PROTAC antibody conjugate and method 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 of related applications This application claims 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 listings submitted as text files via EFS-WEB The official copy of the sequence listing was created on May 20, 2016, and submitted electronically via EFS-Web as an ASCII-formatted sequence listing in a 77-kilobyte file named SEQLIST.TXT, submitted simultaneously with this specification. The sequence listing contained in this ASCII-formatted document is part of this specification, and its entirety is incorporated herein by reference. Sequence numbers 1-6 have been intentionally omitted.

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

[0004] Cell 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 and chromosome segregation. Therefore, such degradation of proteins affects cell proliferation, differentiation, and death.

[0005] Protein inhibitors can block or reduce protein activity in cells, while proteolysis in cells can also reduce activity or completely remove target proteins. Therefore, utilizing cellular proteolysis pathways can provide means to reduce or remove protein activity. One of the major cellular degradation pathways is known as the ubiquitin-proteasome system. In this system, proteins are labeled for degradation by the proteasome by ubiquitination. 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 E3 ubiquitin ligases for attachment to proteins.

[0006] PROTACs have been developed to utilize this degradation pathway. PROTACs combine an E3 ubiquitin ligase with a protein to be labeled for degradation. To facilitate protein degradation by the proteasome, PROTACs contain a group that binds to the E3 ubiquitin ligase and a group that binds to the protein to be degraded. These groups are typically linked by a linker. This molecular structure allows the E3 ubiquitin ligase to be brought into close proximity to the protein so that the protein is ubiquitinated and labeled for degradation.

[0007] Enhanced targeted delivery of PROTACs to cells containing protein targets is a continuing need in the art. Targeted delivery using antibody-PROTAC conjugates can enhance the delivery of PROTACs to specific cells using the specificity of the antibody, and can also enhance the pharmacokinetics of PROTAC delivery to cells compared to other modes of PROTAC administration such as infusion. [Overview of the project]

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

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

[0010] Another aspect of the subject matter described herein is the use of PAC in methods of treating pathological conditions and diseases by administering a pharmaceutical composition containing PAC to a target.

[0011] Another aspect of the subject matter described herein is a method for producing PACs.

[0012] Another aspect of the subject matter described herein is an article comprising a pharmaceutical composition containing PAC, a container, and a package insert or label indicating that the pharmaceutical composition may 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 an E3 ligase binding group covalently bonded to L2. L2 is a linker covalently bonded to E3LB and PB. PB is a protein-binding group that is covalently bonded to L2. Ab is an antibody covalently bound to L1, L1 is a linker covalently bonded to Ab and D. p is a conjugate with values ​​ranging from approximately 1 to approximately 8. (Item 2) E3LB is a group that binds to E3 ligase, and the E3 ligase is the conjugate listed in Tables 13-27, as described in Item 1. (Item 3) E3LB is a group that binds to E3 ligase, and the E3 ligase is von Hippel-Lindau (VHL); Cereblon; XIAP; E3A; MDM2; Late Stage 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 A conjugate as 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) The conjugate described in 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) The conjugate described in item 1, wherein E3LB is selected from the group consisting of compounds that bind to VHL, hydroxyproline compounds that bind to VHL, compounds that bind to MDM2, compounds that bind to cereblon, tetrahydro-benzodiazepinone sodium, thalidomide, lenalidomide, and pomalidomide. (Item 6) E3LB is an XIAP inhibitor that is a tetrahydrobenzodiazepinone having the following formula: [ka] In the formula, R1, R2, R3, R4, and R5 are the conjugates described in item 1, 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 The conjugates described in item 1, which are groups that bind 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 of these variants, mutations, splice variants, indels, and fusions). (Item 8) The conjugate described in item 1, wherein 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 that target human BET bromodomain-containing proteins, aryl hydrocarbon receptors (AHR), REF receptor kinases, FKBP, androgen receptors (AR), estrogen receptors (ER), thyroid hormone receptors, HIV proteases, HIV integrases, HCV proteases, and acyl-protein thioesterases-1 and -2 (APT1 and APT2). (Item 9) PB is a conjugate as described in item 1, which targets the estrogen receptor alpha (ERa). (Item 10) The conjugate according to item 1, wherein Ab is a cysteine-modified antibody or a variant thereof. (Item 11) Ab, DLL3, EDAR, CLL1;BMPR1B;E16;STEAP1;0772P;MPF;NaPi2b;Sema5b;PSCA A conjugate described in item 1 that binds to one or more 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) The conjugate described in item 10, wherein 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. (Item 13) The conjugate described in item 12, wherein 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. (Item 14) The antibody is conjugate as described in item 12, wherein the antibody binds to HER2 or B7-H4. (Item 15) The conjugate according to item 14, wherein the antibody binds to HER2. (Item 16) The conjugate according to item 1, wherein L1 is a peptidomimetic linker. (Item 17) L1 is a peptidomimetic linker represented by the following formula: -Str-(PM)-Sp- In the formula: Str is a stretcher unit covalently bound to Ab, Ab is an antibody, Sp is a binding or spacer unit covalently bound to the PROTAC moiety, PM is a non-peptide chemical moiety selected from the group consisting of:

Chemical formula

[0013] [Figure 1] Western blotting detection of ER-α is shown for PROTAC (without Ab), compound P1, and PROTAC-antibody conjugates (PAC) PAC1 and PAC2. [Figure 2] The quantification of ERα, determined by fluorescence intensity, in Endox-XIAP PACs treated for 3 days with a manipulated HER2-MCF7 line is illustrated. Medium: 10% CS-FBS in phenol red-free RPMI. [Modes for carrying out the invention]

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

[0015] Therefore, the subject matter described herein is directed toward PROTAC-antibody conjugate (PAC) compositions that result in ubiquitination of a target protein and subsequent degradation of the protein. The composition comprises an antibody covalently bound to a linker (L1), the linker covalently bound to the PROTAC at any available linkage point, the PROTAC comprising an E3 ubiquitin ligase binding (E3LB) moiety, the E3LB moiety recognizing 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 for treating diseases and conditions related to protein activity.

[0016] The subject matter of this disclosure is described more fully thereafter. However, a person skilled in the art to which the subject matter of this disclosure belongs, who has an interest in the teachings presented in the following description, will be aware of many modifications and other embodiments of the subject matter of this disclosure described herein. Therefore, it should be understood that the subject matter of this disclosure is not limited to the specific embodiments disclosed, and that 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. If one or more of the incorporated documents, patents, and similar materials, including defined terms, usage of terms, and the techniques described herein, differ from or conflict with this application, this application shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference.

[0017] definition The term "PROTAC" generally refers to a proteolytic target 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,” “part,” or “group” refer to a component that is covalently bonded or linked to another component. For example, “a residue of a PROTAC” refers to a PROTAC that is covalently bonded to one or more groups, such as linker L2, which can optionally further bind to an antibody.

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

[0020] As used herein, the terms “peptide mime” or PM mean the non-peptide chemical moiety. A peptide is a short chain of amino acid monomers linked by a peptide (amide) bond, which is 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, and so on. A peptide mime chemical moiety includes a non-amino acid chemical moiety. A peptide mime chemical moiety may also contain one or more amino acids separated by one or more non-amino acid chemical units. A peptide mime chemical moiety does not contain two or more adjacent amino acids linked by a peptide bond in any part of its chemical structure.

[0021] In this specification, the term “antibody” is used in its broadest sense, specifically encompassing monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be mouse, 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). Target antigens generally have numerous 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. Therefore, one antigen may have two or more corresponding antibodies. The antibodies include full-length immunoglobulin molecules or the immunoactive portion of full-length immunoglobulin molecules, i.e., molecules containing antigen-binding sites that immune-specifically bind to the antigen or a portion of the antigen of interest, such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. The immunoglobulins may originate from any species. However, in one embodiment, the immunoglobulins are of human, mouse, or rabbit origin.

[0022] As used herein, the term “antibody fragment” means a portion of a full-length antibody, generally including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and 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 that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens; as well as multispecific antibodies formed from antibody fragments.

[0023] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for the possibility of native-type mutations that may exist in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which involve different antibodies against different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being able to be synthesized without contamination by other antibodies. The modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, the monoclonal antibodies used in accordance with the subject matter described herein are those of Kohler et al. They can be prepared by the hybridoma method first described by al (1975) Nature, 256:495, or by the recombinant DNA method (see, e.g., US4816567; US5807715). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques described, for example, Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J.Mol.Biol., 222:581-597.

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

[0025] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the rest of the heavy chain and / or light chain originates from a different source or species.

[0026] The "class" of an antibody refers to the type of constant domain or constant region contained in its heavy chain. Antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0027] As used herein, the term “intact antibody” includes the VL and VH domains, as well as the light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be the natural sequence constant domain (e.g., the human natural sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more “effector functions” that refer to biological activities specific to the antibody’s Fc constant region (natural sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors such as B cell receptors and BCRs.

[0028] As used herein, the term “Fc region” means the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes both the native Fc region and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or 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 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 terms “framework” or “FR” refer to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following sequence: 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 antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[0031] "Human antibodies" refer to antibodies produced by humans or human cells, or those derived from non-human sources that utilize the human antibody repertoire, that possess an amino acid sequence corresponding to such an antibody sequence, or another human antibody coding sequence. The definition of human antibodies explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0032] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human HVR and amino acid residues derived from human FR. In certain embodiments, a humanized antibody will contain substantially all of at least one, typically two, variable domains, where all or substantially all HVR (e.g., CDR) corresponds to that of a non-human antibody, and all or substantially all FR corresponds to that of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized” form of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0033] "Isolated antibodies" are those separated from their natural environment. In some embodiments, antibodies are purified to a purity of over 95% or 99%, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for evaluating antibody purification, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0034] "Isolated nucleic acids" refer to nucleic acid molecules that have been separated from their natural environment. While isolated nucleic acids typically refer to nucleic acid molecules contained within cells that normally contain nucleic acid molecules, these nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0035] "An isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, and includes nucleic acid molecules in a single vector or separate vectors, and nucleic acid molecules present in one or more host cells.

[0036] A "naked antibody" refers to an antibody that is not conjugated with a heterogeneous portion (e.g., a cytotoxic portion) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0037] A "natural-type antibody" refers to a naturally occurring immunoglobulin molecule with a different structure. For example, a natural-type IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical disulfide-linked light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a 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 a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ).

[0038] The "amino acid sequence identity percentage (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to achieve the maximum sequence identity percentage, gaps have been introduced where necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved by various methods 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 appropriate parameters for aligning sequences, including any algorithm required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office (Washington DC, 20559) and is registered as 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 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 change.

[0039] In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity percentage of a given amino acid sequence A to, with, or relative to, a given amino acid sequence B (or, alternatively, a given amino acid sequence A that has or contains a certain amino acid sequence identity percentage to, with, or relative to, a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y In the formula, X is the number of amino acid residues scored as identical in the sequences of A and B by the sequence alignment program ALIGN-2, 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, the amino acid sequence identity % for A to B will not be equal to the amino acid sequence identity % for 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 preceding paragraph.

[0040] Depending on the amino acid sequence of the constant domains of their heavy chains, intact antibodies can be assigned to different "classes." Intact immunoglobulin antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are known. The Ig form has hinged or hingeless forms (Roux). et al(1998)J.Immunol.161:4083-4090;Lund This includes 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 representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, human immunoglobulin VL or VH sequences are derived from subgroups of variable domain sequences. Generally, the subgroups of sequences are those described 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. (above). In one embodiment, for VH, the subgroup is subgroup Kappa III in Kabat et al. (above).

[0042] For the purposes of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence, or it may contain changes in the amino acid sequence. 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 sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0043] The terms “variable region” or “variable domain,” as used herein, refer to domains of the antibody heavy or light chain involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (HVRs). (e.g., Kindt et al. Kuby Immunology, 6) th See 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 can be isolated using antibody-derived VH or VL domains to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0044] The terms “hypervariable region” or “HVR,” as used herein, refer to each region of an antibody variable domain whose sequence is hypervariable and / or forms a structurally defined loop (“hypervariable loop”). Generally, native quadruple-chain antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally contain amino acid residues derived from hypervariable loops and / or “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, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 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).) Except for CDR1 in VH, CDRs generally contain amino acid residues that form a hypervariable loop. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that come into contact with the antigen. SDRs are contained within regions of CDRs called 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, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 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. (above).

[0045] "Effector function" refers to the 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 cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); 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 a region within the extracellular domain of HER2 to which the antibody 4D5 (ATCC CRL10463) and trastuzumab bind. This epitope is adjacent to the transmembrane domain of HER2 and is located within domain IV of HER2. To screen for antibodies that bind to the 4D5 epitope, routine cross-blocking assays, such as those described in "Antibody," A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), may be performed. Alternatively, epitope mapping may be performed to assess whether an antibody binds to the 4D5 epitope of HER2 (e.g., any one or more residues in the region approximately 550–610 containing HER2 (SEQ ID NO: 39)).

[0048] The "2C4 epitope" or "2C4 epitope" is a region within the extracellular domain of HER2 to which the antibody 2C4 binds. To screen for antibodies that bind to the 2C4 epitope, routine cross-blocking assays, such as those described in *Antibody*, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), may be performed. Alternatively, epitope mapping may be performed to assess whether an antibody binds to the 2C4 epitope of HER2. The 2C4 epitope contains residues derived from domain II within the extracellular domain of HER2. 2C4 antibodies 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 total strength of non-covalent interactions between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) at the single binding site. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below. In certain embodiments, the antibodies described herein are ≤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, for example, 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 the parental antibody that does not possess such modifications, and such modifications result in an improvement in the affinity of the antibody for the light source.

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

[0052] As used herein, the term "free cysteine amino acid" refers to a cysteine amino acid residue that has been engineered into a parental antibody, has a thiol functional group (-SH), and is not paired as an intramolecular or intermolecular disulfide bridge. As used herein, the term "amino acid" 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] As used herein, the term "linker", "linker unit", or "link" means a chemical moiety that includes a chain of atoms that covalently couples a PROTAC moiety to an antibody or a component of a PROTAC to another component of the 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 can be a "cancer patient", i.e., a patient who has or is at risk of having one or more symptoms of cancer.

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

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

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

[0058] [[ID=I5]] As used herein, "neoadjuvant therapy" or "preoperative therapy" refers to a therapy given before surgery. The purpose of neoadjuvant therapy is to provide immediate systemic treatment and potentially eradicate micrometastases that might otherwise grow if standard surgical procedures were followed after systemic therapy. Neoadjuvant therapy can also help reduce tumor size, thereby allowing complete resection of tumors that were initially inoperable or preservation of part of an organ and its function. Additionally, neoadjuvant therapy allows for in vivo evaluation of drug efficacy and can guide the selection of subsequent therapies.

[0059] In this specification, “adjuvant therapy” refers to therapy administered after final surgery when no trace of residual disease is detected, in order to reduce the risk of disease recurrence. The objective of adjuvant therapy is to prevent cancer recurrence and, therefore, reduce the likelihood of cancer-related death. Adjuvant therapy in this specification explicitly excludes neoadjuvant therapy.

[0060] The term "final surgery" is used as a medical term. A final surgery includes surgical or other procedures that lead to the removal or excision of a tumor, including, for example, those that lead to the removal or excision of all macroscopically visible tumor tissue. A final surgery includes, for example, the complete or curative excision of a tumor, or complete macroscopic excision. A final 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 excision of the tumor. A final surgery includes procedures to remove or excise a tumor that includes the associated organs, parts of organs and tissues, and surrounding organs, such as lymph nodes, parts of organs, or tissues. Removal may be incomplete, and as a result, tumor cells may remain, although they may not be detectable.

[0061] "Survival" refers to the state in which a patient remains alive and includes disease-free survival (DFS), progression-free survival (PFS), and overall survival (OS). Survival can be estimated using the Kaplan-Meier method, and any difference in survival is calculated using a hierarchical log-rank test.

[0062] Progression-free survival (PFS) is defined as the period from day 1 of treatment to the first recorded disease progression (including isolated CNS progression) or death from any cause during the study.

[0063] "Disease-free survival (DFS)" refers to a patient remaining alive without cancer recurrence for a predetermined period, such as approximately 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years, from the start of treatment or initial diagnosis. In one aspect of the subject matter described herein, DFS is analyzed according to the principle of treatment intent, i.e., patients are evaluated based on their assigned therapies. Events used in the analysis of DFS may include local, localized, and distant recurrence of cancer, development of secondary cancers, and death from any cause in patients without prior events (e.g., breast cancer recurrence or secondary primary cancer).

[0064] "Overall survival" refers to a patient remaining alive for a predetermined period, such as approximately 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years, from the start of treatment or the initial diagnosis.

[0065] "Extended survival" refers to an increase in disease-free survival (DFS) and / or overall survival (OS) in treated patients compared to untreated patients or a control treatment protocol. Survival is monitored for at least approximately 6 months, or at least approximately 1 year, or at least approximately 2 years, or at least approximately 3 years, or at least approximately 4 years, or at least approximately 5 years, or at least approximately 10 years, etc., after the initiation of treatment or initial diagnosis.

[0066] "Monotherapy" refers to a treatment regimen that includes only one therapeutic agent for the treatment of cancer or tumors during the course of treatment.

[0067] "Maintenance therapy" refers to a treatment regimen given to reduce the likelihood of disease recurrence or progression. Maintenance therapy can be provided for any period of time, including a long period up to the patient's lifetime. Maintenance therapy can be provided after initial therapy, or in conjunction with initial therapy or additional therapy. The dosage used in maintenance therapy may vary and may include lower dosages compared to the dosages used in 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 acids have been introduced, including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” including primary transformed cells and their offspring, regardless of the number of passages. Offspring may contain mutations, although their nucleic acid content may not be exactly the same as that of the parent cells. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

[0069] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth / proliferation. “Tumor” includes one or more cancerous cells. Examples of cancer are provided elsewhere in this specification.

[0070] "HER2-positive" cancers include cancer cells with HER2 levels higher than normal. 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 Insights hybridization (ISH) amplification of ≥2.0. The term "HER2-positive cell" refers to a cell that expresses HER2 on its surface.

[0071] The terms “early-stage breast cancer (EBC)” or “early breast cancer” are 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 stage I, IIA, IIB, and IIIA breast cancer.

[0072] The references to tumors or cancers as "Stage 0," "Stage I," "Stage II," "Stage III," or "Stage IV," and the various substages within this classification, indicate a classification of tumors or cancers using the Overall Stage Grouping or Roman Numeral Staging methods known in the art. Although the actual disease of cancer depends on the type of cancer, generally, Stage 0 cancer is a situ lesion, Stage I cancer is a small local tumor, Stages II and III cancer are progressive local tumors with local lymph node involvement, and Stage IV cancer represents metastatic cancer. The specific stages of each type of tumor are known to experienced clinicians.

[0073] The term "metastatic breast cancer" refers to a condition of breast cancer in which cancer cells have spread from the primary site to one or more other sites in the body via blood vessels or lymphatics, forming one or more secondary tumors in one or more organs other than the breast.

[0074] "Advanced" cancer is cancer that has spread outside the site or organ of origin by either local invasion or metastasis. Therefore, the term "advanced" cancer includes both locally advanced and metastatic diseases. "Recurrent" cancer is cancer that has grown again in either the primary site or a distant site after responding to initial treatment, such as surgery. "Locally recurrent" cancer is cancer that returns to the same location as the previously treated cancer after treatment. "Operable" or "resectable" cancer is cancer that is confined to the primary organ and suitable for surgery (resection). "Non-resectable" or "unresectable" cancer is cancer that cannot be removed (resected) by surgery.

[0075] As used herein, the term “cytotoxic agent” refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include radioactive isotopes (e.g., At). 211、 I 131、 I 125、 Y 90、 Re 186、 Re 188 Sm 153, Bi 212 , P 32 , Pb 212 ), and radioisotopes 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 inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various antitumor and anticancer drugs disclosed hereinafter, but not limited thereto.

[0076] "Chemotherapy agents" refer to chemical compounds that are useful in treating cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomellamine; acetogenins (especially bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); β-rapacone; lapacol; colchicine; betulinic acid; camptothecin (synthetic analogue topotecan (HYCAMTIN®)), CPT -11 (containing irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (containing its adzeresin, karzeresin, and bizeresin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (synthetic analogs KW-2189 and CB1-TM) Including 1); eryuterobin; pancratistatin; sarcodictyin; spongstatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, fenesterine, prednimustine, trophosphamide, uracil mustard, and other nitrogenous mustards;Nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; engine antibiotics (e.g., calichemycin, especially calichemycin γ1I and calichemycin ω1I (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); the oral α4 integrin inhibitor CDP323; dynemycin including dynemicin A; esperamicin; and neocardinostatin chromophores and related pigment protein engine antibiotic chromophores); aclasinomycin, actinomycin, autramycin, azacerin, bleomycin) Syn, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome method (DOXIL®), liposomal doxorubicin TLC) D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, queramycin, rhodorubicin Antimetabolites such as ubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epotilon, and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, and trimethrexate;Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; frolinic acid Folic acid supplements such as acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabucil; bisanthren; edatraxate; defofamine; demecoltin; diaziquan; elfornithine; eriptinium acetate; epotilon; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; mytansinoids such as mytansin and anthamitosin; mitogluazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products (Eugene, OR)); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 22'2'Trichlorotriethylamine; Trichothecin (especially the T2 toxins verracurin A, loridine A, and anguidin); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Thiotepa; Taxoids, e.g., paclitaxel (TAXOL®), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); Chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum-based drugs such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vinca, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®), which prevent tubulin polymerization from forming microtubules; etoposide (VP-16); ifosfamide; mitoxantrone Leucovorin; Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid containing bexarotene (TARGRETIN®); Clodronate (e.g., BONEFOS® or OSTAC®), Etidronate (DIDROCAL®), NE-58095, Zoledronic acid / Zoledrone (ZOMETA®), Alendronate (FOSAMAX) Bisphosphonates such as (registered trademark), pamidronate (AREDIA®), tildroneate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, such as PKCα, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); THERATOPE® vaccines and gene therapy vaccines. For example, vaccines such as 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); olafenib, ABT510; oblimersen sodium (GENASENSE®, antisense oligonucleotide); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors; serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); ronafarnib (SCH Farnesyltransferase inhibitors such as 6636 and SARASAR®; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above; and two or more combinations 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 of oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin).

[0077] The chemotherapeutic agents as defined herein include “anti-hormone agents” or “endocrine therapies” that act to modulate, reduce, block, or inhibit the action of hormones that may promote cancer growth.These may themselves be hormones, and include anti-estrogen agents having a mixed agonist / antagonist profile, such as tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), doxifen, doroxifen, raloxifene (EVISTA®), trioxyfen, keoxyfen, and selective estrogen receptor modulators (SERMs) such as SERM3; fulvestrant (FASLODEX®) Pure anti-estrogen agents that do not possess agonist properties, such as )) and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); steroid aromatase inhibitors such as formestan and exemestane (AROMASIN®), and nonsteroid aromatase inhibitors such as anastrozole (ARIMIDEX®), letrozole (FEMARA®), and aminoglutethimide. Aromatase inhibitors, including those containing borozol (RIVISOR®), megestrol acetate (MEGASE®), fadrozol, 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; diex Sex steroids, including estrogens such as tylstylbestrol and premarin, and androgens / retinoids such as fluoxymesterone, all trans-retinoic acids, and fenretinide; onapristone; antiprogesterones; estrogen receptor downmodulators (ERDs); antiandrogens such as flutamide, nilutamide, and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0078] When used herein in reference to adjunctive therapy, the term “immunosuppressant” refers to a substance that acts to suppress or shield the immune system of the mammal being treated herein. This includes substances that suppress cytokine production, downregulate or suppress autoantigen expression, or shield MHC antigens. Examples of such drugs include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); anti-inflammatory agents such as glucocorticoids like ganciclovir, tacrolimus, cortisol, or aldosterone, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and leukotriene receptor antagonists; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde (which shields MHC antigens as described in U.S. Patent No. 4,120,649); anti-idiotype antibodies against MHC antigens and MHC fragments; cyclosporine A; corticosteroids such as corticosteroids or glucocorticosteroids, or glucocorticoid analogs, such as prednisone and SOLU-MEDROL®. Methylprednisolone containing methylprednisolone sodium succinate, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); antimalarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; anti-interferon α, beta, or γ antibodies, anti-tumor necrosis factor (TNF) α antibodies (infliximab (REMICADE®) or adalimumab), anti-TNFα immunoadhesins (E Cytokine or cytokine receptor antibodies, including tanercept, anti-TNFβ antibody, anti-interleukin-2 (IL-2) antibody and anti-IL-2 receptor antibody, and anti-interleukin-6 (IL-6) receptor antibody and antagonist (such as ACTEMRA® (tocilizumab)); anti-LFA-1 antibody, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibody; heterologous anti-lymphocyte globulin; pan-T antibody, preferably anti-CD3 or anti-CD4 / CD4a antibody;Soluble peptides containing the LFA-3 binding domain (WO90 / 08187, published July 26, 1990); streptokinase; transforming growth factor β (TGF-β); streptodolase; host-derived RNA or DNA; FK506; RS-61443; chlorambucil; deoxyspagarin; rapamycin; T cell receptor (Cohen et al., U.S. Patent No. 5,114,721); T cell receptor fragments (Offner et al., Science, 251:430-432 (1991); WO90 / 11294; Ianeway, Nature, 341:482 (1989); and WO91 / 01133); BAFF antibodies, and BR3 antibodies, and BAFF antagonists such as zTNF4 antagonists (for an overview, see Mackay and Mackay, Trends) See also Immunol., 23:113-5 (2002), and the definitions below; biological agents that interfere with T cell helper signaling, such as anti-CD40 receptors or anti-CD40 ligands (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 ligands; and T cell receptor antibodies such as T10B9 (EP340, 109). Some preferred immunosuppressants as used herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

[0079] As used herein, “treatment” (and its grammatical variations such as “to treat” or “to treat”) refers to a clinical intervention aimed at altering the natural course of the individual being treated, which may be carried out for preventive purposes or during a clinicopathological process. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or remission of the condition, and remission or improvement of prognosis. In some embodiments, antibodies of the subject matter described herein are used to delay the onset of the disease or to slow the progression of the disease.

[0080] Drugs administered "concurrently" with one or more other drugs are administered on the same treatment day, within the same treatment cycle, and optionally concurrently with one or more other drugs. For example, in the case of cancer therapy administered every three weeks, drugs administered concurrently are each administered on day 1 of the three-week cycle.

[0081] The “effective dose” of a drug, for example, a pharmaceutical formulation, refers to the effective amount in the dosage and duration required to achieve the desired therapeutic or prophylactic outcome. For example, an effective dose of a drug for treating cancer may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., delay, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., delay, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with cancer. A drug may be cell proliferation inhibitory and / or cytotoxic insofar as it can prevent the growth of existing cancer cells and / or kill them. An effective dose may extend progression-free survival (e.g., measured by the Response Assessment of Solid Tumors (RECIST) or CA-125 changes), 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., assessed by FOSI).

[0082] As used herein, the term “therapeutic dose” means any amount that, compared to a corresponding subject not receiving such dose, results in treatment of a disease, disorder, or side effect, or a reduction in the rate of progression of the disease or disorder. This term also includes amounts effective for improving normal physiological function. For therapeutic use, therapeutic doses of PAC, and its salts, may be administered as raw chemicals. Furthermore, 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) described 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 any substitution, including multiple types of substitutions involving one or more substituents, such as one, two, or three. This phrase should not be construed as overlapping with any substitutions described or depicted herein.

[0085] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained therein is effective, and which does not contain any additional components that are unacceptably toxic to the person to whom the preparation is administered.

[0086] A "pharmaceutically acceptable excipient" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target substance. 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, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, sugars, formates, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may include another molecule, such as an acetate ion, a succinate ion, or another counterion. The counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0088] Other pharmaceutically unacceptable salts may be useful in the preparation of the compounds described herein, and these should be considered to form further embodiments of the subject matter. These salts, such as oxalic acid or trifluoroacetate salts, although they themselves are pharmaceutically unacceptable, may be useful as intermediates in the preparation of salts when obtaining the compounds described herein and their pharmaceutically acceptable salts.

[0089] As used herein, the term “plural” refers to two or more conjugates. Each conjugate may be the same as any other conjugate in that plural.

[0090] "Small molecules" or "small molecule compounds" generally refer to organic molecules with a size of less than approximately 5 kilodaltons (Kd). In some embodiments, small molecules are less than approximately 4 Kd, 3 Kd, approximately 2 Kd, or approximately 1 Kd. In some embodiments, small molecules are less than approximately 800 daltons (D), approximately 600 D, approximately 500 D, approximately 400 D, approximately 300 D, approximately 200 D, or approximately 100 D. In some embodiments, small molecules are less than approximately 2000 g / mol, less than approximately 1500 g / mol, less than approximately 1000 g / mol, less than approximately 800 g / mol, or less than approximately 500 g / mol. In some embodiments, small molecules are nonpolymeric. Small molecules are not proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc. 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 through a series of chemical reactions.

[0091] As used herein, the term "alkyl" refers to a group of 1 to 12 carbon atoms (C1-C1). 12) refers to a monovalent saturated linear or branched hydrocarbon group of any length, and the alkyl group may optionally and independently be substituted with one or more substituents listed below. In another embodiment, the alkyl group is 1 to 8 carbon atoms (C1-C8) or 1 to 6 carbon atoms (C1-C6). Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)) CH2CH3), 2-methyl-2-propyl(t-Bu, t-butyl, -C(CH3)3), 1-pentyl(n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl(-CH(CH3)CH2CH2CH3), 3-pentyl(-CH(CH2CH3)2), 2-methyl-2-butyl(-C(CH3)2CH2CH3), 3-methyl-2-butyl(-CH(CH3)CH(CH3)2), 3-methyl-1-butyl(-CH2CH 2CH(CH3)2), 2-methyl-1-butyl(-CH2CH(CH3)CH2CH3), 1-hexyl(-CH2CH2CH2CH2CH2CH3), 2-hexyl(-CH(CH3)CH2CH2CH2CH3), 3-hexyl(-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl(-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl(-CH(CH3)CH(CH3)CH2CH3) Examples include, but are not limited to, 4-methyl-2-pentyl(-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl(-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl(-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl(-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl(-CH(CH3)C(CH3)3), 1-heptyl, 1-octyl, etc.

[0092] When used herein, the term "alkylene" refers to a group of 1 to 12 carbon atoms (C1-C1).12 ) refers to a divalent saturated straight-chain or branched-chain hydrocarbon group of any length, and the alkylene group may optionally and independently be substituted with one or more substituents listed below. In another embodiment, the alkylene group is 1 to 8 carbon atoms (C1-C8) or 1 to 6 carbon atoms (C1-C6). Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), and propylene (-CH2CH2CH2-).

[0093] The term "alkenyl" refers to at least one unsaturated site, i.e., a carbon-carbon sp. 2 This refers to a monovalent linear or branched hydrocarbon group of any length having 2 to 8 carbon atoms (C2-C8) and possessing a double bond, wherein the alkenyl group may optionally and independently be substituted with one or more substituents described herein, and may include groups having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. Examples include, but are not limited to, ethylenyl, vinyl (-CH=CH2), and allyl (-CH2CH=CH2).

[0094] The term "alkenylene" refers to a compound with at least one unsaturated site, i.e., a carbon-carbon sp. 2 This refers to a divalent straight-chain or branched-chain hydrocarbon group of any length having 2 to 8 carbon atoms (C2-C8) and possessing a double bond, wherein the alkenylene group may optionally and independently be substituted with one or more substituents described herein, and may include groups having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (-CH2CH=CH-), etc.

[0095] The term "alkynyl" refers to a monovalent linear or branched hydrocarbon group of any length consisting of 2 to 8 carbon atoms (C2-C8) having at least one unsaturated site, i.e., a carbon-carbon sp triple bond, wherein the alkynyl group may optionally and independently be substituted with one or more substituents described herein. Examples include, but are not limited to, ethynyl (-C≡CH) and propynyl (propargyl, -CH2C≡CH).

[0096] The term "alkynylene" refers to a divalent linear or branched hydrocarbon group of any length consisting of 2 to 8 carbon atoms (C2-C8) having at least one unsaturated site, i.e., a carbon-carbon sp triple bond, wherein the alkynylene group may optionally and independently be substituted with one or more substituents described herein. Examples include, but are not limited to, ethynylene (-C≡C-), propynylene (propargylene, -CH2C≡C-), and others.

[0097] The terms "carbocyclic," "carbocyclyl," "carbocyclic ring," and "cycloalkyl" refer to a monocyclic ring consisting of 3 to 12 carbon atoms (C3-C3). 12This refers to monovalent, non-aromatic saturated or partially unsaturated rings having 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocyclic rings having 7 to 12 atoms may be arranged, for example, as bicyclo[4,5], [5,5], [5,6], or [6,6] systems, and bicyclic carbocyclic rings having 9 or 10 ring atoms may be arranged as bicyclo[5,6] or [6,6] systems, or as bridging 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 carbocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopento-1-enyl, 1-cyclopento-2-enyl, 1-cyclopento-3-enyl, cyclohexyl, 1-cyclohexa-1-enyl, 1-cyclohexa-2-enyl, 1-cyclohexa-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. The carbocyclyl group may optionally be independently substituted with one or more substituents described herein.

[0098] "Aryl" refers to a group of 6 to 20 carbon atoms (C6-C) obtained by removing one hydrogen atom from a single carbon atom in a hydrophilic aromatic ring system. 20 This refers to a monovalent aromatic hydrocarbon group. Some aryl groups are represented as "Ar" in exemplary structures. Aryls include bicyclic groups containing saturated, partially unsaturated, or aromatic rings fused to aromatic carbocyclic rings. Typical aryl groups include, but are not limited to, groups derived from benzene (phenyl), substituted benzenes, naphthalenes, anthracenes, biphenyls, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and others. Aryl groups are optionally independently substituted with one or more substituents described herein.

[0099] "Arylene" is a compound obtained by removing two hydrogen atoms from two carbon atoms in a hydrophilic aromatic ring system, consisting of 6 to 20 carbon atoms (C6-C6).20 Arylenes refer to divalent aromatic hydrocarbon groups. Some arylene groups are represented as "Ar" in exemplary structures. Arylenes include bicyclic groups containing saturated, partially unsaturated, or aromatic rings fused to aromatic carbocyclic rings. Typical arylene groups include, but are not limited to, groups derived from benzene (phenylene), substituted benzenes, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and others. Arylene groups are optionally substituted with one or more substituents as described herein.

[0100] The terms “heterocyclic,” “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 in the ring) carbocyclic group having 3 to about 20 ring atoms, wherein at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms are carbon, and one or more ring atoms are optionally independently substituted with one or more substituents as described below. The heterocyclic ring may be a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S), or a dicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), e.g., bicyclo[4,5],[5,5],[5,6], or [6,6] systems. For complex algebras, see Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WABenjamin, 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 As described in York, 1950 to present, particularly rings 13, 14, 16, 19, and 28; and in J. Am. Chem. Soc. (1960) 82:5566. "Heterocyclyl" also includes groups in which the heterocyclic group is fused to a saturated, partially unsaturated, or aromatic carbocyclic or heterocyclic ring. Examples of heterocyclic rings include morpholine-4-yl, piperidine-1-yl, piperazinyl, piperazine-4-yl-2-one, piperazine-4-yl-3-one, pyrrolidine-1-yl, thiomorpholine-4-yl, S-dioxothiomorpholine-4-yl, azocan-1-yl, azetidine-1-yl, octahydropyrido[1,2-a]pyrazine-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranil, dihydrofuranil, tetrahydrothienyl, tetrahydropyranil, dihydropyranil, tetrahydrothiopyranil, piperidino, morpholino, thiomorpholino, thiooxanil, piperazinyl, homopiperazinyl, azetidine, Examples include, but are not limited to, oxetanil, thietanil, homopiperidinil, oxepanil, thiepanil, oxazepinil, diazepinil, thiazepinil, 2-pyrrolinil, 3-pyrrolinil, indolinil, 2H-pyranil, 4H-pyranil, dioxanil, 1,3-dioxolanil, pyrazolinil, dithianil, dithiolanil, dihydropyranil, dihydrothienyl, dihydrofuranil, pyrazolidinylimidazolinil, imidazolidinil, 3-azabicyco[3.1.0]hexanil, 3-azabicyclo[4.1.0]heptanil, azabicyclo[2.2.2]hexanil, 3H-indollyquinolidinil, and N-pyridylurea. The spiro moiety is also included within the scope of this definition. Examples of heterocyclic groups in which two ring atoms are substituted with an oxo (=O) moiety are pyrimidinol and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein may optionally be independently substituted with one or more substituents as described herein.

[0101] The term "heteroaryl" refers to a monovalent aromatic group of 5, 6, or 7 members, and includes 5 to 20 fused ring systems (at least one of which is aromatic) containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include pyridinyl (e.g., including 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, 1-methyl-1H-benzo[d]imidazole, [1,2,4]triazolo[1,5-a]pyridine, pyrimidinyl (e.g., including 4-hydroxypyridinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, and quinolinyl. These include isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolidinyl, phthalazinyl, pyridadinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, flazanyl, benzoflazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthilidinyl, and phlopyridinyl. The heteroaryl group is optionally independently substituted with one or more substituents described herein.

[0102] Heterocyclic or heteroaryl groups may be carbon (carbon bonded) or nitrogen (nitrogen bonded) bonded, where possible. Examples, though not limited, include carbon-bonded heterocyclic or heteroaryl groups attached at positions 2, 3, 4, 5, or 6 of pyridine, 3, 4, 5, or 6 of pyridazine, 2, 4, 5, or 6 of pyrimidine, 2, 3, 5, or 6 of pyrazine, 2, 3, 5, or 5 of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, 2, 3, 4, or 5 of oxazole, imidazole, or thiazole, 3, 4, or 5 of isoxazole, pyrazole, or isothiazole, 2 or 3 of aziridine, 2, 3, or 4 of azetidine, 2, 3, 4, 5, 6, 7, or 8 of quinoline, or 1, 3, 4, 5, 6, 7, or 8 of isoquinoline.

[0103] As an example, and not an exhaustive list, nitrogen-bonded heterocycles or heteroaryls are linked at position 1 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, position 2 of isoindole or isoindoline, position 4 of morpholine, and position 9 of carbazole or β-carbolin.

[0104] The term "chiral" refers to molecules that cannot be superimposed with their mirror image partners, while the term "achiral" refers to molecules that can be superimposed with those mirror image partners.

[0105] The term "stereoisomer" refers to compounds that have the same chemical structure but differ in the arrangement of atoms or groups in space.

[0106] A "diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated under high-resolution analytical techniques such as electrophoresis and chromatography.

[0107] An "enantiomer" refers to two stereoisomers of a compound that are mirror images of each other and cannot be superimposed.

[0108] The stereochemical definitions and conventions used herein are generally based on SP. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., Stereochemistry. This information follows *Organic Compounds* (1994) by John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, meaning they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around its chiral center(s). The prefixes d and l or (+) and (-) are used to indicate the rotation of plane-polarized light by the compound, with (-) or 1 meaning the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Certain stereoisomers are also sometimes called enantiomers, and mixtures of such isomers are often called enantiomer mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two optically unactive enantiomer species.

[0109] Other terms, definitions, and abbreviations used herein include: wild-type ("WT"); cysteine-modified 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) for the heavy chain; K149C (Kabat numbering) for the light chain. Further definitions and abbreviations are provided elsewhere in this specification.

[0110] II. PROTAC-Antibody Conjugate (PAC) The PROTAC-antibody conjugate (PAC) molecules described herein comprise an antibody conjugated to PROTAC via a linker (L1), the PROTAC comprising a ubiquitin E3 ligase binding group ("E3LB"), a linker ("L2"), and a protein binding group ("PB"). The general formula of PAC is as follows: Ab-(L1-D) p In the formula, D is a PROTAC having the structure E3LB-L2-PB, 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 D, and p has a value of about 1 to about 50. The variable p reflects that the antibody may bind 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 PAC. To obtain PAC with potent efficacy and a desirable therapeutic index, the following components are provided.

[0112] 1. Antibody (Ab) As described herein, antibodies, such as monoclonal antibodies (mABs), are used to deliver PROTACs to target cells, such as cells expressing specific proteins targeted by the antibody. The antibody portion of a PAC can target cells expressing an antigen, thereby allowing antigen-specific PACs to be delivered intracellularly to the target cells, typically via endocytosis. PACs containing antibodies against antigens not found on the cell surface may result in less specific intracellular delivery of the PROTAC portion into the cell, while the PAC may still be affected. The PACs and methods of use described herein utilize antibody recognition on the cell surface and / or endocytosis of the PAC to deliver the PROTAC portion into the cell.

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

[0114] Human antibodies can be prepared by administering an immunosorbent to transgenic animals modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen administration. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci, are extrachromosomal, or are randomly incorporated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Publication No. US2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions derived from intact antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0115] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heterozygous myeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, 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 are also Li This is described in et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, those described in U.S. Patent No. 7,189,826 (describes the production of human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes 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 selected Fv clone variable domain sequences from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0117] b. Antibodies derived from the library Antibodies for use in PACs can be isolated by screening combinatorial libraries for antibodies with desired activity(s). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing desired binding properties. Such methods are outlined, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and for example, 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 certain phage display methods, the repertoire of VH and VL genes can be separately cloned by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phages typically present antibody fragments either as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries derived from immunized sources provide high-affinity antibodies against the immunizer without the need to construct hybridomas. Alternatively, a naive repertoire can be cloned (e.g., from humans) to provide a single source of antibodies against 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 constructed synthetically by cloning an unrearranged V gene region from stem cells, encoding the highly variable CDR3 region using PCR primers containing random sequences, and achieving 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. Patent No. 5,750,373, as well as U.S. Patent Publications 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 human antibodies or human antibody fragments in this specification.

[0120] c. Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes 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 further examples, a chimeric antibody is a "class-switched" antibody, which is an antibody whose class or subclass has changed from that of a parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0121] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity towards humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains, where HVR, e.g., CDR (or part thereof), is derived from the non-human antibody and FR (or part thereof) is derived from the human antibody sequence. The humanized antibody also optionally contains at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues derived from the non-human antibody (e.g., the antibody from which the HVR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0122] Humanized antibodies and methods for producing them are outlined, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al. Further information can be found in al., Methods36:25-34 (2005) (describes SDR (a-CDR) transplantation); Padlan, Mol.Immunol.28:489-498 (1991) (describes "resurfacing"); Dall'Acqua et al., Methods36:43-60 (2005) (describes "FR shuffling"); and Osbourn et al., Methods36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) (describes the "guided selection" approach to FR shuffling).

[0123] Human framework regions that can 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 specific subgroups of light chain 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 mutant) 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 the FR library (see, e.g., Baca et al. This includes, but is not limited to, 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, for example, bispecific antibodies. The term “multispecific antibody,” as used herein, refers to an antibody comprising an antigen-binding domain that has polyepitope specificity (i.e., that can bind to two or more different epitopes on one molecule or to epitopes on two or more different molecules).

[0125] In some embodiments, a multispecific antibody is a monoclonal antibody (e.g., a bispecific antibody) having binding specificity to 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 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 to which the multispecific antibody binds are typically epitopes that are not identically bound by a single monospecific antibody, such as a conventional antibody or a single immunoglobulin monovariate 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, the first antigen-binding domain of a multispecific antibody can bind to one epitope on one protein molecule, while the second antigen-binding domain of a multispecific antibody can 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, where the first VH / VL unit binds to a first epitope and the second VH / VL unit binds to a second epitope, and each VH / VL unit comprises 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 having two or more VL and VH domains, and antibody fragments (e.g., Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, and antibody fragments bound covalently or noncovalently). A VH / VL unit further comprising at least a portion of the heavy chain variable region and / or at least a portion of the light chain variable region may also be referred to as an "arm," or "hemimer," or "half-antibody." In some embodiments, the hemimer contains a heavy chain variable region sufficient to enable the formation of an intermolecular disulfide bond with a second hemimer. In some embodiments, the hemimer includes a knob mutation or a hole mutation to enable heterodimerization with a second hemimer or half-antibody, for example, which includes a complementary hole mutation or knob mutation. Knob mutations and hole mutations are discussed further below.

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

[0128] Techniques for producing multispecific antibodies include the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (Milstein and Cuello, Nature 305:537 (1983), WO93 / 08829, and Traunecker). See et al., EMBO J.10:3655 (1991), as well as “knob-in-hole” operations (see, for example, U.S. Patent No. 5,731,168, WO2009 / 089004, U.S.2009 / 0182127, U.S.2011 / 0287009, Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, and Kontermann (2005) Acta Pharmacol. Sin., 26:1-9). The term “knob-in-hole” or “KnH” technique, as used herein, refers to a technique that directs two polypeptides to form pairs in vitro or in vivo by introducing a bump (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface in which they interact. For example, KnH is introduced at the Fc:Fc binding surface, CL:CH1 binding surface, or VH / VL interface of the 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 during the production of multispecific antibodies. For example, multispecific antibodies having KnH in their Fc regions may further contain a single variable domain bound to each Fc region, or may further contain different heavy chain variable domains that pair with similar or different light chain variable domains. KnH technology can also be used to pair two different receptor extracellular domains together, or any other polypeptide sequence containing different target recognition sequences (e.g., affibodies, peptide bodies, and other Fc fusions).

[0129] As used herein, the term "knob mutation" refers to a mutation that introduces a bump (knob) into a polypeptide at an interface where one polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a whole mutation.

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

[0131] A “protrusion” refers to at least one amino acid side chain that protrudes from the interface of the first polypeptide and is therefore positioned in a complementary cavity at the adjacent interface (i.e., the interface of the second polypeptide), thereby stabilizing the heteromultimer and, for example, preferring heteromultimerization to homomultimerization. Protrusions may be present at the original interface or may be introduced synthetically (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 protrusion. 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 larger side chain amount than the original amino acid residue. It will be understood that there may be more than one original residue and corresponding import residues. Side chain amounts for various amino acid residues are shown, for example, in Table 1 of US2011 / 0287009. Mutations that introduce a “protrusion” may be referred to as “knob mutations.”

[0132] In some embodiments, the import residue for ridge formation is a native 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 ridge formation has a small side chain amount and is alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0133] A “cavity” refers to at least one amino acid side chain that recesses from the interface of the second polypeptide and thus accommodates a corresponding bulge on the adjacent interface of the first polypeptide. Cavities may be present at the original interface or may be introduced synthetically (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 a cavity. To achieve 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 having a smaller side chain length than the original amino acid residue. It will be understood that there may be more than one original residue and corresponding import residues. In some embodiments, the import residue for cavity formation is a native 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 cavity formation has a large side chain and is such as tyrosine, arginine, phenylalanine, or tryptophan. Mutations that introduce a "cavity" may be referred to as "hole mutations."

[0134] The ridges are "situable" within the cavities, meaning that the spatial position of the ridges and cavities on the interface of the first and second polypeptides, respectively, as well as the size of the ridges and cavities, are such that the ridges can be positioned within the cavities without significantly disrupting the normal association of the first and second polypeptides at the interface. Since ridges such as Tyr, Phe, and Trp typically do not extend perpendicularly from the interface axis and have preferred three-dimensional structures, the alignment of the ridges with the corresponding cavities may, in some cases, rely on modeling the ridge / cavity pairs based on three-dimensional structures, such as those obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using techniques 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 includes one or more mutations selected from T366S, L368A, and Y407V (EU numbering). In some embodiments, the hole mutation in the IgG1 constant region includes 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 includes one or more mutations selected from T366S, L368A, and Y407V (EU numbering). In some embodiments, the hole mutation in the IgG4 constant region includes T366S, L368A, and Y407V (EU numbering).

[0137] Multispecific antibodies can also be produced by manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992)); using "diabody" techniques to produce bispecific antibodies (see, e.g., Hollinger et al., Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993)); and using single-stranded Fv(sFv) dimers (see, e.g., Gruber et al. See al., J.Immunol., 152:5368 (1994); and can also be prepared by preparing a triplicate antibody, for example, as described in Tutt et al. J.Immunol. 147:60 (1991).

[0138] Manipulated antibodies having three or more functional antigen-binding sites, including "octopus antibodies" or "bivariable domain immunoglobulins" (DVD), 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 include antigen-binding sites that bind to a target protein as well as another different antigen (see, e.g., US2008 / 0069820).

[0139] e. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For an overview of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For an overview of scFv fragments, see, for example, Pluckthuen, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO93 / 16185, and U.S. Patents 5,571,894 and 5,587,458. For a discussion of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have an increased in vivo half-life, please refer to U.S. Patent No. 5,869,046.

[0140] A diabody is an antibody fragment having two antigen-binding sites that may be bivalent or bispecific. See, for example, EP404,097, WO1993 / 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 part of the light chain variable domains or all or part of the heavy chain variable domains of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc. (Waltham, MA), e.g., U.S. Patent No. 6,248,516B1).

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

[0143] f. Antibody variant In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies 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 the amino acid sequence of the antibody, and / or substitutions of residues therein. Deletions, insertions, and substitutions can be arbitrarily combined to arrive at the final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[0144] i. Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional side-chain induction include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substitutional variations are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below in relation to amino acid side-chain classes. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) or cell-mediated cytotoxicity (CDC).

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

[0146] Non-conservative substitution involves replacing one member of one of these classes with one of another.

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

[0148] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hotspots," i.e., residues encoded by codons that frequently undergo mutations during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction and re-selection 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 one of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Subsequently, a secondary library is constructed. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-targeted 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. CDR-H3 and CDR-L3 are particularly often targeted.

[0149] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made within an HVR. Such modifications may be outside the HVR "hotspot" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unmodified or contains one, two, or three or fewer 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 a light source is affected. Further substitutions may be introduced at amino acid positions that are functionally sensitive to the initial substitution. Alternatively, or additionally, an antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues are targeted or excluded as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

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

[0152] ii. Cysteine-modified antibody variants In certain embodiments, it may be desirable to produce a cysteine-modified antibody, for example, a “THIOMAB® antibody” in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur in the available sites of the antibody. By substituting these residues with cysteine, a reactive thiol group is thereby positioned in the available sites of the antibody and can be used to conjugate the antibody to other parts, such as an L1-PROTAC group, to produce a PAC as further described herein. In certain embodiments, one or more of the following residues may be substituted 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 include HC-A140C (EU numbering) cysteine ​​substitution. In certain embodiments, the antibodies described herein include LC-K149C (Kabat numbering) cysteine ​​substitution. In certain embodiments, the antibodies described herein include HC-A118C (EU numbering) cysteine ​​substitution.

[0153] Cysteine-modified antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0154] In a particular embodiment, the antibody includes one of the following heavy chain cysteine ​​substitutions: [Table 2]

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

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

[0157] PACs include cysteine-modified antibodies in which one or more amino acids of the wild-type or parental antibody are substituted with cysteine ​​amino acids. Any form of antibody can be modified, i.e., mutated, in this way. For example, a parental Fab antibody fragment may be modified to form a cysteine-modified Fab referred to herein as "ThioFab". Similarly, a parental monoclonal antibody may be modified to form a THIOMAB® antibody. Note that a single-site mutation results in a single modified cysteine ​​residue in ThioFab, while due to the dimeric nature of IgG antibodies, a single-site mutation results in two modified cysteine ​​residues in a THIOMAB® antibody. Mutants with substituted ("modified") cysteine ​​(Cys) residues are evaluated for the reactivity of the newly introduced, modified cysteine ​​thiol group. The thiol reactivity value is a relative number in the range of 0 to 1.0 and can be measured for any cysteine-modified antibody. The thiol reactivity values ​​of cysteine-modified antibodies for use in PAC are in the range of 0.6–1.0, 0.7–1.0, or 0.8–1.0.

[0158] To prepare cysteine-modified antibodies by mutagenesis, DNA encoding amino acid sequence variants of the starter polypeptide is prepared by a variety of methods known in the art. These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of DNA encoding a previously prepared polypeptide. Variants of recombinant antibodies can also be constructed by restriction fragment manipulation or by double-extension PCR using synthetic oligonucleotides. Mutagenesis primers encode cysteine ​​codon substitutions. DNA encoding such mutant cysteine-modified antibodies can be generated using standard mutagenesis techniques. 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 manipulated in 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 manipulated cysteine ​​thiol can react with a linker reagent having a thiol-reactive electrophilic group, such as maleimide, an active disulfide (e.g., 4-nitropyridyl), or an alpha-haloamide, or with the linker L1-PROTAC intermediate described herein, to form a PAC with the cysteine-manipulated antibody (THIOMAB® antibody) and PROTAC residue. Therefore, the position of the PROTAC moiety can be designed, controlled, and known. Since the manipulated cysteine ​​thiol group typically reacts with thiol-reactive linker reagents or the linker L1-PROTAC intermediate in high yield, the PROTAC / antibody ratio ("PAR") can be controlled. By manipulating the antibody to introduce cysteine ​​amino acids by single-site substitution on the heavy or light chain, two new cysteines are introduced into a symmetric antibody. A PAR of approximately 2 and almost complete homogeneity of the conjugation product can be achieved.

[0160] Cysteine-modified antibodies preferably retain the antigen-binding ability of their wild-type parent antibodies to their corresponding counterparts. Therefore, cysteine-modified antibodies can preferably bind specifically to antigens. Such antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signaling proteins, cell survival regulators, cell growth 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 associated with (e.g., known or suspected to functionally contribute to) angiogenesis. Tumor-associated antigens may be cluster differentiation factors (i.e., CD proteins). Antigens to which cysteine-modified antibodies can bind may be members of one subset of the above classification, and other subsets of that classification may include other molecules / antigens with specific properties (for the antigen of interest).

[0161] Cysteine-modified antibodies are prepared for conjugation with linker L1 intermediates by reduction and reoxidation of intrachain disulfide groups.

[0162] iii. Glycanization variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying amino acids so that one or more glycosylation sites are created or removed.

[0163] If an antibody contains an Fc region, the carbohydrate bound to it may be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides of a branched chain, generally bound by an N-bond to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose, which binds to GlcNAc at the "stem" of the branched oligosaccharide structure. In some embodiments, modification of oligosaccharides in antibodies may be performed to create antibody variants with certain improved properties.

[0164] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the total of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured by WOMALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (Eu numbering of the Fc region residue) in the Fc region. However, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. For example, see U.S. Patent Publication No. US2003 / 0157108 (Presta, L.) and U.S. Patent Publication No. US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S.2003 / 0157108, WO2000 / 61739, WO2001 / 29246, U.S.2003 / 0115614, U.S.2002 / 0164328, U.S.2004 / 0093621, and U.S.2004 / 013214. Examples include 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), and Yamane-Ohnuki et al. Biotech.Bioeng.87:614(2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), US Patent Application No. US2003 / 0157108A1, Presta, L., and WO2004 / 056312A1, Adams et al., particularly in Example 11), as well as knockout cell lines, e.g., alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al.) See also al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107.

[0165] For example, antibody variants having a bifid oligosaccharide are provided, in which a branched oligosaccharide bound to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have enhanced fucosylation and / or improved 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 U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. 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 variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0167] In certain embodiments, the subject matter described herein is directed towards antibody variants that are desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental, by possessing some, but not all, effector functions. In vivo and / or in vivo cytotoxic assays may be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that an antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only Fc(RIII), while 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 evaluate the ADCC activity of the target molecule are U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l This is described in Acad.Sci.USA82:1499-1502(1985);5,821,337 (see Bruggemann, M. et al., J.Exp. "Med." 166:1351-1361(1987)). Alternatively, non-radioactive assays may be used (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. (Mountain View, CA) and CytoTox96® non-radioactive cytotoxicity assay (Promega (Madison, WI))). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be measured, for example, in Clynes et al. Proc. Nat'l. It can be evaluated in vivo in animal models such as those disclosed in Acad.Sci.USA95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. A CDC assay may also be performed to evaluate complement activity (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 MJ Glennie, Blood 103:2738-2743 (2004). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, S B et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0168] In some embodiments, one or more amino acid modifications may be introduced within the Fc moiety of the antibodies provided herein to increase IgG binding to the neonatal Fc receptor. In certain embodiments, the antibody includes the following three mutations according to EU numbering: M252Y, S254T, and T256E ("YTE mutations") (see also U.S. Patent No. 8,697,650, Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006)). In certain embodiments, the YTE mutation does not affect the antibody's ability to bind to its homologous antigen. In certain embodiments, the YTE mutation increases the serum half-life of the antibody compared to a natural (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation triples the serum half-life of the antibody compared to a natural (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation triples the serum half-life of the antibody compared to a natural (i.e., non-YTE mutant) antibody. Compared to the body, the YTE mutation doubles the serum half-life of the antibody. In some embodiments, the YTE mutation quadruples the serum half-life of the antibody compared to the natural (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation quintuples the serum half-life of the antibody compared to the natural (i.e., non-YTE mutant) antibody. In some embodiments, the YTE mutation tensuple the serum half-life of the antibody compared to the natural (i.e., non-YTE mutant) antibody. See, for example, 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 variant provides a means to modulate the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of an antibody. In certain embodiments, the YTEO variant provides a means to modulate the ADCC activity of a humanized IgG antibody against a human antigen. See, for example, 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, YTE variants enable simultaneous regulation of serum half-life, tissue distribution, and antibody activity (e.g., ADCC activity of IgG antibodies). See, for example, 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 having one or more substitutions at EU numbered Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include so-called "DANA" Fc variants having alanine substitutions at EU numbered residues 265 and 297 (i.e., D265A and N297A), and Fc variants having substitutions at two or more EU numbered amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581). In certain embodiments, the Fc variant includes 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 (EU numbered) (P329) at position 329 of the wild-type human Fc region is replaced with glycine or arginine, or with an amino acid residue large enough to disrupt the proline sandwich formed in the Fc / Fcγ receptor interface between P329 of Fc and the tryptophan residues W87 and W110 of FcgRIII (Sondermann et al.: Nature 406, 267-273 (20 July 2000)). In further embodiments, at least one further 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 further amino acid substitution is L234A and L235A in the human IgG1Fc region, or S228P and L235E in the IgG4Fc region, all of which conform to EU numbering (U.S. Patent No. 8,969,526).

[0173] In certain embodiments, the polypeptide comprises an Fc variant of the wild-type human IgG Fc region, wherein P329 of the human IgG Fc region is substituted with glycine, and the Fc variant comprises at least two further amino acid substitutions in L234A and L235A of the human IgG1Fc region or S228P and L235E of the human IgG4Fc region, the residues numbered according to EU numbering (U.S. Patent No. 8,969,526). In certain embodiments, for downregulation of ADCC to at least 20% of ADCC induced by a polypeptide containing the wild-type human IgG Fc region, and / or for downregulation of ADCP, the polypeptide comprising the P329G, L234A, and L235A (EU numbering) substitution exhibits the suggested affinity to human FcγRIIIA and FcγRIIA (U.S. Patent No. 8,969,526).

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

[0175] A specific antibody variant exhibiting improved or reduced binding to FcR is described. (See, for example, U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

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

[0177] In some embodiments, modifications resulting in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cell injury (CDC) are made within the Fc region, as described, for example, in U.S. Patent No. 6,194,551, WO99 / 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 transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions in 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, for example, substitution of Fc region residue 434 according to EU numbering (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, National Patent No. 5,624,821, and WO94 / 29351.

[0179] g.Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to include further non-proteinoid moieties known and readily available in the art. Suitable moieties for antibody derivatives 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 homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody may vary, and if two or more polymers are bound, they may be the same molecule or different molecules. In general, the number and / or types of polymers used in derivatization may be determined based on considerations including, but not limited to, specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under specified conditions.

[0180] In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including, but is not limited to, wavelengths that heat the non-proteinaceous moiety to a temperature that does not harm normal cells but kills cells adjacent to the antibody-non-proteinaceous moiety.

[0181] h. Tumor-associated antigens Antibodies that may be useful in the treatment of cancer, including but not limited to cysteine-modified antibodies, include but are not limited to antibodies against cell surface receptors and tumor-associated antigens (TAAs). Certain tumor-associated antigens are known in the art and can be prepared for use in the production of antibodies using methods and information known in the art. In attempts to discover effective cell targets for the diagnosis and therapy of cancer, researchers have strived to identify transmembrane polypeptides or alternative tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cells. 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 resulted in the ability to more specifically target cancer cells for destruction via antibody-based therapies.

[0182] Examples of tumor-associated antigens (TAAs) include, but are not limited to, those listed below. For convenience, information on all of these antigens known in the art is listed below, including names, alternative names, Genbank accession numbers, and key references, in accordance with the dissemination and protein sequence identification practices of the National Center for Biotechnology Information (NCBI). The 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 possess at least approximately 70%, 80%, 85%, 90%, or 95% sequence identity to the sequences identified in the references and / or substantially exhibit the same biological properties or characteristics as TAAs having sequences found in the references. For example, TAAs having variant sequences generally bind specifically to antibodies that specifically bind to TAAs having the corresponding sequences. Sequences and disclosures in references specifically cited herein are expressly incorporated by reference.

[0183] i. Recombination methods and compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated antibody encoding the antibody described herein is provided. Such nucleic acid may encode an amino acid sequence including VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, host cells containing such nucleic acid are provided. In one such embodiment, the host cells include (e.g., transformed by these): (1) a vector containing nucleic acid encoding an amino acid sequence including VL of the antibody and an amino acid sequence including VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence including VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence including VH of the antibody. In one embodiment, the host cells are eukaryotic, for example, Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for producing an antibody is provided, which includes culturing host cells containing nucleic acids encoding an antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0184] For recombinant antibody production, the nucleic acid encoding the antibody is isolated, for example, as described above, and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the 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 the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, for the expression of antibody fragments in E. coli.) After expression, antibodies can be isolated from bacterial cell paste into a soluble fraction and further purified.

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

[0187] Host cells suitable for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. In particular, several baculovirus strains have been identified that can be used in conjunction with insect cells for gene transfer of Spodoptera frugiperda cells.

[0188] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0189] Vertebrate cells can 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 the monkey kidney CV1 cell line transformed with 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 cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT060562); e.g., Mather et al., Annals These include TRI cells; MRC5 cells; and FS4 cells, as described in NYAcad.Sci.383:44-68 (1982). 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)), as well as myeloma cell lines such as Y0, NS0, and Sp2 / 0. For an overview of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol.248 (BKCLo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003).

[0190] Next, regarding antibody affinity, in the embodiment, the antibody binds to one or more tumor-associated antigens or cellulite surface receptors selected from (1) to (53): (1) BMPR1B (Bone morphogenetic protein receptor type IB, Genbank contract) 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 Claim 6); WO2003024392 (Claim 2; Figure 112); WO200298358 (Claim 1; page 183); WO200254940 (pages 100-101); WO200259377 (pages 349-350); WO200230268 (Claim 27; page 376); WO200148204 (Example; Figure 4) NP_001194 Osteogenesis Imperative Receptor, IB type / 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; pp. 127-129);WO200286443(Claim 27; pp. 222, 393);WO2003003906(Claim 10; pp. 293 Page);WO200264798(Claim 33; pages 93-95);WO200014228(Claim 5; pages 133-136);US2003224454(Figure 3);WO2003025138(Claim 12; page 150);NP_003477 Solute Transporter Family 7 (Cationic Amino Acid Transporter, y+ System), Member 5 / pid=NP_003477.3-Homo sapiens Cross-reference:MIM:600182;NP_003477.3;NM_015923;NM_003486_1 (3) STEAP1 (six-transmembrane 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; pp. 618-619); WO2003022995 (Example 9; Figure 13A, Example 53; p. 173, Example 2; Figure 2A); NP_036581 Cross-reference of 6 transmembrane epithelial antigens of the prostate: 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, megakaryosphere enhancement factor, mesothelin, Genbank commissioned number NM_005823)Yamaguchi, N., et al Biol.Chem.269(2),805-808(1994),Proc.Natl.Acad.Sci.USA96(20):11531-11536(1999),Proc.Natl.Acad.Sci.USA93(1):136-140(1996),J.Biol.Chem.270(37):21984-21990(1995)) ;WO2003101283(Request 14);(WO2002102235(Request 13; pages 287-288);WO2002101075(Request 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 transporter)ァミリー34 (リン acid ナトリウム), メンバー2, type II ナトリウム dependent リン acid transporter 3b, Genb ank Trustee No. NM_006424)J.Biol.Chem.277(22):19665-19672(2002),Genomics62(2):281-284(1999),Feild,JA,et al(1999)Biochem.Biophys.Res.Commun.258(3):578-582);WO2004022778(Request 2);EP1394274(Implementation Example 11);WO2002102235(Request 13; 326 pages);EP875569(Request 1; 17-19 pages);WO200157188(Request 20; 329 pages);WO2004032842(Implementation Example IV);WO200175177(Request 24; 139-140 pages); Cross-reference:MIM:604217;NP_006415.1;NM_006424_1 (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, Semadomain, 7 thrombospongin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (Semaphorin) 5B, Genbank accession number AB040878) Nagase T., et al. 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. Biol.Chem.272,21589-21596,1997;Verheij JB,et al Am.J.「Med.」Genet.108,223-225,2002;Hofstra RMW,et al Eur.J.Hum.Genet.5,180-1975; Cell79,1257-1266,1994;Attie T.,et al,Hum.Mol.Genet.4,2407-2409,1995;Auricchio A.,et al Hum.Mol.Genet.5:351-354,1996;Amiel J.,et al Hum.Mol.Genet.5,355-357,1996;Hofstra RMW,et al Nat.Genet.12,445-447,1996;Svensson PJ,et al Hum.Genet.103,145-148,1998;Fuchs S.,et al Mol.Med.7,115-124,2001;Pingault V et al(2002)Hum Genet 111 198 206;WO2004045516(Claim 1);WO2004048938(Example 2);WO2004040000(Claim 151);WO2003087768(Claim 1);WO2003016475(Claim 1);WO2003016475(Claim 1);WO200261087(Figure 1);WO2003016494(Figure 6);WO20 03025138 (Claim 12; page 144); WO200198351 (Claim 1; pages 124-125); EP522868 (Claim 8; Figure 2); WO200177172 (Claim 1; pages 297-299); US2003109676; US6518404 (Figure 3); US5773223 (Claim 1a; columns 31-34); WO2004001004; (10) MSG783 (RNF124, virtual protein FLJ20315, Genbank accession number NM_017763); WO2003104275 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim 12); WO2003083074 (Claim 14; page 61); WO2003018621 (Claim 1); WO20030243 92 (Claim 2; Figure 93); WO200166689 (Example 6); Cross-reference: LocusID: 54894; NP_060233.2; NM_017763_1 (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, Prostate cancer-related gene 1, Prostate cancer-related protein 1, Six transmembrane epithelial antigens of the prostate 2, Six membranes Transprostatic prostate protein (Genbank accession number AF455138) Lab.Invest. 82(11):1573-1582(2002)); WO2003087306; US2003064397 (Claim 1; Figure 1); WO200272596 (Claim 13; pp. 54-55); WO200172962 (Claim 1; Figure 4B); WO2003104270 (Claim 11) ;WO2003104270(Claim 16);US2004005598(Claim 22);WO2003042661(Claim 12);US2003060612(Claim 12;Figure 10);WO200226822(Claim 23;Figure 2);WO200216429(Claim 12;Figure 10);Cross-reference:GI:22655488;AAN04080.1;AF455138_1 (12)TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential channel, subfamily M, member 4, Genbank accession number NM_017636) Xu,XZ, et al Proc. Natl. Acad. Sci. USA 98(19):10692-10697 (2001), Cell 109(3):397-407 (2002), J. Biol. Chem. 278(33):30813-30820 (2003)); US2003143557 (Claim 4); WO200040614 (Claim 14; 100~1 Page 3); WO200210382 (Claim 1; Figure 9A); WO2003042661 (Claim 12); WO200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794 (Claim 14; Figures 1A-D); Cross-reference: MIM:606936; NP_060106.2; NM_017636_1 (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number NP_003203 or NM_003212) Ciccodicola, A., et al EMBO J.8(7):1987-1991(1989), Am.J.Hum.Genet.49(3):555-565(1991)); US2003224411 (Claim 1); WO2003083041 (Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; pp. 52-53); WO20 03024392 (Claim 2; Figure 58); WO200216413 (Claim 1; pp. 94-95, 105); WO200222808 (Claim 2; Figure 1); US5854399 (Example 2; columns 17-18); US5792616 (Figure 2); Cross-reference: MIM:187395; NP_003203.1; NM_003212_1 (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792 Genbank accession number M26004) Fujisaku et al (1989) J. Biol. Chem. 264(4):2118-2125; Weis JJ, et al J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad. Sci. USA 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35, 1025-1031, 1998; Weis JJ, et al Proc. Natl. Acad. Sci. USA 83, 5639-5643, 1986; Sinha SK, et al(1993)J.Immunol.150,5311-5320;WO2004045520(Example 4);US2004005538(Example 1);WO2003062401(Claim 9);WO2004045520(Example 4);WO9102536(Figures 9.1-9.9);WO2004020595(Claim 1);Contracted:P20023;Q13866;Q14212;EMBL;M26004;AAA35786.1. (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated 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, p. 102);WO2003062401(Claim 9);WO200278524(Example 2);US2002150573(Claim Claim 5, pp. 15); US5644033; WO2003048202 (Claim 1, pp. 306 and 309); WO99 / 558658, US6534482 (Claim 13, Figure 17A / B); WO200055351 (Claim 11, pp. 1145-1146); Cross-reference: MIM:147245; NP_000617.1; NM_000626_1 (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764, AY358130) Genome Res. 13(10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99(8):2662-2669 (2002), Proc. Natl. Acad. Sci. 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 Trustee No. 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.USA82,6497-6501,1985; Swiercz JM, et al Nature421,756-760,2003;Ehsani A.,et al (1993) Genomics 15, 426-429; WO2004048938 (Implementation Example 2); WO2004027049 (Figure 1I); WO2004009622; WO2003081210; WO2003089904 (Request 9); WO2003016475 (Request 1); US200311859 2; WO2003008537 (Request 1); WO2003055439 (Request 29; Figures 1A-B); WO2003025228 (Request 37; Figure 5C); WO200222636 (Implementation Example 13; Pages 95-107); WO200212341 (Request 68; Figure 7); WO200213847 (Pages 71-74) ;WO200214503 (pages 114-117);WO200153463 (request item 2; pages 41-46);WO200141787 (page 15);WO200044899 (request item 52; picture 7);WO200020579 (request item 3; picture 2);US5869445 (request item 3; columns 31-38);WO963051 4 (Request Item 2; pages 56-61); EP1439393 (Request Item 7); WO2004043361 (Request Item 7); WO2004022709; WO200100244 (Implementation Example 3; Figure 4); Proxy: P04626; EMBL; M11767; AAA35808.1.EMBL; M11761; AAA35808.1. (18)NCA (CEACAM6, Genbank Trustee No. M18728); Barnett T., et al Genomics3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg RL, et al Proc. Natl. Acad. Sci. USA 99: 16899-16903, 2002; WO2004063709; EP1439393 (Request 7); WO2004044178 (Implementation 4); WO2004031238; WO2003042661 (Request 12); WO200278524 (Implementation 2); WO200286443 (Request 27; 427 pages); WO200260317 (Request 2); Proxy: P40199; Q14920; EMBL; M29541; AAA59915.1.EMBL; M18728; (19) MDP(DPEP1Genbank Trustee No. BC017023)Proc.Natl.Acad.Sci.USA99(26):16899-16903(2002));WO2003016475(Request 1);WO200264798(Request 33; 85~87 pages);JP05003790(Pictures 6~8);WO9946284(Picture 9); Cross-reference:MIM:179780;AAH17023.1;BC017023_1 (20)IL20Rα (IL20Ra, ZCYTOR7, Genbank Trustee No. AF184971); Clark HF, et al Genome Res. 13, 2265-2270, 2003; Mungall AJ, et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104,9-19,2001; Dumoutier L., et alJ. Immunol.167,3545-3549,2001; Parrish-Novak J., et alJ. Biol. Chem.277,47517-47523,2002; Pletnev S., et al(2003)Biochemistry42:12617-12624;Sheikh F.,et al(2004)J.Immunol.172,2006-2010;EP1394274(Implementation Example 11);US2004005320(Implementation Example 5);WO2003029262(pp. 74-75);WO2003002717(Request 2; p. 63);WO200222153(pp. 45-47);US2002042366(pp. 20-21);WO200146261(pp. 57-59);WO200146232(pp. 63-65);WO9837193(Request 1; p. 55-59); Proxy:Q9UHF4;Q6UWA9;Q96SH8;EMBL;AF184971;AAF01320.1. (21) BCAN, BEHAB, Genbank (commission number AF229053) Gary SC, et al Gene256, 139-147, 2000; Clark HF, et al Genome Res.13, 2265-2270, 2003; Strausberg RL, et al Proc.Natl.Acad.Sci.USA99, 16899-16903, 2002; US2003186372 (request item 11); US2003186373 (request item 11); US2003119131 (request item 1; illustration 52); US2003119122 (request item 1; illustration 52); US2003119126 (request item 1); Item 1); US2003119121 (Request Item 1; Figure 52); US2003119129 (Request Item 1); US2003119130 (Request Item 1); US2003119128 (Request Item 1; Figure 52); US2003119125 (Request Item 1); WO2003016475 (Request Item 1); WO200202634 (Request Item 1); (22)EphB2R(DRT, ERK, Hek5, EPHT3, Tyro5, Genbank entrusted number NM_004442)Chan, J. and Watt, VM, Oncogene 6(6), 1057-1061(1991)Oncogene 10(5):897-905(1995),Annu.Rev.Neurosci.21:309-345(1998),Int.Rev.Cytol.196:177-244(2000));WO2003042661(request item) 12); WO200053216 (Request 1; page 41); WO2004065576 (Request 1); WO2004020583 (Request 9); WO2003004529 (pages 128-132); WO200053216 (Request 1; page 42); Cross-reference: MIM:600997; NP_004433.2; NM_004442_1 (23) ASLG659 (B7h, Genbank accession number AX092328) US20040101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221 (Figure 3); US2003165504 (Claim 1); US2003124140 (Example 2); US2003065143 (Figure 60); WO2002102235 (Claim 13; page 299); US2003091580 (Example 2); WO200210187 (Claim 6; Figure 10); WO200194641 (Claim 12; Figure 7b); WO200202624 (Claim 1 3; Figures 1A~1B); US2002034749 (Claim 54; pp. 45~46); WO200206317 (Example 2; pp. 320~321, Claim 34; pp. 321~322); WO200271928 (pp. 468~469); WO200202587 (Example 1; Figure 1); WO200140269 (Example 3; pp. 190~192); WO200036107 (Example 2; pp. 205~207); WO2004053079 (Claim 12); WO2003004989 (Claim 1); WO200271928 (pp. 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; p. 288); WO200140309 (Example 1; Figure 17); US2001055751 (Example 1; Figure 1b); WO200032752 (Claim 18; Figure 1); WO9851805 (Claim 17; p. 97); WO9851824 (Claim 10; p. 94); WO9840403 (Claim 2; Figure 1B); Contract: O43653; EMBL; AF043498; AAC39607.1. (25)GEDA (Genbank accession number AY260763); AAP14954 lipoma HMGIC fusion partner-like protein / pid=AAP14954.1-Homo sapiens species: Homo sapiens (human) WO2003054152 (Claim 20); WO2003000842 (Claim 1); WO2003023013 (Example 3, Claim 20); US2003194704 (Claim 45); Cross-reference: GI:30102449;AAP14954.1;AY260763_1 (26) BAFF-R (B cell activator receptor, BlyS receptor 3, BR3, Genbank accession number AF116456); BAFF receptor / pid=NP_443177.1 - Homo sapiens Thompson, JS, et al Science293(5537),2108-2111(2001);WO2004058309;WO2004011611;WO2003045422(Examples;pp. 32-33);WO2003014294(Claim 35;Figure 6B);WO2003035846(Claim 70;pp. 615-616);WO200294852(Columns 136-137);WO200238766(Claim 3;p. 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 (a B cell-specific protein that covalently interacts with CD79A, CD79α, immunoglobulin-associated alpha, and Ig beta (CD79B), and forms a complex on its surface with Ig M molecules, transmitting 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, pp. 13-14); WO9958658 (Claim 13, Figure 16); WO9207574 (Figure 1); US5644033; Ha et al(1992)J.Immunol.148(5):1526-1531;Mueller et al(1992)Eur.J.Biochem.22:1621-1625;Hashimoto et al(1994)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 lymphoma receptor 1, a G protein-binding receptor activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly in 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, p. 269); WO200172830 (pp. 12-13); WO200022129 (Example 1, pp. 152-153; Example 2, pp. 254-256); WO9928468 (Claim 1, p. 38); US5440021 (Example 2, columns 49-52); WO9428931 (pp. 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 to peptides and present them to CD4+ T lymphocytes); 273aa, pI:6.56MW:30820TM:1[P] gene chromosome:6p21.3, Genbank accession number NP_002111.1) Tonnelle et al (1985) EMBO J.4(11):2839-2847; Jonsson et al (1989) Immunogenetics 29(6):411-413; Beck et al (1992) J.Mol.Biol.228:433-441; Strausberg et al (2002) Proc.Natl.Acad.Sci USA 99:16899-16903; Servenius et al(1987)J.Biol.Chem.262:8759-8766;Beck et al(1996)J.Mol.Biol.255:1-13;Naruse et al(2002)Tissue 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 (purine receptor P2X ligand open ion channel 5, an ion channel opened 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 (pp. 51-52, 57-58); WO200075655 (pp. 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), a type I membrane protein of the leucine-rich repeat (LRR) family, which 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, pp. 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, a putative receptor for the immunoglobulin Fc domain containing C2 Ig-like and ITAM domains, which may play a role in B lymphocyte differentiation); 429aa, pI:5.28, MW:46925TM:1[P] gene chromosome:1q21-1q22, Genbank accession number NP_443170.1) WO2003077836; WO200138490 (Claim 6, Figures 18E-1~18-E-2); Davis et al (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003089624 (Claim 8); EP1347046 (Claim 1); WO2003089624 (Claim 7); (35)FCRH5 (IRTA2, immunoglobulin superfamily receptor translocation-related 2, putative immune receptor potentially involved in B cell development and lymphoma formation; gene deregulation due to translocation occurs in some 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); 374aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession number AF179274; AY358907, CAF85723, CQ782436 WO2004074320 (SEQ ID NO: 810); JP2004113151 (SEQ ID NOs: 2, 4, 8); WO2003042661 (SEQ ID NO: 580); WO2003009814 (SEQ ID NO: 411); EP1295944 (pp. 69-70); WO200230268 (p. 329); WO200190304 (SEQ ID NO: 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 (a transmembrane protein having an EGF-like domain and two follistatin-like domains 1; tomolegrin-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, gene locus G6D; Ly6-D, MEGT1); NP_067079.2; NM_021246.2; Mallya, M. et al (2002) Genomics 80(1):113-123; Ribas, G. et al (1999) J. Immunol. 163(1):278-287; (43) LGR5 (Leucine-rich repeat-containing G protein-bound 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, gene 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, AGet al (2003) Int. J. Cancer 103(6):768-774; (46) GPR19 (G protein-bound receptor 19; Mm. 4787); NP_006134.1; NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105(1-2):162-164; O'Dowd, B. Fet 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 (containing aspartate beta-hydroxylase domain 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.Cancer125(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)Nature440(7082):346-351 (51) GPR172A (G protein-binding 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 is a member of the sialic acid-binding immunoglobulin-like lectin family, a 67 kDa glycosylated transmembrane protein. CD33 is expressed on most myeloid and monocytic leukemia cells, as well as on progenitor myelomonocytic and all erythrocytes. It is not found on early pluripotent stem cells, mature granulocytes, lymphocytes, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin. Invest. 75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33 contains two tyrosine residues at its cytoplasmic end, each followed by a hydrophobic residue similar to the immunoreceptor inhibitory tyrosine 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, intercellular signaling, glycoprotein turnover, and inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternative spliced ​​transcriptional variants of this gene have been described, but the full-length properties of some of these variants have 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 expected to bind to either calcium or sugar), a stalk region, a transmembrane domain, and a short cytoplasmic terminal containing an ITIM motif.

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

[0192] As described herein, PAC may include antibodies, for example, antibodies selected from the following:

[0193] Anti-Ly6E antibody In certain embodiments, the PAC may contain 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-bound protein of unknown function, approximately 8.4 kDa, 131 amino acids long, with an unknown binding partner. It was first identified as a transcription expressed in immature thymocytes and 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 containing an anti-Ly6E antibody as described in PCT Publication 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 embodiment, 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 embodiment, 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 embodiment, 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 the 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 embodiment, the subject matter described herein provides a PAC comprising an antibody comprising (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.

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

[0200] In another embodiment, the anti-Ly6E antibody of PAC contains 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, the 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 containing that sequence retains the ability to bind to Ly6E. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-Ly6E antibody contains the VH sequence of SEQ ID NO: 8, which includes post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 14.

[0201] In another embodiment, 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 7 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-Ly6E antibody containing that sequence retains the ability to bind to Ly6E. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-Ly6E antibody includes the VL sequence of SEQ ID NO: 7, which includes post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 9, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 11.

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

[0203] In one embodiment, a PAC is provided, in which 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 embodiment, PACs are provided herein that include antibodies that bind to the same epitopes as the anti-Ly6E antibodies provided herein. For example, in a particular embodiment, PACs are provided that include antibodies that bind to the same epitopes as the anti-Ly6E antibodies, which include the VH sequence of SEQ ID NO: 8 and the VL sequence of SEQ ID NO: 7, respectively.

[0205] In further embodiments, the anti-Ly6E antibody of the PAC according to any of the above embodiments is a monoclonal antibody, which includes a human antibody. In one embodiment, the anti-Ly6E antibody of the PAC is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is substantially a full-length antibody, e.g., an IgG1 antibody, an IgG2a antibody, or other antibody classes or isotypes as defined herein. In some embodiments, the PAC includes 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, PAC includes an anti-HER2 antibody. In one embodiment, the anti-HER2 antibody of PAC includes humanized anti-HER2 antibodies, e.g., 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 a human framework region having a complementarity-determining region of a mouse antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also referred to as trastuzumab and is marketed under the trademark name HERCEPTIN^. In another embodiment, the anti-HER2 antibody of PAC includes a humanized anti-HER2 antibody, e.g., humanized 2C4, as described in US7862817. An exemplary humanized 2C4 antibody is marketed under the trademark name PERJETA (登録商標) This is pertuzumab, which is marketed under the following conditions.

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

[0208] In some embodiments, the PAC described herein includes 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 described herein includes 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 embodiment, a PAC comprising an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 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 is described herein. In one embodiment, 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 is described herein. 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 further embodiments, the antibody comprises (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 23, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 24.

[0210] In another embodiment, 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 is described herein. 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 embodiment, 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 the 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 embodiment, 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 the 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 embodiment, a PAC comprising an antibody comprising (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 is described herein.

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

[0214] In another embodiment, the PAC anti-HER2 antibody contains 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, the 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 the anti-HER2 antibody containing that sequence retains the ability to bind to HER2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 18. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 18. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-HER2 antibody contains the VH sequence of SEQ ID NO: 18, which includes post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 23, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 24.

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

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

[0217] In one embodiment, a PAC containing an antibody is provided, 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 containing an antibody is provided, the antibody containing the humanized 7C2.v2.2.LA(hu7C2)K149C kappa light chain sequence of SEQ ID NO: 30.

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

[0220] In a further embodiment, PACs are provided herein that include antibodies that bind to the same epitopes as the anti-HER2 antibodies provided herein. For example, in a particular embodiment, PACs are provided that include antibodies that bind to the same epitopes as the anti-HER2 antibodies, which include the VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 17, respectively.

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

[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, PACs comprising an anti-MUC16 antibody 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 are described herein.

[0225] In one embodiment, 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, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37 is described herein. 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, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37.

[0226] In another embodiment, 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 is described herein. 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 embodiment, 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 the 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 embodiment, the present invention describes a PAC comprising an antibody containing (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 embodiments described above, the PAC anti-MUC16 antibody is humanized. In one embodiment, the anti-MUC16 antibody comprises an HVR as in any of the embodiments described above, and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework.

[0230] In another embodiment, PAC's anti-MUC16 antibody contains 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, the 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 the anti-MUC16 antibody containing that sequence retains the ability to bind to MUC16. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 39. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 39. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-MUC16 antibody contains the VH sequence of SEQ ID NO: 39, which includes post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 35, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 36, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 37.

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

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

[0233] In one embodiment, a PAC is provided, in which 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 embodiment, PACs are provided herein that include antibodies that bind to the same epitopes as the anti-MUC16 antibodies provided herein. For example, in a particular embodiment, PACs are provided that include antibodies that bind to the same epitopes as the anti-MUC16 antibodies, which include the VH sequence of SEQ ID NO: 39 and the VL sequence of SEQ ID NO: 38, respectively.

[0235] In further embodiments, the anti-MUC16 antibody of PAC according to any of the above embodiments is a monoclonal antibody, which includes a human antibody. In one embodiment, the anti-MUC16 antibody of PAC is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is substantially a full-length antibody, e.g., 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 contains an anti-STEAP 1 antibody.

[0238] In some embodiments, PACs 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 are described herein.

[0239] In one embodiment, 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, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 is described herein. 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, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42.

[0240] In another embodiment, 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 is described herein. 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 embodiment, 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 the 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 embodiment, the present invention describes a PAC comprising an antibody containing (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 embodiments described above, the anti-STEAP-1 antibody of PAC is humanized. In one embodiment, the anti-STEAP-1 antibody comprises an HVR as in any of the embodiments described above, and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework.

[0244] In another embodiment, PAC's anti-STEAP-1 antibody contains 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, the 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 the anti-STEAP-1 antibody containing that sequence retains the ability to bind to STEAP-1. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 46. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 46. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-STEAP-1 antibody contains the VH sequence of SEQ ID NO: 46, which includes post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 40, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 41, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 42.

[0245] In another embodiment, 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 47 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-STEAP-1 antibody containing that sequence retains the ability to bind to STEAP-1. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 47. In certain embodiments, 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 47. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-STEAP-1 antibody contains the VL sequence of SEQ ID NO: 47, which includes post-translational modifications of that sequence. In certain embodiments, the VL contains one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 43, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 44, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 45.

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

[0247] In one embodiment, a PAC is provided, and 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 embodiment, PACs are provided herein that include antibodies that bind to the same epitopes as the anti-STEAP-1 antibodies provided herein. For example, in a particular embodiment, PACs are provided that include antibodies that bind to the same epitopes as the anti-STEAP-1 antibodies, which include the VH sequence of SEQ ID NO: 46 and the VL sequence of SEQ ID NO: 47, respectively.

[0249] In further embodiments, the anti-STEAP-1 antibody of PAC according to any of the above embodiments is a monoclonal antibody, which includes a human antibody. In one embodiment, the anti-STEAP-1 antibody of PAC is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is substantially a full-length antibody, e.g., 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, PACs 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 are described herein.

[0253] In one embodiment, 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, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50 is described herein. 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, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50.

[0254] In another embodiment, 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 is described herein. 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 embodiment, 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 the 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 embodiment, the present invention describes a PAC comprising an antibody containing (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.

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

[0258] In another embodiment, the PAC anti-NaPi2b antibody contains 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, the 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 the anti-NaPi2b antibody containing that sequence retains the ability to bind to NaPi2b. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 54. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 54. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-NaPi2b antibody contains the VH sequence of SEQ ID NO: 54, which includes post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 48, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 49, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 50.

[0259] In another embodiment, 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, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 55 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-NaPi2b antibody containing that sequence retains the ability to bind to anti-NaPi2b. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 55. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 55. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-NaPi2b antibody includes the VL sequence of SEQ ID NO: 55, which includes post-translational modifications of that sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 51, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 52, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 53.

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

[0261] In one embodiment, a PAC is provided, and 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 embodiment, PACs are provided herein that include antibodies that bind to the same epitopes as the anti-NaPi2b antibodies provided herein. For example, in a particular embodiment, PACs are provided that include antibodies that bind to the same epitopes as the anti-NaPi2b antibodies, which include the VH sequence of SEQ ID NO: 54 and the VL sequence of SEQ ID NO: 55, respectively.

[0263] In further embodiments, the anti-NaPi2b antibody of PAC according to any of the above embodiments is a monoclonal antibody, which includes a human antibody. In one embodiment, the anti-NaPi2b antibody of PAC is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is substantially a full-length antibody, e.g., 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, PACs 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 are described herein.

[0266] In one embodiment, 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 is described herein. 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 embodiment, 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 is described herein. 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 embodiment, 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 the 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 embodiment, the present invention describes a PAC comprising an antibody containing (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.

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

[0271] In another embodiment, the anti-CD79b antibody of PAC contains 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, the 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 the anti-CD79b antibody containing that sequence retains the ability to bind to CD79b. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 56. In certain embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 56. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., in the FR). Optionally, the anti-CD79b antibody contains the VH sequence of SEQ ID NO: 8, which includes post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 58, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 59, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 60.

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

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

[0274] In one embodiment, a PAC is provided, in which 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 embodiment, PACs are provided herein that include antibodies that bind to the same epitopes as the anti-CD79b antibodies provided herein. For example, in a particular embodiment, PACs are provided that include antibodies that bind to the same epitopes as the anti-CD79b antibodies, including the VH sequence of SEQ ID NO: 56 and the VL sequence of SEQ ID NO: 57, respectively.

[0276] In further embodiments, the anti-CD79b antibody of PAC according to any of the above embodiments is a monoclonal antibody, which includes a human antibody. In one embodiment, the anti-CD79b antibody of PAC is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is substantially a full-length antibody, e.g., 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 contain 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), whose sequences are shown below. In one embodiment, the anti-CD22 antibody of the PAC comprises the variable light chain sequence of SEQ ID NO. 72 and the variable heavy chain sequence of SEQ ID NO. 73. In one embodiment, the anti-CD22 antibody of the PAC of the present invention comprises the light chain sequence of SEQ ID NO. 74 and the heavy chain sequence of SEQ ID NO. 75. [Table 10-1] [Table 10-2]

[0279] Anti-CD33 antibody In certain embodiments, the PAC may contain 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 anti-CD33 antibody of PAC includes the light chain sequence of SEQ ID NO: 84 and the heavy chain sequence of SEQ ID NO: 85. In one embodiment, the anti-CD33 antibody of PAC includes 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 anti-CD33 antibody of PAC includes the variable light chain sequence of SEQ ID NO: 90 and the variable heavy chain sequence of SEQ ID NO: 91. In one embodiment, the anti-CD33 antibody of PAC includes the variable light chain sequence of SEQ ID NO: 92 and the variable heavy chain sequence of SEQ ID NO: 93. In one embodiment, the anti-CD33 antibody of the present invention includes the variable light chain sequence of SEQ ID NO: 94 and the variable heavy chain sequence of SEQ ID NO: 95. In one embodiment, the anti-CD33 antibody of the present invention includes the variable light chain sequence of SEQ ID NO: 96 and the 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 ≥10 -13 M (for example, 10 -8 Less than M, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) is the answer.

[0282] In one embodiment, Kd is measured by a radiolabeled antigen-binding assay (RIA) performed with the Fab version of the antibody of interest and its antigen, as described by the assay below. The solution binding affinity of Fab to the antigen is determined by adding Fab to the minimum concentration in the presence of a serial titration of the unlabeled antigen. 125 I) The measurement is performed by equilibrating with a labeled antigen and then capturing the bound antigen on a plate coated with anti-Fab antibody (e.g., Chen See et al., J.Mol.Biol.293:865-881 (1999). To establish the conditions for this assay, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2–5 hours at room temperature (approx. 23°C). In non-adsorbent plates (Nunc#269620), 100 pM or 26 pM [ 125I) Mix the antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody, Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although incubation may be continued for a longer time (e.g., about 65 hours) to ensure equilibrium is achieved. Then transfer the mixture to a capture plate for incubation at room temperature (e.g., over 1 hour). Next, remove the solution and wash the plate eight times with 0.1% polysorbate 20 in PBS (TWEEN-20®). Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate on a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select concentrations of each Fab less than 20% of maximum binding for use in competitive binding assays.

[0283] According to another embodiment, Kd is measured at approximately 10 response units (RUs) using a surface plasmon resonance assay with BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc. (Piscataway, NJ)) at 25°C using an immobilized antigen CM5 chip. 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 approximately 10 response units (RUs) of binding protein, the antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) before injection at a flow rate of 5 μl / min. After antigen injection, 1 M ethanolamine is injected into the blocked unreacted groups. For kinetic measurements, 2-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected into PBS at 25°C at a flow rate of approximately 25 μl / min with 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST). The association rate (k on ) and dissociation rate (koff The ratio k is calculated using a simple one-to-one Langmuir coupled model (BIACORE® evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is given by the ratio k off / k on It is calculated as follows. For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The on velocity obtained by the above surface plasmon resonance assay is 10 6 M -1 s -1 If it exceeds this, the on-rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nM, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) in the presence of increasing concentrations of the antigen, measured with a spectrophotometer such as an Aviv Instruments spectrophotometer equipped with stop flow or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirring cuvette.

[0284] 2. Linker (L1) Where otherwise described herein, “linker” (L1) is a bisensitive or multisensitive moiety that can be used to conjugate one or more PROTAC moieties (D) to an antibody (Ab) to form a PAC. In some embodiments, the PAC can be prepared using an L1 having reactive functionality for covalent binding to the PROTAC and the antibody. For example, in some embodiments, a cysteine ​​thiol of the antibody (Ab) can form a bond with the reactive functional group or linker L1-PROTAC group of the linker to produce the 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). Selecting the correct linker affects proper drug delivery to the intended cellular compartment of the target cell.

[0285] Linkers can generally be divided into two categories: cleavable (e.g., peptides, hydrzones, or disulfides) or non-cleavable (e.g., thioethers). Peptide linkers, such as valine-citrulline (Val-Cit), which can be hydrolyzed by lysosomal enzymes (e.g., cathepsin B), are used to link drugs to antibodies (US6,214,345). These are particularly useful, partly due to their relative stability in systemic circulation and their ability to effectively release drugs into tumors. However, the chemical space represented by native 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. Greater diversity in non-peptide structures can result in novel beneficial properties not provided by peptide linkers. Different types of non-peptide linkers for linker L1 that can be cleaved by lysosomal enzymes are provided herein.

[0286] a. Peptide-mimicking linker Different types of non-peptide peptide-mimicking linkers for PACs cleavable by lysosomal enzymes 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] If L1 is a peptide-mimicking linker, it can be represented by the following formula: -Str-(PM)-Sp- During the ceremony, Str is a stretcher unit covalently bonded to Ab, Sp is a bond or spacer unit covalently bonded to the PROTAC portion. PM is a non-peptide chemical moiety selected from the following group: [ka] W is -NH-heterocycloalkyl- or heterocycloalkyl, Y is a heteroaryl, aryl, -C(O)C1-C6 alkylene, C1-C6 alkylene-NH2, C1-C6 alkylene-NH-CH3, C1-C6 alkylene-N-(CH3) 2、 It is a C1-C6 alkenyl or C1-C6 alkylenyl. Each R 1 C1-C 10 Alkyl, C1-C 10 Alkenyl, (C1-C 10 Alkyl)NHC(NH)NH2, or (C1-C 10 It is alkyl)NHC(O)NH2, R 3 and R 2 Each is independent of H, C1-C 10 Alkyl, C1-C 10 It is an alkenyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 They may combine to form a C3-C7 cycloalkyl group. R 4 and R 5 Each is independent of C1-C 10 Alkyl, C1-C 10 Alkenyl, arylalkyl, heteroarylalkyl, (C1-C 10 It is either alkyl)OCH2- or R 4 and R 5 These may combine to form a C3-C7 cycloalkyl ring.

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

[0289] In this embodiment, Y is a heteroaryl, and R 4 and R 5 They come together to form a cyclobutyl ring.

[0290] In this embodiment, Y is a portion selected from the following group: [ka]

[0291] In an embodiment, Str is a chemical part represented by the following formula: [ka] In the formula, R 6 C1-C 10 Alkylene, C1-C 10 Alkenyl, C3-C8 cycloalkyl, (C1-C8 alkylene)O-, and C1-C 10 Alkylene-C(O)N(R) a A molecule is selected from the group consisting of )-C2-C6 alkylenes, and each alkylene may be substituted with 1 to 5 substituents selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C3-C8 cycloalkyl, C4-C7 heterocycloalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl, and each R a The elements are independently H or C1-C6 alkyl, and Sp is -Ar-R b - and in the formula, Ar is aryl or heteroaryl, R b (C1-C 10 It is alkylene (O-).

[0292] In this embodiment, Str has the following formula: [ka] In the formula, R 7 C1-C 10 Alkylene, C1-C 10 Alkenyl, (C1-C 10 Alkylene)O-,N(R) c)-(C2-C6 alkylene)-N(R c ), and N(R c Selected from )-(C2-C6 alkylene), each R c The elements are independently H or C1-C6 alkyl, and Sp is -Ar-R b - and in the formula, Ar is aryl or heteroaryl, R b (C1-C 10 It is either alkylene)O- or Sp-C1-C6alkylene-C(O)NH-.

[0293] In the embodiment, L1 is a non-peptide chemical moiety represented by the following formula, [ka] R 1 These are C1-C6 alkyl, C1-C6 alkenyl, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2. R 3 and R 2 Each is independent of H or C1-C 10 It is alkyl.

[0294] In the embodiment, L1 is a non-peptide chemical moiety represented by the following formula, [ka] R 1 These are C1-C6 alkyl, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2. R 4 and R 5 They combine to form a C3-C7 cycloalkyl ring.

[0295] In the embodiment, L1 is a non-peptide chemical moiety represented by the following formula, [ka] R 1 is C1-C6 alkyl, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2, and W is as defined above.

[0296] In some embodiments, the linker may be a peptide-mimicking linker, such as those described in WO2015 / 095227, WO2015 / 095124, or WO2015 / 095223.

[0297] b. Non-peptide mimic linker In this embodiment, linker L1 forms a disulfide bond with the antibody. In this embodiment, the linker has the following structure, [ka] In the formula, R 1 and R 2 These are independently selected from H and C1-C6 alkyl groups, or R 1 and R 2 It forms a 3, 4, 5, or 6-membered cycloalkyl or heterocyclyl group. The linker is covalently bonded to the antibody and PROTAC as follows: [ka]

[0298] In one embodiment, the carbonyl group of the linker is connected to an amine group in PROTAC. Note that the sulfur atom connected to Ab is a sulfur group derived from cysteine ​​in the antibody. In another embodiment, linker L1 has a functional group that can react with 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 method on page 766 of Bioconjugate Chemistry 15(4):765-773, Klussman, et al (2004), and the examples herein.

[0299] In some embodiments, the linker has a functional group that can react with an electrophilic group present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehydes and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on the antibody to form a covalent bond to the antibody unit. Examples of such reactive functional groups include, but are not limited to, hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides.

[0300] A linker may contain one or more linker components. Exemplary linker components 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"). Various linker components 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-exclusive exemplary cleavable linkers include acid-unstable linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photosensitive linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); US 5208020).

[0302] In a particular embodiment, the linker has the following formula: [ka] In the formula, A is a “stretcher unit” and is an integer between 0 and 1, W is an “amino acid unit” and is between 0 and 12, and Y is a “spacer unit” and is 0, 1, or 2. An exemplary embodiment of such a linker is described in U.S. Patent No. 7,498,298.

[0303] In some embodiments, the linker component includes a “stretcher unit” that binds the antibody to another linker component or to a PROTAC moiety. Non-limiting exemplary stretcher units are shown below (wherein the formulas, the wavy lines indicate sites of covalent binding to the antibody, PROTAC, or further linker component). [ka]

[0304] 3. PROTAC ("D") Useful PROTACs have the general formula shown above. Specific PROTACs are described in US7,208,157, WO2013 / 106643, WO2013 / 106646, and WO2015 / 160845. PROTACs include those having the following components:

[0305] a. E3 ubiquitin ligase binding group (E3LB) E3 ubiquitin ligases (of which over 600 are known in humans) provide substrate specificity for ubiquitination. Known ligands exist 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; Late Stage Accelerator 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 This includes 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 specific E3 ubiquitin ligase is the von Hippel-Lindau (VHL) tumor suppressor, which is the substrate-recognizing subunit of the E3 ligase complex VCB, and also consists of elongin 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 pro-angiogenic growth factor VEGF and the erythropoietin-induced cytokine in response to hypoxic levels. Compounds that bind to VHL may 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 specific E3 ubiquitin ligase is MDM2. An example of a small molecule binding compound to MDM2 is shown in the following structure: Examples of "Nathrin" compounds having this feature include Nathrin 3a and Nathrin 3. [ka]

[0310] Furthermore, MDM2-binding compounds include 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;This also includes those described in Bioorg. Med. Chem. Lett. 16(2006) 3310-3314. Further specific examples of small molecule conjugated compounds for MDM2 intended for use in PAC include RG7112, RG7388, MI773 / SAR405838, AMG232, DS-3032b, RO6839921, RO5045337, RO5503781, Idasanutlin, CGM-097, and MK-8242.

[0311] Another specific E3 ubiquitin ligase is X-linked apoptosis inhibitor (XIAP). XIAP is a protein that stops apoptotic cell death. Deregulation of XIAP has been associated with cancer, neurodegenerative disorders, and autoimmunity. In the development of lung cancer, XIAP overexpression 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 type of inflammatory bowel disease. Mutations in the XIAP gene can also result in a very rare condition called X-linked lymphoproliferative disorder. Degradation of XIAP can enhance apoptosis by preventing XIAP from binding to caspases, which allows normal caspase activity to proceed.

[0312] Examples of small molecule-binding compounds for 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 Examples of compounds disclosed in Review (2010) Expert Opin Ther Pat; 20:251-67 (Flygare & Fairbrother) include all tetrahydro-benzodiazinon compounds of the following formula, as disclosed in WO2015 / 071393. [ka] Other small molecule binding compounds to XIAP include AEG35156, Envelin, TWX006, and TWX024. When the XIAP binding moiety is used as part of a PROTAC, it 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 DNA damage binding protein 1 (DDB1), karin-4A (CUL4A), and karin-1 regulator (ROC1). This complex ubiquitinates numerous other proteins. Cereblon ubiquitination of target proteins results in increased levels of fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10 (FGF10). FGF8 similarly regulates numerous developmental processes, including limb and ear 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 their analogues are known to bind to cereblon. The crystal structures of cereblon with thalidomide and derivative compounds are described in US2015 / 0374678. Other small molecule compounds that bind to cereblon are also known, e.g., compounds disclosed in US2016 / 0058872 and US2015 / 0291562. Furthermore, phthalimide conjugation with a binder of the BET bromodomain, e.g., an antagonist, can provide highly selective cereblon-dependent BET proteolysis in a PROTAC. Winter et al., Science, June 19, 2015, p. 1376. Such a PROTAC can be conjugated with antibodies described herein to form a PAC.

[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 to bind to the PB group. Any protein in a eukaryotic or microbial system, including viruses, bacteria, or fungi as otherwise described herein, is a target of ubiquitination mediated by the compounds described herein.

[0316] The PB group includes, for example, any moiety that specifically binds to a protein (bounds to a target protein), and non-limiting examples of small molecule target protein moieties include: among many, 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 receptors (AHRs). The compositions described below illustrate some of these nine types of small molecule target protein binding moieties. Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvents, and polymorphs of these compositions, as well as other small molecules that can target the protein of interest.

[0317] Generally, target proteins may include, for example, structural proteins, receptors, enzymes, cell surface proteins, proteins involved in cellular integration functions (including proteins involved in catalytic activity, aromatase activity, motility, helicase activity, metabolic processes (anabolism and catabolism), antioxidant activity, proteolysis, and biosynthesis), kinase activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, ligase activity, enzyme regulator activity, signaling factor activity, structural molecular activity, binding activity (proteins, lipids, carbohydrates), receptor activity, cell motility, membrane fusion, intercellular communication, regulation of biological processes, development, cell differentiation, and responses to stimuli. Behavioral proteins, cell adhesion proteins, proteins involved in cell death, and proteins involved in transport (including protein transporter activity, nuclear transport, ion transporter activity, channel transporter activity, carrier activity, permease activity, secretory activity, electron transporter activity, disease-causing activity, chaperone regulatory activity, nucleic acid binding activity, transcription regulatory activity, extracellular matrix formation and biogenesis activity, and translation regulatory activity) may be included. Among the many proteins of interest, eukaryotes and prokaryotes may be derived from humans, other animals including livestock, microorganisms for target determination for antibiotics and other antimicrobial agents, and plants, as well as viruses.

[0318] Therefore, the PB components of PAC are 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, C1 Delta, CK1 Gamma, 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. 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, MAPKPK, FAK, MARK Any peptide or small molecule that binds to target proteins 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 listed proteins).

[0319] Specific 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, immunosuppressive compounds, and compounds targeting human BET bromodomain-containing proteins, aryl hydrocarbon receptors (AHR), REF receptor kinases, FKBP, androgen receptors (AR), estrogen receptors (ER), thyroid hormone receptors, HIV proteases, HIV integrases, HCV proteases, and acyl-protein thioesterases-1 and -2 (APT1 and APT2).

[0320] c. Linker L2 The E3LB and PB groups of the PROTAC described herein can be linked to a linker (L2). In certain embodiments, the linker group L2 is one or more covalent structural units of A (e.g., -A1...A qA group containing -), where A1 is a group bonded to at least one of E3LB, PB, or a combination thereof. In certain embodiments, A1 directly bonds E3LB, PB, or a combination thereof to another E3LB, PB, or a combination thereof. In other embodiments, A1 is A q Through this, EL3B, PB, or a combination thereof is indirectly coupled with another E3LB, PB, or a combination thereof.

[0321] In a particular embodiment, A1~A q Each is independent, combined, CR La R Lb O, S, SO, SO 2、 NR Lc、 SO2NR Lc、 SONR Lc CONR Lc、 NR Lc CONR Ld、 NR Lc SO2NR 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(=CNO2)NR Ld , 0 to 6 R La and / or R Lb C arbitrarily substituted in the base 3-11 Cycloalkyl, 0-6 R La and / or R Lb C arbitrarily substituted in the base 3-11 Heterocycline, 0-6 R La and / or R Lb Aryls arbitrarily substituted with the base, 0 to 6 R La and / or R Lb A heteroaryl compound optionally substituted with a group, where R La Ma R Lb Each of them independently bonds with other A groups, forming 0 to 4 R groups.Le It is possible to form cycloalkyl and / or heterocyclyl moieties that can be further substituted with groups, R La , R Lb , R Lc , R Ld , and R Le These are H, Haro, and C, each independent of the others. 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 Heterocycline, 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, NH2, SH, SO2C 1-8 alkyl, P(O)(OC 1-8 Alkyl)(C 1-8 alkyl), P(O)(OC 1-8 Alkyl)2, CC-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, CO2H, Halogen, CN, CF3, CHF2, CH2F, NO2, SF5, SO2NHC 1-8 Alkyl, SO2N(C 1-8 Alkyl) 2, SONHC 1-8 Alkyl, SON(C 1-8 Alkyl)2, CONHC 1-8 Alkyl, CON(C 1-8 Alkyl)2, N(C 1-8 Alkyl)CONH(C 1-8Alkyl), N(C 1-8 Alkyl)CON(C 1-8 Alkyl)2, NHCONH(C 1-8 Alkyl), NHCON(C 1-8 Alkyl)2, NHCONH2, N(C 1-8 Alkyl)SO2NH(C 1-8 Alkyl), N(C 1-8 Alkyl)SO2N(C 1-8 Alkyl)2,NH₃SO₂NH₃(C 1-8 Alkyl), NH₃SO₂N(C 1-8 It is alkyl)2, NH₃SO₂NH₂.

[0322] In certain embodiments, q is a non-negative integer. In certain embodiments, q is a non-negative integer.

[0323] In a particular embodiment, for example, if q is greater than 2, A q This is a base connected to the E3LB section, and A1 and A q They are connected via structural units of A (number of structural units of A: q-2).

[0324] In a particular embodiment, for example, if q is 2, A q This is a base connected to sections A1 and E3LB.

[0325] In a particular embodiment, for example, when q is 1, the structure of the linker group L2 is -A1-, where A1 is a group connected to the E3LB portion and the PB portion.

[0326] In a further embodiment, q is an integer between 1 and 100, 1 and 90, 1 and 80, 1 and 70, 1 and 60, 1 and 50, 1 and 40, 1 and 30, 1 and 20, or 1 and 10.

[0327] In a particular embodiment, the linker (L2) is selected from the group consisting of the following: [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 about 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 embodiment of the compounds described herein, the linker group may be any preferred part described herein. In one embodiment, the linker is a substituted or unsubstituted polyethylene glycol group having a size in the range of about 1 to about 12 ethylene glycol units, 1 to about 10 ethylene glycol units, about 2 to about 6 ethylene glycol units, about 2 to 5 ethylene glycol units, or about 2 to 4 ethylene glycol units.

[0330] However, the E3LB and PB groups may be covalently bonded to the linker group through any suitable and stable group relative to the linker's chemical structure. The linker may independently covalently bond 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 on the E3LB and PB groups to provide maximum bonding between the E3LB group on the ubiquitin ligase and the PB group on the target protein to be degraded. In a particular embodiment where the PB group is the E3LB group, the target protein for degradation may be the ubiquitin ligase itself. In a particular embodiment, the linker may be bonded to an optionally substituted alkyl, alkylene, alkene, or alkyne group, aryl group, or heterocyclic group on the E3LB and / or PB groups. The E3LB or PB group may need to be derivatized to create a chemically functional group that is reactive with the chemically functional group on the linker. Alternatively, the linker may need to be derivatized to contain chemically functionalized groups that can react with functional groups found on E3LB and / or PB.

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

[0332] In an embodiment, Z is absent (bonded), -(CH2)iO, -(CH2)iS, -(CH2)iNR, (CH2) i -X1Y1 group (wherein X1Y1 forms an amide group, or a urethane group, ester or thioester group), or [ka] In the formula, each R is H, or a C1-C3 alkyl, alkanol group, or heterocycle (containing a water-soluble heterocycle to promote the water solubility of the linker group, preferably a morpholino, piperidine, or piperazine group), each Y is independently a bond, O, S, or NR, and each i is independently 0-100, 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.

[0333] In this embodiment, X is as follows: [ka] (In the equation, each V is independent and is conjugated (absent)), [ka] j 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. k 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, 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 (or possible bond) that bonds Z to X.

[0334] In embodiments, CON is a bond (or absent bond), a heterocycle containing a water-soluble heterocycle such as piperazinyl or another group, or the following groups: [ka] (In the formula, X 2 O, S, NR 4 , S(O), S(O)2, -S(O)2O, -OS(O)2, or OS(O)2O, X 3 O, S, CHR 4 , NR 4 And, R is H, or a C1-C3 alkyl group optionally substituted with one or two hydroxyl groups, or a pharmaceutically acceptable salt, enantiomer, or stereoisomer thereof.

[0335] In an alternative, preferred embodiment, 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, and 2 to 4 ethylene glycol units.

[0336] In the embodiment, CON is [ka] Alternatively, it is an amide group.

[0337] The E3LB and PB groups may be covalently bonded to the linker group through any suitable and stable group relative to the linker's chemical structure, but in a preferred embodiment, the linker independently covalently bonds to the E3LB and PB groups through an amide, ester, thioester, keto group, carbamate (urethane), or ether, and each of these groups may be inserted anywhere on the E3LB and PB groups to allow the binding of the E3LB group to the ubiquitin ligase and the binding of the PB group to the target protein to be degraded. In other words, as shown herein, the linker may be designed and connected to the E3LB and PB to minimize, eliminate, or neutralize any effect its presence may have on the binding of the respective binding partners of the E3LB and PB. In a particular embodiment, the target protein for degradation may be a ubiquitin ligase.

[0338] Further linker L2s are disclosed in U.S. Patent Applications Publication Nos. 2016 / 0058872, 2016 / 0045607, 2014 / 0356322, and 2015 / 0291562, as well as WO2014 / 063061.

[0339] Next, referring to PAC, a PAC can contain a single antibody, and a single antibody can have more than one PROTAC, each PROTAC covalently bound to the antibody through linker L1. "PROTAC load" is the average number of PROTAC portions per antibody. The PROTAC load can range from 1 to 8 PROTAC(D) per antibody(Ab). That is, in a PAC formulation, Ab-(L1-D) p The p has values ​​of approximately 1 to approximately 50, approximately 1 to approximately 8, approximately 1 to approximately 5, approximately 1 to approximately 4, or approximately 1 to approximately 3. Each PROTAC covalently bound to the antibody through linker L1 may be the same or different PROTAC and may have the same or different types of linkers as any other L1 covalently bound to the antibody. In one embodiment, Ab is a cysteine-modified antibody, and p is approximately 2.

[0340] The average number of PROTACs per antibody in a PAC formulation from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and HPLC. The quantitative distribution of PACs with respect to p can also be determined. ELISA can determine the average value of p in a particular formulation of PAC (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 recognizable by antibody-antigen binding and the detection limits of ELISA. Furthermore, ELISA assays for PAC detection do not determine where the PROTAC portion is bound to the antibody, such as heavy chain or light chain fragments, or specific amino acid residues. In some cases, when p is a specific value from PACs with other PROTAC loads, the isolation, purification, and characterization of PACs can be achieved by methods 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, an antibody may have one or more cysteinethiol groups, or one or more sufficiently reactive thiol groups through which a linker can be bound. Another reactive site on Ab for L1-D linking is the amine functional group of a lysine residue. Values ​​of p include about 1 to about 50, about 1 to about 8, about 1 to about 5, about 1 to about 4, and about 1 to about 3, with p 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, during a conjugation reaction, fewer PROTAC moieties than the theoretical maximum are conjugated to the antibody. 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 group can react with the amine-reactive linker reagent. Also, the most reactive cysteinethiol group can react with the thiol-reactive linker reagent or the linker L1-PROTAC group. Generally, the antibody does not contain many, if any, free and reactive cysteinethiol groups that can bind to the PROTAC moiety. Most cysteinethiol residues in the antibody compound exist as disulfide crosslinks and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partially or completely 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) partially or limiting the reduction conditions for cysteinethiol modification.

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

[0344] In certain embodiments, L1 is as described elsewhere in this specification and includes a peptide-mimicking linker. In these embodiments, the L1-PROTAC has the following formula: [ka] During the ceremony, Str is a stretcher unit. Sp is a bond or spacer unit covalently bonded to D, i.e., the PROTAC portion. R 1 C1-C 10 Alkyl, (C1-C 10 Alkyl)NHC(NH)NH2, or (C1-C 10 It is alkyl)NHC(O)NH2, R 4 and R 5 Each is independent of C1-C 10 Alkyl, arylalkyl, heteroarylalkyl, (C1-C 10 It is either alkyl)OCH2- or R 4 and R 5 This may form a C3-C7 cycloalkyl ring. D is the PROTAC part.

[0345] L1-PROTAC compounds can be represented by the following formula: [ka] In the formula, R6 is C1-C 10 It is alkylene, R 4 and R 5 Together they form a C3-C7 cycloalkyl ring, and D is the PROTAC moiety.

[0346] L1-PROTAC compounds can be represented by the following formula: [ka] In the formula, R 1 , R 4 , and R 5 This is as described elsewhere in this specification, and D is the PROTAC portion.

[0347] L1-PROTAC compounds can be represented by the following formula: [ka] During the ceremony, Str is a stretcher unit. Sp is an arbitrary spacer unit covalently bonded to D, i.e., the PROTAC portion. Y is a heteroaryl, aryl, -C(O)C1-C6 alkylene, C1-C6 alkylene-NH2, C1-C6 alkylene-NH-CH3, C1-C6 alkylene-N-(CH3)2, C1-C6 alkenyl, or C1-C6 alkylenyl, and R 1 C1-C 10 Alkyl, (C1-C 10 Alkyl)NHC(NH)NH2, or (C1-C 10 It is alkyl)NHC(O)NH2, R 3 and R 2 Each is independent of H, C1-C 10 It is alkyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 They may together form a C3-C7 cycloalkyl group. D is the PROTAC part.

[0348] L1-PROTAC compounds can be represented by the following formula: [ka] In the formula, R 6 C1-C 10 It is alkylene, R 1 , R 2 , and R 3 This is as described elsewhere in this specification, and D is the PROTAC portion.

[0349] L1-PROTAC compounds can be represented by the following formula: [ka] In the formula, R 1 , R 2 , and R 3 This is as described elsewhere in this specification, and D is the PROTAC portion.

[0350] In any of the above L1-PROTAC compounds, Str can have the following formula: [ka] In the formula, R 6 C1-C 10 Alkylene, C3-C8 cycloalkyl, O-(C1-C8 alkylene), and C1-C 10 Alkylene-C(O)N(R) a A molecule is selected from the group consisting of )-C2-C6 alkylenes, and each alkylene may be substituted with 1 to 5 substituents selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C3-C8 cycloalkyl, C4-C7 heterocycloalkylaryl, arylalkyl, heteroarylalkyl, and heteroaryl, and each R a Each is independently H or C1-C6 alkyl, and Sp is -Ar-R b -Ar is aryl or heteroaryl, R b (C1-C 10 It is alkylene (O-).

[0351] In a certain L1-PROTAC compound, R 6 C1-C 10 It is an alkylene, and Sp is -Ar-R b - and in the formula, Ar is aryl, R b is (C1-C6 alkylene)O- or R6 is -(CH2) q The range is 1 to 10.

[0352] In any of the above L1-PROTAC compounds, Str can have the following formula: [ka] During the ceremony, [ka] This indicates a portion that can be conjugated to an antibody, R 7 C1-C 10 Alkylene, C1-C 10 Alkylene-O,N(R) c )-(C2-C6 alkylene)-N(R c ), and N(R c Selected from )-(C2-C6 alkylene), each R c These are independently H or C1-C6 alkyl groups. Sp is -Ar-R b - and in the formula, Ar is aryl or heteroaryl, R b (C1-C 10 (Alkylene)O- or R 6 C1-C 10 It is an alkylene, and Sp is -Ar-R b - and Ar is aryl, R b It is (C1-C6 alkylene)O-.

[0353] L1-PROTAC can have the following formula, where in each example, D is the PROTAC part. [ka]

[0354] Next, referring to the PB group of PROTAC, in certain embodiments, PB is as described elsewhere in this specification or 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 receptors (AHR), REF receptor kinases, FKBP, androgen receptors (AR), estrogen receptors (ER), thyroid hormone receptors, HIV proteases, HIV integrases, HCV proteases, and acyl-protein thioesterases-1 and -2 (APT1 and APT2).

[0355] In certain embodiments, E3LB is as described elsewhere in this specification and includes groups that bind to XIAP, VHL, Cereblon, and MDM2.

[0356] The subject matter described herein also relates to a method for preparing PACs from L1-PROTAC compounds, the method comprising contacting an antibody, or its variants, mutants, splice variants, indels, and fusions, with an L1-PROTAC under conditions in which the antibody covalently binds to any available binding site on the L1-PROTAC, from which a PAC is prepared. The subject matter described herein also relates to a method for preparing PACs from an Ab-L1 moiety, i.e., an antibody covalently bound to L1, or its variants, mutants, splice variants, indels, and fusions, the method comprising contacting a PROTAC with Ab-L1 under conditions in which the PROTAC covalently binds to any available binding site on Ab-L1, from which a PAC is prepared. The method may further comprise periodic isolation and purification of the PAC.

[0357] Next, referring to PAC and L1-PROTAC compounds, as described herein, they can exist in solid or liquid form. In the solid state, they can exist in crystalline or amorphous form, or as a mixture thereof. Those skilled in the art will understand that pharmaceutically acceptable solvents can be formed for crystalline or amorphous compounds. In crystalline solvents, solvent molecules are incorporated into the crystal lattice during crystallization. The solvent may be, but is not limited to, non-aqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or they may be accompanied by water as the solvent incorporated into the crystal lattice. When water is the solvent incorporated into the crystal lattice, the solvent is typically referred to as a “hydrate.” Hydrates include stoichiometric hydrates, as well as compositions containing a variable amount of water. The subject matter described herein includes all such solvents.

[0358] Those skilled in the art will further understand that certain compounds and PACs (including their various solvents) described herein, existing in crystalline form, may exhibit polymorphism (i.e., the ability to occur in different crystalline 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 the packing, instrumental arrangement, and other descriptive properties of the crystalline solid state. Polymorphs may therefore have different physical properties such as voltness, density, hardness, deformability, stability, and solubility. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffractions, which can be used for identification. Those skilled in the art will understand that different polymorphs can be produced, for example, by changing or adjusting the reaction conditions or reagents used to prepare the compound. For example, changes in temperature, pressure, or solvent can result in polymorphism. Furthermore, one polymorph may spontaneously transform into another under certain conditions.

[0359] The compounds and PACs described herein, or their salts, may exist in stereoisomerized forms (for example, they may 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 should be understood that compounds or salts of formula (I) may exist in tautomers other than those shown in the formula, and these are also included within the scope of the subject matter disclosed herein. It should be 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 mixtures enriched with purified enantiomers or enantiomer / diastereomers. It should be 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 concerning the fact that one or more atoms are substituted by atoms having atomic weights or mass numbers different from those normally found in nature. Examples of isotopes that can be incorporated into the compounds described herein and their pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example. 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 is one example.

[0361] Compounds and PACs disclosed herein, including those containing the aforementioned isotopes and / or other isotopes of other atoms, as well as their pharmaceutically acceptable salts, are within the scope of the subject matter disclosed herein. Isotope-labeled compounds, for example, 3 H, 14 Tritium labeling, i.e., is disclosed herein, which incorporates radioactive isotopes such as 13C, and these are useful in drug and / or substrate tissue distribution assays. 3 H, and carbon-14, i.e. 14 13C isotopes are generally used due to their ease of preparation and detectability. 11 C and 18 The fluorine isotope is useful in PET (positron emission tomography), 125 I isotopes are useful in SPECT (single-photon emission computed tomography), and all of them are useful in brain imaging. Furthermore, deuterium, that is 2 Substitution with heavier isotopes, such as 1H, can provide certain therapeutic benefits, such as improved metabolic stability, increased in vivo half-life, or reduced dosage requirements, and may therefore be preferred in some situations. The isotope-labeled compounds of formula I can generally be prepared by using readily available isotope-labeling reagents in place of non-isotope-labeling reagents, by performing the procedures disclosed in the scheme and / or in the following examples.

[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 an E3 ligase binding group covalently bonded to L2. L2 is a linker covalently bonded to E3LB and PB. PB is a protein-binding group that is covalently bonded to L2. Ab is an antibody covalently bound to L1, L1 is a linker covalently bonded to Ab and D. p is a conjugate with values ​​ranging from approximately 1 to approximately 8.

[0363] 2. The conjugate according to Embodiment 1, wherein E3LB is a group that binds to E3 ligase, and the E3 ligase is listed in Tables 13-27, for example, 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 a group that binds to E3 ligase, and E3 ligase is a group that binds to von Hippel-Lindau (VHL); Cereblon; XIAP; E3A; MDM2; Late Stage 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 A conjugate according to any of the above embodiments, 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 of the above embodiments, 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 of the above embodiments, wherein E3LB is selected from the group consisting of compounds that bind to VHL, hydroxyproline compounds that bind to VHL, compounds that bind to MDM2, compounds that bind to cereblon, tetrahydro-benzodiazepinone sodium, and small molecule binding compounds described herein.

[0367] 6. E3LB is an XIAP inhibitor that is a tetrahydrobenzodiazepinone having the following 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, and which include all the compounds.

[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, 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 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), the conjugates described in any of the above embodiments, which are groups that bind to these.

[0369] 8. A conjugate according to any of the above embodiments, wherein 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 receptors (AHR), REF receptor kinases, FKBP, androgen receptors (AR), estrogen receptors (ER), thyroid hormone receptors, HIV proteases, HIV integrases, HCV proteases, and acyl-protein thioesterases-1 and -2 (APT1 and APT2).

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

[0371] 10. The conjugate described in any of the above embodiments, wherein Ab is selected from Tables 4-12, for example, 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 of the above embodiments, wherein Ab is a cysteine-modified antibody or a variant thereof.

[0373] 12.Ab, DLL3, EDAR, CLL1;BMPR1B;E16;STEAP1;0772P;MPF;NaPi2b;Sema 5b;PSCA A conjugate according to any of the above embodiments, which binds to one or more 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 embodiment above, wherein Ab is bound 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.

[0375] 14. The conjugate according to any of the above embodiments, wherein 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.

[0376] 15. A conjugate according to any of the above embodiments, wherein the antibody binds to HER2 or B7-H4.

[0377] 16. A conjugate according to any of the above embodiments, wherein the antibody binds to Her2.

[0378] 17. The conjugate according to any of the above embodiments, wherein L1 is a peptide-mimicking linker.

[0379] 18. L1 is a peptide-mimicking linker represented by the following formula: -Str-(PM)-Sp- During the ceremony, Str is a stretcher unit covalently bonded to Ab, Sp is a bond or spacer unit covalently bonded to the PROTAC portion. PM is a non-peptide chemical moiety selected from the following group: [ka] W is -NH-heterocycloalkyl- or heterocycloalkyl, Y is a heteroaryl, aryl, -C(O)C1-C6 alkylene, C1-C6 alkylene-NH2, C1-C6 alkylene-NH-CH3, C1-C6 alkylene-N-(CH3) 2、 It is a C1-C6 alkenyl or C1-C6 alkylenyl. Each R 1 C1-C 10 Alkyl, C1-C 10 Alkenyl, (C1-C 10 Alkyl)NHC(NH)NH2, or (C1-C 10 It is alkyl)NHC(O)NH2, R 3 and R 2 Each is independent of H, C1-C 10 Alkyl, C1-C 10 It is an alkenyl, arylalkyl, or heteroarylalkyl, or R 3 and R 2 They may combine to form a C3-C7 cycloalkyl group. R 4 and R 5 Each is independent of C1-C 10 Alkyl, C1-C 10 Alkenyl, arylalkyl, heteroarylalkyl, (C1-C 10 It is either alkyl)OCH2- or R 4 and R 5 The conjugates described in any of the above embodiments may combine to form a C3-C7 cycloalkyl ring.

[0380] 19. Y is a heteroaryl compound, and R 4 and R 5 A conjugate according to any of the above embodiments, wherein the elements combine to form a cyclobutyl ring.

[0381] 20. The conjugate according to any of the above embodiments, wherein Y is a portion selected from the group consisting of the following: [ka]

[0382] 21. Str has a chemical structure represented by the following formula: [ka] In the formula, R 6 C1-C 10 Alkylene, C1-C 10 Alkenyl, C3-C8 cycloalkyl, (C1-C8 alkylene)O-, and C1-C 10 Alkylene-C(O)N(R) a)-C2-C6 alkylenes are selected from the group consisting of C2-C6 alkylenes, and each alkylene may be substituted with 1 to 5 substituents selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C3-C8 cycloalkyl, C4-C7 heterocycloalkyl, heteroarylalkyl, arylarylalkyl, heteroarylalkyl, and heteroaryl, and each R a The elements are independently H or C1-C6 alkyl, and Sp is -C1-C6 alkylene-C(O)NH- or -Ar-R b -Ar is aryl or heteroaryl, R b (C1-C 10 The conjugate described in any of the above embodiments is alkylene)O-.

[0383] 22. Str has the following expression: [ka] In the formula, R 7 C1-C 10 Alkylene, C1-C 10 Alkenyl, (C1-C 10 Alkylene)O-,N(R) c )-(C2-C6 alkylene)-N(R c ), and N(R c Selected from )-(C2-C6 alkylene), each R c These are independently H or C1-C6 alkyl groups. Sp stands for -C1-C6 alkylene-C(O)NH- or -Ar-R b -Ar is aryl or heteroaryl, R b (C1-C 10 The conjugate described in any of the above embodiments is alkylene)O-.

[0384] 23. L1 has the following formula: [ka] R 1 These are C1-C6 alkyl, C1-C6 alkenyl, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2. R 3 and R 2 Each is independent of H, C1-C 10 The conjugate according to any of the above embodiments, wherein Str and Sp are alkyl, and Str and Sp are as defined herein.

[0385] 24. L1 has the following equation: [ka] R 1 is C1-C6 alkyl, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2, and Str and Sp are as defined herein. R 4 and R 5 The conjugates described in any of the above embodiments, which together form a C3-C7 cycloalkyl ring.

[0386] 25. L1 has the following equation: [ka] Str and Sp are as defined herein, R 1 The conjugate according to any of the above embodiments, wherein is C1-C6 alkyl, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2.

[0387] 26. The following formula is obtained: [ka] During the ceremony, Str has a chemical structure represented by the following formula: [ka] R 6 C1-C 10 Alkylene and C1-C 10 Alkylene-C(O)N(R) a A molecule is selected from the group consisting of )-C2-C6 alkylenes, and each alkylene may be substituted with 1 to 5 substituents selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C3-C8 cycloalkyl, C4-C7 heterocycloalkyl, heteroarylalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl, and each R a These are independently H or C1-C6 alkyl groups. Ab and Sp are as defined herein, p is 1, 2, 3, or 4, the conjugate as described in any of the above embodiments.

[0388] 27. The following formula is available: [ka] During the ceremony, Str has a chemical structure represented by the following formula: [ka] R 6 C1-C 10 Alkylene and C1-C 10 Alkylene-C(O)N(R) aA molecule is selected from the group consisting of )-C2-C6 alkylenes, and each alkylene may be substituted with 1 to 5 substituents selected from the group consisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino, cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester, carboxylic acid, alkylthio, C3-C8 cycloalkyl, C4-C7 heterocycloalkyl, aryl, arylalkyl, heteroarylalkyl, and heteroaryl, and each R a These are independently H or C1-C6 alkyl groups. R 1 , R 4 , R 5 Ab, D, and Sp are as defined herein, p is 1, 2, 3, or 4, the conjugate as described in any of the above embodiments.

[0389] 28. Y is a heteroaryl, aryl, or alkenyl, and R 6 However, C1-C 10 The conjugate described in any of the above embodiments is alkylene.

[0390] 29. The conjugate according to any of the above embodiments, wherein Y is as follows: [ka]

[0391] 30. The conjugate described in any of the above embodiments, wherein Y is as follows: [ka]

[0392] 31. The conjugate described in any of the above embodiments, wherein Y is as follows: [ka]

[0393] 32. Str has the following chemical structure: [ka] R 6 It is a C1-C6 alkylene, Sp stands for -C1-C6 alkylene-C(O)NH- or -Ar-R b - and Ar is aryl, R b The conjugate is (C1-C3 alkylene)O-, as described in any of the above embodiments.

[0394] 33. The following formula is obtained: [ka] During the ceremony, Ab, D, R 2 , and R 3 This is as defined herein, R 1 These are C1-C6 alkyl-NH2, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2. p is 1, 2, 3, or 4, the conjugate as described in any of the above embodiments.

[0395] 34. The following formula is obtained: [ka] During the ceremony, Ab and D are as defined herein, p is 1, 2, 3, or 4. R 1 These are C1-C6 alkyl-NH2, (C1-C6 alkyl)NHC(NH)NH2, or (C1-C6 alkyl)NHC(O)NH2. R 4 and R 5 Each of these is independently a C1-C6 alkyl group, and the alkyl group is either unsubstituted or R 4and R 5 The conjugate according to any of the above embodiments, which together with the carbon to which it is bonded, can form a C3-C7 cycloalkyl ring such as cyclobutyl.

[0396] 35. L1 has the following formula: [ka] In the formula, R 1 and R 2 These are independently selected from H and C1-C6 alkyl groups, or R 1 and R 2 The conjugate according to any of the above embodiments, which forms a 3, 4, 5, or 6-membered cycloalkyl or heterocyclyl group.

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

[0398] 37. A conjugate according to any of the above embodiments, wherein the ratio of PROTAC per antibody ("PAR") is approximately 1.5 to approximately 3.

[0399] 38. A conjugate according to any of the above embodiments, wherein the non-PROTAC ratio ("PAR") per antibody is approximately 2.

[0400] 39. A method for treating a disease in a person requiring treatment, comprising administering an effective amount of any of the above embodiments of the conjugate to the person.

[0401] 40. A pharmaceutical composition comprising a conjugate described in any of the above embodiments and a pharmaceutically acceptable excipient.

[0402] 41. A method for treating a disease in a person requiring treatment, comprising administering to the person an effective amount of the pharmaceutical composition described in Embodiment 40.

[0403] 42. A method for treating a disease using a conjugate described in any of the above embodiments, wherein the disease is a benign or malignant solid tumor and hematological disorder, as well as a hyperproliferative disorder involving nerve, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stroma, blastocoel, inflammation, angiogenesis, immunity, and autoimmune conditions.

[0404] 43. A method for treating a disease using a conjugate described in any of the above embodiments, wherein the disease is cancer.

[0405] 44. A method for treating a disease using a conjugate described in any of the above embodiments, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphoid tumor, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, 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...

Claims

1. A conjugate having the following 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 bound to L2. L2 is a linker covalently bonded to E3LB and PB. PB is a group that is covalently bonded to L2 and binds to target proteins intended for degradation. The aforementioned PROTAC can degrade target proteins via ubiquitination, 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, and R １ 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 H and C are independent of each other. １ -C １０ Alkyl, C ２ -C １０ It is an alkenyl, arylalkyl, or heteroarylalkyl, or R ３ and R ２ They are together C ３ -C ７ A cycloalkyl group may be formed. p is an integer from 1 to 8. E3LB is, 【Transformation 60】 And, L2 is, 【Chemistry 60-1】 【Chemistry 60-2】 【Transformation 60-3】 A conjugate selected from the options.

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, CyclinDl, 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 The conjugate according to claim 1, wherein the group is a 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, Mcl1, BRD2, BRD3, BRD4, AR, RAS, ErbB3, EGFR, IRE1, HPK1, RIPK2, and ERα (including all of these variants, mutations, splice variants, indels, and fusions).

3. The conjugate according to claim 1, wherein 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 that target human BET bromodomain-containing proteins, aryl hydrocarbon receptors (AHR), REF receptor kinases, FKBP, androgen receptors (AR), estrogen receptors (ER), thyroid hormone receptors, HIV proteases, HIV integrases, HCV proteases, and acyl-protein thioesterases-1 and -2 (APT1 and APT2).

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

5. The conjugate according to claim 1, wherein Ab is a cysteine-modified 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 The conjugate according to 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 according to claim 5, wherein Ab is bound 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 according to claim 7, wherein 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 according to claim 7, wherein the antibody is bound to HER2 or B7-H4.

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

11. The conjugate according to claim 1, wherein Y is a heteroaryl compound.

12. The conjugate according to claim 1, wherein Y is a portion selected from the group consisting of the following. 【Transformation 62】

13. The conjugate according to claim 1, wherein Y is a heteroaryl, aryl, or alkenyl.

14. The conjugate according to claim 1, wherein Y is as follows: 【Chemistry 72】

15. The conjugate according to claim 1, wherein Y is as follows: 【Transformation 73】

16. The conjugate according to claim 1, wherein Y is as follows: 【Chemistry 74】

17. The conjugate according to claim 1, wherein p is an integer from 1 to 3.

18. The conjugate according to claim 1, wherein p is 2.

19. A conjugate having the following formula, 【Chemical 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 bound to L2. L2 is a linker covalently bonded to E3LB and PB. PB is a group that is covalently bonded to L2 and binds to target proteins intended for degradation. The aforementioned PROTAC can degrade target proteins via ubiquitination, p is 1, 2, 3, or 4. R １ 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 Each is independent of C １ -C ６ It is an alkyl group, and the alkyl group is either unsubstituted or R ４ and R ５ C ３ -C ７ Forming a cycloalkyl ring, Conjugate.

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

21. The conjugate according to claim 19, wherein p is 2.

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

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

24. The conjugate according to claim 22, wherein p is 2.

25. A pharmaceutical composition comprising the conjugate according to claim 1, 19, or 22 and one or more pharmaceutically acceptable excipients.

26. A composition comprising the conjugate according to claim 1, 19, or 22 for treating a disease in a human being requiring treatment.

27. The composition according to claim 26, wherein the disease is cancer.

28. The composition according to claim 27, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphoid tumor, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung, lung cancer including peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, 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.

29. The composition according to claim 28, wherein the cancer is HER2-positive cancer.

30. The composition according to claim 29, wherein the HER2-positive cancer is breast cancer or gastric cancer.

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