Source: http://www.google.fr/patents/US7989598
Timestamp: 2016-07-27 03:44:33
Document Index: 299707329

Matched Legal Cases: ['Application No. 60', 'Application No. 2006', 'Application No. 8319', 'Application No. 2004282491', 'Application No. 10184672', 'Application No. 04793896', 'Application No. 8319']

Brevet US7989598 - Method of targeting specific cell populations using cell-binding agent ... - Google�BrevetsRecherche Images Maps Play YouTube Actualit�s Gmail Drive Plus »Connexion BrevetsThe present invention discloses a method for targeting maytansinoids to a selected cell population, the method comprising contacting a cell population or tissue suspected of containing the selected cell population with a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is...http://www.google.fr/patents/US7989598?utm_source=gb-gplus-shareBrevet US7989598 - Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates and methods of making said conjugates Recherche avanc�e dans les brevets Num�ro de publicationUS7989598 B2Type de publicationOctroi Num�ro de demandeUS 11/927,251 Date de publication2 ao�t 2011 Date de priorit�10 oct. 2003�tat de paiement des fraisPay�Autre r�f�rence de publicationCA2542128A1, CN103223174A, EP1689846A2, EP1689846A4, EP1689846B1, EP2596803A2, EP2596803A3, EP2596804A2, EP2596804A3, EP2612682A2, EP2612682A3, US8088387, US8163888, US8198417, US8563509, US8685920, US20050169933, US20080114153, US20080145374, US20080171856, US20080171865, US20110281856, US20120226025, US20140154804, WO2005037992A2, WO2005037992A3 Num�ro de publication11927251, 927251, US 7989598 B2, US 7989598B2, US-B2-7989598, US7989598 B2, US7989598B2 InventeursRita M. Steeves, Ravi V. J. Chari, Walter A. Blattler Cessionnaire d'origineImmunogen Inc.Exporter la citationBiBTeX, EndNote, RefManCitations de brevets (53), Citations hors brevets (20), R�f�renc� par (15), Classifications (11), �v�nements juridiques (5) Liens externes: USPTO, Cession USPTO, EspacenetMethod of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates and methods of making said conjugates
US 7989598 B2 R�sum�
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1. A process of making a cell-binding agent maytansinoid conjugate, the process comprising:
(a) modifying the cell-binding agent with a cross-linking agent, and
(b) conjugating the modified cell-binding agent with a thiol-containing maytansinoid thereby providing a non-cleavable linker between the cell-binding agent and the thiol-containing maytansinoid to produce a conjugate wherein the non-cleavable linker is a linker that is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, and wherein said cell binding agent is an antibody, a single chain antibody or an antibody fragment that specifically binds to a target cell, wherein the antibody, single chain antibody or antibody fragment comprises a human constant region, provided that the cell-binding agent is not an anti-erbB antibody.
2. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the non-cleavable linker is derived from a maleimido-based moiety.
3. The process of making the cell-binding agent maytansinoid conjugate of claim 2, wherein the non-cleavable linker is derived from a maleimido-based moiety selected from N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidcaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), or a sulfo-succinimidyl variant or an analog thereof.
4. The process of making the cell-binding agent maytansinoid conjugate of claim 3, wherein the non-cleavable linker is SMCC.
5. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the non-cleavable linker is derived from a haloacetyl-based moiety.
6. The process of making the cell-binding agent maytansinoid conjugate of claim 5, wherein the non-cleavable linker is derived from a haloacetyl-based moiety selected from N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), N-succinimidyl 3-(bromoacetamido)propionate (SBAP), or a sulfo-succinimidyl variant or an analog thereof.
7. The process of making the cell-binding agent maytansinoid conjugate of claim 6, wherein the non-cleavable linker is derived from SIAB.
8. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the linker is at any one of the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl or C-20 desmethyl groups of the at least one maytansinoid.
9. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the at least one maytansinoid is an N-methyl-alanine-containing ester of maytansinol.
10. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the at least one maytansinoid is an N-methyl-cysteine-containing ester of maytansinol.
11. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the at least one maytansinoid is represented by formula (II′-L), (II′-D) or (II′-D,L):
(CR7R8)l(CR9═CR10)p(C≡C)qAo(CR5R6)mDu(CR11═CR12)r(C≡C)sBt(CR3R4)nCR1R2S—,
12. The process of making the cell-binding agent maytansinoid conjugate of claim 11, wherein R1 is methyl and R2 is H or R1 and R2 are methyl.
13. The process of making the cell-binding agent maytansinoid conjugate of claim 12, wherein R1 is methyl, R2 is H, R5, R6, R7 and R8 are each H, l and m are each 1, and n is 0; or wherein R1 and R2 are methyl, R5, R6, R7, R8 are each H, 1 and m are 1, and n is 0.
14. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the at least one maytansinoid is represented by formula (II-L), (II-D), or (II-D,L):
Y1 represents (CR7R8)l(CR5R6)m(CR3R4)nCR1R2S—, wherein:
15. The process of making the cell-binding agent maytansinoid conjugate of claim 14, wherein R1 is methyl and R2 is H or R1 and R2 are methyl.
16. The process of making the cell-binding agent maytansinoid conjugate of claim 14, wherein R1 is methyl, R2 is H, R5, R6, R7 and R8 are each H, 1 and m are each 1, and n is 0; or wherein R1 and R2 are methyl, R5, R6, R7, R8 are each H, 1 and m are 1, and n is 0.
17. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the at least one maytansinoid is represented by formula 41′:
(CR7R8)l(CR9═CR10)p(C≡C)qAo(CR5R6)mDu(CR11═C12)r(C≡C)sBt(CR3R4)nCR1R2S—,
18. The process of making the cell-binding agent maytansinoid conjugate of claim 17, wherein R1 is methyl and R2 is H or R1 and R2 are methyl.
19. The process of making the cell-binding agent maytansinoid conjugate of claim 17, wherein R1 is methyl, R2 is H, R5, R6, R7 and R8 are each H, l and m are each 1, and n is 0; or wherein R1 and R2 are methyl, R5, R6, R7, R8 are each H, l and m are 1, and n is 0.
20. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the at least one maytansinoid is represented by formula 41:
Y1 represents (CR7R8)t(CR5R6)m(CR3R4)nCR1R2S—, wherein:
21. The process of making the cell-binding agent maytansinoid conjugate of claim 20, wherein R1 is methyl and R2 is H or R1 and R2 are methyl.
22. The process of making the cell-binding agent maytansinoid conjugate of claim 20, wherein R1 is methyl, R2 is H, R5, R6, R7 and R8 are each H, l and m are each 1, and n is 0; or wherein R1 and R2 are methyl, R5, R6, R7, R8 are each H, l and m are 1, and n is 0.
23. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the at least one maytansinoid is N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1).
24. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the at least one maytansinoid is N2′-deacetyl-N2′-(4-mercapto-1-oxopentyl)-maytansine (DM3).
25. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the at least one maytansinoid is N2′-deacetyl-N2′-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
26. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the cell-binding agent binds to tumor cells; virus infected cells, microorganism infected cells, parasite infected cells, autoimmune cells, activated cells in graft rejection or graft vs. host disease, myeloid cells, activated T-cells, B cells, or melanocytes; cells expressing the CD33, CD19, CanAg, or CALLA_antigen; or cells expressing insulin growth factor receptor or folate receptor.
27. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the cell-binding agent binds to cells selected from breast cancer cells, kidney cancer cells, lung cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cancer cells, small-cell lung cancer cells, testicular cancer cells, neuroblastoma cells, melanoma cells, and cells from cancer of the lymphatic organs or a combination thereof.
28. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced antibody, a resurfaced single chain antibody, or a resurfaced antibody fragment that specifically binds to a target cell.
29. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a humanized antibody, a humanized single chain antibody, or a humanized antibody fragment that specifically binds to a target cell.
30. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a human antibody, a human single chain antibody, or a human antibody fragment that specifically binds to a target cell.
31. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced monoclonal antibody, a resurfaced single chain monoclonal antibody, or a resurfaced monoclonal antibody fragment that specifically binds to a target cell.
32. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a humanized monoclonal antibody, a humanized single chain monoclonal antibody, or a humanized monoclonal antibody fragment that specifically binds to a target cell.
33. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a human monoclonal antibody, a human single chain monoclonal antibody, or a human monoclonal antibody fragment that specifically binds to a target cell.
34. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced monoclonal antibody, a resurfaced single chain monoclonal antibody, or a resurfaced monoclonal antibody fragment that specifically binds to tumor cells.
35. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a humanized monoclonal antibody, a humanized single chain monoclonal antibody, or a humanized monoclonal antibody fragment that specifically binds to tumor cells.
36. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a human monoclonal antibody, a human single chain monoclonal antibody, or a human monoclonal antibody fragment that specifically binds to tumor cells.
37. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced monoclonal antibody, a resurfaced single chain monoclonal antibody, or a resurfaced monoclonal antibody fragment that specifically binds to cancer cells selected from breast cancer cells, kidney cancer cells, lung cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cancer cells, small-cell lung cancer cells, testicular cancer cells, neuroblastoma cells , melanoma cells, and cells from cancer of the lymphatic organs or a combination thereof.
38. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a humanized monoclonal antibody, a humanized single chain monoclonal antibody, or a humanized monoclonal antibody fragment that specifically binds to cancer cells selected from breast cancer cells, kidney cancer cells, lung cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cancer cells, small-cell lung cancer cells, testicular cancer cells, neuroblastoma cells, melanoma cells, and cells from cancer of the lymphatic organs or a combination thereof.
39. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a human monoclonal antibody, a human single chain monoclonal antibody, or a human monoclonal antibody fragment that specifically binds to cancer cells selected from breast cancer cells, kidney cancer cells, lung cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cancer cells, small-cell lung cancer cells, testicular cancer cells, neuroblastoma cells, melanoma cells, and cells from cancer of the lymphatic organs or a combination thereof.
40. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced monoclonal antibody, a resurfaced single chain monoclonal antibody, or a resurfaced monoclonal antibody fragment that specifically binds to breast cancer cells.
41. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a humanized monoclonal antibody, a humanized single chain monoclonal antibody, or a humanized monoclonal antibody fragment that specifically binds to breast cancer cells.
42. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a human monoclonal antibody, a human single chain monoclonal antibody, or a human monoclonal antibody fragment that specifically binds to breast cancer cells.
43. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is an anti-PSMA antibody, an anti-CanAg antibody, an anti-CD19 antibody, an anti-CD33 antibody, an anti-CALLA antibody, an anti-CD56 antibody, or an anti-IGF-IR antibody.
44. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the maytansinoid is N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), N2′-deacetyl-N-2′(4-mercapto-1-oxopentyl)-maytansine (DM3), or N2′-deacetyl-N2′-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
45. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced or humanized My9-6 or N901 antibody or fragment thereof, wherein said My9-6 antibody comprises a heavy chain and a light chain, said heavy chain comprising three complementarity determining regions comprising HCCDR1, HCCDR2 and HCCDR3 of murine antibody My9-6, and wherein said light chain comprises three complementarity determining regions comprising LCCDR1, LCCDR2 and LCCDR3 of murine antibody My9-6; and wherein said resurfaced or humanized N901 antibody or fragment thereof comprises a heavy chain and a light chain, said heavy chain comprising three complementarity determining regions comprising HCCDR1, HCCDR2 and HCCDR3 of murine antibody N901, and said light chain comprising three complementarity determining regions comprising LCCDR1, LCCDR2 and LCCDR3 of murine antibody N901.
46. The process of making the cell-binding agent maytansinoid conjugate of claim 11, wherein the maytansinoid is N2′-deacetyl-N-2′(4-mercapto-1-oxopentyl)-maytansine (DM3) or N2′-deacetyl-N2′-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
47. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced or humanized B4 antibody, or a resurfaced or humanized C242 antibody or fragment thereof, wherein said resurfaced or humanized C242 antibody comprises a heavy chain and a light chain, said heavy chain comprising three complementarity determining regions comprising HCCDR1, HCCDR2 and HCCDR3 of murine antibody C242, and said light chain comprising three complementarity determining regions comprising LCCDR1, LCCDR2 and LCCDR3 of murine antibody C242.
48. The process of making the cell-binding agent maytansinoid conjugate of claim 14, wherein the maytansinoid is N2′-deacetyl-N-2′(4-mercapto-1-oxopentyl)-maytansine (DM3) or N2′-deacetyl-N2′-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
49. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced or humanized B4 antibody.
50. The process of making the cell-binding agent maytansinoid conjugate of claim 1,
wherein the cell-binding agent is a resurfaced or humanized C242 antibody or fragment thereof, wherein said resurfaced or humanized C242 antibody comprises a heavy chain and a light chain, said heavy chain comprising three complementarity determining regions comprising HCCDR1, HCCDR2 and HCCDR3 of murine antibody C242, and said light chain comprising three complementarity determining regions comprising LCCDR1, LCCDR2 and LCCDR3 of murine antibody C242.
51. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the cell-binding agent maytansinoid conjugate has the structure:
52. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein an average of 1 to about 10 maytansinoids is covalently linked to the cell-binding agent.
53. The process of making the cell-binding agent maytansinoid conjugate of claim 1, wherein the cell-binding agent is a humanized antibody, a humanized single chain antibody or a humanized antibody fragment that specifically binds to target cells, the maytansinoid is N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1) and the linker is derived from SMCC.
This application is a divisional of U.S. application Ser. No. 10/960,602, filed Oct. 8, 2004, which claims benefit of Provisional U.S. Patent Application No. 60/509,901, filed Oct. 10, 2003, the entire disclosures of which are incorporated by reference.
In vitro, P388, L1210, and LY5178 murine leukemic cell suspensions have been found to be inhibited by maytansine at doses of 10−3 to 10−1 μg/ml with the P388 line being the most sensitive. Maytansine has also been shown to be an active inhibitor of in vitro growth of human nasopharyngeal carcinoma cells, and the human acute lymphoblastic leukemia line C.E.M. was reported inhibited by concentrations as low as 10−7 μg/ml (Wolpert-DeFillippes et al., 24 Biochem. Pharmacol. 1735-1738 (1975)).
Furthermore, in the area of immunotoxins, conjugates containing linkers with disulfide bridges between monoclonal antibodies and catalytically active protein toxins were shown to be more cytotoxic than conjugates containing other linkers. See, Lambert et al., 260J. Biol. Chem. 12035-12041 (1985); Lambert et al., in Immunotoxins 175-209 (A. Frankel, ed. 1988), and Ghetie et al., 48 Cancer Res. 2610-2617 (1988). This was attributed to the high intracellular concentration of glutathione contributing to the efficient cleavage of the disulfide bond between an antibody molecule and a toxin. More recently, a conjugate of maytansinoids linked to the anti-Her2 breast cancer antibody TA.1 via the non-cleavable linker SMCC was shown to be 200-fold less potent than a conjugate of maytansinoids linked to TA.1 via a linker having a cleavable disulfide bond (Chari et al., 52 Cancer Res. 127-133 (1992)).
Thus, cytotoxic conjugates linked via disulfide-containing cleavable linkers have been sought. Shen et al. described the conversion of methotrexate into a mercaptoethylamide derivative followed by conjugation with poly-D-lysine via a disulfide bond (260 J. Biol. Chem., 10905-10908 (1985)). Preparation of a conjugate of the trisulfide-containing toxic drug calicheamycin with an antibody was also described (Menendez et al., Fourth International Conference on Monoclonal Antibody Immunoconjugates for Cancer, San Diego, Abstract 81 (1989)).
FIG. 5 shows size exclusion chromatography for huC242-SMCC-DM1. Peak 4 represents the monomer fraction of the conjugate, while earlier peaks represent multimer and later peaks represent fragments.
FIG. 11 shows graphically plasma clearance rates of huC242-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.
FIGS. 14A-C show the minimal bystander effect activity of huC242-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.
FIG. 26 shows size exclusion chromatography for trastuzumab-SMCC-DM1. Peak 1 represents multimer, peak 2 represents dimer, and peak 3 represents monomer.
FIG. 29 shows size exclusion chromatography for trastuzumab-SIAB-DM1. Peak 1 represents dimer and peak 2 represents monomer.
(2) C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and
(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chlorides).
U.S. patent application Ser. No. 10/849,136, the entire disclosure of which is hereby incorporated by reference, describes sterically hindered thiol-containing maytansinoids that bear one or two alkyl substituents on the α-carbon bearing the thiol functionality. In addition, the acyl group of the acylated amino acid side chain of the maytansinoid bearing the sulfhydryl group possesses a linear chain length of at least three carbon atoms between the carbonyl group of the amide and the sulfur atom. These novel maytansinoids are suitable for use in the present invention.
(CR7R8)l(CR9═CR10)p(C≡C)qAo(CR5R6)mDu(CR11═CR12)r(C≡C)sBt(CR3R4)nCR1R2SH.
l, m, n, o, p, q, r, s, t, and u are each independently 0 or an integer of from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s, t and u are not both zero.
Y1 represents (CR7R8)l(CR5R6)m(CR3R4)nCR1R2SH.
Examples of heterocycloalkyl radicals include, but are not limited to, dihydrofuryl, tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl, and morpholino.
The monoclonal antibody MY9 is a murine IgG1 antibody that binds specifically to the CD33 antigen (J. D. Griffin et al 8 Leukemia Res., 521 (1984)) and can be used if the target cells express CD33 as in the disease of acute myelogenous leukemia (AML).
Preferred cross-linking reagents that form non-cleavable linkers between the maytansinoid and the cell-binding agent comprise a maleimido- or haloacetyl-based moiety. According to the present invention, such non-cleavable linkers are said to be derived from maleimido- or haloacetyl-based moiety. Cross-linking reagents comprising a maleimido-based moiety include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), which is a “long chain” analog of SMCC (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester [AMAS], succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI) (see FIG. 15 for representative structures of maleimido-based cross-linking reagents). These cross-linking reagents form non-cleavable linkers derived from maleimido-based moieties.
While the active esters described in FIGS. 15 and 16 are comprised of N-succinimidyl and sulfosuccinimidyl esters, other active esters, such as N-hydroxy phthalimidyl esters, N-hydroxy sulfophthalimidyl esters, ortho-nitrophenyl esters, para-nitrophenyl esters, 2,4-dinitrophenyl esters, 3-sulfonyl-4-nitrophenyl esters, 3-carboxy-4-nitrophenyl esters, pentafluorophenyl esters, and sulfonyl tetrafluorophenyl esters can also be used.
HOOC—X1—Yn—Zm-COOH (IV)
wherein X, Y, Z, l, m and n are all defined as for formula (IV) above, and further wherein E together with the carbonyl group forms an active ester such as N-hydroxy succinimidyl and sulfosuccinimidyl esters, N-hydroxy phthalimidyl ester, N-hydroxy sulfophthalimidyl ester, ortho-nitrophenyl ester, para-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxy-4-nitrophenyl ester, pentafluorophenyl ester, and sulfonyl tetrafluorophenyl ester.
Examples of α,ω-dicarboxylic acids of the general formula HOOC—X1—Yn—Zm—COOH include, but are not limited to, adipic acid, glutaric acid, pimelic acid, hexene-1,6-dioc acid, pentene-1,5-dioc acid, cyclohexane-dioic acid, and cyclohexene-dioic acid
Alternatively, as disclosed in U.S. Pat. No. 6,441,163 B1, a thiol-containing maytansinoid can first be modified with a cross-linking reagent, followed by reaction of the modified maytansinoid with a cell-binding agent. For example, the thiol-containing maytansinoid can be reacted with the maleimido compounds described in FIG. 15 or with the haloacetyl compounds described in FIG. 16, to give a maytansinoid thioether bearing an active succinimidyl or sulfosuccinimidyl ester. Reaction of these maytansinoids containing an activated linker moiety with a cell-binding agent provides another method of producing a non-cleavable cell-binding agent maytansinoid conjugate.
Maytansinoid conjugates of antibodies, antibody fragments, protein hormones, protein growth factors and other proteins are made in the same way. For example, peptides and antibodies can be modified with the non-cleavable cross-linking reagents mentioned above. A solution of an antibody in aqueous buffer may be incubated with a molar excess of an antibody-modifying cross-linking reagent such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), sulfo-SMCC, -maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, succinimidyl-iodoacetate, or N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), which is a “long chain” analog of SMCC (LC-SMCC), sulfo-LC-SMCC, κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), sulfo-KMUA, γ-maleimidobutyric acid N-succinimidyl ester (GMBS), sulfo-GMBS, ε-maleimidcaproic acid N-hydroxysuccinimide ester (EMCS), sulfo-EMCS, N-(α-maleimidoacetoxy)-succinimide ester (AMAS), sulfo-AMAS, succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), sulfo-SMPH, N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), sulfo-SMPH, N-(p-maleimidophenyl)isocyanate (PMPI), N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP), as described in the literature. See, Yoshitake et al., 101 Eur. J. Biochem. 395-399 (1979); Hashida et al., J. Applied Biochem. 56-63 (1984); and Liu et al., 18690-697 (1979); Uto et al., 138 J. Immunol. Meth. 87-94 (1991); Rich et al. 18 J. Med. Chem. 1004-1010 (1975); Kitagawa and Aikawa, 79 J. Biochem. (Tohyo) 233-236 (1976); Tanimori et al., 62 J. Immunol. Meth. 123-128 (1983); Hashida et al., 6 J. Appl. Biochem. 56-63 (1984); Thorpe et al., 140 Eur. J. Biochem. 63-71 (1984), Chrisey et al. 24 Nucl. Acid Res. 3031-3039 (1996), Annunziato et al., 4 Bioconjugate Chem. 212-218 (1993), Rector et al., 24 J. Immunol. Meth. 321-336 (1978), and Inman et al. 2 Bioconjugate. Chem. 458-463 (1991).
The modified antibody is then treated with the thiol-containing maytansinoid (1.25 molar equivalent/maleimido or iodoacetyl group) to produce a conjugate. The mixtures are incubated overnight at about 4� C. The antibody-maytansinoid conjugates are purified by gel filtration through a Sephadex G-25 column. The number of maytansinoid molecules bound per antibody molecule can be determined by measuring spectrophotometrically the ratio of the absorbance at 252 nm and 280 nm. Typically, an average of 1-10 maytansinoids per antibody are linked.
The in vitro potency and target specificity of antibody-maytansinoid conjugates of the present invention are shown in FIG. 4. Conjugates of huC242 with DM1 using the cross-linking reagent SMCC are highly potent in destroying antigen positive SKBR3 cells, with an IC50 value of 3.5�10−12 M. In contrast, antigen negative A375 cells are about 800-fold less sensitive demonstrating that maytansinoid conjugates of the present invention are highly potent and specific.
Antibody conjugates with DM1 using the SMCC linker show anti-tumor efficacy against human tumor xenografts in mice (FIG. 10A-C). First, as shown in FIG. 10A, marked inhibition of tumor growth was observed upon treatment of COLO 205 colon tumor xenografts with huC242-SMCC-DM1. In this experiment, one group of five animals bearing established subcutaneous tumors was treated with huC242-SMCC-DM1 at a dose of 150 μg/kg of conjugated DM1. Tumor sizes were measured periodically and graphed vs. time after tumor inoculation. All five treated animals had a complete remission, although three animals relapsed thereafter at different time points, whereas two animals stayed tumor free until termination of the experiment (FIG. 10A). This anti-tumor activity is observed at conjugate doses that have no effect on mouse body weight, a measure of drug toxicity. Second, as shown in FIG. 10B, treatment of mice bearing COLO205 colon carcinoma tumor xenografts with the huC242-SMCC-DM1 conjugate resulted in complete regression of tumors, with some mice remaining free of detectable tumors for over 2 months post-treatment (FIG. 10A). In this experiment, three groups of five animals each bearing established subcutaneous SNU tumors were treated with huC242-SMCC-DM1 at doses of 15 μg/kg, 30 μg/kg, and 60 μg/kg of conjugated DM1, respectively. Tumor sizes were measured periodically and graphed vs. time after tumor inoculation. HuC242-SMCC-DM1 showed a dose-dependent antitumor effect. Again, this activity was obtained at a conjugate concentration that showed no effect on mouse body weight. A trastuzumab-SMCC-DM1 conjugate also showed significant tumor regression, in a mouse tumor xenograft model with the MCF-7 breast carcinoma cell line (FIG. 10C).
Maytansinoid conjugates prepared with non-cleavable linkers such as SMCC show an unexpected increased tolerability in mice compared with conjugates prepared with cleavable disulfide linkers. An acute toxicity test with a single intravenous dose was carried out in female CD-1 mice. A comparison of the tolerability of a huC242-SMCC-DM1 conjugate (non-cleavable) with huC242 conjugates prepared with linkers containing cleavable disulfide bonds was conducted by monitoring the death of mice (FIGS. 12A and B) and signs of toxicity (FIGS. 12C and D) over a series of four escalating doses of each conjugate. The maximum tolerated dose (MTD) for the SMCC-DM1 conjugate was greater than the highest dose tested (150 mg/kg) while the MTD for the disulfide-linked conjugate SPP-DM1 was in the range of 45-90 mg/kg. At 150 mg/kg, all mice in the SMCC-DM1 treated group survived, while lethal toxicity was observed for all mice in the SPP-DM1 treated group by 96 hours post-treatment.
Maytansinoid conjugates are thought to impart their cell destroying activity through the inhibition of microtubule polymerization. This inhibition of microtubule polymerization leads to an arrest of the cell cycle principally at G2/M. The antigen-dependent arrest of cells at G2/M by antibody-maytansinoid conjugates can be monitored by flow cytometry analysis (FIG. 13). Treatment of COL0205 cells with huC242-SPP-DM1 or huC242-SMCC-DM1 conjugate results in a complete G2/M arrest by 6-10 hours. By 30 hours post-treatment however, some of the cells arrested by treatment with the disulfide-linked huC242-SPP-DM1 conjugate escape from cell cycle arrest and reinitiate cell division. Surprisingly, cells treated with the non-cleavable conjugate do not escape from the cell cycle block at this later time point. The difference in the durability of the activity of these two conjugates is also reflected in percentage of dead cells at the 30 hour time point, as judged by a dye exclusion assay using trypan blue. These results demonstrate an unexpected durability of the molecular events induced by treatment with the non-cleavable SMCC linker conjugates.
An additional aspect of conjugates prepared with non-cleavable linkers compared to conjugates that have cleavable disulfide linkers is the absence of activity toward antigen-negative cells when in close proximity to antigen-positive cells, termed here the bystander effect. That is, the conjugates prepared with non-cleavable linkers have minimal bystander activity. Both the huC242-SPP-DM1 (cleavable) and huC242-SMCC (non-cleavable) conjugates show potent cell destroying activity toward the antigen-positive COLO 205 cell line and have no activity toward the antigen-negative cell line, Namalwa, when cultured separately (FIG. 14A-C). However, treatment of co-cultures of COLO 205 and Namalwa cells with huC242-SPP-DM1 reveals dramatic cell destroying activity of the conjugate toward even the antigen-negative Namalwa cells. In contrast, the huC242-SMCC-DM1 conjugate does not demonstrate any such bystander activity under these conditions. No cell destroying activity against Namalwa cells is observed with the huC242-SMCC-DM1 conjugate even when co-cultured with the antigen-positive COLO 205 cells. This minimal bystander activity of the non-cleavable conjugate, as measured in this in vitro assay, may contribute to the increased tolerability of conjugate with non-cleavable linkers observed in acute toxicity studies.
For example, treatment can be carried out as follows. Bone marrow can be harvested from the patient or other individual and then incubated in medium containing serum to which is added the cytotoxic agent of the invention, concentrations range from about 10 pM to 1 nM, for about 30 minutes to about 48 hours at about 37� C. The exact conditions of concentration and time of incubation, i.e., the dose, can be readily determined by one of ordinary skill in the art. After incubation the bone marrow cells can be washed with medium containing serum and returned to the patient intravenously according to known methods. In circumstances where the patient receives other treatment such as a course of ablative chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow cells can be stored frozen in liquid nitrogen using standard medical equipment.
The buffers used in the following experiments were: 50 mM potassium phosphate (KPi)/50 mM sodium chloride (NaCl)/2 mM ethylenediaminetetraacetic acid (EDTA), pH 6.5 (Buffer A); 1� phosphate buffered saline (PBS), pH 6.5 (Buffer B); and 0.1 M KPi buffer/2 mM EDTA at pH 7.5 (Assay Buffer).
SMCC (Product No. 22360, M.W. 334.33 g/mole) and SIAB (Product No. 22329, M.W. 402.15 g/mole) were purchased from Pierce. The huC242 antibody is a humanized form of the monoclonal antibody C242, described in U.S. Pat. No. 5,552,293, for which the hybridoma is deposited with the ECACC Identification Number 90012601). Trastuzumab antibody was obtained from Genentech. DM1 (free thiol form; M.W. 737.5 g/mole) was prepared as described previously in U.S. Pat. Nos. 5,208,020 and 6,333,410 B1.
Chromatography was performed using chromatography columns purchased from Amersham Biosciences (Sephadex G25 NAP-25 prepacked columns (Amersham 17-0852-02); HiPrep 26/10 Desalting Columns, Sephadex G25 fine resin, 3 connected in series (Amersham 17-5087-01)). TSK-GEL G3000SWXL chromatography columns (TOSOH Bioscience, 08541) were also used, with TSK Column Guard SWxl (TOSOH Bioscience 08543).
Preparation of huC242-SMCC-DM1 Conjugate
A 20 mM solution of SMCC (6.69 mg/mL) was prepared in dimethylsulfoxide (DMSO). The solution was diluted 1/40 in Assay Buffer and the absorbance of the samples measured at 302 nm. The concentration of the stock solution was calculated using an extinction coefficient of 602 M−1 cm−1.
A 10 mM solution of DM1 (free thiol form) was prepared in dimethylacetamide (DMA) (7.37 mg/mL) (FIG. 2). The absorbance of dilutions of the stock solution in ethanol (EtOH) was measured at 280 nm. The concentration of stock DM1 was calculated by using an extinction coefficient of 5700 M−1 at 280 nm. The concentration of free sulfhydryl or thiol groups (—SH) in the stock DM1 preparation was measured using Ellman's reagent (DTNB). Dilutions of the stock solution were prepared in Assay buffer made to 3% (v/v) DMA, and then 100 mM DTNB in DMSO ( 1/100th volume) was added. The increase in absorbance at 412 nm was measured against a reagent blank and the concentration was calculated using an extinction coefficient of 14150 M−1 cm−1. The concentration of —SH resulting from the Ellman's assay was used to represent the DM1 stock concentration in calculations for conjugation conditions.
The huC242-SMCC reaction mixtures were gel-filtered through 1.5�4.9 cm pre-packed columns of Sephadex G25 resin equilibrated in Buffer A. The load and elution volumes were according to manufacturer's instructions. The modified antibody elutions were assayed to determine the concentration of the antibody using the extinction co-efficient described above. The yield of modified antibody was 74.6% for the 7.5-fold molar excess SMCC reaction and 81.6% for the 8.5-fold molar excess SMCC reaction.
The conjugation reaction mixtures were gel-filtered through 1.5�4.9 cm pre-packed columns of Sephadex G25 resin equilibrated in Buffer B. The load and elution volumes were according to manufacturer's instructions. The number of DM1 molecules linked per mole of huC242 was determined by measuring absorbance of the eluted material at both 252 nm and 280 nm. The DM1/antibody ratio for the 7.5-fold molar excess SMCC sample was found to be 3.54 and the ratio for the 8.5-fold molar excess SMCC sample was found to be 3.65. The conjugation step yields were 83.7% and 75.4%, respectively. Both conjugates were pooled together, sterile-filtered, and re-assayed for drug and antibody concentrations. The pooled sample was assigned Lot # 1713-146C and analyzed for binding, cytotoxicity, specificity, extent of aggregation and free drug content.
Final DM1 Conc.
DM1/Ab
1713-146C
In vitro Testing of huC242-SMCC-DM1
The binding affinities of huC242 antibody and huC242-SMCC-DM1 were compared using an indirect method on COLO 205 cells, where 5�103 cells per well were used, with three hour primary incubation on ice. The results are shown in FIG. 3. The naked antibody bound with a KD of 5.1�10−10 M and the conjugated version bound with a KD of 5.52�10−10 M. Thus, conjugation with DM1 does not appear to alter the binding affinity of huC242.
The in vitro cytotoxicity and specificity of the huC242-SMCC-DM1 conjugate were evaluated using a continuous exposure clonogenic assay. The results are shown in FIG. 4. HuC242-SMCC-DM1 was effective in destroying the antigen-positive SKBR3 cells (IC50=3.5�10−12 M). Specificity was shown by comparing the IC50 value of the target SKBR3 cells to that of the antigen-negative cell line, A375, in which the IC50 of the conjugate was greater than 3.0�10−9 M.
The conjugate was analyzed using a TSK3000 size exclusion column (FIG. 5). Peak 4 represents the monomer fraction of the conjugate, while earlier peaks represent multimer and later peaks represent fragment. The area under each curve divided by the total peak areas represents the peak's contribution to the sample. The conjugate sample was found to be 96.0% monomer.
Preparation of Trastuzumab-SMCC-DM1 Conjugate
A 10 mM solution of DM1 (free thiol form) was prepared in DMA (7.37 mg/mL) (FIG. 2). The absorbance of dilutions of the stock solution in EtOH was measured at 280 nm. The concentration of stock DM1 was calculated by using a molar extinction coefficient of 5700 M−1 cm−1 at 280 nm. The concentration of free —SH in the stock DM1 preparation was measured using Ellman's reagent (DTNB). Dilutions of the stock solution were prepared in Assay buffer made to 3% (v/v) DMA, and then 100 mM DTNB in DMSO ( 1/100th volume) was added. The increase in absorbance at 412 nm was measured against a reagent blank and the concentration was calculated using an extinction coefficient of 14150 M−1 cm−1. The concentration of —SH resulting from the Ellman's assay was used to represent the DM1 stock concentration in calculations for conjugation conditions.
The trastuzumab-SMCC reaction mixture was gel-filtered through a 1.5�4.9 cm pre-packed column of Sephadex G25 resin equilibrated in Buffer A. The load and elution volumes were according to manufacturer's instructions (Amersham Biosciences). The concentration of the modified antibody solution was assayed spectrophotometrically using the extinction co-efficient described above. The yield of modified antibody was 88% based on protein concentration.
The conjugation reaction mixture was gel-filtered through a 1.5�4.9 cm pre-packed column of Sephadex G25 resin equilibrated in Buffer B. The load and elution volumes were according to manufacturer's instructions (Amersham Biosciences). The number of DM1 molecules linked per mole of trastuzumab was determined by measuring absorbance at both 252 nm and 280 nm of the eluted material. The DM1/antibody ratio was found to be 3.13 and the conjugation step yield was 95.7%. The overall yield of conjugated trastuzumab was 84% based on the starting antibody. The resulting conjugate was analyzed for binding, cytotoxicity, specificity, extent of aggregation and free drug content.
1762-14
In Vitro Testing of Trastuzumab-SMCC-DM1
Binding studies showed that the conjugation of antibody to DM1 did not affect the apparent KD; both naked trastuzumab antibody and trastuzumab-SMCC-DM1 conjugate had the same binding affinity to ECD plates (5.5�10−11 M). Evaluation of the in vitro cytotoxicity of the sample showed that the trastuzumab-SMCC-DM1 conjugate is both highly toxic (IC50 3.6�10−12 M on antigen-positive cell line) and specific (IC50 greater than 3.0�10−9 M on antigen-negative cell line).
The binding affinity of trastuzumab antibody and trastuzumab-SMCC-DM1 were compared using the HER2ECD plate-binding assay provided by Genentech. The results are shown in FIG. 24. Both the naked antibody and conjugated version bind with an apparent KD of 5.5�10−11 M. Thus, conjugation with DM1 does not alter the binding affinity of trastuzumab.
The in vitro cytotoxicity and specificity of the trastuzumab-SMCC-DM1 conjugate were evaluated using a continuous exposure clonogenic assay. The results are shown in FIG. 25. Trastuzumab-SMCC-DM1 was effective in destroying the antigen-positive SKBR3 cells (IC50=3.6�10−12 M). Specificity was shown when comparing the IC50 of the target SKBR3 cells to the antigen-negative cell line, A375, in which the IC50 of the conjugate was greater than 3.0�10−9 M.
The conjugate was analyzed using a TSK3000 size exclusion column (FIG. 26). Peak 1 represents multimer, peak 2 represents dimer, and peak 3 represents monomer. The area under each curve divided by total peak areas represents the peak's contribution to the sample. The conjugate sample was found to be 95.3% monomer.
Preparation of Trastuzumab-SIAB-DM1 Conjugate
An 18 mM solution of SIAB (7.2 mg/mL) was prepared in DMSO. A wavelength scan of the solution diluted into pH 4 buffer was recorded for informational purposes only.
An approximately 30 mM solution of DM1 (free thiol form) was prepared in DMA. The concentration of free —SH in the stock DM1 preparation was measured using Ellman's reagent (DTNB). Dilutions of the stock solution were prepared in Assay buffer made to 3% (v/v) DMA, and then 100 mM DTNB in DMSO ( 1/100th volume) was added. The increase in absorbance at 412 nm was measured against a reagent blank and the concentration was calculated using a molar extinction coefficient of 14150 M−1 cm−1. The concentration of —SH resulting from the Ellman's assay was used to represent the DM1 stock concentration in calculations for conjugation conditions.
d. Modification of Trastuzumab with SLAB Cross Linker
1806-32
In Vitro Testing of Trastuzumab-SIAB-DM1
Binding studies showed that the conjugation of antibody to DM1 did not affect the apparent KD; both naked trastuzumab and trastuzumab-SIAB-DM1 had a similar binding affinities (1.2�10−10 M Ab and 1.9�10−10M apparent KD conjugate). Evaluation of the in vitro cytotoxicity of the sample showed that the trastuzumab-SIAB-DM1 conjugate is both highly toxic (IC50 5�10−12M on antigen-positive cell line SKBR3) and specific (IC50 greater than 3.0�10−9M on antigen-negative cell line, A375).
The binding affinity of trastuzumab antibody and trastuzumab-SIAB-DM1 were compared using the HER2 ECD plate binding assay provided by Genentech. The results are shown in FIG. 27. Naked trastuzumab and trastuzumab-SIAB-DM1 had similar binding affinities (1.2�10−10 M for the antibody and 1.9�10−10 M apparent KD for the conjugate).
Evaluation of the in vitro cytotoxicity of the sample showed that the trastuzumab-SIAB-DM1 conjugate is both highly toxic (IC50=5�10−12 M on antigen-positive cell line, SKBR3) and specific (IC50 greater than 3.0�10−9 M on antigen-negative cell line, A375). See FIG. 28.
The conjugate was analyzed using a TSK3000 size exclusion column (FIG. 29). Peak 1 represents dimer and peak 2 represents monomer. The area under each curve divided by total peak areas represents the peak's contribution to the sample. The conjugate sample was found to be 96.4% monomer.
Conjugation of huC242 with a Cross-Linking Reagent that Forms a Non-S-Containing Non-Cleavable Linker
A stock solution of the cross-linking reagent (see FIG. 21 for structure) was made up in DMA, insoluble precipitate was spun out, and the concentration of the remaining solution was determined using an extinction coefficient of ε280=5700 M−1 cm−1 which is the extinction for DM1 at this wavelength. Since the real extinction coefficient for this material has not been measured this is only an estimate of concentration. It should be noted that the ratio ε252/ε280 for DM1 is 4.7 (in ETOH) while ε252/ε280 for this cross-linking reagent solution (in pH 7.5 buffer) was measured as 1.42 suggesting either different extinctions or impurities.
The conjugation reaction was carried out on a 2 mg scale using 2.8 mg/ml huC242 antibody in 16% DMA in Buffer E, pH 7.5 (Buffer E=50 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA). Based on the estimated cross-linking reagent concentration of the stock solution, 30 equivalents of cross-linker/antibody were used (an earlier experiment using 10 eq. of cross-linker/antibody produced a conjugate with only 0.9 DM1/antibody). The reaction was allowed to go for 3 hours and then the conjugate was purified by passage over a Nap 10 (G25) column. After filtering (Millex GV filter, 0.2 um pore size), the conjugate had 2.56 DM1/antibody (Lot # 1749-119A, antibody recovery=78%). An aliquot of the conjugate was examined by HPLC (HiPrep column) for free DM1 and a sizeable DM1 peak was observed at 12.09′. The sample was therefore dialyzed in Buffer B to get rid of this peak and then reassayed. The final conjugate sample (Lot # 1749-124A) had no free DM1 by HPLC and had 1.84 DM1/antibody. SEC HPLC was carried out on the conjugate to show that it was 97% monomeric antibody.
The inventors carried out binding and cytotoxicity studies on the huC242-non-S-containing non-cleavable linker-DM1 conjugate. First, the binding affinities of huC242 antibody, huC242-SMNP-DM3, and huC242-non-S-containing non-cleavable linker-DM1 to COLO 205 cells were compared. 5�103 cells per well were used, with a three hour primary incubation on ice. The results are shown in FIG. 23. The hu242-non-S-containing non-cleavable linker-DM1 conjugate had about a two-fold higher apparent dissociation constant than free antibody (see FIG. 23). In addition the huC242-non-S-containing non-cleavable linker-DM1 conjugate had an in vitro cytotoxicity comparable to huC242-SMNP-DM3 (IC50 of the non-S-containing non-cleavable linker conjugate=7.0�10−12 M) (see FIG. 22).
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