkaline phosphatase (carcinoma), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinoma), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), These include, but are not limited to, antibodies against leukemia, prostate cancer, PSA (prostate), IL-2 receptor (T cell leukemia and lymphoma), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma) and Neu oncogene product (carcinoma). Certain useful antibodies include, but are not limited to, BR96 mAb (Trail, PA, et al Science (1993) 261, 212-215), BR64 (Trail, PA, et al Cancer Research (1997) 57, 100-105), mAbs against the CD40 antigen, such as S2C6 mAb (Francisco, JA, et al Cancer Res. (2000) 60: 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb, and mAbs against the CD30 antigen, such as AC10. Many other internalizing antibodies that bind tumor-associated antigens can be used and have been reviewed.

[0130] Other antigens that the conjugate can bind include 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD 15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, C D31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD 62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, C ripto protein, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin 3, cadherin 6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, ephrin A4, ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e., CD25), IL-2Rβ (i.e., CD122), IL-2Rγ (i.e., CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R), IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbincluding β3 integrin), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumain, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / Galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostate carcinoma cells, prosteine, Pseudomonas aeruginosa, rabies, sa vivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, respiratory syncytial virus, Rh factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin-C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn including, but not limited to, Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1 and / or XAGE1.

[0131] Antibodies that bind to antigens associated with antigen-presenting cells, such as CD40, OX40L, endoglin, DEC-205, 4-1BBL, CD36, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-L1, BDCA-2, XCR-1, and CCR2, may also be conjugated to a neodegrader.

[0132] The antibodies of the neodegrader conjugates can bind to both receptors or receptor complexes expressed on activated lymphocytes. The receptors or receptor complexes can include immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins, or complement control proteins. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152 / CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103 and CD104. Non-limiting examples of suitable lectins are C-type, S-type and I-type lectins.

[0133] In some embodiments, antibodies that may be useful in the present invention include 3F8, 8H9, abagovomab, abciximab (ReoPro). (登録商標) ), avituzumab, abrazepam, abrilumab, actoxumab, adalimumab (Humira (登録商標)), adecatumumab, aducanumab, afacevicumab, afelimomab, afutuzumab, aracizumab, ALD518, alemtuzumab (campus (登録商標) ), alirocumab (Praluent) (登録商標) ), altumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrukinuzumab, apolizumab, apurutumab, arcitumomab (CEA-SCAN (登録商標) ), ascribclovir, acelizumab, atidortoxumab, atlizumab (tocilizumab, actemra (登録商標) , Roactemra (登録商標) ), atezolizumab (Tecentriq (登録商標) ), atinumab, atrolimumab, avelumab (Bavencio), azintuxizumab, belantamab, bapineuzumab, basiliximab (Simulect (登録商標) ), bavituximab, BCD-100, bectumomab (LYMPHOSCAN (登録商標) ), begelomab, belantamab, belimumab (Benlysta (登録商標) ), bemarituzumab, benralizumab (Fasenra (登録商標) ), Bermekimab, Bersanlimab, Bertilimumab, Besirsomab (SCINITIMUN (登録商標) ), bevacizumab (Avastin (登録商標) ), bezlotoxumab (Geneprab (登録商標) ), bicilumab (FIBRISCINT (登録商標) ), bimagrumab, bimekizumab, viltamimab, bivatuzumab, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, brentuximab, briakinumab, brodalumab (Siliq TM ), brolucizumab (Beovu (登録商標) ), brontixutuzumab, burosumab (Crysvita (登録商標) ), cabilalizumab, caplacizumab (cabrib (登録商標) ), camidanlumab, camrelizumab, canakinumab (Ilaris (登録商標) ), cantuzumab, capromab, carlumab, carotuximab, catumaxomab (REMOVAB (登録商標)), cBR96, CC49, cedelizumab, cemiplimab (libtayo (登録商標) ), sergituzumab, cetrelimab, certolizumab, cetuximab (Erbitux (登録商標) ), civisatamab, cirumtuzumab, sitatuzumab, sitatuzumab, clazakizumab, clenoliximab, clivatuzumab, codrituzumab, cofetuzumab, coltuximab, conatumumab, concizumab, cosfrobiximab, CR6261, crenezumab, crizanlizumab (adaqvio (登録商標) ), cloteduumab, cusatuzumab, dacetuzumab, daclizumab (Zinbryta (登録商標) ), darotuzumab, dapirorizumab, daratumumab (Darazalex (登録商標) ), dectrekumab, demcizumab, denintuzumab, denosumab (Prolia (登録商標) ), depatuximab, dellotuximab, detumomab, desamizumab, dinutuximab (unituximab) (登録商標) ), ziridabu, domagrozumab, dostarlimab, dorlimomab, dorlixizumab, drozitumab, DS-8201, durigotuzumab, dupilumab (Dupixent (登録商標) ), durvalumab (Imfinzi) (登録商標) ), dusigitumab, eclumeximab, eculizumab (Soliris (登録商標) ), edovacomab, edrecolomab (PANOREX (登録商標) ), efalizumab (Raptiva (登録商標) ), efungumab (MYCOGRAB (登録商標) ), eldelumab, elezanumab, elgemtumab, elotuzumab (Empliciti (登録商標) ), elsilimomab, ematuzumab emapalumab (Gamifant (登録商標) ), emibetuzumab, emicizumab (Hemlibra (登録商標) ), enapotamab, enavatuzumab, enfortumab (padceb (登録商標) ), enlimomab, enoblitzumab, enokizumab, enoticumab, encituximab, epitumomab, eptinezumab (Veptine), (登録商標) ), epratuzumab, erenumab (Aimovig (登録商標)), erlizumab, ertumaxomab (REXOMUN (登録商標) ), etaracizumab (ABEGRIN (登録商標) ), etigilimab, etrolizumab, evinacumab, evolocumab (Repatha (登録商標) ), exibirumab, fanolesomab (NEUTROSPEC (登録商標) ), faralimomab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, filibumab, framvotumab, fretikumab, flotetuzumab, fontolizumab (HuZAF (登録商標) ), foralumab, foravirumab, fremanezumab (Ajovy (登録商標) ), fresolimumab, furovoximab, furunevetomab, furanumab, futuximab, galcanezumab (Emgarti (登録商標) ), galiximab, gancotamab, ganitumab, gantenerumab, gavilimomab, gezivumab, gemtuzumab, gevokizumab, gilvetomab, dimcirumab, dilentuximab, glembatumumab, golimumab (simponi (登録商標) ), gomilikimab, guselkumab (Tremfya (登録商標) ), huMy9-6, huMY9-6-IgG4-S228P, ianalumab, ibalizumab (Trogarzo (登録商標) ), IBI308, ibritumomab, icrucumab, idarucizumab (Prizobind) (登録商標) ), ifavotuzumab, igovomab (INDIMACIS-125), iradatuzumab, IMAB362, imalumab, imaprelimab, imicilomab (MYOSCINT (登録商標) ), imgatuzumab, inlacumab, indatuximab, indusatumab, inebilizumab, infliximab (Remicade (登録商標) ), intetumumab, inolimomab, inotuzumab, iomab-B, ipilimumab, iratumumab, isatuximab (Circrisa (登録商標) ), iscalimab, istiratumab, itolizumab, ixekizumab (Toltz) (登録商標) ), keliximab, labetuzumab (CEA-CIDE TM), lacunotuzumab, radilatuzumab, lampalizumab, lanadelumab (taxylo (登録商標) ), landgrozumab, laprituximab, ralcaviximab, lebrikizumab, remaresomab, lendalizumab, lembervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, ribivirumab, rifastuzumab, ligelizumab, rilotomab, lintuzumab, liriru Mab, roderucizumab, loxivetomab, loncastuximab, lorvotuzumab, rosatuximab, lucatumumab, lurizumab, rumiliximab, lumuletuzumab, rupartumab, rutikizumab, mapatumumab, margetuximab, marstacimab, maslimomab, matuzumab, mavrilimumab, mepolizumab (Nucala (登録商標) ), metelemumab, milatuzumab, minletumomab, mirikizumab, mirvetuximab, mitumomab, modotuximab, monalizumab, mogamulizumab (potelizio) (登録商標) ), morolimumab, mosunetuzumab, motavizumab (NUMAX (登録商標) ), moxetumomab (Lumoxiti (登録商標) ), muromonab-CD3 (ORTHOCLONE OKT3 (登録商標) ), nacolomab, namilumab, naptumomab, naratuximab, nalnatumab, natalizumab (Tysabri (登録商標) ), nabicixizumab, nabivumab, naxitamab, nebacumab, necitumumab (Portrazza) (登録商標) ), nemolizumab, NEOD001, nerelimomab, nesbacumab, netakimab, nimotuzumab (THERACIM (登録商標) ), nirsevimab, nivolumab, nofetumomab, obiltoxaximab (Anthim (登録商標) ), obinutuzumab, ocralizumab, ocrelizumab (Ocrevus (登録商標) ), odulimomab, ofatumumab (ARZERRA (登録商標) ), Lartruvo (登録商標) ), olecurumab, orendalizumab, olokizumab, omalizumab (Xolair) (登録商標)), onvertamab, OMS721, onartuzumab, ontecizumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab, oregovomab (OVAREX), olticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozogamicin, ozoralizumab, pagibaximab, palivizumab (Synagis) (登録商標) ), pamrevlumab, panitumumab (Vectibix (登録商標) ), pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab (THERAGYN (登録商標) ), perakizumab, pertuzumab (OMNITARG (登録商標) ), pexelizumab, pidilizumab, pinatuzumab, pintumomab, placumab, polatuzumab (Poraiby), prezalumab, prozalizumab, pogalizumab, ponezumab, polgabiximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO140, kirisumab, racotumomab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranevtomab, ranibizumab (Lucentis) (登録商標) ), ravagalimab, ravulizumab (Ultomiris (登録商標) ), raxibacumab, refanezumab, regavirumab, REGN-EB3, lenatolimab, lemtolumab, reslizumab (Cinqair ( 登録商標) ), rilotumumab, linucumab, risankizumab (Skyrizi) (登録商標) ), Rituximab (Rituxan (登録商標) ), rivavazumab, lumab, lobatumumab, loredumab, romilkimab, romosozumab (evenity (登録商標) ), rontalizumab, rosmantuzumab, rovalpituzumab, rovelizumab (LEUKARREST (登録商標) ), rozanolixizumab, ruplizumab (ANTOVA), SA237, sacituzumab, samolizumab, samlotamab, sarilumab (kevzara) (登録商標) ), satralizumab, satumomab pendetide, secukinumab (Cosentyx)(登録商標) ), cericlerumab, seribantumab, setoxaximab, seturumab, sevirumab, SGN-CD19A, SHP647, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, siltrastuzumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, STI-6129, suresomab (LEUKOSCAN (登録商標) ), sputumab, stimulimab, subizumab, sublatoxumab, tabalumab, tacatuzumab (AFP-CIDE (登録商標) ), tadocizumab, talaxuzumab, talizumab, tamtubetomab, tanezumab, taplitumomab paptox, talexuzumab, taborimab, tefibazumab (AUREXIS (登録商標) ), terimomab, telisotuzumab, tesidolumab, tetraxetan, tetulomab, tenatumomab, teneliximab, teprotunumab (Tepezza (登録商標) ), teplizumab, tezepelumab, TGN1412, tiburizumab, ticilimumab (tremelimumab (登録商標) ), tigatuzumab, timigtuzumab, timolumab, tiragolumab, tiragolumab, tislelizumab, tisotumab, tiuxetan, tildrakizumab (Ilumya (登録商標) ), TNX-650, tocilizumab (atlizumab, actemra (登録商標) ), tomzotuximab, toralizumab, tosatoxumab, tositumomab (Bexar (登録商標) ), tobetumab, tralokinumab, trastuzumab (Herceptin (登録商標) ), TRBS07, tregalizumab, tremelimumab, trevoglumab, tucotuzumab, tuvilumab, urtoxazumab, ustekinumab (Stelara) (登録商標) ), ublituximab, urocupulumab, urelumab, utomilumab, vadatuximab, banalimab, bundletuzumab, vanticutumab, vanucizumab, bapaliximab, valisacumab, varlilumab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab (NUVION (登録商標) ), bovalilizumab, volociximab (HUMASPECT (登録商標)), bonlerolizumab, bopratelimab, borsetuzumab, votumumab, bunakizumab, zentuzumab, XMAB-5574, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), zatuximab, zenoctuzumab, diralimumab, zolbetuximab or zolimomab.

[0134] In certain embodiments, a binding moiety useful in the present invention comprises an anti-CD33 antibody or an antigen-binding portion thereof. CD33 is expressed in approximately 90% of acute myeloid leukemia (AML) cases, demonstrating its usefulness as a target for therapeutic antibodies. High CD33 expression on AML blasts was reported approximately 30 years ago. CD33 has been detected on blasts in 85-90% of patients presenting with AML as well as on normal bone marrow progenitor cells and myelocytes. CD33 is restricted to hematopoietic cells, but not normal hematopoietic stem cells, making it ideal as a therapeutic for the treatment of AML.

[0135] The anti-CD33 antibodies for the conjugates of the present invention can specifically bind to CD33. In some embodiments, the anti-CD33 antibodies described herein have high affinity, e.g., 10 -6 Below, 10 -7 Below, 10 -8 Below, 10 -9 Below, 10 -10 Below, 10 -11 Below, 10 -12 Below, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M or 10 -9 M~10 -7 K of M D and binds to human CD33.

[0136] In some embodiments, the anti-CD33 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) and the light chain comprises a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1 (VH-CDR1), VH-CDR2 and VH-CDR3, and the VL comprises a VL-CDR1, VL-CDR2 and VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the anti-CD33 antibody comprises a VH-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the anti-CD33 antibody comprises a VH-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD33 antibody comprises a VL-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-CD33 antibody comprises a VL-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD33 antibody comprises a VL-CDR3 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the CDR comprises a sequence as shown in Table 1 below. [Table 1]

[0137] In certain embodiments, the anti-CD33 antibody heavy chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the anti-CD33 antibody light chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0138] In certain embodiments, the anti-CD33 antibody comprises a heavy chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:4, and a light chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0139] In certain embodiments, the anti-CD33 antibody heavy chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9 or SEQ ID NO:11. In certain embodiments, the anti-CD33 antibody comprises a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:10 or SEQ ID NO:12. [Table 2]

[0140] In certain embodiments, the anti-CD33 antibody comprises a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9, and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:10. The term "CD33AB" includes the heavy chain set forth in SEQ ID NO:9 and the light chain set forth in SEQ ID NO:10.

[0141] In certain embodiments, the anti-CD33 antibody comprises a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:11, and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:12.

[0142] In certain embodiments, the anti-CD33 antibodies are disclosed in US Pat. No. 5,585,089, US Pat. No. 5,693,762, each of which is expressly incorporated herein by reference.

[0143] In some embodiments, the anti-CD33 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) and the light chain comprises a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1 (VH-CDR1), VH-CDR2 and VH-CDR3, and the VL comprises a VL-CDR1, VL-CDR2 and VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-CD33 antibody comprises a VH-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the anti-CD33 antibody comprises a VH-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the anti-CD33 antibody comprises a VL-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the anti-CD33 antibody comprises a VL-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD33 antibody comprises a VL-CDR3 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the CDR comprises a sequence as shown in Table 3 below. [Table 3]

[0144] In certain embodiments, the anti-CD33 antibody heavy chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the anti-CD33 antibody light chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0145] In certain embodiments, the anti-CD33 antibody comprises a heavy chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:16, and a light chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0146] In certain embodiments, the anti-CD33 antibody heavy chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 21. In certain embodiments, the anti-CD33 antibody comprises a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO:22. [Table 4]

[0147] In certain embodiments, the anti-CD33 antibody comprises a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 21 and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 22. The anti-CD33 antibody comprises a heavy chain set forth in SEQ ID NO: 21 and a light chain set forth in SEQ ID NO:22.

[0148] An antibody that "binds" to a molecular target or antigen of interest is one that can bind to that antigen with sufficient affinity that the antibody is useful for targeting cells expressing that antigen.

[0149] In the present invention, the group "Bm" can be conjugated to more than one neodegrader. In some embodiments, "Bm" can be conjugated to 1-10 neodegraders. In some embodiments, "Bm" can be conjugated to 1-9 neodegraders. In some embodiments, "Bm" can be conjugated to 1-8 neodegraders. In some embodiments, "Bm" can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 neodegraders. In some embodiments, "Bm" can be conjugated to 7 or 8 neodegraders. In some embodiments, "Bm" is conjugated to 5 neodegraders. In some embodiments, "Bm" is conjugated to 6 neodegraders. In some embodiments, "Bm" is conjugated to 7 neodegraders. In some embodiments, "Bm" is conjugated to 8 neodegraders. In some embodiments, "Bm" is conjugated to 9 neodegraders.

[0150] IV. Compositions and Methods of Use The conjugates and / or compounds described herein may be in the form of a pharma- ceutically or pharma-ceutically acceptable salt. In some embodiments, such salts are derived from inorganic or organic salts.

[0151] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, cinnamate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate salts.

[0152] Examples of suitable base addition salts include ammonium salts; alkali metal salts, such as sodium salts and potassium salts; alkaline earth metal salts, such as calcium salts and magnesium salts; salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts with amino acids, such as arginine, lysine, and the like.

[0153] For example, Berge lists the following FDA-approved commercially available salts of the anions acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycolyl arsanilate (glycolamide phenylarsonate), hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-hydroxyphenyl)-2-propanediol ... the organic cations benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and the metallic cations aluminium, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0154] Berge further lists the following non-FDA approved commercially available (outside the United States) salts: anionic adipate, alginate, aminosalicylate, anhydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, cinnamate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylene bis(salicylate), napadisilate (1,5-naphthalenedisulfonate), and oxalate. salts, pectinates, persulfates, phenylethylbarbiturates, picrates, propionates, thiocyanates, tosylates and undecanoates; the organic cations benethamine (N-benzylphenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), diethylamine, piperazine and tromethamine (tris(hydroxymethyl)aminomethane); and the metal cations barium and bismuth.

[0155] Pharmaceutical compositions containing the neodegrader conjugates described herein may also contain suitable carriers, additives and adjuvants, which may vary depending on the method of administration.

[0156] In some embodiments, the pharmaceutical composition may be formulated into a suitable parenteral dosage form. The formulation may be prepared by a variety of methods known in the art. The pharmaceutical composition may be administered directly into the bloodstream, into muscle, or directly into an organ. Suitable means of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle syringes, needleless syringes, and infusion techniques.

[0157] Parenteral compositions are typically aqueous solutions which may contain additives such as salts, carbohydrates, and buffers. However, the compositions can also be formulated as sterile non-aqueous solutions or a dry form for use with a suitable vehicle such as sterile pyrogen-free water.

[0158] The preparation of parenteral compositions under sterile conditions, such as by lyophilization, may be readily accomplished using standard techniques well known to those skilled in the art.

[0159] Compositions for parenteral administration can be formulated for immediate and / or modified release.Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.Thus, the composition can be formulated as a solid, semi-solid or thixotropic liquid for administration as an implanted depot that provides modified release of active agent.

[0160] The parenteral formulation may be mixed with other suitable pharma- ceutically acceptable additives used in parenteral dosage forms, such as, but not limited to, preservatives.

[0161] In other embodiments, the pharmaceutical composition may be formulated as a suitable oral dosage form such as a tablet, capsule, powder, pellet, suspension, solution, emulsion, etc. Other suitable carriers may be present such as disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, bulking agents, anti-adherents, etc.

[0162] The oral administration formulations may also contain other suitable pharmaceutical additives such as sweeteners, vehicles / wetting agents, colorants, flavoring agents, preservatives, thickening / thickening agents, and the like.

[0163] The neodegrader conjugates described herein may be used to treat various cancers. Some conjugates of the present invention may be useful as medicines because they may be superior in terms of efficacy, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., aqueous solubility), interactions with other drugs (e.g., drug-metabolizing enzyme inhibition), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central nervous toxicity) and / or stability (e.g., chemical stability, stability against enzymes).

[0164] The neodegrader conjugates of the invention are useful in treating diseases, such as cancers, including colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumors), pharyngeal cancer, laryngeal cancer, esophageal cancer, stomach / gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestinal cancer, breast cancer, Cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., ovarian epithelial carcinoma, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, non-hormone-dependent prostate cancer, castration-resistant prostate cancer), liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic bile duct cancer), thyroid cancer (e.g., medullary thyroid carcinoma), kidney cancer (e.g., For example, renal cell carcinoma (e.g., clear cell renal cell carcinoma), renal pelvis and ureter transitional cell carcinoma), uterine cancer (e.g., cervical cancer, uterine carcinoma, uterine sarcoma), gestational choriocarcinoma, brain tumors (e.g., medulloblastoma, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., basal cell carcinoma, malignant melanoma), sarcomas (e.g., rhabdomyosarcoma, smooth muscle cell carcinoma, gli ... The compounds may be used as pharmaceuticals for the prevention or treatment of cancers including myosinoma, soft tissue sarcoma, spindle cell sarcoma, malignant bone tumors, bladder cancer, hematological / blood cancers (e.g., multiple myeloma, leukemia (e.g., acute myeloid leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorders), and cancers of unknown primary origin; cancer growth inhibitors; cancer metastasis inhibitors; apoptosis promoters; and drugs for treating precancerous lesions (e.g., myelodysplastic syndromes).

[0165] In certain embodiments, the neodegrader conjugates of the present invention can be used as medicaments for the treatment of breast cancer, gastric cancer, ovarian cancer, uterine cancer, lung cancer, pancreatic cancer, liver cancer, lymphoma, or hematological cancer.

[0166] Furthermore, the neodegrader conjugates of the present invention may be used simultaneously with, before or after non-pharmacological therapies, namely, the conjugates may be combined with non-pharmacological therapies such as (1) surgery, (2) hypertensive therapy, such as with angiotensin II, (3) gene therapy, (4) hyperthermia, (5) cryotherapy, (6) laser ablation, and (7) radiation therapy.

[0167] For example, use of the neodegrader conjugate of the present invention before or after the above-mentioned surgery can provide effects such as preventing the development of resistance, extending disease-free survival, inhibiting cancer metastasis or recurrence, and extending life span.

[0168] Furthermore, treatment with the neodegrader conjugates of the present invention can be combined with supportive therapies such as (i) administration of antibiotics (e.g., β-lactams such as pansporin, macrolides such as clarithromycin) for various concurrent infectious diseases, (ii) administration of high-calorie infusions, amino acid preparations or general vitamin preparations to improve nutritional disorders, (iii) administration of morphine to relieve pain, (iv) administration of drugs to reduce side effects such as nausea, vomiting, eating disorders, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, liver damage, kidney damage, DIC, fever, etc., and (v) administration of drugs to suppress multidrug resistance in cancer.

[0169] In some embodiments, the neodegrader or neodegrader conjugate of the present invention may be used in combination with standard therapy, e.g., one or more therapeutic agents (e.g., anti-cancer agents and / or immunomodulatory agents). Thus, in some embodiments, the method of treating tumors disclosed herein comprises administering a combination of the neodegrader or neodegrader conjugate of the present invention and one or more additional therapeutic agents. In some embodiments, the neodegrader or neodegrader conjugate of the present invention may be used in combination with one or more anti-cancer agents, e.g., so that multiple elements of the immune pathway can be targeted. In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocking signaling through a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that may be used in the present methods include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies), or combinations thereof.

[0170] In some embodiments, the neodegrader or neodegrader conjugate of the present invention is administered to the subject before or after administration of the additional therapeutic agent. In other embodiments, the neodegrader or neodegrader conjugate of the present invention is administered to the subject simultaneously with the additional therapeutic agent. In some embodiments, the neodegrader or neodegrader conjugate of the present invention and the additional therapeutic agent can be administered simultaneously as one composition in a pharma- ceutically acceptable carrier. In other embodiments, the neodegrader or neodegrader conjugate of the present invention and the additional therapeutic agent are administered simultaneously as separate compositions.

[0171] In some embodiments, the subject that can be treated with the neodegrader or neodegrader conjugate of the present invention is a non-human animal, such as a rat or a mouse. In some embodiments, the subject that can be treated is a human.

[0172] V. Methods for Making Neodegraders and Compositions The present invention provides a method for producing a neodegrader conjugate, comprising combining a binding moiety with a compound of formula (I-1): [ka] [During the ceremony, L' is [ka] is a linker precursor selected from: where [ka] is the point of attachment to the nitrogen atom. or a pharma- ceutically acceptable salt thereof.

[0173] As described herein, the linker precursor comprises a heterobifunctional group that binds to a binding moiety.

[0174] In some embodiments, the binding moiety is pretreated prior to reaction with the compound of formula (I-1). In some embodiments, the compound of formula (I-1) is reacted with a binding moiety that comprises an antibody or an antigen-binding portion thereof. In embodiments where the binding moiety is an antibody, the antibody may be pretreated to reduce interchain disulfides prior to reaction with the compound of formula (I-1). EXAMPLES

[0175] General Synthetic Methods and Intermediates The compounds of the present invention can be prepared by one of ordinary skill in the art in light of this disclosure and knowledge in the art and / or by reference to the following schemes and synthetic examples. Example synthetic routes are shown in the following schemes and examples. It should be understood that variables (e.g., "R" groups) used in the following schemes and examples are to be interpreted independently from those shown elsewhere in this specification. One of ordinary skill in the art will readily understand how the following schemes and examples illustrate the preparation of the compounds described herein.

[0176] Abbreviations used in the schemes follow conventions commonly used in the art. Chemical abbreviations used in the specification and examples are defined as follows: Abbreviations used in the schemes follow conventions commonly used in the art. Chemical abbreviations used in the specification and examples are defined as follows: "Me" is methyl; "Bu" is butyl; "Ph" is phenyl; "TFA" is trifluoroacetic acid; "DCM" is dichloromethane; "HATU" is N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; "THF" is tetrahydrofuran; "BOC" or "Boc" is tributoxycarbonyl; "TEA" is triethylamine; "EtOH" is ethanol; "DMF" is N,N-dimethylformamide; "PE" is petroleum ether; "EtOAc" is ethyl acetate; "DIEA" is diisopropylethylamine; "MeOH" is "methanol"; h is hours; min is minutes; "Ac" is acetic acid; "EDCI" is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; "HOBT" is 1-hydroxybenzotriazole hydrate; "ACN" is acetonitrile; "TCEP" is (tris(2-carboxyethyl)phosphine); "DMA" is N,N-dimethylacetamide;

[0177] [ka] Scheme 1: Preparation of Neodegrader P1

[0178] [ka] Example 1: Synthesis of Neodegrader P1 [ka] Step 1: Synthesis of Compound 2 To a stirred solution of 2-chloro-4-nitrophenyl)acetic acid (compound 1, 5.00 g, 23.19 mmol, 1.00 equiv.) in THF (75.00 mL) was added BH3-Me2S (10 M in THF) (5.80 mL, 58.0 mmol, 2.50 equiv.) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at 70° C. for 2 h under nitrogen atmosphere. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=1:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (3 g, 64%) as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.0 Hz, 1H), 8.10-8.05 (m, 1H), 7.50 (d, J = 8.0 Hz, 1H), 3.99-3.91 (m, 2H), 3.16-3.09 (m, 2H)

[0179] [ka] Step 2: Synthesis of Compound 3 To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 2, 5.00 g, 24.800 mmol, 1.00 equiv.) and tert-butyl 2-bromoacetate (29.0 mL, 148.28 mmol, 8.00 equiv.) in toluene (150.00 mL) was added Bu4NHSO4 (6.74 g, 19.84 mmol, 0.80 equiv.). To the above mixture was added NaOH (5M in H2O) (500.00 mL) dropwise for 40 min at 0° C. The resulting mixture was stirred for another 2 h at 25° C. The resulting mixture was extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (400 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=4:1) to give tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (8 g, 65%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.23 ​​(d, J = 4.0 Hz, 1H), 8.10-8.04 (m, 1H), 7.60 (d, J = 8.0 Hz, 1H), 4.09 (s, 2H), 3.83-3.80 (m, 2H), 3.17-3.14(m, 2H), 1.45(s, 9H)

[0180] [ka] Step 3: Synthesis of Compound 4 To a stirred solution of tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (compound 3, 8.00 g, 16.14 mmol, 1.00 equiv, 63.7%) in DCM (80.00 mL) was added dropwise at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (500 mL). The mixture was extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (6.5 g, crude) as a yellow oil. LCMS (ESI): 517 (2M-H) -

[0181] [ka] Step 4: Synthesis of Compound 5 To a stirred solution of [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 4, 6.30 g, 21.84 mmol, 1.00 equiv, 90%) and HATU (12.46 g, 32.76 mmol, 1.50 equiv) in DMF (65.00 mL) was added CH3NH2.HCl (1.77 g, 26.21 mmol, 1.20 equiv) and DIEA (15.20 g, 117.8 mmol, 4.00 equiv) dropwise at room temperature. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was diluted with water. The resulting mixture was extracted with EtOAc (2×100 mL). The combined organic layers were washed with brine (50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=10:1) to give 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (10 g, purity: 50%, yield: 84%) as a yellow oil. LCMS (ESI): 273.28 (M+H) +

[0182] [ka] Step 5: Synthesis of Compound 6 To a stirred solution of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 5, 3.3 g, 12.10 mmol, 1.00 equiv.) in THF (35.00 mL) was added BH3-THF (1 M in THF) (12.10 mL, 12.10 mmol, 1.00 equiv.) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 70° C. for 2 h under nitrogen atmosphere. The reaction was quenched with MeOH. The residue was acidified to pH 6 with 1N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was basified to pH 8 with saturated NaHCO3 (sat., aq.). The resulting mixture was extracted with EtOAc (3×100 mL), washed with brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (2.5 g, 80%) as a yellow oil. LCMS (ESI): 259.26 (M+H).+

[0183] [ka] Step 6: Synthesis of Compound 7 To a stirred solution of [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (compound 6, 2.50 g, 9.69 mmol, 1.00 equiv.) and Boc2O (2.53 g, 11.6 mmol, 1.20 equiv.) in THF (40 mL) was added TEA (1.17 g, 11.6 mmol, 1.20 equiv.) dropwise at 25° C. The mixture was stirred at 25° C. for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=5:1) to give tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (1.70 g, 50%) as a yellow oil. LCMS (ESI): 359.36 (M+H) +

[0184] [ka] Step 7: Synthesis of Compound 8 To a stirred solution of tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 7, 1.70 g, 4.74 mmol, 1.00 equiv.) and NH4Cl (750 mg, 14.2 mmol, 3.00 equiv.) in EtOH (85 mL) and HO (17 mL) at 25° C. was added Fe (1.3 g, 23.7 mmol, 5.00 equiv.). The mixture was stirred at 80° C. for 2 h. The mixture was cooled to room temperature. The resulting mixture was filtered and the filter cake was washed with EtOH (3×50 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=4:1) to give tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (900 mg, 58%) as a yellow oil. LCMS (ESI): 329.33 (M+H) +

[0185] [ka] Step 8: Synthesis of Compound 9 To a stirred solution of tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 8, 500 mg, 1.52 mmol, 1.00 equiv.) in THF (10 mL) was added diphosgene (601 mg, 3.04 mmol, 2.00 equiv.) dropwise at 25° C. The mixture was stirred at 25° C. for 1 h. The resulting mixture was concentrated under reduced pressure and redissolved in DMF (5 mL). To a stirring mixture of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, prepared as below, 499 mg, 1.82 mmol, 1.20 equiv.) and TEA (1.56 g, 15.45 mmol, 10.00 equiv.) in DMF (20 mL) at 25° C., the above solution was added dropwise. The mixture was stirred at 25° C. for 1 h. The resulting mixture was diluted with 40 mL of ice water. The resulting mixture was extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (5×40 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=10:1) to give tert-butyl (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenetoxy)ethyl)(methyl)carbamate (670 mg, 70%) as a white solid. LCMS: (ESI): 628.63(M+H) +

[0186] [ka] Step 9: Synthesis of Neodegrader P1 To a stirring solution of tert-butyl N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-N-methylcarbamate (compound 9, 670 mg, 1.07 mmol, 1 equiv.) in DCM (10 mL) was added TFA (2.5 mL) dropwise at 0° C. The mixture was stirred at 25° C. for 1 h. The resulting mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC using the following conditions: column, SunFire C18 OBD Prep column, 100 μm, 19×250 mm; mobile phase, water (0.05% TFA) and ACN (5% phase B to 60% within 30 min); detector, UV 220 nm. The collected fractions were lyophilized to give 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (500 mg, 89%) as a white solid. LCMS (ESI): 528.53 (M+H). + . 1 H NMR (400 MHz, methanol-d4) δ 7.77 (d, J = 8.0 Hz, 1H), 7.57-7.53 (m, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 4.0 Hz, 2H), 5.19-5.1 (m, 1H), 4.55-4.41 (m, 4H), 3.75-3.67 (m, 4H), 3.21-3.15 (m,2H), 3.03-3.96 (m, 2H), 2.96-2.84 (m, 1H), 2.83-2.73 (m, 2H), 2.69 (s, 3H), 2.55-2.42 (m, 1H), 2.21-2.12 (m, 1H)

[0187] [ka] Scheme 2: Synthesis of Neodegrader P1-β-glucuronide linker conjugate (compound (Ia))

[0188] [ka] Example 2: Synthesis of compound (Ia) [ka] Step 1: Synthesis of Compound 12 To a stirring solution of 5-formyl-2-hydroxybenzoic acid, 10 (20 g, 120.38 mmol, 1.00 equiv.) in DMF (200 mL), EDCI (28 g, 144.44 mmol, 1.20 equiv.), HOBT (20 g, 144.46 mmol, 1.20 equiv.) and tert-butyl N-(2-aminoethyl)carbamate, 11 (23 g, 144.46 mmol, 1.20 equiv.) were added portionwise under nitrogen atmosphere at 0° C. The resulting mixture was stirred at room temperature overnight under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was quenched with water and extracted with ethyl acetate (3×200 mL). The combined organics were washed with brine (200 mL) and dried over Na2SO4. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (2:3) to give tert-butyl N-[2-[(5-formyl-2-hydroxyphenyl)formamido]ethyl]carbamate, 12 (23 g, 53%) as a white solid. LCMS (ES, m / z): 209 [M+H-100] + , 309 [M+H] + , 331 [M+Na] +

[0189] [ka] Step 2: Synthesis of compound 14 To a stirring solution of tert-butyl N-[2-[(5-formyl-2-hydroxyphenyl)formamido]ethyl]carbamate, 12 (23 g, 74.59 mmol, 1.00 equiv.) in ACN (600 mL) was added Ag2O (34.57 g, 149.17 mmol, 2.00 equiv.) and methyl (2S,3S,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-bromooxane-2-carboxylate, 13 (32.6 g, 82.05 mmol, 1.10 equiv.) in portions at 0° C. under nitrogen atmosphere. The resulting mixture was stirred at room temperature overnight under nitrogen atmosphere. LCMS showed the reaction was complete. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (1:4) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-([2-[(tert-butoxycarbonyl)amino]ethyl]carbamoyl)-4-formylphenoxy]oxane-2-carboxylate, 14 (38 g, 76%) as a green solid. LCMS (ES, m / z): 525 [M+H-100] + , 625 [M+H] + , 647 [M+Na] +

[0190] [ka] Step 3: Synthesis of compound 15 To a stirred solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butoxycarbonyl)amino]ethyl}carbamoyl)-4-formylphenoxy]oxane-2-carboxylate, 14 (20 g, 32.02 mmol, 1.00 equiv) in EA (200 mL) was added Pd / C (4.0 g, 10%) portionwise at room temperature. The resulting mixture was stirred under hydrogen atmosphere overnight at room temperature. LCMS showed the reaction was complete. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (19:1) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butoxycarbonyl)amino]ethyl}carbamoyl)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate, 15 (12.5 g, 58%) as a white solid. LCMS (ES, m / z): 527 [M+H-100] + , 627 [M+H] + , 649 [M+Na] +

[0191] [ka] Step 4: Synthesis of compound 16 To a stirring solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butoxycarbonyl)amino]ethyl}carbamoyl)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate, 15 (5.0 g, 7.98 mmol, 1.00 equiv) in DMF (50 mL) was added bis(4-nitrophenyl)carbonate (2.67 g, 8.77 mmol, 1.10 equiv) and DIEA (2.0 g, 15.94 mmol, 2.00 equiv) in portions under nitrogen atmosphere at 0° C. The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. LCMS showed the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 70% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butoxycarbonyl)amino]ethyl}carbamoyl)-4-{[(4-nitrophenoxycarbonyl)oxy]methyl}phenoxy]oxane-2-carboxylate, 16 (5.3 g, 78%) as a white solid. LCMS (ES, m / z): 692 [M+H-100] + , 792 [M+H] + , 814 [M+Na] +

[0192] [ka] Step 5: Synthesis of compound 17 A mixture of methyl(2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butoxycarbonyl)amino]ethyl}carbamoyl)-4-{[(4-nitrophenoxycarbonyl)oxy]methyl}phenoxy]oxane-2-carboxylate, 16 (550 mg, 0.69 mmol, 1.00 equiv.) and 1-(3-chlorophenyl)-2-(2-phenylpropanediol, 1.00 eq.) was stirred for 1 hour. To a solution of -4-{2-[2-(methylamino)ethoxy]ethyl}phenyl)-3-{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}urea, P1 (367 mg, 0.69 mmol, 1.00 equiv.) in DMF (6.0 mL), DIEA (180 mg, 1.38 mmol, 2.00 equiv.) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at room temperature. LCMS showed the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 70% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butoxycarbonyl)amino]ethyl}carbamoyl)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy]oxane-2-carboxylate, 17 (670 mg, 77%) as a green solid. LCMS (ES, m / z): 1080 [M+H-100] + , 1180 [M+H] + , 1202 [M+Na] +

[0193] [ka] Step 6: Synthesis of Compound 18 To a stirring solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butoxycarbonyl)amino]ethyl}carbamoyl)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy]oxane-2-carboxylate, 17 (660 mg, 0.56 mmol, 1.00 equiv) in THF (12 mL) was added HCl (12 mL, 6.0 N) in portions at 45° C. under nitrogen atmosphere. The resulting mixture was stirred at 45° C. for 4 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 40% gradient in 30 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-{2-[(2-aminoethyl)carbamoyl]-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid (320 mg, 54%), 18, as a white solid. LCMS (ES, m / z): 940 [M+H] + , 962 [M+Na] +

[0194] [ka] Step 7: Synthesis of compound (Ia) (2S,3S,4S,5R,6S)-6-{2-[(2-aminoethyl)carbamoyl]-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-tetrahydrofuran To a solution of trihydroxyoxane-2-carboxylic acid, 18 (100 mg, 0.11 mmol, 1.00 equiv.) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoic acid, 19 (36 mg, 0.12 mmol, 1.10 equiv.) in DMF (1.00 mL), DIEA (27 mg, 0.21 mmol, 2.0 equiv.) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was purified by preparative HPLC using the following conditions: Xselect CSH F-Phenyl OBD column, 19×250 mm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 25 mL / min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-({2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]ethyl}carbamoyl)phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid, compound (Ia) (46 mg, 37%) as a white solid. LCMS (ES, m / z): 568 [M / 2+H] + , 1133 [M+H] + , 1155 [M+Na] + . 1H-NMR (300MHz, DMSO-d6): 10.98 (s, 1H), 8.87 (br s, 1H), 8.30 (t, J=6Hz, 1H), 7.86 (t, J=3Hz, 1H), 7.77 (s, 1H), 7.70-7.69 (m, 2H), 7.51-7.42 (m, 3H), 7.30-7.10 (m, 3H), 6.99 (s, 2H), 6.93 (br s, 1H), 5.79-5.77 (m, 1H), 5.36 (d, J=4.2Hz,1H), 5.13-5.02 (m, 4H), 4.47-4.28 (m, 4H), 3.95 (d, J=9.0Hz,1H), 3.540-3.49 (m, 4H), 3.39-3.36(m, 4H), 3.24-3.22(m, 2H), 2.86-2.81(m, 7H), 2.62-2.51(m, 1H), 2.49-2.41(m, 3H), 2.06-1.99(m, 3H), 1.49-1.42(m, 4H), 1.23-1.16(m, 2H)

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[0198] スキーム4: Manufacturing of compound (Ic)

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[0200] Process 1: Synthesis of Compound 25 To a stirring mixture of 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanoic acid (compound 24, 5.00 g, 16.06 mmol, 1.00 equiv.) was added SOCl2 (25 mL) at room temperature. The resulting mixture was stirred for 16 h at 80° C. The desired product was detected by LCMS (derivative with MeOH MS=326). LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure to give 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 25, 7.5 g, crude) as a yellow oil. The crude product was used directly in the next step without further purification. 1 H-NMR analysis showed it to be the desired product (derivative with MeOH). 1 H-NMR (300 MHz, CDCl3) δ 7.81-7.77 (m, 2H), 7.63-7.59 (m, 2H), 7.46-7.40 (m, 2H), 7.40-7.31 (m, 2H), 5.33 (s, 1H), 4.42 (d, J=3.0 Hz, 2H), 4.24 (t, J=6.0 Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J=3.0 Hz, 2H), 2.59 (t, J=6.0 Hz, 2H)

[0201] Step 2: Synthesis of compound 28 To a stirred solution of 4-formyl-2-nitrophenol (compound 27, 4.21 g, 25.19 mmol, 1.00 equiv.) and Ag2O (7.00 g, 30.20 mmol, 1.20 equiv.) in ACN (100 mL, 190.24 mmol, 75.00 equiv.) was added compound 26 (10.00 g, 25.17 mmol, 1.00 equiv.) in portions at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature overnight under N2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (50 ml x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (PE:EA=1:2) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 28, 10.5 g, 86%) as a white solid. 1 H-NMR analysis showed it to be the desired product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR (300 MHz, CDCl3) δ 10.00 (s, 1H), 8.34 (s, 1H), 8.13-8.09 (m, 1H), 7.52 (d, J=3.0 Hz, 1H), 5.47-5.29 (m, 4H), 4.37-4.35 (m, 1H), 3.75-3.73 (m, 3H), 2.17-2.06 (m, 9H)

[0202] Step 3: Synthesis of compound 29 To a stirring solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 28, 6.00 g, 12.41 mmol, 1.00 equiv.) in MeOH (50 mL) was added NaBH4 (0.47 g, 12.42 mmol, 1.00 equiv.) in portions at RT under N2 atmosphere. The resulting mixture was stirred at RT for 2 h under N2 atmosphere. LCMS showed completion of the reaction. At RT, the reaction was quenched with water. The resulting material was dried over Na2SO4. The resulting mixture was filtered and the filter cake was washed with DCM. The resulting mixture was concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 29, 5.5 g, 91%) as a solid. LCMS (ES, m / z): 486 [M+H]+

[0203] Step 4: Synthesis of compound 30 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 29, 5.50 g, 11.33 mmol, 1.00 equiv) in EA (60 mL) was added Pd / C (1.10 g, 10%) portionwise at room temperature. The resulting mixture was stirred at room temperature for 16 h under H2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered, the filter cake was washed with DCM and MeOH, and the filtrate was concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 30, 4.0 g, 77%) as a solid. The crude product was used directly in the next step without further purification. LCMS (ES, m / z): 456 [M+H] +

[0204] Step 5: Synthesis of Compound 31 To a stirred solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 30, 1.00 g, 2.19 mmol, 1.00 equiv.) and NaHCO3 (0.20 g, 2.40 mmol, 1.1 equiv.) in THF (10 mL) was added compound 25 (0.87 g, 2.62 mmol, 1.20 equiv.) in portions at 0° C. under N2 atmosphere. The resulting mixture was stirred at 0° C. for 6 h under N2 atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The resulting mixture was extracted with DCM. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (EA=100%) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 31, 1.1 g, 66%) as a light yellow solid. LCMS (ES, m / z): 749 [M+H] +

[0205] Step 6: Synthesis of compound 33 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 31, 1.50 g, 2.00 mmol, 1.00 equiv.) and bis(4-nitrophenyl)carbonate (compound 32, 0.68 g, 2.24 mmol, 1.12 equiv.) in DMF (15 mL) was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv.) in portions at 0° C. under a N2 atmosphere. The resulting mixture was stirred at room temperature overnight under a nitrogen atmosphere. LCMS indicated the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 90% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 33, 1.4 g, 48%) as a yellow solid. LCMS (ES, m / z): 914 [M+H] +

[0206] [ka] Scheme 5B: Synthesis of Neodegrader P1-β-glucuronide linker conjugate

[0207] Step 7: Synthesis of compound 34 A mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 33, 1.00 g, 1.09 mmol, 1.00 equiv.) and 1-(3-chloro-4-[2-[2-(methylamino)ethoxy To a mixture of 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (Neodegrader P1, 0.58 g, 1.09 mmol, 1.00 equiv.) in DMF (10 mL) under N2 atmosphere, HOBT (1.18 g, 8.72 mmol, 8.00 equiv.) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equiv.) were added portionwise at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature for 16 h under N2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]oxane-2-carboxylate (compound 34, 800 mg, 56%) as a solid. LCMS (ES, m / z): 1302 [M+H] +

[0208] Step 8: Synthesis of Compound 35 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]oxane-2-carboxylate (compound 34, 800.00 mg, 0.61 mmol, 1.00 equiv) in THF (80 mL) was added HCl (6N, 80 mL) in portions at room temperature under N2 atmosphere. The resulting mixture was stirred at 50° C. for 3 h under nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 35, 230 mg, 32%) as a white solid. LCMS (ES, m / z): 1162 [M+H] +

[0209] Step 9: Synthesis of Compound 36 To a stirring solution of (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 35, 230 mg, 0.2 mmol, 1.00 equiv) in DMF (2 mL) was added piperidine (0.4 mL) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 10 min. LCMS indicated completion of the reaction. The resulting mixture was analyzed under the following conditions (column: XSelect CSH Prep C18 OBD column, 19 × 250 mm, 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20B to 40B in 7 min; 220 nm; RT This was used directly for further purification by preparative HPLC with 1:5.78 min) to give (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 36, 35 mg, 18%) as a white solid. LCMS (ES, m / z): 940 [M+H]+

[0210] Step 10: Synthesis of compound (Ie) To a stirring solution of (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 36, 30 mg, 0.03 mmol, 1.00 equiv.) in DMF (3 mL), DIEA (13 mg, 0.10 mmol, 3.00 equiv.) and compound 37 (30 mg, 0.10 mmol, 3.00 equiv.) were added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 h. LCMS showed the reaction was complete. The resulting mixture was purified by preparative HPLC using the following conditions: (Column: Xselect CSH OBD column 30×150 mm 5 μm, Mobile phase A: Water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 21B to 36B in 10 min; 220 nm; RT 1:11.15 min). The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-(methyl)carbamoyl]oxy)methyl]-2-[3-[6-(2,5-dioxopyrrol-1-yl)hexanamido]propanamido]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound (Ie), 10.5 mg, 28%)) as a white solid. LCMS (ES, m / z): 1133 [M+H] + . 1H-NMR (300 MHz, DMSO-d6) δ 10.9 (s, 1H), 9.13 (s, 1H), 8.16 (s, 1H), 7.92-7.68 (m, 4H), 7.52 (s, 1H), 7.44 (d, J=3.0 Hz, 1H), 7.18-6.99 (m, 7H), 5.76 (s, 1H), 5.20-5.10 (m, 2H), 4.98 (br s, 2H), 4.76-4.74 (m, 1H), 4.42-4.33 (m, 4H), 3.65 (br s, 1H), 3.58-3.54 (m, 5H), 3.35 (d, J=6 Hz, 2H), 2.90-2.83 (m, 7H), 2.57-2.55 (m, 3H), 2.45-2.30 (m, 1H), 2.02-1.98 (m, 4H), 1.48-1.42 (m, 5H), 1.40-1.20 (m, 3H)

[0211] [ka] Scheme 6: Synthesis of Neodegrader P1-β-glucuronide linker conjugate (compound (Ih))

[0212] [ka] Example 6: Synthesis of compound (Ih) Step 1: Synthesis of compound 63 To a stirring mixture of 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoic acid (compound 62, 5.00 g, 16.06 mmol, 1.00 equiv.) was added SOCl2 (25 mL) at room temperature. The resulting mixture was stirred for 16 h at 80° C. The desired product was detected by LCMS (derivative with MeOH MS=326). LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure to give 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 63, 7.5 g, crude) as a yellow oil. The crude product was used directly in the next step without further purification. 11 H NMR analysis showed it to be the desired product (derivative with MeOH). 1 H-NMR (300 MHz, CDCl3) δ 7.81-7.77 (m, 2H), 7.63-7.59 (m, 2H), 7.46-7.40 (m, 2H), 7.40-7.31 (m, 2H), 5.33 (s, 1H), 4.42 (d, J=3.0 Hz, 2H), 4.24 (t, J=6.0 Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J=3.0 Hz, 2H), 2.59 (t, J=6.0 Hz, 2H)

[0213] Step 2: Synthesis of compound 66 To a stirred solution of 4-formyl-2-nitrophenol (compound 65, 4.21 g, 25.19 mmol, 1.00 equiv.) and Ag2O (7.00 g, 30.20 mmol, 1.20 equiv.) in ACN (100 mL, 190.24 mmol, 75.00 equiv.), methyl (2S,3S,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-bromooxane-2-carboxylate (compound 64, 10.00 g, 25.17 mmol, 1.00 equiv.) was added portionwise at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature overnight under N2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (50 mL x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (PE:EA=1:2) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 66, 10.5 g, 86%) as a white solid. 1 H-NMR analysis showed it to be the desired product. LCMS (ES, m / z): 484 [M+1] + . 1H-NMR (300 MHz, CDCl3) δ 10.00 (s, 1H), 8.34 (s, 1H), 8.13-8.09 (m, 1H), 7.52 (d, J=3.0 Hz, 1H), 5.47-5.29 (m, 4H), 4.37-4.35 (m, 1H), 3.75-3.73 (m, 3H), 2.17-2.06 (m, 9H)

[0214] Step 3: Synthesis of compound 67 To a stirring solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 66, 6.00 g, 12.41 mmol, 1.00 equiv.) in MeOH (50 mL) was added NaBH4 (0.47 g, 12.42 mmol, 1.00 equiv.) at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature for 2 h under N2 atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The resulting material was dried over Na2SO4. The resulting mixture was filtered and the filter cake was washed with DCM. The resulting mixture was concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 67, 5.5 g, 91%) as a solid. LCMS (ES, m / z): 486 [M+H] +

[0215] Step 4: Synthesis of compound 68 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 67, 5.50 g, 11.33 mmol, 1.00 equiv) in EA (60 mL) was added Pd / C (1.10 g, 10%) portionwise at room temperature. The resulting mixture was stirred at room temperature for 16 h under H2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered, the filter cake was washed with DCM and MeOH, and the filtrate was concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 68, 4.0 g, 77%) as a solid. The crude product was used directly in the next step without further purification. LCMS (ES, m / z): 456 [M+H] +

[0216] Step 5: Synthesis of Compound 70 To a stirred solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 68, 1.00 g, 2.19 mmol, 1.00 equiv.) and NaHCO3 (0.20 g, 2.40 mmol, 1.1 equiv.) in THF (10 mL) was added 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 69, 0.87 g, 2.62 mmol, 1.20 equiv.) in portions at 0° C. under N2 atmosphere. The resulting mixture was stirred at 0° C. for 6 h under N2 atmosphere. LCMS showed the reaction was complete. At room temperature, the reaction was quenched with water. The resulting mixture was extracted with DCM. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (EA=100%) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 70, 1.1 g, 66%) as a light yellow solid. LCMS (ES, m / z): 749 [M+H] +

[0217] Step 6: Synthesis of compound 72 To a stirring mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 70, 1.50 g, 2.00 mmol, 1.00 equiv.) and bis(4-nitrophenyl)carbonate (compound 71, 0.68 g, 2.24 mmol, 1.12 equiv.) in DMF (15 mL) was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv.) in portions at 0° C. under a N2 atmosphere. The resulting mixture was stirred at room temperature overnight under a nitrogen atmosphere. LCMS indicated the reaction was complete. The reaction mixture was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 90% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 72, 1.4 g, 48%) as a yellow solid. LCMS (ES, m / z): 914 [M+H] +

[0218] Step 7: Synthesis of compound 73 A mixture of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (Compound 72, 1.00 g, 1.09 mmol, 1.00 equiv.) and 1-(3-chloro-4-[2-[2-(methylamino)ethoxy To a mixture of 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (Neodegrader P1, 0.58 g, 1.09 mmol, 1.00 equiv.) in DMF (10 mL) under N2 atmosphere, HOBT (1.18 g, 8.72 mmol, 8.00 equiv.) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equiv.) were added portionwise at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature for 16 h under N2 atmosphere. LCMS showed the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under reduced pressure to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]oxane-2-carboxylate (compound 73 (800 mg, 56%) as a solid. LCMS (ES, m / z): 1302 [M+H] +

[0219] Step 8: Synthesis of compound 74 Methyl(2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]oxane-2-carboxylate under stirring To a mixture of 73 (800.00 mg, 0.61 mmol, 1.00 equiv) in THF (80 mL) under N2 atmosphere, HCl (6N, 80 mL) was added portionwise at room temperature. The resulting mixture was stirred at 50 °C under nitrogen atmosphere for 3 h. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 0% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 74, 230 mg, 32%) as a white solid. LCMS (ES, m / z): 1162 [M+H] +

[0220] Step 9: Synthesis of Compound 75 To a stirred solution of (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid, 74 (230 mg, 0.2 mmol, 1.00 equiv) in DMF (2 mL) was added piperidine (0.4 mL) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 10 min. LCMS indicated completion of the reaction. The resulting mixture was analyzed under the following conditions (column: XSelect CSH Prep C18 Further purification directly by preparative HPLC using an OBD column, 19×250 mm, 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20B to 40B in 7 min; 220 nm; RT1: 5.78 min) afforded (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 75, 35 mg, 18%) as a white solid. LCMS (ES, m / z): 940 [M+H] +

[0221] Step 10: Synthesis of compound (Ih) (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy]-3,4 To a solution of ,5-trihydroxyoxane-2-carboxylic acid (compound 75, 110 mg, 0.12 mmol, 1.00 equiv.) and bis(2,5-dioxopyrrolidin-1-yl)pentanedioate (compound 76, 46 mg, 0.14 mmol, 1.2 equiv.) in DMF (2.0 mL), DIEA (30 mg, 0.23 mmol, 2.0 equiv.) was added portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 h under nitrogen atmosphere. LCMS showed the reaction was complete. The reaction mixture was purified by preparative HPLC using the following conditions (column: Kinetex EVO prep C18, 30 × 150, 5 μm; Mobile phase A: water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 21% B to 41% B in 7 min, 41% B; Wavelength: 254 nm; RT1 (min): 5.8. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3 H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-(3-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanamido}propanamido)phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid (compound (Ih), 48 mg, 34%) was obtained as a white solid. LCMS (ES, m / z): 1151 [M+H] + , 1173 [M+Na] + . 1H-NMR (300MHz, DMSO-d6): 12.80 (br s, 1H), 10.98 (s, 1H), 9.08 (s, 1H), 8.79 (s, 1H), 8.18 (s, 1H), 7.96 (s, 1H), 7.68-7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J=8.1 Hz,1H), 7.25-7.00 (m, 4H), 6.82-6.80 (m, 1H), 5.86 (s, 1H), 5.39-5.30 (m, 2H), 5.14-5.07 (m, 1H), 4.97 (s, 2H), 4.84 (d, J=7.2 Hz,1H), 4.47-4.27 (m, 4H), 3.90 (d, J=9.6 Hz, 1H), 3.56-3.48 (m, 4H), 3.45-3.36 (m, 6H), 2.95-2.80 (m, 8H), 2.75-2.65 (m, 3H), 2.62-2.55 (m, 2H), 2.49-2.35 (m, 1H), 2.21-2.16 (m, 2H), 2.01-1.95 (m, 1H), 1.85-1.80 (m, 2H)

[0222] Example 7: General method for preparation and characterization of neodegrader conjugates [ka] Scheme 7: Synthesis of CD33AB-compound (Ia)

[0223] Synthesis of CD33AB-compound (Ia) 2.25 molar equivalents of TCEP were added to a 7.8 mg / mL solution of CD33AB in 50 mM EPPS, 5 mM EDTA pH 7.0, and the mixture was incubated for 2 hours at 37° C. After cooling the partially reduced antibody to ambient temperature, 8 molar equivalents of compound (Ia) were added as a stock solution in DMA to a final antibody concentration of 7.0 mg / mL and a final DMA concentration of 10% (v / v). The reaction was incubated at ambient temperature for 1 hour. The resulting conjugate was purified by gel filtration using a Zeba 40K desalting column followed by dialysis using a Slide-a-Lyzer cassette (10K MWCO) into 20 mM sodium succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer. Purified AnDC was found to have 100% monomer by SEC, an average drug loading of 3.1 drug / antibody by reduced RPLC-MS and <1.5% unconjugated compound (Ia) by RPLC.

[0224] [ka] Scheme 8: Synthesis of CD33AB-compound (Ib)

[0225] Synthesis of CD33AB-compound (Ib) 3.8 molar equivalents of compound (Ib) were added as a stock solution in DMC to a final antibody concentration of 6.4 mg / mL and a final DMA concentration of 10% (v / v) in CD33AB in 50 mM EPPS pH 8.0 buffer. The reaction was incubated at ambient temperature for 3 hours. The resulting conjugate was purified by gel filtration using a Zeba 40K desalting column followed by dialysis using a Slide-a-Lyzer cassette (10K MWCO) into 20 mM sodium succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer. The purified AnDC was found to have 100% monomer by SEC, an average drug loading of 3.1 drug / antibody by intact RPLC-MS and intact RPLC-MS by RPLC.

[0226] Concentrations and monomers were determined by size-exclusion chromatography using a 7.8 x 300 mm TSKGel 3000SWXL column (Tosoh Bioscience) with 5 μm particles, run at 0.5 mg / mL for 30 min, and eluted isocratically with a 400 mM sodium perchlorate, 50 mM sodium phosphate, 5% (v / v) isopropanol mobile phase. Neodegrader conjugates were quantified from an antibody standard curve detected at 214 nm.

[0227] Drug-to-antibody ratios (DAR) were determined by hydrophobic interaction chromatography using a 4.6 x 35 mm TSKgel Butyl-NPR column with 2.5 μm particles. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate pH 7.0. Mobile phase B was 25 mM sodium phosphate pH 7.0, 25% (v / v) isopropanol. Analytes were eluted with a linear gradient of 0-100% B in 12 min at a flow rate of 0.6 mL / min. Detection was at 214 nm.

[0228] Free linker-payload was determined by mixed-mode chromatography using a 4.6 x 250 mm HISEP column (Supelco) with 2.5 μm particles. Mobile phase A was 100 mM ammonium acetate. Mobile phase B was 100% acetonitrile. Analytes were eluted with a gradient of 25-40% B for 25 min, then 40-100% B in 2 min at a flow rate of 0.7 mL / min. Column temperature was 35°C. Free linker-payload was quantified using an external standard curve with detection at 254 nm.

[0229] Additional neodegrader conjugates can be prepared using the methods described above, substituting the appropriate linker-neodegrader with a lysine or cysteine ​​reactive conjugation handle.

[0230] Example 8: Treatment of Acute Myeloid Leukemia (AML) with Anti-CD33 Antibody-Neodegrader Conjugates The CD33AB-neodegrader compound was administered to athymic nude mice (Crl:NU(NCr)-Foxn1 nu , Charles River). 7 MV411 human acute monocytic leukemia cells (ATCC (登録商標) CRL-5991 TM ) was injected subcutaneously into the flank of the mice (0.1 mL / mouse). 3 Once the average size of the tumors had been reached, mice were administered anti-CD33 antibody-neodegrader conjugate, non-targeting neodegrader conjugate, and vehicle control.

[0231] Stock solutions of CD33AB-Compound (Ia), CD33AB-Compound (Ib) were diluted with vehicle to obtain 0.302 and 0.294 mg / mL dosing solutions, which were adjusted for each animal's weight at 3.02 and 2.94 mg / kg, resulting in a dosing volume of 10 mL / kg (0.2 mL / 20 g mouse). This dosing strategy ensured delivery of the same amount of payload in each test group. Mylotarg was diluted to 0.01 mg / mL with 0.9% sodium chloride solution, resulting in a dosing volume of 10 mL / kg (0.2 mL / 20 g mouse) at 3 mg / kg. Venetoclax was formulated in a vehicle consisting of 60% PG, 30% PEG400, 10% ethanol by sonication to obtain a 5 mg / mL dosing suspension, which delivered 50 mg / kg when administered at a volume of 10 mL / kg. CC-90009 was centrifuged to collect the powder at the bottom; then N-methyl-2-pyrrolidinone (NMP), PEG400 and saline were added and mixed well one by one to give a 0.5 mg / mL dosing solution in 5% NMP, 45% PEG400 and 50% saline, which delivered 5 mg / kg when administered in a volume of 10 mL / kg.

[0232] Mice were divided into 6 treatment groups (N=9 / group): 1) vehicle; 2) CD33AB-compound (Ia) (3.02 mg / kg, iv, qd x 1); 3) CD33AB-compound (Ib) (2.94 mg / kg, iv, qd x 1); 4) Mylotarg (0.1 mg / kg, iv, qd x 1); 5) Venetoclax (50 mg / kg, po, qd x 21); 6) CC-90009 (5 mg / kg, ip, bid x 10). Test articles in groups 1-4 were administered intravenously (iv) in a single dose (qd x 1) with a volume adjusted for body weight (0.200 mL / 20 g mouse). Venetoclax was administered orally (po) while CC-90009 was administered intraperitoneally (ip) in a dose volume of 10 mL / kg (0.2 mL / 20 g mouse) adjusted for the BW of each animal.

[0233] Tumors were measured twice weekly using calipers and each animal was cultured until the tumor reached the endpoint volume (2,000 mm 3 ) or on the final day of the study (day 45), whichever occurred first. MTV(n) was defined as the median tumor volume on the final day of the study among the remaining number of animals (n) whose tumors had not reduced to the endpoint volume.

[0234] As shown in Figure 1A, both neodegrader conjugates resulted in slower tumor growth over time compared to vehicle.

[0235] To further confirm activity and evaluate alternative conjugation and linker-releasing modalities, a panel of CD33AB-based conjugates were tested and compared with Mylotarg, a clinically approved CD33-targeting ADC at clinical dose levels, and CC-90009, a small molecule GSPT1 degrader in clinical trials, respectively. As shown in Figure 1B, consistent with the in vitro observations, in vivo treatment of CD33-positive AML model tumors (MV4-11) with CD33AB-based conjugates releasing neodegrader P1 resulted in tumor regression, with the most robust effect seen with conjugates containing beta-glucuronide release triggers and cysteine ​​conjugation. Comparison of the two variants with beta-glu linkers (compound (Ie) vs. compound (Ia)) showed that the compound (Ia) conjugate showed a longer-lasting response compared to compound (Ie).

[0236] Example 9: Treatment of human leukemia models with anti-CD33 antibody-neodegrader conjugates To confirm activity across a range of CD33 positive and CD33 negative models, the efficacy of CD33AB-compound (Ia) conjugates to induce tumor cell killing in a panel of human leukemia models (including CD33 positive AML and CD33 negative malignancies) in vitro was evaluated. The cytotoxicity of the test articles (TAs) was measured using a panel of CD33 positive acute myeloid leukemia cell lines and a panel of non-AML CD33 negative cells. Cells were seeded in 96-well plates at the designated concentrations and after overnight incubation at 37°C / 5% CO2, serial dilutions of each test article (TA) were added to the cells. Cells were incubated with the test articles for 72 hours and viability was detected with CellTiter-Glo® reagent (Promega). Luminescence values ​​were normalized to each cell line and IC 50 was calculated using Prizm software. The results are shown in Figure 2. The conjugate showed good activity on several CD33+ cells, but advantageously, the conjugate was inactive in a CD33-negative cell model.

[0237] Example 10: Cytotoxicity of anti-CD33 antibody-Neodegrader conjugates The cytotoxicity of the conjugates was measured using MV4-11 CD33-positive acute myeloid leukemia cells. Cells were seeded in 96-well plates in assay medium, and serial dilutions of CD33AB-compound I(a) conjugates, venetoclax, Mylotarg, CC-885, CC-90009, CD33AB (unconjugated antibody) or non-binding AnDC control prepared in assay medium were added to the cells. Cells were incubated for 72 hours, and then cell viability was detected with Cell Counting kit-8 (Dojindo) or CellTiter-Glo® reagent (Promega). As shown in Figure 3, the results showed that the conjugates showed overall in vitro efficacy on CD33-positive AML cells comparable to CC885 or Mylotarg - in some cases, the efficacy was superior.

[0238] Example 11: Activity of anti-CD33 antibody-neodegrader conjugates on cells from AML patients The CD33AB-compound (Ia) conjugate was evaluated in vitro on several AML patient primary blast cells. Frozen bone marrow samples from adult AML patients collected after the last treatment were thawed and seeded into 96-well plates containing serial dilutions of the conjugate, Mylotarg or CC-90009. The plates were incubated for 48 hours at 37°C / 5% CO2, then red blood cells in each well were lysed and the remaining cells were stained with blast marker antibodies (the arbitrarily selected blast antibodies for each donor were pre-determined) along with Annexin V. Samples were analyzed by flow cytometry. Tumor cell survival was determined by the absolute number of viable tumor cells in each sample and normalized to the number in untreated wells. As shown in Figure 4, the anti-CD33 antibody-neodegrader conjugate showed superior activity, as measured by cytotoxicity, on patient-derived cells compared to standard of care treatment (Mylotarg) or the exploratory small molecule GSPT1 degrader (CC-90009).

[0239] Example 12: Activity of anti-CD33 antibody-neodegrader conjugates on human progenitor CFC proliferation Because CD33 is expressed on normal myeloid progenitor cell populations and normal cell myelosuppression is an adverse event (AE) observed with Mylotarg, the activity of CD33AB-Compound (Ia) conjugate, Mylotarg and CC-90009 on normal erythroid, myeloid and megakaryocytic progenitor cells was measured in a colony formation assay. Normal human bone marrow low density cells were thawed on the day of the experiment, washed and seeded into 24-well plates in XVivo 15 medium supplemented with rhIL-3 (10 ng / mL), rhGMCSF (10 ng / mL) and rhSCF (50 ng / mL). CD33AB-Compound I(a) conjugate, Mylotarg and CC-90009 were added to the wells and the cells were incubated for 72 hours. After incubation, 400 μL of each cell suspension was added to tubes containing methylcellulose-based medium supplemented with rhIL-3 (10 ng / mL), rh SCF (50 ng / mL), rhGM-CSF (10 ng / mL) and rhEpo (3 U / mL) for myeloid and erythroid progenitor cells. For megakaryocytic progenitor cells, cells were added to 35 mm dishes containing a semi-solid, collagen-based matrix supplemented with rhIL-3 (10 ng / mL), rhIL-6 (10 ng / mL) and rhTpo (50 ng / mL). After 14 days of incubation, colonies of myeloid and erythroid progenitor cells were evaluated microscopically. Colonies were divided into several categories based on size: CFU-E (colony forming unit-erythroid; this colony forming cell produces small colonies containing less than 200 erythroblasts) and BFU-E-erythroid progenitor (burst forming unit-erythroid; this is a more primitive colony forming cell that produces large colonies containing more than 200 erythroblasts), CFU-GM-granulocyte-monocyte progenitor (colony forming unit-granulocyte, macrophage; this bone marrow colony forming cell can produce colonies of 40 or more granulocyte-monocyte and / or macrophage cells) and CFU-GEMM-multipotent progenitor (colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte; this primitive colony forming cell can produce colonies containing erythroid cells and 20 or more granulocytes, macrophages and megakaryocytes).

[0240] For megakaryocytes, after 14 days of incubation, cells were transferred to glass slides, fixed, and stained with CD41 antibody and phosphodetection system. Colonies were divided into three categories according to size: CFU-Mk(3-20), CFU-Mk(21-49), and CFU-Mk(≧50). Results were normalized by colony counts in untreated samples and IC 50 was calculated using Prizm software, and the results are shown in Figure 5.

[0241] Example 13: Activity of anti-CD33 antibody-neodegrader conjugates in AML tumors compared to current standard of care Subcutaneous tumor model MV4-11 human acute myeloid leukemia cells (1 × 10 in 0.1 mL) 6 The mice were inoculated subcutaneously into the right flank of female athymic nude mice. 3 The mice were treated with TA either by intravenous injection into the lateral tail vein, intraperitoneal injection, oral gavage or a combination thereof, beginning when the tumor reached 100%. Tumor size and mouse weight were measured twice weekly. As shown in Figure 6, the CD33AB-Compound (Ia) conjugate demonstrated superior efficacy to the best available treatment options.

[0242] Example 14: Activity of anti-CD33 antibody-neodegrader conjugates in a disseminated AML model Because AML is a disease that has lesions in multiple bone marrow niches, circulates in the bloodstream, and is often disseminated throughout the patient, the activity of CD33AB-Compound (Ia) conjugates in disseminated AML in an in vivo model was tested.

[0243] Disseminated model MV4-11 cells (3 × 10 in 0.2 mL) 6 Cells) were injected intravenously into the lateral tail vein of female NCG mice. Treatment was initiated 13 days after tumor cell injection. Mice were checked daily for morbidity, mortality and clinical findings. Body weights were measured twice weekly. Weekly imaging analysis of tumor progression was performed under anesthesia by injection of 5 μL / g D-luciferin 10 min prior to bioluminescence imaging.

[0244] Disseminated model OCI-AML2 human acute myeloid leukemia cells (1 × 10 in 0.2 mL) 7 Cells) were injected intravenously into the lateral tail vein of female NOG mice. Treatment was initiated 9 days after tumor cell injection. Mice were checked daily for morbidity, mortality and clinical findings. Body weights were measured twice weekly. Weekly imaging analysis of tumor progression was performed under anesthesia by injection of 5 μL / g D-luciferin 10 min prior to bioluminescence imaging.

[0245] As shown in FIG. 7, a robust single-dose response was observed in both models (MV4-11 and OCI-AML2) that was accompanied by a sustained attenuation of luciferin signal, a marker for tumor cells.

[0246] Example 15: Degradation of GSPT1 by CD33AB-Compound (Ia) conjugate The mechanism of action of CD33AB-compound (Ia) conjugate was confirmed by monitoring the degradation of GSPT1 by Western blot. Whole cell lysates were prepared from MV4-11 CD33-positive AML cells treated with CD33AB-compound (Ia) conjugate, Neodegrader P1, CC-90009 or Mylotarg for 6, 12 and 18 hours, then proteins were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. GSPT1 was probed with a commercial rabbit-anti-GSPT1 antibody (Abcam) followed by an anti-rabbit HRP-conjugated secondary antibody (Cell Signaling Technology) and detected with a chemiluminescent substrate (ThermoFisher). The PVDF membrane was then stripped and re-probed with beta-Actin-HRP-conjugated antibody (Cell Signaling Technology). The results are shown in Figure 8. These data support that conjugation of Neodegrader P1 to CD33AB enhances intracellular exposure that drives selective GSPT1 degradation. Similar dose-dependent reductions in GSPT1 levels were seen with the conjugate and small molecule, Neodegrader P1 and CC-90009, as short as 6 hours post-administration. In contrast to the transient depletion seen with Neodegrader P1 and CC-90009 - showing a rebound in GSPT1 levels at 12 hours - treatment with the conjugate showed sustained depletion up to 18 hours post-administration. These data support prolonged exposure of the active payload following administration - consistent with a much broader response period and the potential for less frequent administration.

[0247] Example 16: Pharmacokinetic and pharmacodynamic efficacy of anti-CD33 antibody-Neodegrader conjugates The exposure and pharmacodynamic activity of the CD33AB-Compound (Ia) conjugate was evaluated at doses previously observed to establish tumor regression. In a subcutaneous tumor model, MV4-11 human acute myeloid leukemia cells (1×10 in 0.1 mL) were 6Cells) were inoculated subcutaneously into the right flank of female athymic nude mice. Mice were treated with CD33AB-Compound (Ia) at 0.5 mg / kg, 1 mg / kg, and 3 mg / kg via intravenous medial tail vein injection. Terminal cardiac puncture blood and tumors were collected from subcutaneous MV4-11 tumor model mice at pre-dose, 10 min, 30 min, 1 h, 6 h, 24 h, 72 h, and 120 h post-dose. Blood was processed to EDTA plasma and tumors were flash frozen in liquid nitrogen.

[0248] Pharmacokinetic analysis: Conjugate payload levels were quantified in plasma samples by LC-MS / MS. Processing included b-glucuronidase digestion to release the Neodegrader P1 payload from the conjugate, followed by LC-MS / MS analysis using protein precipitation and MRM acquisition to collect the free and released payload. The method was validated and met the accepted criteria for non-GLP bioanalytical quantitative LC-MS / MS for small molecule analysis according to linearity, specificity, carryover, precision and accuracy. Relevant pharmacokinetic parameters were calculated using WinNonLin (V8.3).

[0249] Pharmacodynamic analysis - Tumor tissues were homogenized using RIPA lysis buffer with protease and phosphatase inhibitors and Western blotting was performed using a polyclonal rabbit anti-GSPT1 antibody (Abcam ab126090). Consistent with the sustained depletion of GSPT1 seen in vitro, a single in vivo administration of the conjugate was sufficient to maintain reduced GSPT1 levels for up to 120 hours (Figure 9, top). Furthermore, conjugation of neodegrader P1 to CD33AB resulted in a sustained half-life of P1 of over 64 hours (Figure 9, bottom), supporting the potential for sustained exposure and infrequent dosing of the payload.

[0250] Example 17: Activity of anti-CD33 neodegrader conjugates against Mylotarg-insensitive cell lines The in vitro cytotoxicity of the test articles (TA) was measured using a panel of CD33-positive acute myeloid leukemia cell lines (AML193 and Kasumi-6) known to be Mylotarg-insensitive. Cells were seeded into 96-well plates at the designated concentrations and after overnight incubation at 37°C / 5% CO2, serial dilutions of each test article (TA) were added to the cells. Cells were incubated with the test articles for 72 hours and viability was detected with CellTiter-Glo® reagent (Promega). Luminescence values ​​were normalized for each cell line and IC 50 was calculated using Prizm software.

[0251] As shown in Figures 10A and 10B, the conjugate had good activity against both cell lines.

[0252] It is recognized that the Detailed Description section, and not the Summary and Abstract sections, are intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more, but not all, example aspects of the invention as contemplated by the inventors, and thus are not intended to limit the scope of the invention and the appended claims in any manner.

[0253] The present invention has been described above with the aid of functional components that illustrate the performance of certain functions and their interrelationships. The boundaries of these functional components have been arbitrarily defined herein for convenience of description. Other boundaries may be defined so long as the specified functions and their relationships are appropriately performed.

[0254] The above description of the specific embodiments fully reveals the generality of the invention so that others may easily modify and / or adapt such specific embodiments to various applications by application of knowledge within the skill of the art without undue experimentation without departing from the general concept of the invention. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments based on the teachings and guidance set forth herein. It should be understood that the expressions or terms herein are intended to be illustrative, not limiting, as the terms or terms herein should be interpreted by those skilled in the art in light of the teachings and guidance.

[0255] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. Formula (I): 【Chemical 1】 [wherein, a is from 1 to 10; L is 【Chemical Formula 2】 ; here 【Chemical Formula 3】 is the bonding point to the nitrogen atom; and 【Chemical Formula 4】 is the bonding point to Bm; and Bm is an antibody comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 9 and a light chain containing the amino acid sequence shown in SEQ ID NO: 10] or a pharmaceutically acceptable salt thereof.

2. The conjugate according to claim 1, wherein the conjugate is a compound of formula (I).

3. The conjugate according to claim 1, wherein the conjugate is a pharmaceutically acceptable salt of formula (I).

4. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein a is from 2 to 8.

5. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein a is from 3 to 5.

6. Formula (I): 【Chemical Formula 5】 [wherein, a is from 1 to 10; L is 【Chemical Formula 6】 ; here [Chemical Formula 7] is the bonding point to the nitrogen atom; and 【Chemical Formula 8】 is the bonding point to Bm; and Bm is an antibody or an antigen-binding portion thereof that can specifically bind to CD33] or a pharmaceutically acceptable salt thereof.

7. a is from 2 to 8, L is 【Chemical Formula 9】 ; The conjugate according to claim 6 or a pharmaceutically acceptable salt thereof.

8. The conjugate according to claim 7 or a pharmaceutically acceptable salt thereof, wherein a is from 3 to 5.

9. Bm is an antibody or an antigen-binding portion thereof comprising a heavy chain variable region (VH) complementarity determining region (CDR) 1 (VH-CDR1) containing the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, light chain variable region (VL) CDR1 (VL-CDR1) containing the amino acid sequence shown in SEQ ID NO: 5, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 6, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 7, the conjugate according to claim 6, 7, or 8 or a pharmaceutically acceptable salt thereof.

10. Bm is an antibody or an antigen-binding portion thereof comprising a VH containing the amino acid sequence shown in SEQ ID NO: 4 and a VL containing the amino acid sequence shown in SEQ ID NO: 8, the conjugate according to claim 6, 7, or 8 or a pharmaceutically acceptable salt thereof.

11. Bm is an antibody comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 9 and a light chain containing the amino acid sequence shown in SEQ ID NO: 10, the conjugate according to claim 6, 7, or 8 or a pharmaceutically acceptable salt thereof.

12. The conjugate according to claim 6, 7, or 8, wherein the conjugate is a compound of formula (I).

13. The conjugate according to claim 6, 7, or 8, wherein the conjugate is a pharmaceutically acceptable salt of formula (I).

14. The conjugate according to claim 6, 7, or 8, wherein L is covalently bonded to Bm via a sulfur atom in Bm.

15. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

16. A pharmaceutical composition for treating acute myeloid leukemia, comprising the conjugate according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

17. A pharmaceutical composition for treating myelodysplastic syndrome, comprising the conjugate according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical composition for treating cancer, comprising the conjugate according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

19. The pharmaceutical composition according to claim 18, wherein the cancer is breast cancer, gastric cancer, lymphoma, acute myeloid leukemia, multiple myeloma, head and neck cancer, squamous cell carcinoma, or hepatocellular carcinoma.

20. The conjugate according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof for use as a medicament.

21. A compound of formula (II): 【Chemical Formula 10】 or a pharmaceutically acceptable salt thereof.

22. The compound according to claim 21, wherein the compound has a structure of the formula: 【Chemical 11】

23. A compound of formula (III): or a pharmaceutically acceptable salt thereof. 【Chemical Formula 12】

24. The compound according to claim 23, wherein the compound has a structure of the formula:

25. 【Chemical 13】 A compound selected from the following group of compounds: [wherein, ND is a neodegrader compound and Bm is an antibody or an antigen-binding portion thereof] [wherein, ND is a neodegrader compound] 【Chemical 14】 and 【Chemical Formula 15】 【Chemical Formula 16】 【Chemical 17】 【Chemical Formula 18】 【Chemical Formula 19】 【Chemical 20】 or a pharmaceutically acceptable salt of any of them. 【Chemical Formula 21】 【Chemical 22】 【Chemical 23】

26. 【Chemical 24】 An antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

10.

27. A method for producing the conjugate according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, comprising reacting an antibody or an antigen-binding portion thereof with a compound of formula (I-1): [wherein, L' is 【Chemical 25】 ; here 【Chemical 26】 is a bonding point to a nitrogen atom] or a pharmaceutically acceptable salt thereof. 【Chemical 27】

28. ​ ​ The method of claim 27, further comprising reducing the antibody or antigen-binding portion thereof prior to the reaction of the antibody or antigen-binding portion thereof with the compound of formula (I-1).

29. L' is 【Chemical Formula 28】 The method of claim 27 or 28, wherein

30. Furthermore, Compound 18: 【Chemical 29】 and Compound 19: 【Chemical 30】 reacting to produce a compound of formula (I-1) or a pharmaceutically acceptable salt thereof, the method of claim 29.

31. Reaction Scheme 2: 【Chemical Formula 31】 The method of claim 29, further comprising producing a compound of formula (I-1) or a pharmaceutically acceptable salt thereof by a process comprising

32. (a) reacting Compound 12: 【Chemical 32】 with Compound 13: 【Chemical 33】 to produce Compound 14: 【Chemical 34】 ; (b) converting Compound 14: 【Chemical 35】 to Compound 15: 【Chemical 36】 ; (c) reacting Compound 15: 【Chemical 37】 with a compound having the formula: 【Chemical 38】 to produce Compound 16: 【Chemical Formula 39】 ; (d) reacting Compound 16: 【Chemical 40】 with a compound having the formula: 【Chemical Formula 41】 to produce Compound 17: 【Chemical 42】 ; and (e) converting Compound 17: 【Chemical 43】 to Compound 18: 【Chemical 44】 a production method selected from the group consisting of

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