Specific sites for modifying antibodies to make immunoconjugates

Abstract

The present invention provides specific sites for modifying antibodies or antibody fragments by replacing at least one native amino acid in the constant region of a parental antibody or antibody fragment with cysteine, which can be used as a site of attachment for a payload or linker-payload combination.

Claims

1. An immunoconjugate comprising a modified antibody or antigen binding fragment thereof, wherein said modified antibody or antigen binding fragment comprises a substitution of the amino acid at position 152 of the heavy chain constant region of said antibody or antigen binding fragment thereof with a cysteine, wherein said position is numbered according to the EU system.

2. The immunoconjugate of claim 1, wherein said antibody or antigen binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 10.

3. An immunoconjugate comprising a modified antibody or antigen binding fragment thereof, wherein said modified antibody or antigen binding fragment comprises two amino acid substitutions with a cysteine, wherein the substitutions are selected from: a. positions 152 and 375 of the heavy chain constant region of said antibody or antigen binding fragment thereof, wherein said positions are numbered according to the EU system; or b. position 107 of the light chain constant region of said antibody or antigen binding fragment thereof, wherein said light chain is a kappa light chain, and position 360 of the heavy chain constant region of said antibody or antigen binding fragment thereof, wherein said positions are numbered according to the EU system.

4. The immunoconjugate of claim 1, wherein the antibody or antigen binding fragment thereof comprises an E152C substitution; wherein the position is numbered according to the EU system.

5. The immunoconjugate of claim 1, wherein the antibody or antigen binding fragment thereof is an IgG1 antibody.

6. The immunoconjugate of claim 3, wherein the antibody or antigen binding fragment thereof comprises substitutions selected from: a) E152C and S375C in the heavy chain constant region; or b) K107C in the kappa light chain constant region and K360C in the heavy chain constant region; wherein the positions are numbered according to the EU system.

7. The immunoconjugate of claim 3, wherein the antibody or antigen binding fragment thereof is an IgG1 antibody.

8. The immunoconjugate of claim 1 wherein the modified antibody or antigen binding fragment thereof is attached to a drug moiety, directly or indirectly through a linker, by the sulfur atom of said cysteine.

9. The immunoconjugate of claim 1 wherein the modified antibody or antigen binding fragment thereof is attached to a drug moiety, directly or indirectly through a linker, by a thiolmaleimide linkage, a —S—CH2-C(═O)— linkage, or a disulfide linkage.

10. The immunoconjugate of claim 1, wherein said antibody or antigen binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully humanized antibody, a bispecific antibody, or a multi-specific antibody.

11. A composition comprising the immunoconjugate of claim 1 and a pharmaceutically acceptable carrier.

12. A modified antibody or antigen binding fragment thereof comprising a substitution of the amino acid at position 152 of the heavy chain constant region of said antibody or antigen binding fragment with a cysteine, wherein said position is numbered according to the EU system.

13. The modified antibody or antigen binding fragment thereof of claim 12, wherein said antibody or antigen binding fragment thereof comprises the amino acid sequence of SEQ ID NO:10.

14. A modified antibody or antigen binding fragment thereof comprising two amino acid substitutions with cysteine, wherein the substitutions are selected from: a. positions 152 and 375 of the heavy chain constant region of said antibody or antigen binding fragment thereof, wherein said positions are numbered according to the EU system; or b. position 107 of the light chain constant region of said antibody or antigen binding fragment thereof, wherein said light chain is a kappa light chain, and position 360 of the heavy chain constant region of said antibody or antigen binding fragment thereof, wherein said positions are numbered according to the EU system.

15. The modified antibody or antigen binding fragment thereof of claim 12, wherein the antibody or antigen binding fragment thereof comprises an E152C substitution; wherein the position is numbered according to the EU system.

16. The modified antibody or antigen binding fragment thereof of claim 12, wherein the antibody or antigen binding fragment thereof is an IgG1 antibody.

17. The modified antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof comprises substitutions selected from: a) E152C and S375C in the heavy chain constant region; or b) K107C in the kappa light chain constant region and K360C in the heavy chain constant region; wherein the positions are numbered according to the EU system.

18. The modified antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof is an IgG1 antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-FIG. 1B. Surface accessibility plot of amino acid residues in human IgG1 heavy chain (A) and kappa light chain (B). Surface accessibility was calculated using Surface Racer 5.0 and is expressed as Angstrom square [Å.sup.2].

(2) FIG. 2. Location of selected 92 TAG mutations in the structure of a human IgG1 with a kappa light chain. Selected residues for TAG mutations are shown in black on only one of the two heavy chains and for one of the two kappa light chains (1HZH.pdb). Structures are shown using PyMOL, an open-source molecular modeling package (The PyMOL Molecular Graphics System, Version 1.5.0. Schrödinger. LLC).

(3) FIG. 3. The amino acid sequence alignment of the heavy chain constant regions of trastuzumab and antibody 14090. Residues mutated to Cys in the trastuzumab antibody and in antibody 14090 are underlined. Amino acid residues in heavy chain are numbered by Eu numbering system (Edelman et al., 1969).

(4) FIG. 4. Amino acid sequence alignment of constant regions of trastuzumab, human IgG1, IgG2, IgG3 and IgG4.

(5) FIG. 5A-FIG. 5B. The amino acid sequence alignment of the constant regions of human kappa and lambda light chains. A. Residues mutated to Cys in the kappa light chain of trastuzumab and in the lambda light chain of antibody 14090 are underlined. B. Residues selected for Cys mutations are shown in a PyMOL structure model of a human lambda light chain (Protein Structure Databank entry 3G6D.pdb)

(6) FIG. 6. Analysis of trastuzumab Cys antibodies by non-reducing SDS-PAGE.

(7) FIG. 7. Size exclusion chromatography of the trastuzumab LC-S156C mutant antibody (dashed line) and wild-type trastuzumab (solid line).

(8) FIG. 8A-FIG. 8B. Analysis of wild-type trastuzumab (A) and the trastuzumab LC-E158C mutant antibody (B) by reverse phase high pressure liquid chromatography (RP-HPLC).

(9) FIG. 9. MS analysis of trastuzumab LC-R108C mutant antibody after Protein A purification (intact MS).

(10) FIG. 10. Structure of MC-MMAF.

(11) FIG. 11A-FIG. 11D. Analysis of conjugation mixtures of trastuzumab Cys antibodies with MC-MMAF by RP-HPLC. RP-HPLC traces of the conjugation mixtures are shown as dashed lines. RP-HPLC traces of unmodified antibodies are shown as solid lines. A. LC-R108C-MMAF, B. HC-360C-MMAF, C. LC-S156C-MMAF, and D. IC-S275C-MMAF ADC.

(12) FIG. 12A-FIG. 12B. Analysis of conjugation mixtures of trastuzumab Cys antibodies with MC-MMAF by RP-HPLC. RP-HPLC traces of the conjugation mixtures are shown as dashed lines. RP-HPLC traces of unmodified antibodies are shown as solid lines. A. HC-S134C-MMAF, and B. HC-S136C-MMAF ADC.

(13) FIG. 13. Analysis of trastuzumab Cys-MMAF ADCs by analytical size-exclusion chromatography (AnSEC). Trastuzumab HC-K290C-MMAF ADC (short dashed line), trastuzumab LC-R142C-MMAF ADC (dashed line), and trastuzumab LC-L154C-MMAF ADC (dotted line) are compared to unmodified wild-type trastuzumab (solid line).

(14) FIG. 14. Thermal melting curve of unmodified wild-type trastuzumab and trastuzumab HC-T335C-MMAF, trastuzumab HC-S337C-MMAF and trastuzumab HC-K360C-MMAF ADCs.

(15) FIG. 15A-FIG. 15C. Cell proliferation assays for trastuzumab LC-S159C-MMAF with A. HCC1954, B. MDA-MB231 clone 16 and C. MDA-MB231 clone 40 cells.

(16) FIG. 16. IC.sub.50 of trastuzumab Cys-MMAF ADCs in MDA-MB231 clone 16 cell proliferation assay.

(17) FIG. 17A-FIG. 17B. Cell proliferation assays for Antibody 14090 HC-S375C-MMAF ADC with A. CMK11-5 and B. Jurkat cells.

(18) FIG. 18A-FIG. 18E. Pharmacokinetics study of trastuzumab LC-Cys-MMAF ADCs displaying no significant drug lost. A. Wild-type unconjugated trastuzumab, B. LC-K107C-MMAF, C. LC-R108C-MMAF, D. LC-L154C-MMAF, and E. LC-S159C-MMAF ADC.

(19) FIG. 19A-FIG. 19D. Pharmacokinetics study of trastuzumab HC-Cys-MMAF ADCs displaying no significant drug lost. A. HC-K121C-MMAD, B. HC-L174C-MMAF, C. HC-E258C-MMAF, and D. HC-R292C-MMAF ADC.

(20) FIG. 20A-FIG. 20D. Pharmacokinetics study of trastuzumab Cys-MMAF ADCs displaying significant drug lost. A. LC-T129C-MMAF, B. LC-E143C-MMAF, C. HC-K246C-MMAF, and D. HC-R344C-MMAF ADC.

(21) FIG. 21A-FIG. 21B. Pharmacokinetics study of two trastuzumab Cys-MMAF ADCs displaying fast clearance in vivo. A. HC-T335C-MMAF and B. HC-S337C-MMAF ADC.

(22) FIG. 22. In vivo efficacy studies of trastuzumab Cys-MMAF ADCs in MDA-MB231 clone 16 xenograft mouse model.

(23) FIG. 23A-FIG. 23B: Retention times of trastuzumab Pcl MMAF DAR 2 ADCs as measured by Hydrophobic Interaction Chromotography. ABA-MMAF is attached at a Pcl residue substituted for the indicated HC or LC residue. A) HC conjugated ADCs. B) LC conjugated ADCs. The retention time of unconjugated wild-type antibody is indicated (WT).

(24) FIG. 24A-FIG. 24C. Location of selected payload sites in the structure of a human IgG1 with a kappa light chain. Selected residues are shown in black on only one of the two heavy chains and for one of the two kappa light chains (1HZH.pdb). Three rotations of the structure are shown using PyMOL, an open-source molecular modeling package (The PyMOL Molecular Graphics System, Version 1.5.0. Schrödinger, LLC).

(25) FIG. 25A-FIG. 25L. Pharmacokinetics study of trastuzumab and antibody 14090 Cys-MMAF ADCs with DAR 4, 6 and 8 prepared with antibodies with 2, 3 or 4 Cys mutations. DAR 4 trastuzumab ADCs: HC-E258C-LC-S159C-MMAF (A), HC-S375C-LC-S159C-MMAF (B), HC-E258C-LC-E165C-MMAF (C), HC-S375C-LC-E165C-MMAF (D), HC-E152C-LC-R142C-MMAF (E), HC-P171C-LC-R142C-MMAF, and HC-E152C-LC-S159C-MMAF (G); DAR 4 antibody 14090 ADCs: HC-S375C-LC-A143C-MMAF (H), HC-K360C-LC-V159C-MMAF (I), and HC-S375C-LC-V159C-MMAF (J); K. DAR 6 trastuzumab ADCs HC-K334C-S375C-LC-E165C-MMAF and HC-K334C-K392C-LC-E165C-MMAF; L. DAR 8 trastuzumab ADCs HLC-K334C-K360C-S375C-LC-E165C-MMAF, HC-K334C-K360C-K392C-LC-E165C-MMAF and HC-K334C-S375C-K392C-LC-E165C-MMAF. Antibody 14090 is mouse cross-reactive and therefore is cleared more rapidly that then the trastuzumab ADCs which do not bind to any mouse antigens.

DETAILED DESCRIPTION

(26) The present invention provides methods of site-specific labeling of antibodies or antibody fragments by replacing one or more amino acids of a parental antibody or antibody fragment at specific positions with cysteine amino acids (“Cys”), such that the engineered antibodies or antibody fragments are capable of conjugation to various agents (e.g., cytotoxic agents). The present invention also provides immunoconjugates that are produced by using the methods described herein.

(27) When a cysteine is engineered into a parental antibody or antibody fragment, the modified antibody or antibody fragment is first recovered from the expression medium with cysteine or glutathione (GSH) attached at the engineered cysteine site(s) via a disulfide linkage (Chen et al., (2009) mAbs 16, 353-571). The attached cysteine or GSH is then removed in a reduction step, which also reduces all native inter-chain disulfide bonds of the parental antibody or antibody fragment. In a second step these disulfide bonds are re-oxidized before conjugation occurs. The present disclosure shows that when cysteine is engineered at certain sites, the re-oxidation step does not proceed well, presumably due to formation of the incorrect disulfide bonds. Accordingly, the present invention provides unique sets of sites on the antibody heavy chain constant region and antibody light chain constant region, respectively, where Cys substitution as described herein produces modified antibodies or antibody fragments that perform well in the re-oxidation process, and also produce stable and well behaved immunoconjugates.

(28) The site-specific antibody labeling according to the present invention can be achieved with a variety of chemically accessible labeling reagents, such as anti-cancer agents, fluorophores, peptides, sugars, detergents, polyethylene glycols, immune potentiators, radio-imaging probes, prodrugs, and other molecules.

(29) Accordingly, the present invention provides methods of preparation of homogeneous immunoconjugates with a defined drug-to-antibody ratio for use in cancer therapy and other indications as well as imaging reagents. The present invention also provides immunoconjugates prepared thereby, as well as pharmaceutical compositions comprising these immunoconjugates. The methods of the instant invention can be used in combination with other conjugation methods known in the art.

(30) The following enumerated embodiments represent certain aspects and variations of the invention:

(31) ##STR00006## wherein Ab represents an antibody or antibody fragment comprising at least one cysteine residue at one of the preferred cysteine substitution sites described herein; LU is a linker unit as described herein; X is a payload or drug moiety; and n is an integer from 1 to 16. In these embodiments, n is preferably about 2, about 4, about 6, or about 8. LU is typically a group of formula -L.sub.1-L.sub.2-L.sub.3-L.sub.4-L.sub.5-L.sub.6-, wherein L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5 and L.sub.6 are independently selected from -A.sub.1-, -A.sub.1X.sup.2— and —X.sup.2—; wherein: A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —(O(C(R.sup.4).sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((C(R.sup.4).sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nNHC(═O)—, —(C(R.sup.4).sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —NHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —C(═O)NH(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —S(C(R.sup.4).sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —C(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(C(R.sup.4).sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, or —(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8,

(32) ##STR00007## ##STR00008## ##STR00009## ##STR00010##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; R.sup.8 is independently selected from

(33) ##STR00011## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

(34) In some of these embodiments, the immunoconjugate comprises a group of the formula

(35) ##STR00012##

(36) wherein the sulfur atom is the sulfur of a cysteine residue in a modified antibody or antibody fragment and is located at one of the substitution sites identified herein.

(37) In any of the foregoing embodiments, the cysteine substitution site may be a position that corresponds to one of the sites identified by a position number, even though the position of the site in the sequence has been changed by a modification or truncation of the full-length antibody. Corresponding sites can be readily identified by alignment of an antibody or fragment with a full-length antibody.

1. Site-Specific Cysteine Engineered Antibodies

Site-Specific Labeling

(38) The antibodies (e.g., a parent antibody, optionally containing one or more non-canonical amino acids) of the present invention are numbered according to the EU numbering system as set forth in Edelman et al., (1969) Proc. Natl. Acad. USA 63:78-85, except that the lambda light chain is numbered according to the Kabat numbering system as set forth in Kabat et al., (1991) Fifth Edition. NIH Publication No. 91-3242. Human IgG1 constant region is used as a representative throughout the application. However, the invention is not limited to human IgG1; corresponding amino acid positions can be readily deduced by sequence alignment. For example, FIG. 4 shows sequence alignment of human IgG1, IgG2, IgG3 and IgG4 heavy chain constant regions, so that an identified Cys engineering site in the IgG1 constant region can be readily identified for IgG2, IgG3, and IgG4 as shown in FIG. 4. For the light chain constant region, IgG1, IgG2, IgG3 and IgG4 are the same. Table 1 below lists the amino acid positions in the constant region of the heavy chain of an antibody that can be replaced by a cysteine. Table 2 lists the amino acid positions in the constant region of the kappa light chain of an antibody that can be replaced by a cysteine. Table 3 lists the amino acid positions in the constant region of the lambda light chain of an antibody that can be replaced by a cysteine.

(39) TABLE-US-00001 TABLE 1 Identified cysteine substitution sites in the heavy chain constant region of human IgG1 (Sites numbered according to EU numbering system). Surface EU accessibility Selected SEQ ID number Residue [Å.sup.2] HC Cys NO. 117 SER 128.0 HC-S117C 2 119 SER 79.1 HC-S119C 3 121 LYS 135.9 HC-K121C 4 124 SER 40.2 HC-S124C 5 132 SER 34.4 HC-S132C 6 134 SER 123.3 HC-S134C 7 136 SER 182.9 HC-S136C 8 139 THR 32.9 HC-T139C 9 152 GLU 52.1 HC-E152C 10 153 PRO 89.1 HC-P153C 11 155 THR 69.0 HC-T155C 12 157 SER 39.0 HC-S157C 13 164 THR 125.4 HC-T164C 14 165 SER 183.2 HC-S165C 15 169 THR 60.0 HC-T169C 16 171 PRO 33.3 HC-P171C 17 174 LEU 68.1 HC-L174C 18 176 SER 161.9 HC-S176C 19 177 SER 68.1 HC-S177C 20 189 PRO 86.4 HC-P189C 21 191 SER 126.8 HC-S191C 22 195 THR 111.3 HC-T195C 23 197 THR 89.8 HC-T197C 24 205 LYS 217.1 HC-K205C 25 207 SER 50.0 HC-S207C 26 212 ASP 97.0 HC-D212C 27 246 LYS 55.1 HC-K246C 28 258 GLU 42.1 HC-E258C 29 269 GLU 189.2 HC-E269C 30 274 LYS 137.8 HC-K274C 31 286 ASN 119.4 HC-N286C 32 288 LYS 181.8 HC-K288C 33 290 LYS 177.0 HC-K290C 34 292 ARG 251.5 HC-R292C 35 293 GLU 83.3 HC-E293C 36 294 GLN 73.5 HC-E294C 37 320 LYS 55.0 HC-K320C 38 322 LYS 78.3 HC-K322C 39 326 LYS 212.7 HC-K326C 40 330 ALA 96.3 HC-A330C 41 333 GLU 84.7 HC-E333C 42 334 LYS 49.6 HC-K334C 43 335 THR 70.1 HC-T335C 44 337 SER 15.1 HC-S337C 45 344 ARG 98.2 HC-R344C 46 355 ARG 249.4 HC-R355C 47 360 LYS 113.9 HC-K360C 48 362 GLN 40.8 HC-Q362C 49 375 SER 28.9 HC-S375C 50 382 GLU 21.8 HC-E382C 51 389 ASN 189.5 HC-N389C 52 390 ASN 36.4 HC-N390C 53 392 LYS 81.8 HC-K392C 54 393 THR 35.8 HC-T393C 55 398 LEU 110.9 HC-L398C 56 400 SER 81.3 HC-S400C 57 413 ASP 79.6 HC-D413C 58 415 SER 69.0 HC-S415C 59 422 VAL 80.8 HC-V422C 60

(40) TABLE-US-00002 TABLE 2 Identified cysteine substitution sites in the kappa light chain constant region of human IgG1 (Sites numbered according to EU numbering system). Surface EU accessibility Selected SEQ ID number Residue [Å.sup.2] LC Cys NO. 107 LYS 90 LC-K107C 61 108 ARG 49 LC-R108C 62 109 THR 148 LC-T109C 63 112 ALA 50 LC-A112C 64 114 SER 39 LC-S114C 65 122 ASP 90 LC-D122C 66 123 GLU 51 LC-E123C 67 129 THR 41 LC-T129C 68 142 ARG 55 LC-R142C 69 143 GLU 117 LC-E143C 70 145 LYS 160 LC-K145C 71 152 ASN 157 LC-N152C 72 154 LEU 117 LC-L154C 73 156 SER 122 LC-S156C 74 159 SER 22 LC-S159C 75 161 GLU 66 LC-E161C 76 165 GLU 74 LC-E165C 77 168 SER 170 LC-S168C 78 169 LYS 241 LC-K169C 79 170 ASP 48 LC-D170C 80 182 SER 59 LC-S182C 81 183 LYS 131 LC-K183C 82 188 LYS 201 LC-K188C 83 190 LYS 167 LC-K190C 84 191 VAL 58 LC-V191C 85 197 THR 38 LC-T197C 86 199 GLN 127 LC-Q199C 87 203 SER 110 LC-S203C 88 206 THR 70 LC-T206C 89

(41) TABLE-US-00003 TABLE 3 Identified cysteine substitution sites on the lambda light chain of human IgG1. Surface Kabat accessibility Selected SEQ ID number Residue [Å.sup.2] LC Cys NO. 143 ALA 82 LC-A143C 92 145 THR 106 LC-T145C 93 147 ALA 14 LC-A147C 94 156 LYS 233 LC-K156C 95 159 VAL 28 LC-V159C 96 163 THR 157 LC-T163C 97 168 SER 166 LC-S168C 98

(42) Because of the high sequence homology of constant regions of IgG1, IgG2, IgG3 and IgG4 antibodies, findings of the invention are not limited to any specific antibodies or antibody fragments.

(43) In one embodiment, the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids on its heavy chain constant region chosen from positions identified in Table 1. In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region chosen from positions 121, 124, 152, 171, 174, 258, 292, 333, 334, 360, 375, and 392 of the heavy chain. For example, an immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with cysteine on its constant region chosen from positions 121 and 124, 121 and 152, 121 and 171, 121 and 174, 121 and 258, 121 and 292, 121 and 333, 121 and 334, 121 and 360, 121 and 375, 121 and 392, 124 and 152, 124 and 171, 124 and 174, 124 and 258, 124 and 292, 124 and 333, 124 and 334, 124 and 360, 124 and 375, 124 and 392,152 and 171, 152 and 174, 152 and 258, 152 and 292, 152 and 333, 152 and 334, 152 and 360, 152 and 375, 152 and 392, 171 and 174, 171 and 258, 171 and 292, 171 and 333, 171 and 360, 171 and 375, 174 and 258, 174 and 292, 174 and 333, 174 and 334, 174 and 360, 174 and 375, 174 and 392, 258 and 292, 258 and 333, 258 and 334, 258 and 360, 258 and 375, 258 and 392, 292 and 333, 292 and 334, 292 and 360, 292 and 375, 292 and 392, 333 and 334, 333 and 360, 333 and 375, 333 and 392; 334 and 360, 334 and 375, 334 and 392, 360 and 375, 360 and 392, or 375 and 392 of the heavy chain.

(44) In another embodiment, an immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of three amino acids with cysteine on its constant region chosen from positions 121, 124 and 152; 121, 124 and 171; 121, 124 and 174; 121, 124 and 258; 121, 124 and 292; 121, 124 and 333; 121, 124 and 334; 121, 124 and 360; 121, 124 and 375; 121, 124 and 392; 121, 152 and 171; 121, 152 and 174; 121, 152 and 258; 121, 152 and 292; 121, 152 and 333; 121, 152 and 334; 121, 152 and 360; 121, 152 and 375; 121, 152 and 392; 121, 171 and 174; 121, 171 and 258; 121, 171 and 292; 121, 171 and 333; 121, 171 and 334; 121, 171 and 360; 121, 171 and 375; 121, 171 and 392; 121, 174 and 258, 121, 174 and 292; 121, 174 and 333; 121, 174 and 334; 121, 174 and 360; 121, 174 and 375; 121, 174 and 392; 121, 258 and 292; 121, 258 and 333; 121, 258 and 334; 121, 258 and 360; 121, 258 and 375; 121, 258 and 392; 121, 292 and 333; 121, 292 and 334; 121, 292 and 360; 121, 292 and 375; 121, 292 and 392; 121, 333 and 334; 121, 333 and 360; 121, 333 and 375; 121, 333 and 392; 121, 334 and 360; 121, 334 and 375; 121, 334 and 392; 121, 360 and 375; 121, 360 and 392; 121, 375 and 392; 124, 152 and 171; 124, 152 and 174; 124, 152 and 258; 124, 152 and 292; 124, 152 and 333; 124, 152 and 334; 124, 152 and 360; 124, 152 and 375; 124, 152 and 392; 124, 171 and 174; 124, 171 and 258; 124, 171 and 292; 124, 171 and 333; 124, 171 and 334; 124, 171 and 360; 124, 171 and 375; 124, 171 and 392; 124, 174 and 258; 124, 174 and 292; 124, 174 and 333; 124, 174 and 334; 124, 174 and 360; 124, 174 and 375; 124, 174 and 392; 124, 258 and 292; 124, 258 and 333; 124, 258 and 334; 124, 258 and 360; 124, 258 and 375; 124, 258 and 392; 124, 292 and 333; 124, 292 and 334; 124, 292 and 360; 124, 292 and 375; 124, 292 and 392; 124, 333 and 360; 124, 333 and 334; 124, 333 and 375; 124, 333 and 392; 124, 334 and 360; 124, 334 and 375; 124, 334 and 392; 124, 360 and 375; 124, 360 and 392; 124, 375 and 392; 152, 171 and 174; 152, 171 and 258; 152, 171 and 292; 152, 171 and 333; 152, 171 and 334; 152, 171 and 360; 152, 171 and 375; 152, 171 and 392; 152, 174 and 258; 152, 174 and 292; 152, 174 and 333; 152, 174 and 334; 152, 174 and 360; 152, 174 and 375; 152, 174 and 392; 152, 258 and 292; 152, 258 and 333; 152, 258 and 334; 152, 258 and 360; 152, 258 and 375; 152, 258 and 392; 152, 292 and 333; 152, 292 and 334; 152, 292 and 360; 152, 292 and 375; 152, 292 and 392; 152, 333 and 334; 152, 333 and 360; 152, 333 and 375; 152, 333 and 392; 152, 334 and 360; 152, 334 and 375; 152, 334 and 392; 152, 360 and 375; 152, 360 and 392; 152, 375 and 392; 171, 174 and 258; 171, 174 and 292; 171, 174 and 333; 171, 174 and 334; 171, 174 and 360; 171, 174 and 375; 171, 174 and 392; 171, 258 and 292; 171, 258 and 292; 171, 258 and 333; 171, 258 and 334; 171, 258 and 360; 171, 258 and 375; 171, 258 and 392; 171, 292 and 333; 171, 292 and 334; 171, 292 and 360; 171, 292 and 375; 171, 292 and 392; 171, 333 and 334; 171, 333 and 360; 171, 333 and 375; 171, 333 and 392; 171, 334 and 360; 171, 334 and 392; 171, 360 and 375; 171, 360 and 392; 171, 375 and 392; 174, 258 and 292; 174, 258 and 333; 174, 258 and 334; 174, 258 and 360; 174, 258 and 375; 174, 258 and 392; 174, 292 and 333; 174, 292 and 334; 174, 292 and 360; 174, 292 and 375; 174, 292 and 392; 174, 333 and 334; 174, 333 and 360; 174, 333 and 375; 174, 333 and 392; 174, 334 and 360; 174, 334 and 375; 174, 334 and 392; 174, 360 and 375; 174, 360 and 392; 174, 375 and 392; 258, 292 and 333; 258, 292 and 334; 258, 292 and 360; 258, 292 and 375; 258, 292 and 392; 258, 333 and 360; 258, 333 and 375; 258, 333 and 392; 258, 334 and 360; 258, 334 and 375; 258, 334 and 392; 258, 360 and 375; 258, 360 and 392; 258, 375 and 392; 292, 333 and 334; 292, 333 and 360; 292, 333 and 375; 292, 333 and 392; 292, 334 and 360; 292, 334 and 375; 292, 334 and 392; 292, 360 and 375; 292, 360 and 392; 292, 375 and 392; 333, 334 and 360; 333, 334 and 375; 333, 334 and 392; 333, 360 and 375, 333, 360 and 392; 333, 375 and 392; 334, 360 and 375; 334, 360 and 392; or 360, 375 and 392 of the heavy chain.

(45) In an embodiment, an immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of four amino acids with cysteine on its constant region chosen from positions 152, 333, 375 and 392; or 152, 334, 375 and 392 of the heavy chain.

(46) In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NO: 2, 3, 9, 11, 12, 13, 14, 16, 21, 25, 26, 28, 30, 31, 32, 33, 34, 36, 38, 39, 40, 43, 44, 45, 46, 47, 51, 53, 54, 56, 57, or 60. In another specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NO: 6, 7, 8, 15, 19, 20, 22, 23, 24, 27, 36, 37, 41, 49, 52, 55, 58, or 59.

(47) In another embodiment, the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) on its light chain constant region chosen from positions identified in Table 2. In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region chosen from positions 107, 108, 142, 145, 159, 161, and 165 of the light chain, wherein said light chain is human kappa light chain. For example, an immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with cysteine on its constant region chosen from positions 107 and 108; 107 and 142; 107 and 145; 107 and 159; 107 and 161; 107 and 165; 108 and 142; 108 and 145; 108 and 159; 108 and 161; 108 and 165; 142 and 145; 142 and 159; 142 and 161; 142 and 165; 145 and 159; 145 and 161; 145 and 165; 159 and 161; 159 and 165; 161 and 165 of the light chain, wherein said light chain is human kappa light chain. In another embodiment, an immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of three amino acids with cysteine on its constant region chosen from positions 107, 108 and 142; 107, 108 and 145; 107, 108 and 159; 107, 108 and 161; 107, 108 and 165; 107, 142 and 145; 107, 142 and 159; 107, 142 and 161; 107, 142 and 165; 107, 145 and 159; 107, 145 and 161; 107, 145 and 165; 107, 159 and 161; 107, 159 and 165; 107, 161 and 165; 108, 142 and 145; 108, 142 and 159; 108, 142 and 161; 108, 142 and 165; 108, 145 and 159; 108, 145 and 161; 108, 145 and 165; 108, 159 and 161; 108, 159 and 165; 108, 161 and 165; 142, 145 and 159; 142, 145 and 161; 142, 145 and 165; 142, 159 and 161; 142, 159 and 165; 142, 161 and 165; 145, 159 and 161; 145, 159 and 165; 145, 161 and 165; or 159, 161 and 165 of the light chain, wherein said light chain is human kappa light chain.

(48) In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NO: 63, 65, 68, 70, 72, 73, 74, 78, 79, 80, 81, 82, 83, 86, 87, or 88. In another specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NO: 64, 66, 67, 84, 85, or 89 63, 64, 65, 66, 67, 68, 70, 72, 73, 74, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89.

(49) In another embodiment, the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids on its light chain constant region chosen from positions identified in Table 3. In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region chosen from positions 143, 147, 159, 163, and 168 of the light chain, wherein said light chain positions are numbered according to the Kabat system, and wherein said light chain is human lambda light chain. For example, an immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with cysteine on its constant region chosen from positions 143 and 147; 143 and 159; 143 and 163; 143 and 168; 147 and 159; 147 and 163; 147 and 168; 159 and 163; 159 and 168; or 163 and 168 of the light chain, wherein said light chain positions are numbered according to the Kabat system, and wherein said light chain is human lambda light chain. In another embodiment, an immunoconjugate of the invention comprises a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of three amino acids with cysteine on its constant region chosen from positions 143, 147 and 159; 143, 147 and 163; 143, 147 and 168; 143, 159 and 163; 143, 159 and 168; 143, 163 and 168; 147, 159 and 163; 147, 159 and 168; 147, 163 and 168; or 159, 163 and 168 of the light chain, wherein said light chain positions are numbered according to the Kabat system, and wherein said light chain is human lambda light chain.

(50) In an embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NO: 92, 94, 96, 97 or 98. In another specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NO: 93 or 95.

(51) In an embodiment, the immunoconjugate can have an DAR of about 2 or about 4. In an embodiment, the present invention provides immunoconjugates comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment comprises a Cys substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids on its heavy chain constant region chosen from positions identified in Table 1, and a Cys substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids on its light chain constant region chosen from positions identified in Table 2 or Table 3. In one embodiment, the present invention provides immunoconjugates comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment comprises a Cys substitution of one or more amino acids in its heavy chain constant region chosen from positions 121, 124, 152, 171, 174, 258, 292, 333, 334, 360, 375 and 392; and a Cys substitution of one or more amino acids in its light chain constant region chosen from positions 107, 108, 142, 145, 159, 161, and 165, wherein said light chain is human kappa light chain. In an embodiment, a modified antibody or antibody fragment according to the present invention may comprise a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 121 of a heavy chain, and a Cy s substitution on position 145 of a human kappa light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cy s substitution on position 152 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 152 of a heavy chain, and a Cy s substitution on position 108 of a human kappa light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 107 of a human kappa light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 108 of a human kappa light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 142 of a human kappa light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 145 of a human kappa light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 159 of a human kappa light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 161 of a human kappa light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain. In a embodiment, a modified antibody or antibody fragment according to the present invention comprises a Cys substitution on position 375 and on position 392 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain. In an embodiment a modified antibody or antibody fragment according to the present invention may comprise a Cys substitution on position 334 and on position 375 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain. In another example, a modified antibody or antibody fragment according to the present invention may comprise a Cys substitution on position 334 and on position 392 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain. In an embodiment, an immunoconjugates of those combinations can have a DAR of about 4 or about 6.

(52) In an embodiment, a modified antibody or antibody fragment according to the present invention may comprise a Cys substitution on position 334, on position 375 and on position 392 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain. In an embodiment, a modified antibody or antibody fragment according to the present invention may comprise a Cys substitution on position 333, on position 375 and on position 392 of a heavy chain, and a Cys substitution on position 165 of a human kappa light chain. In an embodiment, those combinations can have a DAR of about 4, 6, or 8.

(53) In an embodiment, the present invention provides immunoconjugates comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment comprises a Cys substitution of one or more amino acids in its heavy chain constant region chosen from positions 121, 124, 152, 171, 174, 258, 292, 333, 334 360, 375 and 392; and a Cys substitution of one or more amino acids in its light chain constant region chosen from positions 143, 147, 159, 163, and 168, wherein said light chain is human lambda light chain. For example, a modified antibody or antibody fragment according to the present invention may comprise a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 121 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 124 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 152 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 171 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 174 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 258 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 292 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 333 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 334 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 360 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 375 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 143 of a human lambda light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 147 of a human lambda light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 159 of a human lambda light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 163 of a human lambda light chain; or a Cys substitution on position 392 of a heavy chain, and a Cys substitution on position 168 of a human lambda light chain;

(54) In an embodiment of the invention, the amino acid substitution described herein is cysteine comprising a thiol group. In some aspects of the invention, the thiol group is utilized for chemical conjugation, and is attached to a linker unit (LU) and/or drug moiety. In some embodiments, the immunoconjugates of the invention comprise a drug moiety selected from the group consisting of a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, proteasome inhibitors, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, an kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor. In some embodiments, the immunoconjugates of the invention comprise a drug moiety that is an anti-cancer agent. The modified antibody or antibody fragments of the present invention can be any formats known in the art, such as a monoclonal, chimeric, humanized, fully human, bispecific, or multispecific antibody or antibody fragment thereof.

(55) According to the present invention, the modified antibody heavy chain and/or light chain (or antibody fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more cysteine substitutions in its constant regions. In one embodiment, the modified antibodies or antibody fragments contain 2, 4, 6, 8, or more cysteine substitutions in its constant regions. In some embodiments, the modified antibody, antibody fragment or immunoconjugate thereof comprises 2 or 4 Cys substitutions.

(56) In one embodiment, the parental antibody (antibody without cysteine substitution) is an IgG, IgM, IgE, or IgA antibody. In a specific embodiment, the parental antibody is an IgG1 antibody. In another specific embodiment, the parental antibody is an IgG2, IgG3, or IgG4 antibody.

(57) The present invention also provides modified antibodies or antibody fragments thereof comprising a substitution of one or more amino acids on its heavy chain constant region chosen from positions identified in Table 1. In some embodiments, the present invention provides modified antibodies or antibody fragments thereof comprising a substitution of one or more amino acids on its light chain constant region chosen from positions identified in Table 2 or Table 3.

(58) In certain embodiments, the modified antibodies or antibody fragments provided herein are labeled using the methods of the invention in combination with other conjugation methods known in the art including, but not limited to, chemoselective conjugation through lysine, histidine, tyrosine, formyl-glycine, pyrrolysine, pyrroline-carboxy-lysine, unnatural amino acids, and protein tags for enzyme-mediated conjugation (e.g., S6 tags).

2. Conjugation Chemistry

(59) The conjugated antibody or antibody fragment thereof provided herein is produced by post-translational modification of at least one cysteine residue that was incorporated into the antibody or antibody fragment thereof as described above by site-specific labeling methods. The conjugated antibody or antibody fragment can be prepared by methods known in the art for conjugation of a payload of interest to cysteine residues that occur naturally in proteins, and by methods described for conjugation to proteins engineered to contain an additional cysteine residue substituted for another amino acid of a natural protein sequence.

(60) In certain embodiments the modified antibodies or antibody fragment thereof provided herein are conjugated using known methods wherein the incorporated cysteine (cys) is conjugated to a maleimide derivative as Scheme Ia below. Modified antibodies of the invention that undergo this type of conjugation contain a thiol-maleimide linkage.

(61) ##STR00013##
where:
LU is a Linker Unit (LU), and
X is a payload or drug moiety.

(62) In other embodiments, the Cys incorporated into the modified antibodies or antibody fragment is conjugated by reaction with an alpha-halo carbonyl compound such as a chloro-, bromo-, or iodo-acetamide as shown in Scheme Ib below. It is understood that other leaving groups besides halogen, such as tosylate, triflate and other alkyl or aryl sulfonates, can be used as the leaving group Y. While Scheme Ib depicts reaction of a Cys thiol with an alpha-halo acetamide, the method includes any alkylation of a sulfur of an incorporated Cys with a group of the formula Y—CHR—C(═O)—, where R is H or C.sub.1-4alkyl, Y is a leaving group (typically Cl, Br, or I, and optionally an alkylsulfonate or arylsulfonate); it is not limited to amides.

(63) ##STR00014##

(64) Alternatively, the Cys incorporated into the modified antibodies or antibody fragment can be conjugated by reaction with an external thiol under conditions that induce formation of a disulfide bond between the external thiol and the sulfur atom of the incorporated cysteine residue as shown in Scheme Ic below. In these examples, R can be H; however, compounds where one or both R groups represent an alkyl group, e.g., Methyl, have been found to increase the stability of the disulfide.

(65) ##STR00015##

(66) By way of example only, such post-translational modifications are illustrated in Schemes (Ia)-(Ic) above, where the starting structure represents a cysteine incorporated into a light chain or heavy chain of an antibody at one of the specific sites identified herein. Methods for performing each of these conjugation methods are well known in the art. An antibody can be modified by these methods in its light chains, or its heavy chains, or in both light and heavy chains. An antibody in which each light chain or each heavy chain has been modified to contain a single incorporated cysteine will generally contain two conjugation sites, since an antibody typically contains two light and two heavy chains.

(67) Upon conjugation, the modified antibodies of the invention typically contain 1-12, frequently 2-8, and preferably 2, 4 or 6-LU-X (Linker Unit-Payload) moieties. In some embodiments, an antibody light or heavy chain is modified to incorporate two new Cys residues at two of the specific sites identified herein for Cys substitutions (or alternatively one Cys is incorporated in the light chain and one in the heavy chain), so the tetrameric antibody ultimately contains four conjugation sites. Similarly the antibody can be modified by replacement of 3 or 4 of its native amino acids with Cys at the specific sites identified herein, in light chain or heavy chain or a combination thereof, resulting in 6 or 8 conjugation sites in the tetrameric antibody.

(68) X in these conjugates represents a payload, which can be any chemical moiety that is useful to attach to an antibody. In some embodiments, X is a drug moiety selected from a cytotoxin, an anti-cancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, an immune potentiator, and an anesthetic agent or any other therapeutic, or biologically active moiety or drug moiety. In other embodiments, X is a label such as a biophysical probe, a fluorophore, an affinity probe, a spectroscopic probe, a radioactive probe, a spin label, or a quantum dot. In other embodiments, X is a chemical moiety that modifies the antibody's physicochemical properties such as a lipid molecule, a polyethylene glycol, a polymer, a polysaccharide, a liposome, or a chelator. In other embodiments, X is a functional or detectable biomolecule such as a nucleic acid, a ribonucleic acid, a protein, a peptide (e.g., an enzyme or receptor), a sugar or polysaccharide, an antibody, or an antibody fragment. In other embodiments, X is an anchoring moiety such as a nanoparticle, a PLGA particle, or a surface, or any binding moiety for specifically binding the conjugate to another moiety, such as a histidine tag, poly-G, biotin, avidin, streptavidin, and the like. In other embodiments, X is a reactive functional group that can be used to attach the antibody conjugate to another chemical moiety, such as a drug moiety, a label, another antibody, another chemical moiety, or a surface.

(69) The Linker Unit (LU) can be any suitable chemical moiety that covalently attaches the thiol-reactive group (e.g., maleimide, alpha-halo carbonyl, vinyl carbonyl (e.g., acrylate or acrylamide), vinyl sulfone, vinylpyridine, or thiol) to a payload. Many suitable LUs are known in the art. For example, LU can be comprised of one, two, three, four, five, six, or more than six linkers referred to herein as L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5 and L.sub.6. In certain embodiments the LU comprises a linker selected from a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or any combination thereof, and the LU optionally contains a self-immolative spacer.

(70) In some embodiments, LU is a group of the formula -L.sub.1-L.sub.2-L.sub.3-L.sub.4- or -L.sub.1-L-L.sub.3-L.sub.4-L.sub.5-L.sub.6-. Linking groups L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5 and L.sub.6 for use in LU include alkylene groups —(CH.sub.2).sub.n— (where n is 1-20, typically 1-10 or 1-6), ethylene glycol units (—CH.sub.2CH.sub.2O—).sub.n (where n is 1-20, typically 1-10 or 1-6), amides —C(═O)—NH— or —NH—C(═O)—, esters —C(═O)—O— or —O—C(═O)—, rings having two available points of attachment such as divalent phenyl, C.sub.3-8cycloalkyl or C.sub.4-8 heterocyclyl groups, amino acids NH—CHR*—C═O— or C(═O)—CHR*—NH—, where R* is the side chain of a known amino acid (frequently one of the canonical amino acids, but also including e.g. norvaline, norleucine, homoserine, homocysteine, phenylglycine, citrulline, and other named alpha-amino acids), polypeptides of known amino acids (e.g., dipeptides, tripeptides, tetrapeptides, etc.), thiol-maleimide linkages (from addition of —SH to maleimide). —S—CR.sub.2— and other thiol ethers such as —S—CR.sub.2—C(═O)— or —C(═O)—CR.sub.2—S—, where R is as defined above for Scheme Ic, —CH.sub.2—C(═O)—, and disulfides (—S—S—), as well as combinations of any of these with other linkers described below, e.g., a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a linker that comprises a self-immolative spacer.

(71) In some embodiments when LU is -L.sub.1-L-L.sub.3-L.sub.4-L.sub.5-L.sub.6-, L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5 and L.sub.6 can be selected from: -A.sub.1-, -A.sub.1X.sup.2— and —X.sup.2—; wherein: A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —(O(C(R.sup.4).sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((C(R.sup.4).sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nNHC(═O)—, —(C(R.sup.4).sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —NHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —C(═O)NH(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —S(C(R.sup.4).sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —C(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(C(R.sup.4).sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, or —(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8,

(72) ##STR00016## ##STR00017## ##STR00018## ##STR00019##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine;

(73) R.sup.8 is independently selected from

(74) ##STR00020## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

(75) In some embodiments, at least one of L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5 and L.sub.6 is a stable, or non-cleavable, linker. In some embodiments, at least one of L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5 and L.sub.6 is a cleavable linker, which may be chemically cleavable (hydrazones, disulfides) or enzymatically cleavable. In some embodiments, the enzymatically cleavable linker is one readily cleaved by a peptidase: The Val-Cit linker (valine-citrulline), a dipeptide of two known amino acids, is one such linker. In other embodiments, the enzymatically cleavable linker is one that is triggered by activity of a glucuronidase:

(76) ##STR00021##
is an example of such a linker, which also comprises a self-immolative spacer that falls apart spontaneously under physiological conditions once glucuronidase cleaves the glycosidic linkage.

(77) In some embodiments, the immunoconjugate of the invention comprises a modified cysteine residue of the formula IIA or IIB:

(78) ##STR00022##
wherein —CH.sub.2—S— represents the side chain of Cys incorporated at one of the selected Cys substitution sites described herein, and L.sub.2-L.sub.6 and X represent linking groups and payloads, respectively, as further described herein. In some embodiments of IIA, L.sub.2 is a bond. In some embodiments of IIB, L.sub.2 is NH or O. In some embodiments of both IIA and IIB, L.sub.3 is selected from (CH.sub.2).sub.1-10 and (CH.sub.2CH.sub.2O).sub.1-6. L.sub.4, L.sub.5 and L.sub.6 are additional optional linkers selected from those described herein. In certain embodiments, L.sub.6 can be a carbonyl (C═O) or a linker that comprises a self-immolative spacer.

(79) In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein: L.sub.1 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker; L.sub.2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker; L.sub.3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker, and L.sub.4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a linker that comprises a self-immolative spacer.

(80) In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein L.sub.1 is a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker; L.sub.2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker; L.sub.3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker, and L.sub.4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a linker that comprises a self-immolative spacer.

(81) In some of the embodiments of LU at least one of L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5 and L.sub.6 is a cleavable linker, and LU is considered cleavable. Similarly, in some of the embodiments of LU at least one of L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5 and L.sub.6 is a non-cleavable linker. In certain of these embodiments, each linker of LU is non-cleavable, and LU is considered non-cleavable.

(82) In some of the foregoing embodiments wherein LU is-L.sub.1-L.sub.2-L.sub.3-L.sub.4-, at least one of L.sub.1, L.sub.2, L.sub.3 and L.sub.4 is a linker selected from -A.sub.1-, -A.sub.1X.sup.2— and —X.sup.2—; wherein: A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —(O(C(R.sup.4).sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((C(R.sup.4).sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nNHC(═O)—, —(C(R.sup.4).sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —NHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —C(═O)NH(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —S(C(R.sup.4).sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —C(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(C(R.sup.4).sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.a).sub.mNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, or —(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8,

(83) ##STR00023## ##STR00024## ##STR00025## ##STR00026##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; R.sup.8 is independently selected

(84) ##STR00027## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

(85) In these embodiments, the other linkers of LU are independently selected from a bond, -A.sup.1-, -A.sub.1X.sup.2—, —X.sup.2—, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker and a linker that comprises a self-immolative spacer.

(86) In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2— or —X.sup.2—; where: A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —(O(C(R.sup.4).sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.n—, —((C(R.sup.4).sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nNHC(═O)—, —(C(R.sup.4).sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —NHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —C(═O)NH(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —S(C(R.sup.4).sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —C(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(C(R.sup.4).sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, or —(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8

(87) ##STR00028## ##STR00029## ##STR00030## ##STR00031##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; R.sup.8 is independently selected from

(88) ##STR00032## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; L.sub.2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker; L.sub.3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker, and L.sub.4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a linker that comprises a self-immolative spacer. In certain embodiments, L.sub.1 is C(═O)—CH.sub.2CH.sub.2—NH—C(═O)—CH.sub.2CH.sub.2—S—, so LU is —C(═O)—CH.sub.2CH.sub.2—NH—C(═O)—CH.sub.2CH.sub.2—S-L.sub.2-L.sub.3-L.sub.4-. In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2— or —X.sup.2—; where: A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, or —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8

(89) ##STR00033## ##STR00034## ##STR00035##
CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; R.sup.8 is independently selected

(90) ##STR00036## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each in is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; L.sub.2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker; L.sub.3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker; L.sub.4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a linker that comprises a self-immolative spacer.

(91) In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2— or —X.sup.2—; where: A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —NHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n— or —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8

(92) ##STR00037## ##STR00038## ##STR00039##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; R.sup.8 is independently selected

(93) ##STR00040## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each in is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; L.sub.2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker; L.sub.3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker or a photo-cleavable linker, and L.sub.4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a linker that comprises a self-immolative spacer.

(94) In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L-, wherein L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2— or —X.sup.2—; where: A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —NHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n— or —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8

(95) ##STR00041## ##STR00042## ##STR00043##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; R.sup.8 is independently selected from

(96) ##STR00044## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each in is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; L.sub.2 is a bond, a non-enzymatically cleavable linker or a non-cleavable linker; L.sub.3 is a bond, a non-enzymatically cleavable linker or a non-cleavable linker; L.sub.4 is a bond, an enzymatically cleavable linker or a linker that comprises a self-immolative spacer.

(97) In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein

(98) L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2— or —X.sup.2—;

(99) L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2—;

(100) L.sub.3 is a bond, -A.sub.3-, or -A.sub.3X.sup.2—;

(101) L.sub.4 is a bond, -A.sub.4-, -A.sub.4X.sup.2—,

(102) ##STR00045## ##STR00046## A.sub.1 is —C(═O)NH—, —NHC(═O)—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —(O(C(R.sup.4).sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((C(R.sup.4).sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nNHC(═O)—, —(C(R.sup.4).sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —NHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —C(═O)NH(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —S(C(R.sup.4).sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —C(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(C(R.sup.4).sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).sub.2).sub.n—, (O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, or —(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—; A.sub.2 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —(O(C(R.sup.4).sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((C(R.sup.4).sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(C(R.sup.4).sub.2).sub.nC(═O)NR.sup.4—, —(CH.sub.2).sub.nNHC(═O)—, —(C(R.sup.4).sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —NHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —C(═O)NH(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —S(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nS—, —(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.n—, —S(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNH—, —(C(R.sup.4).sub.2).sub.nNH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —C(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(C(R.sup.4).sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(═O)NH(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(═O)NH(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2)).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, — (O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—,

(103) ##STR00047## A.sub.3 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —(O(C(R.sup.4).sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((C(R.sup.4).sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nNHC(═O)—, —(C(R.sup.4).sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —NHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —C(═O)NH(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —S(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nS—, —(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.n—, —S(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —C(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(C(R.sup.4).sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(═O)NH(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(═O)NH(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(═O)—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mC(═O)—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mC(═O)—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—,

(104) ##STR00048## A.sub.4 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —(O(C(R.sup.4).sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((C(R.sup.4).sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —(C(R.sup.4).sub.2).sub.nC(═O)NH—, —(CH.sub.2).sub.nNHC(═O)—, —(C(R.sup.4).sub.2).sub.nNHC(═O)—, —NHC(═O)(CH.sub.2).sub.n—, —NHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —C(═O)NH(C(R.sup.4).sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —S(C(R.sup.4).sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)NH(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —C(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(C(R.sup.4).sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNHC(═O)(C(R.sup.4).sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, or —(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(═O)(C(R.sup.4).sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8

(105) ##STR00049## ##STR00050## ##STR00051##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; R.sup.8 is independently selected from

(106) ##STR00052## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.
In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein
L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2— or —X.sup.2—;
L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2—;
L.sub.3 is a bond, -A.sub.3-, or -A.sub.3X.sup.2—;
L.sub.4 is a bond, -A.sub.4-, -A.sub.4X.sup.2—,

(107) ##STR00053## A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH(CH.sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2)˜NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n— or —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—; A.sub.2 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH(CH.sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —(CH.sub.2)˜C(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n— or

(108) ##STR00054## A.sub.3 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH(CH.sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n— or

(109) ##STR00055## A.sub.4 —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)NH—, —NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH(CH.sub.2).sub.nS—, —S(CH.sub.2).sub.nC(═O)NH—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —C(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nC(═O)—, —(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2)˜NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n— or —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—; each X.sup.2 is independently selected from a bond,

(110) ##STR00056##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.
In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein
L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2— or —X.sup.2—;
L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2—;
L.sub.3 is a bond, -A.sub.3-, or -A.sub.3X.sup.2—;
L.sub.4 is a bond, -A.sub.4-, -A.sub.4X.sup.2—,

(111) ##STR00057## A.sub.1 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n— or —(O(CH.sub.2)).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—; A.sub.2 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n— or

(112) ##STR00058## A.sub.3 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n— or

(113) ##STR00059## A.sub.4 is —C(═O)NH—, —C(═O)NH(CH.sub.2).sub.n—, —C(═O)NH(CH.sub.2).sub.nS—, —(O(CH.sub.2).sub.n).sub.m—, —((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n—, —NHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH(CH.sub.2).sub.nNHC(═O)(CH.sub.2).sub.n—, —(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n— or —(O(CH.sub.2).sub.n).sub.mNHC(═O)(CH.sub.2).sub.n—; each X.sup.2 is independently selected from a bond, R.sup.8

(114) ##STR00060## ##STR00061## ##STR00062##
—CHR.sup.4(CH.sub.2).sub.nC(═O)NH—, —CHR.sup.4(CH.sub.2).sub.nNHC(═O)—, —C(═O)NH— and —NHC(═O)—; each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, —C(═O)OH and —OH, each R.sup.5 is independently selected from H, C.sub.1-4alkyl, phenyl or C.sub.1-4alkyl substituted with 1 to 3 —OH groups; each R.sup.6 is independently selected from H, fluoro, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C.sub.1-4alkoxy substituted with —C(═O)OH and C.sub.1-4alkyl substituted with —C(═O)OH; R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; R.sup.8 is independently selected from

(115) ##STR00063## R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

(116) In one embodiment, L.sub.1 is —(CH.sub.2).sub.1-10—C(═O)—, e.g., —(CH.sub.2).sub.5—C(═O)—; and L.sub.2, L.sub.3 and L.sub.4 each represent a bond.

(117) In certain embodiments LU comprises a val-cit linker of this formula, wherein X represents a payload, typically a drug moiety such as one having anticancer activity:

(118) ##STR00064##
When L.sub.4-L.sub.5-L.sub.6 is a val-cit linker as shown above, L.sub.3 is preferably —(CH.sub.2).sub.2-6—C(═O)—.

(119) In certain embodiments the X group is a maytansinoid such as DM1 or DM4, or a dolastatin analog or derivative such as dolastatin 10 or 15 and auristatins MMAF or MMAE, or a calicheamicin such as N-acetyl-γ-calicheamicin, or a label or dye such as rhodamine or tetramethylrhodamine.

(120) As used herein, a “linker” is any chemical moiety that is capable of connecting an antibody or a fragment thereof to an X group (payload) to form an immunoconjugate. Linkers can be susceptible to cleavage, such as, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Alternatively, linkers can be substantially resistant to cleavage. A linker may or may not include a self-immolative spacer.

(121) Non-limiting examples of the non-enzymatically cleavable linkers as used herein to conjugate an X.sup.1 group to the modified antibodies or antibody fragment thereof provided herein include, acid-labile linkers, linkers containing a disulfide moiety, linkers containing a triazole moiety, linkers containing a hydrazone moiety, linkers containing a thioether moiety, linkers containing a diazo moiety, linkers containing an oxime moiety, linkers containing an amide moiety and linkers containing an acetamide moiety.

(122) Non-limiting examples of the enzymatically cleavable linkers as used herein to conjugate an X group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, linkers that are cleaved by a protease, linkers that are cleaved by an amidase, and linkers that are cleaved by β-glucuronidase or another glycosidase.

(123) In certain embodiments, such enzyme cleavable linkers are linkers which are cleaved by cathepsin, including cathepsin Z, cathepsin B, cathepsin H and cathepsin C. In certain embodiments the enzymatically cleavable linker is a dipeptide cleaved by cathepsin, including dipeptides cleaved by cathepsin Z, cathepsin B, cathepsin H or cathepsin C. In certain embodiments the enzymatically cleavable linker is a cathepsin B-cleavable peptide linker. In certain embodiments the enzymatically cleavable linker is a cathepsin B-cleavable dipeptide linker. In certain embodiments the enzymatically cleavable dipeptide linker is valine-citrulline or phenylalanine-lysine. Other non-limiting examples of the enzymatically cleavable linkers as used herein conjugate an X group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, linkers which are cleaved by β-glucuronidase, e.g.,

(124) ##STR00065##
See Ducry et al, Bioconjugate Chem, (2010) vol. 21(1), 5-13.

(125) “Self-immolative spacers” are bifunctional chemical moieties covalently linked at one terminus to a first chemical moiety and at the other terminus to a second chemical moiety, thereby forming a stable tripartite molecule. A linker can comprise a self-immolative spacer bonded to a third chemical moiety that is cleavable from the spacer either chemically or enzymatically. Upon cleavage of a bond between the self-immolative spacer and the first chemical moiety or the third chemical moiety, self-immolative spacers undergo rapid and spontaneous intramolecular reactions and thereby separate from the second chemical moiety. These intramolecular reactions generally involve electronic rearrangements such as 1,4, or 1,6, or 1,8 elimination reactions or cyclizations to form highly favored five- or six-membered rings. In certain embodiments of the present invention, the first or third moiety is an enzyme cleavable group, and this cleavage results from an enzymatic reaction, while in other embodiments the first or third moiety is an acid labile group and this cleavage occurs due to a change in pH. As applied to the present invention, the second moiety is the “Payload” group as defined herein. In certain embodiments, cleavage of the first or third moiety from the self-immolative spacer results from cleavage by a proteolytic enzyme, while in other embodiments it results from cleaved by a hydrolase. In certain embodiments, cleavage of the first or third moiety from the self-immolative spacer results from cleavage by a cathepsin enzyme or a glucuronidase.

(126) In certain embodiments, the enzyme cleavable linker is a peptide linker and the self-immolative spacer is covalently linked at one of its ends to the peptide linker and covalently linked at its other end to a drug moiety. This tripartite molecule is stable and pharmacologically inactive in the absence of an enzyme, but which is enzymatically cleavable by enzyme at a bond covalently linking the spacer moiety and the peptide moiety. The peptide moiety is cleaved from the tripartite molecule which initiates the self-immolating character of the spacer moiety, resulting in spontaneous cleavage of the bond covalently linking the spacer moiety to the drug moiety, to thereby effect release of the drug in pharmacologically active form.

(127) In other embodiments, a linker comprises a self-immolative spacer that connects to the peptide, either directly or indirectly at one end, and to a payload at the other end; and the spacer is attached to a third moiety that can be cleaved from the spacer enzymatically, such as by a glucuronidase. Upon cleavage of the third moiety, the spacer degrades or rearranges in a way that causes the payload to be released. An example of a linker with this type of self-immolative spacer is this glucuronidase-cleavable linker, where hydrolysis of the acetal catalyzed by glucuronidase releases a phenolic compound that spontaneously decomposes under physiological conditions:

(128) ##STR00066##

(129) Non-limiting examples of the self-immolative spacer optionally used in the conjugation of an X.sup.1 group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, moieties which include a benzyl carbonyl moiety, a benzyl ether moiety, a 4-aminobutyrate moiety, a hemithioaminal moiety or a N-acylhemithioaminal moiety.

(130) Other examples of self-immolative spacers include, but are not limited to, p-aminobenzyloxycarbonyl groups, aromatic compounds that are electronically similar to the p-aminobenzyloxycarbonyl group, such as 2-aminoimidazol-5-methanol derivatives and ortho or para-aminobenzylacetals. In certain embodiments, self-immolative spacers used herein which undergo cyclization upon amide bond hydrolysis, include substituted and unsubstituted 4-aminobutyric acid amides and 2-aminophenylpropionic acid amides.

(131) In certain embodiments, the self-immolative spacer is

(132) ##STR00067##
while in other embodiments the self-immolative spacer is

(133) ##STR00068##
where n is 1 or 2. In other embodiments the self-immolative spacer is

(134) ##STR00069##
where n is 1 or 2. In other embodiments the self-immolative spacer is

(135) ##STR00070##
where n is 1 or 2. In other embodiments the self-immolative spacer is

(136) ##STR00071##
where n is 1 or 2. In other embodiments the self-immolative spacer is

(137) ##STR00072##
where n is 1 or 2.

(138) Schemes (2a-2c) illustrate the post-translational modification of the modified antibodies or antibody fragment thereof provided herein wherein the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, and L1 in each case is the group that reacts with the new Cys.

(139) ##STR00073##

(140) ##STR00074##

(141) ##STR00075##

(142) In each of Schemes 2ab 2c, the starting material is the replacement Cys residue in an antibody or antibody fragment modified as described herein, where the dashed bonds indicate connection to adjoining residues of the antibody or antibody fragment; each R is H or C.sub.1-4alkyl, typically H or methyl; L.sub.2, L.sub.3 and L.sub.4 are components of the linking unit LU, such as those described above; X is the payload; and the group connecting L.sub.2 to the sulfur of the substitute Cys of the invention is L.sub.1.

(143) In some embodiments of the invention, X is a reactive functional group that can be used to connect the conjugated antibody to another chemical moiety, by interacting with a suitable complementary functional group. Table 4 depicts some examples of reactive functional groups that X can represent, along with a complementary functional group that can be used to connect a conjugate comprising X to another compound. Methods for using X to connect to the corresponding complementary functional group are well known in the art. Connections using azide are typically done using ‘Click’ or copper-free click chemistry; reactions involving hydrazines, alkoxyamines or acyl hydrazines typically proceed through the formation of a Schiff base with one of the carbonyl functional groups.

(144) TABLE-US-00004 TABLE 4 Complementary Reactive X Functional Group for X a thiol a thiol, a maleimide, a haloacetamide, a vinyl sulfone, or a vinylpyridine an azide an alkene, alkyne, a phosphine-(thio)ester, a cyclooctyne, a cyclooctene or an oxanobornadiene a phosphine-(thio)ester) an azide an oxanobornadiene an azide or a tetrazine an alkyne an azide or a tetrazine an alkene a tetrazine a cyclooctyne an azide or a tetrazine a cyclooctene a tetrazine a norbornene a tetrazine a tetrazine a norbornene, an alkene, alkyne, a cyclooctyne or an oxanobornadiene an aldehyde a hydroxylamine, a hydrazine or NH.sub.2—NH—C(═O)— a ketone a hydroxylamine, a hydrazine or NH.sub.2—NH—C(═O)— a hydroxylamine an aldehyde or a ketone a hydrazine an aldehyde or a ketone NH.sub.2—NH—C(═O)— an aldehyde or a ketone a haloacetamide a thiol a thiol a thiol a maleimide a thiol a vinyl sulfone a thiol a vinylpyridine a thiol
Exemplary products of the connections made using these components are depicted in Table 5, where Y.sup.1 represents an antibody of the invention, A.sub.1 represents a linking unit (LU) connecting the antibody to payload X.sup.a, -L.sub.2-L.sub.3-L.sub.4- in Formula II-a represents a linker unit that can be present in a molecule to be connected to the conjugated antibody via X.sup.a, and X.sup.1 represents a payload. Payload X.sup.a is a reactive functional group, and X.sup.b on Formula II-a is the corresponding complementary functional group, and Formula II-a itself represents a molecule to be connected to the conjugated antibody. The third column in Table 5 depicts a product from reaction of X.sup.a with X.sup.b.

(145) TABLE-US-00005 TABLE 5 X.sup.b—L.sub.2—L.sub.3—L.sub.4—X.sup.1 Y.sup.1—A.sub.1—X.sup.a Formula (II-a) Y.sup.1—A.sub.1—X.sup.2—L.sub.2—L.sub.3—L.sub.4—X.sup.1 Y.sup.1—A.sub.1—N.sub.3 HC≡C—L.sub.2—L.sub.3—L.sub.4—X.sup.1 Y.sup.1—A.sub.1—N.sub.3 HC≡C—L.sub.2—L.sub.3—L.sub.4—X.sup.1 Y.sup.1—A.sub.1—C≡CH N.sub.3—L.sub.2—L.sub.3—L.sub.4—X.sup.1 Y.sup.1—A.sub.1—C≡CH N.sub.3—L.sub.2—L.sub.3—L.sub.4—X.sup.1 0 NH.sub.2—O—L.sub.2—L.sub.3—L.sub.4—X.sup.1 NH.sub.2—O—L.sub.2—L.sub.3—L.sub.4—X.sup.1 CH.sub.3C(═O)—L.sub.2—L.sub.3—L.sub.4—X.sup.1 HC(═O)—L.sub.2—L.sub.3—L.sub.4—X.sup.1 HS—L.sub.2—L.sub.3—L.sub.4—X.sup.1 0 00 01 02 03 04 05 NH.sub.2—NH—C(═O)—L.sub.2—L.sub.3—L.sub.4—X.sup.1 06 07 NH.sub.2—NH—C(═O)—L.sub.2—L.sub.3—L.sub.4—X.sup.1 08 09 R.sub.5C(═O)—L.sub.2—L.sub.3—L.sub.4—X.sup.1 0 HC(═O)—L.sub.2—L.sub.3—L.sub.4—X.sup.1 HS—L.sub.2—L.sub.3—L.sub.4—X.sup.1 Y.sup.1—A.sub.1—N.sub.3 N.sub.3—L.sub.2—L.sub.3—L.sub.4—X.sup.1 Y.sup.1—A.sub.1—N.sub.3 0 N.sub.3—L.sub.2—L.sub.3—L.sub.4—X.sup.1 Y.sup.1—A.sub.1—N.sub.3 N.sub.3—L.sub.2—L.sub.3—L.sub.4—X.sup.1 0 N.sub.3—L.sub.2—L.sub.3—L.sub.4—X.sup.1 0 Y.sup.1—A.sub.1—N.sub.3 Y.sup.1—A.sub.1—N.sub.3 0 N.sub.3—L.sub.2—L.sub.3—L.sub.4—X.sup.1

(146) In certain embodiments, the modified antibody or antibody fragment thereof provided herein is conjugated with an “X group-to-antibody” (payload to antibody) ratio between about 1 and 16, such as 1-12, or 1, 2, 3, 4, 5, 6, 7, or 8, wherein the modified antibody or antibody fragment thereof contains 1, 2, 3, 4, 5, 6, 7, or 8 cysteine residues incorporated at the specific sites disclosed herein. For example, an “X group-to-antibody” ratio of 4 can be achieved by incorporating two Cys residues into the heavy chain of an antibody, which will contain 4 conjugation sites, two from each heavy chain. Immunoconjugates of such antibodies will contain up to 4 payload groups, which may be alike or different and are preferably all alike. In another example, an “X group-to-antibody” ratio of 4 can be achieved by incorporating one Cys residue into the heavy chain and a second Cys residue into the light chain of an antibody resulting in 4 conjugation sites, two in the two heavy chains and two in the two light chains. A ratio 6, 8 or higher can be achieved by combinations of 3, 4 or more cysteine substitutions of the invention in heavy and light chain of the antibody. Substituting multiple cysteine groups into an antibody can lead to inappropriate disulfide formation and other problems. Thus for loading more than 4 payload groups onto one antibody molecule, the methods of the invention can alternatively be combined with methods that do not rely upon reactions at cysteine sulfur, such as acylations at lysine, or conjugation via S6 tags or Pcl methodology.

(147) While the payload to antibody ratio has an exact value for a specific conjugate molecule, it is understood that the value will often be an average value when used to describe a sample containing many molecules, due to some degree of inhomogeneity, typically in the conjugation step. The average loading for a sample of an immunoconjugate is referred to herein as the drug to antibody ratio, or DAR. In some embodiments, the DAR is between about 1 and about 16, and typically is about 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, at least 50% of a sample by weight is compound having the average ratio plus or minus 2, and preferably at least 50% of the sample is a conjugate that contains the average ratio plus or minus 1. Preferred embodiments include immunoconjugates wherein the DAR is about 2 or about 8, e.g., about 2, about 4, about 6 or about 8. In some embodiments, a DAR of ‘about n’ means the measured value for DAR is within 10% of n (in Formula (I)).

3. Further Alteration of the Framework of Fc Region

(148) The present invention provides site-specific labeled immunoconjugates. The immunoconjugates of the invention may comprise modified antibodies or antibody fragments thereof that further comprise modifications to framework residues within V.sub.H and/or V.sub.L, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “back-mutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be “back-mutated” to the germline sequence by, for example, site-directed mutagenesis. Such “back-mutated” antibodies are also intended to be encompassed by the invention.

(149) Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

(150) In addition or alternative to modifications made within the framework or CDR regions, antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

(151) In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

(152) In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.

(153) In yet other embodiments, the Fe region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in, e.g., U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

(154) In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in, e.g., U.S. Pat. No. 6,194,551 by Idusogie et al.

(155) In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g., the PCT Publication WO 94/29351 by Bodmer el al. In a specific embodiment, one or more amino acids of an antibody or antibody fragment thereof of the present invention are replaced by one or more allotypic amino acid residues, such as those shown in FIG. 4 for the IgG1 subclass and the kappa isotype. Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgG1, IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et al., MAbs. 1:332-338 (2009).

(156) In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids. This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

(157) In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for “antigen.” Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

(158) Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

(159) In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, or T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or C.sub.L region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fe region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

4. Antibody Conjugates

(160) The present invention provides site-specific labeling methods, modified antibodies and antibody fragments thereof, and immunoconjugates prepared accordingly. Using the methods of the invention, a modified antibody or antibody fragments thereof can be conjugated to a label, such as a drug moiety, e.g., an anti-cancer agent, an autoimmune treatment agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent, or an imaging reagent, such as a chelator for PET imaging, or a fluorescent label, or a MRI contrast reagent. An antibody or antibody fragments can also be conjugated using several identical or different labeling moieties combining the methods of the invention with other conjugation methods.

(161) In certain embodiments, the immunoconjugates of the present invention comprise a drug moiety selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, proteasome inhibitors, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder, topoisomerase inhibitors, RNA synthesis inhibitors, kinesin inhibitors, inhibitors of protein-protein interactions, and a DHFR inhibitor.

(162) Further, the modified antibodies or antibody fragments of the present invention may be conjugated to a drug moiety that modifies a given biological response. Drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be an immune modulator, such as an immune potentiator, a small molecule immune potentiator, a TLR agonist, a CpG oligomer, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope peptide or a like. The drug moiety may also be an oligonucleotide, a siRNA, a shRNA, a cDNA or a like. Alternatively, the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response modifier such as, for example, a lymphokine.

(163) In one embodiment, the modified antibodies or antibody fragments of the present invention are conjugated to a drug moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Examples of cytotoxin include but not limited to, taxanes (see, e.g., International (PCT) Patent Application Nos. WO 01/38318 and PCT/US03/02675), DNA-alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansinoids, and cytotoxic agents comprising a reactive polyethylene glycol moiety (see, e.g., Sasse el al., J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa el al., Bioorg. Med. Chem., 8, 2175-84 (2000), Ichimura et al., J. Antibiot. (Tokyo), 44, 1045-53 (1991), Francisco et al., Blood (2003) (electronic publication prior to print publication), U.S. Pat. Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application Publication No. 2001/0036923 A1, Pending U.S. patent application Ser. Nos. 10/024,290 and 10/116,053, and International (PCT) Patent Application No. WO 01/49698), taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, anti-metabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). (See e.g., Seattle Genetics US20090304721).

(164) Other examples of therapeutic cytotoxins that can be conjugated to the modified antibodies or antibody fragments of the invention include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg™; Wyeth-Ayerst).

(165) For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito et al., (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al., (2003) Cancer Immunol. Immunother. 52:328-337; Payne, (2003) Cancer Cell 3:207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev. 53:247-264.

(166) According to the present invention, modified antibodies or antibody fragments thereof can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine.sup.131, indium.sup.111, yttrium.sup.90, and lutetium.sup.177. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (DEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al., (1998) Clin. Cancer Res. 4(10):2483-90; Peterson et al., (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman el al., (1999) Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.

(167) The present invention further provides modified antibodies or fragments thereof that specifically bind to an antigen. The modified antibodies or fragments may be conjugated or fused to a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. In particular, the invention provides fusion proteins comprising an antibody fragment described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a V.sub.H domain, a V.sub.H CDR, a V.sub.L domain or a V.sub.L CDR) and a heterologous protein, polypeptide, or peptide.

(168) In some embodiments, modified antibody fragments without antigen binding specificity, such as but not limited to, modified Fc domains with engineered cysteine residue(s) according to the present invention, are used to generate fusion proteins comprising such an antibody fragment (e.g., engineered Fc) and a heterologous protein, polypeptide, or peptide.

(169) Additional fusion proteins may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends Biotechnol. 16(2):76-82; Hansson el al., (1999) J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24(2):308-313 (each of these patents and publications are hereby incorporated by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

(170) Moreover, the modified antibodies or antibody fragments thereof of the present invention can be conjugated to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., (1989) Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson el al., (1984) Cell 37:767), and the “FLAG” tag (A. Einhauer et al., J. Biochem. Biophys. Methods 49: 455-465, 2001). According to the present invention, antibodies or antibody fragments can also be conjugated to tumor-penetrating peptides in order to enhance their efficacy.

(171) In other embodiments, modified antibodies or antibody fragments of the present invention are conjugated to a diagnostic or detectable agent. Such immunoconjugates can be useful for monitoring or prognosing the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials, such as, but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and acquorin; radioactive materials, such as, but not limited to, iodine (.sup.131I, .sup.125I, .sup.123I, and .sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In, and .sup.111In), technetium (.sup.99Tc), thallium (.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, 47Sc, .sup.186Re, .sup.188Re, .sup.142 Pr, .sup.105Rh, .sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P, .sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se, .sup.64Cu, .sup.113Sn, and .sup.117Sn; and positron emitting metals using various positron emission tomographies, and non-radioactive paramagnetic metal ions.

(172) Modified antibodies or antibody fragments of the invention may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

5. Pharmaceutical Composition

(173) To prepare pharmaceutical or sterile compositions including immunoconjugates, the immunoconjugates of the invention are mixed with a pharmaceutically acceptable carrier or excipient. The compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing cancer (breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors (e.g., schwannoma), head and neck cancer, bladder cancer, esophageal cancer, Barretts esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynacomastica, and endometriosis).

(174) Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, N Y, 1993; Lieberman, el al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, N Y, 1990; Lieberman, et al. (eds.) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N Y, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y., 2000).

(175) Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules is available (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, U K, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y., 1991; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y., 1993; Baert el al., New Engl. J. Med. 348:601-608, 2003; Milgrom el al., New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl. J. Med. 344:783-792, 2001; Beniaminovitz et al., New Engl. J. Med. 342:613-619, 2000; Ghosh el al., New Engl. J. Med. 348:24-32, 2003; Lipsky el al., New Engl. J. Med. 343:1594-1602, 2000).

(176) Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.

(177) Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.

(178) Compositions comprising antibodies or fragments thereof of the invention can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.

(179) For the immunoconjugates of the invention, the dosage administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight. The dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight. The dosage of the antibodies or fragments thereof of the invention may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.

(180) Doses of the immunoconjugates the invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In a specific embodiment, does of the immunoconjugates of the invention are repeated every 3 weeks.

(181) An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see, e.g., Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, U K, 2001).

(182) The route of administration may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerobrospinal, intralesional, or by sustained release systems or an implant (see, e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference their entirety.

(183) A composition of the present invention may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Selected routes of administration for the immunoconjugates of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, a composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In one embodiment, the immunoconjugates of the invention is administered by infusion. In another embodiment, the immunoconjugates of the invention is administered subcutaneously.

(184) If the immunoconjugates of the invention are administered in a controlled release or sustained release system, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574, 1989). Polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; see also Levy et al., Science 228:190, 1985; During el al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 7 1:105, 1989; U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), poly anhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), poly acrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

(185) Controlled release systems are discussed in the review by Langer, Science 249:1527-1533, 1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more immunoconjugates of the invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al., Radiotherapy & Oncology 39:179-189, 1996; Song et al., PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, 1997; and Lam el al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, 1997, each of which is incorporated herein by reference in their entirety.

(186) If the immunoconjugates of the invention are administered topically, they can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some instances, greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as Freon™) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.

(187) If the compositions comprising the immunoconjugates are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

(188) Methods for co-administration or treatment with a second therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation, are known in the art (see, e.g., Hardman et al., (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of therapeutic may decrease the symptoms by at least 10%; by at least 20%; at least about 30%; at least 40%, or at least 50%.

(189) Additional therapies (e.g., prophylactic or therapeutic agents), which can be administered in combination with the immunoconjugates of the invention may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the immunoconjugates of the invention. The two or more therapies may be administered within one same patient visit.

(190) In certain embodiments, the immunoconjugates of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa el al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (Bloeman et al., (1995) FEBS Lett. 357:140; Owais el al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

(191) The invention provides protocols for the administration of pharmaceutical composition comprising immunoconjugates of the invention alone or in combination with other therapies to a subject in need thereof. The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the present invention can be administered concomitantly or sequentially to a subject. The therapy (e.g., prophylactic or therapeutic agents) of the combination therapies of the present invention can also be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies (e.g., agents) to avoid or reduce the side effects of one of the therapies (e.g., agents), and/or to improve, the efficacy of the therapies.

(192) The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the invention can be administered to a subject concurrently.

(193) The term “concurrently” is not limited to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather it is meant that a pharmaceutical composition comprising antibodies or fragments thereof the invention are administered to a subject in a sequence and within a time interval such that the antibodies of the invention can act together with the other therapy or therapies to provide an increased benefit than if they were administered otherwise. For example, each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route. In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 15 minutes, less than 30 minutes, less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other embodiments, two or more therapies (e.g., prophylactic or therapeutic agents) are administered to a within the same patient visit.

(194) The prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

(195) The invention having been fully described, it is further illustrated by the following examples and claims, which are illustrative and are not meant to be further limiting.

EXAMPLES

Example 1. Selection of Surface Accessible Sites for Cys Mutation in Human IgG1 Heavy Chain and Kappa Light Chain

(196) Surface exposed residues in the constant region of human IgG1 heavy and human kappa light chains were identified in a crystal structure of an hIgG1/kappa antibody (Protein Databank structure entry 1HZH.pdb, Table 6, Table 7. FIG. 1) using the computer program Surface Racer 5.0, as described by Tsodikov et al, “A novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature,” J. Comput. Chem., 23, 600-609 (2002). 88 residues were selected for Cys substitution, 59 sites in hIgG heavy chain and 29 in human kappa light chain, based on the following criteria: 1) select residues in CH1, CH2 and CH3 domains of the constant regions of heavy chain and the constant regions of light chain; 2) select surface exposed residues but circumvent globally exposed residues and the C-terminal region to avoid inter-antibody dimer formation; 3) focus on polar or charged residues such as Ser, Thr, Lys, Arg, Glu, and Asp; and 4) exclude residues in FcRn binding domain, Protein A binding domain, and heavy chain hinge region.

(197) Criterion 1) namely the selection of Cys substitution sites in the constant region of the antibody, assures transferability of the conjugation sites to many different antibodies. Criterion 2) is based on observation of inter-antibody dimer formation for Cys substitutions of prominently exposed residues (residues excluded based on this criteria are listed in Table 6). Based on the IgG crystal structure, the putative orientation of the Cys side chain was taking into consideration: residues for which the Cys side chain may be partially shielded from interactions with another antibody but may still be reactive with a small molecular payload, were favored over residues with larger surface accessibility but with an orientation that may enable interactions with a large macromolecule such as dimer formation. Criterion 3) was implemented to favor conservative mutations in order to minimize destabilizing effects of the mutations on the antibody. Similarly, criterion 4) was used to avoid functional changes to the antibody such as effects on FcRn and Protein A binding which may affect the antibody's pharmacokinetic properties or may result in the loss of a purification handle, respectively. Residues excluded based on criterion 4 are listed in Table 6. The location of the 88 selected mutation sites in the structure model of hIgG1/kappa indicates that the selected sites are surface accessible (FIG. 2).

(198) TABLE-US-00006 TABLE 6 Surface accessibility of amino acid residues in human IgG1 heavy chain. Surface accessibility was calculated using Surface Racer 5.0 and is expressed as Angstrom square [Å.sup.2]. “Excluded sites” indicate the sites that are excluded from selection due to the reasons mentioned in example 1. “Selected sites” are the sites that are selected for substitution to Cys in the invention. Surface Reason for Eu accessibility exclusion Selected number Residue [Å.sup.2] (if applicable) sites 117 SER 128 HC-S117C 118 ALA 2 119 SER 79 HC-S119C 120 THR 71 121 LYS 136 HC-K121C 122 GLY 21 123 PRO 2 124 SER 40 HC-S124C 125 VAL 0 126 PHE 1 127 PRO 0 128 LEU 0 129 ALA 0 130 PRO 0 131 SER 0 132 SER 34 HC-S132C 133 LYS 87 134 SER 123 HC-S134C 135 THR 1 136 SER 183 HC-S136C 137 GLY 84 138 GLY 40 139 THR 33 HC-T139C 140 ALA 0 141 ALA 0 142 LEU 0 143 GLY 0 144 CYS 0 145 LEU 0 146 VAL 0 147 LYS 0 148 ASP 1 149 TYR 0 150 PHE 0 151 PRO 0 152 GLU 52 HC-E152C 153 PRO 89 HC-P153C 154 VAL 10 155 THR 69 HC-T155C 156 VAL 0 157 SER 39 HC-S157C 158 TRP 0 159 ASN 4 160 SER 164 Dimer 161 GLY 35 Dimer 162 ALA 115 Dimer 163 LEU 17 164 THR 125 HC-T164C 165 SER 183 HC-S165C 166 GLY 20 167 VAL 12 168 HIS 5 169 THR 60 HC-T169C 170 PHE 0 171 PRO 33 HC-P171C 172 ALA 9 173 VAL 0 174 LEU 68 HC-L174C 175 GLN 0 176 SER 162 HC-S176C 177 SER 68 HC-S177C 178 GLY 8 179 LEU 0 180 TYR 6 181 SER 0 182 LEU 2 183 SER 0 184 SER 0 185 VAL 0 186 VAL 0 187 THR 30 188 VAL 0 189 PRO 86 HC-P189C 190 SER 21 191 SER 127 HC-S191C 192 SER 17 193 LEU 0 194 GLY 18 195 THR 111 HC-T195C 196 GLN 79 197 THR 90 HC-T197C 198 TYR 0 199 ILE 25 200 CYS 0 201 ASN 8 202 VAL 0 203 ASN 22 204 HIS 0 205 LYS 217 HC-K205C 206 PRO 66 207 SER 50 HC-S207C 208 ASN 91 209 THR 24 210 LYS 234 Dimer 211 VAL 30 212 ASP 97 HC-D212C 213 LYS 70 214 LYS 146 215 ALA 0 216 GLU 79 217 PRO 0 218 LYS 4 219 SER 149 220 CYS 7 221 ASP 0 Hinge 222 LYS 208 Hinge 223 THR 112 Hinge 224 HIS 1 Hinge 225 THR 22 Hinge 226 CYS 12 Hinge 227 PRO 22 Hinge 228 PRO 133 Hinge 229 CYS 7 Hinge 230 PRO 84 Hinge 231 ALA 114 Hinge 232 PRO 49 Hinge 233 GLU 114 Hinge 234 LEU 90 235 LEU 88 236 GLY 9 237 GLY 46 238 PRO 14 239 SER 9 240 VAL 0 241 PHE 0 242 LEU 0 243 PHE 1 244 PRO 34 245 PRO 0 246 LYS 55 HC-K246C 247 PRO 18 248 LYS 47 249 ASP 1 250 THR 0 FcRn binding 251 LEU 0 252 MET 53 Protein A, FcRn binding 253 ILE 155 Protein A binding 254 SER 157 Protein A, FcRn binding 255 ARG 103 256 THR 86 FcRn binding 257 PRO 0 FcRn binding 258 GLU 42 HC-E258C 259 VAL 0 FcRn binding 260 THR 0 261 CYS 0 262 VAL 0 263 VAL 0 264 VAL 0 265 ASP 11 FcRn binding 266 VAL 0 267 SER 10 268 HIS 79 269 GLU 189 HC-E269C 270 ASP 23 271 PRO 20 272 GLU 152 273 VAL 19 274 LYS 138 HC-K274C 275 PHE 2 276 ASN 1 277 TRP 0 278 TYR 14 279 VAL 0 280 ASP 66 281 GLY 72 282 VAL 141 283 GLU 80 284 VAL 25 285 HIS 133 286 ASN 119 HC-N286C 287 ALA 67 288 LYS 182 HC-K288C 289 THR 5 290 LYS 177 HC-K290C 291 PRO 51 292 ARG 252 HC-R292C 293 GLU 83 HC-E293C 294 GLU 73 HC-E294C 295 GLN 170 296 TYR 29 297 ASN 61 Glycosylation 298 SER 125 Glycosylation 299 THR 2 Glycosylation 300 TYR 28 301 ARG 18 302 VAL 0 303 VAL 10 304 SER 0 305 VAL 17 306 LEU 0 307 THR 27 FcRn binding 308 VAL 0 FcRn binding 309 LEU 122 310 HIS 4 Protein A binding 311 GLN 145 Protein A, FcRn binding 312 ASP 14 313 TRP 0 314 LEU 6 Protein A binding 315 ASN 151 Protein A binding 316 GLY 12 317 LYS 81 318 GLU 49 319 TYR 0 320 LYS 55 HC-K320C 321 CYS 0 322 LYS 78 HC-K322C 323 VAL 0 324 SER 0 325 ASN 0 326 LYS 213 HC-K326C 327 ALA 10 328 LEU 9 329 PRO 158 330 ALA 96 HC-A330C 331 PRO 44 332 ILE 32 333 GLU 85 HC-E333C 334 LYS 50 HC-K334C 335 THR 70 HC-T335C 336 ILE 13 337 SER 15 HC-S337C 338 LYS 0 339 ALA 37 340 LYS 217 Protein A binding 341 GLY 37 342 GLN 235 343 PRO 42 344 ARG 98 HC-R344C 345 GLU 105 346 PRO 0 347 GLN 24 348 VAL 3 349 TYR 3 350 THR 0 351 LEU 0 352 PRO 38 353 PRO 0 354 SER 0 355 ARG 249 HC-R355C 356 ASP 53 357 GLU 0 358 LEU 36 359 THR 144 Dimer 360 LYS 114 HC-K360C 361 ASN 155 362 GLN 41 HC-Q362C 363 VAL 0 364 SER 0 365 LEU 0 366 THR 0 367 CYS 0 368 LEU 0 369 VAL 0 370 LYS 1 371 GLY 0 372 PHE 0 373 TYR 23 374 PRO 0 375 SER 29 HC-S375C 376 ASP 9 377 ILE 11 378 ALA 11 379 VAL 4 380 GLU 18 FcRn binding 381 TRP 0 382 GLU 22 HC-E382C 383 SER 1 384 ASN 147 385 GLY 102 Dimer 386 GLN 161 387 PRO 99 388 GLU 4 389 ASN 189 HC-N389C 390 ASN 36 HC-N390C 391 TYR 44 392 LYS 82 HC-K392C 393 THR 36 HC-T393C 394 THR 0 395 PRO 72 396 PRO 47 397 VAL 9 398 LEU 111 HC-L398C 399 ASP 0 400 SER 81 HC-S400C 401 ASP 68 402 GLY 29 403 SER 0 404 PHE 22 405 PHE 0 406 LEU 0 407 TYR 0 408 SER 0 409 LYS 0 410 LEU 0 411 THR 0 412 VAL 0 413 ASP 80 HC-D413C 414 LYS 83 415 SER 69 HC-S415C 416 ARG 53 417 TRP 0 418 GLN 108 419 GLN 177 420 GLY 39 421 ASN 35 422 VAL 81 HC-V422C 423 PHE 0 424 SER 2 425 CYS 0 426 SER 0 427 VAL 0 428 MET 0 FcRn binding 429 HIS 0 430 GLU 14 431 ALA 22 432 LEU 1 433 HIS 227 Protein A binding 434 ASN 126 Protein A, FcRn binding 435 HIS 28 436 TYR 54 437 THR 36 438 GLN 82 439 LYS 12 440 SER 62 441 LEU 2 442 SER 34 443 LEU 101 444 SER 70 Dimer

(199) TABLE-US-00007 TABLE 7 Surface accessibility of amino acid residues in human kappa light chain. Surface accessibility was calculated using Surface Racer 5.0 and is expressed as Angstrom squared [Å.sup.2]. “Selected sites” indicate the sites selected for substitution to Cys in the invention. Surface EU accessibility Selected number Residue [Å.sup.2] sites 107 LYS 90 LC-K107C 108 ARG 49 LC-R108C 109 THR 148 LC-T109C 110 VAL 77 111 ALA 16 112 ALA 50 LC-A112C 113 PRO 2 114 SER 39 LC-S114C 115 VAL 0 116 PHE 0 117 ILE 0 118 PHE 0 119 PRO 0 120 PRO 0 121 SER 0 122 ASP 90 LC-D122C 123 GLU 51 LC-E123C 124 GLN 0 125 LEU 21 126 LYS 230 127 SER 101 128 GLY 12 129 THR 41 LC-T129C 130 ALA 0 131 SER 0 132 VAL 0 133 VAL 0 134 CYS 0 135 LEU 0 136 LEU 0 137 ASN 5 138 ASN 18 139 PHE 0 140 TYR 0 141 PRO 3 142 ARG 55 LC-R142C 143 GLU 117 LC-E143C 144 ALA 7 145 LYS 160 LC-K145C 146 VAL 11 147 GLN 22 148 TRP 0 149 LYS 48 150 VAL 0 151 ASP 59 152 ASN 157 LC-N152C 153 ALA 51 154 LEU 117 LC-L154C 155 GLN 26 156 SER 122 LC-S156C 157 GLY 114 158 ASN 19 159 SER 22 LC-S159C 160 GLN 36 161 GLU 66 LC-E161C 162 SER 8 163 VAL 14 164 THR 5 165 GLU 74 LC-E165C 166 GLN 8 167 ASP 13 168 SER 170 LC-S168C 169 LYS 241 LC-K169C 170 ASP 48 LC-D170C 171 SER 1 172 THR 0 173 TYR 0 174 SER 0 175 LEU 0 176 SER 0 177 SER 0 178 THR 0 179 LEU 0 180 THR 13 181 LEU 21 182 SER 59 LC-S182C 183 LYS 131 LC-K183C 184 ALA 32 185 ASP 52 186 TYR 0 187 GLU 77 188 LYS 201 LC-K188C 189 HIS 42 190 LYS 167 LC-K190C 191 VAL 58 LC-V191C 192 TYR 0 193 ALA 0 194 CYS 0 195 GLU 12 196 VAL 0 197 THR 38 LC-T197C 198 HIS 4 199 GLN 127 LC-Q199C 200 GLY 11 201 LEU 17 202 ARG 343 203 SER 110 LC-S203C 204 PRO 69 205 VAL 30 206 THR 70 LC-T206C 207 LYS 44 208 SER 47 209 PHE 5 210 ASN 44 211 ARG 89 212 GLY 15 213 GLU 107 214 CYS 58

Example 2. Preparation of Trastuzumab Cys Mutant Antibodies

(200) DNA encoding variable regions of heavy and light chains of trastuzumab were chemically synthesized and cloned into two mammalian expression vectors, pOG-HC and pOG-LC that contain constant regions of human IgG1 and human kappa light chain, resulting in two wild-type constructs, pOG-trastuzumab HC and pOG-trastuzumab LC, respectively. In the vectors the expression of antibody heavy and light chain constructs in mammalian cells is driven by a CMV promoter. The vectors contain a synthetic 24 amino acid signal sequence: MKTFILLLWVLLLWVIFLLPGATA (SEQ ID NO: 99), in the N-terminal of heavy chain or light chain to guide their secretion from mammalian cells. The signal sequence has been validated to be efficient in directing protein secretion in hundreds of mammalian proteins in 293 Freestyle™ cells. Oligonucleotide directed mutagenesis was employed to prepare Cys mutant constructs in trastuzumab. 88 pairs of mutation primers (Table 8) were chemically synthesized that correspond to the 88 Cys mutation sites selected in the constant regions of human IgG1 heavy chain and kappa light chain as described in Example 1. The sense and anti-sense mutation primer pairs were mixed prior to PCR amplification. PCR reactions were performed by using PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pOG-trastuzumab HC and pOG-trastuzumab LC as the templates. After PCR reactions, the PCR products were confirmed on agarose gels, and treated with DPN I followed by transformation in DH5a cells (Klock el al., (2009) Methods Mol Biol. 498:91-103).

(201) Sequences of 88 Cys mutant constructs were confirmed by DNA sequencing. The full length amino acid sequence of wild-type trastuzumab heavy chain is shown as SEQ ID NO:1 and that of the light chain as SEQ ID NO:90. The encoded protein sequence of the constant region of 59 trastuzumab HC Cys mutant constructs (SEQ ID NO:2 through SEQ ID NO:60) and 29 trastuzumab LC Cys mutant constructs (SEQ ID NO:61 to SEQ ID NO:89) are shown in Table 9 and Table 10, respectively. Amino acid residues in human IgG1 heavy chain and human kappa light chain are numbered by Eu numbering system (Edelman et al, (1969) Proc Natl Acad Sci USA, 63:78-85).

(202) TABLE-US-00008 TABLE 8 DNA sequences of mutation primers used to prepare 88 Cys mutations  heavy and light chains of human IgG1. Mutation Primer SEQ ID sites name Sequence NO. LC-K107C LC-CYS-S1 GTGGAGATCTGTCGAACGGTGGCCGCTC 100 CCAGCGTGTTCA LC-CYS-A1 ACCGTTCGACAGATCTCCACCTTGGTACC 101 CTGTCCGAAC LC-R108C LC-CYS-S2 GGAGATCAAATGCACGGTGGCCGCTCCC 102 AGCGTGTTCATCT LC-CYS-A2 GCCACCGTGCATTTGATCTCCACCTTGGT 103 ACCCTGTCCGA LC-T109C LC-CYS-S3 GATCAAACGATGTGTGGCCGCTCCCAGC 104 GTGTTCATCTTCC LC-CYS-A3 GCGGCCACACATCGTTTGATCTCCACCTT 105 GGTACCCTGTC LC-A112C LC-CYS-S4 ACGGTGGCCTGTCCCAGCGTGTTCATCTT 106 CCCCCCCAGCGA LC-CYS-A4 CACGCTGGGACAGGCCACCGTTCGTTTG 107 ATCTCCACCTTG LC-S114C LC-CYS-S5 GCCGCTCCCTGCGTGTTCATCTTCCCCCC 108 CAGCGACGAGCA LC-CYS-A5 ATGAACACGCAGGGAGCGGCCACCGTTC 109 GTTTGATCTCCA LC-D122C LC-CYS-S6 CCCCCAGCTGTGAGCAGCTGAAGAGCGG 110 CACCGCCAGCGT LC-CYS-A6 CAGCTGCTCACAGCTGGGGGGGAAGATG 111 AACACGCTGGGA LC-E123C LC-CYS-S7 CCCAGCGACTGTCAGCTGAAGAGCGGCA 112 CCGCCAGCGTG LC-CYS-A7 TTCAGCTGACAGTCGCTGGGGGGGAAGA 113 TGAACACGCTG LC-T129C LC-CYS-S10 AGAGCGGCTGTGCCAGCGTGGTGTGCCT 114 GCTGAACAACTT LC-CYS-A10 CACGCTGGCACAGCCGCTCTTCAGCTGCT 115 CGTCGCTGGGG LC-R142C LC-CYS-S11 TCTACCCCTGTGAGGCCAAGGTGCAGTG 116 GAAGGTGGACAA LC-CYS-A11 TTGGCCTCACAGGGGTAGAAGTTGTTCA 117 GCAGGCACACCA LC-E143C LC-CYS-S12 TACCCCCGGTGTGCCAAGGTGCAGTGGA 118 AGGTGGACAAC LC-CYS-A12 ACCTTGGCACACCGGGGGTAGAAGTTGT 119 TCAGCAGGCACA LC-K145C LC-CYS-S13 CGGGAGGCCTGCGTGCAGTGGAAGGTGG 120 ACAACGCCCTGC LC-CYS-A13 CACTGCACGCAGGCCTCCCGGGGGTAGA 121 AGTTGTTCAGCA LC-N152C LC-CYS-S14 AAGGTGGACTGTGCCCTGCAGAGCGGCA 122 ACAGCCAGGAGA LC-CYS-A14 TGCAGGGCACAGTCCACCTTCCACTGCAC 123 CTTGGCCTCCC LC-L154C LC-CYS-S15 GACAACGCCTGTCAGAGCGGCAACAGCC 124 AGGAGAGCGTCA LC-CYS-A15 TGCCGCTCTGACAGGCGTTGTCCACCTTC 125 CACTGCACCTTG LC-S156C LC-CYS-S16 GCCCTGCAGTGTGGCAACAGCCAGGAGA 126 GCGTCACCGAGCA LC-CYS-A16 GCTGTTGCCACACTGCAGGGCGTTGTCCA 127 CCTTCCACTGCA LC-S159C LC-CYS-S18 AGCGGCAACTGTCAGGAGAGCGTCACCG 128 AGCAGGACAGCAA LC-CYS-A18 CTCTCCTGACAGTTGCCGCTCTGCAGGGC 129 GTTGTCCACCT LC-E161C LC-CYS-S19 AACAGCCAGTGCAGCGTCACCGAGCAGG 130 ACAGCAAGGACT LC-CYS-A19 GTGACGCTGCACTGGCTGTTGCCGCTCTG 131 CAGGGCGTTGT LC-E165C LC-CYS-S20 GAGCGTCACCTGTCAGGACAGCAAGGAC 132 TCCACCTACAGC LC-CYS-A20 CTGTCCTGACAGGTGACGCTCTCCTGGCT 133 GTTGCCGCTCT LC-S168C LC-CYS-S21 GAGCAGGACTGCAAGGACTCCACCTACA 134 GCCTGAGCAGCA LC-CYS-A21 GAGTCCTTGCAGTCCTGCTCGGTGACGCT 135 CTCCTGGCTGT LC-K169C LC-CYS-S22 CAGGACAGCTGTGACTCCACCTACAGCC 136 TGAGCAGCACC LC-CYS-A22 GTGGAGTCACAGCTGTCCTGCTCGGTGAC 137 GCTCTCCTGG LC-D170C LC-CYS-S23 ACAGCAAGTAGTCCACCTACAGCCTGAG 138 CAGCACCCTGAC LC-CYS-A23 TAGGTGGACTACTTGCTGTCCTGCTCGGT 139 GACGCTCTCCT LC-S182C LC-CYS-S24 TGACCCTGTGCAAGGCCGACTACGAGAA 140 GCATAAGGTGTA LC-CYS-A24 GTCGGCCTTGCACAGGGTCAGGGTGCTG 141 CTCAGGCTGTAG LC-K183C LC-CYS-S25 GACCCTGAGCTGTGCCGACTACGAGAAG 142 CATAAGGTGTAC LC-CYS-A25 TAGTCGGCACAGCTCAGGGTCAGGGTGC 143 TGCTCAGGCTGT LC-K188C LC-CYS-S26 GACTACGAGTGCCATAAGGTGTACGCCT 144 GCGAGGTGAC LC-CYS-A26 ACCTTATGGCACTCGTAGTCGGCCTTGCT 145 CAGGGTCAGG LC-K190C LC-CYS-S27 GAGAAGCATTGCGTGTACGCCTGCGAGG 146 TGACCCACCAG LC-CYS-A27 GGCGTACACGCAATGCTTCTCGTAGTCGG 147 CCTTGCTCAGG LC-V191C LC-CYS-S28 AGCATAAGTAGTACGCCTGCGAGGTGAC 148 CCACCAGGGCT LC-CYS-A28 CAGGCGTACTACTTATGCTTCTCGTAGTC 149 GGCCTTGCTCA LC-T197C LC-CYS-S29 GCGAGGTGTGTCACCAGGGCCTGTCCAG 150 CCCCGTGACCAA LC-CYS-A29 CCCTGGTGACACACCTCGCAGGCGTACA 151 CCTTATGCTTCT LC-Q199C LC-CYS-S30 GTGACCCACTGTGGCCTGTCCAGCCCCGT 152 GACCAAGAGCT LC-CYS-A30 GACAGGCCACAGTGGGTCACCTCGCAGG 153 CGTACACCTTAT LC-S203C LC-CYS-S31 GGCCTGTCCTGTCCCGTGACCAAGAGCTT 154 CAACAGGGGCGA LC-CYS-A31 GTCACGGGACAGGACAGGCCCTGGTGGG 155 TCACCTCGCAGG LC-T206C LC-CYS-S32 CAGCCCCGTGTGCAAGAGCTTCAACAGG 156 GGCGAGTGCTAA LC-CYS-A32 AAGCTCTTGCACACGGGGCTGGACAGGC 157 CCTGGTGGGTC HC-S117C HC-CYS-S1  CCGTCTCCTGCGCTAGCACCAAGGGCCC 158 CAGCGTGTTC HC-CYS-A1 GGTGCTAGCGCAGGAGACGGTGACCAGG 159 GTTCCTTGAC HC-S119C HC-CYS-S2 TCCTCGGCTTGTACCAAGGGCCCCAGCGT 160 GTTCCCCCTGG HC-CYS-A2 CCCTTGGTACAAGCCGAGGAGACGGTGA 161 CCAGGGTTCCTT HC-K121C HC-CYS-S3 CTAGCACCTGTGGCCCCAGCGTGTTCCCC 162 CTGGCCCCCA HC-CYS-A3 GCTGGGGCCACAGGTGCTAGCCGAGGAG 163 ACGGTGACCAG HC-S124C HC-CYS-S4 AGGGCCCCTGTGTGTTCCCCCTGGCCCCC 164 AGCAGCAAGA HC-CYS-A4 GGGGAACACACAGGGGCCCTTGGTGCTA 165 GCCGAGGAGACG HC-S132C HC-CYS-S5 CCCCCAGCTGCAAGAGCACCAGCGGCGG 166 CACAGCCGCCCT HC-CYS-A5 GGTGCTCTTGCAGCTGGGGGCCAGGGGG 167 AACACGCTGGGG HC-S134C HC-CYS-S6 AGCAGCAAGTGTACCAGCGGCGGCACAG 168 CCGCCCTGGGCT HC-CYS-A6 CCGCTGGTACACTTGCTGCTGGGGGCCA 169 GGGGGAACACG HC-S136C HC-CYS-S7 AGAGCACCTGTGGCGGCACAGCCGCCCT 170 GGGCTGCCTGGT HC-CYS-A7 GTGCCGCCACAGGTGCTCTTGCTGCTGGG 171 GGCCAGGGGGA HC-T139C HC-CYS-S8 AGCGGCGGCTGTGCCGCCCTGGGCTGCC 172 TGGTGAAGGACT HC-CYS-A8 CAGGGCGGCACAGCCGCCGCTGGTGCTC 173 TTGCTGCTGGGG HC-E152C HC-CYS-S9 TACTTCCCCTGTCCCGTGACCGTGTCCTG 174 GAACAGCGGA HC-CYS-A9 GGTCACGGGACAGGGGAAGTAGTCCTTC 175 ACCAGGCAGC HC-P153C HC-CYS-S10 TCCCCGAGTGCGTGACCGTGTCCTGGAAC 176 AGCGGAGCCCT HC-CYS-A10 CACGGTCACGCACTCGGGGAAGTAGTCC 177 TTCACCAGGCAG HC-T155C HC-CYS-S11 GAGCCCGTGTGCGTGTCCTGGAACAGCG 178 GAGCCCTGACCT HC-CYS-A11 CAGGACACGCACACGGGCTCGGGGAAGT 179 AGTCCTTCACCA HC-S157C HC-CYS-S12 TGACCGTGTGCTGGAACAGCGGAGCCCT 180 GACCTCCGGCGT HC-CYS-A12 CTGTTCCAGCACACGGTCACGGGCTCGG 181 GGAAGTAGTCCT HC-T164C HC-CYS-S13 GGAGCCCTGTGCTCCGGCGTGCACACCTT 182 CCCCGCCGTGCT HC-CYS-A13 ACGCCGGAGCACAGGGCTCCGCTGTTCC 183 AGGACACGGTCA HC-S165C HC-CYS-S14 CCCTGACCTGTGGCGTGCACACCTTCCCC 184 GCCGTGCTGCA HC-CYS-A14 TGTGCACGCCACAGGTCAGGGCTCCGCT 185 GTTCCAGGACAC HC-T169C HC-CYS-S15 GCGTGCACTGCTTCCCCGCCGTGCTGCAG 186 AGCAGCGGCCT HC-CYS-A15 GGCGGGGAAGCAGTGCACGCCGGAGGTC 187 AGGGCTCCGCTG HC-P171C HC-CYS-S16 CACACCTTCTGTGCCGTGCTGCAGAGCAG 188 CGGCCTGTACA HC-CYS-A16 CAGCACGGCACAGAAGGTGTGCACGCCG 189 GAGGTCAGGGCT HC-L174C HC-CYS-S17 CCGCCGTGTGTCAGAGCAGCGGCCTGTA 190 CAGCCTGTCCA HC-CYS-A17 GCTGCTCTGACACACGGCGGGGAAGGTG 191 TGCACGCCGGAG HC-S176C HC-CYS-S18 TGCTGCAGTGCAGCGGCCTGTACAGCCT 192 GTCCAGCGTGGT HC-CYS-A18 ACAGGCCGCTGCACTGCAGCACGGCGGG 193 GAAGGTGTGCACG HC-S177C HC-CYS-S19 CTGCAGAGCTGTGGCCTGTACAGCCTGTC 194 CAGCGTGGTGA HC-CYS-A19 TACAGGCCACAGCTCTGCAGCACGGCGG 195 GGAAGGTGTGCA HC-P189C HC-CYS-S21 TGACAGTGTGCAGCAGCAGCCTGGGCAC 196 CCAGACCTACAT HC-CYS-A21 CTGCTGCTGCACACTGTCACCACGCTGGA 197 CAGGCTGTACA HC-S191C HC-CYS-S22 TGCCCAGCTGCAGCCTGGGCACCCAGAC 198 CTACATCTGCAA HC-CYS-A22 CCCAGGCTGCAGCTGGGCACTGTCACCA 199 CGCTGGACAGGCT HC-T195C HC-CYS-S23 GCCTGGGCTGTCAGACCTACATCTGCAAC 200 GTGAACCACAA HC-CYS-A23 GTAGGTCTGACAGCCCAGGCTGCTGCTG 201 GGCACTGTCACCA HC-T197C HC-CYS-S24 GCACCCAGTGCTACATCTGCAACGTGAA 202 CCACAAGCCCA HC-CYS-A24 GCAGATGTAGCACTGGGTGCCCAGGCTG 203 CTGCTGGGCACT HC-K205C HC-CYS-S25 TGAACCACTGTCCCAGCAACACCAAGGT 204 GGACAAGAGAGT HC-CYS-A25 TGTTGCTGGGACAGTGGTTCACGTTGCAG 205 ATGTAGGTCTGG HC-5207C HC-CYS-S26 ACAAGCCCTGCAACACCAAGGTGGACAA 206 GAGAGTGGAGC HC-CYS-A26 CTTGGTGTTGCAGGGCTTGTGGTTCACGT 207 TGCAGATGTAG HC-D212C HC-CYS-S27 ACCAAGGTGTGCAAGAGAGTGGAGCCCA 208 AGAGCTGCGACA HC-CYS-A27 CACTCTCTTGCACACCTTGGTGTTGCTGG 209 GCTTGTGGTTCA HC-K246C HC-CYS-S28 TCCCCCCCTGTCCCAAGGACACCCTGATG 210 ATCAGCAGGA HC-CYS-A28 GTCCTTGGGACAGGGGGGGAACAGGAAC 211 ACGGAGGGTCCG HC-E258C HC-CYS-S29 AGGACCCCCTGCGTGACCTGCGTGGTGG 212 TGGACGTGAG HC-CYS-A29 CAGGTCACGCAGGGGGTCCTGCTGATCA 213 TCAGGGTGTCCT HC-E269C HC-CYS-S30 TGAGCCACTGTGACCCAGAGGTGAAGTT 214 CAACTGGTACG HC-CYS-A30 CTCTGGGTCACAGTGGCTCACGTCCACCA 215 CCACGCAGGTC HC-K274C HC-CYS-S32 CCAGAGGTGTGCTTCAACTGGTACGTGG 216 ACGGCGTGGAGG HC-CYS-A32 CCAGTTGAAGCACACCTCTGGGTCCTCGT 217 GGCTCACGTCCA HC-N286C HC-CYS-S35 GAGGTGCACTGTGCCAAGACCAAGCCCA 218 GAGAGGAGCAGT HC-CYS-A35 GGTCTTGGCACAGTGCACCTCCACGCCGT 219 CCACGTACCAGT HC-K288C HC-CYS-S36 CACAACGCCTGTACCAAGCCCAGAGAGG 220 AGCAGTACAACA HC-CYS-A36 GGCTTGGTACAGGCGTTGTGCACCTCCAC 221 GCCGTCCACGT HC-K290C HC-CYS-S37 GCCAAGACCTGTCCCAGAGAGGAGCAGT 222 ACAACAGCACCT HC-CYS-A37 CTCTCTGGGACAGGTCTTGGCGTTGTGCA 223 CCTCCACGCCGT HC-R292C HC-CYS-S38 ACCAAGCCCTGTGAGGAGCAGTACAACA 224 GCACCTACAGGGT HC-CYS-A38 CTGCTCCTCACAGGGCTTGGTCTTGGCGT 225 TGTGCACCTCCA HC-E293C HC-CYS-S39 CAAGCCCAGATGCGAGCAGTACAACAGC 226 ACCTACAGGGTG HC-CYS-A39 GTACTGCTCGCATCTGGGCTTGGTCTTGG 227 CGTTGTGCACCT HC-E294C HC-CYS-S40 GCCCAGAGAGTGTCAGTACAACAGCACC 228 TACAGGGTGGT HC-CYS-A40 TTGTACTGACACTCTCTGGGCTTGGTCTT 229 GGCGTTGTGCA HC-K320C HC-CYS-S41 CAAGGAATACTGCTGCAAGGTCTCCAAC 230 AAGGCCCTGCCA HC-CYS-A41 GACCTTGCAGCAGTATTCCTTGCCGTTCA 231 GCCAGTCCTGGT HC-K322C HC-CYS-S42 TACAAGTGCTGCGTCTCCAACAAGGCCCT 232 GCCAGCCCCCA HC-CYS-A42 GTTGGAGACGCAGCACTTGTATTCCTTGC 233 CGTTCAGCCAGT HC-K326C HC-CYS-S43 GGTCTCCAACTGTGCCCTGCCAGCCCCCA 234 TCGAAAAGACC HC-CYS-A43 GGCAGGGCACAGTTGGAGACCTTGCACT 235 TGTATTCCTTGC HC-A330C HC-CYS-S44 GCCCTGCCATGTCCCATCGAAAAGACCA 236 TCAGCAAGGCCA HC-CYS-A44 TTCGATGGGACATGGCAGGGCCTTGTTG 237 GAGACCTTGCACT HC-E333C HC-CYS-S45 GCCCCCATCTGCAAGACCATCAGCAAGG 238 CCAAGGGCCAGC HC-CYS-A45 GATGGTCTTGCAGATGGGGGCTGGCAGG 239 GCCTTGTTGGAGA HC-K334C HC-CYS-S46 CCCATCGAATGCACCATCAGCAAGGCCA 240 AGGGCCAGCCA HC-CYS-A46 GCTGATGGTGCATTCGATGGGGGCTGGC 241 AGGGCCTTGTTG HC-T335C HC-CYS-S47 TCGAAAAGTGCATCAGCAAGGCCAAGGG 242 CCAGCCACGGGA HC-CYS-A47 CTTGCTGATGCACTTTTCGATGGGGGCTG 243 GCAGGGCCTTGT HC-S337C HC-CYS-S48 AGACCATCTGCAAGGCCAAGGGCCAGCC 244 ACGGGAGCCCCA HC-CYS-A48 CCTTGGCCTTGCAGATGGTCTTTTCGATG 245 GGGGCTGGCAGG HC-R344C HC-CYS-S50 GGCCAGCCATGCGAGCCCCAGGTGTACA 246 CCCTGCCTCCAT HC-CYS-A50 CTGGGGCTCGCATGGCTGGCCCTTGGCCT 247 TGCTGATGGTCT HC-R355C HC-CYS-S51 CTCCATCCTGCGACGAGCTGACCAAGAA 248 CCAGGTGTCCCT HC-CYS-A51 CAGCTCGTCGCAGGATGGAGGCAGGGTG 249 TACACCTGGGGCT HC-K360C HC-CYS-S52 AGCTGACCTGCAACCAGGTGTCCCTGAC 250 CTGTCTGGTGA HC-CYS-A52 CACCTGGTTGCAGGTCAGCTCGTCCCGGG 251 ATGGAGGCAGG HC-Q362C HC-CYS-S53 CCAAGAACTGCGTGTCCCTGACCTGTCTG 252 GTGAAGGGCTT HC-CYS-A53 TCAGGGACACGCAGTTCTTGGTCAGCTCG 253 TCCCGGGATGGA HC-5375C HC-CYS-S54 TTCTACCCCTGCGACATCGCCGTGGAGTG 254 GGAGAGCAACG HC-CYS-A54 GGCGATGTCGCAGGGGTAGAAGCCCTTC 255 ACCAGACAGGTCA HC-E382C HC-CYS-S55 TGGAGTGGTGCAGCAACGGCCAGCCCGA 256 GAACAACTACA HC-CYS-A55 GGCCGTTGCTGCACCACTCCACGGCGAT 257 GTCGCTGGGGTAG HC-N389C HC-CYS-S56 AGCCCGAGTGCAACTACAAGACCACCCC 258 CCCAGTGCTGGA HC-CYS-A56 CTTGTAGTTGCACTCGGGCTGGCCGTTGC 259 TCTCCCACTCCA HC-N390C HC-CYS-S57 CCCGAGAACTGCTACAAGACCACCCCCC 260 CAGTGCTGGACA HC-CYS-A57 GGTCTTGTAGCAGTTCTCGGGCTGGCCGT 261 TGCTCTCCCACT HC-K392C HC-CYS-S58 GAACAACTACTGCACCACCCCCCCAGTG 262 CTGGACAGCGAC HC-CYS-A58 GGGGTGGTGCAGTAGTTGTTCTCGGGCTG 263 GCCGTTGCTCT HC-T393C HC-CYS-S59 AACTACAAGTGTACCCCCCCAGTGCTGG 264 ACAGCGACGGCA HC-CYS-A59 TGGGGGGGTACACTTGTAGTTGTTCTCGG 265 GCTGGCCGTTG HC-L398C HC-CYS-S60 CCCCAGTGTGTGACAGCGACGGCAGCTT 266 CTTCCTGTACA HC-CYS-A60 GTCGCTGTCACACACTGGGGGGGTGGTC 267 TTGTAGTTGTTCT HC-5400C HC-CYS-S61 TGCTGGACTGCGACGGCAGCTTCTTCCTG 268 TACAGCAAGCT HC-CYS-A61 GCTGCCGTCGCAGTCCAGCACTGGGGGG 269 GTGGTCTTGTAGT HC-D413C HC-CYS-S62 TGACCGTGTGCAAGTCCAGGTGGCAGCA 270 GGGCAACGTGTT HC-CYS-A62 ACCTGGACTTGCACACGGTCAGCTTGCTG 271 TACAGGAAGAAG HC-S415C HC-CYS-563 TGGACAAGTGCAGGTGGCAGCAGGGCAA 272 CGTGTTCAGCT HC-CYS-A63 CTGCCACCTGCACTTGTCCACGGTCAGCT 273 TGCTGTACAGG HC-V422C HC-CYS-564 AGGGCAACTGCTTCAGCTGCAGCGTGAT 274 GCACGAGGCCCT HC-CYS-A64 GCAGCTGAAGCAGTTGCCCTGCTGCCAC 275 CTGGACTTGTCCA

(203) TABLE-US-00009 TABLE 9  Amino acid sequences of the constant region of Cys mutant constructs  in human IgG1 heavy chain. SEQ ID NO: 1 is the sequence for  full-length trastuzumab (human IgG1). SEQ ID NO: 2 to SEQ ID NO: 60 indicate the sequence ID numbers for 59 Cys mutant constructs in human  IgG1 heavy  chain, showing only the sequences of the constant region. SEQ ID NO: 1 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGF YAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 2 CASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 3 SACTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 4 SASTCGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 23 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGCQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 24 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQCYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 25 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHCPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO :26 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPCNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 27 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVCKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 28 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPCPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 29 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPCVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 30 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHCDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 31 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVCFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 32 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHCAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 33 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 34 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTCPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 35 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPCEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 36 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPRCEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 37 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPRECQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 38 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYCCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 39 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCCVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 40 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNCALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 41 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP CPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 42 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APICKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 43 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMEALHNH YTQKSLSLSPGK SEQ ID NO: 44 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKCISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 45 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTICKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 46 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPCEPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 47 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSCEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 48 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTCNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 49 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNCVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 50 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 51 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWCS NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 52 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPECNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 53 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 54 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 55 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKCTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 56 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVCDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 57 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 58 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVCKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 59 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKCRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 60 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNCFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 290 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEC TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO: 291 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEC TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYCTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK SEQ ID NO: 292 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEC TISKAKGQPREPQVYTLPPSREEMTCNQVSLTCLVKGFYPCDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO: 293 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIECTISKAKGQP REPQVYTLPPSREEMTCNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK SEQ ID NO: 294 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIECTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPE NNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK SEQ ID NO: 295 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

(204) TABLE-US-00010 TABLE 10 Amino acid sequences of the constant region of 29 human kappa light  chain Cys mutant constructs. SEQ ID NO: 61 is the sequence of the  constant region of wild-type human kappa light chain SEQ ID NO: 62 to  SEQ ID NO: 90 indicate the sequence ID numbers for 29 Cys mutant constructs in the constant region of human kappa light chain SEQ ID NO: 61 CRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 62 KCTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 63 KRCVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 64 KRTVACPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 65 KRTVAAPCVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 66 KRTVAAPSVFIFPPSCEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 67 KRTVAAPSVFIFPPSDCQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 68 KRTVAAPSVFIFPPSDEQLKSGCASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 69 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPCEAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 70 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRCAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 71 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREACVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 72 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDCALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 73 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNACQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 74 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQCG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 75 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NCQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 76 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQCSVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 77 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTCQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 78 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDCKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 79 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSCDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 80 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKCSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 81 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLCKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 82 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSCADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 83 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYECHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 84 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHCVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 85 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKCYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 86 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVCHQGLSSPVTKSF NRGEC SEQ ID NO: 87 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHCGLSSPVTKSF NRGEC SEQ ID NO: 88 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSCPVTKSF NRGEC SEQ ID NO: 89 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCKSF NRGEC SEQ ID NO: 90 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSAS FLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

Example 3. Transfer of the Trastuzumab Heavy Chain and Light Chain Cys Mutations to Different Antibodies

(205) For trastuzumab, all Cys mutations for the attachment of drug payloads were chosen to be in the constant region of its human IgG1 heavy and human kappa light chain. Because constant regions of antibodies are highly conserved in primary sequence and structure, Cys mutant residues that are identified as good payload attachment sites in the context of trastuzumab will also serve as preferred attachment residues in other antibodies. To demonstrate the transferability of these generic conjugation sites to other antibodies, we cloned a set of Cys mutations into antibody 14090. Antibody 14090 is an antibody with a human IgG1 heavy chain and a human lambda light chain that binds to a different target protein than trastuzumab. The DNA encoding variable region of antibody 14090 was cloned into seven selected pOG trastuzumab HC Cys mutant plasmid constructs (SEQ ID NO listed in Table 11) to replace the variable regions of trastuzumab constructs in the plasmids as described in Example 2. As result, the amino acid sequences of the heavy chain constant regions in corresponding seven Cys constructs of antibody 14090 and trastuzumab are identical (FIG. 3). Subsequent examples show that these sites can be conjugated readily. Conversely, due to a high degree of similarity in primary sequences and in tertiary structures for different human IgG isotypes (FIG. 4), Cys mutations on the kappa light chain of trastuzumab can readily be transferred to equivalent light chains on human antibodies containing different isotype heavy chains. In the same way, the sites identified in the constant region of IgG1 may be transferred to IgG2, IgG3 and IgG4.

Example 4. Cysteine Mutations in Human Lambda Light Chains

(206) Human lambda and kappa light chains have little amino acid sequence similarity (FIG. 5A). Mutations in the lambda light chain of antibody 14090 were selected based on the approximate similarity of the locations of the residues in a protein crystal structure model (Protein Databank structure entry 3G6D.pdb) of a Fab containing the human lambda light chain in reference to the desirable residues in the kappa light chain of trastuzumab (FIGS. 5 A and B). Seven additional Cys mutant constructs were generated in antibody 14090-lambda light chain plasmid using oligonucleotide directed mutagenesis (Higuchi et al. 1988) in combination with PIPE cloning strategy (Klock and Lesley, 2009). The mutation primers used to generate Cys point mutations in the lambda light chain are listed in Table 12. The secretion of antibody 14090 is also directed by the synthetic 24 amino acid signal sequence: MKTFILLLWVLLLWVIFLLPGATA (SEQ ID NO: 99). Sequences of antibody 14090 Cys constructs were confirmed by DNA sequencing. The sequence for the constant region of human wild-type lambda light chain is shown as SEQ ID NO:91. The encoded protein sequences of seven Cys mutant constructs in the light chain (SEQ ID NO:92 to SEQ ID NO:98) are shown in Table 13. Subsequent examples will show that these Cys mutants are efficiently conjugated with an ADC payload. Because all of these mutants are in the constant region of the human lambda light chain, these conjugation sites can readily be transferred to other antibodies with lambda light chains.

(207) TABLE-US-00011 TABLE 11 Sequence ID numbers of trastuzumab heavy chain Cys constructs used for cloning of the variable region of antibody 14090. Sequence ID NO: of trastuzumab HC Cys construct SEQ ID NO: 5 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 18 SEQ ID NO: 48 SEQ ID NO: 50

(208) TABLE-US-00012 TABLE 12 Nucleotide sequences of primers used in mutagenesis of seven Cys  mutant constructs in lambda light chain of human IgG1. SEQ Mutation Primer ID sites name Sequence NO. LC-A143C Seq-0017 CCGGGATGCGTGACAGTGGCCTGG 276 AAGGCAGATAGC Seq-0018 TGTCACGCATCCCGGGTAGAAGTCA 277 CTTATGAGACA LC-T145C Seq-0019 GCCGTGTGTGTGGCCTGGAAGGCA 278 GATAGCAGCCCC Seq-0020 GGCCACACACACGGCTCCCGGGTA 279 GAAGTCACTTAT LC-A147C Seq-0021 ACAGTGTGTTGGAAGGCAGATAGC 280 AGCCCCGTCAAG Seq-0022 CTTCCAACACACTGTCACGGCTCCC 281 GGGTAGAAGTC LC-K156C Seq-0023 CCCGTCTGTGCGGGAGTGGAGACC 282 ACCACACCCTCC Seq-0024 TCCCGCACAGACGGGGCTGCTATCT 283 GCCTTCCAGGC LC-V159C Seq-0025 GCGGGATGTGAGACCACCACACCC 284 TCCAAACAAAGC Seq-0026 GGTCTCACATCCCGCCTTGACGGGG 285 CTGCTATCTGC LC-T163C Seq-0027 ACCACCTGTCCCTCCAAACAAAGCA 286 ACAACAAGTAC Seq-0028 GGAGGGACAGGTGGTCTCCACTCC 287 CGCCTTGACGGG LC-5168C Seq-0029 AAACAATGCAACAACAAGTACGCG 288 GCCAGCAGCTAT Seq-0030 GTTGTTGCATTGTTTGGAGGGTGTG 289 GTGGTCTCCAC

(209) TABLE-US-00013 TABLE 13 Amino acid sequence of the constant region of Cys mutant constructs  in antibody 14090 lambda light chain. SEQ ID NO: 91 is the sequence  for the constant region of wild-type human lambda light chain SEQ ID  NO: 91 to SEQ ID NO: 98 indicate the sequences of the 7 Cys mutants  in the constant region of human lambda light chain of antibody 14090. SEQ ID NO: 91 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS SEQ ID NO: 92 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGCVTVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS SEQ ID NO: 93 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVCVAWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS SEQ ID NO: 94 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVCWKADSSPVKAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS SEQ ID NO: 95 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVCAG VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS SEQ ID NO: 96 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG CETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS SEQ ID NO: 97 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTCPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS SEQ ID NO: 98 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG VETTTPSKQCNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS

Example 5. Expression and Purification of Cys Mutant Antibodies in 293 Freestyle™ Cells

(210) Cys mutants of the trastuzumab antibody were expressed in 293 Freestyle™ cells by co-transfecting heavy chain and light chain plasmids using transient transfection method as described previously (Meissner, et al., Biotechnol Bioeng. 75:197-203 (2001)). The DNA plasmids used in co-transfection were prepared using Qiagen plasmid preparation kit according to manufacturer's protocol. 293 Freestyle™ cells were cultured in suspension in Freestyle™ expression media (Invitrogen) at 37° C. under 5% CO.sub.2. On the day before transfection, cells were split to 0.7×10.sup.6 cells/ml into fresh media. On the day of transfection, the cell density typically reached 1.5×10.sup.6 cells/ml. The cells were transfected with a mixture of heavy chain and light chain plasmids at the ratio of 1:1 using PEI method (Meissner et al., 2001). The transfected cells were further cultured for five days. The media from the culture was harvested by centrifugation of the culture at 2000×g for 20 min and filtered through 0.2 micrometer filters. The expressed antibodies were purified from the filtered media using Protein A-Sepharose™ (GE Healthcare Life Sciences). Antibody IgGs were eluted from the Protein A-Sepharose™ column by the elution buffer (pH 3.0) and immediately neutralized with 1 M Tris-HCl (pH 8.0) followed by a buffer exchange to PBS.

(211) Expression levels of 88 Cys trastuzumab mutant antibodies in transiently transfected 293 Freestyle™ are similar to that of wild-type trastuzumab, with an average yield at 18.6 mg/L+/−9.5 mg/L (Table 14), suggesting that single point mutations in the selected sites did not significantly alter retention of the expressed antibody by the cells' secretion machinery. Analysis of the purified trastuzumab Cys mutant antibodies using non-reducing SDS PAGE indicates that the Cys mutant antibodies did not form oligomers disulfide-linked by the engineered cysteines (FIG. 6). Size exclusion chromatography (FIG. 7) further supported the conclusion that all Cys mutant trastuzumab antibodies are monomeric. HPLC reverse phase analysis of the mutant antibodies also suggests that majority of the Cys mutant antibodies are indistinguishable from wild-type trastuzumab in terms of retention time and homogeneity (FIG. 8). Analysis of non-reduced deglycosylated full length trastuzumab LC-R108C by mass spectrometry (intact LC-MS) revealed that the majority of the antibody was modified by two cysteines (FIG. 9 and Table 15). These observations are consistent with a previous publication indicating that the thiol group of the engineered cysteine in the trastuzumab Cys mutant antibodies is modified by cysteine when expressed in 293 Freestyle™ cells, and that the modification needs to be removed by reducing reagents before conjugation with any thiol reactive reagents (Chen, et al., mAbs 1:6, 563-571, 2009).

(212) The Cys mutants of antibody 14090 were also expressed in in 293 Freestyle™ cells by co-transfecting HC and LC plasmids using PEI method as described (Meissner et al., 2001). The expression levels of the Cys mutants of antibody 14090 are similar to that of wild-type antibody 14090 (Table 16).

(213) TABLE-US-00014 TABLE 14 Yield of trastuzumab Cys mutant antibodies transiently expressed in 293 Freestyle ™ cells. Yields were measured by UV absorbance at 280 nm after Protein A purification. trastuzumab Cys mutant Purified Ab (mg/L) HC-S117C 46.9 HC-S119C 22.5 HC-K121C 22.1 HC-S124C 17.8 HC-S132C 30.9 HC-S134C 18.6 HC-S136C 21.2 HC-T139C 25.9 HC-E152C 13.0 HC-P153C 10.8 HC-T155C 18.4 HC-S157C 16.9 HC-T164C 20.2 HC-S165C 20.6 HC-T169C 8.2 HC-P171C 24.6 HC-L174C 15.2 HC-S176C 13.4 HC-S177C 30.0 HC-P189C 11.7 HC-K205C 13.3 HC-S207C 2.5 HC-D212C 26.5 HC-K246C 12.0 HC-E258C 18.7 HC-E269C 6.3 HC-K273C 20.7 HC-N286C 15.0 HC-K288C 20.9 HC-K290C 20.0 HC-R292C 21.0 HC-E293C 31.2 HC-E294C 37.5 HC-K320C 23.6 HC-K322C 35.1 HC-K326C 28.0 HC-A330C 27.1 HC-E333C 10.3 HC-K334C 14.0 HC-T335C 7.0 HC-S337C 6.9 HC-R344C 32.6 HC-R355C 30.1 HC-K360C 32.0 HC-Q362C 20.7 HC-S375C 33.3 HC-E382C 35.3 HC-N389C 28.7 HC-N390C 34.5 HC-K392C 28.2 HC-T393C 6.6 HC-L398C 5.1 HC-S400C 4.1 HC-D413C 27.6 HC-S415C 10.6 HC-V422C 5.0 LC-K107C 11.0 LC-R108C 27.0 LC-T109C 13.1 LC-A112C 10.5 LC-S114C 21.2 LC-D122C 25.5 LC-E123C 20.1 LC-T129C 7.1 LC-R142C 14.6 LC-E143C 10.0 LC-K145C 13.0 LC-N152C 12.0 LC-L154C 13.1 LC-S156C 12.0 LC-S159C 26.6 LC-E161C 20.0 LC-E165C 5.0 LC-S168C 12.0 LC-K169C 4.0 LC-D170C 5.0 LC-S182C 8.8 LC-K183C 12.6 LC-K188C 12.0 LC-K190C 5.2 LC-V191C 29.9 LC-T197C 19.0 LC-Q199C 16.8 LC-S203C 26.2 LC-T206C 27.8

(214) TABLE-US-00015 TABLE 15 Theoretical and observed mass for trastuzumab LC-R108C antibody after purification from 293 Freestyle ™ cells. Theoretical Observed Antibody species mass (Da) mass (Da) LC-R108C 145063 145071 LC-R108C-Cys +1 Cys 145181 145189 adduct LC-R108C-Cys +2 Cys 145299 145311 double adduct

(215) TABLE-US-00016 TABLE 16 Yield of antibody 14090 Cys mutants transiently expressed in 293 Freestyle ™ cells. Antibody 14090 Cys mutant Ab yield (mg/L) HC-S124C 4.72 HC-S136C 3.64 HC-T139C 4.59 HC-E152C 2.93 HC-L174C 5.26 HC-E258C 5.86 HC-K360C 4.86 LC-A143C 4.63 LC-T145C 6.98 LC-A147C 8.37 LC-K156C 5.74 LC-V159C 9.67 LC-T163C 9.98 LC-S168C 5.61

Example 6. Reduction, Re-Oxidation and Conjugation of Cys Mutant Antibodies with MC-MMAF

(216) Because engineered Cys in antibodies expressed in mammalian cells are modified by adducts (disulfides) such as glutathione (GSH) and/or Cysteine during their biosynthesis (Chen et al. 2009), the modified Cys in the product as initially expressed is unreactive to thiol reactive reagents such as maleimido or bromo- or iodo-acetamide groups. To conjugate the engineered cysteine after expression, the glutathione or cysteine adducts need to be removed by reducing these disulfides, which generally entails reducing all of the disulfides in the expressed protein. This can be accomplished by first exposing the antibody to a reducing agent such as dithiothreitol (DTT) followed by a procedure that allows for the re-oxidation of all native disulfide bonds of the antibody to restore and/or stabilize the functional antibody structure. Accordingly, in order to reduce all native disulfide bonds and the disulfide bound between the cysteine or GSH adducts of the engineered cysteine residue, freshly prepared DTT was added to previously purified Cys mutants of trastuzumab and antibody 14090, to a final concentration of 20 mM. After the antibody incubation with DTT at 37° C. for 1 hour, the mixtures were dialyzed at 4° C. against PBS for three days with daily buffer exchange to remove DTT and re-oxidize the native disulfide bonds. An alternative method is to remove the reducing reagents through a desalting column, Sephadex G-25. Once the protein is fully reduced, 1 mM oxidized ascorbate (dehydro-ascorbic acid) is added to the desalted samples and the re-oxidation incubations are carried out for 20 hours. Both methods have produced similar results. However, attempts to follow the re-oxidation protocols previously described in the literature using CuSO.sub.4 resulted in protein precipitation. All examples herein use the dialysis protocol described above. Reoxidation restores intra-chain disulfides, while dialysis allows cysteines and glutathiones connected to the newly-introduced cysteine(s) to dialyze away.

(217) After re-oxidation, the antibodies are ready for conjugation. Maleimide-MMAF (MC-MMAF, 10 equivalents relative to the antibody, FIG. 10) was added to re-oxidized antibodies in PBS buffer (pH7.2). The incubations were carried out from 1 hour to 24 hours. The conjugation process was monitored by reverse-phase HPLC, which is able to separate conjugated antibodies from non-conjugated ones. The conjugation reaction mixtures were analyzed on a PRLP-S 4000A column (50 mm×2.1 mm, Agilent) heated to 80° C. and elution of the column was carried out by a linear gradient of 30-60% acetonitrile in water containing 0.1% TFA at a flow rate of 1.5 ml/min. The elution of proteins from the column was monitored at 280 nm, 254 nm and 215 nm. The reverse-phase HPLC trace of a typical conjugation mixture is shown in FIG. 11.

(218) When the conjugation mixtures were analyzed by reverse-phase HPLC, many Cys sites generated homogenous conjugation products, as suggested by uniform, single peak elution profiles (FIG. 11), while some Cys sites generated heterogeneous conjugation products (FIG. 12). The procedures described above involve reduction and re-oxidation of native disulfide bonds as well as the reduction of bonds between the cysteine and GSH adducts of the engineered cysteine residues. During the re-oxidation process, the engineered cysteine residue may interfere with reforming of the proper native disulfide bonds through a process of disulfide shuffling. This may lead to the formation of mismatched disulfide bonds, either between the engineered cysteine and a native cysteine residue or between incorrectly matched native disulfide bonds. Such mismatched disulfide bonds may affect the retention of the antibody on the reverse-phase HPLC column. The mismatch processes may also result in unpaired cysteine residues other than the desired engineered cysteine. Attachment of the maleimide-MMAF to different positions on the antibody affects the retention time differently (see discussion of homogenously conjugated ADCs below). In addition, incomplete re-oxidation will leave the antibody with native cysteine residues that will react with maleimide-MMAF in addition to the desired conjugation with the engineered cysteine residue. Any process that hinders proper and complete formation of the native disulfide bonds will result in a complex HPLC profile (FIG. 11) upon conjugation to Maleimide-MMAF. The yield of the uniform ADC as measured by UV absorption of the unpurified reaction mixtures, varied depending on the Cys mutations (Table 17). Using the reduction/re-oxidation protocol and conjugation procedures described above 65 of the 88 Cys mutant trastuzumab antibodies resulted in homogeneous conjugation products and these sites are advantageous sites for Cys replacements to be made when making cysteine-engineered antibodies for conjugation.

(219) These 65 Cys-MMAF ADCs were analyzed in details in various assays: Differential scanning fluorimetry (DSF) was used to measure thermal stability. Analytical size exclusion chromatograph (AnSEC) was used to measure aggregation. In vitro antigen dependent cell killing potency was measured by cell viability assays and pharmacokinetics behavior was measured in mice. These assays and the respective results are described in more detail below.

(220) To evaluate the aggregation state of trastuzumab Cys-MMAF ADCs, the ADCs were analyzed in a size exclusion chromatography column (GE, Superdex200, 3.2/30) at a flow rate of 0.1 ml/min in PBS. All 65 Cys-MMAF ADCs were monomeric. The majority of the ADCs contain less than 10% oligomer (FIG. 13, Table 18), indicating that conjugation of MC-MMAF to trastuzumab Cys mutant constructs at the selected sites did not cause aggregation of the antibody.

(221) TABLE-US-00017 TABLE 17 Yield of MMAF ADCs generated with trastuzumab Cys mutant constructs. “Hetero” indicates a heterogeneous mixture of species shown in reverse phase HPLC with different retention times. trastuzumab Yield Cys Yield Cys-MMAF ADC (mg/L) constract (mg/L) HC-S117C 6.9 HC-R344C 33.4  HC-S119C 15.3 HC-R355C 24.3  HC-K121C 4.4 HC-K360C 26.5  HC-S124C 13.2 HC-Q362C hetero HC-S132C Hetero HC-S375C 34.3  HC-S134C Hetero HC-E382C 34.9  HC-S136C Hetero HC-N389C hetero HC-T139C 11.1 HC-N390C 33.1  HC-E152C 7.8 HC-K392C 20.8  HC-P153C 8.2 HC-T393C hetero HC-T155C 12.9 HC-L398C 3.4 HC-S157C 13.5 HC-S400C 1.7 HC-T164C 13.7 HC-D413C hetero HC-S165C Hetero HC-S415C hetero HC-T169C 4.7 HC-V422C 3.6 HC-P171C 14.7 LC-K107C 1.6 HC-L174C 9.1 LC-R108C 12.2  HC-S176C Hetero LC-T109C 8.4 HC-S177C Hetero LC-A112C hetero HC-P189C 7.7 LC-S114C 16.9 HC-S191C Hetero LC-D122C Hetero HC-T195C Hetero LC-E123C Hetero HC-T197C Hetero LC-T129C 4.0 HC-K205C 11.3 LC-R142C 11.3  HC-S207C 1.0 LC-E143C 4.0 HC-D212C Hetero LC-K145C 8.7 HC-K246C 9.0 LC-N152C 7.2 HC-E258C 10.1 LC-L154C 1.3 HC-E269C 5.6 LC-S156C 7.2 HC-K274C 15.3 LC-S159C 12.3  HC-N286C 12.9 LC-E161C 12.0  HC-K288C 14.4 LC-E165C 2.0 HC-K290C 8.0 LC-S168C 3.1 HC-R292C 10.3 LC-K169C 2.5 HC-E293C 15.0 LC-D170C 2.2 HC-E294C Hetero LC-S182C 7.9 HC-K320C 18.9 LC-K183C 3.8 HC-K322C 29.1 LC-K188C 7.2 HC-K326C 22.8 LC-K190C Hetero HC-A330C Hetero LC-V191C Hetero HC-E333C 7.4 LC-T197C 16.4  HC-K334C 11.2 LC-Q199C 10.3  HC-T335C 5.2 LC-S203C 13.5  HC-S337C 1.4 LC-T206C Hetero

(222) TABLE-US-00018 TABLE 18 Percentage of oligomer in trastuzumab Cys-MMAF ADC preparations as determined by analytical size-exclusion chromatography. trastuzumab Oligomer Conjugation Oligomer Cys-MMAF ADC (%) site (%) HC-S117C b.d. HC-R344C 9.5 HC-S119C 3.2 HC-R355C b.d. HC-K121C b.d. HC-K360C b.d. HC-S124C b.d. HC-S375C b.d. HC-T139C 4.8 HC-E382C b.d. HC-E152C b.d. HC-N390C b.d. HC-P153C b.d. HC-K392C b.d. HC-T155C b.d. HC-L398C b.d. HC-S157C b.d. HC-S400C 9.2 HC-T164C b.d. HC-V422C b.d. HC-T169C b.d. LC-K107C b.d. HC-P171C b.d. LC-R108C b.d. HC-L174C b.d. LC-T109C b.d. HC-P189C b.d. LC-S114C b.d. HC-K205C b.d. LC-T129C b.d. HC-S207C b.d. LC-R142C b.d. HC-K246C b.d. LC-E143C 13.1  HC-E258C b.d. LC-K145C b.d. HC-E269C b.d. LC-N152C b.d. HC-K274C 11.7  LC-L154C 7.3 HC-N286C 9.2 LC-S156C 6.1 HC-K288C b.d. LC-S159C 2.8 HC-K290C b.d. LC-E161C b.d. HC-R292C b.d. LC-E165C b.d. HC-E293C b.d. LC-S168C b.d. HC-K320C b.d. LC-K169C b.d. HC-K322C b.d. LC-D170C b.d. HC-K326C b.d. LC-S182C 6.9 HC-E333C b.d. LC-K183C b.d. HC-K334C b.d. LC-K188C b.d. HC-T335C b.d. LC-T197C b.d. HC-S337C b.d. LC-Q199C 6.3 LC-S203C b.d. b.d.: Below detection limit.

Example 7. In Vitro Thermal Stability Assay of Trastuzumab Cys-MMAF ADCs

(223) Conjugation of MMAF payload to trastuzumab may stabilize or destabilize the antibody, leading to changes in melting temperature of the antibody, which can be determined by differential scanning fluorimetry (DSF) that is based on temperature induced denaturation monitored by an environmentally sensitive dye, such as sypro orange. ADC samples were aliquoted in triplicate to 384-well plates into PBS (6.7 mM sodium phosphate pH7.2; 150 mM NaCl). In each well, 8 μl of 0.25 mg/ml antibody was mixed with 2 μl 25× sypro orange dye (Invitrogen). Plates were sealed and analyzed in a Roche LightCycler 480 system with a temperature ramp from 30 to 85° C. with 20 fluorescence scans recorded per degree C. Melting temperatures were determined from the first derivative of the fluorescence intensity vs. time curves.

(224) A typical thermal shift assay for wild-type trastuzumab revealed two melting transitions (Tm), Tm1 at 69.7° C. and Tm2 at 81.2° C., respectively (Table 19). When trastuzumab Cys-MMAF ADCs were subjected to protein thermal stability assays, it was evident that conjugation of MC-MMAF to the antibodies induced different Tm changes depending on the sites of conjugation (Table 19). When MC-MMAF was conjugated to majority of the Cys sites in either CH1 or CH3 domains, the resulting ADCs, for example HC-K356C-MMAF, showed a similar pattern as that of wild-type anti-Her with little changes in Tm1 and Tm2. However, when MC-MMAF was conjugated to Cys sites located in the CH2 domain, a decrease in Tm1 was observed for most sites while the Tm2 remained largely unchanged. The Tm1 decrease observed for most CH2 domain Cys-MMAF conjugates ranged from 5° C. to 26° C. The two ADCs with the largest decrease in Tm1 are HC-T335C-MMAF and HC-S337C-MMAF, with Tm1 at 42° C. and 45° C., respectively (FIG. 14). The results indicate that the location of MC-MMAF conjugation can have a significant impact on the stability of the ADCs.

(225) TABLE-US-00019 TABLE 19 Melting temperatures Tm1 and Tm2 of trastuzumab Cys-MMAF ADCs observed by differential scanning fluorimetry (DSF). trastuzumab HC Cys-MMAF ADC domain Tm1 [° C.] Tm2 [° C.] wild-type n.a. 69.71 81.18 antibody HC-S117C CH1 69.09 79.85 HC-S119C CH1 69.28 78.58 HC-K121C CH1 69.63 78.52 HC-S124C CH1 69.27 80.56 HC-T139C CH1 69.09 80.74 HC-E152C CH1 69.63 80.83 HC-P153C CH1 69.71 78.52 HC-T155C CH1 69.27 80.83 HC-S157C CH1 69.72 80.81 HC-T164C CH1 69.17 80.7  HC-T169C CH1 68.74 80.47 HC-P171C CH1 69.27 77.18 HC-L174C CH1 69.89 80.03 HC-P189C CH1 69.09 81.27 HC-K205C CH1 69.54 80.65 HC-S207C CH1 69.00 80.65 HC-K246C CH2 64.65 80.74 HC-E258C CH2 65.32 81.03 HC-E269C CH2 65.36 81.01 HC-K274C CH2 67.14 81.09 HC-N286C CH2 67.22 81.09 HC-K288C CH2 65.54 80.83 HC-K290C CH2 69.00 80.65 HC-R292C CH2 67.49 80.56 HC-E293C CH2 64.34 81.03 HC-K320C CH2 60.60 80.59 HC-K322C CH2 62.41 80.70 HC-K326C CH2 63.05 80.74 HC-E333C CH2 63.67 80.92 HC-K334C CH2 64.65 80.47 HC-T335C CH2 42.93 80.04 HC-S337C CH2 45.56 80.48 HC-R344C CH3 69.50 80.92 HC-R355C CH3 68.18 81.25 HC-K360C CH3 69.28 80.92 HC-S375C CH3 68.20 81.36 HC-E382C CH3 69.36 80.74 HC-N390C CH3 68.73 80.92 HC-K392C CH3 67.05 80.92 HC-L398C CH3 68.47 81.36 HC-S400C CH3 68.65 81.27 HC-V422C CH3 69.98 81.45 LC-K107C n.a. 69.45 80.29 LC-R108C n.a. 70.10 n.d..sup.1 LC-T109C n.a. 68.47 80.21 LC-T129C n.a. 68.47 80.12 LC-R142C n.a. 69.00 78.61 LC-E143C n.a. 69.83 80.59 LC-K145C n.a. 69.00 80.65 LC-N152C n.a. 67.49 81.09 LC-L154C n.a. 68.47 80.65 LC-S156C n.a. 68.83 80.47 LC-S159C n.a. 69.50 79.93 LC-E161C n.a. 68.65 80.12 LC-E165C n.a. 69.27 79.76 LC-S168C n.a. 69.54 79.67 LC-K169C n.a. 69.09 80.29 LC-D170C n.a. 68.83 80.12 LC-S182C n.a 69.18 80.29 LC-K183C n.a 69.09 80.47 LC-K188C n.a 68.74 80.65 LC-T197C n.a 69.63 80.74 LC-Q199C n.a 69.54 80.21 LC-S203C n.a 68.84 80.92 n.d. Not determined because a broad transition in Tm2 prevented accurate Tm determination., n.a. Not applicable

Example 8. Cell Proliferation Assays to Measure In Vitro Cell Killing Potency of Cys ADCs

(226) Cells that naturally express target antigens or cell lines engineered to express target antigens are frequently used to assay the activity and potency of ADCs. For evaluation of the cell killing potency of trastuzumab ADCs in vitro, two engineered cell lines, MDA-MB231 clone 16 and clone 40, and HCC1954 cells were employed (Clinchy B, Gazdar A, Rabinovsky R, Yefenof E, Gordon B, Vitetta E S. Breast Cancer Res Treat. (2000) 61:217-228). MDA-MB231 clone 16 cells stably express high copy numbers (˜5×10.sup.5 copies/cell) of recombinant human Her2 while clone 40 expresses low copy numbers (˜5×10.sup.3 copies/cell) of human Her2. HCC1954 cells endogenously express high level (˜5×10.sup.5 copies/cell) of human Her2 in the surface. For determination of the cell killing potency of antibody 14090 ADCs, CMK11-5 and Jurkat cells were used. While CMK11-5 cells express a high level of the antigen for antibody 14090 in the cell surface there is no detectable antigen expression in Jurkat cells. An antigen dependent cytotoxic effect should only kill cells that express sufficient antigen in the cell surface and not cells lacking the antigen. The cell proliferation assays were conducted with Cell-Titer-Glo™ (Promega) five days after cells were incubated with various concentrations of ADCs (Riss et al., (2004) Assay Drug Dev Technol. 2:51-62). In some studies, the cell based assays are high throughput and conducted in an automated system (Melnick et al., (2006) Proc Natl Acad Sci USA. 103:3153-3158).

(227) Trastuzumab Cys-MMAF ADCs specifically killed MDA-MB231 clone 16 and HCC1954 but not MDA-MB231 clone 40 cells (FIG. 15). IC.sub.50 of the trastuzumab Cys-MMAF ADCs in MDA-MB231 clone 16 cell assays ranges from 30 pM to 200 pM (Table 20, FIG. 16). Similarly, antibody 14090 Cys-MMAF ADC displayed antigen dependent cell killing in cell proliferation assays. The antibody 14090 Cys-MMAF ADCs killed antigen expressing CMK11-5 cells but not antigen negative Jurkat cells (FIG. 17). The IC.sub.50 of the antibody 14090-MMAF ADC in CMK11-5 proliferation assay is in the range of 400 pM to 1 nM (Table 21).

(228) TABLE-US-00020 TABLE 20 IC.sub.50 of trastuzumab Cys-MMAF ADCs in MDA-MB231 clone 16 Her2.sup.+ cell proliferation assay. trastuzumab Cys-MMAF ADC IC.sub.50 (μM) HC-S117C 1.55E−04 HC-S119C 1.18E−04 HC-K121C 1.06E−04 HC-S124C 9.78E−05 HC-T139C 1.48E−04 HC-E152C 7.62E−05 HC-P153C 9.27E−05 HC-T155C 1.33E−04 HC-S157C 1.47E−04 HC-T164C 1.34E−04 HC-T169C 1.98E−04 HC-P171C 1.33E−04 HC-L174C 1.19E−04 HC-P189C 1.82E−04 HC-K205C 1.02E−04 HC-S207C 2.16E−04 HC-K246C 9.54E−05 HC-E258C 9.40E−05 HC-E269C 8.98E−05 HC-K274C 9.99E−05 HC-N286C 9.94E−05 HC-K288C 7.47E−05 HC-K290C 3.55E−04 HC-R292C 9.69E−05 HC-E293C 1.10E−04 HC-K320C 9.79E−05 HC-K322C 1.16E−04 HC-K326C 1.73E−04 HC-E333C 1.28E−04 HC-K334C 1.43E−04 HC-T335C 8.69E−05 HC-S337C 7.79E−05 HC-R344C 2.75E−04 HC-R355C 1.21E−04 HC-K360C 1.92E−04 HC-S375C 1.42E−04 HC-E382C 2.53E−04 HC-N390C 1.58E−04 HC-K392C 1.43E−04 HC-L398C 1.08E−04 HC-S400C 1.43E−04 HC-V422C 1.72E−04 LC-K107C 2.59E−05 LC-R108C 2.96E−05 LC-T109C 8.12E−05 LC-S114C 3.37E−05 LC-T129C 2.73E−05 LC-R142C 2.64E−05 LC-E143C 3.76E−05 LC-K145C 3.87E−05 LC-N152C 2.14E−05 LC-L154C 3.52E−05 LC-S156C 4.28E−05 LC-S159C 4.34E−05 LC-E161C 3.62E−05 LC-E165C 4.68E−05 LC-S168C 2.50E−04 LC-K169C 7.74E−05 LC-D170C 1.64E−04 LC-S182C 9.07E−05 LC-K183C 8.39E−05 LC-K188C 9.71E−05 LC-T197C 1.07E−04 LC-Q199C 1.31E−04 LC-S203C 1.18E−04

(229) TABLE-US-00021 TABLE 21 IC.sub.50 of antibody 14090 Cys-MMAF ADCs in CMK11-5 cell proliferation assay Antibody 14090 Cys-MMAF ADC IC.sub.50 (μM) HC-S124C 9.26E−04 HC-T139C 1.22E−03 HC-E152C 4.60E−04 HC-L174C 6.02E−04 HC-K360C 8.56E−04 HC-S375C 4.38E−04 LC-A143C 7.09E−04 LC-A147C 1.14E−03 LC-V159C 5.41E−04 LC-T163C 6.38E−04 LC-S168C 1.06E−03

Example 9. Pharmacokinetic Study of Trastuzumab Cys-MMAF ADCs

(230) It has been demonstrated that a long serum half-life is critical for high in vivo efficacy of ADCs (Hamblett, et al., “Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate,” Clin Cancer Res., 10:7063-7070 (2004); Alley et al., Bioconjug Chem. 19:759-765 (2008)). Attaching an usually hydrophobic drug payload to an antibody could significantly affect the properties of an antibody, and this may lead to a fast clearance of the ADCs in vivo (Hamblett et al., 2004) and poor in vivo efficacy. To evaluate the effects of different conjugation site on clearance of MMAF ADCs in vivo, pharmacokinetic studies in non-tumor bearing mice were carried out with 65 trastuzumab Cys-MMAF ADCs. To detect MMAF containing ADCs in murine plasma, an anti-MMAF antibody was generated. ELISA assays for the detection of ADCs were developed using the extracellular domain of human HER2 to capture trastuzumab IgG molecules from the plasma and an anti-human IgG (anti-hIgG) antibody and the anti-MMAF antibody for signal generation in two separate assays. The two ELISA assays measure the serum concentration of the trastuzumab antibody and the “intact” ADC respectively as discussed in more detail below.

(231) Three mice per group were administered with a single dose of a trastuzumab Cys-MMAF ADC at 1 mg/kg. Ten plasma samples were collected over the course of two weeks and assayed by ELISA using the extracellular domain of human HER2 to capture all trastuzumab IgG molecules including trastuzumab Cys-MMAF ADCs and trastuzumab lacking MMAF. An anti-MMAF and an anti-hIgG antibody were then used for detection in two separate assays. The anti-MMAF antibody ELISA measures the concentration of trastuzumab MMAF conjugates only and the anti-hIgG ELISA quantitates both trastuzumab Cys-MMAF conjugates and trastuzumab antibodies that lack MMAF. Standard curves were generated for each ADC separately using the same material as injected into the mice. The assays with anti-MMAF and anti-hIgG should therefore yield identical concentration readouts if no changes to the drug loading of the trastuzumab Cys-MMAF ADC occur after injection into mice. For trastuzumab Cys-MMAF ADCs that lost some of the MMAF payload, the ELISA assay with the anti-MMAF antibody will measure a lower concentration than the anti-hIgG ELISA. A comparison of the two concentration readouts therefore allows to measure drug-release from trastuzumab Cys-MMAF ADCs during in vivo incubation in the mouse.

(232) As measured by anti-hIgG ELISA, 63 out of 65 ADCs displayed a pharmacokinetic profile similar to unconjugated wild-type trastuzumab antibody (FIGS. 18, 19, 20), indicating that MC-MMAF payload conjugation to these sites did not significantly affect the antibody's clearance. The two exceptions are HC-T335C and HC-S337C. Conjugation of MC-MMAF to these two sites results in rapid clearance of the ADCs as measured by the anti-MMAF and the anti-hIgG ELISA (FIG. 21). The protein thermal shift assay revealed that the Tm1 for trastuzumab HC-T335C-MMAF and trastuzumab HC-S337C-MMAF decreased from 69° C. in wild-type trastuzumab antibody to 42° C. and 45° C., respectively (FIG. 14). Conjugation of MC-MMAF to the two sites dramatically reduces the thermal stability of the ADC (by 27° C. and 24° C., respectively). For the 63 ADCs that show a pharmacokinetic profile similar to unconjugated antibody, Tm1 changes were smaller than 8° C. suggesting that fast clearance may possibly correlate with low thermal stability of the ADC.

(233) To determine the chemical stability of linkage between the MMAF payload and the antibody at the various Cys sites, the concentrations of trastuzumab Cys-MMAF ADC as measured by the anti-MMAF ELISA and of all trastuzumab molecules as measured by the anti-hIgG ELISA were compared to each other for each sample. Many trastuzumab Cys-MMAF ADCs, within the error of the measurements, displayed a good overlap between the two concentrations over the course of two weeks, suggesting that the bond between MC-MMAF and the cysteine introduced at these sites was stable during circulation in mice over this period (FIGS. 18, 19). In contrast, some trastuzumab Cys-MMAF ADCs displayed a significant drug loss as indicated by the higher anti-hIgG readout than the anti-MMAF readout (FIG. 20). For some trastuzumab Cys-MMAF ADCs, the concentration of ADC was about 50% of that of hIgG. These results suggest that there are significant differences in stability of a thiol-maleimide bond of drug payloads conjugated to different sites as has been suggested previously (Shen et al. Nat. Biotechnol. 2012, 30 (2):184-9). Sites having good stability are preferred sites for use of preparing ADCs as described herein.

(234) In pharmacokinetic studies, the area-under-the-plasma-concentration-versus-time-curve (AUC) is an important parameter in estimating total clearance and bioavailability of an administered drug. In our pharmacokinetic studies, for each trastuzumab Cys-MMAF ADC two AUC values, AUC-MMAF and AUC-hIgG, were calculated separately from measurements with the anti-MMAF and the anti-hIgG ELISA. The ratios of AUC-MMAF to AUC-hIgG for all trastuzumab Cys-MMAF ADCs varied from 0.4 to 1.2 (Table 20). FIGS. 18, 19 and 20 include PK curves for ADCs over the full range of observed AUC-MMAF/AUC-hIgG ratios and illustrate the variability and uncertainty of the measurements. Ratios of AUC-MMAF to AUC-hIgG>1 (Table 20) suggest uncertainties of >25% since the ratio should remain near 1 if no drug loss occurs. As shown in Table 20, out of 63 trastuzumab Cys-MMAF ADCs with measurable AUCs from both ELISAs, 40 ADCs show a ratio of AUC-MMAF/AUC-hIgG>0.7, indicating that within the accuracy of the measurement, little MMAF drug loss was observed at these after administration in mice. However, 23 ADCs displayed a ratio of AUC-MMAF/AUC-hIgG<0.7, suggesting that the amount of MMAF payload conjugate at these 23 sites decreased significant during the in vivo incubation in the mouse.

(235) Differences in stability of the maleimide linkage at different conjugation sites has previously been reported for Cys engineered ADCs (see Shen et al., (2012) Nat Biotechnol. 22; 30(2):184-9 for discussion and references). For the preferred sites that exhibit enhanced serum stability, the antibody environment likely catalyzes the hydrolysis of the succinimide ring formed by the reaction of maleimide with cysteine. The hydrolyzed form cannot revert back and cannot release the maleimide drug. As such, the ability of the antibody environment to catalyze the ring hydrolysis cannot be predicted and is an unexpected property of certain engineered Cys sites. Sites in Table 22 having an AUC(MMAF)/AUC(hIgG) ratio greater than 0.7 are therefore particularly suitable sites for cysteine substitution based on this criterion, and sites having a ratio of about 0.9 or above are especially preferred cysteine substitution sites for purposes of the invention when applying. These include heavy chain sites 322, 334, 121, 288, 171, 139, 360, 117, 392, 375, 292, 333, 174, 258, 337, 422, 320, 390, and 335; and light chain sites 107, 203, 108 and 114.

(236) TABLE-US-00022 TABLE 22 AUC-MMAF and AUC-hIgG of trastuzumab Cys-MMAF ADCs in mice trastuzumab AUC-MMAF AUC-hIgG AUC(MMAF)/ Cys-MMAF ADC (hrs*μg/ml) (hrs*μg/ml) AUC(hIgG) HC-K246C-MMAF 1515 3587 0.4 HC-K205C-MMAF 2109 4893 0.4 LC-S168C-MMAF 1688 3619 0.5 LC-E143C-MMAF 1589 3254 0.5 HC-E382C-MMAF 1364 2541 0.5 HC-T155C-MMAF 2930 5308 0.6 HC-S119C-MMAF 2230 4045 0.6 LC-T129C-MMAF 2375 4332 0.6 LC-T109C-MMAF 1588 2716 0.6 LC-K169C-MMAF 2858 4855 0.6 HC-S400C-MMAF 2363 3922 0.6 HC-R355C-MMAF 2344 3777 0.6 HC-R344C-MMAF 1994 3215 0.6 LC-Q199C-MMAF 2042 3261 0.6 LC-S182C-MMAF 2434 3722 0.7 HC-P153C-MMAF 2201 3402 0.7 HC-N286C-MMAF 2286 3535 0.7 HC-T169C-MMAF 2113 3190 0.7 LC-K183C-MMAF 2014 3053 0.7 LC-T197C-MMAF 2126 3177 0.7 LC-K145C-MMAF 2339 3454 0.7 HC-L398C-MMAF 2063 2979 0.7 HC-P189C-MMAF 2042 2968 0.7 HC-S157C-MMAF 2625 3640 0.7 HC-E269C-MMAF 2373 3293 0.7 LC-S159C-MMAF 2063 2809 0.7 LC-E161C-MMAF 1974 2632 0.8 LC-E165C-MMAF 2481 3244 0.8 HC-T164C-MMAF 2514 3290 0.8 LC-R142C-MMAF 2903 3786 0.8 LC-S156C-MMAF 2217 2847 0.8 HC-S207C-MMAF 2378 3001 0.8 LC-N152C-MMAF 2303 2862 0.8 HC-E152C-MMAF 3403 4202 0.8 LC-L154C-MMAF 1959 2387 0.8 LC-K188C-MMAF 2230 2680 0.8 HC-K326C-MMAF 2621 3157 0.8 LC-D170C-MMAF 2048 2420 0.9 HC-K290C-MMAF 2668 3090 0.9 HC-E293C-MMAF 2167 2523 0.9 HC-S124C-MMAF 2107 2463 0.9 HC-K274C-MMAF 3080 3554 0.9 HC-K322C-MMAF 3108 3437 0.9 HC-K334C-MMAF 4527 5048 0.9 HC-K121C-MMAF 2647 2952 0.9 HC-K288C-MMAF 2681 2902 0.9 HC-P171C-MMAF 2312 2481 0.9 LC-K107C-MMAF 2621 2817 0.9 HC-T139C-MMAF 2951 3186 0.9 HC-K360C-MMAF 3791 4014 0.9 HC-S117C-MMAF 2661 2828 0.9 LC-S203C-MMAF 2730 2919 0.9 HC-K392C-MMAF 3148 3302 1.0 HC-S375C-MMAF 2593 2644 1.0 HC-R292C-MMAF 2816 2806 1.0 HC-E333C-MMAF 3850 3796 1.0 HC-L174C-MMAF 2604 2541 1.0 HC-E258C-MMAF 3941 3732 1.1 HC-S337C-MMAF 34.38 32.14 1.1 HC-V422C-MMAF 2662 2424 1.1 HC-K320C-MMAF 3181 2776 1.2 HC-N390C-MMAF 3627 3105 1.2 LC-R108C-MMAF 3711 2992 1.2 LC-S114C-MMAF n.a. 2567 n.a. HC-T335C-MMAF 6.71 n.a. n.a. n.a: not applicable.

Example 10: Combination of Cys Sites to Produce Antibody Drug Conjugates with Drug-to-Antibody-Ratios Greater than 2

(237) Antibody conjugates produced through conjugation to lysine residues or partially reduced native disulfide bonds often feature drug-to-antibody-ratios (DAR) of between 3 and 4. Cys engineered antibodies more typically feature a DAR of 2. For certain indications, it may be desirable to produce ADCs with higher DAR which can in principle be achieved by introducing multiple Cys mutations in the antibody. As the number of Cys mutation increases, the likelihood that such mutations interfere with the required re-oxidation process during ADC preparation and hence result in heterogeneous products also increases. In this study, a large number of single site heavy and light chain Cys mutants with good re-oxidation behavior were identified.

(238) To demonstrate that several conjugation sites can be combined for the production of ADCs with DAR greater than two, several preferred single site Cys constructs of light and heavy chain of trastuzumab and antibody 14090 (Table 23) were coexpressed in 293 Freestyle™ cells as described in Example 5. Purified antibodies which all contain one Cys mutation on the heavy chain and one Cys mutation on the light chain were reduced, re-oxidized and conjugated with MC-MMAF as described in Example 6. Reverse phase high-pressure liquid chromatography demonstrated a single defined elution peak suggesting efficient re-oxidation of the native disulfide bonds. Reverse phase high-pressure liquid chromatography after MC-MMAF conjugation also showed predominantly a single elution peak for the DAR 4 ADC species. The DAR of all ADCs in Table 23 was confirmed to be 4 by mass spectrometry. Production yields varied from 16 to 24 mg/L transient cell culture. The ADCs were predominantly monomeric as determined by analytical size exclusion chromatography; only for 2 of the 8 antibodies could small amounts of aggregates be detected (Table 23). Trastuzumab and 14090 ADCs exhibited antigen-dependent cell killing in MDA-MB231 clone 16 and CMK1105 cell proliferation assays, respectively (Table 23).

(239) TABLE-US-00023 TABLE 23 Properties of Cys engineered MMAF ADCs with DAR of 4. IC.sub.50 MDA- IC.sub.50 MB231-16 CMK11-5 Cys-MMAF ADC LC SEQ HC SEQ Yield AnSEC AnSEC cells cells (DAR = 4) ID NO ID NO (mg/L) % Monomer % Multimer (μM) (μM) trastuzumab 75 29 17.3 100 Not 4.91e−4 No potency LC-S159C- detected HC-E258C trastuzumab 75 50 17.8 100 Not 2.44e−4 No potency LC-S159C- detected HC-S375C trastuzumab 77 29 16.5 100 Not 3.24e−4 No potency LC-E165C- detected HC-E258C trastuzumab 77 50 16.9 100 Not 2.15e−4 No potency LC-E165C- detected HC-S375C Antibody 92 48 16.1 94.8 5.2 No potency 4.92e−4 14090 LC-A143C- HC-K360C Antibody 92 50 21.8 100 Not No potency 4.76e−4 14090 detected LC-A143C- HC-S375C Antibody 96 48 24.0 100 Not No potency 4.55e−4 14090 detected LC-V159C- HC-K360C Antibody 96 50 21.7 97.1 2.9 No potency 3.99e−4 14090 LC-V159C- HC-S375C n.d.: not detectable, no potency: no sign of cell killing at highest concentration evaluated (66 nM) SEQ ID NOs only specify constant regions of antibody sequences.

Example 11. Selection of Cys Sites Based on ADC Hydrophobicity

(240) To further optimize the selection Cys mutants and mutant combinations for the preparation of ADCs with DAR 2, 4, 6 and 8, the properties of MMAF ADCs prepared with single site trastuzumab Cys and Pcl mutants (Preparation of Pcl ADCs is described in patent application 55573) were analyzed, and accessibility and solvent exposure of conjugation sites was inspected in the crystal structures of IgG.

(241) One of the most informative data was the observation that the hydrophobicity of trastuzumab Pcl-MMAF ADCs varied greatly when the payload was attached different sites (FIG. 23). The hydrophobicity of these ADCs was measured by hydrophobic interaction chromatography (HIC) using a TSKgel Phenyl-5PW column (Tosoh Bioscience, TSKgel Phenyl-5PW, 13 □m, 21×150 mm, stainless steel, Cat #07656; running buffer A: 1.5 M ammonium sulfate in 20 mM NaPi (pH7.4); buffer B: 20% isopropanol in 20 mM NaPi (pH7.4); flow rate 5 ml/min; linear gradient from 20% to 80% buffer B over 90 mins; monitored by UV absorbance at 280 nm). Surprisingly, it was observed that retention times of the DAR 2 species varied greatly among ADCs although the only difference is the site of ABA-MMAF attachment (FIG. 23). HIC separates molecules on the basis of the hydrophobicity. All DAR 2 ADCs have a HIC retention time larger than that of unconjugated antibody (WT=45 min, FIG. 23) which is to be expected when a hydrophobic drug molecule such as ABA-MMAF is attached to an antibody. However, attaching the payload at different sites increases the hydrophobicity of the ADC to various extends.

(242) The surprisingly large differences in retention times can be rationalized from the inspection of location of the attachment sites on the structure of an antibody (FIG. 24): The retention times are higher if the drug payload is attached at an exposed site on the outside of an antibody, for example at HC-K288Pcl, HC-N286Pcl, HC-V422Pcl, HC-L398Pcl and HC-S415Pcl where retention time between 87 and 94 min were measured for the respective ADCs (FIG. 23). Conversely, if the payload is attached at an interior site such as the cavity formed between variable and CH1 domains (for examples, HC-P153Pcl, HC-E152Pcl, HC-L174Pcl, HC-P171Pcl, LC-R142Pcl, LC-E161Pcl, LC-E165Pcl, LC-S159Pcl) or the large opening between CH2 and CH3 domains of the antibody (for examples, HC-K246C, HC-S375Pcl, HC-T393Pcl, HC-K334Pcl), the HIC retention time increased to only 47 to 57 mins because the payload is partially sequestered from interacting with solvent and the HIC column. For other sites, for example, the relatively exposed sites, LC-K107Pcl and HC-K360Pcl, intermediate retention time of 70 and 83 min were measured.

(243) Reducing hydrophobicity of a protein drug is generally considered beneficial because it may reduce aggregation and clearance from circulation. We propose that the HIC data presented in FIG. 23 enables selection of preferred payload attachment sites. Conjugating drug payloads at sites where they are sequestered from solvent interactions and attachment minimally increases the hydrophobicity of the antibody upon drug attachment should be beneficial independent of the conjugation chemistry and payload class. Carefully selecting attachment sites that result in minimal changes in hydrophobicity may be particularly beneficial when 4, 6 or 8 drugs are attached per antibody, or when particularly hydrophobic payloads are used.

(244) Cys Sites Selected for ADCs with Low Hydrophobicity:

(245) To minimize hydrophobicity of ADCs, sites were chosen that would point toward the interior of the various protein domains of the antibody. Selection was based on analysis of the antibody structure and behavior of the existing ADCs with DAR=2 where applicable (behavior=retention time on HIC and/or delayed retention time on AnSEC with conjugates that interact with SEC resins). Of the Cys sites identified in Table 1 and Table 2, sites listed in Table 24 fulfill the above criteria.

(246) All ADCs were analyzed by hydrophobic interaction chromatography (HIC). Trastuzumab MMAF ADCs conjugated at the exposed sites HC-K360C, LC-K107C, HC-E258C and HC-R292C were used for comparison purposes. The results are shown in Table 25. The trastuzumab Cys-MMAF ADCs and unconjugated, wild-type antibody were analyzed on a TSKgel Butyl-NPR column as described below. For comparison, HIC data previously obtained for Pcl-MMAF ADCs on a TSKgel Phenyl-5PW (FIG. 23) are also listed. Despite different instrumentation and protocols, and although some variability is expected due to the different geometry and structures of the two linker, the ratio of retention times for the ADC conjugated at the same position but through different conjugation methods remains nearly constant. The HIC data suggests that retention times are indeed a measure of how well a payload is sequestered in the interior of the antibody independent of attachment chemistry and linker structure. As expected the relative ranking of the different attachment sites remains largely identical for Pcl-MMAF and Cys-MMAF ADCs.

(247) Attachment to site selected in Table 24, HC-E333C, HC-K392C, and HC-K326C results in MMAF ADCs that have HIC retention times that are similar to the exposed site ADCs LC-K107C-MMAF, HC-E258C-MMAF, HC-R292C-MMAF and HC-K360C-MMAF (Table 28). Attachment to the HC-E152C, LC-E165C, HC-P171C, LC-R142C, LC-E161C, HC-L174C and HC-S124C sites increases the retention time of the resulting ADC by less than 15% compared to the unconjugated, wild-type antibody. These sites are all located on in the CH1 domain or on the light chain (LC) and HIC retention time data suggests them as preferred attachment sites. Of the CH3 domain sites, HC-K334C and HC-S375C exhibit to lowest increase in hydrophobicity upon conjugation making them preferred attachment sites.

(248) TABLE-US-00024 TABLE 24 Cys mutant sites Cys mutant site Site (EU No.) LC-R142C 142 LC-S159C 159 LC-E161C 161 LC-E165C 165 HC-S124C 124 HC-E152C 152 HC-P171C 171 HC-L174C 174 HC-K326C 326 HC-E333C 333 HC-K334C 334 HC-S375C 375 HC-K392C 392

(249) TABLE-US-00025 TABLE 25 Hydrophobic interaction chromatography (HIC) retention time of the DAR 2 species of trastuzumab MMAF ADCs. Comparing Cys and Pcl conjugation chemistry, the two sets agree well: Sites that hide the drug conjugated by one chemistry also tend to hide the drug when conjugated by the other chemistry. Some variability is expected due to the different geometry of the two linker systems. DAR2 DAR2 HIC re- HIC re- tention.sup.a tention.sup.b Trastuzumab ADC (min) Trastuzumab ADC (min) Ratio WT 19.5 WT 45 0.43 HC-E152C-MMAF 20.4 HC-E152Pcl-MMAF 50 0.41 LC-E165C-MMAF 20.8 LC-E165Pcl-MMAF 55 0.38 HC-P171C-MMAF 21.0 HC-P171Pcl-MMAF 51 0.41 HC-K334C-MMAF 21.5 HC-K334Pcl-MMAF 56 0.38 HC-S375C-MMAF 21.6 HC-S375Pcl-MMAF 52 0.42 LC-R142C-MMAF 21.7 LC-R142Pcl-MMAF 51 0.42 LC-E161C-MMAF 22.0 LC-E161Pcl-MMAF 55 0.40 HC-L174C-MMAF 22.0 HC-L174Pcl-MMAF 50 0.44 HC-S124C-MMAF 22.4 HC-S124Pcl-MMAF 59 0.38 HC-E333C-MMAF 23.1 HC-E333Pcl-MMAF 63 0.37 HC-K392C-MMAF 23.1 HC-K392Pcl-MMAF 60 0.38 HC-R292C-MMAF 23.8 HC-R292Pcl-MMAF 69 0.35 HC-K326C-MMAF 24.5 HC-K326Pcl-MMAF 72 0.34 LC-K107C-MMAF 24.8 LC-K107Pcl-MMAF 70 0.35 HC-E258C-MMAF 24.9 HC-E258Pcl-MMAF 69 0.36 HC-K360C-MMAF 26.8 HC-K360Pcl-MMAF 83 0.32 .sup.aAnalytical HIC: Tosoh Bioscience (King of Prussia, PA, USA) TSKgel Butyl-NPR column (100 mm × 4.6 mm, 2.5 μm), running buffer A: 50 mM sodium phosphate, 1.5M ammonium sulfate, pH 7.0; buffer B: 50 mM sodium phosphate, pH 7.0; gradient consisted of 5 min holding at 100% A, followed by a linear gradient of 20 to 100% B over 40 min; monitored by UV absorption at 280 nm. .sup.bSemi-prep HIC: Tosoh Bioscience (King of Prussia, PA, USA), TSKgel Phenyl-5PW, 13 μm, 21 × 150 mm; running buffer A: 1.5M ammonium sulfate in 20 mM NaPi (pH 7.4); buffer B: 20% isopropanol in 20 mM NaPi (pH 7.4); flow rate 5 ml/min; linear gradient from 20% to 80% buffer B over 90 mins; monitored by UV absorption at 280 nm.
Analytical HIC Protocol in Detail:

(250) Analytical HIC data for trastuzumab Cys-MMAF ADCs were collected using a Tosoh Bioscience (King of Prussia, Pa., USA) TSKgel Butyl-NPR column (100 mm×4.6 mm, 2.5 μm) installed on a Dionex UltiMate 3000 HPLC (Sunnyvale, Calif., USA). The method consisted of a binary gradient of buffer A (50 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0) and buffer B (50 mM sodium phosphate, pH 7.0). Samples were prepared by diluting approximately 50 μg of antibody (PBS) with an equal volume of 3 M ammonium sulfate. The gradient consisted of 5 min holding at 100% A, followed a linear gradient of 20 to 100% B over 40 min and finally re-equilibrating at initial conditions for 10 min prior to the next injection. The separation was monitored by UV absorption at 280 nm.

(251) Preparation and Characterization of DAR 4, 6 and 8 Cys ADCs

(252) Cys mutations can be combined for the preparation of DAR 4, 6 and 8 ADCs. In general, the preferred combination is a combination of two Cys mutations resulting in ADCs with DAR 4. Some examples that involve combining a heavy chain (HC) Cys mutant with a light chain (LC) Cys mutant for the preparation of DAR 4 ADCs are shown in Example 10 for trastuzumab and for antibody 14090. Additional data is provided in Table 26. Based on the HIC data and the inspection of attachment sites in the IgG crystal structures, additional Cys combinations were prepared using the protocols described in Examples 2, 5 and 6. Data for selected examples of MMAF ADCs are shown in Table 26. In addition, selected heavy chain sites were combined and double Cys mutations of the heavy chain were cloned following protocols listed in Example 2. Antibodies featuring two HC Cys mutations were prepared and conjugated following protocols described in Example 5 and 6.

(253) For the preparation of DAR 4 ADCs, combinations include single site mutations listed in Table 24. Combinations of single sites resulted in ADCs with low hydrophobicity (Table 25). In thesome combination, one Cys mutation is located in the CH1 domain or on the light chain and the second site is located in the CH3 domain. Examples of such combinations are antibodies featuring Cys mutant combinations of HC-E152C and HC-S375C, and LC-E165C and HC-S375C, and HC-E152C and HC-K334C, and LC-E165C and IC-K334C.

(254) ADCs with DAR 6 and 8 can also be prepared when three or four Cys mutations are combined in one antibody. Selected heavy chain combinations were combined for the preparations of DAR 4, 6 and 8 ADCs. Double and triple Cys mutations of the heavy chain were cloned following protocols listed in Example 2. Antibodies featuring two, three and four Cys mutations were prepared and conjugated following protocols described in Example 5 and 6. The characteristics of some DAR 4, DAR 6 and DAR 8 ADC examples are summarized in Table 26. Some of these ADCs have surprisingly good PK properties as shown in FIG. 25. Antibody 14090 is mouse cross-reactive and therefore, antibody 14090 ADCs, as expected, are cleared more rapidly than trastuzumab ADCs which do not bind to any mouse antigens.

(255) Combinations include those with three and four of the single site mutations listed in Table 24. Combinations include those sites that resulted in ADCs with low hydrophobicity (Table 25). Combinations include one Cys mutation is located in the CH1 domain or on the light chain (LC), and optionally an additional one to three sites are in the CH3 domain. Examples of such combinations include antibodies featuring Cys mutant combinations of HC-E152C or LC-E165C, with HC-S375C, with HC-K334C, and/or HC-K392C. Preferred combinations for the preparation of DAR 6 and DAR 8 ADCs are shown in Table 27 and Table 28 respectively.

(256) With a few of exceptions, attachment of MMAF at all Cys sites studied results in ADCs with high thermal stability (Example 7, Table 19), low propensity to aggregate (Example 6, Table 18) and good pharmacokinetic properties of DAR 2 ADCs (Example 9, Table 22, FIG. 18). Differences in ADC hydrophobicity apparently do not translate into large differences in biophysical and pharmacokinetic properties when a relatively soluble payload such as MMAF is used. In fact, as is shown above, DAR 4, DAR 6 and DAR 8 MMAF ADCs with acceptable pharmacokinetic properties can be prepared even using exposed, “hydrophobic” sites such as HC-K360C in combination with more preferred attachment sites. However, when less well behaved, more hydrophobic payloads are used, carefully selecting attachment sites that result in minimal changes in hydrophobicity may be essential to allow the preparation of non-aggregating ADCs with good pharmacokinetic properties. For such hydrophobic payloads, using combination of sites that reduce hydrophobicity increases might be beneficial when 4, 6 or 8 drugs are attached per antibody.

(257) TABLE-US-00026 TABLE 26 Characterization of selected DAR 4, 6 and 8 MMAF ADCs prepared with combinations of Cys mutations. AUC % MMAF/ Multimer AUC Cys-MMAF ADC name DAR AnSEC hIgG trastuzumab-HC-E258C-LC-S159C-MMAF 4.0 n.d. 0.9 trastuzumab-HC-S375C-LC-S159C-MMAF 4.0 n.d. 0.8 trastuzumab-HC-E258C-LC-E165C-MMAF 4.0 n.d. 0.9 trastuzumab-HC-S375C-LC-E165C-MMAF 4.0 n.d. 0.8 trastuzumab-HC-E152C-LC-R142C-MMAF 3.8 n.d. 0.9 trastuzumab-HC-P171C-LC-R142C-MMAF 3.8 0.1 1.1 trastuzumab-HC-E152C-LC-S159C-MMAF 3.8 n/a 0.7 Antibody 14090-HC-S375C-LC-A143C- 4.0 n.d. 0.9 MMAF Antibody 14090-HC-K360C-LC-V159C- 4.0 n.d. 1.0 MMAF Antibody 14090-HC-S375C-LC-V159C- 4.0 2.9 1.0 MMAF trastuzumab-HC-K334C-S375C-LC- 6.0 n.d. 0.8 E165C-MMAF trastuzumab-HC-K334C-K392C-LC- 5.8 11   0.4 E165C-MMAF trastuzumab-HC-K334C-K360C-S375C- 8.0 5   0.6 LC-E165C-MMAF trastuzumab-HC-K334C-K360C-K392C- 7.8 n.d. 0.8 LC-E165C-MMAF trastuzumab-HC-K334C-S375C-K392C- 8.0 n.d. 0.7 LC-E165C-MMAF *AUC calculations based on mouse PK measurements with anti-MMAF and anti-IgG ELISA assays. n.d.; not detected, below limited of quantitation.

(258) TABLE-US-00027 TABLE 27 Preferred combinations of Cys sites for the preparation of DAR 6 ADCs. ADC combination Site 1 Site 2 Site 3 1 HC-E152C HC-S375C HC-K392C 2 HC-E152C HC-S375C HC-K334C 3 HC-E152C HC-K334C HC-K392C 4 LC-E165C HC-S375C HC-K392C 5 LC-E165C HC-S375C HC-K334C 6 LC-E165C HC-K334C HC-K392C

(259) TABLE-US-00028 TABLE 28 Preferred combinations of Cys sites for the preparation of DAR 8 ADCs. ADC combination Site 1 Site 2 Site 3 Site 4 1 HC-E152C HC-S375C HC-K334C HC-K392C 2 HC-E152C HC-S375C HC-E333C HC-K392C 3 LC-E165C HC-S375C HC-K334C HC-K392C 4 LC-E165C HC-S375C HC-E333C HC-K392C

Example 12. In Vivo Efficacy Studies of Trastuzumab Cys-MMAF ADCs

(260) In vivo xenograft tumor models simulate biological activity observed iby grafting relevant and well characterized human primary tumors or tumor cell lines into immune-deficient nude mice. Studies on treatment of tumor xenograft mice with anti-cancer reagents have provided valuable information regarding in vivo efficacy of the tested reagents (Sausville and Burger, 2006). Since MDA-MB231 clone 16 cells were sensitive to trastuzumab Cys-MMAF ADCs in antigen dependent manner (FIG. 15), the cell line was chosen as the in vivo model to evaluate the trastuzumab Cys-MMAF ADCs. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication; National Academy Press, 8.sup.th edition, 2001). MDA-MB231 clone 16 cells were implanted in nu/nu mice subcutaneously (Morton and Houghton, 2007). After the tumor size reached ˜200 mm.sup.3, trastuzumab Cys-MMAF ADCs were administered into the mice by IV injection in a single dose at 3 mg/kg. The tumor growth was measured weekly after ADC injection. Each treatment group included 9 mice. An example of the in vivo efficacy study is indicated in FIG. 22 with three trastuzumab Cys-MMAF ADCs. Treatment of mice with 3 mg/kg trastuzumab Cys-MMAF ADCs caused tumor regression for all three tested Cys-MMAF ADCs (FIG. 22). No weight loss was observed associated with the ADC treatment. The results confirmed that with a single dose treatment at 3 mg/kg, trastuzumab Cys-MMAF ADCs effectively caused regression of MDA-MB231 clone 16 tumors.