Protac-antibody conjugates and methods of use

Abstract

To provide antibody-PROTAC conjugates (PACs), pharmaceutical compositions containing them, and their use in the treatment of diseases and conditions in which degradation of targeted protein is beneficial.SOLUTION: In one aspect, the subject matter described herein is directed to a PROTAC-antibody conjugate (PAC) of the formula Ab-(L1-D)p in which D is a PROTAC having the structure E3LB-L2-PB; in which E3LB is an E3 ligase binding group covalently bound to L2; L2 is a linker covalently bound to E3LB and PB; PB is a protein binding group covalently bound to L2; Ab is an antibody covalently bound to L1; L1 is a linker, covalently bound to Ab and to D; and p has a value from about 1 to about 8.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 339,257, filed May 20, 2016, the contents of which are incorporated herein by reference.

[0002] Reference to sequence listing submitted as a text file via EFS-WEB An official copy of the sequence listing has been submitted electronically via EFS-Web as an ASCII format sequence listing in a file named SEQLIST.TXT having a size of 77 kilobytes, created on May 20, 2016, and is being submitted contemporaneously herewith. The sequence listing contained in this ASCII format document is a part of the present specification and is incorporated herein by reference in its entirety. SEQ ID NOs: 1-6 have been intentionally omitted.

[0003] The subject matter described herein relates generally to antibody-(proteolysis-targeting chimera) (PROTAC) conjugate molecules that are useful for facilitating the intracellular degradation of targeted proteins. [Background technology]

[0004] Cellular maintenance and normal function require the controlled degradation of cellular proteins. For example, the degradation of regulatory proteins triggers events in the cell cycle, such as DNA replication, chromosome segregation, etc. Thus, such degradation of proteins affects cell proliferation, differentiation, and death.

[0005] While protein inhibitors can block or reduce protein activity in cells, protein degradation in cells can also reduce activity or completely eliminate target proteins. Thus, harnessing the cell's protein degradation pathways can provide a means to reduce or eliminate protein activity. One of the major degradation pathways in cells is known as the ubiquitin-proteasome system. In this system, proteins are labeled for degradation by the proteasome by ubiquitinating them. Protein ubiquitination is achieved by E3 ubiquitin ligases, which bind to proteins and add ubiquitin molecules to them. E3 ubiquitin ligases are part of a pathway that includes E1 and E2 ubiquitin ligases, which make ubiquitin available to the E3 ubiquitin ligase for addition to proteins.

[0006] To take advantage of this degradation pathway, PROTACs have been developed. PROTACs combine an E3 ubiquitin ligase with a protein that is to be marked for degradation. To facilitate the protein for degradation by the proteasome, PROTACs contain a group that binds to the E3 ubiquitin ligase and a group that binds to the protein that is desired to be degraded. These groups are typically connected by a linker. This molecular structure can bring the E3 ubiquitin ligase into close proximity with the protein so that the protein is ubiquitinated and marked for degradation.

[0007] There is a continuing need in the art for enhanced targeted delivery of PROTACs to cells containing protein targets.Targeted delivery using antibody-PROTAC conjugates can enhance the delivery of PROTACs to specific cells using the specificity of antibodies, and can also enhance the pharmacokinetics of the delivery of PROTACs to cells compared to other administration modes of PROTACs, such as injection. Summary of the Invention

[0008] In one aspect, the subject matter described herein is directed to a PROTAC-antibody conjugate (PAC) having the formula: Ab-(L1-D) p wherein D is a PROTAC having the structure E3LB-L2-PB, E3LB is an E3 ligase binding group covalently attached to L2, L2 is a linker covalently attached to E3LB and PB, PB is a protein binding group covalently attached to L2, Ab is an antibody covalently attached to L1, L1 is a linker covalently attached to Ab and D, and p has a value of about 1 to about 8.

[0009] Another aspect of the subject matter described herein is a pharmaceutical composition comprising a PAC and one or more pharma- ceutically acceptable excipients.

[0010] Another aspect of the subject matter described herein is the use of a PAC in a method for treating conditions and diseases by administering to a subject a pharmaceutical composition comprising the PAC.

[0011] Another aspect of the subject matter described herein is a method of making a PAC.

[0012] Another aspect of the subject matter described herein is an article comprising a pharmaceutical composition comprising a PAC, a container, and a package insert or label indicating that the pharmaceutical composition can be used to treat a disease or condition. In certain embodiments, for example, the following are provided: (Item 1) A conjugate having the following chemical structure: Ab-(L1-D) p During the ceremony, D is a PROTAC having the structure E3LB-L2-PB, E3LB is the E3 ligase binding group covalently attached to L2; L2 is a linker covalently linking E3LB and PB, PB is a protein-binding group covalently linked to L2; Ab is an antibody covalently linked to L1; L1 is a linker covalently connecting Ab and D; A conjugate, wherein p has a value of from about 1 to about 8. (Item 2) 2. The conjugate according to item 1, wherein E3LB is a group that binds to an E3 ligase, and the E3 ligase is listed in Tables 13 to 27. (Item 3) E3LB is a group that binds to an E3 ligase, the E3 ligase being selected from the group consisting of von Hippel-Lindau (VHL); cereblon; XIAP; E3A; MDM2; anaphase-promoting complex (APC); UBR5 (EDD1); SOCS / BC-box / eloBC / CUL5 / RING; LNXp80; CBX4; CBLL1; HACE1; HECTD1; HECTD2; HECTD3; HECW1; HECW2; HERC1; HERC2; HERC3; HERC4; HUWE1; ITCH; NEDD4; NEDD4L; PPIL2; PRPF19; PIAS1; PIAS2; PIAS3; PIAS4; RANBP2; RNF4; RBX1; SMURF1; SMURF2; STUB1; TOPORS;TRIP12;UBE3A;UBE3B;UBE3C;UBE4A;UBE4B;UBOX5;UBR5;WWP1;WWP2;Parkin;A20 / TNFAIP3;AMFR / gp78;ARA54;Beta-TrCP1 / BTRC;BRCA1;CBL;CHIP / STUB1 ;E6;E6AP / UBE3A;F-box protein 15 / FBXO15;FBXW7 / Cdc4;GRAIL / RNF128;HOIP / RNF31;cIAP-1 / HIAP-2;cIAP-2 / HIAP-1;cIAP(pan);ITCH / AIP4;KAP1;MARCH8;;Mind The conjugate described in item 1, selected from the group consisting of Bomb 1 / MIB1; Mind Bomb 2 / MIB2; MuRF1 / TRIM63; NDFIP1; NEDD4; NleL; Parkin; RNF2; RNF4; RNF8; RNF168; RNF43; SART1; Skp2; SMURF2; TRAF-1; TRAF-2; TRAF-3; TRAF-4; TRAF-5; TRAF-6; TRIM5; TRIM21; TRIM32; UBR5; and ZNRF3. (Item 4) 2. The conjugate according to item 1, wherein E3LB is a group that binds to an E3 ligase selected from the group consisting of XIAP, VHL, cereblon, and MDM2. (Item 5) 2. The conjugate according to item 1, wherein E3LB is selected from the group consisting of a compound that binds to VHL, a hydroxyproline compound that binds to VHL, a compound that binds to MDM2, a compound that binds to cereblon, the tetrahydro-benzodiazepinone nutrine, thalidomide, lenalidomide, and pomalidomide. (Item 6) E3LB is a XIAP inhibitor that is a tetrahydro-benzodiazepinone having the formula: [ka] The conjugate according to item 1, wherein R1, R2, R3, R4, and R5 are as described in WO / 2015 / 071393. (Item 7) PB, FoxOl, HDAC, DP-1, E2F, ABL, AMPK, BRK, BRSK I, BRSK2, BTK, CAMKK1, CAMKK, CAMKK, Rb, Suv39HI, SCF, p19INK4D, GSK-3, pi 8INK4, myc, ring E, CDK2, CDK9, CDG4 / 6, ring D, pl6 INK4A, cdc25A, BMI1, SCF, Akt, CHKl / 2, C1, CK1, C2, CLK2 CSK, DDR2, DYRK1A / 2 / 3, EF2K, EPH-A2 / A4 / B1 / B2 / B3 / B4, EIF2A 3, Smad2, Smad3, Smad4, Smad7, p53, p2 Cipl, PAX, Fyn, CAS, C3G, SOS, Tal, Raptor, RACK-1, CRK, Rapl, Rac, KRas, NRas, HRas, GRB2, FAK, PI 3K, Spred, Spry, mTOR, MPK, LKBl, PAK1 / 2 / 4 / 5 / 6, PDGFRA, PYK2, Src, SRPK1, PLC, PKC, PKA, and PKB / Pacific, PKC, PKD, PLKl, PRAK, PRK2, R IPK2, WAVE-2, TSC2, DAPKl, BAD, IMP, C-TAK1, TAK l TAOl TBK1 TESK1 TGFBR1 TIE2 TLK1 TrkA TSSK1 TTBK1 / 2 TTK Tpl2 / cotl MEK1 MEK2 PLDL Erkl, Erk2, Erk5, Erk8, p90RSK, PEA-15, SRF, p27KIP1, TIF la, HMGN1, ER81, MKP-3, c-Fos, FGF-R1, GCK, GSK3, HER4, HIPK1 / 2 / 3 / , IGF-1R, cdc25, UBF. LAMTOR2, Statl, StaO, CREB, JAK, Src, PTEN, NF-カパB, HECTH9, Bax, HSP70, HSP90, Apaf-1, Cyto c, BCL-2, Bcl-xL, Smac, XIAP, Libra-9 Plate-3, Plate-6, Plate-7, CDC37, TAB, IKK TRADD, TRAF2, R1P1, FLIP, TAKl, JNKl / 2 / 3, Lck, A-Raf, B-Raf, C-Raf, MOS, MLKl / 3, MNl / 2, MSKl, MST2 / 3 / 4, MPSK1, MEKKl, ME K4, MEL, ASK1, MINK1, MKK1 / 2 / 3 / 4 / 6 / 7, NE2a / 6 / 7, NUAK1, OSR1, SAP, STK33, Syk, Lyn, PDK1, PHK, PIM1 / 2 / 3, Ataxin-1, mTORCl, MDM2, p21Wafl, Cyclin Dl, Lamln A, Tpl2, Myc, Catenin, Wnt, IKK-beta, IKK-gamma, IKK-alpha, IKK-epsilon, ELK, p65RelA, IRAKI, IRA2, IRAK4, IRR, FADD, TRAF6, TRAF3, MKK3, MKK6, ROCK2, RSK1 / 2, SGK1, SmMLCK, SIK2 / 3, ULK1 / 2, VEGFR1, WNKl, YES1, ZAP70, MAP4K3, MAP4K5, MAPKlb, MAPKAP-K2 2. The conjugate according to item 1, wherein the group binds to K3, p38 alpha / beta / delta / gamma MAPK, Aurora A, Aurora B, Aurora C, MCAK, Clip, MAPKAPK, FAK, MARK1 / 2 / 3 / 4, Mucl, SHC, CXCR4, Gap-1, Myc, beta-catenin / TCF, Cbl, BRM, Mcl1, BRD2, BRD3, BRD4, AR, RAS, ErbB3, EGFR, IRE1, HPK1, RIPK2, and ERα (including all variants, mutations, splice variants, indels, and fusions thereof). (Item 8) 2. The conjugate according to item 1, wherein the PB is selected from the group consisting of heat shock protein 90 (HSP90) inhibitors, kinase and phosphatase inhibitors, MDM2 inhibitors, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting human BET bromodomain-containing proteins, aryl hydrocarbon receptor (AHR), REF receptor kinase, FKBP, androgen receptor (AR), estrogen receptor (ER), thyroid hormone receptor, HIV protease, HIV integrase, HCV protease, and acyl-protein thioesterase-1 and -2 (APT1 and APT2). (Item 9) 2. The conjugate according to item 1, wherein PB is a group that targets estrogen receptor alpha (ERa). (Item 10) 2. The conjugate of claim 1, wherein the Ab is a cysteine ​​engineered antibody or a variant thereof. (Item 11) Ab, DLL3, EDAR, CLL1;BMPR1B;E16;STEAP1;0772P;MPF;NaPi2b;Sema5b;PSCA The conjugate described in item 1, which binds to one or more of the polypeptides selected from the group consisting of hlg; ETBR; MSG783; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2; B7-H4; HER2; NCA; MDP; IL20Rα; brevican; EphB2R; ASLG659; PSCA; GEDA; BAFF-R; CD22; CD79a; CXCR5; HLA-DOB; P2X5; CD72; LY64; FcRH1; IRTA2; TENB2; PMEL17; TMEFF1; GDNF-Ra1; Ly6E; TMEM46; Ly6G6D; LGR5; RET; LY6K; GPR19; GPR54; ASPHD1; tyrosinase; TMEM118; GPR172A; MUC16 and CD33. (Item 12) 11. The conjugate of item 10, wherein the Ab binds to one or more of the polypeptides selected from the group consisting of CLL1, STEAP1, NaPi2b, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, B7-H4, HER2, CD22, CD79a, CD72, LY64, Ly6E, MUC16, and CD33. (Item 13) 13. The conjugate of item 12, wherein the Ab is an antibody that binds to one or more of the polypeptides selected from the group consisting of B7-H4, HER2, CLL1, CD33, CD22, and NaPi2b. (Item 14) 13. The conjugate according to item 12, wherein the antibody binds to HER2 or B7-H4. (Item 15) 15. The conjugate according to item 14, wherein the antibody binds to HER2. (Item 16) 2. The conjugate according to item 1, wherein L1 is a peptidomimetic linker. (Item 17) L1 is a peptidomimetic linker represented by the formula: -Str-(PM)-Sp- During the ceremony, Str is the Stretcher unit covalently attached to Ab, Ab is an antibody, Sp is a linkage or spacer unit covalently attached to the PROTAC moiety; PM is a non-peptide chemical moiety selected from the group consisting of: [ka] W is -NH-heterocycloalkyl- or heterocycloalkyl; Y is heteroaryl, aryl, -C(O)C 1 -C 6 Alkylene, C 1 -C 6 Alkylene-NH 2 , C 1 -C 6 Alkylene-NH-CH 3 , C 1 -C 6 Alkylene-N-(CH 3 ) 2 , C 1 -C 6 Alkenyl, or C 1 -C 6 is alkylenyl, Each R 1 is independent, C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , (C 1 -C 6 (alkyl)NHC(O)NH2 , (C 1 -C 10 (alkyl)NHC(NH)NH 2 , or (C 1 -C 10 (alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H, C 1 -C 10 Alkyl, C 1 -C 10 alkenyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 Let's get together and 3 -C 7 may form a cycloalkyl, R 4 and R 5 are each independently 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, arylalkyl, heteroarylalkyl, (C 1 -C 10 Alkyl)OCH 2 - or R 4 and R 5 Let's get together and 3 -C 7 17. The conjugate according to item 16, which may form a cycloalkyl ring. (Item 18) Y is heteroaryl and R 4 and R 5 18. The conjugate according to item 17, wherein together form a cyclobutyl ring. (Item 19) 18. The conjugate according to item 17, wherein Y is a moiety selected from the group consisting of: [ka] (Item 20) Str is a chemical moiety represented by the formula: [ka] In the formula, R 6 is C 1 -C 10 Alkylene, C 1 -C 10 Alkenyl, C 3 -C 8 Cycloalkyl, (C 1 -C 8 Alkylene)O- and C 1 -C 10 Alkylene-C(O)N(R a )-C 2 -C 6 alkylene, each alkylene being selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Each R is optionally substituted with 1 to 5 substituents selected from the group consisting of heterocycloalkyl, heteroarylalkyl, arylarylalkyl, heteroarylalkyl, and heteroaryl. a are independently H or C 1 -C 6 is alkyl, Sp is -C 1 -C 6 Alkylene-C(O)NH- or -Ar-R b -, Ar is aryl or heteroaryl, and R b is (C 1 -C 10 18. The conjugate according to item 17, wherein the alkylene group is O-. (Item 21) Str has the formula: [ka] In the formula, R 7 is C 1 -C10 Alkylene, C 1 -C 10 Alkenyl, (C 1 -C 10 alkylene)O-, N(R c )-(C 2 -C 6 alkylene)-N(R c ), and N(R c )-(C 2 -C 6 alkylene), each R c are independently H or C 1 -C 6 is alkyl, Sp is -C 1 -C 6 Alkylene-C(O)NH- or -Ar-R b -, Ar is aryl or heteroaryl, and R b is (C 1 -C 10 18. The conjugate according to item 17, wherein the alkylene group is O-. (Item 22) L1 has the formula: [ka] In the formula, R 1 is C 1 -C 6 Alkyl, C 1 -C 6 Alkenyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 or (C 1 -C 6 alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H, C 1 -C 10 18. The conjugate according to item 17, wherein the alkyl is alkyl. (Item 23) L1 has the formula: [ka] R 1 is C 1 -C 6 Alkyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 (alkyl)NHC(O)NH 2 and R 4 and R 5 Let's get together and 3 -C 7 18. The conjugate according to item 17, which forms a cycloalkyl ring. (Item 24) L1 has the formula: [ka] R 1 is C 1 -C 6 Alkyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 18. The conjugate according to item 17, (Item 25) having the formula [ka] During the ceremony, Sp is a linkage or spacer unit covalently attached to the PROTAC moiety D; Y is heteroaryl, aryl, -C(O)C 1 -C 6 Alkylene, C 1 -C 6 Alkylene-NH 2 , C 1 -C 6 Alkylene-NH-CH 3, C 1 -C 6 Alkylene-N-(CH 3 ) 2 , C 1 -C 6 Alkenyl, or C 1 -C 6 is alkylenyl, R 1 is independent, C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , (C 1 -C 6 alkyl)NHC(O)NH 2 , (C 1 -C 10 (alkyl)NHC(NH)NH 2 , or (C 1 -C 10 alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H, C 1 -C 10 Alkyl, C 1 -C 10 alkenyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 Let's get together and 3 -C 7 may form a cycloalkyl, Str is a chemical moiety represented by the formula: [ka] R 6 is C 1 -C 10 Alkylene and C 1 -C 10 Alkylene-C(O)N(R a )-C 2 -C 6alkylene, each alkylene being selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Each R is optionally substituted with 1 to 5 substituents selected from the group consisting of heterocycloalkyl, heteroarylalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl. a are independently H or C 1 -C 6 2. The conjugate according to claim 1, wherein n is alkyl and p is 1, 2, 3, or 4. (Item 26) Having [ka] During the ceremony, Sp is a linkage or spacer unit covalently attached to the PROTAC moiety D; R 4 and R 5 are each independently 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, arylalkyl, heteroarylalkyl, (C 1 -C 10 Alkyl)OCH 2 - or R 4 and R 5 Let's get together and 3 -C 7 may form a cycloalkyl ring, R 1 is independent, C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , (C 1 -C6 alkyl)NHC(O)NH 2 , (C 1 -C 10 (alkyl)NHC(NH)NH 2 , or (C 1 -C 10 alkyl)NHC(O)NH 2 and Str is a chemical moiety represented by the formula: [ka] R 6 is C 1 -C 10 Alkylene and C 1 -C 10 Alkylene-C(O)N(R a )-C 2 -C 6 alkylene, each alkylene being selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Each R is optionally substituted with 1 to 5 substituents selected from the group consisting of heterocycloalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl. a are independently H or C 1 -C 6 2. The conjugate according to claim 1, wherein n is alkyl and p is 1, 2, 3, or 4. (Item 27) Y is heteroaryl, aryl, or alkenyl; R 6 But, C 1 -C 10 26. The conjugate according to item 25, which is alkylene. (Item 28) 26. The conjugate according to item 25, wherein Y is: [ka] (Item 29) 26. The conjugate according to item 25, wherein Y is: [ka] (Item 30) 26. The conjugate according to item 25, wherein Y is: [ka] (Item 31) Str is a chemical moiety represented by the formula: [ka] R 6 is C 1 -C 6 is alkylene, Sp is -C 1 -C 6 Alkylene-C(O)NH- or -Ar-R b -, Ar is aryl, and R b is (C 1 -C 3 26. The conjugate according to item 25, wherein the alkylene)O- is aryl, alkylene)O-. (Item 32) having the formula [ka] During the ceremony, Y is heteroaryl, aryl, -C(O)C 1 -C 6 Alkylene, C 1 -C 6 Alkylene-NH 2 , C 1 -C 6 Alkylene-NH-CH 3 , C 1 -C 6 Alkylene-N-(CH 3 ) 2 , C1 -C 6 Alkenyl, or C 1 -C 6 is alkylenyl, R 1 is C 1 -C 6 Alkyl-NH 2 , (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H, C 1 -C 10 Alkyl, C 1 -C 10 alkenyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 Let's get together and 3 -C 7 may form a cycloalkyl, 2. The conjugate according to item 1, wherein p is 1, 2, 3, or 4. (Item 33) having the formula [ka] During the ceremony, p is 1, 2, 3, or 4; R 1 is C 1 -C 6 Alkyl-NH 2 , (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 and R 4 and R 5 are each independently 1 -C 6alkyl, said alkyl being unsubstituted or R 4 and R 5 is C 3 -C 7 The conjugate according to item 1, which may form a cycloalkyl ring. (Item 34) L1 has the formula: [ka] In the formula, R 1 and R 2 are independently H and C 1 -C 6 alkyl or R 1 and R 2 The conjugate according to item 1, wherein: (Item 35) 2. The conjugate according to item 1, selected from the group consisting of PAC1, PAC2, PAC3, PAC4 and PAC5. (Item 36) 2. The conjugate according to item 1, wherein p is about 1.0 to about 3. (Item 37) 2. The conjugate according to item 1, wherein p is about 2. (Item 38) 2. A pharmaceutical composition comprising the conjugate according to item 1 and one or more pharma- ceutically acceptable excipients. (Item 39) Item 40. A method for treating a disease in a human in need of treatment, comprising administering to said human an effective amount of the conjugate described in item 1 or the composition described in item 38. 40. The method of claim 39, wherein the disease is cancer. (Item 41) 41. The method of claim 40, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphoid neoplasms, lung cancer including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. (Item 42) 42. The method of claim 41, wherein the cancer is a HER2-positive cancer. (Item 43) 43. The method of claim 42, wherein the HER2-positive cancer is breast cancer or gastric cancer. (Item 44) 40. The method of claim 39, wherein the disease is an autoimmune disease. (Item 45) 45. The method of claim 44, wherein the autoimmune disease is selected from the group consisting of rheumatic disorders, osteoarthritis, autoimmune gastrointestinal and liver disorders, vasculitis, autoimmune neurological disorders, renal disorders, autoimmune skin disorders, blood disorders, atherosclerosis, uveitis, autoimmune hearing disorders, Behcet's disease, Raynaud's syndrome, organ transplantation, autoimmune endocrine disorders, Addison's disease, and autoimmune thyroid diseases. (Item 46) 46. ​​The method of claim 45, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis. [Brief description of the drawings]

[0013] [Figure 1] Detection of ER-α by Western blot is shown for PROTAC (no Ab), compound P1, and PROTAC-antibody conjugates (PAC) PAC1 and PAC2. [Diagram 2] Illustrates quantification of ERα as determined by fluorescence intensity for Endox-XIAP PACs treated for 3 days in engineered HER2-MCF7 lines. Media: 10% CS-FBS in RPMI without phenol red. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Disclosed herein are antibody-proteolysis targeting chimeric conjugates, herein referred to as PROTAC-antibody conjugates (PACs), useful for targeted protein degradation and the treatment of associated diseases and disorders. The subject matter described herein utilizes antibody targeting to direct the PROTAC to a target cell or tissue. As described herein, it has been shown that by linking an antibody with a PROTAC to form a PAC, the PROTAC is delivered to a target cell or tissue. As shown herein, for example in Examples 1 and 2, cells expressing an antigen can be targeted by an antigen-specific PAC, whereby the PROTAC portion of the PAC is delivered intracellularly to the target cell. Also shown herein, a PAC containing an antibody against an antigen not found on a cell does not result in significant intracellular delivery of the PROTAC to the cell.

[0015] Thus, the subject matter described herein is directed to a PROTAC-antibody conjugate (PAC) composition that results in the ubiquitination of a target protein and subsequent degradation of the protein. The composition comprises an antibody covalently linked to a linker (L1), which is covalently linked to a PROTAC at any available attachment point, and the PROTAC comprises an E3 ubiquitin ligase binding (E3LB) moiety that recognizes an E3 ubiquitin ligase protein and a protein binding moiety (PB) that recognizes the target protein. The subject matter described herein is useful for regulating protein activity and treating diseases and conditions associated with protein activity.

[0016] The subject matter of the present disclosure will be described more fully hereinafter. However, many modifications and other embodiments of the subject matter of the present disclosure described herein will occur to those skilled in the art to which the subject matter of the present disclosure belongs, having the benefit of the teachings presented in the following description. Therefore, it should be understood that the subject matter of the present disclosure is not limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein encompasses all alternatives, modifications, and equivalents. In the event that one or more of the incorporated documents, patents, and similar materials, including defined terms, term usage, described techniques, and the like, differ from or conflict with this application, this application shall take precedence. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0017] definition The term "PROTAC" generally refers to a proteolytically targeted chimeric molecule having three components: an E3 ubiquitin ligase binding group (E3LB), a linker L2, and a protein binding group (PB).

[0018] The terms "residue," "moiety," or "group" refer to a component that is covalently bound or linked to another component. For example, a "residue of a PROTAC" refers to a PROTAC covalently bound to one or more groups, such as a linker, L2, which itself may optionally be further bound to an antibody.

[0019] The terms "covalently bound" or "covalently linked" refer to a chemical bond formed by the sharing of one or more electron pairs.

[0020] As used herein, the term "peptidomimetic" or PM refers to a non-peptide chemical moiety. A peptide is a short chain of amino acid monomers linked by peptide (amide) bonds, a covalent chemical bond formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid. The shortest peptide is a dipeptide, consisting of two amino acids linked by a single peptide bond, followed by tripeptides, tetrapeptides, etc. A peptidomimetic chemical moiety includes a non-amino acid chemical moiety. A peptidomimetic chemical moiety may also include one or more amino acids separated by one or more non-amino acid chemical units. A peptidomimetic chemical moiety does not contain two or more adjacent amino acids linked by peptide bonds at any part in its chemical structure.

[0021] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has multiple binding sites, also called epitopes, that are recognized by CDRs (complementarity determining regions) on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may 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 may be derived from any species. However, in one embodiment, the immunoglobulins are of human, murine, or rabbit origin.

[0022] As used herein, the term "antibody fragment(s)" includes a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab') 2and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al (2004) Protein Eng. Design & Sel. 17(4):315-323), fragments produced by Fab expression libraries, anti-idio (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens; and multispecific antibodies formed from antibody fragments.

[0023] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible natural mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the subject matter described herein may be any of the monoclonal antibodies described in Kohler et al. Monoclonal antibodies may be produced by the hybridoma method first described by Clackson et al (1991) Nature, 256:495, or by recombinant DNA methods (see, for example, US4816567; US5807715). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597.

[0024] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homogeneous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class, and the remainder of the chain(s) is identical or homogeneous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, so long as they exhibit the desired biological activity (US4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0025] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remaining portions of the heavy and / or light chain are derived from a different source or species.

[0026] The "class" of an antibody refers to the type of constant domain or region carried by its heavy chain. There are five major classes of antibodies, IgA, IgD, IgE, IgG, and IgM, some of which are further subdivided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0027] As used herein, the term "intact antibody" includes VL and VH domains, as well as the light chain constant domain (CL) and the 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. An intact antibody may have one or more "effector functions," which refer to biological activities unique to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors such as B cell receptors and BCR.

[0028] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0029] As used herein, the term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains, FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0030] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that contains an Fc region as defined herein.

[0031] A "human antibody" refers to an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire, or other human antibody coding sequence. The definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0032] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized" form of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0033] An "isolated antibody" is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purification, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0034] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from its natural environment. Isolated nucleic acid refers to a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0035] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), and includes nucleic acid molecule(s) in a single vector or separate vectors, and nucleic acid molecule(s) present in one or more locations in a host cell.

[0036] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in a pharmaceutical formulation.

[0037] "Native antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0038] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0039] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's sequences of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0040] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact immunoglobulin 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 that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are known. Ig forms include hinge-modified or hingeless forms (Roux et al., 1999). et al(1998)J.Immunol.161:4083-4090;Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US2005 / 0048572; US2004 / 0229310).

[0041] The term "human consensus framework" as used herein refers to a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the human immunoglobulin VL or VH sequence is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup kappa III in Kabat et al. (supra).

[0042] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence as it, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0043] The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated using the VH or VL domain from an antibody to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0044] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs), the latter of which have the highest variability and / or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35B of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) With the exception of CDR1 in VH, CDRs generally include amino acid residues that form hypervariable loops. CDRs also include "specificity determining residues" or "SDRs," which are residues that contact the antigen. The SDRs are contained within regions of the CDRs referred to as abbreviated-CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3.(See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered according to Kabat et al. (supra).

[0045] "Effector function" refers to a biological activity specific to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0046] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.

[0047] "Epitope 4D5", or "4D5 epitope", or "4D5" is the region in the extracellular domain of HER2 to which antibody 4D5 (ATCC CRL10463) and trastuzumab bind. This epitope is proximal to the transmembrane domain of HER2 and is within domain IV of HER2. To screen for antibodies that bind to the 4D5 epitope, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed. Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the region from about residue 550 to about residue 610, including HER2 (SEQ ID NO: 39)).

[0048] "Epitope 2C4" or "2C4 epitope" is the region in the extracellular domain of HER2 to which antibody 2C4 binds. To screen for antibodies that bind to the 2C4 epitope, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed. Alternatively, epitope mapping can be performed to assess whether an antibody binds to the 2C4 epitope of HER2. Epitope 2C4 comprises residues from domain II in the extracellular domain of HER2. The 2C4 antibody and pertuzumab bind to the extracellular domain of HER2 at the junction of domains I, II, and III (Franklin et al. Cancer Cell 5:317-328 (2004)).

[0049] "Affinity" refers to the strength of the total number of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Certain illustrative and exemplary embodiments for measuring binding affinity are described below. In certain embodiments, the antibodies described herein have a dissociation constant of ≦1 μM, ≦100 nM, ≦10 nM, ≦5 nm, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 Less than M, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) has a dissociation constant (Kd)

[0050] An "affinity matured" antibody refers to an antibody that has one or more modifications in one or more hypervariable regions (HVRs), compared to a parent antibody that does not possess such modifications, which modifications result in improved affinity of the antibody for the antigen.

[0051] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0052] The term "free cysteine ​​amino acid" as used herein refers to a cysteine ​​amino acid residue engineered into a parent antibody, bearing a thiol functional group (-SH) and not paired as an intramolecular or intermolecular disulfide bridge. The term "amino acid" as used herein means glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, or citrulline.

[0053] The term "linker," "linker unit," or "link," as used herein, refers to a chemical moiety that comprises a chain of atoms that covalently bonds a PROTAC moiety to an antibody or a component of a PROTAC to another component of a PROTAC. In various embodiments, the linker is a divalent group designated as L1 or L2.

[0054] A "patient" or "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the patient, individual, or subject is a human. In some embodiments, the patient may be a "cancer patient," i.e., a patient suffering from or at risk of suffering from one or more symptoms of cancer.

[0055] "Patient population" refers to a group of cancer patients. Such populations can be used to demonstrate statistically significant efficacy and / or safety of a drug.

[0056] A "relapsed" patient is one who has signs or symptoms of cancer after remission. Optionally, the patient has relapsed after adjuvant or neoadjuvant therapy.

[0057] A cancer or biological sample that "exhibits HER expression, amplification, or activation" is one that expresses (including overexpresses) a HER receptor, has an amplified HER gene, and / or otherwise demonstrates activation or phosphorylation of a HER receptor in a diagnostic test.

[0058] "Neoadjuvant therapy" or "preoperative therapy" herein refers to therapy given before surgery. The purpose of neoadjuvant therapy is to provide immediate systemic treatment and potentially eradicate micrometastases that may otherwise grow if standard surgical procedures followed by systemic therapy are followed. Neoadjuvant therapy may also help reduce tumor size, thereby allowing complete removal of initially unresectable tumors, or preservation of parts of organs and their functions. Additionally, neoadjuvant therapy may allow in vivo evaluation of drug efficacy to guide subsequent treatment selection.

[0059] "Adjuvant therapy" as used herein refers to therapy given after definitive surgery when no evidence of residual disease is detected, to reduce the risk of disease recurrence. The purpose of adjuvant therapy is to prevent cancer recurrence, thus reducing the likelihood of cancer-related death. Adjuvant therapy as used herein specifically excludes neoadjuvant therapy.

[0060] "Definitive surgery" is used as a term in the medical community. Definitive surgery includes, for example, surgical or other procedures that lead to the removal or resection of a tumor, including those that lead to the removal or resection of all visible tumors. Definitive surgery includes, for example, complete or curative resection of a tumor, or complete macroscopic resection. Definitive surgery includes procedures that occur in one or more stages, for example, multi-stage surgical procedures in which one or more surgical or other procedures are performed before the resection of a tumor. Definitive surgery includes procedures to remove or resect a tumor, including associated organs, parts of organs and tissues, and surrounding organs, such as lymph nodes, parts of organs, or tissues. Removal may be incomplete, so that tumor cells may remain undetected.

[0061] "Survival" refers to a patient remaining alive and includes disease-free survival (DFS), progression-free survival (PFS), and overall survival (OS). Survival can be estimated by the Kaplan-Meier method, and any differences in survival are calculated by a hierarchical log-rank test.

[0062] "Progression-free survival" (PFS) is the time from day 1 of treatment to documented disease progression (including isolated CNS progression) or death from any cause on study, whichever occurs first.

[0063] "Disease-free survival (DFS)" refers to a patient remaining alive without cancer returning for a predefined period of time, such as about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, etc., from the start of treatment or from initial diagnosis. In one embodiment of the subject matter described herein, DFS is analyzed according to the intention-to-treat principle, i.e., patients are evaluated based on their assigned therapy. Events used in the analysis of DFS can include local, regional, and distant recurrence of cancer, occurrence of secondary cancer, and death from any cause in patients without prior events (e.g., breast cancer recurrence or second primary cancer).

[0064] "Overall survival" refers to a patient remaining alive for a defined period of time, e.g., about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, etc., from the start of treatment or from initial diagnosis.

[0065] By "prolonged survival" is meant an increase in DFS and / or OS in a treated patient relative to an untreated patient or relative to a control treatment protocol. Survival is monitored for at least about 6 months, or at least about 1 year, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc., after initiation of treatment or initial diagnosis.

[0066] "Monotherapy" means a treatment regimen that includes only a single therapeutic agent for the treatment of a cancer or tumor during the treatment period.

[0067] "Maintenance therapy" refers to a treatment regimen given to reduce the possibility of disease recurrence or progression. Maintenance therapy can be provided for any period of time, including up to the lifetime of a subject. Maintenance therapy can be provided after initial therapy, or in conjunction with initial therapy or additional therapy. The dosage used for maintenance therapy can vary and can include smaller dosages compared to the dosages used for both other types of therapy.

[0068] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0069] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. A "tumor" contains one or more cancerous cells. Examples of cancer are provided elsewhere herein.

[0070] "HER2 positive" cancers include cancer cells with higher than normal levels of HER2. HER2 positive cancers include HER2 positive breast cancer and HER2 positive gastric cancer. Optionally, HER2 positive cancers have an immunohistochemistry (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification of ≧2.0. The term "HER2 positive cell" refers to a cell that expresses HER2 on its surface.

[0071] The term "early stage breast cancer (EBC)" or "early breast cancer" is used herein to refer to breast cancer that has not spread beyond the breast or axillary lymph nodes. This includes ductal carcinoma in situ, as well as stages I, IIA, IIB, and IIIA breast cancer.

[0072] Reference to a tumor or cancer as "Stage 0", "Stage I", "Stage II", "Stage III", or "Stage IV", and various substages within this classification, indicates the classification of the tumor or cancer using the Overall Stage Grouping or Roman Numeral Staging methods known in the art. Although the actual stage of the cancer depends on the type of cancer, generally, stage 0 cancers are in situ lesions, stage I cancers are small localized tumors, stage II and stage III cancers are aggressive localized tumors exhibiting regional lymph node involvement, and stage IV cancers represent metastatic cancers. The specific stages of each type of tumor are known to the skilled clinician.

[0073] The term "metastatic breast cancer" refers to a condition of breast cancer in which cancer cells have spread by blood or lymph from the primary site to one or more other sites in the body and have formed one or more secondary tumors in one or more organs other than the breast.

[0074] An "advanced" cancer is one that has spread outside the primary site or organ, either by local invasion or metastasis. Thus, the term "advanced" cancer includes both locally advanced and metastatic disease. A "recurrent" cancer is one that has regrowth, either at the primary site or at a distant site, after responding to initial treatment, e.g., surgery. A "locally recurrent" cancer is one that returns, after treatment, to the same location as the previously treated cancer. An "operable" or "resectable" cancer is one that is confined to the organ of origin and is suitable for surgery (resection). A "non-resectable" or "unresectable" cancer cannot be removed (resected) by surgery.

[0075] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211、 I 131、 I 125、 Y 90、 Re 186、 Re 188 , Sm 153, Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleases; antibiotics; small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various anti-tumor and anti-cancer agents disclosed below.

[0076] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analog topotecan (HYCAMTIN®), CPT -11 (including irinotecan, CAMPTOSAR®, acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs KW-2189 and CB1-TM 1); erytherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, nitrogen mustards such as uracil mustard;nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin γ1I and calicheamicin ω1I (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); the oral α4 integrin inhibitor CDP323; the dynemicins, including dynemicin A; esperamicins; and neocarzinostatin chromophore and related enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, doxorubicin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposomal (DOXIL®), liposomal doxorubicin TLC D-99 (including MYOCET®, pegylated liposomal doxorubicin (CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, ubicin), streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; frolinic acid folic acid supplements such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids such as maytansine and ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, rosoxantrone, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex (JHS Natural Products (Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 22'2' trichlorotriethylamine; trichothecines (especially the T2 toxins verracurin A, roridin A and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin engineered nanoparticle formulations of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas that prevent tubulin polymerization to form microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone. ; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), (registered trademark), bisphosphonates such as pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways implicated in cell proliferation disorders, such as, for example, PKCα, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); THERATOPE® vaccines and gene therapy vaccines, Vaccines such as, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341);Bcl-2 inhibitors such as bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; oblimersen sodium (GENASENSE®, an antisense oligonucleotide); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors; serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); lonafarnib (SCH 6636, SARASAR™); and pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (an abbreviation for a treatment regimen with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™));

[0077] Chemotherapeutic agents, as defined herein, include "anti-hormonal agents" or "endocrine therapy" agents that act to regulate, reduce, block, or inhibit the action of hormones that may promote the growth of cancer.They may themselves be hormones, such as antiestrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxyfene, ketoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; fulvestrant (FASLODEX®); )) and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrozole (ARIMIDEX®), letrozole (FEMARA®), and aminoglutethimide. Aromatase inhibitors, including but not limited to vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazole; luteinizing hormone releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; progestins, such as megestrol acetate and medroxyprogesterone acetate, diethromycin, erythromycin, and cefotaxime; These include, but are not limited to, sex steroids, including estrogens such as tilstilbestrol and premarin, and androgens / retinoids such as fluoxymesterone, all-trans retinoic acid, and fenretinide; onapristone; antiprogesterone agents; estrogen receptor down-modulators (ERDs); antiandrogens such as flutamide, nilutamide, and bicalutamide; and pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0078] The term "immunosuppressant" as used herein for adjunctive therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This includes substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); anti-inflammatory agents such as ganciclovir, tacrolimus, glucocorticoids such as cortisol or aldosterone, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, leukotriene receptor antagonists; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde (to mask MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies against MHC antigens and MHC fragments; cyclosporine A; corticosteroids such as corticosteroids or glucocorticosteroids, or glucocorticoid analogs, e.g., prednisone, SOLU-MEDROL®, methylprednisolone, including methylprednisolone sodium succinate, and dexamethasone; dihydrofolate reductase inhibitors, such as methotrexate (oral or subcutaneous); antimalarials, such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; anti-interferon alpha, beta, or gamma antibodies, anti-tumor necrosis factor (TNF) alpha antibodies (infliximab (REMICADE®) or adalimumab), anti-TNF alpha immunoadhesins (Ebola, erythrocyte sedimentation syndrome (ESS), erythrocyte sedimentation syndrome (EC ... tanercept), anti-TNFβ antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptor antibodies and antagonists (such as ACTEMRA™ (tocilizumab)); anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; xenogeneic antilymphocyte globulins; pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies;Soluble peptides containing the LFA-3 binding domain (WO 90 / 08187 published July 26, 1990); streptokinase; transforming growth factor beta (TGF-β); streptodornase; RNA or DNA derived from a host; FK506; RS-61443; chlorambucil; deoxyspergualin; rapamycin; T cell receptors (Cohen et al., U.S. Pat. No. 5,114,721); T cell receptor fragments (Offner et al., Science, 251:430-432 (1991); WO 90 / 11294; Ianeway, Nature, 341:482 (1989); and WO 91 / 01133); BAFF antibodies, and BAFF antagonists such as BR3 antibodies, and zTNF4 antagonists (for review, see Mackay and Mackay, Trends Immunol., 23:113-5 (2002), and see also definitions below; biological agents that interfere with T cell helper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD154) (e.g., Durie et al., Science, 261:1328-30 (1993); Mohan et al., J. Immunol., 154:1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265:1225-7 (1994)), including blocking antibodies against CD40-CD40 ligand; and T cell receptor antibodies such as T10B9 (EP340,109). Some preferred immunosuppressants herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.;

[0079] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention aimed at altering the natural history of the individual being treated, and may be performed for prophylaxis or during the clinical pathology process. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or remission of disease, and remission or improvement of prognosis. In some embodiments, the subject antibodies described herein are used to delay the onset of disease or slow the progression of disease.

[0080] A drug that is administered "concurrently" with one or more other drugs is administered on the same treatment day as the one or more other drugs, during the same treatment cycle, and, optionally, simultaneously with the one or more other drugs. For example, in the case of a cancer therapy given every three weeks, the drugs that are administered simultaneously are each administered on day 1 of a three-week cycle.

[0081] An "effective amount" of a drug, e.g., a pharmaceutical preparation, refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic or preventive result. For example, an effective amount of a drug for treating cancer can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that a drug can prevent the growth of existing cancer cells and / or kill them, the drug can be cytostatic and / or cytotoxic. An effective amount can prolong progression-free survival (e.g., as measured by Response Evaluation in Solid Tumors (RECIST) or CA-125 change), produce an objective response (including partial response (PR) or complete response (CR)), increase overall survival, and / or improve one or more symptoms of cancer (e.g., as measured by FOSI).

[0082] As used herein, the term "therapeutically effective amount" refers to any amount that causes a treatment of a disease, disorder, or side effect, or reduces the rate of progression of a disease or disorder, compared to a corresponding subject that does not receive such amount. This term also includes within its scope an amount that is effective for improving normal physiological function. For use in therapy, a therapeutically effective amount of PAC, as well as its salts, may be administered as raw chemicals. In addition, the active ingredient may be presented as a pharmaceutical composition.

[0083] As used herein, unless otherwise defined in the claims, the term "optionally" means that the event(s) recited thereafter may or may not occur, and includes both the event(s) that occur and the event(s) that do not occur.

[0084] As used herein, unless otherwise defined, the phrases "optionally substituted," "substituted," or variations thereof, refer to optional substitution, including multiple types of substitution with one or more, e.g., one, two, or three, substituents. This phrase should not be construed as redundant with the substitutions described and depicted herein.

[0085] The term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective, and that does not contain any additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0086] "Pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, carriers, stabilizers, or preservatives.

[0087] The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of a molecule. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counter ion. A counter ion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter ions.

[0088] Other salts that are not pharmaceutically acceptable may be useful in the preparation of the compounds described herein and should be considered as forming further aspects of the subject matter. These salts, such as oxalic acid or trifluoroacetic acid salts, while not themselves pharmaceutically acceptable, may be useful in the preparation of salts as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable salts.

[0089] As used herein, the term "plurality" refers to two or more conjugates. Each conjugate may be the same or different from any other conjugate in the plurality.

[0090] A "small molecule" or "small molecule compound" generally refers to an organic molecule that is less than about 5 kilodaltons (Kd) in size. In some embodiments, a small molecule is less than about 4 Kd, 3 Kd, about 2 Kd, or about 1 Kd. In some embodiments, a small molecule is less than about 800 Daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is non-polymeric. A small molecule is not a protein, polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide, polysaccharide, glycoprotein, proteoglycan, or the like. A derivative of a small molecule refers to a molecule that shares the same structural core as the original small molecule, but can be prepared from the original small molecule by a series of chemical reactions.

[0091] The term "alkyl" as used herein refers to an alkyl group having 1 to 12 carbon atoms (C 1 -C 12 In another embodiment, an alkyl group is an alkyl group having 1 to 8 carbon atoms (C 1 -C 8 ) or 1 to 6 carbon atoms (C 1 -C 6 Examples of alkyl groups are methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-Butyl (n-Bu, n-Butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2 CH(CH 3 ) 2 ), 2-Butyl (s-Bu, s-Butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl-2-butyl (-C(CH 3 ) 2 CH 2 CH 3 ), 3-methyl-2-butyl (-CH(CH 3 )CH(CH 3 ) 2 ), 3-methyl-1-butyl (-CH 2 CH 2 CH(CH 3 ) 2 ), 2-methyl-1-butyl (-CH 2 CH(CH 3 )CH 2 CH 3 ), 1-hexyl (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 ), 3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 )), 2-methyl-2-pentyl (-C(CH3 ) 2 CH 2 CH 2 CH 3 ), 3-methyl-2-pentyl (-CH(CH 3 )CH(CH 3 )CH 2 CH 3 ), 4-methyl-2-pentyl (-CH(CH 3 )CH 2 CH(CH 3 ) 2 ), 3-methyl-3-pentyl (-C(CH 3 )(CH 2 CH 3 ) 2 ), 2-methyl-3-pentyl (-CH(CH 2 CH 3 )CH(CH 3 ) 2 ), 2,3-dimethyl-2-butyl (-C(CH 3 ) 2 CH(CH 3 ) 2 ), 3,3-dimethyl-2-butyl (-CH(CH 3 )C(CH 3 ) 3 Examples of aryl groups include, but are not limited to, 1-heptyl, 1-octyl, and the like.

[0092] The term "alkylene" as used herein refers to an alkylene having 1 to 12 carbon atoms (C 1 -C 12 In another embodiment, an alkylene group is a divalent saturated straight or branched chain hydrocarbon group of any length from 1 to 8 carbon atoms (C 1 -C 8 ) or 1 to 6 carbon atoms (C 1 -C 6 An example of an alkylene group is methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2-), but are not limited to these.

[0093] The term "alkenyl" refers to an alkyl group having at least one site of unsaturation, i.e., a carbon-carbon sp 2 2-8 carbon atoms (C 2 -C 8 "Alkenyl" refers to a monovalent straight or branched chain hydrocarbon radical of any length, which may be optionally substituted independently with one or more substituents described herein, including radicals having "cis" and "trans" orientations, or, alternatively, "E" and "Z" orientations. Examples include ethylenyl or vinyl (-CH=CH 2 ), allyl (-CH 2 CH=CH 2 ), but are not limited to these.

[0094] The term "alkenylene" refers to an alkyl group having at least one site of unsaturation, i.e., a carbon-carbon sp 2 2-8 carbon atoms (C 2 -C 8 "Alkenylene" refers to a divalent straight or branched chain hydrocarbon radical of any length, which may be optionally substituted independently with one or more substituents described herein, and includes radicals having "cis" and "trans" orientations, or, alternatively, "E" and "Z" orientations. Examples include ethylenylene or vinylene (-CH=CH-), allyl (-CH 2 Examples include, but are not limited to, CH=CH-.

[0095] The term "alkynyl" refers to an alkynyl group having 2 to 8 carbon atoms (C 2 -C 8 ) which is an alkynyl group that may be optionally substituted independently with one or more substituents described herein. Examples include ethynyl (-C≡CH), propynyl (propargyl, -CH 2C≡CH), but are not limited to these.

[0096] The term "alkynylene" refers to an alkynylene having 2 to 8 carbon atoms (C 2 -C 8 alkynylene refers to a divalent straight or branched chain hydrocarbon radical of any length, which may be optionally substituted independently with one or more substituents described herein. Examples include ethynylene (-C≡C-), propynylene (propargylene, -CH 2 C≡C-), but are not limited to these.

[0097] The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring”, and “cycloalkyl” refer to rings having 3 to 12 carbon atoms (C 3 -C 12 ), or as a bicyclic ring. Bicyclic carbocycles having 7 to 12 atoms may be arranged, for example, as bicyclo[4,5], [5,5], [5,6], or [6,6] systems, and bicyclic carbocycles having 9 or 10 ring atoms may be arranged as bicyclo[5,6] or [6,6] systems, or as bridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. Spiro moieties are also included within the scope of this definition. Examples of monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, etc. Carbocyclyl groups are optionally substituted independently with one or more substituents described herein.

[0098] "Aryl" means an aromatic ring system having 6 to 20 carbon atoms (C 6 -C 20 ) monovalent aromatic hydrocarbon radical. Some aryl groups are represented in the exemplary structures as "Ar". Aryl includes bicyclic groups containing an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic ring. Typical aryl groups include, but are not limited to, groups derived from benzene (phenyl), substituted benzene, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. Aryl groups are optionally substituted independently with one or more substituents described herein.

[0099] "Arylene" means an aryl group of 6 to 20 carbon atoms (C 6 -C 20 ) divalent aromatic hydrocarbon radical. Some arylene groups are represented in the exemplary structures as "Ar". Arylene includes bicyclic groups that contain an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic ring. Typical arylene groups include, but are not limited to, groups derived from benzene (phenylene), substituted benzene, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionally substituted with one or more substituents described herein.

[0100] The terms "heterocycle", "heterocyclyl", and "heterocyclic ring" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic group of 3 to about 20 ring atoms, in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms are C, and one or more ring atoms are optionally independently substituted with one or more substituents described below. A heterocycle can be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S), or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), such as a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7, and 9; "The "Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley&Sons, New York, 1950 to present), particularly rings 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. "Heterocyclyl" also includes groups in which a heterocyclic group is fused to a saturated, partially unsaturated ring, or an aromatic carbocyclic or heterocyclic ring. Examples of heterocyclic rings include morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, Examples of the aryl group include, but are not limited to, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl imidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl, and N-pyridyl urea. Spiro moieties are also included within the scope of this definition. Examples of heterocyclic groups in which two ring atoms are substituted with oxo (=O) moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.

[0101] The term "heteroaryl" refers to a monovalent aromatic group of 5, 6, or 7 rings, including fused ring systems of 5 to 20 atoms (at least one of which is aromatic) containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, 1-methyl-1H-benzo[d]imidazole, [1,2,4]triazolo[1,5-a]pyridine, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, and quinolinyl. Heteroaryl groups are optionally substituted or unsubstituted with one or more independently selected heteroaryl groups, such as isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

[0102] The heterocycle or heteroaryl group may be carbon (carbon-linked) or nitrogen (nitrogen-linked) linked, where possible. By way of example and not limitation, the carbon-linked heterocycle or heteroaryl is bonded at the 2-, 3-, 4-, 5-, or 6-position of pyridine, the 3-, 4-, 5-, or 6-position of pyridazine, the 2-, 4-, 5-, or 6-position of pyrimidine, the 2-, 3-, 5-, or 6-position of pyrazine, the 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2-, 4-, or 5-position of oxazole, imidazole, or thiazole, the 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole, the 2- or 3-position of aziridine, the 2-, 3-, or 4-position of azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline.

[0103] By way of example and not limitation, nitrogen-linked heterocycles or heteroaryls are bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or β-carboline.

[0104] The term "chiral" refers to molecules that have the property of not being superimposable on their mirror image partners, while the term "achiral" refers to molecules that are superimposable on their mirror image partners.

[0105] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0106] "Diastereomer" refers to a stereoisomer with two or more centers of asymmetry and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can separate under high resolution analytical techniques such as electrophoresis and chromatography.

[0107] "Enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0108] The stereoscientific definitions and conventions used herein are generally those set forth in S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to indicate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. Compounds with a (+) or d prefix are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0109] Other terms, definitions, and abbreviations herein include: wild type ("WT"); cysteine ​​engineered mutant antibody ("Thio"); light chain ("LC"); heavy chain ("HC"); 6-maleimidocaproyl ("MC"); maleimidopropanoyl ("MP"); valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyl ("PAB"), and p-aminobenzyloxycarbonyl ("PABC"); A118C (EU numbering) = A121C (sequential numbering) = A114C (Kabat numbering) in the heavy chain and K149C (Kabat numbering) in the light chain. Still further definitions and abbreviations are provided elsewhere herein.

[0110] II. PROTAC-Antibody Conjugates (PACs) The PROTAC-antibody conjugate (PAC) molecules described herein comprise an antibody conjugated via a linker (L1) to a PROTAC, where the PROTAC comprises a ubiquitin E3 ligase binding group ("E3LB"), a linker ("L2"), and a protein binding group ("PB"). The general formula of a PAC is: Ab-(L1-D) p where D is a PROTAC having the structure E3LB-L2-PB, E3LB is an E3 ligase binding group covalently attached to L2, L2 is a linker covalently attached to E3LB and PB, PB is a protein binding group covalently attached to L2, Ab is an antibody covalently attached to L1, L1 is a linker covalently attached to Ab and D, and p has a value of about 1 to about 50. The variable p reflects that an antibody may be attached to one or more L1-D groups. In one embodiment, p is about 1 to 8. In another embodiment, p is about 2.

[0111] The following sections describe the components that make up the PAC. In order to obtain a PAC with potent efficacy and a desirable therapeutic index, the following components are provided:

[0112] 1. Antibody (Ab) As described herein, antibodies, e.g., monoclonal antibodies (mABs), are used to deliver PROTACs to target cells, e.g., cells expressing a specific protein targeted by the antibody. The antibody portion of the PAC can target cells expressing the antigen, whereby the antigen-specific PAC is delivered intracellularly to the target cell, typically through endocytosis. PACs that include antibodies against antigens not found on the cell surface may result in less specific intracellular delivery of the PROTAC moiety into the cell, while the PAC may still be pinocytosed. The PACs and their methods of use described herein advantageously utilize antibody recognition of cell surfaces and / or endocytosis of the PAC to deliver the PROTAC moiety to the interior of the cell.

[0113] A human antibody In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in detail in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. .Immunol.20:450-459(2008).

[0114] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). (See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Application Publication No. US2007 / 0061900, which describes VELOCIMOUSE® technology.) The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0115] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies produced by human B-cell hybridoma technology have also been described by Li et al. et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0116] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0117] b. Library-derived antibodies Antibodies for use in PACs can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and are described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0118] In one particular phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene portions from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0119] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0120] c. Chimeric and Humanized Antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody, which is an antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0121] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, in which the HVRs, e.g., CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0122] Humanized antibodies and methods for making them are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0123] Human framework regions that may be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0124] d. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. The term "multispecific antibody" as used herein refers to an antibody that contains an antigen-binding domain with polyepitopic specificity (i.e., capable of binding to two or more different epitopes on one molecule or capable of binding to epitopes on two or more different molecules).

[0125] In some embodiments, a multispecific antibody is a monoclonal antibody (e.g., a bispecific antibody) that has binding specificities for at least two different antigen-binding sites. In some embodiments, the first and second antigen-binding domains of a multispecific antibody may bind to two epitopes within one and the same molecule (intramolecular binding). For example, the first and second antigen-binding domains of a multispecific antibody may bind to two different epitopes on the same protein molecule. In certain embodiments, the two different epitopes that a multispecific antibody binds are epitopes that are not normally bound identically by one monospecific antibody, such as a conventional antibody or one immunoglobulin single variable domain. In some embodiments, the first and second antigen-binding domains of a multispecific antibody may bind to epitopes located within two different molecules (intermolecular binding). For example, a first antigen-binding domain of a multispecific antibody may bind to one epitope on one protein molecule, while a second antigen-binding domain of the multispecific antibody may bind to another epitope on a different protein molecule, thereby cross-linking the two molecules.

[0126] In some embodiments, the antigen-binding domain of a multispecific antibody (e.g., a bispecific antibody) comprises two VH / VL units, a first VH / VL unit that binds a first epitope and a second VH / VL unit that binds a second epitope, each VH / VL unit comprising a heavy chain variable domain (VH) and a light chain variable domain (VL). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, and antibody fragments (e.g., Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked antibody fragments). A VH / VL unit that further comprises at least a portion of a heavy chain variable region and / or at least a portion of a light chain variable region may also be referred to as an "arm", or a "hemimer", or a "half antibody". In some embodiments, a hemimer comprises sufficient heavy chain variable region to allow it to form an intermolecular disulfide bond with a second hemimer. In some embodiments, a hemimer contains a knob or hole mutation, e.g., to allow heterodimerization with a second hemimer or half antibody that contains a complementary hole or knob mutation. Knob and hole mutations are discussed further below.

[0127] In certain embodiments, the multispecific antibodies provided herein are bispecific antibodies. The term "bispecific antibodies" as used herein refers to multispecific antibodies that contain antigen-binding domains that can bind to two different epitopes on one molecule or that can bind to epitopes on two different molecules. Bispecific antibodies may also be referred to herein as having "dual specificity" or being "dual specific." Exemplary bispecific antibodies may bind to both a protein and any other antigen. In certain embodiments, one of the binding specificities is for a protein and the other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certain embodiments, bispecific antibodies may bind to two different epitopes on the same protein molecule. In certain embodiments, bispecific antibodies may bind to two different epitopes on two different protein molecules. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing the protein. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0128] Techniques for producing multispecific antibodies include recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (Milstein and Cuello, Nature 305:537 (1983), WO93 / 08829, and Traunecker et al., J. Immunol. 1999, 14:131-132 (2003)). et al., EMBO J. 10:3655 (1991)), as well as "knobs-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168, WO 2009 / 089004, US 2009 / 0182127, US 2011 / 0287009, Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, and Kontermann (2005) Acta Pharmacol. Sin., 26:1-9). The term "knobs-in-hole" or "KnH" technology, as used herein, refers to a technique that directs the pairing of two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface where they interact. For example, KnH has been introduced at the Fc:Fc binding surface, the CL:CH1 binding surface, or the VH / VL interface of an antibody (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, Zhu et al., 1997, Protein Science 6:781-788, and WO2012 / 106587). In some embodiments, KnH drives the pairing of two different heavy chains together during the production of multispecific antibodies. For example, multispecific antibodies with KnH in their Fc regions may further comprise a single variable domain attached to each Fc region, or may further comprise different heavy chain variable domains paired with similar or different light chain variable domains. The KnH technology can also be used to pair two different receptor extracellular domains together, or any other polypeptide sequence that contains different target recognition sequences (e.g., affibodies, peptibodies, and other Fc fusions).

[0129] The term "knob mutation," as used herein, refers to a mutation that introduces a protuberance (knob) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

[0130] The term "hole mutation," as used herein, refers to a mutation that introduces a cavity (hole) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0131] A "bulge" refers to at least one amino acid side chain that protrudes from the interface of a first polypeptide and thus can be positioned in a complementary cavity in an adjacent interface (i.e., the interface of a second polypeptide) to stabilize a heteromultimer, thereby, for example, favoring heteromultimer formation over homomultimer formation. The bulge can be present in the original interface or can be synthetically introduced (e.g., by modifying the nucleic acid encoding the interface). In some embodiments, the nucleic acid encoding the interface of the first polypeptide is modified to encode a bulge. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with a nucleic acid encoding at least one "import" amino acid residue having a side chain mass greater than the original amino acid residue. It will be understood that there may be more than one original residue and a corresponding import residue. The side chain masses of various amino acid residues are shown, for example, in Table 1 of US2011 / 0287009. A mutation that introduces a "bulge" may be referred to as a "knob mutation."

[0132] In some embodiments, the import residue for formation of the bulge is a naturally occurring amino acid residue selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, the import residue is tryptophan or tyrosine. In some embodiments, the original residue for formation of the bulge has a small side chain mass, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0133] A "cavity" refers to at least one amino acid side chain that is recessed from the interface of the second polypeptide and thus accommodates a corresponding protuberance on the adjacent interface of the first polypeptide. The cavity may be present in the original interface or may be synthetically introduced (e.g., by modifying the nucleic acid encoding the interface). In some embodiments, the nucleic acid encoding the interface of the second polypeptide is modified to encode the cavity. To accomplish this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one "import" amino acid residue that has a smaller side chain mass than the original amino acid residue. It will be understood that there may be more than one original residue and a corresponding import residue. In some embodiments, the import residue for the formation of the cavity is a naturally occurring amino acid residue selected from alanine (A), serine (S), threonine (T), and valine (V). In some embodiments, the import residue is serine, alanine, or threonine. In some embodiments, the original residue for the formation of the cavity has a large side chain mass, such as tyrosine, arginine, phenylalanine, or tryptophan. A mutation that introduces a "cavity" may be referred to as a "hole mutation."

[0134] The protuberance is "positionable" in the cavity, which means that the spatial location of the protuberance and the cavity on the interface of the first and second polypeptides, respectively, and the size of the protuberance and the cavity are such that the protuberance can be positioned in the cavity without significantly disrupting the normal association of the first and second polypeptides at the interface.Since protuberances such as Tyr, Phe, and Trp typically do not extend perpendicularly from the interface axis and have preferred conformations, the alignment of the protuberance with the corresponding cavity may in some cases rely on modeling the protuberance / cavity pair based on three-dimensional structures, such as those obtained by X-ray crystallography or nuclear magnetic resonance (NMR).This can be achieved using techniques that are widely accepted in the art.

[0135] In some embodiments, the knob mutation in the IgG1 constant region is T366W (EU numbering). In some embodiments, the hole mutation in the IgG1 constant region comprises one or more mutations selected from T366S, L368A, and Y407V (EU numbering). In some embodiments, the hole mutation in the IgG1 constant region comprises T366S, L368A, and Y407V (EU numbering).

[0136] In some embodiments, the knob mutation in the IgG4 constant region is T366W (EU numbering). In some embodiments, the hole mutation in the IgG4 constant region comprises one or more mutations selected from T366S, L368A, and Y407V (EU numbering). In some embodiments, the hole mutation in the IgG4 constant region comprises T366S, L368A, and Y407V (EU numbering).

[0137] Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibodies (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 1999, 11:1112-1114 (1993)). al., J. Immunol., 152:5368 (1994)); as well as by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0138] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies" or "dual variable domain immunoglobulins" (DVDs) are also included herein (see, e.g., US2006 / 0025576A1, and Wu et al. Nature Biotechnology (2007)). The antibodies or fragments herein also include "dual acting FAbs" or "DAFs" that contain antigen binding sites that bind to the target protein as well as another distinct antigen (see, e.g., US2008 / 0069820).

[0139] e. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include Fab, Fab', Fab'-SH, F(ab') 2 Antibody fragments include, but are not limited to, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthuen, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185, and U.S. Patent Nos. 5,571,894 and 5,587,458. Fab and F(ab') fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives 2 For a discussion of fragments, see US Pat. No. 5,869,046.

[0140] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0141] A single domain antibody is an antibody fragment that contains all or a portion of the light chain variable domain or all or a portion of the heavy chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc. (Waltham, MA), see, e.g., U.S. Patent No. 6,248,516 B1).

[0142] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0143] f. Antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues therein of the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0144] i. Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional variance include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." More substitution-type changes are provided in Table 1 under the heading of "exemplary substitutions" and are further described below in relation to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved antibody-dependent cellular cytotoxicity (ADCC) or cell-dependent cytotoxicity (CDC).

[0145] [Table 1-1] [Table 1-2] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0146] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0147] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have an alteration (e.g., improvement) in certain biological properties compared to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for certain biological properties (e.g., binding affinity).

[0148] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that undergo high frequency mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0149] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within the HVRs. Such modifications may be outside of HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0150] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody's interaction with the light source is affected. Further substitutions may be introduced in amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, antigen-antibody complexes are used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues are targeted or eliminated as candidates for substitution. The variants may be screened to determine whether they contain the desired properties.

[0151] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed prodrug therapy (ADEPT)), or a polypeptide which increases the serum half-life of the antibody.

[0152] ii. Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "THIOMAB™ antibodies," in which one or more residues of an antibody are replaced with a cysteine ​​residue. In certain embodiments, the replaced residues occur at available sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby positioned at available sites on the antibody, which can be used to conjugate the antibody to other moieties, such as L1-PROTAC groups, to generate PACs, as further described herein. In certain embodiments, one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain; A140 (EU numbering) of the heavy chain; L174 (EU numbering) of the heavy chain; Y373 (EU numbering) of the heavy chain; K149 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. In certain embodiments, the antibodies described herein comprise an HC-A140C (EU numbering) cysteine ​​substitution. In certain embodiments, the antibodies described herein comprise an LC-K149C (Kabat numbering) cysteine ​​substitution. In certain embodiments, the antibodies described herein comprise an HC-A118C (EU numbering) cysteine ​​substitution.

[0153] Cysteine ​​engineered antibodies can be generated, for example, as described in US Pat. No. 7,521,541.

[0154] In certain embodiments, the antibody comprises one of the following heavy chain cysteine ​​substitutions: [Table 2]

[0155] In certain embodiments, the antibody comprises one of the following light chain cysteine ​​substitutions: [Table 3]

[0156] A non-limiting exemplary hu7C2.v2.2.LA light chain (LC) K149C THIOMAB™ antibody has the heavy and light chain amino acid sequences of SEQ ID NOs: 26 and 30, respectively. A non-limiting exemplary hu7C2.v2.2.LA heavy chain (HC) A118C THIOMAB™ antibody has the heavy and light chain amino acid sequences of SEQ ID NOs: 31 and 25, respectively.

[0157] PACs include cysteine ​​engineered antibodies in which one or more amino acids of a wild-type or parent antibody are replaced with a cysteine ​​amino acid. Any form of antibody may be so engineered, i.e., mutated. For example, a parent Fab antibody fragment may be engineered to form a cysteine ​​engineered Fab, referred to herein as a "ThioFab." Similarly, a parent monoclonal antibody may be engineered to form a THIOMAB™ antibody. Note that a single-site mutation results in a single engineered cysteine ​​residue in a ThioFab, whereas a single-site mutation results in two engineered cysteine ​​residues in a THIOMAB™ antibody, due to the dimeric nature of IgG antibodies. Mutants with a substituted ("engineered") cysteine ​​(Cys) residue are assessed for the reactivity of the engineered cysteine ​​thiol group introduced at the new position. The thiol reactivity value is a relative numerical value ranging from 0 to 1.0 and can be measured for any cysteine ​​engineered antibody. Thiol reactivity values ​​of cysteine ​​engineered antibodies for use in PAC range from 0.6-1.0, 0.7-1.0, or 0.8-1.0.

[0158] To prepare cysteine ​​engineered antibodies by mutagenesis, DNA encoding amino acid sequence variants of the starting polypeptide is prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of DNA encoding a previously prepared polypeptide. Recombinant antibody variants can also be constructed by restriction fragment manipulation or by overlap extension PCR using synthetic oligonucleotides. The mutagenic primer codes for the cysteine ​​codon substitution(s). Standard mutagenesis techniques can be used to generate DNA encoding such mutant cysteine ​​engineered antibodies. General guidance can be found in Sambrook et al Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0159] Cysteine ​​amino acids may be engineered into antibodies at reactive sites that do not form intrachain or intramolecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; US7521541; US7723485; WO2009 / 052249; Shen et al (2012) Nature Biotech., 30(2):184-191; Junutula et al (2008) Jour of Immun. Methods 332:41-52). The engineered cysteine ​​thiol can be reacted with a linker reagent bearing a thiol-reactive electrophilic group, such as maleimide, activated disulfide (e.g., 4-nitropyridyl), or alpha-haloamide, or with a linker-L1-PROTAC intermediate described herein, to form a PAC with the cysteine ​​engineered antibody (THIOMAB™ antibody) and the PROTAC residue. Thus, the location of the PROTAC moiety can be designed, controlled, and known. The engineered cysteine ​​thiol group typically reacts with a thiol-reactive linker reagent or linker-L1-PROTAC intermediate in high yield, allowing control of the PROTAC / antibody ratio ("PAR"). By engineering an antibody to introduce a cysteine ​​amino acid by substitution at a single site on the heavy or light chain, two new cysteines are introduced into the symmetric antibody. A PAR of about 2 and near homogeneity of the conjugation product can be achieved.

[0160] Cysteine ​​engineered antibodies preferably retain the antigen binding ability of their wild-type parent antibody counterparts. Thus, cysteine ​​engineered antibodies can bind, preferably specifically, to antigens. Such antigens include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signaling proteins, cell survival regulators, cell proliferation regulators, molecules associated with (e.g., known or suspected to functionally contribute to) cell growth or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis, and molecules related to (e.g., known or suspected to functionally contribute to) angiogenesis. The tumor-associated antigen may be a cluster differentiation factor (i.e., CD protein). The antigens to which the cysteine ​​engineered antibodies can bind may be members of one subset of the above-mentioned classes, other subset(s) of which may include other molecules / antigens with unique properties (relative to the antigen of interest).

[0161] The cysteine ​​engineered antibody is prepared for conjugation with the linker L1 intermediate by reduction and reoxidation of the intrachain disulfide groups.

[0162] iii. Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by modifying amino acids such that one or more glycosylation sites are created or removed.

[0163] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, generally linked by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc at the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibody may be performed to generate antibody variants with certain improved properties.

[0164] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, measured by WOMALDI-TOF mass spectrometry, for example as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (Eu numbering of Fc region residues) in the Fc region. However, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., at positions 294-300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.) and US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 013214, and US2005 / 0101111. 0, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L, and WO2004 / 056312A1, Adams et al., especially in Example 11), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107.

[0165] Further provided are antibody variants with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have improved fucosylation and / or ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US2005 / 0123546 (Umana et al.). Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju S), and WO1999 / 22764 (Raju S).

[0166] iv. Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0167] In certain embodiments, the subject matter described herein is directed to antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications where in vivo antibody half-life is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vivo and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express only Fc(RIII, whereas monocytes express Fc(RI, Fc(RII, and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest include those described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)). Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and the CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be determined by measuring the activity of the molecule of interest using a method such as that described in, e.g., Clynes et al. Proc. Nat'l. Acad. Sci. USA 95:652-656 (1998) may be evaluated in vivo. C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activity, CDC assays may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and See, M. J. Glennie, Blood 103:2738-2743 (2004). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0168] In some embodiments, one or more amino acid modifications may be introduced into the Fc portion of the antibodies provided herein to increase IgG binding to neonatal Fc receptors. In certain embodiments, the antibody comprises the following three mutations according to EU numbering: M252Y, S254T, and T256E ("YTE mutations") (U.S. Pat. No. 8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006). In certain embodiments, the YTE mutations do not affect the ability of the antibody to bind to its cognate antigen. In certain embodiments, the YTE mutations increase the serum half-life of the antibody as compared to a native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutations increase the serum half-life of the antibody by 3-fold as compared to a native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutations increase the serum half-life of the antibody by 3-fold as compared to a native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by 2-fold compared to the native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by 4-fold compared to the native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by 5-fold compared to the native (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation increases the serum half-life of the antibody by 10-fold compared to the native (i.e., non-YTE mutant) antibody. See, e.g., U.S. Patent No. 8,697,650. See also Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

[0169] In certain embodiments, the YTE variants provide a means of modulating the antibody-dependent cellular cytotoxicity (ADCC) activity of antibodies. In certain embodiments, the YTEO variants provide a means of modulating the ADCC activity of humanized IgG antibodies against human antigens. See, e.g., U.S. Patent No. 8,697,650. See also Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524(2006).

[0170] In certain embodiments, the YTE variants allow for simultaneous modulation of serum half-life, tissue distribution, and antibody activity (e.g., ADCC activity of IgG antibodies). See, e.g., U.S. Patent No. 8,697,650. See also Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

[0171] Antibodies with reduced effector cells include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering (US Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc variants with substitutions of residues 265 and 297 with alanine according to EU numbering (i.e., D265A and N297A according to EU numbering) (US Pat. No. 7,332,581). In certain embodiments, the Fc variant comprises the following two amino acid substitutions: D265A and N297A. In certain embodiments, the Fc variant consists of the following two amino acid substitutions: D265A and N297A.

[0172] In certain embodiments, the proline at position 329 (EU numbering) (P329) of the wild-type human Fc region is substituted with glycine or arginine, or an amino acid residue large enough to disrupt the proline sandwich in the Fc / Fcγ receptor interface formed between P329 of Fc and tryptophan residues W87 and W110 of FcgRIII (Sondermann et al.: Nature 406, 267-273 (20 July 2000)). In a further embodiment, the at least one additional amino acid substitution in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S, and in yet another embodiment, the at least one additional amino acid substitution is L234A and L235A in a human IgG1 Fc region, or S228P and L235E in an IgG4 Fc region, all according to EU numbering (U.S. Pat. No. 8,969,526).

[0173] In certain embodiments, the polypeptide comprises an Fc variant of a wild-type human IgG Fc region, where P329 of the human IgG Fc region is replaced with glycine, and the Fc variant comprises at least two additional amino acid substitutions at L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, where the residues are numbered according to EU numbering (US Pat. No. 8,969,526). In certain embodiments, for downregulation of ADCC to at least 20% of that induced by a polypeptide comprising a wild-type human IgG Fc region and / or for downregulation of ADCP, the polypeptide comprising the P329G, L234A, and L235A (EU numbering) substitutions exhibits the proposed affinity for human FcγRIIIA and FcγRIIA (US Pat. No. 8,969,526).

[0174] In certain embodiments, a polypeptide comprising an Fc variant of a wild-type human Fc polypeptide comprises a triple mutation: an amino acid substitution at position Pro329, an L234A and an L235A mutation (P329 / LALA) according to EU numbering (U.S. Pat. No. 8,969,526). In certain embodiments, the polypeptide comprises the following amino acid substitutions: P329G, L234A, and L235A according to EU numbering.

[0175] Certain antibody variants have been described that have improved or diminished binding to FcRs. (See, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0176] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and 334 of the Fc region (EU numbering).

[0177] In some embodiments, modifications are made within the Fc region that result in altered (i.e., either improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0178] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve access of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., substitution at Fc region residue 434 according to EU numbering (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0179] g.Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0180] In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation.In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc.Natl.Acad.Sci.USA102:11600-11605(2005)).The radiation can be of any wavelength, including but not limited to, a wavelength that does not harm normal cells but heats the non-proteinaceous moiety to a temperature that kills cells adjacent to the antibody-non-proteinaceous moiety.

[0181] h. Tumor-associated antigens Antibodies, including but not limited to cysteine ​​engineered antibodies, that may be useful in the PACs provided herein in the treatment of cancer include, but are not limited to, antibodies against cell surface receptors and tumor associated antigens (TAA). Certain tumor associated antigens are known in the art and can be prepared for use in generating antibodies using methods and information known in the art. In an attempt to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cell(s). Often, such tumor associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells. The identification of such tumor associated cell surface antigen polypeptides has led to the ability to more specifically target cancer cells for destruction via antibody-based therapy.

[0182] Examples of tumor-associated antigens TAAs include, but are not limited to, those listed below. For convenience, information on these antigens, all of which are known in the art, is listed below, including names, alternative names, Genbank accession numbers, and primary reference(s) in accordance with the National Center for Biotechnology Information (NCBI) diffusion and protein sequence identification conventions. Nucleic acid and protein sequences corresponding to the TAAs listed below are available in public databases, such as GenBank. Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms that bear at least about 70%, 80%, 85%, 90%, or 95% sequence identity to the sequences identified in the cited references and / or exhibit substantially the same biological properties or characteristics as the TAA having the sequence found in the cited reference. For example, a TAA having a variant sequence will generally bind specifically to an antibody that specifically binds to a TAA having the corresponding sequence listed. The sequences and disclosures in the references specifically cited herein are expressly incorporated by reference.

[0183] i. Recombinant Methods and Compositions Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated antibody is provided that encodes an antibody described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or a lymphocytic cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of making an antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as described above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0184] For recombinant production of an antibody, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in host cells, e.g., as described above. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0185] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector cells are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated in a soluble fraction from the bacterial cell paste and further purified.

[0186] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including bacterial and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0187] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0188] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0189] Vertebrate cells may also be used as host cells. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); e.g., Mather et al., Annals TRI cells as described in NYAcad.Sci.383:44-68(1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc.Natl.Acad.Sci.USA77:4216(1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol.248 (BKC Lo,ed.,Humana Press,Totowa,NJ),pp.255-268(2003).

[0190] Referring now to antibody affinity, in embodiments, the antibody binds to one or more tumor-associated antigens or cell surface receptors selected from (1)-(53): (1) BMPR1B (bone morphogenetic protein receptor type IB, Genbank accession) Number NM_001203) ten Dijke, P., et al. Science264(5155):101-104(1994), Oncogene14(11):1377-1382(1997)); WO2004063362 (Claim 2); WO2003042661 (Claim 12); US200 3134790-A1 (pages 38-39); WO2002102235 (claim 13; page 296); WO2003055443 (pages 91-92); WO200299122 (Example 2; pages 528-530); WO2003029421 (claim item 6); WO2003024392 (claim 2; figure 112); WO200298358 (claim 1; page 183); WO200254940 (pages 100-101); WO200259377 (pages 349-350); WO200230268 (claim 27; page 376); WO200148204 (example; figure 4) NP_001194 Bone morphogenetic protein receptor, type IB / pid=NP_001194.1 - Cross reference: MIM:603248; NP_001194.1; AY065994 (2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486) Biochem. Biophys. Res. Commun. 255(2), 283-288(1999), Nature 395(6699):288-291(1998), Gaugitsch, HW, et al. al (1992) J. Biol. Chem. 267(16):11267-11273; WO2004048938 (Example 2); WO2004032842 (Example IV); WO2003042661 (Claim 12); WO2003016475 (Claim 1); WO200278524 (Example 2); WO200299074 (Claim 19; pages 127-129); WO200286443 (Claim 27; pages 222, 393); WO2003003906 (Claim 10; pages 293 p.); WO200264798 (claim 33; p. 93-95); WO200014228 (claim 5; p. 133-136); US2003224454 (fig. 3); WO2003025138 (claim 12; p. 150); NP_003477 Solute transporter family 7 (cationic amino acid transporters, y+ system), member 5 / pid=NP_003477.3 - Homo sapiens Cross References: MIM:600182; NP_003477.3; NM_015923; NM_003486_1 (3) STEAP1 (six-transmembrane epithelial antigen of the prostate, Genbank accession number NM_012449) Cancer Res. 61(15), 5857-5860 (2001), Hubert, RS, et al (1999) Proc. Natl. Acad. Sci. USA 96(25): 14523-14528; WO2004065577 (claim 6); WO2004027049 (Figure 1L); EP1394274 (Example 11); WO2004016225 (claim 2); WO2003042661 (claim 12); US2003157089 (Example 5); US 2003185830 (Example 5); US2003064397 (Figure 2); WO200289747 (Example 5; pages 618-619); WO2003022995 (Example 9; Figure 13A, Example 53; page 173, Example 2; Figure 2A); NP_036581 Six-transmembrane epithelial antigen of the prostate Cross references: MIM:604415; NP_036581.1; NM_012449_1 (4)0772P(CA125, MUC16, Genbank accession number AF361486)J.Biol.Chem.276(29):27371-27375(2001));WO2004045 553 (Claim 14); WO200292836 (Claim 6; Figure 12); WO200283866 (Claim 15; pages 116 to 121); US2003124140 (Example 16); US798959. Cross reference: GI:34501467;AAK74120.3;AF361486_1 (5)MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession number NM_005823) Yamaguchi, N., et al Biol.Chem. 269(2), 805 - 808(1994), Proc.Natl.Acad.Sci.U.S.A. 96(20):11531 - 11536(1999), Proc.Natl.Acad.Sci.U.S.A. 93(1):136 - 140(1996), J.Biol.Chem. 270(37):21984 - 21990(1995)); WO2003101283 (claim 14); (WO2002102235 (claim 13; pages 287 - 288); WO2002101075 (claim 4; pages 308 - 309); WO200271928 (pages 320 - 321); WO9410312 (pages 52 - 57); cross - reference: MIM:601051; NP_005814.2; NM_005823_1 (6)Napi2b (Napi3b, NAPI - 3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium - dependent phosphate transporter 3b, Genbank accession number NM_006424) J.Biol.Chem. 277(22):19665 - 19672(2002), Genomics 62(2):281 - 284(1999), Feild, J.A., et al(1999) Biochem.Biophys.Res.Commun. 258(3):578 - 582); WO2004022778 (claim 2); EP1394274 (example 11); WO2002102235 (claim 13; page 326); EP875569 (claim 1; pages 17 - 19); WO200157188 (claim 20; page 329); WO2004032842 (example IV); WO200175177 (claim 24; pages 139 - 140); cross - reference: MIM:604217; NP_006415.1; NM_006424_1 (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, 7 thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession number AB040878) Nagase T., et al(2000)DNA Res.7(2):143-150); WO2004000997 (Claim 1); WO2003003984 (Claim 1); WO200206339 (Claim 1; Page 50); WO200188133 (Claim 1; Pages 41-43 , pages 48-58); WO2003054152 (Claim 20); WO2003101400 (Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1.Genew; HGNC: 10737; (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene, Genbank accession number AY358628); Ross et al (2002) Cancer Res.62:2546-2553;US2003129192 (Claim 2);US2004044180 (Claim 12);US2004044179 (Claim 11);US2003096961 (Claim 11);US2003232056 (Example 5);WO2003105758 (Claim 12);US2003206918 (Example 5);EP1347046 (Claim 1);WO2003025148 (Claim 20);Cross Reference:GI:37182378;AAQ88991.1;AY358628_1 (9)ETBR(Nakamuta M., et al Biochem.Biophys.Res.Commun.177,139-139;Ogawa et al., 1999;B al Biochem.Biophys.Res.Commun.178,248-255,1991;Arai H.,et al Jpn.Circ.J.56,1303-1307,1992;Arai H.,et alJ.Biol.Chem.268,3463-3470,1993; Metal Biochem.Biophys.Res.Commun.178,656-663,1991;Elshourbagy NA,et alJ.Biol.Chem.268,3873-3879,1993;Haendler B.,et alJ.Cardiovasc.Pharmacol.20,s1-S4,1992; M.,et al Gene228,43-49,1999;Strausberg RL,et al Proc.Natl.Acad.Sci.USA99,16899-16903,2002;Bourgeois J.,et alJ.Clin.Endocrinol.Metab.82,3116-31939,Okamo, et al. 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Immunol. 150, 5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim 9); WO2004045520 (Example 4); WO9102536 (Figures 9.1-9.9); WO2004020595 (Claim 1); Deposit: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1. (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related beta), B29, Genbank accession number NM_000626 or 11038674) Proc. Natl. Acad. Sci. USA (2003) 100(7):4126-4131, Blood (2002) 100(9):3068-3076, Muller et al. al (1992) Eur. J. Immunol. 22(6):1621-1625; WO2004016225 (claim 2, figure 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (example 2); US2002150573 (claim item 5, page 15); US5644033; WO2003048202 (claim 1, pages 306 and 309); WO99 / 558658, US6534482 (claim 13, figure 17A / B); WO200055351 (claim 11, pages 1145-1146); cross references: MIM:147245; NP_000617.1; NM_000626_1 (16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchor protein 1a containing SH2 domain), SPAP1B, SPAP1C, Genbank accession numbers NM_030764, AY358130) Genome Res. 13(10):2265-2270(2003), Immunogenetics 54(2):87-95(2002), Blood 99(8):2662-2669(2002), Proc. Natl. Acad. Sci. USA 98(17):9772-9777(2001), Xu, MJ, et al. al(2001)Biochem.Biophys.Res.Commun.280(3):768-775;WO2004016225(Claim 2);WO2003077836;WO200138490(Claim Item 5; Figures 18D-1 to 18D-2); WO2003097803 (Claim 12); WO2003089624 (Claim 25); Cross reference: MIM: 606509; NP_110391.2; NM_030764_1 (17) HER2 (ErbB2, Genbank accession number M11730); Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T., et al Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985; Swiercz J.M., et al J. Cell Biol. 165, 869-880, 2004; Kuhns J.J., et al J. Biol. 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Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003) Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004005320 (Example 5); WO2003029262 (pages 74-75); WO2003002717 (Claim 2; page 63); WO200222153 (pages 45-47); US2002042366 (pages 20-21); WO200146261 (pages 57-59); WO200146232 (pages 63-65); WO9837193 (Claim 1; pages 55-59); Deposit: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1. (21) Brevican (BCAN, BEHAB, Genbank accession number AF229053) Gary S.C., et al Gene256, 139-147, 2000; Clark H.F., et al Genome Res.13, 2265-2270, 2003; Strausberg R.L., et al Proc.Natl.Acad.Sci.U.S.A.99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; Figure 52); US2003119122 (claim 1; Figure 52); US2003119126 (claim 1); US2003119121 (claim 1; Figure 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; Figure 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1); (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442) Chan, J. and Watt, V.M., Oncogene6(6), 1057-1061(1991) Oncogene10(5):897-905(1995), Annu.Rev.Neurosci.21:309-345(1998), Int.Rev.Cytol.196:177-244(2000)); WO2003042661 (claim 12); WO200053216 (claim 1; page 41); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (pages 128 - 132); WO200053216 (claim 1; page 42); Cross-reference: MIM:600997; NP_004433.2; NM_004442_1 (23) ASLG659 (B7h, Genbank Accession No. AX092328) US20040101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221 (Figure 3); US2003165504 (Claim 1); US2003124140 (Example 2); US2003065143 (Figure 60); WO2002102235 (Claim 13; page 299); US2003091580 (Example 2); WO200210187 (Claim 6; Figure 10); WO200194641 (Claim 12; Figure 7b); WO200202624 (Claim 1 3; Figures 1A-1B); US2002034749 (claim 54; pages 45-46); WO200206317 (Example 2; pages 320-321, claim 34; pages 321-322); WO200271928 (pages 468-469); WO200202587 (Example 1; Figure 1); WO200140269 (Example 3; pages 190-192); WO200036107 (Example 2; pages 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (pages 233-234, 452-453); WO0116318; (24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436) Reiter RE, et al Proc. Natl. Acad. Sci. USA95, 1735-1740, 1998; Gu Z., et al Oncogene19,1288-1296,2000;Biochem.Biophys.Res.Commun.(2000)275(3):783-788;WO2004022709;E P1394274 (Example 11); US2004018553 (Claim 17); WO2003008537 (Claim 1); WO200281646 (Claim 1; page 164); WO200300390 6 (claim 10; page 288); WO200140309 (example 1; figure 17); US2001055751 (example 1; figure lb); WO200032752 (claim 18; figure 1); WO9851805 (claim 17; page 97); WO9851824 (claim 10; page 94); WO9840403 (claim 2; figure 1B); Accession: O43653; EMBL; AF043498; AAC39607.1. (25) GEDA (Genbank Accession No. AY260763); AAP14954 Lipoma HMGIC Fusion Partner-Like Protein / pid=AAP14954.1 - Homo sapiens species: Homo sapiens (human) WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (Example 3, claim 20); US2003194704 (claim 45); Cross References: GI:30102449; AAP14954.1; AY260763_1 (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456); BAFF receptor / pid=NP_443177.1-Homo sapiens Thompson, JS, et al. Science 293(5537), 2108-2111(2001);WO2004058309;WO2004011611;WO2003045422 (Examples; pages 32-33);WO2003014294 (Claim 35; Figure 6B);WO2003035846 (Claim 70; pages 615-616);WO200294852 (Columns 136-137);WO200238766 (Claim 3; page 133);WO200224909 (Example 3; Figure 3);Cross Reference:MIM:606269;NP_443177.1;NM_052945_1;AF132600 (27)CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number AK026467); Wilson et al(1991)J.Exp.Med.173:137-146;WO2003072036(Claim 1; Figure 1);Cross reference:MIM:107266;NP_001762.1;NM_001771_1 (28) CD79a (CD79A, CD79α, immunoglobulin-related alpha, B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces signals involved in B cell differentiation), pI: 4.84, MW: 25028 TM: 2[P] gene chromosome: 19q13.2, Genbank accession number NP_001774.10) WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, Figure 16); WO9207574 (Figure 1); US5644033; Ha et al. al(1992)J.Immunol.148(5):1526-1531;Mueller et al(1992)Eur.J.Biochem.22:1621-1625;Hashimoto et al(1994)Immunogenetics40(4):287-295;Preud'homme et al al(1992)Clin.Exp.Immunol.90(1):141-146;Yu et al(1992)J.Immunol.148(2)633-637;Sakaguchi et al(1988)EMBO J.7(11):3457-3464; (29) CXCR5 (Burkitt's lymphoma receptor 1, G protein-coupled receptor activated by CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly the development of AIDS, lymphoma, myeloma, and leukemia); 372aa, pI: 8.54MW: 41959TM: 7[P] gene chromosome: 11q23.3, Genbank accession number NP_001707.1) WO2004040000; WO2004015426; U S2003105292 (Example 2); US6555339 (Example 2); WO200261087 (Figure 1); WO200157188 ​​(Claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (Example 1, pages 152-153, Example 2, pages 254-256); WO9928468 (Claim 1, page 38); US5440021 (Example 2, columns 49-52); WO9428931 (pages 56-58); WO9217497 (Claim 7, Figure 5); Dobner et al(1992)Eur.J.Immunol.22:2795-2799;Barella et al(1995)Biochem.J.309:773-779; (30) HLA-DOB (beta subunit of MHC class II molecules (Ia antigens) that bind peptides and present them to CD4+ T lymphocytes); 273 aa, pI: 6.56 MW: 30820 TM: 1 [P] gene chromosome: 6p21.3, Genbank accession number NP_002111.1) Tonnelle et al (1985) EMBO J. 4(11): 2839-2847; Jonsson et al (1989) Immunogenetics 29(6): 411-413; Beck et al (1992) J. Mol. Biol. 228: 433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99: 16899-16903; Servenius et al al(1987)J.Biol.Chem.262:8759-8766;Beck et al(1996)J.Mol.Biol.255:1-13;Naruse et al(2002)Tissue Antigens59:512-519; WO9958658 (Claim 13, Figure 15); US6153408 (Columns 35-38); US5976551 (Columns 168-170); US6011146 (Columns 145-146); Kasahara et al(1989) Immunogenetics30(1):66-68;Larhammar et al(1985)J.Biol.Chem.260(26):14111-14119; (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422aa), pI: 7.63, MW: 47206TM: 1[P] gene chromosome: 17p13.3, Genbank accession number NP_002552.2) Le et al (1997) FEBS Lett. 418(1-2): 195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al (2000) Genome Res.10:165-173; WO200222660 (Claim 20); WO2003093444 (Claim 1); WO2003087768 (Claim 1); WO2003029277 (Page 82); (32) CD72 (B cell differentiation antigen CD72, Lyb-2) protein sequence complete maeaity...tafrfpd(1..359;359aa), pI:8.66, MW:40225TM:1[P] gene chromosome:9p13.3, Genbank accession number NP_001773.1) WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et al(1990)J.Immunol.144(12):4870-4877;Strausberg et al(2002)Proc.Natl.Acad.Sci USA99:16899-16903; (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine-rich repeat (LRR) family, regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661aa, pI: 6.20, MW: 74147TM: 1[P] gene chromosome: 5q12, Genbank accession number NP_005573.1) US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al (1996) Genomics 38(3): 299-304; Miura et al (1998) Blood 92: 2815-2822; WO2003083047; WO9744452 (claim 8 57~61 pages);WO200012130(24~26 pages); (34) FcRH1 (Fc receptor-like protein 1, putative receptor for immunoglobulin Fc domains containing C2-type Ig-like and ITAM domains, may play a role in B lymphocyte differentiation); 429aa, pI: 5.28, MW: 46925 TM: 1 [P] gene chromosome: 1q21-1q22, Genbank accession number NP_443170.1) WO2003077836; WO200138490 (claim 6, Figures 18E-1 to 18-E-2); Davis et al (2001) Proc. Natl. Acad. Sci USA 98 (17): 9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (Claim 7); (35) FCRH5 (IRTA2, Immunoglobulin superfamily receptor translocation associated 2, a putative immune receptor with a possible role in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in several B cell malignancies); 977aa, pI: 6.88MW: 106468TM: 1[P] gene chromosome: 1q21, Genbank accession numbers Human: AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse: AK089756, AY158090, AY506558; NP_112571.1WO2003024392 (claim 2, figure 97); Nakayama et al. al(2000)Biochem.Biophys.Res.Commun.277(1):124-127;WO2003077836;WO200138490 (Claim 3, Figures 18B-1 to 18B-2); (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF / heregulin family of growth factors and follistatin); 374 aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession no. AF179274; AY358907, CAF85723, CQ782436 WO2004074320 (sequence number 810); JP2004113151 (sequence numbers 2, 4, 8); WO2003042661 (sequence number 580); WO2003009814 (sequence number 411); EP1295944 (pages 69-70); WO200230268 (page 329); WO200190304 (sequence number 2706); US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579; Horie et al (2000) Genomics 67:146-152; Uchida et al. al(1999)Biochem.Biophys.Res.Commun.266:593-602;Liang et al(2000)Cancer Res.60:4907-12;Glynne-Jones et al(2001)Int J Cancer.Oct15;94(2):178-84; (37)PMEL17(silver homologue;SILV;D12S53E;PMEL17;SI;SIL);ME20;gp100)BC001414;BT007202;M32295;M77348;NM006928;McGlinchey RP et al(2009)Proc Natl.Acad.Sci.USA106(33),13731-13736;Kummer,MPet al(2009)J.Biol.Chem.284(4),2296-2306; (38) TMEFF1 (transmembrane protein with EGF-like domain and two follistatin-like domains 1; tomoregulin-1); H7365; C9orf2; C9ORF2; U19878; X83961; NM_080655; NM_003692; Harms, PW (2003) Genes Dev. 17 (21), 2624-2629; Gery, S. et al(2003)Oncogene22(18):2723-2727;(39)GDNF-Ra1(GDNF family receptor alpha 1;GFRA1;GDNFR;GDNFRA;RETL1;TRNR1;RET1L;GDNFR-alpha 1;GFR-ALPHA-1);U95847;BC014962;NM_145793NM_005264;Kim,MHet al(2009)Mol.Cell.Biol.29(8),2264-2277;Treanor,JJet al(1996)Nature382(6586):80-83; (40)Ly6E (l lymphocyte antigen 6 complex, locus E;Ly67, RIG-E, SCA-2, TSA-1);NP_002337.1;NM_002346.2;de Nooij-van Dalen,AGet al(2003)Int.J.Cancer103(6),768-774;Zammit,DJet al(2002)Mol.Cell.Biol.22(3):946-952;WO2013 / 17705; (41)TMEM46(shisa homolog 2 (Xenopus laevis);SHISA2);NP_001007539.1;NM_001007538.1;Furushima,K.et al(2007)Dev.Biol.306(2),480-492;Clark,HFet al(2003)Genome Res.13(10):2265-2270; (42)Ly6G6D (Lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); NP_067079.2; NM_021246.2; Mallya, M. et al (2002) Genomics 80(1): 113-123; Ribas, G. et al (1999) J. Immunol. 163(1): 278-287; (43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et al (2009) Am. J. Epidemiol. 170(5): 537-545; Yamamoto, Y. et al (2003) Hepatology 37(3): 528-533; (44)RET(ret proto-oncogene;MEN2A;HSCR1;MEN2B;MTC1;PTC;CDHF12;Hs.168114;RET51;RET-ELE1);NP_066124.1;NM_020975.4;Tsukamoto,H.et al(2009)Cancer Sci.100(10):1895-1901;Narita,N.et al(2009)Oncogene28(34):3058-3068; (45)LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226); NP_059997.3; NM_017527.3; Ishikawa, N. et al (2007) Cancer Res. 67(24): 11601-11611; de Nooij-van Dalen, A Get al (2003) Int. J. Cancer 103(6): 768-774; (46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_006134.1; NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105(1-2): 162-164; O'Dowd, BF et al (1996) FEBS Lett. 394(3): 325-329; (47)GPR54(KISS1 receptor;KISS1R;GPR54;HOT7T175;AXOR12);NP_115940.2;NM_032551.4;Navenot,JMet al(2009)Mol.Pharmacol.75(6):1300-1306;Hata,K.et al(2009)Anticancer Res.29(2):617-623; (48) ASPHD1 (Aspartate beta-hydroxylase domain containing 1; LOC253982); NP_859069.2; NM_181718.3; Gerhard, DSet al(2004)Genome Res.14(10B):2121-2127; (49) tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); NP_000363.1; NM_000372.4; Bishop, DT et al (2009) Nat. Genet. 41 (8): 920-925; Nan, H. et al (2009) Int. J. Cancer 125 (4): 909-917; (50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627); NP_001103373.1; NM_001109903.1; Clark, HF et al (2003) Genome Res. 13 (10): 2265-2270; Scherer, SE et al (2006) Nature 440 (7082): 346-351 (51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, TA et al (2003) Proc. Natl. Acad. Sci. USA 100(11): 6759-6764; Takeda, S. et al (2002) FEBS Lett. 520(1-3): 97-101. (52) CD33, a member of the sialic acid-binding immunoglobulin-like lectin family, is a 67 kDa glycosylated transmembrane protein. CD33 is expressed on most myeloid and monocytic leukemia cells, as well as on precursor myelomonocytic and erythroid pan-cells. It is not found on earliest pluripotent stem cells, mature granulocytic, lymphocytic, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin. Invest. 75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33 contains two tyrosine residues on its cytoplasmic tail, each of which is followed by a hydrophobic residue similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) found in many inhibitory receptors. (53) CLL-1 (CLEC12A, MICL, and DCAL2), C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, including roles in cell adhesion, cell-cell signaling, glycoprotein turnover, and inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced ​​transcript variants of this gene have been described, although the full-length nature of some of these variants has not been determined. This gene is closely linked to other CTL / CTLD superfamily members in the natural killer gene complex region on chromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9(5):585-90; van Rhenen A, et al., (2007) Blood 110(7):2659-66; Chen CH, et al. (2006) Blood 107(4):1459-67; Marshall AS, et al. (2006) Eur. J. Immunol. 36(8):2159-69; Bakker AB, et al (2005) Cancer Res. 64(22):8443-50; Marshall AS, et al (2004) J. Biol. Chem. 279(15):14792-802). CLL-1 has been shown to be a type II transmembrane receptor containing a single C-type lectin-like domain (not predicted to bind either calcium or sugar), a stalk region, a transmembrane domain, and a short cytoplasmic tail containing ITIM motifs.

[0191] In one embodiment, the antibody of the PAC is an antibody against a protein found on many cell or tissue types. Examples of such antibodies include gD and EpCAM. In other words, the PAC can be used to deliver PROTACs to many cells or tissues, rather than to a specific cell or tissue type, as is the case when using targeting antibodies.

[0192] As described herein, the PAC can comprise an antibody, for example an antibody selected from the following:

[0193] Anti-Ly6E antibody In certain embodiments, the PAC may comprise an anti-Ly6E antibody. Lymphocyte antigen 6 complex, locus E (Ly6E), also known as retinoic acid-induced gene E (RIG-E) and stem cell antigen 2 (SCA-2), is a GPI-linked, 131 amino acid long, approximately 8.4 kDa protein of unknown function with no known binding partners. It was first identified as a transcript expressed in immature thymocytes, thymic medullary epithelial cells in mice (Mao, et al. (1996) Proc. Natl. Acad. Sci. USA 93:5910-5914). In some embodiments, the subject matter described herein provides a PAC comprising an anti-Ly6E antibody described in PCT Publication No. WO2013 / 177055.

[0194] In some embodiments, the subject matter described herein provides a PAC comprising an anti-Ly6E antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0195] In one aspect, the subject matter described herein provides a PAC comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0196] In another aspect, the subject matter described herein provides a PAC comprising an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:9, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:10, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:11. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:9, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:10, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:11.

[0197] In another aspect, the PAC comprises an antibody comprising: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 14; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0198] In another aspect, the subject matter described herein provides a PAC comprising an antibody comprising (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14, (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0199] In any of the above embodiments, the anti-Ly6E antibody of the PAC is humanized. In one embodiment, the anti-Ly6E antibody comprises a HVR as in any of the above embodiments and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework.

[0200] In another aspect, the anti-Ly6E antibody of the PAC comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 8 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Ly6E antibody comprising the sequence retains the ability to bind to Ly6E. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-Ly6E antibody comprises a VH sequence of SEQ ID NO: 8, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0201] In another aspect, an anti-Ly6E antibody of PAC is provided, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 7 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-Ly6E antibody comprising the sequence retains the ability to bind to Ly6E. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-Ly6E antibody comprises a VL sequence of SEQ ID NO: 7, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

[0202] In another aspect, a PAC is provided comprising an anti-Ly6E antibody, the antibody comprising a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above.

[0203] In one embodiment, a PAC is provided, wherein the antibody comprises the VH and VL sequences of SEQ ID NO:8 and SEQ ID NO:7, respectively, including post-translational modifications of those sequences.

[0204] In a further aspect, provided herein is a PAC comprising an antibody that binds to the same epitope as an anti-Ly6E antibody provided herein. For example, in certain embodiments, a PAC is provided comprising an antibody that binds to the same epitope as an anti-Ly6E antibody comprising the VH sequence of SEQ ID NO:8 and the VL sequence of SEQ ID NO:7, respectively.

[0205] In a further aspect, the anti-Ly6E antibody of the PAC according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-Ly6E antibody of the PAC is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another embodiment, the antibody is a substantially full length antibody, e.g., an IgG1 antibody, an IgG2a antibody, or other antibody class or isotype as defined herein. In some embodiments, the PAC comprises an anti-Ly6E antibody comprising heavy and light chains of amino acid sequences 16 and 15, respectively. [Table 4-1] [Table 4-2]

[0206] Anti-HER2 antibody In certain embodiments, the PAC comprises an anti-HER2 antibody. In one embodiment, the anti-HER2 antibody of the PAC comprises a humanized anti-HER2 antibody, such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8, as described in Table 3 of US5821337. These antibodies contain human framework regions with the complementarity determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also referred to as trastuzumab and is commercially available under the trade name HERCEPTIN^. In another embodiment, the anti-HER2 antibody of the PAC comprises a humanized anti-HER2 antibody, such as humanized 2C4, as described in US7862817. An exemplary humanized 2C4 antibody is available under the trade name PERJETA. (登録商標) The new drug is pertuzumab, which is marketed under the National Cancer Institute (NICI).

[0207] In another embodiment, the anti-HER2 antibody of the PAC comprises a humanized 7C2 anti-HER2 antibody. The humanized 7C2 antibody is an anti-HER2 antibody.

[0208] In some embodiments, described herein is a PAC comprising an anti-HER2 antibody comprising one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, 27, or 28; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24 or 29; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the subject matter described herein is a PAC comprising an anti-HER2 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

[0209] In one aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:23, 27, or 28, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:24 or 29. In one aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:23, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:24. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:68, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:23, 27, or 28, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:24 or 29. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0210] In another aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

[0211] In another aspect, the PAC comprises an antibody comprising: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, 27, or 28; and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 24 or 29; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In another aspect, the PAC comprises an antibody comprising: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 24; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

[0212] In another aspect, a PAC is described herein comprising an antibody comprising (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:23, 27, or 28, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:24 or 29, (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:19, (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:20, and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:21. In another aspect, a PAC is described herein comprising an antibody comprising (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:23, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:24, (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:19, (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:20, and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:21.

[0213] In any of the above embodiments, the anti-HER2 antibody of the PAC is humanized. In one embodiment, the anti-HER2 antibody of the PAC comprises an HVR as in any of the above embodiments and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework.

[0214] In another aspect, the anti-HER2 antibody of the PAC comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 18 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-HER2 antibody comprising the sequence retains the ability to bind to HER2. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 18. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 18. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-HER2 antibody comprises a VH sequence of SEQ ID NO: 18, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0215] In another aspect, a PAC anti-HER2 antibody is provided, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 17 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-HER2 antibody comprising the sequence retains the ability to bind to HER2. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 17. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 17. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-HER2 antibody comprises a VL sequence of SEQ ID NO: 17, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

[0216] In another aspect, a PAC is provided that comprises an anti-HER2 antibody, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above.

[0217] In one embodiment, a PAC is provided that comprises an antibody, the antibody comprising the VH and VL sequences of SEQ ID NO: 18 and SEQ ID NO: 17, respectively, including post-translational modifications of those sequences.

[0218] In one embodiment, a PAC comprising an antibody is provided, wherein the antibody comprises the humanized 7C2.v2.2.LA (hu7C2) K149C kappa light chain sequence of SEQ ID NO:30.

[0219] In one embodiment, a PAC is provided comprising an antibody, wherein the antibody comprises the Hu7C2 A118C IgG1 heavy chain sequence of SEQ ID NO:31.

[0220] In a further aspect, provided herein is a PAC comprising an antibody that binds to the same epitope as an anti-HER2 antibody provided herein. For example, in certain embodiments, a PAC is provided comprising an antibody that binds to the same epitope as an anti-HER2 antibody comprising the VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 17, respectively.

[0221] In a further aspect, the anti-HER2 antibody of the PAC according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-HER2 antibody of the PAC is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another embodiment, the PAC comprises a substantially full length antibody, for example an IgG1 antibody, an IgG2a antibody, or an antibody of another antibody class or isotype as defined herein.

[0222] [Table 5-1] [Table 5-2] [Table 5-3]

[0223] Anti-MUC16 antibody In certain embodiments, the PAC comprises an anti-MUC16 antibody.

[0224] In some embodiments, described herein are PACs comprising anti-MUC16 antibodies comprising one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0225] In one aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37.

[0226] In another aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0227] In another aspect, the PAC comprises an antibody comprising: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 37; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0228] In another aspect, a PAC is described herein comprising an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0229] In any of the above embodiments, the anti-MUC16 antibody of the PAC is humanized. In one embodiment, the anti-MUC16 antibody comprises an HVR as in any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0230] In another aspect, the anti-MUC16 antibody of the PAC comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 39 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-MUC16 antibody comprising that sequence retains the ability to bind to MUC16. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 39. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 39. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-MUC16 antibody comprises a VH sequence of SEQ ID NO: 39, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37.

[0231] In another aspect, a PAC anti-MUC16 antibody is provided, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 38 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-MUC16 antibody comprising that sequence retains the ability to bind to MUC16. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 38. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 38. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-MUC16 antibody comprises a VL sequence of SEQ ID NO: 38, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0232] In another aspect, a PAC is provided that comprises an anti-MUC16 antibody, the antibody comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

[0233] In one embodiment, a PAC is provided, wherein the antibody comprises the VH and VL sequences of SEQ ID NO: 39 and SEQ ID NO: 38, respectively, including post-translational modifications of those sequences.

[0234] In a further aspect, provided herein is a PAC comprising an antibody that binds to the same epitope as an anti-MUC16 antibody provided herein, for example, in certain embodiments, a PAC is provided comprising an antibody that binds to the same epitope as an anti-MUC16 antibody comprising the VH sequence of SEQ ID NO: 39 and the VL sequence of SEQ ID NO: 38, respectively.

[0235] In a further aspect, the anti-MUC16 antibody of the PAC according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-MUC16 antibody of the PAC is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another embodiment, the antibody is a substantially full length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody class or isotype as defined herein.

[0236] [Table 6]

[0237] Anti-STEAP-1 antibody In certain embodiments, the PAC comprises an anti-STEAP 1 antibody.

[0238] In some embodiments, described herein is a PAC comprising an anti-STEAP-1 antibody comprising one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0239] In one aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42.

[0240] In another aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0241] In another aspect, the PAC comprises an antibody comprising: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 42; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0242] In another aspect, a PAC is described herein comprising an antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0243] In any of the above embodiments, the anti-STEAP-1 antibody of the PAC is humanized. In one embodiment, the anti-STEAP-1 antibody comprises an HVR as in any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0244] In another aspect, the anti-STEAP-1 antibody of PAC comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 46. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 46 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-STEAP-1 antibody comprising the sequence retains the ability to bind to STEAP-1. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 46. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 46. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-STEAP-1 antibody comprises a VH sequence of SEQ ID NO: 46, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42.

[0245] In another aspect, an anti-STEAP-1 antibody of PAC is provided, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 47. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 47 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-STEAP-1 antibody comprising the sequence retains the ability to bind to STEAP-1. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 47. In certain embodiments, 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 47. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-STEAP-1 antibody comprises a VL sequence of SEQ ID NO: 47, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0246] In another aspect, a PAC is provided comprising an anti-STEAP-1 antibody, the antibody comprising a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above.

[0247] In one embodiment, a PAC is provided, wherein the antibody comprises the VH and VL sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, including post-translational modifications of those sequences.

[0248] In a further aspect, provided herein is a PAC comprising an antibody that binds to the same epitope as an anti-STEAP-1 antibody provided herein, for example, in certain embodiments, a PAC is provided comprising an antibody that binds to the same epitope as an anti-STEAP-1 antibody comprising the VH sequence of SEQ ID NO: 46 and the VL sequence of SEQ ID NO: 47, respectively.

[0249] In a further aspect, the anti-STEAP-1 antibody of the PAC according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-STEAP-1 antibody of the PAC is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another embodiment, the antibody is a substantially full length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody class or isotype as defined herein.

[0250] [Table 7-1] [Table 7-2]

[0251] Anti-NaPi2b antibody In certain embodiments, the PAC comprises an anti-NaPi2b antibody.

[0252] In some embodiments, described herein is a PAC comprising an anti-NaPi2b antibody comprising one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53.

[0253] In one aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50.

[0254] In another aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:51, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:52, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:53. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:51, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:52, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:53.

[0255] In another aspect, the PAC comprises an antibody comprising: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 50; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53.

[0256] In another aspect, a PAC is described herein comprising an antibody comprising: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50, (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51, (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52, and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53.

[0257] In any of the above embodiments, the anti-NaPi2b antibody of the PAC is humanized. In one embodiment, the anti-NaPi2b antibody comprises a HVR as in any of the above embodiments and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework.

[0258] In another aspect, the anti-NaPi2b antibody of the PAC comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 54. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 54 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-NaPi2b antibody comprising the sequence retains the ability to bind to NaPi2b. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 54. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 54. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-NaPi2b antibody comprises a VH sequence of SEQ ID NO: 54, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50.

[0259] In another aspect, an anti-NaPi2b antibody of PAC is provided, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 55. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 55 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-NaPi2b antibody comprising the sequence retains the ability to bind to anti-NaPi2b. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 55. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 55. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-NaPi2b antibody comprises a VL sequence of SEQ ID NO: 55, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53.

[0260] In another aspect, a PAC is provided comprising an anti-NaPi2b antibody, the antibody comprising a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above.

[0261] In one embodiment, a PAC is provided, wherein the antibody comprises the VH and VL sequences of SEQ ID NO: 54 and SEQ ID NO: 55, respectively, including post-translational modifications of those sequences.

[0262] In a further aspect, provided herein is a PAC comprising an antibody that binds to the same epitope as an anti-NaPi2b antibody provided herein. For example, in certain embodiments, a PAC is provided comprising an antibody that binds to the same epitope as an anti-NaPi2b antibody comprising the VH sequence of SEQ ID NO: 54 and the VL sequence of SEQ ID NO: 55, respectively.

[0263] In a further aspect, the anti-NaPi2b antibody of the PAC according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-NaPi2b antibody of the PAC is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another embodiment, the antibody is a substantially full length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody class or isotype as defined herein. [Table 8]

[0264] Anti-CD79b antibody In certain embodiments, the PAC comprises an anti-CD79b antibody.

[0265] In some embodiments, described herein is a PAC comprising an anti-CD79b antibody comprising one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

[0266] In one aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60. In a further embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60.

[0267] In another aspect, described herein is a PAC comprising an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

[0268] In another aspect, the PAC comprises an antibody comprising: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 60; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

[0269] In another aspect, a PAC is described herein comprising an antibody comprising: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63.

[0270] In any of the above embodiments, the anti-CD79b antibody of the PAC is humanized. In one embodiment, the anti-CD79b antibody comprises an HVR as in any of the above embodiments and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0271] In another aspect, the anti-CD79b antibody of the PAC comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 56. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 56 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-CD79b antibody comprising that sequence retains the ability to bind to CD79b. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 56. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 56. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD79b antibody comprises the VH sequence of SEQ ID NO: 8, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60.

[0272] In another aspect, a PAC anti-CD79b antibody is provided, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 57. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 57 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-Ly6E antibody comprising the sequence retains the ability to bind to CD79b. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 57. In certain embodiments, a total of 1-5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 57. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CD79b antibody comprises a VL sequence of SEQ ID NO:57, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:61, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:62, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:63.

[0273] In another aspect, provided herein is a PAC comprising an anti-CD79b antibody, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.

[0274] In one embodiment, a PAC is provided, wherein the antibody comprises the VH and VL sequences of SEQ ID NO: 56 and SEQ ID NO: 57, respectively, including post-translational modifications of those sequences.

[0275] In a further aspect, provided herein is a PAC comprising an antibody that binds to the same epitope as an anti-CD79b antibody provided herein, for example, in certain embodiments, a PAC is provided comprising an antibody that binds to the same epitope as an anti-CD79b antibody comprising the VH sequence of SEQ ID NO: 56 and the VL sequence of SEQ ID NO: 57, respectively.

[0276] In a further aspect, the anti-CD79b antibody of the PAC according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-CD79b antibody of the PAC is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another embodiment, the antibody is a substantially full length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody class or isotype as defined herein.

[0277] [Table 9-1] [Table 9-2]

[0278] Anti-CD22 antibody In certain embodiments, the PAC may comprise an anti-CD22 antibody comprising three light chain hypervariable regions (HVR-L1, HVR-L2, and HVR-L3) and three heavy chain hypervariable regions (HVR-H1, HVR-H2, and HVR-H3). In one embodiment, the anti-CD22 antibody of the PAC comprises three light chain hypervariable regions and three heavy chain hypervariable regions (SEQ ID NOs: 66-71), the sequences of which are shown below. In one embodiment, the anti-CD22 antibody of the PAC comprises a variable light chain sequence of SEQ ID NO: 72 and a variable heavy chain sequence of SEQ ID NO: 73. In one embodiment, the anti-CD22 antibody of the PAC of the invention comprises a light chain sequence of SEQ ID NO: 74 and a heavy chain sequence of SEQ ID NO: 75. [Table 10-1] [Table 10-2]

[0279] Anti-CD33 antibody In certain embodiments, the PAC may comprise an anti-CD33 antibody comprising three light chain hypervariable regions and three heavy chain hypervariable regions, the sequences of which are shown below (SEQ ID NOs: 76-81). In one embodiment, the anti-CD33 antibody of the PAC comprises the variable light chain sequence of SEQ ID NO: 82 and the variable heavy chain sequence of SEQ ID NO: 83. [Table 11]

[0280] In one embodiment, the PAC anti-CD33 antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 85. In one embodiment, the PAC anti-CD33 antibody comprises three light chain hypervariable regions and three heavy chain hypervariable regions, the sequences of which (SEQ ID NOs: 84-89) are shown below. In one embodiment, the PAC anti-CD33 antibody comprises a variable light chain sequence of SEQ ID NO: 90 and a variable heavy chain sequence of SEQ ID NO: 91. In one embodiment, the PAC anti-CD33 antibody comprises a variable light chain sequence of SEQ ID NO: 92 and a variable heavy chain sequence of SEQ ID NO: 93. In one embodiment, the anti-CD33 antibody of the invention comprises a variable light chain sequence of SEQ ID NO: 94 and a variable heavy chain sequence of SEQ ID NO: 95. In one embodiment, the anti-CD33 antibody of the invention comprises a variable light chain sequence of SEQ ID NO: 96 and a variable heavy chain sequence of SEQ ID NO: 97. [Table 12-1] [Table 12-2]

[0281] antibody affinity In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦50 nM, ≦10 nM, ≦5 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM, and optionally, a dissociation constant of ≧10 -13 M (e.g., 10 -8 Less than M, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M).

[0282] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed on the Fab version of the antibody of interest and its antigen as described by the following assay: The solution binding affinity of the Fab for the antigen is determined by binding the Fab to a minimal concentration ( 125 I) Equilibration with labeled antigen followed by capture of bound antigen on a plate coated with an anti-Fab antibody (e.g., Chen et al., (See, e.g., E. et al., J. Mol. Biol. 293:865-881 (1999)). To establish conditions for this assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I] Antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although incubation may be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., over 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 in PBS (TWEEN-20®). Once the plate is dry, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). A concentration of each Fab that is 20% or less of maximum binding is selected for use in the competitive binding assay.

[0283] According to another embodiment, Kd is measured at 25° C. using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip at about 10 response units (RU). Briefly, a carbomethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. To achieve a binding protein of about 10 response units (RU), the antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) before injection at a flow rate of 5 μl / min. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately 25 μl / min. The association rate (k on ) and dissociation rate (koff The equilibrium dissociation constant (Kd) is calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated using the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate by the surface plasmon resonance assay described above is 10 6 M -1 s -1 If it exceeds , the on-rate can be determined using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nM, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) in the presence of increasing concentrations of antigen measured with a spectrometer such as a spectrophotometer equipped with a stopped-flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer with a stirred cuvette (ThermoSpectronic).

[0284] 2. Linker (L1) As described herein, a "linker" (L1) is a bi- or multi-functional moiety that can be used to attach one or more PROTAC moieties (D) to an antibody (Ab) to form a PAC. In some embodiments, a PAC can be prepared using L1, which has reactive functionalities for covalently binding to a PROTAC and an antibody. For example, in some embodiments, a cysteine ​​thiol of an antibody (Ab) can form a bond with a reactive functional group of the linker or a linker L1-PROTAC group to create a PAC. In particular, the chemical structure of the linker can have a significant impact on both the efficacy and safety of the PAC (Ducry & Stump, Bioconjugate Chem, 2010, 21, 5-13). Choosing the correct linker influences proper drug delivery to the intended cellular compartment of the target cell.

[0285] Linkers can generally be divided into two categories: cleavable (e.g., peptide, hydrzone, or disulfide) or non-cleavable (e.g., thioether). Peptide linkers such as valine-citrulline (Val-Cit), which can be hydrolyzed by lysosomal enzymes (e.g., cathepsin B), have been used to connect drugs to antibodies (US 6,214,345). These are particularly useful due in part to their relative stability in the systemic circulation and their ability to effectively release drugs into tumors. However, the chemical space represented by natural peptides is limited, and therefore it is desirable to have a variety of non-peptide linkers that act like peptides and can be effectively cleaved by lysosomal proteases. More diversity in non-peptide structures may bring novel beneficial properties not offered by peptide linkers. Different types of non-peptide linkers for linker L1 that can be cleaved by lysosomal enzymes are provided herein.

[0286] a. Peptidomimetic Linker Different kinds of non-peptide peptidomimetic linkers for lysosomal enzyme cleavable PAC are provided herein. For example, the amide bond in the middle of a dipeptide (e.g., Val-Cit) is replaced with an amide mimetic, and / or the entire amino acid (e.g., the valine amino acid in the Val-Cit dipeptide) is replaced with a non-amino acid moiety (e.g., a cycloalkyldicarbonyl structure (e.g., ring size=4 or 5)).

[0287] When L1 is a peptidomimetic linker, it is represented by the formula: -Str-(PM)-Sp- During the ceremony, Str is the Stretcher unit covalently attached to Ab, Sp is a linkage or spacer unit covalently attached to the PROTAC moiety; PM is a non-peptide chemical moiety selected from the group consisting of: [ka] W is -NH-heterocycloalkyl- or heterocycloalkyl; Y is heteroaryl, aryl, -C(O)C 1 -C 6 Alkylene, C 1 -C 6 Alkylene-NH 2 , C 1 -C 6 Alkylene-NH-CH 3 , C 1 -C 6 Alkylene-N-(CH 3 ) 2、 C 1 -C 6 Alkenyl, or C 1 -C 6 is alkylenyl, Each R 1 is independent, C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, (C 1 -C 10 (alkyl)NHC(NH)NH 2 , or (C 1 -C 10 alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H, C 1 -C 10 Alkyl, C 1 -C 10 alkenyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 Let's get together and 3 -C 7 may form a cycloalkyl, R 4 and R 5 are each independently 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, arylalkyl, heteroarylalkyl, (C1 -C 10 Alkyl)OCH 2 - or R 4 and R 5 Let's get together and 3 -C 7 A cycloalkyl ring may also be formed.

[0288] Note that L1 can be connected to the PROTAC through either the E3LB, L2, or PB group.

[0289] In an embodiment, Y is heteroaryl and R 4 and R 5 taken together form a cyclobutyl ring.

[0290] In an embodiment, Y is a moiety selected from the group consisting of: [ka]

[0291] In an embodiment, Str is a chemical moiety represented by the formula: [ka] In the formula, R 6 is C 1 -C 10 Alkylene, C 1 -C 10 Alkenyl, C 3 -C 8 Cycloalkyl, (C 1 -C 8 Alkylene)O- and C 1 -C 10 Alkylene-C(O)N(R a )-C 2 -C 6 alkylene, each alkylene being selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C3 -C 8 Cycloalkyl, C 4 -C 7 Each R is optionally substituted with 1 to 5 substituents selected from the group consisting of heterocycloalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl. a are independently H or C 1 -C 6 alkyl, and Sp is -Ar-R b -, where Ar is aryl or heteroaryl, and R b is (C 1 -C 10 alkylene)O-.

[0292] In an embodiment, Str has the formula: [ka] In the formula, R 7 is C 1 -C 10 Alkylene, C 1 -C 10 Alkenyl, (C 1 -C 10 alkylene)O-, N(R c )-(C 2 -C 6 alkylene)-N(R c ), and N(R c )-(C 2 -C 6 alkylene), each R c are independently H or C 1 -C 6 alkyl, and Sp is -Ar-R b -, where Ar is aryl or heteroaryl, and R b is (C 1 -C 10 Alkylene)O- or Sp-C 1 -C 6 It is alkylene -C(O)NH-.

[0293] In an embodiment, L1 is a non-peptide chemical moiety represented by the formula: [ka] R 1 is C 1 -C 6 Alkyl, C 1 -C 6 Alkenyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H or C 1 -C 10 It is an alkyl group.

[0294] In an embodiment, L1 is a non-peptide chemical moiety represented by the formula: [ka] R 1 is C 1 -C 6 Alkyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 or (C 1 -C 6 alkyl)NHC(O)NH 2 and R 4 and R 5 Let's get together and 3 -C 7 It forms a cycloalkyl ring.

[0295] In an embodiment, L1 is a non-peptide chemical moiety represented by the formula: [ka] R1 is C 1 -C 6 Alkyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 where W is as defined above.

[0296] In some embodiments, the linker can be a peptidomimetic linker, such as those described in WO2015 / 095227, WO2015 / 095124, or WO2015 / 095223.

[0297] b. Non-peptide mimetic linkers In an embodiment, the linker L1 forms a disulfide bond with the antibody. In an embodiment, the linker has the following structure: [ka] In the formula, R 1 and R 2 are independently H and C 1 -C 6 alkyl or R 1 and R 2 forms a 3-, 4-, 5-, or 6-membered cycloalkyl or heterocyclyl group. The linker is covalently attached to the antibody and the PROTAC as follows: [ka]

[0298] In one embodiment, the carbonyl group of the linker is connected to an amine group in the PROTAC. It should also be noted that the sulfur atom connected to Ab is a sulfur group derived from a cysteine ​​in the antibody. In another embodiment, the linker L1 has a functional group that can react with a free cysteine ​​present on the antibody to form a covalent bond. Non-limiting examples of such reactive functional groups include maleimides, haloacetamides, α-haloacetyls, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, for example, the conjugation methods in Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, p. 766, and the Examples herein.

[0299] In some embodiments, the linker has a functional group that can react with an electrophilic group present on an antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on an antibody to form a covalent bond to an antibody unit. Examples of such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

[0300] The linker may include one or more linker moieties. Exemplary linker moieties include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("MCC"). A variety of linker moieties are known in the art, some of which are described below.

[0301] The linker may be a "cleavable linker" that facilitates the release of the PROTAC. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); US5208020).

[0302] In certain embodiments, the linker has the formula: [ka] where A is a "stretcher unit" and is an integer from 0 to 1, W is an "amino acid unit" and w is 0 to 12, and Y is a "spacer unit" and y is 0, 1, or 2. Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298.

[0303] In some embodiments, a linker component comprises a "stretcher unit" that attaches an antibody to another linker component or to a PROTAC moiety. Non-limiting exemplary stretcher units are shown below (where the wavy line indicates the site of covalent attachment to an antibody, PROTAC, or further linker component): [ka]

[0304] 3. PROTAC ("D") Useful PROTACs have the general formula above. Specific PROTACs are described in US 7,208,157, WO 2013 / 106643, WO 2013 / 106646, and WO 2015 / 160845. PROTACs include those having the following components:

[0305] a. E3 ubiquitin ligase binding group (E3LB) E3 ubiquitin ligase (of which more than 600 are known in humans) provides substrate specificity for ubiquitination. There are known ligands that bind to these ligases. As described herein, E3 ubiquitin ligase binding groups are peptides or small molecules that can bind to E3 ubiquitin ligases.

[0306] Specific E3 ubiquitin ligases include von Hippel-Lindau (VHL), cereblon, XIAP, E3A; MDM2; anaphase-promoting complex (APC); UBR5 (EDD1); SOCS / BC-box / eloBC / CUL5 / RING; LNXp80; CBX4; CBLL1; HACE1; HECTD1; HECTD2; HECTD3; HECW1; HECW2; HERC1; HERC2; HERC3; HERC4; HUWE1; ITCH; NEDD4; NEDD4L; PPIL2; PRPF19; PIAS1; PIAS2; PIAS3; PIAS4; RANBP2; RNF4; RBX1; SMURF1; SMURF2; STUB1; TOPORS;TRIP12;UBE3A;UBE3B;UBE3C;UBE4A;UBE4B;UBOX5;UBR5;WWP1;WWP2;Parkin;A20 / TNFAIP3;AMFR / gp78;ARA54;Beta-TrCP1 / BTRC;BRCA1;CBL;CHIP / STUB1 ;E6;E6AP / UBE3A;F-box protein 15 / FBXO15;FBXW7 / Cdc4;GRAIL / RNF128;HOIP / RNF31;cIAP-1 / HIAP-2;cIAP-2 / HIAP-1;cIAP(pan);ITCH / AIP4;KAP1;MARCH8;;Mind Bomb 1 / MIB1; Mind Bomb 2 / MIB2; MuRF1 / TRIM63; NDFIP1; NEDD4; NleL; Parkin; RNF2; RNF4; RNF8; RNF168; RNF43; SART1; Skp2; SMURF2; TRAF-1; TRAF-2; TRAF-3; TRAF-4; TRAF-5; TRAF-6; TRIM5; TRIM21; TRIM32; UBR5; and ZNRF3.

[0307] Tables 13-27 below list certain E3 ligases. [Table 13-1] [Table 13-2] [Table 14-1]

Table 14-2

Table 14-3

Table 14-4

Table 14-5

Table 14-6

Table 14-7

Table 14-8

Table 15

Table 16-1

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23-1

Table 23-2

Table 23-3

Table 24

Table 25-1

Table 25-2

Table 25-3

Table 26

Table 27

[0308] A particular E3 ubiquitin ligase is the von Hippel-Lindau (VHL) tumor suppressor, which is a substrate recognition subunit of the E3 ligase complex VCB, which also consists of elongins B and C, Cul2, and Rbxl. The main substrate of VHL is hypoxia-inducible factor lα (HIF-lα), a transcription factor that upregulates genes such as the proangiogenic growth factor VEGF and the erythrocyte-inducing cytokine erythropoietin in response to low oxygen levels. Compounds that bind to VHL can be hydroxyproline compounds such as those disclosed in WO2013 / 106643, as well as other compounds described in US2016 / 0045607, WO2014187777, US20140356322, and US9,249,153.

[0309] Another particular E3 ubiquitin ligase is MDM2. An example of a small molecule binding compound for MDM2 is the following structure: Examples of "nutrin" compounds include nutrin 3a and nutrin 3, having the formula: [ka]

[0310] In addition, MDM2 binding compounds include those described in WO2012 / 121361; WO2014 / 038606;WO2010 / 082612;WO2014 / 044401;WO2009 / 151069;WO2008 / 072655;W02014 / 100065;W02014 / 100071;W02014 / 123882;W02014 / 120748;W02013 / 096150;W02015 / 161032;W02012 / 155066;W02012 / 065022;W02011 / 060049;W02008 / 036168;W02006 / 091646;W02012 / 155066;W02012 / 065022;W02011 / 153509;W02013 / 049250;W02014 / 151863;W02014 / 130470;W02014 / 134207;W02014 / 200937;W02015 / 070224;W02015 / 158648;W02014 / 082889;W02013 / 178570;W02013 / 135648;W02012 / 116989;W02012 / 076513;W02012 / 038307;W02012 / 034954;W02012 / 022707;W02012 / 007409;W02011 / 134925;W02011 / 098398;W02011 / 101297;W02011 / 067185;W02011 / 061139;W02011 / 045257;W02010 / 121995;W02010 / 091979;W02010 / 094622;W02010 / 084097;W02009 / 115425;W02009 / 080488;W02009 / 077357;W02009 / 047161A1、W02008 / 141975A1、W02008 / 141917A1、W02008 / 125487A1、W02008 / 034736A2、W02008 / 055812A1;W02007 / 104714A1;W02007 / 104664A1;W02007 / 082805A1;W02007 / 063013A1、W02006 / 136606A2、W02006 / 097261A1、W02005 / 123691A1、W02005 / 110996A1、W02005 / 003097A1、W02005 / 002575A1;W02004 / 080460A1、W02003 / 051360A1、W02003 / 051359A1、W01998 / 001467;W02011 / 023677;W02011 / 076786;W02012 / 066095;W02012 / 175487;W02012 / 175520;W02012 / 176123;W02013 / 080141;W020 13 / 111105;W02013 / 175417;W02014 / 115080;W02014 / 115077;W02014 / 191896;W02014 / 198266;W02016 / 028391A9;W02016 / 028391A2;W02016 / 026937;W02016 / 001376;W02015 / 189799;W02015 / 155332A1;W02015 / 004610A8;W02013 / 105037A1;W0 2012 / 155066A3;W02012 / 155066A2;W02012 / 033525A3;W02012 / 047587A2;W02012 / 033525A2;W02011 / 106650A3、W02011 / 1 06650A2、W02011 / 005219A1、W02010 / 058819A1;W02010 / 028862A1;W02009 / 037343A1、W02009 / 037308A1、W02008 / 130614A 3、W02009 / 019274A1、W02008 / 130614A2;W02008 / 106507A3;W02008 / 106507A2;W02007 / 107545A1;W02007 / 107543A1;W02007 06032631A1、W02000 / 015657A1;W01998 / 001467A2;W01997 / 009343A3;W01997 / 009343A2;W01996 / 002642A1;US2007 / 0129 416;Med.Chem.Lett,2013,4,466-469;J.Med.Chem.,2015,58,1038-1052;Bioorg.Med.Chem.Lett.25(2015)3621-3625;Also included are those described in Bioorg.Med.Chem.Lett.16(2006)3310-3314. Further specific examples of small molecule binding compounds to MDM2 contemplated for use in PAC include RG7112, RG7388, MI773 / SAR405838, AMG232, DS-3032b, RO6839921, RO5045337, RO5503781, Idasanutlin, CGM-097, MK-8242.

[0311] Another specific E3 ubiquitin ligase is X-linked inhibitor of apoptosis (XIAP). XIAP is a protein that stops apoptotic cell death. Deregulation of XIAP has been linked to cancer, neurodegenerative disorders, and autoimmunity. In the development of lung cancer, overexpression of XIAP inhibits caspases. In the development of prostate cancer, XIAP is one of four IAPs that are overexpressed in the epithelium of the prostate. Mutations in the XIAP gene can result in a severe and rare form of inflammatory bowel disease. Vascularization in the XIAP gene can also result in a very rare condition called X-linked lymphoproliferative disease. Degradation of XIAP can enhance apoptosis by preventing XIAP from binding to caspases. This allows normal caspase activity to proceed.

[0312] Examples of small molecule binding compounds against XIAP include US9,096,544; WO2015187998; WO2015071393; US9,278,978; US9,249,151; US20160024055; US20150307499; US20140135270; US20150284427; US20150259359; US20150266879; US20150246882; US20150252072; US20150225449; US8,883,771, J. Med. Chem., 2015, 58(16)6574-6588, and Small-molecule Pan-IAP Antagonists: A Patent Review (2010) Expert Opin Ther Pat;20:251-67 (Flygare & Fairbrother). Specific compounds include all tetrahydro-benzodiazinones of the following formula, as disclosed in WO2015 / 071393: [ka] Other small molecule binding compounds to XIAP include AEG35156, embelin, TWX006, and TWX024. When a XIAP binding moiety is used as part of a PROTAC, the XIAP binding moiety can bind to the BIR2 or BIR3 domain of XIAP, or both.

[0313] Another specific E3 ubiquitin ligase is cereblon. Cereblon is a protein that forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), cullin-4A (CUL4A), and regulator of cullin 1 (ROC1). This complex ubiquitinates a number of other proteins. Cereblon ubiquitination of target proteins leads to increased levels of fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10 (FGF10). FGF8 similarly regulates a number of developmental processes, such as limb and otic vesicle formation. In the absence of cereblon, DDB1 forms a complex with DDB2, which functions as a DNA damage binding protein.

[0314] Thalidomide, lenalidomide, pomalidomide, and analogs thereof are known to bind to cereblon. The crystal structure of cereblon with thalidomide and derivative compounds is described in US2015 / 0374678. Other small molecule compounds that bind to cereblon are also known, such as those disclosed in US2016 / 0058872 and US2015 / 0291562. Furthermore, phthalimide conjugation with a binder, such as an antagonist, of the BET bromodomain can provide highly selective cereblon-dependent BET protein degradation to the PROTAC. Winter et al., Science, June 19, 2015, p. 1376. Such PROTACs can be conjugated to the antibodies described herein to form PACs.

[0315] b. Protein binding group (PB) The PB component is a group that binds to the target protein intended for degradation. The term "protein" includes oligopeptides and reference polypeptide sequences of sufficient length that they can be bound to the PB group. Any protein in a eukaryotic or microbial system, including a virus, bacteria, or fungus as otherwise described herein, is a target for ubiquitination mediated by the compounds described herein.

[0316] PB groups include, for example, any moiety that specifically binds to a protein (binds to a target protein), including the following non-limiting examples of small molecule target protein moieties: Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting human BET bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and aryl hydrocarbon receptor (AHR), among many others. The compositions described below exemplify some groups of these nine small molecule target protein binding moieties. Such small molecule target protein binding moieties also include pharma- ceutically acceptable salts, enantiomers, solvents, and polymorphs of these compositions, as well as other small molecules that can target the protein of interest.

[0317] In general, target proteins may include, for example, proteins having structural proteins, receptors, enzymes, cell surface proteins, proteins associated with the integral functions of a cell (including proteins involved in catalytic activity, aromatase activity, motility activity, helicase activity, metabolic processes (anabolism and catrabolism), antioxidant activity, protein degradation, biosynthesis), kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulator activity, signal transduction factor activity, structural molecule activity, binding activity (proteins, lipids carbohydrates), receptor activity, cell motility, membrane fusion, cell-cell signaling, regulation of biological processes, development, cell differentiation, response to stimuli. Proteins of interest may include behavioral proteins, cell adhesion proteins, proteins involved in cell death, proteins involved in transport (including protein transporter activity, nuclear transport, ion transporter activity, channel transporter activity, carrier activity, permease activity, secretion activity, electron transporter activity, pathogenesis, chaperone regulator activity, nucleic acid binding activity, transcription regulator activity, extracellular matrix modification and biogenesis activity, translation regulator activity. Proteins of interest may include proteins from eukaryotes and prokaryotes, including humans as targets for drug therapy, other animals, including livestock animals, microorganisms for determination of targets for antibiotics and other antimicrobial agents, and plants, as well as viruses, among many others.

[0318] Thus, the PB components of PAC include FoxOl, HDAC, DP-1, E2F, ABL, AMPK, BRK, BRSK I, BRSK2, BTK, CAMKK1, CAMKK alpha, CAMKK beta, Rb, Suv39HI, SCF, p19INK4D, GSK-3, pi 8 INK4, myc, cyclin E, CDK2, CDK9, CDG4 / 6, cyclin D, pl6 INK4A, cdc25A, BMI1, SCF, Akt, CHKl / 2, C 1 delta, CK1 gamma, C 2, CLK2, CSK, DDR2, DYRK1A / 2 / 3, EF2K, EPH-A2 / A4 / B1 / B2 / B3 / B4, EIF2A 3, Smad2, Smad3, Smad4, Smad7, p53, p2 Cipl, PAX, Fyn, CAS, C3G, SOS, Tal, Raptor, RACK-1, CRK, Rapl, Rac, KRas, NRas, HRas, GRB2, FAK. PI3K, spread, Spry, mTOR, MPK, LKBl, PAK1 / 2 / 4 / 5 / 6, PDGFRA, PYK2, Src, SRPK1, PLC, PKC, PKA, PKB PKC PKD PLKl PRAK PR K2, WAVE-2, TSC2, DAPKl, BAD, IMP, C-TAK1, TAKl TAOl, TBK1, TESK1, TGFBR1, TIE2, TLK1, TrkA, TSSK1, TTBK1 / 2, TTK, Tpl2 / cotl, MEK1, MEK2, PLDL Erkl, Erk2, Erk5, Erk8, p90RSK, PEA-15, SRF, p27 KIP1, TIF la, HMGN1, ER81, MKP-3, c-Fos, FGF-R1, GCK, GSK3, HER4, HIPK1 / 2 / 3 / , IGF-1R, cdc25, UBF. LAMTOR2, Statl, StaO, CREB, JAK, Src, PTEN, NF-カパB, HECTH9, Bax, HSP70, HSP90, Apaf-1, Cyto c, BCL-2, Bcl-xL, Smac, XIAP, Libra-9 Plate-3, Plate-6, Plate-7, CDC37, TAB, IKK TRADD, TRAF2, R1P1, FLIP, TAKl, JNKl / 2 / 3, Lck, A-Raf, B-Raf, C-Raf, MOS, MLKl / 3, MN l / 2、MSKl、MST2 / 3 / 4、MPSK1、MEKKl、ME K4、MEL、ASK1、MINK1、MKK 1 / 2 / 3 / 4 / 6 / 7、NE 2a / 6 / 7 NUAK1 OSR1 SAP STK33 Syk Lyn PDK1 PHK PIM 1 / 2 / 3 アキシン-1 mTORCl MDM2 p21 Wafl イインDl Lamln A, Tpl2, Myc, Wnt, IKK-link, IKK-link, IK K-Qaeda, IKK-Qaeda, ELK, p65RelA, IRAQ, IRA2, IRAK4, IRR, FADD, TRAF6, TRAF3, MKK3, MKK6, ROCK2, RSK1 / 2, SGK1, SmMLCK, SIK2 / 3, ULK1 / 2, VEGFR1, WNK1, YES1, ZAP70, MAP4K3, MAP4K5, MAPKlb, MAPKAP-K2 K3, p38 alpha / beta / delta / gamma MAPK, Aurora A, Aurora B, Aurora C, MCAK, Clip, MAPKAPK, FAK, MARK The peptide or small molecule is any peptide or small molecule that binds to a target protein such as .1 / 2 / 3 / 4, Mucl, SHC, CXCR4, Gap-1, Myc, beta-catenin / TCF, Cbl, BRM, Mcl-1, BRD2, BRD3, BRD4, AR, RAS, ErbB3, EGFR, IRE1, HPK1, RIPK2, and ERα (including all variants, mutations, splice variants, indels, and fusions of these proteins listed).

[0319] Particular PB groups are small molecule compounds such as those disclosed in US2014 / 0356322 and US2016 / 0045607. The compounds disclosed therein can be classified as heat shock protein 90 (HSP90) inhibitors, kinase and phosphatase inhibitors, MDM2 inhibitors, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressant compounds, and compounds targeting human BET bromodomain-containing proteins, aryl hydrocarbon receptor (AHR), REF receptor kinase, FKBP, androgen receptor (AR), estrogen receptor (ER), thyroid hormone receptor, HIV protease, HIV integrase, HCV protease, acyl-protein thioesterase-1 and -2 (APT1 and APT2).

[0320] c. Linker L2 The E3LB and PB groups of the PROTACs described herein can be connected with a linker (L2). In certain embodiments, the linker group L2 is linked to one or more covalently linked structural units of A (e.g., -A 1 ...A q-), wherein A 1 is a group attached to at least one of ELB, PB, or a combination thereof. 1 directly binds an E3LB, PB, or a combination thereof to another E3LB, PB, or a combination thereof. 1 A q An EL3B, PB, or combination thereof is indirectly linked to another E3LB, PB, or combination thereof via

[0321] In certain embodiments, A 1 ~A q each independently represents a bond, CR La R Lb , O, S, SO, SO 2、 NR Lc、 SO 2 NR Lc、 SONR Lc ,CONR Lc、 NR Lc CONR Ld、 NR Lc SO 2 NR Ld、 CO, CR La= CR Lb、 C≡C, SiR La R Lb、 P(O)R La、 P(O)OR La、 NR Lc C(=NCN)NR Ld、 NR Lc C(=NCN), NR Lc C(=CNO 2 )NR Ld , 0 to 6 R La and / or R Lb C optionally substituted with a group 3-11 Cycloalkyl, 0 to 6 R La and / or R Lb C optionally substituted with a group 3-11 Heterocyclyl, 0 to 6 R La and / or R Lb aryl optionally substituted with a group; 0 to 6 R La and / or R Lbheteroaryl optionally substituted with a group, La M Lb each independently represents 0 to 4 R Le R can form cycloalkyl and / or heterocyclyl moieties which can be further substituted with groups. La , R Lb , R Lc , R Ld , and R Le are each independently H, halo, or C 1-8 Alkyl, OC 1-8 Alkyl, SC 1-8 Alkyl, NHC 1-8 Alkyl, N(C 1-8 Alkyl) 2 , C 3-11 Cycloalkyl, aryl, heteroaryl, C 3-11 Heterocyclyl, OC 1-8 Cycloalkyl, SC 1-8 Cycloalkyl, NHC 1-8 Cycloalkyl, N(C 1-8 Cycloalkyl) 2 , N(C 1-8 Cycloalkyl)(C 1-8 alkyl), OH, NH 2 , SH, SO 2 C 1-8 Alkyl, P(O)(OC 1-8 Alkyl)(C 1-8 alkyl), P(O)(OC 1-8 Alkyl) 2 , C.C.-C. 1-8 Alkyl, CCH, CH=CH(C 1-8 alkyl), C(C 1-8 Alkyl)=CH(C 1-8 alkyl), C(C 1-8 Alkyl)=C(C 1-8 Alkyl) 2 , Si(OH) 3 , Si(C 1-8 Alkyl) 3 , Si(OH)(C 1-8 Alkyl) 2 , COC 1-8 Alkyl, CO 2 H, halogen, CN, CF 3, CHF 2 , C.H. 2 F, NO 2 ,SCIENCE FICTION 5 , S.O. 2 NHC 1-8 Alkyl, SO 2 N(C 1-8 Alkyl) 2 ,SONHC 1-8 Alkyl, SON(C 1-8 Alkyl) 2 , CONH.C. 1-8 Alkyl, CON(C 1-8 Alkyl) 2 , N(C 1-8 alkyl)CONH(C 1-8 alkyl), N(C 1-8 Alkyl)CON(C 1-8 Alkyl) 2 , NHCONH(C 1-8 alkyl), NHCON(C 1-8 Alkyl) 2 , N.H.C.O.N. 2 , N(C 1-8 Alkyl)SO 2 NH(C 1-8 alkyl), N(C 1-8 Alkyl)SO 2 N(C 1-8 Alkyl) 2 , NHSO 2 NH(C 1-8 alkyl), NH SO 2 N(C 1-8 Alkyl) 2 , NHSO 2 NH 2 It is.

[0322] In certain embodiments, q is an integer greater than or equal to 0. In certain embodiments, q is an integer greater than or equal to 1.

[0323] In certain embodiments, for example, when q is greater than 2, A q is the group attached to the E3LB moiety, and A 1 and A q are connected via structural units of A (the number of such structural units of A is: q-2).

[0324] In certain embodiments, for example, when q is 2, A q A 1 and the group attached to the E3LB moiety.

[0325] In certain embodiments, for example, when q is 1, the structure of the linker group L2 is -A 1 - and A 1 is the group connecting the E3LB and PB moieties.

[0326] In further embodiments, q is an integer from 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10.

[0327] In certain embodiments, the linker (L2) is selected from the group consisting of: [ka] [ka]

[0328] In further embodiments, the linker group is an optionally substituted (poly)ethylene glycol having 1 to about 100 ethylene glycol units, about 1 to about 50 ethylene glycol units, 1 to about 25 ethylene glycol units, about 1 to 10 ethylene glycol units, 1 to about 8 ethylene glycol units, and 1 and 6 ethylene glycol units, 2 to 4 ethylene glycol units, or an optionally substituted alkyl group interdispersed with optionally substituted O, N, S, P, or Si atoms. In certain embodiments, the linker is substituted with an aryl, phenyl, benzyl, alkyl, alkylene, or heterocyclic group. In certain embodiments, the linker may be asymmetric or symmetric.

[0329] In any of the embodiments of the compounds described herein, the linker group can be any suitable moiety described herein. In one embodiment, the linker is a substituted or unsubstituted polyethylene glycol group ranging in size from about 1 to about 12 ethylene glycol units, from 1 to about 10 ethylene glycol units, from about 2 to about 6 ethylene glycol units, from about 2 to 5 ethylene glycol units, or from about 2 to 4 ethylene glycol units.

[0330] However, the E3LB and PB groups may be covalently linked to the linker group through any group that is suitable and stable to the chemical structure of the linker. The linker is independently covalently linked to the E3LB and PB groups, preferably through an amide, ester, thioester, keto group, carbamate (urethane), carbon, or ether, and each of these groups may be inserted anywhere in the E3LB and PB groups to provide maximum binding between the E3LB group on the ubiquitin ligase and the PB group on the target protein to be degraded. In certain embodiments where the PB group is an E3LB group, the target protein for degradation may be the ubiquitin ligase itself. In certain embodiments, the linker may be attached to an optionally substituted alkyl, alkylene, alkene, or alkyne group, an aryl group, or a heterocyclic group on the E3LB and / or PB group. The E3LB or PB group may need to be derivatized to create a chemical functional group that is reactive with the chemical functional group on the linker. Alternatively, the linker may need to be derivatized to contain chemical functional groups capable of reacting with functional groups found on the E3LB and / or PB.

[0331] L2 can also be expressed by the following formula: [ka] wherein Z is a group that bonds E3LB to X, and X is a group that bonds Z to the group PB.

[0332] In embodiments, Z is absent (a bond), -(CH2 )iO, -(CH 2 )iS, -(CH 2 )iNR, (CH 2 ) i -X 1 Y 1 group (in the formula, X 1 Y 1 forms an amide group, or a urethane group, ester or thioester group, or [ka] In the formula, each R is H or C 1 -C 3 and each i is independently 0 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4, or 5.

[0333] In an embodiment, X is: [ka] wherein each V is independently a bond (absent); [ka] j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2–35, 3–30, 1–15, 1–10, 1–8, 1–6, 1, 2, 3, 4, or 5; k is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4, or 5, preferably k is 1, 2, 3, 4, or 5; m' is 1-100, 1-75, 1-60, 1-55, 1-50, 1-45, 1-40, 2-35, 3-30, 1-15, 1-10, 1-8, 1-6, 1, 2, 3, 4, or 5; n is 1-100, 1-75, 1-60, 1-55, 1-50, 1-45, 1-40, 2-35, 3-30, 1-15, 1-10, 1-8, 1-6, 1, 2, 3, 4, or 5; X 1 is O, S, or NR, preferably O; Y is the same as above, CON, when present in a linker group, is a connector group (which may be a bond) that connects Z to X.

[0334] In embodiments, CON is a bond (absent), a heterocycle, including a water soluble heterocycle, such as piperazinyl or other group, or the following groups: [ka] (In the formula, X 2 are O, S, and NR 4 , S(O), S(O) 2 , -S(O) 2 O, -OS(O) 2 , or OS(O) 2 O, X 3 is O, S, CHR 4 , N.R. 4 and R is H or C optionally substituted with one or two hydroxyl groups. 1 -C 3 alkyl group, or a pharma- ceutically acceptable salt, enantiomer, or stereoisomer thereof.

[0335] In alternative preferred embodiments, the linker group is a (poly)ethylene glycol having 1 to about 100 ethylene glycol units, about 1 to about 50 ethylene glycol units, 1 to about 25 ethylene glycol units, about 1 to 10 ethylene glycol units, 1 to about 8 ethylene glycol units, and 1 and 6 ethylene glycol units, 2 to 4 ethylene glycol units.

[0336] In an embodiment, CON is [ka] Or an amide group.

[0337] Although the E3LB and PB groups may be covalently linked to the linker group through any group that is suitable and stable to the chemical structure of the linker, in preferred embodiments, the linker is independently covalently linked to the E3LB and PB groups through an amide, ester, thioester, keto group, carbamate (urethane), or ether, each of which may be inserted anywhere in the E3LB and PB groups to allow binding of the E3LB group to a ubiquitin ligase and binding of the PB group to a target protein to be degraded. In other words, as shown herein, the linker may be designed and connected to the E3LB and PB groups to minimize, eliminate, or neutralize any effect that its presence may have on the binding of the E3LB and PB to their respective binding partners. In certain embodiments, the target protein for degradation may be a ubiquitin ligase.

[0338] Further linkers L2 are disclosed in U.S. Application Publication Nos. 2016 / 0058872, 2016 / 0045607, 2014 / 0356322, and 2015 / 0291562, as well as WO2014 / 063061.

[0339] Referring now to PACs, a PAC can include a single antibody, and a single antibody can have more than one PROTAC, with each PROTAC covalently attached to the antibody through a linker L1. "PROTAC loading" is the average number of PROTAC moieties per antibody. PROTAC loading can range from 1 to 8 PROTACs (D) per antibody (Ab). That is, in a PAC formulation, Ab-(L1-D) p and p has a value of about 1 to about 50, about 1 to about 8, about 1 to about 5, about 1 to about 4, or about 1 to about 3. Each PROTAC covalently attached to the antibody through a linker L1 can be the same or a different PROTAC and can have the same or a different type of linker as any other L1 covalently attached to the antibody. In one embodiment, Ab is a cysteine ​​engineered antibody and p is about 2.

[0340] The average number of PROTACs per antibody in a formulation of PAC from a conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, electrophoresis, and HPLC. The quantitative distribution of PACs in terms of p can also be determined. The average value of p in a particular formulation of PAC can be determined by ELISA (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p values ​​is not discernible due to the antibody-antigen binding and detection limits of ELISA. Also, ELISA assays for detection of PACs do not determine where the PROTAC moiety is attached to the antibody, such as to the heavy or light chain fragment, or to a specific amino acid residue. In some instances, separation, purification, and characterization of PACs where p is a certain value from PACs with other PROTAC loadings can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0341] For some PACs, p may be limited by the number of binding sites on the antibody. For example, the antibody may have one or several cysteine ​​thiol groups, or one or several sufficiently reactive thiol groups through which a linker may be attached. Another reactive site on the Ab for attaching L1-D is the amine functionality of a lysine residue. Values ​​of p include values ​​of about 1 to about 50, about 1 to about 8, about 1 to about 5, about 1 to about 4, about 1 to about 3, where p is equal to 2. In some embodiments, the subject matter described herein is directed to any PAC where p is about 1, 2, 3, 4, 5, 6, 7, or 8.

[0342] Generally, less than the theoretical maximum amount of PROTAC moieties are conjugated to the antibody during the conjugation reaction. The antibody may contain many lysine residues that do not react with, for example, the linker L1-PROTAC group (L1-D) or the linker reagent. Only the most reactive lysine groups can react with amine-reactive linker reagents. Also, the most reactive cysteine ​​thiol groups can react with thiol-reactive linker reagents or the linker L1-PROTAC group. Generally, the antibody does not contain many, if any, free and reactive cysteine ​​thiol groups that can be bound to the PROTAC moiety. Most cysteine ​​thiol residues in the antibody of the compound exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partial or total reducing conditions. However, the PROTAC loading of PAR (PROTAC / antibody ratio, "PAR") can be controlled in several different ways, including (i) limiting the molar excess of the linker L1-PROTAC group or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reducing conditions for cysteine ​​thiol modification.

[0343] III.L1-PROTAC compounds The PROTACs described herein can be covalently linked to a linker, L1, to prepare the L1-PROTAC group. These compounds have the general formula: L1-D where D is a PROTAC having the structure E3LB-L2-PB, E3LB is an E3 ligase binding group covalently attached to L2, L2 is a linker covalently attached to E3LB and PB, PB is a protein binding group covalently attached to L2, and L1 is a linker covalently attached to D. Useful groups for these components are described above.

[0344] In certain embodiments, L1 is as described elsewhere herein and comprises a peptidomimetic linker. In these embodiments, the L1-PROTAC has the formula: [ka] During the ceremony, Str is the stretcher unit, Sp is a linkage or spacer unit covalently attached to D, the PROTAC moiety; R 1 is C 1 -C 10 Alkyl, (C 1 -C 10 (alkyl)NHC(NH)NH 2 , or (C 1 -C 10 alkyl)NHC(O)NH 2 and R 4 and R 5 are each independently 1 -C 10 Alkyl, arylalkyl, heteroarylalkyl, (C 1 -C 10 Alkyl)OCH 2 - or R 4 and R 5 is C 3 -C 7 may form a cycloalkyl ring, D is a PROTAC moiety.

[0345] The L1-PROTAC compound can be represented by the formula: [ka] In the formula, R 6 is C 1 -C 10 alkylene, R 4 and R 5 Let's go together 3 -C 7 forming a cycloalkyl ring, and D is a PROTAC moiety.

[0346] The L1-PROTAC compound can be represented by the formula: [ka] In the formula, R 1 , R 4 , and R 5 is as described elsewhere herein and D is a PROTAC moiety.

[0347] The L1-PROTAC compound can be represented by the formula: [ka] During the ceremony, Str is the stretcher unit, Sp is an optional spacer unit covalently linked to D, the PROTAC moiety; Y is heteroaryl, aryl, -C(O)C 1 -C 6 Alkylene, C 1 -C 6 Alkylene-NH 2 , C 1 -C 6 Alkylene-NH-CH 3 , C 1 -C 6 Alkylene-N-(CH 3 ) 2 , C 1 -C 6 Alkenyl, or C 1 -C 6 alkylenyl, R1 is C 1 -C 10 Alkyl, (C 1 -C 10 (alkyl)NHC(NH)NH 2 , or (C 1 -C 10 alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H, C 1 -C 10 alkyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 Let's go together 3 -C 7 may form a cycloalkyl, D is a PROTAC moiety.

[0348] The L1-PROTAC compound can be represented by the formula: [ka] In the formula, R 6 is C 1 -C 10 alkylene, R 1 , R 2 , and R 3 is as described elsewhere herein and D is a PROTAC moiety.

[0349] The L1-PROTAC compound can be represented by the formula: [ka] In the formula, R 1 , R 2 , and R 3 is as described elsewhere herein and D is a PROTAC moiety.

[0350] In any of the above L1-PROTAC compounds, Str may have the formula: [ka] In the formula, R 6 is C 1 -C 10 Alkylene, C 3 -C 8 Cycloalkyl, O-(C 1 -C 8 alkylene), and C 1 -C 10 Alkylene-C(O)N(R a )-C 2 -C 6 alkylene, each alkylene being selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Each R is optionally substituted with 1 to 5 substituents selected from the group consisting of heterocycloalkylaryl, arylalkyl, heteroarylalkyl, and heteroaryl. a are each independently H or C 1 -C 6 alkyl, and Sp is -Ar-R b -, Ar is aryl or heteroaryl, and R b is (C 1 -C 10 alkylene)O-.

[0351] In a particular L1-PROTAC compound, R 6 is C 1 -C 10 alkylene, and Sp is -Ar-R b -, where Ar is aryl and R b is (C 1 -C 6 alkylene)O- or R6 is -(CH 2 ) q is 1 to 10.

[0352] In any of the above L1-PROTAC compounds, Str may have the formula: [ka] During the ceremony, [ka] indicates a moiety that can be conjugated to an antibody, R 7 is C 1 -C 10 Alkylene, C 1 -C 10 Alkylene-O,N(R c )-(C 2 -C 6 alkylene)-N(R c ), and N(R c )-(C 2 -C 6 alkylene), each R c are independently H or C 1 -C 6 is alkyl, Sp is -Ar-R b -, where Ar is aryl or heteroaryl, and R b is (C 1 -C 10 alkylene)O- or R 6 is C 1 -C 10 alkylene, and Sp is -Ar-R b -, Ar is aryl, and R b is (C 1 -C 6 alkylene)O-.

[0353] The L1-PROTAC may have the following formula, where in each instance, D is a PROTAC moiety: [ka]

[0354] Referring now to the PB group of the PROTAC, in certain embodiments, the PB is as described elsewhere herein or is selected from the group consisting of heat shock protein 90 (HSP90) inhibitors, kinase and phosphatase inhibitors, MDM2 inhibitors, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressant compounds, and compounds targeting human BET bromodomain-containing proteins, aryl hydrocarbon receptor (AHR), REF receptor kinase, FKBP, androgen receptor (AR), estrogen receptor (ER), thyroid hormone receptor, HIV protease, HIV integrase, HCV protease, acyl-protein thioesterase-1 and -2 (APT1 and APT2).

[0355] In certain embodiments, E3LB is as described elsewhere herein and includes groups that bind to XIAP, VHL, cereblon, and MDM2.

[0356] The subject matter described herein is also directed to a method of preparing a PAC from an L1-PROTAC compound, comprising contacting an antibody, or variants, mutants, splice variants, indels, and fusions thereof, with an L1-PROTAC under conditions in which the antibody is covalently attached to any available attachment point on the L1-PROTAC, whereby a PAC is prepared. The subject matter described herein is also directed to a method of preparing a PAC from an Ab-L1 moiety, i.e., an antibody covalently attached to L1, or variants, mutants, splice variants, indels, and fusions thereof, comprising contacting a PROTAC with Ab-L1 under conditions in which the PROTAC is covalently attached to any available attachment point on the Ab-L1, whereby a PAC is prepared. The method may further comprise periodic isolation and purification of the PAC.

[0357] Referring now to the PAC and L1-PROTAC compounds, as described herein, they can exist in solid or liquid form. In the solid state, it can exist in crystalline or non-crystalline form, or as a mixture thereof. Those skilled in the art will understand that pharma- ceutically acceptable solvents can be formed for crystalline or non-crystalline compounds. In crystalline solvents, the solvent molecules are incorporated into the crystal lattice during crystallization. The solvents can involve non-aqueous solvents, such as, but not limited to, ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or they can involve water as the solvent incorporated into the crystal lattice. Solvents where the water is incorporated into the crystal lattice are typically referred to as "hydrates". Hydrates include stoichiometric hydrates, as well as compositions containing variable amounts of water. The subject matter described herein includes all such solvents.

[0358] Those skilled in the art will further appreciate that certain compounds and PACs described herein that exist in crystalline form (including their various solvents) may exhibit polymorphism (i.e., the ability to occur in different crystal structures). These different crystalline forms are typically known as "polymorphs". The subject matter disclosed herein includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, instrumental arrangement, and other descriptive properties of the crystalline solid state. Polymorphs may therefore have different physical properties such as volume, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction, which may be used for identification. Those skilled in the art will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents used to make the compound. For example, changes in temperature, pressure, or solvent may result in polymorphism. Additionally, one polymorph may spontaneously transform into another polymorph under certain conditions.

[0359] The compounds and PACs or their salts described herein may exist in stereoisomeric forms (e.g., they contain one or more asymmetric carbon atoms). Individual stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of the subject matter disclosed herein. Similarly, it is understood that the compounds or salts of formula (I) may exist in tautomeric forms other than those shown in the formula, which are also included within the scope of the subject matter disclosed herein. It is understood that the subject matter disclosed herein includes all combinations and subsets of the specific groups described herein. The scope of the subject matter disclosed herein includes mixtures of stereoisomers, as well as purified enantiomers or enantiomerically / diastereomerically enriched mixtures. It is understood that the subject matter disclosed herein includes all combinations and subsets of the specific groups defined herein above.

[0360] The subject matter disclosed herein also includes isotopically labeled forms of the compounds described herein, but with respect to the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be incorporated into the compounds described herein and their pharma- ceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, e.g., 2 H, 3 H, 11 C. 13 C. 14 C. 15 N, 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 I can be mentioned.

[0361] Compounds and PACs disclosed herein that contain the aforementioned isotopes and / or other isotopes of other atoms, as well as pharma- ceutically acceptable salts thereof, are within the scope of the presently disclosed subject matter. Isotopically labeled compounds, e.g., 3 H, 14 Disclosed herein are those into which radioactive isotopes such as C are incorporated, which are useful in drug and / or substrate tissue distribution assays. Tritium-labeled, i.e. 3 H, and carbon-14, i.e. 14 C isotopes are commonly used due to their ease of preparation and detectability. 11 C and 18 F isotopes are useful in PET (positron emission tomography), 125 I isotopes are useful in SPECT (single photon emission computed tomography), all of which are useful in brain imaging. In addition, deuterium, i.e. 2 Substitution with heavier isotopes such as H can confer certain therapeutic advantages resulting from better metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. Isotopically labeled compounds of formula I can generally be prepared by carrying out the procedures disclosed in the schemes and / or in the examples below by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents.

[0362] The subject matter described herein includes the following embodiments. 1. A conjugate having the following chemical structure: Ab-(L1-D) p During the ceremony, D is a PROTAC having the structure E3LB-L2-PB, E3LB is the E3 ligase binding group covalently attached to L2; L2 is a linker covalently linking E3LB and PB, PB is a protein-binding group covalently linked to L2; Ab is an antibody covalently linked to L1; L1 is a linker covalently connecting Ab and D; A conjugate, wherein p has a value of from about 1 to about 8.

[0363] 2. The conjugate of embodiment 1, wherein E3LB is a group that binds to an E3 ligase, and the E3 ligase is listed in Tables 13-27, e.g., Table 13, Table 14, Table 15, Table 16, Table 17, Table 18, Table 19, Table 20, Table 21, Table 22, Table 23, Table 24, Table 25, Table 26, or Table 27.

[0364] 3. E3LB is the group that binds to E3 ligase, which in turn binds von Hippel-Lindau (VHL); cereblon; XIAP; E3A; MDM2; anaphase-promoting complex (APC); UBR5 (EDD1); SOCS / BC-box / eloBC / CUL5 / RING; LNXp80; CBX4; CBLL1; HACE1; HECTD1; HECTD2; HECTD3; HECW1; HECW2; HERC1; HERC2; HERC3; HERC4; HUWE1; ITCH; NEDD4; NEDD4L; PPIL2; PRPF19; PIAS1; PIAS2; PIAS3; PIAS4; RANBP2; RNF4; RBX1; SMURF1; SMURF2; STUB1; TOPO RS;TRIP12;UBE3A;UBE3B;UBE3C;UBE4A;UBE4B;UBOX5;UBR5;WWP1;WWP2;Parkin;A20 / TNFAIP3;AMFR / gp78;ARA54;Beta-TrCP1 / BTRC;BRCA1;CBL;CHIP / STUB1;E6;E6 AP / UBE3A;F-box protein 15 / FBXO15;FBXW7 / Cdc4;GRAIL / RNF128;HOIP / RNF31;cIAP-1 / HIAP-2;cIAP-2 / HIAP-1;cIAP(pan);ITCH / AIP4;KAP1;MARCH8;MDM2 / HDM2;Mind The conjugate described in any of the above embodiments, wherein the conjugate is selected from the group consisting of Bomb 1 / MIB1; Mind Bomb 2 / MIB2; MuRF1 / TRIM63; NDFIP1; NEDD4; NleL; Parkin; RNF2; RNF4; RNF8; RNF168; RNF43; SART1; Skp2; SMURF2; TRAF-1; TRAF-2; TRAF-3; TRAF-4; TRAF-5; TRAF-6; TRIM5; TRIM21; TRIM32; UBR5; and ZNRF3.

[0365] 4. The conjugate according to any preceding embodiment, wherein E3LB is a group that binds to an E3 ligase selected from the group consisting of XIAP, VHL, cereblon, and MDM2.

[0366] 5. The conjugate according to any preceding embodiment, wherein E3LB is selected from the group consisting of a compound that binds to VHL, a hydroxyproline compound that binds to VHL, a compound that binds to MDM2, a compound that binds to cereblon, a tetrahydro-benzodiazepinone nutrine, and a small molecule binding compound as described herein.

[0367] 6. E3LB is a XIAP inhibitor that is a tetrahydro-benzodiazepinone having the formula: [ka] A conjugate according to any of the above embodiments, wherein R1, R2, R3, R4, and R5 are as described in WO / 2015 / 071393, including all compounds therein.

[0368] 7.PB, FoxOl, HDAC, DP-1, E2F, ABL, AMPK, BRK, BRSK I, BRSK2, BTK, CAMKK1, CAMKK, CAMKK, Rb, Suv39HI, SCF, p19INK4D, GSK-3, pi 8 INK4, myc, glucose E, CDK2, CDK9, CDG4 / 6, glucose D, pl6 INK4A, cdc25A, BMI1, SCF, Akt, CHKl / 2, C.S 1. CK1, C2, CLK2, CSK, DDR2, DYRK1A / 2 / 3, EF2K, EPH-A2 / A4 / B1 / B2 / B3 / B4, EIF2A 3, Smad2, Smad3, Smad4, Smad7, p53, p2 Cipl, PAX, Fyn, CAS, C3G, SOS, Tal, Raptor, RACK-1, CRK, Rapl, Rac, KRas, NRas, HRas, GRB2, FAK, PI 3K, Spred, Spry, mTOR, MPK, LKBl, PAK1 / 2 / 4 / 5 / 6, PDGFRA, PYK2, Src, SRPK1, PLC, PKC, PKA, and PKB / Pacific, PKC, PKD, PLKl, PRAK, PRK2, R IPK2, WAVE-2, TSC2, DAPKl, BAD, IMP, C-TAK1, TAK l TAOl TBK1 TESK1 TGFBR1 TIE2 TLK1 TrkA TSSK1 TTBK1 / 2 TTK Tpl2 / cotl MEK1 MEK2 PLDL Erkl, Erk2, Erk5, Erk8, p90RSK, PEA- 15, SRF, p27 KIP1, TIF la, HMGN1, ER81, MKP-3, c-Fos, FGF-R1, GCK, GSK3, HER4, HIPK1 / 2 / 3 / , IGF-1R, cdc25, UBF. LAMTOR2, Statl, StaO, CREB, JAK, Src, PTEN, NF-カパB, HECTH9, Bax, HSP70, HSP90, Apaf-1, Cyto c, BCL-2, Bcl-xL, Smac, XIAP, Libra-9 Plate-3, Plate-6, Plate-7, CDC37, TAB, IKK TRADD, TRAF2, R1P1, FLIP, TAKl, JNKl / 2 / 3, Lck, A-Raf, B-Raf, C-Raf, MOS, MLKl / 3, MNl / 2, MSKl, MST2 / 3 / 4, MPSK1, MEKKl, ME K4, MEL, ASK1, MINK1, MKK 1 / 2 / 3 / 4 / 6 / 7, NE 2a / 6 / 7, NUAK1, OSR1, SAP, STK33, Syk, Lyn, PDK1, PHK, PIM 1 / 2 / 3, Ataxin-1, mTORCl, MDM2, p21 Wafl, Cyclin Dl, Lamln A, Tpl2, Myc, Catenin, Wnt, IKK-beta, IKK-gamma, IKK-alpha, IKK-epsilon, ELK, p65RelA, IRAKI, IRA 2, IRAK4, IRR, FADD, TRAF6, TRAF3, MKK3, MKK6, ROCK2, RSK1 / 2, SGK 1, SmMLCK, SIK2 / 3, ULK1 / 2, VEGFR1, WNK 1, YES1, ZAP70, MAP4K3, MAP4K5, MAPKlb, MAPKAP-K2 K3, p38 alpha / beta / delta / gamma MAPK, Aurora A, Aurora B, Aurora C, MCAK, Clip, MAPKAPK, FAK, MARK 1 / 2 / 3 / 4, Mucl, SHC, CXCR4, Gap-1, Myc, beta-catenin / TCF, Cbl, BRM, Mcl1, BRD2, BRD3, BRD4, AR, RAS, ErbB3, EGFR, IRE1, HPK1, RIPK2, and Era (including all variants, mutations, splice variants, indels, and fusions thereof).

[0369] 8. The conjugate according to any of the above embodiments, wherein the PB is selected from the group consisting of heat shock protein 90 (HSP90) inhibitors, kinase and phosphatase inhibitors, MDM2 inhibitors, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressant compounds, and compounds targeting human BET bromodomain-containing proteins, aryl hydrocarbon receptor (AHR), REF receptor kinase, FKBP, androgen receptor (AR), estrogen receptor (ER), thyroid hormone receptor, HIV protease, HIV integrase, HCV protease, and acyl-protein thioesterase-1 and -2 (APT1 and APT2).

[0370] 9. The conjugate according to any preceding embodiment, wherein PB is a compound that targets estrogen receptor alpha (ERα).

[0371] 10. The conjugate according to any preceding embodiment, wherein Ab is selected from Tables 4-12, e.g., Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, or Table 12.

[0372] 11. The conjugate according to any preceding embodiment, wherein the Ab is a cysteine ​​engineered antibody or variant thereof.

[0373] 12.Ab, DLL3, EDAR, CLL1;BMPR1B;E16;STEAP1;0772P;MPF;NaPi2b;Sema 5b;PSCA The conjugate according to any of the above embodiments, which binds to one or more of the polypeptides selected from the group consisting of: hlg; ETBR; MSG783; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2; B7-H4; HER2; NCA; MDP; IL20Rα; brevican; EphB2R; ASLG659; PSCA; GEDA; BAFF-R; CD22; CD79a; CXCR5; HLA-DOB; P2X5; CD72; LY64; FcRH1; IRTA2; TENB2; PMEL17; TMEFF1; GDNF-Ra1; Ly6E; TMEM46; Ly6G6D; LGR5; RET; LY6K; GPR19; GPR54; ASPHD1; tyrosinase; TMEM118; GPR172A; MUC16 and CD33.

[0374] 13. The conjugate according to any preceding embodiment, wherein the Ab binds to one or more of the polypeptides selected from the group consisting of CLL1, STEAP1, NaPi2b, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, B7-H4, HER2, CD22, CD79a, CD72, LY64, Ly6E, MUC16, and CD33.

[0375] 14. The conjugate according to any preceding embodiment, wherein Ab is an antibody that binds to one or more of the polypeptides selected from the group consisting of B7-H4, Her2, CLL1, CD33, CD22, and NaPi2b.

[0376] 15. The conjugate according to any preceding embodiment, wherein the antibody binds to HER2 or B7-H4.

[0377] 16. The conjugate according to any preceding embodiment, wherein the antibody binds to Her2.

[0378] 17. The conjugate according to any preceding embodiment, wherein L1 is a peptidomimetic linker.

[0379] 18. L1 is a peptidomimetic linker represented by the formula: -Str-(PM)-Sp- During the ceremony, Str is the Stretcher unit covalently attached to Ab, Sp is a linkage or spacer unit covalently attached to the PROTAC moiety; PM is a non-peptide chemical moiety selected from the group consisting of: [ka] W is -NH-heterocycloalkyl- or heterocycloalkyl; Y is heteroaryl, aryl, -C(O)C 1 -C 6 Alkylene, C 1 -C 6 Alkylene-NH 2 , C 1 -C 6 Alkylene-NH-CH 3 , C 1 -C 6 Alkylene-N-(CH 3 ) 2、 C 1 -C 6 Alkenyl, or C 1-C 6 is alkylenyl, Each R 1 is independent, C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, (C 1 -C 10 (alkyl)NHC(NH)NH 2 , or (C 1 -C 10 alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H, C 1 -C 10 Alkyl, C 1 -C 10 alkenyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 Let's get together and 3 -C 7 may form a cycloalkyl, R 4 and R 5 are each independently 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, arylalkyl, heteroarylalkyl, (C 1 -C 10 Alkyl)OCH 2 - or R 4 and R 5 Let's get together and 3 -C 7 The conjugate according to any of the above embodiments, which may form a cycloalkyl ring.

[0380] 19. Y is heteroaryl and R 4 and R 5 The conjugate according to any preceding embodiment, wherein together form a cyclobutyl ring.

[0381] 20. The conjugate according to any preceding embodiment, wherein Y is a moiety selected from the group consisting of: [ka]

[0382] 21. Str is a chemical structure represented by the formula: [ka] In the formula, R 6 is C 1 -C 10 Alkylene, C 1 -C 10 Alkenyl, C 3 -C 8 Cycloalkyl, (C 1 -C 8 Alkylene)O- and C 1 -C 10 Alkylene-C(O)N(R a )-C 2 -C 6 alkylene, each alkylene being selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Each R is optionally substituted with 1 to 5 substituents selected from the group consisting of heterocycloalkyl, heteroarylalkyl, arylarylalkyl, heteroarylalkyl, and heteroaryl. a are independently H or C 1 -C 6 alkyl, and Sp is -C 1 -C 6 Alkylene-C(O)NH- or -Ar-R b -, Ar is aryl or heteroaryl, and R b is (C 1 -C 10The conjugate according to any preceding embodiment, wherein R is 0, 1 or 2;

[0383] 22. Str has the formula: [ka] In the formula, R 7 is C 1 -C 10 Alkylene, C 1 -C 10 Alkenyl, (C 1 -C 10 alkylene)O-, N(R c )-(C 2 -C 6 alkylene)-N(R c ), and N(R c )-(C 2 -C 6 alkylene), each R c are independently H or C 1 -C 6 is alkyl, Sp is -C 1 -C 6 Alkylene-C(O)NH- or -Ar-R b -, Ar is aryl or heteroaryl, and R b is (C 1 -C 10 The conjugate according to any preceding embodiment, wherein R is 0, 1 or 2;

[0384] 23. L1 has the formula: [ka] R 1 is C 1 -C 6 Alkyl, C 1 -C 6 Alkenyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6alkyl)NHC(O)NH 2 and R 3 and R 2 are each independently H, C 1 -C 10 The conjugate according to any preceding embodiment, wherein R is alkyl, and Str and Sp are as defined herein.

[0385] 24. L1 has the formula: [ka] R 1 is C 1 -C 6 Alkyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 where Str and Sp are as defined herein; R 4 and R 5 Let's get together and 3 -C 7 The conjugate according to any of the above embodiments, which forms a cycloalkyl ring.

[0386] 25. L1 has the formula: [ka] Str and Sp are as defined herein; R 1 is C 1 -C 6 Alkyl, (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 The conjugate according to any preceding embodiment,

[0387] 26. Having the formula: [ka] During the ceremony, Str is a chemical structure represented by the formula: [ka] R 6 is C 1 -C 10 Alkylene and C 1 -C 10 Alkylene-C(O)N(R a )-C 2 -C 6 alkylene, each alkylene being selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Each R is optionally substituted with 1 to 5 substituents selected from the group consisting of heterocycloalkyl, heteroarylalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl. a are independently H or C 1 -C 6 is alkyl, Ab and Sp are as defined herein; The conjugate according to any preceding embodiment, wherein p is 1, 2, 3, or 4.

[0388] 27. Having the formula: [ka] During the ceremony, Str is a chemical structure represented by the formula: [ka] R 6 is C 1 -C 10 Alkylene and C 1 -C 10 Alkylene-C(O)N(R a )-C 2 -C 6 alkylene, each alkylene being selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamido, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C 3 -C 8 Cycloalkyl, C 4 -C 7 Each R is optionally substituted with 1 to 5 substituents selected from the group consisting of heterocycloalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl. a are independently H or C 1 -C 6 is alkyl, R 1 , R 4 , R 5 , Ab, D, and Sp are as defined herein; The conjugate according to any preceding embodiment, wherein p is 1, 2, 3, or 4.

[0389] 28. Y is heteroaryl, aryl, or alkenyl, and R 6 But, C 1 -C 10 The conjugate according to any preceding embodiment, which is alkylene.

[0390] 29. The conjugate according to any preceding embodiment, wherein Y is: [ka]

[0391] 30. The conjugate according to any preceding embodiment, wherein Y is: [ka]

[0392] 31. The conjugate according to any preceding embodiment, wherein Y is: [ka]

[0393] 32. Str is a chemical structure represented by the formula: [ka] R 6 is C 1 -C 6 is alkylene, Sp is -C 1 -C 6 Alkylene-C(O)NH- or -Ar-R b -, Ar is aryl, and R b is (C 1 -C 3 The conjugate according to any preceding embodiment, wherein R is 0, 1 or 2;

[0394] 33. Having the formula: [ka] During the ceremony, Ab, D, R 2 , and R 3 is as defined herein; R 1 is C 1 -C 6 Alkyl-NH 2 , (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6alkyl)NHC(O)NH 2 and The conjugate according to any preceding embodiment, wherein p is 1, 2, 3, or 4.

[0395] 34. Having the formula: [ka] During the ceremony, Ab and D are as defined herein; p is 1, 2, 3, or 4; R 1 is C 1 -C 6 Alkyl-NH 2 , (C 1 -C 6 (alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 and R 4 and R 5 are each independently 1 -C 6 alkyl, which is unsubstituted or R 4 and R 5 Together with the carbon to which it is attached, it forms a C such as cyclobutyl. 3 -C 7 The conjugate according to any of the above embodiments, which is capable of forming a cycloalkyl ring.

[0396] 35. L1 has the formula: [ka] In the formula, R 1 and R 2 are independently H and C 1 -C 6 alkyl or R 1 and R 2The conjugate according to any of the above embodiments, wherein:

[0397] 36. The conjugate according to any preceding embodiment, selected from the group consisting of PAC1, PAC2, PAC3, PAC4, and PAC5.

[0398] 37. The conjugate of any preceding embodiment, wherein the ratio of PROTAC per antibody ("PAR") is from about 1.5 to about 3.

[0399] 38. The conjugate of any preceding embodiment, wherein the non-PROTAC per antibody ("PAR") is about 2.

[0400] 39. A method of treating a disease in a human in need thereof, comprising administering to the human an effective amount of a conjugate according to any of the above embodiments.

[0401] 40. A pharmaceutical composition comprising a conjugate according to any of the above embodiments and a pharma- ceutically acceptable excipient.

[0402] 41. A method for treating a disease in a human in need thereof, comprising administering to said human an effective amount of the pharmaceutical composition of embodiment 40.

[0403] 42. A method of treating a disease with a conjugate according to any preceding embodiment, wherein the disease is a hyperproliferative disorder, including benign or malignant solid tumors and hematological diseases, as well as disorders involving neuronal, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stromal, blastocoel, inflammatory, angiogenic, immune, and autoimmune conditions.

[0404] 43. A method of treating a disease with a conjugate according to any of the above embodiments...

Claims

1. A conjugate having the formula: 【Transformation 70】 During the ceremony, Ab is an antibody; D is a PROTAC having the structure E3LB-L2-PB-; E3LB is a XIAP inhibitor that is a tetrahydrobenzodiazepinone covalently linked to L2; L2 is a linker covalently connecting E3LB and PB; PB is a group covalently linked to L2 that binds to a target protein intended for degradation; The PROTAC is capable of ubiquitination-mediated degradation of a target protein; Y is heteroaryl, aryl, —C(O)C 1 -C 6 alkylene, C 1 -C 6 alkylene-NH 2 , C 1 -C 6 alkylene-NH—CH 3 , C 1 -C 6 alkylene-N—(CH 3 ) 2 , C 2 -C 6 alkenyl, or C 2 -C 6 alkylenyl; R 1 is C 1 -C 6 alkyl-NH 2 , (C 1 -C 6 alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 ; R 3 and R 2 are each independently H, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 may together form C 3 -C 7 cycloalkyl; p is an integer from 1 to 8; E3LB is, 【Transformation 60】 and L2 is 【Chemistry 60-1】 【Chemistry 60-2】 【Transformation 60-3】 The conjugate is selected from: 【Contents 2】 THが、DAYS、DYS、H11、 H20、DAYH、DHASH、SHAKE 、SHAKE2、SHAK、HASHA1、HASHAアルファ、HASHAベータLIKE4 、90、サイクリンH、CH2、 10.9.10.16.00.00.16 CHAIR、DAY25、CHAR1、SYS、 ROY2、SA1デルタ、SA1ガンマ、SA2、SALO2 、SYS、SHY2、DYSH123、HY20、 ROY2040000000000000000000000000000000,000,000,000,0 3、SY2、SY3、SY4、SY7、D53、2 1000000.00000000000000000000000000000000000 、SAY、SYS、SIGNIFICANT、SHAKE 1. LIKE. SHY、SHY、HY2、HY、 3、Share、Share、XY、9 RH、CHARH、HAR1124456、 CHRONIC、SHY2、SYS 1000000000000000000000000000000000000 / 0 Yベータ アルファ / ガンマ / ゼータ、DYH、HYK、HYHY、HYH2、S ROSH2、SHASH2、SHA2、SH TH、HYH、HYH、HYS1、S 、SHAY、SHA1、SHA1、3 CHA1、SHA2、SHA11、SYS、2 SYS1、SYS12、SYS、S 2 LIKE、CH1、SH22 BYZ、BY2、HY5、HY8、N900 SYS、SYS15、SYS、SY2SYS1、S WH、NO11、SH81、DYS3 、DAS、CH11、SHA、S 33ベータ、HAR4、HHH12 3、NJ10、FY25、20、 LOVE YOU、SYSY、 SHY、SYS、SYSY、9 No. カッパ70、SHAY00、DY11、NカスパーゼH9、カスパーゼ、カスパーゼ16、カスパーゼ77 THIS、THE THIS2、THE 101、THE THIS、THE THIS、WHY123 、LIKE、HAY、HAYY、HYS、HYYY、N9l / 2, MSKl, MST2 / 3 / 4, MPSK1, MEKKl, ME K4, MEL, ASK1, MINK1, MKK1 / 2 / 3 / 4 / 6 / 7, NE2a / 6 / 7, NUAK1, OSR1, SAP, STK33, Syk, Lyn, PDK1, PHK, PIM1 / 2 / 3, ataxin-1, mTORCl, MDM2, p21Wafl, cyclin Dl, Lamln A, Tpl2, Myc, catenin, Wnt, IKK-beta, IKK-gamma, IKK-alpha, IKK-epsilon, ELK, p65RelA, IRAKI, IRA2, IRAK4, IRR, FADD, TRAF6, TRAF3, MKK3, MKK6, ROCK2, RSK1 / 2, SGK1, SmMLCK, SIK2 / 3, ULK1 / 2, VEGFR1, WNK1, YES1, ZAP70, MAP4K3, MAP4K5, MAPKlb, MAPKAP-K2 2. The conjugate of claim 1, wherein the group that binds to K3, p38 alpha / beta / delta / gamma MAPK, Aurora A, Aurora B, Aurora C, MCAK, Clip, MAPKAPK, FAK, MARK1 / 2 / 3 / 4, Mucl, SHC, CXCR4, Gap-1, Myc, beta-catenin / TCF, Cbl, BRM, Mcll, BRD2, BRD3, BRD4, AR, RAS, ErbB3, EGFR, IRE1, HPK1, RIPK2, and ERα (including all variants, mutations, splice variants, indels, and fusions thereof).

3. The conjugate of claim 1, wherein the PB is selected from the group consisting of heat shock protein 90 (HSP90) inhibitors, kinase and phosphatase inhibitors, MDM2 inhibitors, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting human BET bromodomain-containing proteins, aryl hydrocarbon receptor (AHR), REF receptor kinase, FKBP, androgen receptor (AR), estrogen receptor (ER), thyroid hormone receptor, HIV protease, HIV integrase, HCV protease, and acyl-protein thioesterase-1 and -2 (APT1 and APT2).

4. The conjugate described in claim 1, wherein PB is a group that targets estrogen receptor alpha (ERa).

5. The conjugate described in claim 1, wherein the Ab is a cysteine-engineered antibody or a variant thereof.

6. Ab is DLL3, EDAR, CLL1; BMPR1B; E16; STEAP1; 0772P; MPF; NaPi2b; Sema5b; PSCA hlg; ETBR; MSG783; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2; B7-H4; HER2; NCA; MDP; IL20Rα; Brevica EphB2R; ASLG659; PSCA; GEDA; BAFF-R; CD22; CD79a; CXCR5; HLA-DOB; P2X5; CD72; LY64; FcRH1; IRT A conjugate as described in claim 1, which binds to one or more polypeptides selected from the group consisting of: A2; TENB2; PMEL17; TMEFF1; GDNF-Ra1; Ly6E; TMEM46; Ly6G6D; LGR5; RET; LY6K; GPR19; GPR54; ASPHD1; tyrosinase; TMEM118; GPR172A; MUC16 and CD33.

7. The conjugate of claim 5, wherein the Ab binds to one or more polypeptides selected from the group consisting of CLL1, STEAP1, NaPi2b, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, B7-H4, HER2, CD22, CD79a, CD72, LY64, Ly6E, MUC16, and CD33.

8. The conjugate of claim 7, wherein the Ab is an antibody that binds to one or more polypeptides selected from the group consisting of B7-H4, HER2, CLL1, CD33, CD22, and NaPi2b.

9. The conjugate of claim 7, wherein the antibody binds to HER2 or B7-H4.

10. The conjugate of claim 9, wherein the antibody binds to HER2.

11. The conjugate of claim 1, wherein Y is heteroaryl.

12. The conjugate of claim 1, wherein Y is a moiety selected from the group consisting of: 【Transformation 62】 13. The conjugate of claim 1, wherein Y is heteroaryl, aryl, or alkenyl.

14. The conjugate of claim 1, wherein Y is: 【Chemistry 72】 15. The conjugate of claim 1, wherein Y is: 【Transformation 73】 16. The conjugate of claim 1, wherein Y is: 【Chemistry 74】 17. The conjugate of claim 1, wherein p is an integer from 1 to 3.

18. The conjugate of claim 1, wherein p is 2.

19. A conjugate having the formula: 【Chemical Formula 77】 During the ceremony, Ab is an antibody; D is a PROTAC having the structure E3LB-L2-PB-; E3LB is a XIAP inhibitor that is a tetrahydrobenzodiazepinone covalently linked to L2; L2 is a linker covalently connecting E3LB and PB; PB is a group covalently linked to L2 that binds to a target protein intended for degradation; The PROTAC is capable of ubiquitination-mediated degradation of a target protein; p is 1, 2, 3, or 4; R 1 is C 1 -C 6 alkyl-NH 2 , (C 1 -C 6 alkyl)NHC(NH)NH 2 , or (C 1 -C 6 alkyl)NHC(O)NH 2 ; R 4 and R 5 are each independently C 1 -C 6 alkyl, said alkyl being unsubstituted or R 4 and R 5 form a C 3 -C 7 cycloalkyl ring; Conjugates.

20. The conjugate of claim 19, wherein p is an integer from 1 to 3.

21. The conjugate of claim 19, wherein p is 2.

22. A conjugate having the formula: 【Transformation 78】 During the ceremony, Ab is an antibody; p is 1, 2, 3, or 4; Conjugates.

23. The conjugate of claim 22, wherein p is an integer from 1 to 3.

24. The conjugate of claim 22, wherein p is 2.

25. The conjugate of claim 22, wherein the conjugate is PAC1, PAC2, PAC3, PAC4, or PAC5.

26. A pharmaceutical composition comprising the conjugate of claim 1, 19 or 22 and one or more pharmaceutically acceptable excipients.

27. A composition comprising a conjugate according to claim 1, 19 or 22 for treating a disease in a human in need thereof.

28. The composition described in claim 27, wherein the disease is cancer.

29. The composition of claim 28, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphoid malignancies, lung cancer including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

30. The composition described in claim 29, wherein the cancer is a HER2-positive cancer.

31. The composition described in claim 30, wherein the HER2-positive cancer is breast cancer or gastric cancer.

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