SPECIFIC SITES FOR MODIFYING ANTIBODIES TO MAKE IMMUNOCONJUGATES

Abstract

The present application 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. In one embodiment, the antibodies are modified with cysteines at positions 152 and 375 of the heavy chain constant region, as defined by EU numbering format. In another embodiment, the antibodies are modified with cysteines at position 360 of the heavy chain constant region, and position 107 of the kappa light chain constant region, as defined by EU numbering format.

Claims

1. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of substitution of two or more amino acids with cysteine on its constant regions wherein the combinations comprise substitutions selected from position 360 of an antibody heavy chain, and position 107 of an antibody kappa light chain, wherein said positions are numbered according to the EU system.

2. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of substitution of two or more amino acids with cysteine on its constant regions wherein the combinations comprise substitutions selected from positions 152 and 375 of an antibody heavy chain, wherein said positions are numbered according to the EU system.

3. An immunoconjugate comprising a modified antibody or antibody fragment thereof comprising a heavy chain constant region of SEQ ID NO: 48 and a kappa light chain constant region comprising SEQ ID NO: 61.

4. An immunoconjugate comprising a modified antibody or antibody fragment thereof comprising a heavy chain constant region of SEQ ID NO: 131.

5. The immunoconjugates of any of claims 1-4 wherein the immunoconjugate further comprises a drug moiety.

6. The immunoconjugates of any of claims 1-5 wherein the drug antibody ratio is about 4.

7. The immunoconjugate of any of claim 1-6, wherein said drug moiety is attached to the modified antibody or antibody fragment through the sulfur of said cysteine and an optional linker.

8. The immunoconjugate of claim 7, wherein said drug moiety is connected to said sulfur of said cysteine through a cleavable or non-cleavable linker.

9. The immunoconjugate of claim 8, wherein said drug moiety is connected to said sulfur of said cysteine through a non-cleavable linker.

10. The immunoconjugate of claim 7-9, wherein said immunoconjugate comprises a thiol-maleimide linkage.

11. The immunoconjugate of claim 10, wherein said immunoconjugate comprises a SCH.sub.2C(O) linkage or a disulfide linkage.

12. The immunoconjugate of claim 11, wherein said drug moiety is a cytotoxic agent.

13. The immunoconjugate of claim 12, wherein said drug moiety is selected from the group consisting of taxanes, DNA-alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansinoids, and Eg5 inhibitors.

14. The immunoconjugate of any of claims 1-13, wherein said antibody is a monoclonal antibody.

15. The immunoconjugate of any of claims 1-13, wherein said antibody is a chimeric antibody.

16. The immunoconjugate of claim 1-13, wherein said antibody is a humanized or fully human antibody.

17. The immunoconjugate of any of claims 14-16, wherein said antibody is a bispecific or multi-specific antibody.

18. The immunoconjugate of any of claims 1-17, wherein said antibody or antibody fragment specifically binds to a cell surface marker on a tumor.

19. A pharmaceutical composition comprising the immunoconjugate of any of claims 1-18.

20. The modified antibody or antibody fragment of any of claims 1-19, further comprising at least one Pcl or unnatural amino acid substitution or a peptide tag for enzyme-mediated conjugation and/or combinations thereof.

21. A nucleic acid encoding the modified antibody or antibody fragment of any of claims 1-4.

22. A host cell comprising the nucleic acid of claim 21.

23. A method of producing a modified antibody or antibody fragment comprising incubating the host cell of claim 22 under suitable conditions for expressing the antibody or antibody fragment, and isolating said antibody or antibody fragment.

24. A method to produce an immunoconjugate, which comprises attaching a Linker Unit (LU) or a Linker Unit-Payload combination (-LU-X) to a cysteine residue in an antibody or antibody fragment of any of claims 1-4

25. The method of claim 24, wherein the immunoconjugate is of Formula (I): ##STR00163## 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.

26. A modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of substitution of two or more amino acids with cysteine on its constant regions wherein the combinations comprise substitutions selected from position 360 of an antibody heavy chain, and position 107 of an antibody kappa light chain, wherein said positions are numbered according to the EU system.

27. A modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a combination of substitution of two or more amino acids with cysteine on its constant regions wherein the combinations comprise substitutions selected from positions 152 and 375 of an antibody heavy chain, wherein said positions are numbered according to the EU system.

28. A modified antibody or antibody fragment thereof comprising a heavy chain constant region of SEQ ID NO: 48 and a kappa light chain constant region comprising SEQ ID NO: 61.

29. A modified antibody or antibody fragment thereof comprising a heavy chain constant region of SEQ ID NO:131.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0148] FIG. 1 depicts an amino acid sequence alignment of constant regions of trastuzumab (SEQ ID NO:155), human IgG1 (SEQ ID NO:151), IgG2 (SEQ ID NO:152), IgG3 (SEQ ID NO:153) and IgG4 (SEQ ID NO: 154).

[0149] FIG. 2 is a graph depicting cell killing activity of antibody drug conjugates comprising a cKIT antibody that has two cys mutations in its constant regions on cells that express cKIT. The antibody is conjugated to a linker-payload complex that inhibits Eg5. The square data points are for immunoconjugates comprising Compound A in Table 5; the triangle data points are for immunoconjugates comprising Compound B in Table 5; the square data points are for immunoconjugates comprising Compound C in Table 5.

[0150] FIG. 3 depicts graphs illustrating the activity of immunoconjugates comprising cysteine-engineered cKIT antibodies in H526 tumor mouse xenograft models at dosages of 5 mg/kg (FIG. 3A) and 10 mg/kg (FIG. 3B) and an immunoconjugate comprising wild type cKIT antibody administered at dosages of 5.9 mg/kg (FIG. 3A) and 11.8 mg/kg (FIG. 3B).

[0151] FIG. 4 is a graph depicting in vivo efficacy of anti-Her2 immunoconjugates conjugated with Eg5 inhibitor in a Her2 positive MDA-MB-231 clone 16 breast cancer xenograft model in mice.

[0152] FIG. 5 is a graph depicting in vivo efficacy of anti-Her2 immunoconjugates conjugated with Eg5 inhibitor in a Her2 positive MDA-MB-453 breast cancer xenograft model in mice.

[0153] FIG. 6 is a graph depicting in vivo efficacy of anti-Her2 immunoconjugates conjugated with Eg5 inhibitor in a Her2 positive HCC1954 breast cancer xenograft model in mice.

[0154] FIG. 7 is a graph depicting results from an in vivo efficacy study of anti-cKIT ADCs conjugated with Compound F, in H526 tumor xenograft model in mice. Compound F was conjugated to cysteine-engineered or wild type cKIT antibodies. An anti-Her2 immunoconjugate was included as a non-binding control.

[0155] FIG. 8 is a graph depicting results from pharmacokinetic studies of antibody anti-cKIT-HC-E152C-S375C-Compound F (FIG. 8A) and antibody anti-cKIT-Compound F (FIG. 8B) ADCs in nave mice at a dose of 1 mg/kg.

[0156] FIG. 9 is a graph depicting in vivo efficacy of anti-cKIT immunoconjugates conjugated to two different compounds to two different cysteine-engineered antibodies in a H526 tumor xenograft model in mice.

DETAILED DESCRIPTION

[0157] The present application 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 application also provides immunoconjugates that are produced by using the methods described herein.

[0158] 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 application 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.

[0159] The site-specific antibody labeling according to the present application 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.

[0160] Accordingly, the present application 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 application also provides immunoconjugates prepared thereby, as well as pharmaceutical compositions comprising these immunoconjugates. The methods of the instant application can be used in combination with other conjugation methods known in the art.

[0161] The following enumerated embodiments represent certain aspects and variations of the application:

##STR00006## [0162] wherein Ab represents an antibody or antibody fragment comprising at least one cysteine residue at one of the preferred cysteine substitution sites described herein; [0163] LU is a linker unit as described herein; [0164] X is a payload or drug moiety; [0165] 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: [0166] 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()(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, [0167] or (O(C(R.sup.4).sub.2).sub.n).sub.mNHC(O)(C(R.sup.4).sub.2).sub.n; [0168] each X.sup.2 is independently selected from a bond, R.sup.8,

##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); [0169] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0170] 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; [0171] 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; [0172] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0173] R.sup.8 is independently selected from

##STR00011## [0174] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0175] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0176] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0177] In some of these embodiments, the immunoconjugate comprises a group of the formula

##STR00012##

[0178] 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.

[0179] 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

[0180] The antibodies (e.g., a parent antibody, optionally containing one or more non-canonical amino acids) of the present application 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 present application is not limited to human IgG1; corresponding amino acid positions can be readily deduced by sequence alignment. For example, FIG. 1 shows sequence alignment of antibody trastuzumab wild type heavy chain constant region (the sequence of which is STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO:155)), human IgG1 (the sequence of which is STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG (SEQ ID NO: 151)), IgG2 (the sequence of which is STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLN GKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG (SEQ ID NO: 152)), IgG3 (the sequence of which is STKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEP KSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPP MLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 152)), and IgG4 (the sequence of which is STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG (SEQ ID NO: 153)) 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. 1. For the light chain constant region, IgG1, IgG2, IgG3 and IgG4 are the same (the full-length wild type light chain sequence of human antibody trastuzumab is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO:90)).

[0181] 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 2A 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 2B lists the amino acid positions in the constant region of the lambda light chain of an antibody that can be replaced by a cysteine.

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 [.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

TABLE-US-00002 SEQIDNO:1 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV KGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:2 CASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:3 SACTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:4 SASTCGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:5 SASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:6 SASTKGPSVFPLAPSCKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:7 SASTKGPSVFPLAPSSKCTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:8 SASTKGPSVFPLAPSSKSTCGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:9 SASTKGPSVFPLAPSSKSTSGGCAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:10 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:11 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPECVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:12 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVCVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:13 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVCWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:14 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALCSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:15 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTCGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:16 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHCFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:17 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFCAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:18 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVCQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:19 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQCSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:20 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSCGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:21 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVCSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:22 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSCSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:23 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGCQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:24 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQCYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:25 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHCPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:26 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPCNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:27 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVCKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:28 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPCPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:29 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPCVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:30 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHCDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:31 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVCFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:32 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHCAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:33 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNACTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:34 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTCPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:35 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:36 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRCEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:37 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRECQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:38 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYCCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:39 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCCVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:40 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNCALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:41 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPCPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:42 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPICKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:43 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:44 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKCISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:45 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTICKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:46 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPCEPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:47 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSCEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:48 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTCNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:49 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNCVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:50 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:51 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWCSNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQIDNO:52 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPECNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:53 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENCYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:54 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:55 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKCTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQIDNO:56 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVCDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQIDNO:57 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDCDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQIDNO:58 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVCKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQIDNO:59 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKCRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQIDNO:60 SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNCFSCSVMHEALHNHYTQKS LSLSPGK

TABLE-US-00003 TABLE 2A Identified cysteine substitution sites in the kappa light chain constant region of human IgG1 (Sites numbered according to EU numbering system). Surface EU accessibility SEQ ID number Residue [.sup.2] Selected 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

TABLE-US-00004 SEQIDNO:61 CRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:62 KCTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:63 KRCVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:64 KRTVACPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:65 KRTVAAPCVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:66 KRTVAAPSVFIFPPSCEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:67 KRTVAAPSVFIFPPSDCQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:68 KRTVAAPSVFIFPPSDEQLKSGCASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:69 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPCEAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:70 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRCAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:71 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREACVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:72 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDCALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:73 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNACQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:74 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQCGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:75 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNCQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:76 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQCSVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:77 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTCQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:78 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDCK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:79 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSCD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:80 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKC STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:81 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLCKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:82 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSCADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:83 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYECHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:84 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHCVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:85 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKCYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:86 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVCHQGLSSPVTKSFNRGEC SEQIDNO:87 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHCGLSSPVTKSFNRGEC SEQIDNO:88 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSCPVTKSFNRGEC SEQIDNO:89 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCKSFNRGEC

TABLE-US-00005 TABLE 2B Identified cysteine substitution sites on the lambda light chain of human IgG1. Surface Kabat accessibility SEQ ID number Residue [.sup.2] Selected 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

TABLE-US-00006 (ConstantRegionofhumanlambdaligtchain) SEQIDNO:91 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS SEQIDNO:92 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGCVTVAWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS SEQIDNO:93 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVCVAWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS SEQIDNO:94 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVCWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS SEQIDNO:95 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVCA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS SEQIDNO:96 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GCETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS SEQIDNO:97 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GVETTCPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS SEQIDNO:98 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GVETTTPSKQCNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS

[0182] Because of the high sequence homology of constant regions of IgG1, IgG2, IgG3 and IgG4 antibodies, findings of the present application are not limited to any specific antibodies or antibody fragments.

[0183] In one embodiment, the present application 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

[0184] In an embodiment of the present application, the amino acid substitution described herein is cysteine comprising a thiol group. In some aspects of the present application, 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 present application 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, an Eg5 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 present application comprise a drug moiety that is an anti-cancer agent. The modified antibody or antibody fragments of the present application can be any formats known in the art, such as a monoclonal, chimeric, humanized, fully human, bispecific, or multispecific antibody or antibody fragment thereof.

[0185] According to the present application, 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 four or more Cys substitutions.

[0186] 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.

[0187] The present application 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 application 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 2A or Table 2B. In other embodiments, the modified antibodies or antibody fragment thereof comprise one or more amino acids on its heavy chain constant region chosen from positions identified in Table 1 and one or more amino acids on its light chain constant region chosen from positions identified in Table 2A.

[0188] In certain embodiments, the modified antibodies or antibody fragments provided herein are labeled using the methods of the present application 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

[0189] 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.

[0190] 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 present application that undergo this type of conjugation contain a thiol-maleimide linkage.

##STR00013##

where:

LU is a Linker Unit (LU), and

[0191] X is a payload or drug moicly.

[0192] 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 YCHRC(O), where R is H or C.sub.1-4 alkyl, Y is a leaving group (typically Cl, Br, or I, and optionally an alkylsulfonate or arylsulfonate); it is not limited to amides.

##STR00014## [0193] Y is a leaving group (CI, Br, I, OTs, OTf. and the like) [0194] LU is a linker unit [0195] X is a payload or drug moiety

[0196] 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.

##STR00015## [0197] each R is independently H or C.sub.1-4 alkyl [0198] LU is a linker unit [0199] X is a payload or drug moiety

[0200] 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.

[0201] Upon conjugation, the modified antibodies of the present application 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.

[0202] 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.

[0203] 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.

[0204] 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 NHC(O), esters C(O)O or OC(O), rings having two available points of attachment such as divalent phenyl, C.sub.3-8 cycloalkyl or C.sub.4-8 heterocyclyl groups, amino acids NHCHR*CO 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), SCR.sub.2 and other thiol ethers such as SCR.sub.2C(O) or C(O)CR.sub.2S, where R is as defined above for Scheme Ic, CH.sub.2C(O), and disulfides (SS), 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.

[0205] In some embodiments when LU is -L.sub.1-L.sub.2-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: [0206] -A.sub.1-, -A.sub.1X.sup.2 and X.sup.2; wherein: [0207] 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, [0208] or (O(C(R.sup.4).sub.2).sub.n).sub.mNHC(O)(C(R.sup.4).sub.2).sub.n; [0209] each X.sup.2 is independently selected from a bond, R.sup.8,

##STR00016## ##STR00017## ##STR00018##

CHR.sup.4(CH.sub.2).sub.nC(O)NH, CHR.sup.4(CH.sub.2).sub.nNHC(O), C(O)NH and NHC(N); [0210] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0211] 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; [0212] 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; [0213] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0214] R.sup.8 is independently selected from

##STR00019##

[0215] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0216] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0217] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0218] 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:

##STR00020##

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.

[0219] In some embodiments, the immunoconjugate of the present application comprises a modified cysteine residue of the formula IIA or IIB:

##STR00021##

wherein CH.sub.2S 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 (CO) or a linker that comprises a self-immolative spacer.

[0220] In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein: [0221] 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; [0222] 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; [0223] 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 [0224] 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.

[0225] In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0226] 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; [0227] 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; [0228] 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 [0229] 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.

[0230] 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.

[0231] 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: [0232] 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, [0233] or (O(C(R.sup.4).sub.2).sub.n).sub.mNHC(O)(C(R.sup.4).sub.2).sub.n; [0234] each X.sup.2 is independently selected from a bond, R.sup.8,

##STR00022## ##STR00023## ##STR00024##

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); [0235] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0236] 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; [0237] 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; [0238] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0239] R.sup.8 is independently selected

##STR00025## [0240] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0241] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0242] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0243] 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.

[0244] In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0245] L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2 or X.sup.2; where: [0246] 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), 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; [0247] each X.sup.2 is independently selected from a bond, R.sup.8

##STR00026## ##STR00027## ##STR00028##

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); [0248] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0249] 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; [0250] 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; [0251] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0252] R.sup.8 is independently selected from

##STR00029##

[0253] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0254] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0255] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0256] 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; [0257] 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 [0258] 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.

[0259] In certain embodiments, L.sub.1 is C(O)CH.sub.2CH.sub.2NHC(O)CH.sub.2CH.sub.2S, so LU is C(O)CH.sub.2CH.sub.2NHC(O)CH.sub.2CH.sub.2S-L.sub.2-L.sub.3-L.sub.4-.

[0260] In certain embodiments the Linker Unit (LU) is -L.sub.1-L-L.sub.3-L.sub.4-, wherein [0261] L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2 or X.sup.2; where: [0262] 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; [0263] each X.sup.2 is independently selected from a bond, R.sup.8

##STR00030## ##STR00031## ##STR00032##

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); [0264] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0265] 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; [0266] 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; [0267] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0268] R.sup.8 is independently selected

##STR00033## [0269] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0270] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0271] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0272] 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; [0273] 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; [0274] 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.

[0275] In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0276] L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2 or X.sup.2; where: [0277] 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; [0278] each X.sup.2 is independently selected from a bond, R.sup.8

##STR00034## ##STR00035## ##STR00036## ##STR00037##

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); [0279] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0280] 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; [0281] 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; [0282] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0283] R.sup.8 is independently selected

##STR00038##

[0284] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0285] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0286] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0287] 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; [0288] 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 [0289] 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.

[0290] In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0291] L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2 or X.sup.2; where: [0292] 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; [0293] each X.sup.2 is independently selected from a bond, R.sup.8

##STR00039## ##STR00040## ##STR00041## ##STR00042##

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); [0294] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0295] 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; [0296] 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; [0297] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0298] R.sup.8 is independently selected from

##STR00043## [0299] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0300] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0301] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0302] L.sub.2 is a bond, a non-enzymatically cleavable linker or a non-cleavable linker; [0303] L.sub.3 is a bond, a non-enzymatically cleavable linker or a non-cleavable linker; [0304] L.sub.4 is a bond, an enzymatically cleavable linker or a linker that comprises a self-immolative spacer.

[0305] In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0306] L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2 or X.sup.2; [0307] L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2; [0308] L.sub.3 is a bond, -A.sub.3-, or -A.sub.3X.sup.2; [0309] L.sub.4 is a bond, -A.sub.4-, -A.sub.4X.sup.2,

##STR00044## [0310] 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; [0311] 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)C(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,

##STR00045## [0312] 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,

##STR00046## [0313] 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; [0314] each X.sup.2 is independently selected from a bond, R.sup.8

##STR00047## ##STR00048## ##STR00049## ##STR00050##

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); [0315] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0316] 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; [0317] 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; [0318] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0319] R.sup.8 is independently selected from

##STR00051## [0320] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0321] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0322] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0323] In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0324] L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2 or X.sup.2; [0325] L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2; [0326] L.sub.3 is a bond, -A.sub.3-, or -A.sub.3X.sup.2; [0327] L.sub.4 is a bond, -A.sub.4-, -A.sub.4X.sup.2,

##STR00052## [0328] 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).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; [0329] 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).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

##STR00053## [0330] 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

##STR00054## [0331] 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).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; [0332] each X.sup.2 is independently selected from a bond,

##STR00055##

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); [0333] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0334] 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; [0335] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0336] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0337] In certain embodiments the Linker Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0338] L.sub.1 is a bond, -A.sub.1-, -A.sub.1X.sup.2 or X.sup.2; [0339] L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2; [0340] L.sub.3 is a bond, -A.sub.3-, or -A.sub.3X.sup.2; [0341] L.sub.4 is a bond, -A.sub.4-, -A.sub.4X.sup.2,

##STR00056## [0342] 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), (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; [0343] 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, ((CH.sub.2).sub.n).sub.mNHC(O)(CH.sub.2).sub.n or

##STR00057## [0344] 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, ((CH.sub.2).sub.n).sub.mNHC(O)(CH.sub.2).sub.n or

##STR00058## [0345] 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; [0346] each X.sup.2 is independently selected from a bond, R.sup.8

##STR00059## ##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); [0347] each R.sup.4 is independently selected from H, C.sub.1-4alkyl, side chains of known amino acids, C(O)OH and OH, [0348] 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; [0349] 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; [0350] R.sup.7 is independently selected from H, C.sub.1-4alkyl, phenyl, pyrimidine and pyridine; [0351] R.sup.8 is independently selected from

##STR00063##

[0352] R.sup.9 is independently selected from H and C.sub.1-6haloalkyl; [0353] each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0354] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0355] In one embodiment, L.sub.1 is (CH.sub.2).sub.1-10C(O), e.g., (CH.sub.2).sub.5C(O); and L.sub.2, L.sub.3 and L.sub.4 each represent a bond.

[0356] 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:

##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.2-6C(O).

[0357] 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--calicheamicin, or a label or dye such as rhodamine or tetramethylrhodamine.

[0358] 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.

[0359] 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.

[0360] 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 -glucuronidase or another glycosidase.

[0361] 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 -glucuronidase, e.g.,

##STR00065##

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

[0362] 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 tripartate 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 application, 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 application, 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.

[0363] 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 tripartate 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.

[0364] 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 glucoronidase releases a phenolic compound that spontaneously decomposes under physiological conditions:

##STR00066##

[0365] 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.

[0366] 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.

[0367] In certain embodiments, the self-immolative spacer is or

##STR00067##

while in other embodiments the self-immolative spacer is

##STR00068##

where n is 1 or 2. In other embodiments the self-immolative spacer is

##STR00069##

where n is 1 or 2. In other embodiments the self-immolative spacer is

##STR00070##

where n is 1 or 2. In other embodiments the self-immolative spacer is

##STR00071##

where n is 1 or 2. In other embodiments the self-immolative spacer is

##STR00072##

where n is 1 or 2.

[0368] 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.

##STR00073##

##STR00074##

##STR00075##

In each of Schemes 2a-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.1-4 alkyl, 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 present application is L.sub.1.

[0369] In some embodiments of the present application, 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 3 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.

TABLE-US-00007 TABLE 3 X Complementary Reactive 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.2NHC(O) a ketone a hydroxylamine, a hydrazine or NH.sub.2NHC(O) a hydroxylamine an aldehyde or a ketone a hydrazine an aldehyde or a ketone NH.sub.2NHC(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 4, where Y.sup.1 represents an antibody of the present application, 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 4 depicts a product from reaction of X.sup.a with X.sup.b.

TABLE-US-00008 TABLE 4 X.sup.bL.sub.2L.sub.3L.sub.4X.sup.1 Y.sup.1A.sub.1X.sup.a Formula (II-a) Y.sup.1A.sub.1X.sup.2L.sub.2L.sub.3L.sub.4X.sup.1 Y.sup.1A.sub.1N.sub.3 HCCL.sub.2L.sub.3L.sub.4X.sup.1 [00076] Y.sup.1A.sub.1N.sub.3 HCCL.sub.2L.sub.3L.sub.4X.sup.1 [00077] Y.sup.1A.sub.1CCH N.sub.3L.sub.2L.sub.3L.sub.4X.sup.1 [00078] Y.sup.1A.sub.1CCH N.sub.3L.sub.2L.sub.3L.sub.4X.sup.1 [00079] [00080] NH.sub.2OL.sub.2L.sub.3L.sub.4X.sup.1 [00081] [00082] NH.sub.2OL.sub.2L.sub.3L.sub.4X.sup.1 [00083] [00084] CH.sub.3C(O)L.sub.2L.sub.3L.sub.4X.sup.1 [00085] [00086] HC(O)L.sub.2L.sub.3L.sub.4X.sup.1 [00087] [00088] HSL.sub.2L.sub.3L.sub.4X.sup.1 [00089] [00090] [00091] [00092] [00093] [00094] [00095] [00096] [00097] [00098] [00099] [00100] [00101] [00102] [00103] [00104] [00105] [00106] [00107] [00108] [00109] [00110] [00111] R.sub.5C(O)L.sub.2L.sub.3L.sub.4X.sup.1 [00112] [00113] HC(O)L.sub.2L.sub.3L.sub.4X.sup.1 [00114] [00115] HSL.sub.2L.sub.3L.sub.4X.sup.1 [00116] Y.sup.1A.sub.1N.sub.3 [00117] [00118] [00119] N.sub.3L.sub.2L.sub.3L.sub.4X.sup.1 [00120] Y.sup.1A.sub.1N.sub.3 [00121] [00122] [00123] N.sub.3L.sub.2L.sub.3L.sub.4X.sup.1 [00124] Y.sup.1A.sub.1N.sub.3 [00125] [00126] [00127] N.sub.3L.sub.2L.sub.3L.sub.4X.sup.1 [00128] [00129] [00130] [00131] [00132] [00133] [00134] [00135] [00136] [00137] [00138] [00139] [00140] [00141] N.sub.3L.sub.2L.sub.3L.sub.4X.sup.1 [00142] Y.sup.1A.sub.1N.sub.3 [00143] [00144] [00145] [00146] [00147] [00148] [00149] [00150] Y.sup.1A.sub.1N.sub.3 [00151] [00152] [00153] N.sub.3L.sub.2L.sub.3L.sub.4X.sup.1 [00154]

[0370] 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 present application 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 present application 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.

[0371] 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 in homogeneity, 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 4 to about 16, and typically is about 4, 5, 6, 7, 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

[0372] The present application provides site-specific labeled immunoconjugates. The immunoconjugates of the present application 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 present application.

[0373] 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.

[0374] In addition or alternative to modifications made within the framework or CDR regions, antibodies of the present application 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 present application 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.

[0375] 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.

[0376] 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.

[0377] In yet other embodiments, the Fc 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.

[0378] 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.

[0379] 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 et al. In a specific embodiment, one or more amino acids of an antibody or antibody fragment thereof of the present application are replaced by one or more allotypic amino acid residues. 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).

[0380] 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 Fc 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 FcRI, FcRII, FcRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0381] 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.

[0382] 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 present application 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, Lec13 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).

[0383] 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 Fc 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

[0384] The present application provides site-specific labeling methods, modified antibodies and antibody fragments thereof, and immunoconjugates prepared accordingly. Using the methods of the present application, 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 present application with other conjugation methods.

[0385] In certain embodiments, the immunoconjugates of the present application 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, an Eg5 inhibitor, and a DHFR inhibitor.

[0386] Further, the modified antibodies or antibody fragments of the present application 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, -interferon, 3-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.

[0387] In one embodiment, the modified antibodies or antibody fragments of the present application 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 et al., J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa et 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, meiphalan, 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).

[0388] Other examples of therapeutic cytotoxins that can be conjugated to the modified antibodies or antibody fragments of the present application include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg; Wyeth-Ayerst).

[0389] 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.

[0390] According to the present application, 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.77. 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 present application. 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 et al., (1999) Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.

[0391] The present application 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 present application 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.

[0392] 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 application, are used to generate fusion proteins comprising such an antibody fragment (e.g., engineered Fc) and a heterologous protein, polypeptide, or peptide.

[0393] 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 present application 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 et 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.

[0394] Moreover, the modified antibodies or antibody fragments thereof of the present application 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 et 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 application, antibodies or antibody fragments can also be conjugated to tumor-penetrating peptides in order to enhance their efficacy.

[0395] In other embodiments, modified antibodies or antibody fragments of the present application 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 aequorin; 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.142Pr, .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.

[0396] Modified antibodies or antibody fragments of the present application 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

[0397] To prepare pharmaceutical or sterile compositions including immunoconjugates, the immunoconjugates of the present application 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).

[0398] 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, NY, 1993; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman, et al. (eds.) Pharmaceutical Dosage Forms: Disperse Systems, Inc., New York, N.Y., 2000).

[0399] 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, UK, 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 et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et 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 et al., New Engl. J. Med. 348:24-32, 2003; Lipsky et al., New Engl. J. Med. 343:1594-1602, 2000).

[0400] 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.

[0401] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present application 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 application 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.

[0402] Compositions comprising antibodies or fragments thereof of the present application 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.

[0403] For the immunoconjugates of the present application, 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 present application may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.

[0404] Doses of the immunoconjugates the present application 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 present application are repeated every 3 weeks.

[0405] 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, UK, 2001).

[0406] The route of administration may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, 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.

[0407] A composition of the present application 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 present application 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 present application 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 present application is administered by infusion. In another embodiment, the immunoconjugates of the present application is administered subcutaneously.

[0408] If the immunoconjugates of the present application 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 present application (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 et al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 7 1:105, 1989; U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 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), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, 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).

[0409] 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 present application. 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 et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, 1997, each of which is incorporated herein by reference in their entirety.

[0410] If the immunoconjugates of the present application 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.

[0411] 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 application 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.

[0412] 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%.

[0413] Additional therapies (e.g., prophylactic or therapeutic agents), which can be administered in combination with the immunoconjugates of the present application 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 present application. The two or more therapies may be administered within one same patient visit.

[0414] In certain embodiments, the immunoconjugates of the present application 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 present application 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 et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (Bloeman et al., (1995) FEBS Lett. 357:140; Owais et 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.

[0415] The present application provides protocols for the administration of pharmaceutical composition comprising immunoconjugates of the present application 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 application can be administered concomitantly or sequentially to a subject. The therapy (e.g., prophylactic or therapeutic agents) of the combination therapies of the present application 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.

[0416] The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the present application can be administered to a subject concurrently.

[0417] 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 present application are administered to a subject in a sequence and within a time interval such that the antibodies of the present application 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.

[0418] 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.

[0419] The present application 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

Payload Compounds

[0420] Table 5 below lists structures of various payload compounds used in making antibody drug conjugates as described in the Examples in this application. Compounds A-E and methods of synthesizing the compounds, are disclosed, for example, in PCT/US2014/024795, and Compound F is disclosed, for example, in PCT/US2014/070800, both of which are incorporated herein by reference in their entirety. A synthetic method for Compound G is disclosed below.

Compound G

Synthetic Procedure

Synthesis of (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (MC-MMAF, Compound G)

[0421] ##STR00155##

MMAF-OMe (135 mg, Concortis Biosystems) was dissolved in CH3CN (10 mL). To the resulting clear solution was added 5 mL water, followed by 0.375 mL of IN aqueous sodium hydroxide (certified, Fisher Scientific). The reaction mixture was stirred magnetically at 21 C. for 18 hours, at which time LCMS analysis indicated a complete reaction. The reaction mixture mixture was frozen and lyophilized, affording MMAF sodium salt. LCMS retention time 0.911 minutes. MS (ESI+) m/z 732.5 (M+1). The whole MMAF sodium salt thus obtained in previous reaction was dissolved in 10 mL DMSO. In a separate reaction vessel, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (95 mg) was treated with HATU (165 mg) and DIEA (0.126 mL) in 3.0 mL DMSO at at 21 C. for 25 min. The whole reaction mixture of the activated ester was added to the solution of MMAF sodium salt, and The reaction mixture was stirred at the same temperature for 3 hours. The reaction mixture mixture was partitioned between 40 mL of EtOAc and 20 mL of 5% aqueous citric acid. The organic layer was separated, and the aqueous layer was extracted with 20 mL of EtOAc. The combined organic layers were washed with 10 mL saturated aqueous NaCl, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was purified on an ISCO CombiFlash instrument using an ISCO C18gold 15.5 g column. The desired material was eluted with 50% CH.sub.3CN in H.sub.2O. The fractions containing the desired product was combined and lyophilized, affording compound as white solid. LCMS retention time 1.392 minutes. MS (ESI+) m/z 925.6 (M+1).

TABLE-US-00009 TABLE 5 Linker-payloads tested. Compound Structure Compound A (Eg5 inhibitor) [00156] Compound B (Eg5 inhibitor) [00157] Compound C (Eg5 inhibitor) [00158] Compound D (Eg5 inhibitor) [00159] Compound E (Eg5 inhibitor) [00160] Compound F (cytotoxic peptide) [00161] Compound G (MMAF) [00162]

Example 2

Preparation of Trastuzumab Cys Mutant Antibodies

[0422] DNA encoding variable regions of heavy and light chains of trastuzumab, an anti-HER2 antibody (the terms trastuzumab, anti-HER2 antibody, and TBS are used interchangeably herein), 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.

[0423] Oligonucleotide directed mutagenesis was employed to prepare Cys mutant constructs in trastuzumab. Pairs of mutation primers were chemically synthesized for each Cys mutation site (Table 6). 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 DH10b cells (Klock et al., (2009) Methods Mol Biol. 498:91-103).

TABLE-US-00010 TABLE6 DNAsequencesofmutationprimersusedtoprepare11individual CysmutationsheavyandlightchainsofhumanIgG SEQ Mutation Primer ID sites name Sequence NO. LC-K107C LC-CYS-S1 GTGGAGATCTGTCGAACGGTGGCCGCTCCCAGCGTGTTCA 100 LC-CYS-A1 ACCGTTCGACAGATCTCCACCTTGGTACCCTGTCCGAAC 101 LC-S159C LC-CYS-S18 AGCGGCAACTGTCAGGAGAGCGTCACCGAGCAGGACAG 102 CAA LC-CYS-A18 CTCTCCTGACAGTTGCCGCTCTGCAGGGCGTTGTCCACCT 103 LC-E165C LC-CYS-S20 GAGCGTCACCTGTCAGGACAGCAAGGACTCCACCTACAGC 104 LC-CYS-A20 CTGTCCTGACAGGTGACGCTCTCCTGGCTGTTGCCGCTCT 105 HC-E152C HC-CYS-S9 TACTTCCCCTGTCCCGTGACCGTGTCCTGGAACAGCGGA 106 HC-CYS-A9 GGTCACGGGACAGGGGAAGTAGTCCTTCACCAGGCAGC 107 HC-P171C HC-CYS-S16 CACACCTTCTGTGCCGTGCTGCAGAGCAGCGGCCTGTACA 109 HC-CYS-A16 CAGCACGGCACAGAAGGTGTGCACGCCGGAGGTCAGGGCT 110 HC-P247C HC-CYS-S247 CTGTTCCCACCCAAGTGTAAGGACACCCTGATGATCAG 111 HC-CYS-A247 CTTGGGTGGGAACAGGAACACGGAGGGTCCGCCCAG 112 HC-A327C HC-CYS-S327 TGCAAGGTCTCCAACAAGTGTCTGCCAGCCCCCATCGA 113 AAAG HC-CYS-A327 GTTGGAGACCTTGCACTTGTATTCCTTGCCGTTCAGCCAG 114 HC-K334C HC-CYS-S46 CCCATCGAATGCACCATCAGCAAGGCCAAGGGCCAGCCA 115 HC-CYS-A46 GCTGATGGTGCATTCGATGGGGGCTGGCAGGGCCTTGTTG 116 HC-A339C HC-CYS-S339 CTTGCTGATGGTCTTTTCGATGGGGGCTGGCAGGGCCTTG 117 HC-CYS-A339 AAGACCATCAGCAAGTGTAAGGGCCAGCCACGGGAG 118 HC-K360C HC-CYS-S52 AGCTGACCTGCAACCAGGTGTCCCTGACCTGTCTGGTGA 119 HC-CYS-A52 CACCTGGTTGCAGGTCAGCTCGTCCCGGGATGGAGGCAGG 120 HC-Y373C HC-CYS-S373 CTGGTGAAGGGCTTCTGTCCCAGCGACATCGCCGTGGAGTG 121 HC-CYS-A373 GAAGCCCTTCACCAGACAGGTCAGGGACACCTGGTTCTTG 122 HC-5375C HC-CYS-S54 TTCTACCCCTGCGACATCGCCGTGGAGTGGGAGAGCAACG 123 HC-CYS-A54 GGCGATGTCGCAGGGGTAGAAGCCCTTCACCAGACAGGTCA 124 HC-Y391C HC-CYS-S391 AACAACTGTAAGACCACACCTCCAGTGCTGGACAGCGAC 125 HC-CYS-A391 GGTCTTACAGTTGTTCTCGGGCTGGCCGTTGCTCTCCCAC 126 HC-P396C HC-CYS-S396 ACACCTTGTGTGCTGGACAGCGACGGCAGCTTCTTCCTG 127 HC-CYS-A396 CAGCACACAAGGTGTGGTCTTGTAGTTGTTCTCGGGCTG 128

[0424] In some cases, two or more mutations were made in the same chain of trastuzumab. Oligonucleotide directed mutagenesis was employed to prepare the multiple Cys mutant constructs using the same method as above but using a pOG-trastuzumab-Cys mutant plasmid as the template for serial rounds of mutagenesis.

[0425] Sequences of all Cys mutant constructs were confirmed by DNA sequencing. The encoded protein sequence of the constant region of the HC and LC Cys mutant IgG1 constructs are shown in Table 7 and Table 8, 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).

TABLE-US-00011 TABLE7 AminoacidsequencesoftheconstantregionofCysmutant constructsinhumanIgG1heavychain. SEQIDNO:1 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK SEQIDNO:10(Cysteinesubstitutionatposition152) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:18(Cysteinesubstitutionatposition174) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVCQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:42(Cysteinesubstitutionatposition333) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPICKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:48(Cysteinesubstitutionatposition360) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTC NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:50(Cysteinesubstitutionatposition375) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO129:(Cysteinesubstitutionatpositions334and375) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:130(Cysteinesubstitutionatpositions334and392) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:131(Cysteinesubstitutionatpositions152and375) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:132(Cysteinesubstitutionatpositions339and396) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKCKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPCVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:133(Cysteinesubstitutionatpositions152and171) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGA LTSGVHTFCAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQIDNO:134(Cysteinesubstitutionatpositions334and396) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIECTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPCVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:135(Cysteinesubstitutionatposition396) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPCVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:136(Cysteinesubstitutionatpositions375and396) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKT TPCVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:137(Cysteinesubstitutionatpositions375and391) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNCKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:138(Cysteinesubstitutionatpositions391and396) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNCKT TPCVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:139(Cysteinesubstitutionatpositions152and396) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPCVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQIDNO:140(Cysteinesubstitutionatpositions327and339) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKCLPAPIEKTISKCKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:141(Cysteinesubstitutionatposition391) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNCKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:142(Cysteinesubstitutionatpositions152and339) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKCKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQIDNO:143(Cysteinesubstitutionatpositions339and375) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKCKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:144(Cysteinesubstitutionatpositions152and327) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKCLPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQIDNO:145(Cysteinesubstitutionatposition373) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFCPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:146(Cysteinesubstitutionatpositions327and375) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKCLPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:147(Cysteinesubstitutionatposition247) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKCKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQIDNO:150(Constantregionofthewildtypeheavychain ofanti-cKITandanti-Her2antibodies) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 1 is the sequence for full-length trastuzumab with the constant region underlined. Additional sequences are Cys mutant constructs in human IgG1 heavy chain, showing only the sequences of the constant region. The mutant cys positions are shown by bold and underlined text.

TABLE-US-00012 TABLE8 Aminoacidsequencesoftheconstantregionof3humankappa lightchainCysmutantconstructs. SEQIDNO:90(anti-Her2lightchain) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL LIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTP PTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:61(Cysteinesubstitutionatposition107) CRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC SEQIDNO:75(Cysteinesubstitutionatposition159) KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNCQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC SEQIDNO:77(Cysteinesubstitutionatposition165) KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTCQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC SEQIDNO:148(Cysteinesubstitutionatpositions159and165) KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNCQESVTCQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVCKSFNRGEC SEQIDNO:149(Constantregionofwildtypelightchainfor anti-Her2andanti-cKIT) KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC SEQ ID NO: 90 sequence for full-length trastuzumab (human kappa light hain) with the constant region underlined. Additional sequences are the sequence ID numbers for Cys mutant constructs in the constant region of human kappa light chain.

Example 3

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

[0426] 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 an anti-cKIT antibody. The anti-cKIT antibody is an antibody with a human IgG1 heavy chain and a human kappa light chain that binds to the cKIT protein. The DNA encoding variable regions of antibody anti-cKIT were cloned into three selected pOG trastuzumab HC Cys mutant plasmid constructs and two selected pOG trastuzumab LC Cys mutant plasmid constructs (SEQ ID NOs listed in Table 9) to replace the variable regions of trastuzumab constructs in the plasmids as described in Example 2. As a result, the amino acid sequences of the heavy chain and light chain constant regions in corresponding five Cys constructs of the anti-cKIT antibody and of trastuzumab are identical. 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, 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.

TABLE-US-00013 TABLE 9 Sequence ID numbers of trastuzumab Cys constructs used for cloning of the variable region of the anti-cKIT antibody. Sequence ID NO: of trastuzumab Cys construct SEQ ID NO: 48 SEQ ID NO: 61 SEQ ID NO: 77 SEQ ID NO: 129 SEQ ID NO: 131

Example 4

Expression and Purification of Cys Mutant Antibodies in 293 Freestyle Cells

[0427] 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. To demonstrate that several conjugation sites can be combined for the production of ADCs with DAR greater than two, several single site Cys constructs of light and heavy chain of trastuzumab and anti-cKIT antibody (Table 10) were co-expressed in 293 Freestyle cells.

TABLE-US-00014 TABLE 10 Sequence IDs for constant regions of Cys engineered antibodies resulting in ADCs with DAR of 4 or 6 LC HC Target .Cys-drug ADC (DAR = 4 to 6) SEQ ID NO SEQ ID NO DAR anti-cKIT-HC-E152C-S375C 149 131 4 trastuzumab-HC-E152C-S375C 149 131 4 anti-cKIT-HC-K360C-LC-K107C 61 48 4 trastuzumab-HC-K360C-LC-K107C 61 48 4 trastuzumab-HC-A339C-P396C 149 132 4 trastuzumab-HC-E152C-LC-E165C 77 10 4 trastuzumab-HC-E152C-LC-S159C- 148 10 6 E165C trastuzumab-HC-E152C-P171C 149 133 4 trastuzumab-HC-K334C-P396C 149 134 4 trastuzumab-HC-K334C-S375C 149 129 4 anti-cKIT-HC-K334C-S375C-LC- 77 129 6 E165C trastuzumab-HC-P396C-LC-E165C 77 135 4 trastuzumab-HC-S375C-P396C 149 136 4 trastuzumab-HC-S375C-Y391C 149 137 4 trastuzumab-HC-Y391C-P396C 149 138 4 trastuzumab-LC-S159C-E165C 148 150 4

[0428] Cys mutant 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. Three days before transfection, cells were split to 0.2510.sup.6 cells/ml into fresh media. On the day of transfection, the cell density typically reached 1.5-210.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., Biotechnol Bioeng. 75:197-203 (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.

[0429] Expression levels of trastuzumab and anti-cKIT Cys mutant antibodies in transiently transfected 293 Freestyle are similar to that of wild type antibodies, with yields ranging from 12-25 mg/L, suggesting that single to triple point mutations in the selected sites did not significantly alter retention of the expressed antibody by the cells' secretion machinery. Analysis of the purified Cys mutant antibodies using non-reducing SDS PAGE indicates that the Cys mutant antibodies did not form oligomers disulfide-linked by the engineered cysteines.

Example 5

Reduction, Re-Oxidation and Conjugation of Cys Mutant Antibodies with Various Payloads

[0430] Because engineered Cys in antibodies expressed in mammalian cells are typically 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 purified Cys mutants of trastuzumab and anti-cKIT antibody, to a final concentration of 10 or 20 mM DTT. After the antibody incubation with DTT at 37 C. for 1 hour, the mixtures were dialyzed against PBS for three days with daily buffer exchange to remove DTT and re-oxidize the native disulfide bonds. The re-oxidation process was monitored by reverse-phase HPLC, which is able to separate full IgG from individual heavy and light chain molecules. The conjugation reaction mixtures were analyzed on a PRLP-S 4000A column (50 mm2.1 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. Dialysis was allowed to continue until reoxidation was complete. Reoxidation restores intra-chain disulfides, while dialysis allows cysteines and glutathiones connected to the newly-introduced cysteine(s) to dialyze away.

[0431] After re-oxidation, the antibodies are ready for conjugation. Maleimide-containing compounds were added to re-oxidized antibodies in PBS buffer (pH 7.2) at ratios of typically 1.5:1, 2:1, or 10:1. 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 in most cases. The elution of proteins from the column was monitored by UV absorbance at wavelengths of 280 nm, 254 nm and 215 nm.

[0432] 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, while some Cys sites generated heterogeneous conjugation products or showed only peaks matching the unconjugated antibodies.

[0433] 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-compound 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-compound 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 upon conjugation to Cys reactive compounds. Although sites were chosen to be surface exposed, there may also be heterogeneity in the final product if the introduced free cysteine is not accessible to or is otherwise unable to interact productively with the maleimide-drug in some or all conformations of the antibody. If the free cysteine is non-reactive, the final DAR will be lowered and the product likely to be a heterogenous mixture of fully, partially, and unmodified Cys mutant antibody. In the case of an antibody with two or more introduced free cysteines, there can be additional complexity introduced if drug attachment at one site interferes (i.e. by steric crowding) with the binding of a second drug at a second site. Such competition will lead to lower final DAR and a heterogeneous product. If two introduced cysteines are very close and properly oriented, then they may also form a non-native disulfide bond rather than forming two free cysteines. In this case, the antibody will not be reactive towards a maleimide-drug compound and the result will be a lower final DAR or even a uniform unconjugated product. The yield of the uniform ADC as measured by UV absorption by RP-HPLC the unpurified reaction mixtures, varied depending on the Cys mutations as well as the linker-payload compound used. Using the reduction/re-oxidation protocol and conjugation procedures described above 26 of 45 multiple Cys mutant trastuzumab or anti-cKIT antibodies described here resulted in homogeneous conjugation products of acceptable final DAR (DAR 3.4-4.4 for double Cys mutant, 5.1-6.0 for triple Cys mutant, Table 11) for such small test conjugations. These Cys sites and drug combinations are advantageous when making ADCs.

TABLE-US-00015 TABLE 11 DAR calculated from RP-HPLC analysis and verified by LCMS of intact, reduced, deglycosylated antibody chains for 45 multiple Cys mutant antibody samples conjugated to various drug by the methods described above. Linker- Ob- payload Expected served Cys mutant antibody compound DAR DAR trastuzumab-HC-A339C-P396C Compound A 4.0 2.0 trastuzumab-HC-A329C-P396C Compound G 4.0 3.6 trastuzumab-HC-A339C-P396C Compound G 4.0 3.6 trastuzumab-HC-E152C-LC-E165C Compound A 4.0 3.0 trastuzumab-HC-E152C-LC-E165C Compound E 4.0 3.0 trastuzumab-HC-E152C-LC-E165C Compound F 4.0 3.7 trastuzumab-HC-E152C-LC-E165C Compound G 4.0 2.9 trastuzumab-HC-E152C-LC- Compound A 6.0 4.0 S159C-E165C trastuzumab-HC-E152C-LC- Compound E 6.0 5.2 S159C-E165C trastuzumab-HC-E152C-LC- Compound G 6.0 5.2 S159C-E165C trastuzumab-HC-E152C-P174C Compound A 4.0 1.9 trastuzumab-HC-E152C-P174C Compound F 4.0 3.7 anti-cKIT-HC-E152C-S375C Compound A 4.0 3.9 anti-cKIT-HC-E152C-S375C Compound F 4.0 3.8 trastuzumab-HC-E152C-S375C Compound A 4.0 3.7 trastuzumab-HC-E152C-S375C Compound G 4.0 3.7 trastuzumab-HC-K334C-P396C Compound A 4.0 0.6 trastuzumab-HC-K334C-P396C Compound F 4.0 3.7 trastuzumab-HC-K334C-P396C Compound G 4.0 3.5 trastuzumab-HC-K334C-S375C Compound A 4.0 2.6 trastuzumab-HC-K334C-S375C Compound F 4.0 3.8 trastuzumab-HC-K334C-S375C Compound G 4.0 3.0 anti-cKIT-HC-K334C-S375C-LC- Compound E 6.0 5.8 E165C anti-cKIT-HC-K334C-S375C-LC- Compound G 6.0 5.2 E165C trastuzumab-HC-K334C-S375C- Compound E 6.0 6.0 LC-E165C trastuzumab-HC-K334C-S375C- Compound G 6.0 6.0 LC-E165C anti-KIT-HC-K360C-LC-K107C Compound A 4.0 4.0 anti-KIT-HC-K360C-LC-K107C Compound F 4.0 4.0 trastuzumab-HC-K360C-LC-K107C Compound A 4.0 4.0 trastuzumab-HC-K360C-LC-K107C Compound G 4.0 3.9 trastuzumab-HC-P396C-LC-E165C Compound A 4.0 1.6 trastuzumab-HC-P396C-LC-E165C Compound F 4.0 3.8 trastuzumab-HC-P396C-LC-E165C Compound G 4.0 3.4 trastuzumab-HC-S375C-P396C Compound A 4.0 0.0 trastuzumab-HC-S375C-P396C Compound F 4.0 0.0 trastuzumab-HC-S375C-P396C Compound G 4.0 0.0 trastuzumab-HC-S375C-Y391C Compound A 4.0 2.3 trastuzumab-HC-S375C-Y391C Compound G 4.0 3.2 trastuzumab-HC-Y391C-P396C Compound A 4.0 0.0 trastuzumab-HC-Y391C-P396C Compound F 4.0 3.6 trastuzumab-HC-Y391C-P396C Compound G 4.0 2.9 trastuzumab-LC-S159C-E165C Compound A 4.0 2.0 trastuzumab-LC-S159C-E165C Compound D 4.0 3.5 trastuzumab-LC-S159C-E165C Compound E 4.0 3.6

[0434] A subset of the 45 ADC samples in Table 11 were analyzed in details in various assays: Differential scanning fluorimetry (DSF) was used to measure thermal stability. Analytical size exclusion chromatograph (AnSEC) and multi-angle light scattering (MALS) were used to measure aggregation. In vitro antigen dependent cell killing potency was measured by cell viability assays and pharmacokinetics behavior was measured in mice. In general, the multiple Cys mutant ADCs showed thermal stability similar to single Cys mutant ADCs. The ADCs were predominantly monomeric as determined by analytical size exclusion chromatography.

Example 6

Preparation of Anti-cKIT and Trastuzumab Cys Mutant ADCs Conjugated with Various Compounds

[0435] Antibody drug conjugates of trastuzumab and anti-cKIT cys mutant antibodies HC-E152C-S375C and HC-K360C-LC-K107C were prepared using several payloads as described above. Some of the properties of these ADCs are shown in Table 12. The in vitro cell killing potency of these ADCs was tested as described in Example 7 and the results are summarized in Table 13 and Table 14. The compounds were further subjected to pharmacokinetic (PK) studies in naive mice as described in Example 8. The PK properties are summarized in Table 15 and Table 16.

TABLE-US-00016 TABLE 12 Properties of anti-Her2 Cys mutant ADCs conjugated with various compounds. Aggregation ADC name.sup.a DAR.sup.b (%).sup.c trastuzumab-HC-E152C-S375C-Compound A 3.8 0.4 trastuzumab-HC-E152C-S375C-Compound B 4.0 BLQ trastuzumab-HC-E152C-S375C-Compound C 3.7 0.6 trastuzumab-HC-E152C-S375C-Compound F 3.8 0.3 trastuzumab-HC-K360C-LC-K107C-Compound A 3.9 3.8 trastuzumab-HC-K360C-LC-K107C-Compound B 3.8 2.0 trastuzumab-HC-K360C-LC-K107C-Compound C 4.0 5.1 trastuzumab-HC-K360C-LC-K107C-Compound F 3.8 3.5 anti-cKIT-HC-E152C-S375C-Compound A 3.9 BLQ anti-cKIT-HC-E152C-S375C-Compound F 3.8 1.5 anti-cKIT-HC-K360C-LC-K107C-Compound A 4 BLQ anti-cKIT-HC-K360C-LC-K107C-Compound F 4 1.5 .sup.aName consists of a description of the mutated antibody and the name of the compound used in the chemical conjugation step. .sup.bDrug-to-antibody ratio according to reverse-phase HPLC. .sup.cAggregation measured by analytical size exclusion chromatography; includes dimeric and oligomeric species. BLQ = below limit of quantitation.

Example 7

Cell Proliferation Assays to Measure In Vitro Cell Killing Potency of Cys Mutant ADCs

[0436] 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 (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 (510.sup.5 copies/cell) of recombinant human Her2 while clone 40 expresses low copy numbers (510.sup.3 copies/cell) of human Her2. HCC1954 cells endogenously express high level (510.sup.5 copies/cell) of human Her2 in the surface. For determination of the cell killing potency of anti-cKIT ADCs, H526, KU812, CMK11-5 and Jurkat cells were used. While CMK11-5, H526 and KU812 cells express a high level of the antigen for the anti-cKIT antibody 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).

[0437] Trastuzumab Cys mutant ADCs specifically killed Her2 expressing MDA-MB231 clone 16 and HCC1954 but not MDA-MB231 clone 40 cells that express Her2 at very low levels (Table 13). Trastuzumab ADCs prepared with Compound F also killed JimT1 cells. IC.sub.50 of the trastuzumab Cys mutant ADCs varied by cell type and depending on the compound used (Table 13). Similarly, anti-cKIT Cys mutant ADCs displayed antigen-dependent cell killing in cell proliferation assays. Anti-cKIT Cys-drug ADCs killed antigen expressing NCI-H526, KU812 and CMK115 cells but not antigen negative Jurkat cells. The IC.sub.50 of the anti-cKIT ADCs varied with cell type and compound used (Table 14).

TABLE-US-00017 TABLE 13 In vitro cell killing potency of anti-Her2 ADCs conjugated with various compounds. IC.sub.50 (M).sup.b ADC name.sup.a MDA231-40 HCC1954 JimT1 MDA231-16 trastuzumab-HC-E152C-S375C-Compound A 6.7E02 1.4E04 4.8E02 6.7E02 trastuzumab-HC-E152C-S375C-Compound B 6.7E02 1.6E04 6.7E02 2.6E04 trastuzumab-HC-E152C-S375C-Compound C 6.7E02 1.8E04 5.6E02 3.6E04 trastuzumab-HC-E152C-S375C-Compound F 6.7E02 1.6E04 1.7E04 1.8E04 trastuzumab-HC-K360C-LC-K107C-Compound A 6.7E02 1.7E04 6.7E02 4.5E02 trastuzumab-HC-K360C-LC-K107C-Compound B 6.7E02 8.3E05 6.7E02 6.7E02 trastuzumab-HC-K360C-LC-K107C-Compound C 6.7E02 1.7E04 6.7E02 4.5E02 trastuzumab-HC-K360C-LC-K107C-Compound F 6.7E02 5.4E05 1.3E04 8.1E05 .sup.aName consists of a description of the mutated antibody and the name of the compound used in the chemical conjugation step. .sup.bThe highest concentration used in the assay was 6.7E02 M. IC.sub.50 values of 6.7E02 M therefore refer to inactivity of the ADC in the assay.

TABLE-US-00018 TABLE 14 In vitro cell killing activity of anti-cKIT ADCs conjugated with various compounds. IC.sub.50 (M).sup.b ADC name.sup.a Jurkat H526 KU812 CMK115 anti-cKIT-HC-E152C-S375C-Compound A 6.7E02 1.9E04 6.7E02 6.7E02 anti-cKIT-HC-E152C-S375C-Compound F 6.7E02 5.3E05 5.7E05 6.1E05 anti-cKIT-HC-K360C-LC-K107C-Compound A 6.7E02 2.0E04 6.7E02 6.7E02 anti-cKIT-HC-K360C-LC-K107C-Compound F 5.2E02 5.7E05 6.1E05 9.9E05 .sup.aName consists of a description of the mutated antibody and the name of the compound used in the chemical conjugation step. .sup.bThe highest concentration used in the assay was 6.7E02 M. IC.sub.50 values of 6.7E02 M therefore refer to inactivity of the ADC in the assay.

Example 8

Pharmacokinetic Study of Cys Mutant ADCs

[0438] 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 a usually hydrophobic drug payload to an antibody can 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 sites on clearance of multi-Cys-drug ADCs in vivo, pharmacokinetic studies in non-tumor bearing mice were carried out. To detect drug containing ADCs in murine plasma, an anti-MMAF antibody was generated. ELISA assays for the detection of ADCs were developed on a Gyros platform using an anti-human IgG (anti-hIgG) to capture human IgG molecules from the plasma and an anti-human IgG (anti-hIgG) antibody and an anti-MMAF antibody for signal generation in two separate assays. The two ELISA assays measure the serum concentration of the antibody and the intact ADC respectively as discussed in more detail below. Three mice per group were administered with a single dose of the Cys ADCs at 1 mg/kg. Eight plasma samples were collected over the course of three weeks and assayed by ELISA as described above. Standard curves were generated for each ADC separately using the same material as was injected into the mice. As measured by anti-hIgG ELISA, the Cys mutant ADCs (Tables 15 and 16) displayed a pharmacokinetic profile similar to unconjugated wild type antibodies, indicating that mutation and payload conjugation to these sites did not significantly affect the antibody's clearance. To determine the chemical stability of the linkage between the maleimide payload and the antibody at the various Cys sites during circulation in mouse, ADC concentrations as measured by the anti-MMAF ELISA assay and as measured by the anti-hIgG ELISA assay were compared to each other for ADCs prepared with Compound F which is readily detected with the anti-MMAF ELISA (Tables 15 and 16). For these ADCs, values for the area-under-the-plasma-concentration-versus-time-curve (AUC) were calculated from measurements with the anti-MMAF (AUC-MMAF) and the anti-hIgG ELISA (AUC-IgG). Previous results for similar analyses suggest uncertainties of >25%. Since the ratio should remain near 1 if no drug loss occurs, a ratio >0.7 indicates that within the accuracy of the measurement, little drug loss was observed after administration in mice for trastuzumab and anti-cKIT ADCs prepared with Compound F (Tables 15 and 16).

[0439] To further understand the retention of ADC drug payloads especially for payloads that are not detectable by the anti-MMAF ELISA (such as Compounds A-E), samples were analyzed by immuno-precipitation mass spectrometry (IP-MS). In particular, ADCs were affinity-purified from mouse serum collected through terminal bleeding and the drug payloads attached to ADCs were analyzed by MS analysis. In a typical process, 200 l of plasma was diluted with an equal amount of PBS containing 10 mM EDTA. To the dilution, 10 l of affinity resin (IgG Select Sepharose 6 Fast flow; GE Healthcare 17-0969-01; 50% slurry) was added. Incubation of the resin with the diluted plasma samples was performed for 1 hr at room temperature by applying mild agitation to avoid resin settling. The resin was then filtered off and washed two times with 200 l of PBS. To deglycosylate the antibody, 10 l of PNGase F (1 mg/mL, TBS pH 7.4, 2.5 mM EDTA, 50% Glycerol) diluted with 10 l of PBS was added to the resin and the mixtures were incubated for 2-3 hrs at 37 C. After PNGase F was removed by washing the affinity resin twice with 200 l PBS, the sample was eluted twice from the affinity resin by adding 20 l of 1% formic acid and filtering off the resin. The combined eluates were diluted with 20 l of 6 M guanidine hydrochloride and 5 l of reduction buffer (0.66 M TCEP, 3.3 M ammonium acetate, pH 5). To effectively reduce the antibody, samples were incubated for at least 30 min at room temperature before analysis. LCMS was performed with an Agilent Technologies 6550-iFunnel QTOF MS/Agilent 1260 HPLC system. A standard reversed-phase chromatography was used for sample desalting with a PLRS column (8 m, 2.150 mm, 1000 , Agilent) at a flow rate of 0.5 ml/min at 80 C. Elution was carried out using a linear gradient of 20%- to 60%-acetonitrile containing 0.1% formic acid in 6 min. Agilent Qualitative Analysis was used for processing of the spectral record and spectral deconvolution. For analysis the spectral record was summed over the time interval covering elution of all relevant species. Summed spectra were deconvoluted in charge state and images of the deconvoluted spectra were recorded. The values of peak intensity were extracted for assignable species. Assignments of DAR state and fragment species were made based on values of calculated mass from the sequence of the analyzed antibodies and the expected mass shifts of the conjugates with drug molecules. The average DAR was calculated using the relative peak heights of all DAR states across a distribution. Average antibody DAR was calculated as the sum of DARs from 2 average light chains and 2 average heavy chains.

[0440] The average DAR of ADCs purified from plasma after three weeks in mouse circulation, as measured by MS, was compared to the DAR in the original ADC preparations. Payload retention (Tables 15 and 16) was calculated from the ratio of the two DARs (DAR of ADC isolated from mouse plasma divided by the DAR of original ADC preparation), and represent the percentage of payloads retained on the ADC after three weeks in mouse circulation. Payload retention of ADCs as measured by MS is largely in agreement with results obtained by the aforementioned ELISA assay for ADCs prepared with Compound F (Tables 15 and Table 16). The data indicate a high degree of stability of the drug-antibody linkage during circulation in mice over a three week period for the Cys ADCs described herein

TABLE-US-00019 TABLE 15 Pharmacokinetic properties of anti-cKIT Cys mutant ADCs conjugated with various compounds. AUC ratio AUC (Payload Payload.sup.b AUC IgG.sup.c AUC/IgG Payload ADC name.sup.a (g/ml*h) (g/ml*h) AUC) retention.sup.d anti-cKit-HC-E152C-S375C-Compound A n.a. 7016 n.a. 0.82 anti-cKit-HC-E152C-S375C-Compound F 2565 3912 0.66 0.64 anti-cKit-HC-K360C-LC-K107C-Compound A n.a. 5112 n.a. 0.88 anti-cKit-HC-K360C-LC-K107C-Compound F 4582 5051 0.91 0.78 .sup.aName consists of a description of the mutated antibody and the name of the compound used in the chemical conjugation step. .sup.bAUC readout by anti-MMAF ELISA. .sup.cAUC readout by anti-IgG ELISA. .sup.dPayload retention as measured by IP-MS after 3 weeks of circulation in mouse. n.a: not applicable. Anti-MMAF ELISA does not detect payload.

TABLE-US-00020 TABLE 16 Pharmacokinetic properties of anti-Her2 Cys mutant ADCs conjugated with various compounds. AUC ratio AUC (Payload Payload.sup.b AUC IgG.sup.c AUC/IgG Payload ADC name.sup.a (g/ml*h) (g/ml*h) AUC) retention.sup.d trastuzumab-HC-E152C-S375C-Compound A n.a. 1859 n.a. 0.74 trastuzumab-HC-E152C-S375C-Compound B n.a. 2172 n.a. 0.73 trastuzumab-HC-E152C-S375C-Compound C n.a. 2506 n.a. 0.76 trastuzumab-HC-E152C-S375C-Compound F 1367 2414 0.57 0.50 trastuzumab-HC-K360C-LC-K107C- n.a. 3870 n.a. 0.86 Compound A trastuzumab-HC-K360C-LC-K107C- n.a. 3280 n.a. 0.91 Compound B trastuzumab-HC-K360C-LC-K107C- n.a. 3258 n.a. 0.84 Compound C trastuzumab-HC-K360C-LC-K107C- 2622 3842 0.68 0.82 Compound F .sup.aName consists of a description of the mutated antibody and the name of the compound used in the chemical conjugation step. .sup.bAUC readout by anti-MMAF ELISA. .sup.cAUC readout by anti-IgG ELISA. .sup.dPayload retention as measured by IP-MS after 3 weeks of circulation in mouse. n.a: not applicable. Anti-MMAF ELISA does not detect payload.

Example 9

Preparation and Trastuzumab and Anti-cKIT ADC Conjugated with Eg5 Inhibitor

[0441] Engineered Cys ADCs have been reported to be better tolerated in mice and rat animal models than ADCs made by conjugation to partially reduced native disulfides or through native lysine residues (Junutula et al., (2008) Nat Biotechnol. 26(8):925-932). To evaluate differences in in vivo efficacy between ADCs conjugated through engineered Cys antibodies and ADCs conjugated to partially reduced native disulfide bonds (Doronina et al., (2003) Nat. Biotechnol. 21, 778-784), Cys mutants of trastuzumab and the anti-cKIT antibody were expressed in 293 Freestyle cells and purified as described in Example 4 and ADCs were prepared as described in Example 5.

[0442] Eg5 linker-payload Compound A in Table 5 was conjugated to antibody anti-cKIT-HC-E152C-S375C double mutant (also referred to as cKITB, the immunoconjugates are referred to as cKitB-Cmpd A or cKitB-5B) and anti-cKIT-HC-K360C-LC-K107C double mutant (also referred to as cKitC, immunoconjugates are referred to as cKitC-Cmpd A or cKitC-5B) as well as wild type anti-cKIT antibody (immunoconjugates also referred to as cKitA-Cmpd A or cKitA-5B). (Residue Numbers are EU numbers). The sequences of the constant regions of the antibodies are set forth in Table 17.

TABLE-US-00021 TABLE17 Sequenceinformationforwildtypeandcys-substitutedconstant regionsofantibodies. SEQIDNO:150(Constantregionoftheheavychainwildtype ofantibodyanti-cKIT) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:149(Constantregionofthelightchainwildtype ofantibodyanti-cKIT) KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:50(Constantregionofthemutantheavychainof antibodyanti-cKITwithmutationHC-5375C) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:61(Constantregionofthemutantlightchainof antibodyanti-cKITwithmutationLC-K107C) CRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:131(Constantregionofthemutantheavychainof antibodyanti-cKITwithmutationsHC-E152C-5375C) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQIDNO:48(Constantregionofmutantheavychainantibody (anti-cKITwithmutationatHC-K360C) SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTCNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0443] Specifically, reoxidized antibodies were conjugated with Compound A by incubating 5 mg/ml antibody with 0.35 mM Compound A for 1 hour in 50 mM sodium phosphate buffer (pH 7.2). The completeness of the reaction was monitored by RP-HPLC and a DAR of 3.9 and 4.0 were obtained for the cKitB and cKitC conjugates, respectively. DAR measurements were further verified by MS. ADCs were shown to be potent and in vitro cell killing assays and had pharmacokinetics properties similar to unconjugated antibody in non-tumor bearing mice.

[0444] The ADC with Compound A conjugated to the native disulfide bonds of cKitA was prepared as follows in a 2-step process. The antibody at a concentration of 5-10 mg/ml in PBS containing 2 mM EDTA, was first partially reduced for 1 hour at 37 C. with 50 mM mercaptoethylamine (added as a solid). After desalting and addition of 1% w/v PS-20 detergent, the partially reduced antibody (1-2 mg/ml) was reacted overnight at 4 C. with an amount of 0.5-1 mg of compound A, dissolved at 10 mg/ml in DMSO, per 10 mg antibody. The ADC was purified by Protein A chromatography. After base-line washing with PBS, the conjugate was eluted with 50 mM citrate, pH 2.7, 140 mM NaCl, neutralized and sterile filtered. The average DAR was 3.2.

TABLE-US-00022 TABLE 18 Properties of three anti-cKIT ADCs conjugated with Compound A. ADC name.sup.a DAR.sup.b Aggregation.sup.c Anti-cKIT-Compound A (cKitA-5B) 3.2 0.8% anti-cKIT-HC-E152C-S375C-Compound 3.9 1.5% A (cKitB-5B) anti-cKIT-HC-K360C-LC-K107C- 4.0 3.2% Compound A (cKitC-5B) .sup.aName consists of a description of the mutated antibody and the name of the compound used in the chemical conjugation step. .sup.bDrug-to-antibody ratio according to reverse-phase HPLC or HIC. .sup.cAggregation measured by analytical size exclusion chromatography; includes dimeric and oligomeric species.

[0445] Similarly, immunoconjugates with the following combinations of payloads with anti-cKIT and trastuzumab mutated antibodies having cysteine substitutions were prepared and characterized by the same methods. Note that the engineered antibodies consistently provided DAR near 4, the expected loading if the four added cysteine residues per antibody complex are all conjugated to payload (Tables 18 and 19):

TABLE-US-00023 TABLE 19 Summary of anti-cKIT and trastuzumab Cys mutant ADCs with Eg5 inhibitor payloads. Conc. Endotoxin ADC name.sup.a (mg/ml) DAR.sup.b Aggregation (%).sup.c (Eu/mg) trastuzumab-HC-E152C-S375C- 2 3.9 3.4 <0.1 Compound A (TBS-5B) trastuzumab-HC-E152C-S375C- 2 3.8 2 <0.1 Compound B (TBS-5E) trastuzumab-HC-E152C-S375C- 2 4 5.1 <0.1 Compound C (TBS-5D) anti-cKIT-HC-E152C-S375C- 4 3.8 0 <0.1 Compound A (cKitB-5B) anti-cKIT-HC-E152C-S375C- 4 3.9 0.1 <0.1 Compound B (cKitB-5E) anti-cKIT-HC-E152C-S375C- 4 3.9 0.2 0.2 Compound C (cKitB-5D) trastuzumab-HC-K360C-LC-K107C- 4 3.8 0.4 <0.1 Compound A (5B) trastuzumab-HC-K360C-LC-K107C- 4 4 0 0.1 Compound B (5E) trastuzumab-HC-K360C-LC-K107C- 3 3.7 0.6 <0.1 Compound C (5D) .sup.aName consists of a description of the mutated antibody and the name of the compound used in the chemical conjugation step. .sup.bDrug-to-antibody ratio according to reverse-phase HPLC. .sup.cAggregation was measured analytical size exclusion chromatography; includes dimeric and oligomeric species.

Example 10

In Vitro Potency and In Vivo Efficacy of ADCs Prepared with Eg5 Inhibiting Payloads

[0446] Immunoconjugates were prepared from each of the Eg5 inhibiting linker-payload compounds shown in Table 5 conjugated with an anti-cKIT antibody (also referred to as cKitA) and HC-E152C-S375C Cys-mutated versions of anti-cKIT antibody (cKitB). The constant region for anti-cKIT (cKitA) wild type and Cys-substituted mutants are shown in Table 17 above. Conjugates having a drug to antibody ratio (DAR) between 3.5 and 4.0 were prepared for each payload by the methods described above. The immunoconjugates were tested for activity in a cell line expected to be recognized by antibodies to cKit.

[0447] FIG. 2 shows inhibition of cell growth by immunoconjugates with the HC-E152C-S375C Cys-substituted cKIT immunoconjugates comprising Compounds A, B, and C. Jurkat cells are a cKIT negative cell line, and were not sensitive to the three anti-cKIT (cKitA) immunoconjugates. However, proliferation of H526 cells, a cKIT positive cell line, was inhibited by all three anti-cKIT (cKitA) conjugates with IC.sub.50s ranging from 100 to 500 pM. The H526 cell line was selected as a xenograft model for in vivo efficacy studies.

[0448] In vivo xenograft tumor models simulate biological activity observed in humans and consist of 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) Cancer Res. 66:3351-3354).

[0449] 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). H526 cells were implanted in nu/nu mice subcutaneously (Morton and Houghton, Nat Protoc. 2007; 2:247-250). After the tumor size reached 200 mm.sup.3, ADCs were administered into the mice by i.v. injection in a single dose. Tumor growth was measured periodically after ADC injection. An example of such an in vivo efficacy study is shown in FIG. 3.

[0450] FIG. 3 summarizes the activity of two ADCs made with cysteine-engineered anti-cKIT antibodies, namely anti-cKIT-HC-E152C-S375C-Compound A (cKitB-5B) and anti-cKIT-HC-K360C-LC-K107C-Compound A (cKitC-5B), which inhibited growth of H526 tumor xenografts in mice at doses of 5 mg/kg (FIG. 3A) and 10 mg/kg (FIG. 3B). Anti-cKIT-Compound A (cKitA-5B) prepared with the wild type antibody through partial reduction, because of lower DAR, was administered at higher doses to match the molar payload dose. 6 mice were injected per group for each ADC tested. No significant weight loss was observed associated with the ADC treatment in any group suggesting low systemic toxicity.

[0451] The cysteine-engineered anti-cKIT ADCs of Compound A were more active than the ADC prepared through partial reduction of the wild type antibody Anti-cKIT-Compound A (cKitA-5B). Thus, while immunoconjugates of Eg5 inhibitors were active with various cKit antibodies including unmodified ones, this demonstrates that protein engineering to introduce new cysteine residues into the constant region and using the new cysteine residues as attachment points for the payload/linker group can provide improved immunoconjugates.

[0452] The Cys-substituted cKitA immunoconjugates were also tested in murine xenograft model. Both of the Cys substituted immunoconjugates showed greater activity than the nonsubstituted immunoconjugates, as measured by tumor volume post-implant.

Example 11

Dose Dependent In Vivo Efficacy of an Anti-Her2 ADC Conjugated with an Eg5 Inhibitor in the Her2 Positive MDA-MB-231 Clone 16 Breast Cancer Model in Mice

[0453] The anti-tumor efficacy of the anti-Her2 ADC trastuzumab-HC-E152C-S375C-Compound A was prepared by conjugating trastuzumab HC-E152C-S375C Cys mutant antibody with Eg5 inhibitor Compound A was evaluated in the Her2 positive MDA-MB-231 HER2 clone 16 breast cancer xenograft model. Female athymic nude-Foxn1 mice were implanted subcutaneously with 510.sup.6 cells containing 50% Matrigel (BD Biosciences) in phosphate-buffered saline (PBS) solution. The total injection volume containing cells in suspension was 200 l. Mice were enrolled in the study 13 days post implantation of tumor cells with average tumor volumes of 220 mm.sup.3. After being randomly assigned to one of eight groups (n=5/group), mice were administered a single i.v. dose of PBS, a non-specific isotype control-HC-E152C-S375C-Compound A (10 mg/kg) or trastuzumab-HC-E152C-S375C-Compound A (2.5, 5 or 10 mg/kg). Tumor volumes (FIG. 4) and body weights were measured at least twice weekly.

[0454] On Day 40 post-tumor cell implant, mice treated with a single administration of 2.5 mg/kg of trastuzumab-HC-E152C-S375C-Compound A had tumors that showed a percent mean change in tumor volume compared to the vehicle control (T/C) of 8.22%. Mice treated with a single administration of 5 mg/kg and 10 mg/kg of trastuzumab-HC-E152C-S375C-Compound A had tumors that showed a regression in volume of 74.14% and 76.35%, respectively, both of which were statistically different from the vehicle alone and non-specific isotype ADC controls (p<0.05, ANOVA, Tukey's post-hoc test). The treatments were well tolerated at all dose levels.

TABLE-US-00024 TABLE 20 TBS-HC-E152C-S375C-Compound A dose response in the Her2 positive MDA-MB-231clone 16, breast cancer model in mice on Day 40. Tumor Response Mean change of Host Response tumor Mean change Mean volume vs of tumor change of Survival Dose control Regression volume body weight (Survivors/ Drug (mg/kg) Schedule (T/C) (%) (%) (mm3 SEM) (% SEM) total) Vehicle 0 Single 100 1112.82 254.74 7.15 6.63 5/5 dose IV Isotype 10 Single 81.54 907.35 246.84 2.78 2.05 5/5 Control-HC- dose E152C-S375C- IV Compound A TBS-HC- 2.5 Single 8.22 91.47 99.08 2.92 0.80 5/5 E152C-S375C- dose Compound A IV TBS-HC- 5 Single 74.14 151.46 25.52 4.01 0.78 5/5 E152C-S375C- dose Compound A IV TBS-HC- 10 Single 76.35 163.29 20.63 0.52 1.80 5/5 E152C-S375C- dose Compound A IV

Example 12

In Vivo Efficacy of an Anti-Her2 ADC Conjugated with an Eg5 Inhibitor in the Her2 Positive MDA-MB-453 Human Breast Cancer Xenograft Mouse Model

[0455] The anti-tumor efficacy of the anti-Her2 trastuzumab-HC-E152C-S375C-Compound A ADC was also evaluated in the Her2 positive MDA-MB-453 human breast cancer xenograft model. Female SCID beige mice were implanted subcutaneously with 510.sup.6 cells containing 50% Matrigel (BD Biosciences) in phosphate-buffered saline (PBS) solution. The total injection volume containing cells in suspension was 200 l. Mice were enrolled in the study seven days post implantation of tumor cells with tumor volumes of approximately 168 mm.sup.3-216 mm.sup.3. After being randomly assigned to one of four groups (n=6/group), mice were administered a single i.v. dose of PBS, a non-specific isotype control-HC-E152C-S375C-Compound A (10 mg/kg) or trastuzumab-HC-E152C-S375C-Compound A (10 mg/kg). Tumor volumes (FIG. 5) and body weights were measured at least twice weekly.

[0456] On Day 45 post-implant, mice treated with trastuzumab-HC-E152C-S375C-Compound A (10 mg/kg) had tumors that showed a regression in volume of 71.2%, which was statistically different from the vehicle alone and non-specific isotype ADC controls (p<0.05, ANOVA, Tukey's post-hoc test). The treatments were well tolerated at all dose levels.

TABLE-US-00025 TABLE 21 ADC efficacy of of trastuzumab-HC-E152C-S375C-Compound A at 10 mg/kg in the Her2 positive MDA-MB-453 human breast cancer xenograft mouse model on Day 45. Tumor Response Mean change of tumor Host Response volume Mean vs Mean change change of control of tumor body Survival Dose (T/C) Regression volume weight (Survivors/ Drug (mg/kg) Schedule (%) (%) (mm3 SEM) (% SEM) total) Vehicle 0 Single 100 654 69.5 12 1.93 6/6 dose IV Isotype Control- 10 Single 128.3 827.9 96.7 11.1 0.85 6/6 HC-E152C- dose IV S375C- Compound A Trastuzumab-HC- 10 Single 71.2 138.5 22.2 11.7 3.8 6/6 E152C-S375C- dose IV Compound A

Example 13

In Vivo Efficacy of an Anti-Her2 ADC Conjugated with an Eg5 Inhibitor in the Her2 Positive HCC1954 Human Breast Cancer Xenograft Mouse Model

[0457] The anti-tumor efficacy of the anti-Her2 trastuzumab-HC-E152C-S375C-Compound A ADC was further evaluated in the Her2 positive HCC1954 breast cancer xenograft model. Female athymic nude-Foxn1 mice were implanted subcutaneously with 510.sup.6 cells containing 50% Matrigel (BD Biosciences) in phosphate-buffered saline (PBS) solution. The total injection volume containing cells in suspension was 200 l. Mice were enrolled in the study 11 days post implantation with tumor volumes of approximately 148 mm.sup.3-216 mm.sup.3. After being randomly assigned to one of four groups (n=6/group), mice were administered a single i.v. dose of PBS, a non-specific isotype control-HC-E152C-S375C-Compound A (10 mg/kg) or trastuzumab-HC-E152C-S375C-Compound A (10 mg/kg. Tumor volumes (FIG. 6) and body weights were measured at least twice weekly.

[0458] On Day 45 post-implant, mice treated with trastuzumab-HC-E152C-S372C-Compound A (10 mg/kg) had tumors that showed a regression in volume of 63.0%, which was statistically different from the vehicle alone and non-specific isotype ADC controls (p<0.05, ANOVA, Tukey's post-hoc test). The treatments were well tolerated at all dose levels.

TABLE-US-00026 TABLE 22 ADC efficacy of trastuzumab-HC-E152C-S375C-Compound A at 10 mg/kg in the Her2 positive HCC1954 human breast cancer xenograft mouse model on Day 45. Tumor Response Mean Host Response change of Mean tumor Mean change change of volume vs of tumor body Survival Dose control Regression volume weight (Survivors/ Drug (mg/kg) Schedule (T/C) (%) (%) (mm3 SEM) (% SEM) total) Vehicle 0 Single 100 555.2 122.5 5.9 1.4 6/6 dose IV Isotype control 10 Single 117.9 654.6 200.1 8.6 0.9 6/6 antibody-HC- dose IV E152C-S375C- Compound A trastuzumab- 10 Single 63.0 112.0 15.95 8.0 1.4 6/6 HC-E152C- dose IV S375C- Compound A

Example 14

In Vivo Efficacy Study Comparing Anti-cKIT Cys Mutant ADCs to ADCs Prepared by Partial Reduction of a Non-Engineered Antibody

[0459] The in vivo efficacy of two anti-cKIT ADCs: anti-cKIT-HC-E152C-S375C-Compound F and anti-cKIT-Compound F, were compared in the H526 xenograft mouse model (FIG. 7). The two ADCs were prepared with the same payload; Compound F (Table 5), conjugated to different Cys sites using two different methods. Conjugate anti-cKIT-HC-E152C-S375C-Compound F was prepared with a Cys mutant antibody, as described in Example 5 with Compound F conjugated to engineered Cys residues, HC-E152C and HC-S375C. Conjugate anti-cKIT-Compound F was prepared by applying the partial reduction method described in Example 9 to wild type anti-cKIT antibody with Compound F conjugated to native Cys residues. Anti-cKIT-Compound F had a slightly higher DAR (DAR 4.6) and aggregation (2.9%) than anti-cKIT-HC-E152C-S375C-Compound F (DAR 3.9, 0.6% aggregation). Pharmacokinetic studies in non-tumor bearing mice showed that the two ADCs retained the same payload to a very different extent during three weeks of circulation in mouse: As illustrated by ELISA (FIG. 8A, FIG. 8B) and as determined by IP-MS (see Example 8), anti-cKIT-HC-E152C-S375C-Compound F displayed much better payload retention (56%) than anti-cKIT-Compound F (20%).

[0460] In the H526 xenograft model, the same dosage of anti-cKIT-HC-E152C-S375C-Compound F is more efficacious in inhibiting tumors than anti-cKIT-Compound F (FIG. 7). Anti-Her2-HC-E152C-S375C-Compound F (see Table 12 for properties), whose antigen is not expressed in H526 cells, was included as control and did not show any tumor inhibiting activity.

Example 15

In Vivo Efficacy Study Comparing Anti-cKIT Cys Mutant ADCs Conjugated at Different Sites with Compound F and Compound A

[0461] In another example, the in vivo efficacy of anti-cKIT-HC-E152C-S375C-Compound F, anti-cKIT-HC-K360C-LC-K107C-Compound F, anti-cKIT-HC-E152C-S375C-Compound A, and anti-cKIT-HC-K360C-LC-K107C-Compound A ADCs were compared in the H526 xenograft model (FIG. 9). The two payloads, Compound F and Compound A, were conjugated to different Cys sites using two different antibodies as described in Example 7. The properties of the ADCs are summarized in Table 12. The DAR measured was close to the theoretical DAR of 4 for all four conjugates and little aggregation was observed for the resulting ADCs (Table 12). Single doses of 3.5 mg/kg of the ADCs were injected i.v. into animals bearing H526 tumors. The results of tumor volume measurements in the H526 xenograft model are shown in FIG. 9. In this model, the same dosage of anti-cKIT-Compound F ADC was more efficacious in inhibiting tumors than ADCs prepared with Compound A. There is not statistically significant difference in tumor inhibiting activity between ADCs conjugated to the two different sets of Cys mutants.

Example 16

Hydrophobicity of Trastuzumab ADCs Conjugated with Compound G

[0462] To further optimize the selection of Cys mutants and mutant combinations for the preparation of ADCs with DAR 2, 4, 6 and greater, the properties of trastuzumab Cys mutant ADCs were analyzed with respect to hydrophobicity. Cys mutants ADCs conjugated with Compound G (MC-MMAF) were prepared as described above. The final DAR as determined experimentally as described were generally close to the target and are listed in Table 23 below. The hydrophobicity of these ADCs was measured by hydrophobic interaction chromatography as follows.

[0463] Analytical HIC data for trastuzumab Cys-MMAF ADCs were collected using a Tosoh Bioscience (King of Prussia, Pa., USA) TSKgel Butyl-NPR column (100 mm4.6 mm, 2.5 m) installed on an Agilent 1260 LC system (Santa Clara, Calif., USA). The method consisted of a binary gradient of buffer A (20 mM His-HCl, 1.5 M ammonium sulfate, pH 6.0) and buffer B (20 mM His-HCl, 15% isopropanol, pH 6.0). Samples were prepared by diluting approximately 20 g of antibody (PBS) with 0.5 volume of 3 M ammonium sulfate. The gradient consisted of 5 min holding at 100% A, followed a linear gradient of 0 to 100% B over 30 min, a return to 100% A over 5 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 and analyzed using Chromelion software (Dionex).

[0464] Surprisingly, it was observed that the retention times of the DAR 4 ADCs varied greatly although the only difference is the site of Compound G attachment. In addition, the range of retention times overlapped substantially with the range observed for DAR 2 ADCs included for comparison (Table 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 (retention time=45 min) which is to be expected when a hydrophobic drug molecule such as Compound G is attached to an antibody. However, attaching the payload at different sites increases the hydrophobicity of the ADC to various extents.

TABLE-US-00027 TABLE 23 DAR and analytical HIC retention times for trastuzumab-Cys-Compound G ADCs with DAR = 2, 4, or 6. Retention time Trastuzumab Cys mutation site DAR (min) HC-E152C 1.8 15.5 HC-K334C-P396C 3.5 15.8 HC-P396C 2.0 15.9 HC-E152C-P396C 3.3 16.1 HC-E152C-LC-E165C 2.9 16.2 HC-A327C-A339C 3.5 16.2 LC-E165C-HC-P396C 3.4 16.3 HC-Y391C 2.0 16.4 HC-E152C-S375C 3.7 16.5 LC-E165C-HC-S375C 4.0 16.7 HC-E152C-A339C 3.7 17.1 HC-E152C-LC-R142C 3.8 17.1 HC-A339C-S375C 3.3 17.2 HC-E333C 1.9 17.2 HC-E152C-A327C 3.7 17.3 LC-E165C-HC-L174C 3.4 17.4 HC-S375C-Y391C 3.2 17.4 HC-A339C-P396C 3.6 17.5 LC-S159C-HC-E152C 3.8 17.5 HC-Y373C 2.0 17.7 LC-E165C-HC-K334C-S375C 6.0 18.1 HC-A327C-S375C 3.8 18.2 LC-E165C-HC-K334C-K392C 5.8 18.2 HC-P247C 2.0 18.9 LC-K107C-HC-K360C 3.9 21.5

[0465] 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: 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-P247C where retention time of almost 19 min were measured. Conversely, if the payload is attached at an interior site such as the cavity formed between variable and CH1 domains (for example, HC-E152C) or the large opening between CH2 and CH3 domains of the antibody (for example, HC-P396C), the HIC retention time is below 16 min because the payload is partially sequestered from interacting with solvent and the HIC column. Likewise, for DAR 4 ADCs that include two relatively interior sites (for examples HC-E152C-P396C and HC-E152C-S375C), the retention time remains short, on the order of 15.5-16.5 min, while DAR 4 ADCs that include very exposed sites (for example, LC-K107C-HC-K360C) can show retention times greater than 21 min.

[0466] Reducing hydrophobicity of a protein drug including ADCs is generally considered beneficial because it may reduce aggregation and clearance from circulation. 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 more drugs are attached per antibody, or when particularly hydrophobic payloads are used.

Example 17

Hydrophobicity of Anti-Her2 Cys Mutant ADCs Conjugated with Various Compounds

[0467] A subset of the trastuzumab-HC-E152C-S375C and trastuzumab-HC-K360C-LC-K107C ADCs prepared in Example 7 (see Table 12 for properties) were also characterized by hydrophobic interaction chromatograpy as described in detail below (Table 24). ADCs conjugated to the combination of exposed Cys residues (positions HC-K360C-LC-K107C) are more hydrophobic than ADCs with drugs attached to the HC-E152C-S375C antibody. The effect is more pronounced for the Eg5 inhibitor payloads Compound A and Compound C compared to the cytotoxic peptide Compound F.

[0468] As discussed in Example 16, attaching drugs at sites where they are sequestered from solvent interactions such as HC-E152C-S375C appears to increase the hydrophobicity of the antibody to a lesser degree than when attached at more solvent exposed positions such as HC-K360C and LC-K107C. Although beneficial for many applications, particularly for the attachment of hydrophobic payloads, conjugating payloads at more solvent exposed positions will have beneficial utility in other applications.

TABLE-US-00028 TABLE 24 Hydrophobicity scores of various Cys mutant anti-Her2-ADCs conjugated with different payloads ADC name.sup.a Hydrophobicity score.sup.b trastuzumab-HC-E152C-S375C-Compound F 0.91 trastuzumab-HC-E152C-S375C-Compound A 0.90 trastuzumab-HC-E152C-S375C-Compound C 0.87 trastuzumab-HC-K360C-LC-K107C-Compound F 0.51 trastuzumab-HC-K360C-LC-K107C-Compound A 0.33 trastuzumab-HC-K360C-LC-K107C-Compound C 0.31 .sup.aName consists of a description of the mutated antibody and a description of the compound used in the chemical conjugation step. .sup.bHydrophobic Interaction Chromatography (HIC) measurements: The separation of the different species was carried out on a TSKgel Butyl-NPR column (4.6 mm ID 35 mm L, Tosoh Bioscience) connected to an Agilent 1260 Infinity LC System (Agilent Technologies). The system was equilibrated firstly with mobile phase B (17 mM His/HCl, pH 6.0 containing 15% isopropanol) and subsequently with mobile phase A (20 mM His/HCl pH 6.0, containing 1.5M (NH.sub.4).sub.2SO.sub.4) until a stable baseline was reached. 10 to 50 ug of sample, stored at 4 C. in the auto-sampler, was injected and separated at a flow rate of 1.0 mL/min at a constant temperature of 25 C. Elution of species with different hydrophobicity was achieved using a gradient from 100% mobile phase A to 100% mobile phase B within 30 column volumes. Eluting species were detected at 280 nm and the retention time of the peak maximum was used to calculate the hydrophobicity index. This index is determined with respect to the linear regression (plot retention time vs. hydrophobicity index) of three reference molecules of defined hydrophobicity. This procedure allows compensating for potential run-to-run variability and variations due to differences between column batches and is independent of the exact absolute ammonium sulfate concentration. The lower the hydrophobicity index (=late elution from HIC), the more hydrophobic is the molecule and the higher is the risk of unfavorable behavior during production or storage of the drug substance and drug product.

[0469] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.