SEQUENCE LISTING

In accordance with 37 CFR 1.52(e)(5), a Sequence Listing in the form of an ASCII text file (entitled “PC72358A_Seq_Listing_ST25.txt,” created on Aug. 14, 2018, and 40.0 KB in size) is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to antibodies, and antigen-binding fragments thereof, engineered to introduce amino acids for site-specific conjugation.

BACKGROUND OF THE INVENTION

Antibodies have been conjugated to a variety of cytotoxic drugs, including small molecules that alkylate DNA (e.g., duocarmycin and calicheamicin), disrupt microtubules (e.g., maytansinoids and auristatins) or bind DNA (e.g., anthracyclins). One such antibody-drug conjugate (ADC) comprising a humanized anti-CD33 antibody conjugated to calicheamicin—Mylotarg™ (gemtuzumab ozogamicin)—has been approved for treating acute myeloid leukemia. Adcetris™ (brentuximab vedotin), an ADC comprising a chimeric antibody to CD30 conjugated to the auristatin monomethyl auristatin E (MMAE) has been approved for treatment of Hodgkin's lymphoma and anaplastic large cell lymphoma.

Although ADCs hold promise for cancer therapy, cytotoxic drugs are generally conjugated to the antibodies via lysine side chains or by reducing inter-chain disulfide bonds present in the antibodies to provide activated cysteine sulfhydryl groups. This non-specific conjugation approach, however, has numerous drawbacks. For example, drug conjugation to antibody lysine residues is complicated by the fact that there are many lysine residues (˜30) in an antibody available for conjugation. As a result, lysine conjugation often generates a very heterogeneous profile, and this may make the drug to antibody ratio (DAR) sub-optimally high. Furthermore, many lysines are located in critical antigen binding sites of CDR region and drug conjugation may lead to a reduction in antibody affinity. On the other hand, while thiol mediated conjugation mainly targets the eight cysteines involved in hinge disulfide bonds, it is still difficult to predict and identify which four of eight cysteines are consistently conjugated among the different preparations.

Recently, genetic engineering of free cysteine residues has enabled site-specific conjugation with thiol-based chemistries. The site-specific ADCs have homogeneous profiles and well-defined conjugation sites, and showed potent in vitro cytotoxicity and strong in vivo antitumor activity.

WO 2013/093809 discloses engineered antibody constant regions (Fc, Cγ, Cκ, Cλ), or a fragment thereof, that comprise amino acid substitutions at specific sites to introduce a cysteine residue for conjugation. A number of Cys-mutation sites in IgG heavy chain and lambda/kappa light chain constant regions are disclosed.

The success of using introduced Cys residues for site-specific conjugation relies on the ability to select sites in which Cys-substitution does not alter protein structure or function. Further, using different conjugation sites can result in different characteristics, such as biological stability of the ADC. Therefore, a site-specific conjugation strategy which generates an ADC with a defined conjugation site and desired ADC characteristics would be highly useful.

SUMMARY OF THE INVENTION

The invention relates to polypeptides, antibodies, and antigen-binding fragments thereof, that comprise an engineered cysteine for site-specific conjugation.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (E).

E1. An antibody drug conjugate of formula Ab-(L-D), wherein:

(a) Ab is an antibody or an antigen binding fragment comprising an antibody heavy chain constant domain containing an engineered cysteine residue at position 334, according to the numbering of the Eu index of Kabat; and

(b) L-D is a linker-drug moiety that is attached to the Ab via the engineered cysteine residue, wherein L is a linker and D is a drug.

E2. An antibody drug conjugate of formula Ab-(L-D), wherein:

(a) Ab is an antibody or an antigen binding fragment comprising an antibody heavy chain constant domain containing an engineered cysteine residue at a position corresponding to residue 104 of SEQ ID NO: 25, when said constant domain is aligned with SEQ ID NO:25; and
(b) L-D is a linker-drug moiety that is attached to the Ab via the engineered cysteine residue, wherein L is a linker and D is a drug.
E3. An antibody drug conjugate of formula Ab-(L-D), wherein:
(a) Ab is an antibody or an antigen binding fragment comprising an antibody heavy chain constant domain containing an engineered cysteine residue at position 392, according to the numbering of the EU index of Kabat; and
(b) L-D is a linker-drug moiety that is attached to the Ab via the engineered cysteine residue, wherein L is a linker and D is a drug.
E4. An antibody drug conjugate of formula Ab-(L-D), wherein:
(a) Ab is an antibody or an antigen binding fragment comprising an antibody heavy chain constant domain containing an engineered cysteine residue at a position corresponding to residue 162 of SEQ ID NO: 26, when said constant domain is aligned with SEQ ID NO: 26; and
(b) L-D is a linker-drug moiety that is attached to the Ab via the engineered cysteine residue, wherein L is a linker and D is a drug.
E5. The antibody drug conjugate of any one of E1-E4, wherein said constant domain comprises an IgG, IgA, IgD, IgE, or IgM heavy chain domain.
E6. The antibody drug conjugate of E1-E5, wherein said constant domain comprises an IgG1, IgG2, IgG3, or IgG4heavy chain domain.
E7. The antibody drug conjugate of E1-E5, wherein said constant domain comprises an IgA1or IgA2heavy chain domain.
E8. The antibody drug conjugate of any one of E1-E7, wherein said constant domain is a human antibody constant domain.
E9. The antibody drug conjugate of any one of E1-E8, wherein said constant domain further comprises one or more engineered cysteine residues at a position selected from the group consisting of: 118, 246, 249, 265, 267, 270, 276, 278, 283, 290, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 375, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 according to the numbering of the Eu index of Kabat.
E10. The antibody drug conjugate of any one of E1-E9, wherein said constant domain further comprises one or more engineered cysteine residues at a position selected from the group consisting of: 290, 334, 347, and 392 according to the numbering of the Eu index of Kabat.
E11. The antibody drug conjugate of any one of E1-E10, wherein said constant domain comprises an engineered cysteine residue at position 334 and at position 392, according to the numbering of the Eu index of Kabat.
E12. The antibody drug conjugate of E9-E11, wherein said constant domain comprises an IgG heavy chain CH2domain and an IgG heavy chain CH3domain.
E13. The antibody drug conjugate of any one of E1-E12, wherein said Ab further comprises an antibody light chain constant region comprising (i) an engineered cysteine residue at position 110, 111, 125, 149, 155, 158, 161, 183, 185, 188, 189, 191, 197, 205, 206, 207, 208, 210, or any combination thereof, according to the numbering of Kabat; (ii) an engineered cysteine residue at a position corresponding to residue 4, 42, 76, 81, 100, 103, or any combination thereof, of SEQ ID NO:30, when said constant domain is aligned with SEQ ID NO:30 (kappa light chain); or (iii) an engineered cysteine residue at a position corresponding to residue 4, 5, 19, 43, 49, 52, 55, 78, 81, 82, 84, 90, 96, 97, 98, 99, 101, or any combination thereof, of SEQ ID NO:31, when said constant domain is aligned with SEQ ID NO:31 (lambda light chain).
E14. The antibody drug conjugate of E13, wherein said light chain constant region comprises (i) an engineered cysteine residue at position 183, according to the numbering of Kabat; or (ii) an engineered cysteine residue at a position corresponding to residue 76 of SEQ ID NO:30, when said constant domain is aligned with SEQ ID NO:30.
E15. The antibody drug conjugate of E13 or E14, wherein said light chain constant region comprises a kappa light chain constant domain (CLκ).
E16. The antibody drug conjugate of E13 or E14, wherein said light chain constant region comprises a lambda light chain constant domain (CLλ).
E17. The antibody drug conjugate of any one of E1-E16, wherein said antibody or antigen binding fragment thereof is selected from the group consisting of a chimeric antibody, a human antibody, and a humanized antibody.
E18. The antibody drug conjugate of any one of E1-E17, wherein said antibody or antigen binding fragment binds to an antigen disclosed in paragraphs [54]-[58].
E19. The antibody drug conjugate of any one of E1-E17, wherein said antibody or antigen binding fragment binds to an antigen selected from the group consisting of: HER2, HER3, HER4, CD22, and CD33.
E20. The antibody drug conjugate of any one of E1-E19, wherein said antibody or antigen binding fragment binds to CD33.
E21. The antibody drug conjugate of any one of E1-E20, wherein said antibody comprises
(i) a heavy chain variable region (VH) that comprises:

(a) a VH complementarity determining region one (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 5 or 6,

(b) a VH CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9 or 10,

(c) a VH CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and

(ii) a light chain variable region (VL) that comprises:

(a) a VL complementarity determining region one (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 17,

(b) a VL CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19; and

(c) a VL CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

E22. The antibody drug conjugate of any one of E1-E21, comprising (i) a VH that comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1; and (ii) a VL that comprises an amino acid sequence at least 90% identical to SEQ ID NO: 3.

E23. The antibody drug conjugate of any one of E1-E22, comprising (i) a heavy chain that comprises an amino acid sequence of SEQ ID NOs: 32, 33, or 34 or an amino acid sequence at least 90% identical to SEQ ID NOs: 32, 33 or 34; and (ii) a light chain that comprises an amino acid sequence of SEQ ID NO: 23; or an amino acid sequence at least 90% identical to SEQ ID NO: 23.
E24. The antibody drug conjugate of any one of E1-E23, wherein the percent of drug-to-antibody ratio (DAR) loss in plasma, at 37° C. under 5% CO2at 72 hours, is no more than about 20%, no more than about 15%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, or no more than about 1%.
E25. The antibody drug conjugate of any one of E1-E24, wherein the percent of drug-to-antibody ratio (DAR) loss in plasma, at 37° C. under 5% CO2at 72 hours is no more than about 15%.
E26. The antibody drug conjugate of any one of E1-E24, wherein the percent of drug-to-antibody ratio (DAR) loss in plasma, at 37° C. under 5% CO2at 72 hours is no more than about 5%.
E27. The antibody drug conjugate of any one of E1-E26, wherein the linker comprises valine-citrulline (val-cit; vc), 6-maleimidocaproyl (mc), methoxy-polyethylene glycol maleimide 6 (MalPeg6), p-aminobenzylcarbamate (PABC), dimethylaminoethanol (DMAE), maleimidopropanoyl (MP), hydrolyzed Peg-maleimides, m(H20)c, m(H20)cvc, alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-Succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1carboxylate (SMCC), N-Succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), or 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB) or a combination thereof.
E28. The antibody drug conjugate of any one of E1-E27, wherein the linker comprises a combination of MalPeg6, vc, PABC, and DMAE.
E29. The antibody drug conjugate of any one of E1-E28, wherein the linker comprises MalPeg6-vc-PABC-DMAE.
E30. The antibody drug conjugate of any one of E1-E29, wherein the drug is selected from the group consisting of: a cytotoxic agent, a cytostatic agent, a chemotherapeutic agent, a toxin, a radionuclide, a DNA, an RNA, an siRNA, a microRNA, a peptide nucleic acid, a non-natural amino acid, a peptide, an enzyme, a fluorescent tag, biotin, and any combination thereof.
E31. The antibody drug conjugate of any one of E1-E30, wherein the drug is a DNA alkylating agent.
E32. The antibody drug conjugate of any one of E1-E31, wherein the drug is a DNA minor groove binding alkylating agent.
E33. The antibody drug conjugate of any one of E1-E32, wherein the drug is a CPI dimer, a CTI dimer, or a CBI dimer.
E34. The antibody drug conjugate of any one of E1-E33, wherein the drug is a CPI dimer.
E35. The antibody drug conjugate of any one of E1-E34, wherein the drug is a CPI dimer and the CPI dimer is a compound of the formula:
F1-DL-F2(Formula I)
or a pharmaceutically acceptable salt or solvate thereof, wherein:
F1is a CPI monomer comprising:


wherein
R1is H, —OH, —O-acyl, azido, halo (F, Cl, Br, I), sulfonate (—OSO2R), cyanate, thiocyanate, isocyanate, or thioisocyanate;
R2is R2is H, —OH, —SH, NHR, acyl, acetate, phosphate, glucuronide, or galactoside;
R3is H, —C1-C5substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, —C(O)OR, —C(O)SR, —C(O)NHN(R)2, or —C(O)N(R)2;
F2is a CPI monomer comprising:


wherein
R4is H, —OH, —O-acyl, azido, halo (F, Cl, Br, I), sulfonate, cyanate, thiocyanate, isocyanate, or thioisocyanate;
R5is H, —OH, —SH, NHR, acyl, acetate, phosphate, glucuronide, or galactoside;
R6is H, —C1-C5substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, —C(O)OR, —C(O)SR, —C(O)NHN(R)2, or —C(O)N(R)2;
R is selected from the group consisting of H, —C1-C20alkyl, —C2-C6alkenyl, —C2-C6alkynyl, halo, hydroxyl, alkoxy, —NH2, —NH(C1-C8alkyl), —N(C1-C8alkyl)2, —NO2, —C6-C14aryl and —C6-C14heteroaryl, wherein two or more R optionally join to form a ring or rings, and wherein said —C6-C14aryl and —C6-C14heteroaryl are optionally substituted with 1 to 5 substituents independently from —C1-C10alkyl, —C1-C10alkoxy, halo, —C1-C10alkylthio, trifluoromethyl, —NH2, —NH(C1-C8alkyl), —N(C1-C8alkyl)2, —C1-C10alkyl-N(C1-C8alkyl)2, —C1-C3alkylthio, —NO2, or —C1-C10heterocyclyl for each ring system in which R appears;
DL is a dimer-linker comprising:
R7—X—R8, wherein
R7and R8are each independently selected from a direct bond, carbonyl, or a carbonyl acyl group bonded to F1or F2at the free nitrogen atom of the saturated five-membered ring; and X is a —C1-C20substituted or unsubstituted alkyl chain, a —C1-C20substituted or unsubstituted fused or bridged cycloalkyl, or a substituted or unsubstituted saturated or aromatic heterocyclic ring comprised of from 3-20 atoms selected from C, N, O, and/or S.
E36. The antibody drug conjugate of E35, wherein the CPI dimer is:


E37. The antibody drug conjugate of E36, wherein R1is a halo (F, Cl, Br, I) or sulfonate (—OSO2R), R2is —OH, phosphate, glucuronide or galactoside, R3is a C1-C5unsubstituted or substituted alkyl, R4is a halo (F, Cl, Br, I) or suphonate (—OSO2R), R5is —OH, phosphate, glucuronide or galactoside, and R6is a C1-C5unsubstituted or substituted alkyl.
E38. The antibody drug conjugate of E37, wherein R1is a halo (F, Cl, Br, I), R2is—OH or phosphate, R3is a C3or a C5unsubstituted or substituted alkyl, R4is a halo (F, Cl, Br, I), R5is —OH or phosphate, and R6is a C3or a C5unsubstituted or substituted alkyl.
E39. The antibody drug conjugate of E38, wherein the CPI dimer is selected from the group consisting of:


E40. The antibody drug conjugate of any one of E35-E39, wherein R7and R8each independently is a carbonyl, and X is a —C1-C20substituted or unsubstituted alkyl chain, a —C1-C20substituted or unsubstituted fused or bridged cycloalkyl, or a substituted or unsubstituted saturated or aromatic heterocyclic ring comprised of from 3-20 atoms selected from C, N, O, and/or S.
E41. The antibody drug conjugate of E40, wherein R7and R8each independently is a carbonyl, and X is a —C5-C10substituted or unsubstituted alkyl chain, a —C5-C10substituted or unsubstituted fused or bridged cycloalkyl, or a substituted or unsubstituted saturated or aromatic heterocyclic ring comprised of from 5-10 atoms selected from C, N, O, and/or S.
E42. The antibody drug conjugate of E41, wherein R7and R8each independently is a carbonyl, and X is a C5or a C8substituted or unsubstituted alkyl chain, a C5or a C8substituted or unsubstituted fused or bridged cycloalkyl, or a substituted or unsubstituted saturated or aromatic heterocyclic ring comprised of 5 or 8 atoms selected from C, N, O, and/or S.
E43. The antibody drug conjugate of any one of E1-E42, wherein the drug is a CPI dimer and is selected from the group consisting of:


E44. The antibody drug conjugate of any one of E1-E43, wherein the drug is a CPI dimer and is selected from the group consisting of:


E45. The antibody drug conjugate of any one of E1-E44, wherein the linker comprises MalPeg6-vc-PABC-DMAE, and wherein D is a CPI dimer selected from the group consisting of:


E46. The antibody drug conjugate of E33, wherein the drug is a CTI dimer, and the CTI dimer is selected from the group consisting of:


E47. The antibody drug conjugate of E33, wherein the drug is a CBI dimer and the CBI dimer is selected from the group consisting of:


E48. The antibody drug conjugate of E47, wherein the CBI dimer is:


E49. The antibody drug conjugate of any one of E1-E48, wherein L-D is selected from the group consisting of:(i) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(5-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}thiophen-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide;(ii) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(5-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}thiophen-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](ethyl)amino}ethyl) (ethyl)carbamoyl]oxy}methyl) phenyl]-L-ornithinamide;(iii) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-(4-{7-[({(1S)-1-(chloromethyl)-3-[(5-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}thiophen-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl]-4-(2-methoxyethyl)-3-oxo-2,10-dioxa-4,7-diazaundec-1-yl}phenyl)-L-ornithinamide;(iv) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N-(4-{7-[({(1S)-1-(chloromethyl)-3-[(5-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}thiophen-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl]-4-(2-methoxyethyl)-3-oxo-2,10-dioxa-4,7-diazaundec-1-yl}phenyl)-L-alaninamide;(v) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(4-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}pentacyclo[4.2.0.02,5.03,8.04,7]oct-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide;(vi) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(3-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide;(vii) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide;(viii) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(8S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4 yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)-phenyl]-L-ornithinamide;(ix) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-6-{5-[(8S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-5-oxopentanoyl}-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide; and(x) N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-6-{(8S)-8-(chloromethyl)-4-(beta-D-glucopyranuronosyloxy)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-5-oxopentanoyl}-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl) phenyl]-L-ornithinamide.
E50. The antibody drug conjugate of E49, wherein said L-D comprises N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide.
E51. The antibody drug conjugate of any one of E1-E50, wherein drug-to-antibody ratio (DAR) at position 334 and/or at position 392 in plasma, at 37° C. at 72 hours is at least 1.6, at least 1.7, at least 1.8, at least 1.9 or at least 2.
E52. The antibody drug conjugate of any one of E1-E51, wherein said antibody drug conjugate has a melting transition temperature greater than at least 60° C., at least 65° C., at least 70° C., at least 75° C., at least 80° C., or at least 85° C.
E53. The antibody drug conjugate of E52, wherein said antibody drug conjugate has a melting transition temperature greater than about 65° C.
E54. The antibody drug conjugate of any one of E1-E53, wherein said antibody drug conjugate has an IC50 value of no more than about 180 ng/ml, no more than about 160 ng/ml, no more than about 150 ng/ml, no more than about 100 ng/ml, no more than about 50 ng/ml, no more than about 25 ng/ml, no more than about 20 ng/ml, no more than about 15 ng/ml, no more than about 10 ng/ml, no more than about 5 ng/ml, or no more than about 2.5 ng/ml.
E55. The antibody drug conjugate of E54, wherein said antibody drug conjugate has an IC50 value of no more than about 10 ng/ml, no more than about 5 ng/ml, or no more than about 2.5 ng/ml.
E56. The antibody drug conjugate of E54 or E55, wherein said antibody or antigen binding fragment thereof binds to CD33 and said IC50 values are determined in CD33 expressing cells.
E57. The antibody drug conjugate of any one of E1-E56, wherein said antibody drug conjugate reduces mean tumor volume to less than about 150 mm3, less than about 125 mm3, less than about 100 mm3, less than about 75 mm3by about day 4, about day 6, about day 8, about day 10, about day 12, about day 14, about day 16, or about day 18 of treatment with said conjugate in a HL60 AML tumor xenograft model.
E58. The antibody drug conjugate of any one of E1-E56, wherein said antibody drug conjugate reduces mean tumor volume by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to mean tumor volume in untreated controls in a HL60 AML tumor xenograft model.
E59. The antibody drug conjugate of any one of E1-E56, wherein said antibody drug conjugate reduces mean tumor volume to less than about 150 mm3, less than about 125 mm3, less than about 100 mm3, less than about 75 mm3by about day 4, about day 6, about day 8, about day 10, about day 12, about day 14, about day 16, or about day 18 of treatment with said conjugate in a TF1 AML tumor xenograft model.
E60. The antibody drug conjugate of any one of E1-E56, wherein said antibody drug conjugate reduces mean tumor volume by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to mean tumor volume in untreated controls in a TF1 AML tumor xenograft model.
E61. The antibody drug conjugate of any one of E1-E56, wherein said antibody drug conjugate reduces mean tumor volume to less than about 150 mm3, less than about 125 mm3, less than about 100 mm3, less than about 75 mm3by about day 4, about day 6, about day 8, about day 10, about day 12, about day 14, about day 16, or about day 18 of treatment with said conjugate in a Her2 expressing tumor xenograft model.
E62. The antibody drug conjugate of any one of E1-E56, wherein said antibody drug conjugate reduces mean tumor volume by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to mean tumor volume in untreated controls in a Her2 expressing tumor xenograft model.
E63. A pharmaceutical composition comprising the antibody drug conjugate of any one of E1-E62; and a pharmaceutically acceptable carrier.
E64. A method of treating cancer, an autoimmune disease, an inflammatory disease, or an infectious disease, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody drug conjugate of any one of E1-E62, or the composition of E63.
E65. The antibody drug conjugate of any one of E1-E62, or the composition of E63, for use in treating cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.
E66. Use of the antibody drug conjugate of any one of E1-E62, or the composition of E63, for treating cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.
E67. Use of the antibody drug conjugate of any one of E1-E62, or the composition of E63, in the manufacture of a medicament for treating cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.
E68. A nucleic acid encoding the antibody moiety of the antibody drug conjugate of any one of E1-E62.
E69. The nucleic acid of E68, wherein the nucleic acid encodes any one of SEQ ID NO: 1-34.
E70. A host cell comprising the nucleic acid of E68 or E69.
E71. A method of producing an antibody, comprising culturing the host cell of E70 under suitable conditions for expressing said antibody, and isolating said antibody.
E72. A method of making an antibody drug conjugate comprising conjugating an antibody or an antigen binding fragment comprising an antibody heavy chain constant domain containing an engineered cysteine residue at position 334 and/or at position 392, according to the numbering of the Eu index of Kabat to a linker and a drug so as to form an antibody drug conjugate as set forth in any one of E1-E62.
E73. The method of E72, wherein the antibody is conjugated to the linker which is then conjugated to the drug.
E74. The method of E72, wherein the linker is conjugated to the drug which is then conjugated to the antibody.

