HK1260858A1

HK1260858A1 - Cell-binding agent maytansinoid conjugates linked via a non-cleavable linker

Info

Publication number: HK1260858A1
Application number: HK19120698.6A
Authority: HK
Inventors: R‧斯蒂夫斯; R‧卢茨; R‧查里; H‧谢; Y‧科夫通
Original assignee: 伊缪诺金公司
Priority date: 2003-10-10
Filing date: 2008-02-27
Publication date: 2019-12-20

Description

Cell-binding agent maytansinoid conjugates with uncleavable linkers

The application is a divisional application of Chinese patent application 200480026459.3 with the application date of 2004, 10 and 12. This application claims the benefit of U.S. provisional application serial No. 60/509,901 filed on 10/2003, the entire disclosure of which is incorporated herein by reference.

Technical Field

One method consistent with the present invention involves targeting specific cell populations with cell-binding agent maytansinoid conjugates (cell-binding agent maytansinoid conjugates) linked via a non-cleavable linker. Another method consistent with the present invention is a method of preparing the conjugate. One composition consistent with the present invention relates to novel cell-binding agent maytansinoid conjugates, wherein the maytansinoid is linked to the cell-binding agent via a non-cleavable linker. Another composition in accordance with the present invention relates to novel maytansinoid esters (maytansinoid esters).

Background

Maytansinoids (maytansinoids) are highly cytotoxic drugs. Maytansine (maytansine) was first isolated from the east African shrub maytansine (Maytenus serrata) by Kupchan et al, and is 100 to 1000 times more cytotoxic than traditional cancer chemotherapeutics such as methotrexate, daunorubicin, and vincristine (U.S. Pat. No. 3,896,111). Subsequently, it was found that some microorganisms also produce maytansinoids, such as maytansinol (maytansinol) and C-3 esters of maytansinol (U.S. Pat. No. 4,151,042). Synthetic C-3 maytansinol esters and analogues of maytansinol have also been reported (Kupchan et al, 21J. Med. chem.31-37 (1978); Higashide et al, 270Nature721-722 (1977); Kawai et al, 32chem. pharm. Bull.3441-3451 (1984)). Examples of maytansinol analogues for the preparation of C-3 esters include maytansinol which has modifications in the aromatic ring (e.g., dechlorination) or modifications at C-9, C-14 (e.g., hydroxylated methyl), C-15, C-18, C-20 and C-4, 5.

Naturally occurring and synthetic C-3 esters can be divided into two groups:

(a) c-3 esters with simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348 and 4,331,598), and

(b) c-3 esters with N-methyl-L-alanine derivatives (U.S. Pat. Nos. 4,137,230 and 4,260,608; and Kaiwa et al, 32chem. pharm. Bull.3441-3451 (1984)).

The esters of group (b) were found to be more cytotoxic than the esters of group (a).

Maytansine is a mitotic inhibitor. It has been reported that treatment of L1210 cells with maytansine in vivo results in 67% of the cells accumulating in mitosis. Untreated control cells are reported to exhibit mitotic indices ranging from 3.2% to 5.8% (Sieber et al 43Bibl. Haematol.495-500 (1976)). Experiments with sea urchin eggs and clam eggs have shown that maytansine interferes with microtubule formation by inhibiting the polymerization of tubulin (microtubule), the tubulin, thereby inhibiting mitosis (Remillard et al, 189Science 1002-1005 (1975)).

In vitro, P388, L1210 and LY5178 murine leukemia cell suspensions were found to be inhibited by maytansine at a dose of 10^-3To 10^-1μ g/ml, of which the P388 cell line is the most sensitive. Maytansine has also been shown to be an active inhibitor of the in vitro growth of human nasopharyngeal carcinoma cells, and human acute lymphocytic leukemia line c.e.m. has been reported to be as low as 10^-7μ g/ml (Wolpert-DeFillippes et al, 24biochem. Pharmacol.1735-1738 (1975)).

Maytansine has also been shown to be active in vivo. Tumor growth in the P388 lymphocytic leukemia system was inhibited in the 50-fold to 100-fold dose range, indicating a high therapeutic index; significant inhibitory activity was also shown with the L1210 murine leukemia system, the human Lewis lung carcinoma system and the human B-16 melanoma system (Kupchan,33ped. Proc.2288-2295 (1974)).

Since maytansinoids are highly cytotoxic, they are expected to be useful in the treatment of many diseases such as cancer. This expectation is yet to be achieved. Clinical trials of maytansine have not been satisfactory due to a number of side effects (Issel et al, 5Cancer treat. Rev.199-207 (1978)). Adverse effects on central nervous system and gastrointestinal symptoms (overturse effect) are the reason for some patients to reject further treatment (Issel at 204), and maytansine appears to be associated with peripheral neuropathy, which may be cumulative (Issel at 207).

Thus, targeting techniques are used to selectively deliver drugs to target cells. Cleavable and non-cleavable linkers were studied for several drugs, but in most cases, including maytansinoids, in vitro cytotoxicity experiments revealed that antibody-drug conjugates rarely achieve the same cytotoxic capacity as free, non-linked drugs. Thus, it is generally accepted that for targeted delivery of maytansinoids to be effective, the linkage between the maytansinoid and the cell-binding agent must be cleavable.

Furthermore, in the field of immunotoxins (immunotoxins), conjugates containing linkers with disulfide bridges between the monoclonal antibody and the catalytically active protein toxin (toxins) have been shown to be more cytotoxic than conjugates containing other linkers. See, Lambert et al, 260J.biol.chem.12035-12041 (1985); lambert et al, Immunotoxins 175-. This is due to the high concentration of intracellular glutathione, which contributes to efficient cleavage of the disulfide bond between the antibody molecule and the toxin. Recently, maytansinoid conjugates linked to the anti-Her 2 breast Cancer antibody TA.1 via a non-cleavable linker SMCC showed 200-fold less potency than maytansinoid conjugates linked to TA.1 via a linker with a cleavable disulfide bond (Chari et al, 52Cancer Res.127-133 (1992)).

Therefore, attempts were made to find cytotoxic conjugates linked by disulfide-containing cleavable linkers. Shen et al describe the conversion of methotrexate to a mercaptoacetamide derivative, followed by disulfide linkage to poly-D-lysine (poly-D-lysine) (260j. biol. chem.10905-10908 (1985)). The preparation of conjugates of the toxic trisulfide-containing drug calicheamicin (calicheamicin) and antibodies is also described (Menendez et al, Fourth International Conference on Monoclonal Antibody antibodies for Cancer, San Diego, Abstract 81 (1989)).

U.S. Pat. Nos. 5,208,020 and 5,416,064, the entire disclosures of which are expressly incorporated herein by reference, disclose cytotoxic conjugates comprising a cell-binding agent linked to a specific maytansinoid derivative via a cleavable linker, such as a disulfide group-containing linker, an acid labile group-containing linker, a photolabile group-containing linker, a peptidase labile group-containing linker, and an esterase labile group-containing linker.

U.S. Pat. No. 6,333,410B1, the entire disclosure of which is specifically incorporated herein by reference, discloses a process for preparing and purifying thiol-containing maytansinoids for attachment to cell-binding agents, and U.S. Pat. No. 6,441,163B1, the entire disclosure of which is specifically incorporated herein by reference, discloses a one-step method for preparing cytotoxic conjugates of maytansinoids and cell-binding agents, wherein the linker is a cleavable linker that contains a disulfide.

Furthermore, U.S. Pat. No. 5,208,020 teaches an antibody-maytansinoid conjugate with a non-cleavable linker, wherein the linker comprises a maleimide group (maleimido group). However, there is no experimental data in this reference demonstrating that such conjugates are effective in treating disease.

Unexpectedly, it has now been found that cytotoxic conjugates of a maytansinoid and a cell-binding agent linked via a non-cleavable linker are very effective and, in many cases, have unexpected advantages over conjugates of a maytansinoid and a cell-binding agent having a cleavable linker.

Summary of The Invention

The illustrative, non-limiting embodiments of the invention described below overcome the above disadvantages and other disadvantages not described above. Also, the present invention is not required to overcome the disadvantages described above, and the illustrative, non-limiting embodiments of the present invention described below may not overcome any of the problems described above.

In one aspect of the invention, a method of targeting maytansinoids (target) to a selected cell population (selected cell population) comprises contacting a cell population or tissue suspected of containing cells from said selected cell population with a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are linked to the cell-binding agent by a non-cleavable linker.

Another aspect of the invention is a method of treating tumors, autoimmune diseases, transplant rejection, graft-versus-host disease, viral infections, parasitic infections and other diseases that can be treated by targeted therapy, wherein the targeting agent is a cell-binding agent, comprising administering to a patient in need of treatment an effective amount of a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are linked to a cell-binding agent, or a pharmaceutically acceptable formulation (formulation) or solvate (solvate) of said conjugate.

Another aspect of the invention is a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are linked to the cell-binding agent via a non-cleavable linker.

Another aspect of the invention is a composition comprising the above conjugate.

Another aspect of the invention is a method of preparing the above conjugate.

Another aspect of the invention is a novel maytansinoid ester.

The present invention provides the following:

1. a method of targeting a maytansinoid 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 are covalently linked to the cell-binding agent by a non-cleavable linker and the cell-binding agent binds to cells in the selected cell population.

2. The method of item 1, wherein the non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, or disulfide bond cleavage.

3. The method of clause 1, wherein the non-cleavable linker does not have a sulfur atom.

4. The method of item 3, wherein the linker is derived from a dicarboxylic acid-based moiety.

5. The method of item 4, wherein the linker is derived from a moiety based on an α, ω -dicarboxylic acid, wherein the α, ω -dicarboxylic acid is of the general formula HOOC-X_l-Y_n-Z_m-COOH, wherein X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a ring having 3 to 10 carbon atomsAlkyl or cycloalkenyl, Z is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group, wherein the heteroatom is selected from N, O or S, and wherein each of l, m and n is 0 or 1, provided that they are not both 0 at the same time.

6. The method of item 5, wherein the α, ω -dicarboxylic acid is adipic acid, glutaric acid, pimelic acid, hexene-1, 6-diacid, pentene-1, 5-diacid, cyclohexane-diacid, or cyclohexene-diacid.

7. The method of clause 1, wherein the non-cleavable linker bears a sulfur atom.

8. The method of clause 7, wherein the non-cleavable linker is derived from a maleimide-based moiety.

9. The method of item 8, wherein the non-cleavable linker is derived from a maleimide-based moiety selected from the group consisting of: n-succinimidyl-4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate) (LC-SMCC), N-succinimidyl kappa-maleimidoundecanoate (KMUA), N-succinimidyl gamma-maleimidobutyrate (GMBS), N-hydroxysuccinimidyl epsilon-maleimidohexanoate (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N- (. alpha. -maleimidoacetoxy) -succinimidyl ester (AMAS), succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI).

10. The method of item 9, wherein the non-cleavable linker is derived from SMCC.

11. The method of item 7, wherein the non-cleavable linker is derived from a haloacetyl-based moiety.

12. The method of item 11, wherein the non-cleavable linker is derived from a haloacetyl-based moiety selected from the group consisting of N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl Bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido) propionate (SBAP).

13. The method of item 12, wherein the non-cleavable linker is derived from a SIAB.

14. The method of item 1, wherein the linker is located at any one of the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl, or C-20 demethylation of at least one maytansinoid.

15. The process of clause 1, wherein the at least one maytansinoid is an N-methyl-alanine-containing ester of maytansinol.

16. The process of item 1, wherein the at least one maytansinoid is an N-methyl-cysteine-containing ester of maytansinol.

17. The process of item 1, wherein the at least one maytansinoid is represented by formula (II ' -L), (II ' -D), or (II ' -D, L):

wherein:

Y₁' is representative of

(CR₇CR₈)_l(CR₉＝CR₁₀)_p(C≡C)_qA_o(CR₅CR₆)_mD_u(CR₁₁＝CR₁₂)_r(C≡C)_sB_t(CR₃CR₄)_nCR₁R₂S-，

Wherein:

R₁to R₁₂Each independently is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl group, and further, R is₂To R₁₂May be H;

A. b and D, each independently is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a simple or substituted aryl, or a heterocyclic aryl or heterocycloalkyl group;

each of l, m, n, o, p, q, r, s, t, and u is independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s, t, and u are not simultaneously 0; and

may represents a maytansinoid bearing a side chain at the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethylation.

18. The method of item 17, wherein R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

19. The method of item 17, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

20. The process of item 1, wherein the at least one maytansinoid is represented by formula (II-L), (II-D), or (II-D, L):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

R₁to R₈Each independently is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl group, and further, R is₂To R₈May be H;

each of l, m, and n is independently an integer from 1 to 5, and further, n may be 0; and

21. The method of clause 20, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

22. The method of clause 20, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

23. The process of item 1, wherein the at least one maytansinoid is represented by formula 41':

wherein:

Y₁' is representative of

Wherein:

A. b and D, each independently is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a simple or substituted aryl, or a heterocyclic aryl or heterocycloalkyl group; and

each of l, m, n, o, p, q, r, s, t, and u is independently 0 or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s, t, and u are not simultaneously 0.

24. The method of clause 23, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

25. The method of clause 23, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

26. The method of clause 1, wherein the at least one maytansinoid is represented as 41:

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

R₁to R₈Each independently is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl group, and further, R is₂To R₈May be H; and

each of l, m, and n is independently an integer from 1 to 5, and further, n may be 0.

27. The method of clause 26, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

28. The method of clause 26, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

29. The method of clause 1, wherein the at least one maytansinoid is DM 1.

30. The method of clause 1, wherein the at least one maytansinoid is DM 3.

31. The method of clause 1, wherein the at least one maytansinoid is DM 4.

32. The process of item 1, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to give a compound of formula 5:

wherein X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, Z is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group wherein the heteroatom is selected from N, O or S, and each of l, m and n is 0 or 1, provided that they are not both 0 at the same time; and E together with the carbonyl group forms an active ester such as N-hydroxysuccinimidyl and thiosuccinimidyl esters, N-hydroxyphthalimido esters, N-hydroxythiophthalimidyl esters o-nitrophenyl esters, p-nitrophenyl esters, 2, 4-dinitrophenyl esters, 3-sulfonyl-4-nitrophenyl esters, 3-carboxy-4-nitrophenyl esters, pentafluorophenyl esters, and sulfonyl tetrafluorophenyl esters.

33. The process of item 1, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to give a compound of formula 6:

wherein N represents an integer from 3 to 24, and E together with the carbonyl group forms an active ester such as N-hydroxysuccinimide and thiosuccinimide esters, N-hydroxyphthalimidyl esters, N-hydroxythiophthalimidyl esters o-nitrophenyl esters, p-nitrophenyl esters, 2, 4-dinitrophenyl esters, 3-sulfonyl-4-nitrophenyl esters, 3-carboxy-4-nitrophenyl esters, pentafluorophenyl esters, and sulfonyl tetrafluorophenyl esters.

34. The process of item 1, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to give a compound of formula 7:

wherein R is H or SO₃ ^-Na⁺。

35. The method of item 1, wherein the cell-binding agent binds to a tumor cell; a virally infected cell, a microbially infected cell, a parasite infected cell, an autoimmune cell, an activated cell, a bone marrow cell, an activated T cell, a B cell, or a melanocyte; cells expressing CD33, CD19, CanAg, cala, or Her-2 antigen; or cells expressing insulin growth factor receptor, epidermal growth factor receptor or folate receptor.

36. The method of item 1, wherein the cell-binding agent binds to a cell selected from the group consisting of: breast cancer cells, prostate cancer cells, ovarian cancer cells, rectal cancer cells, stomach cancer cells, squamous cancer cells, small cell lung cancer cells, testicular cancer cells, and neuroblastoma cells.

37. The method of item 1, wherein the cell-binding agent is an antibody, single chain antibody, antibody fragment that specifically binds to a target cell, a monoclonal antibody, single chain monoclonal antibody, monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, chimeric antibody fragment that specifically binds to a target cell, a structural region antibody, structural region antibody fragment that specifically binds to a target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient transport molecule.

38. The method of item 1, wherein the cell binding agent is interferon, IL2, IL3, IL4, IL6, insulin, thyroid stimulating hormone releasing hormone, melanocyte stimulating hormone, steroid hormone, growth hormone release inhibiting factor, EGF, TGF- α, FGF, G-CSF, VEGF, MCSF, GM-CSF, folate, transferrin, estrogen analog, androgen, or androgen analog.

39. The method of item 1, wherein the cell-binding agent is an antibody, single chain antibody, or antibody fragment that specifically binds to a target cell.

40. The method of item 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.

41. The method of item 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.

42. The method of item 1, wherein the cell-binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell.

43. The method of item 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.

44. The method of item 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.

45. The method of item 1, wherein the cell-binding agent is a chimeric antibody, a chimeric antibody fragment, a structural region antibody, or a structural region antibody fragment that specifically binds to a target cell.

46. The method of item 1, wherein the cell binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to tumor cells.

47. The method of item 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.

48. The method of item 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.

49. The method of item 1, wherein the cell-binding agent is a chimeric antibody, a chimeric antibody fragment, a structural region antibody, or a structural region antibody fragment that specifically binds to a tumor cell.

50. The method of item 1, wherein the cell binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a rectal cancer cell or a breast cancer cell.

51. The method of item 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 rectal cancer cells or breast cancer cells.

52. The method of item 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 rectal cancer cell or a breast cancer cell.

53. The method of item 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.

54. The method of item 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.

55. The method of any one of items 1 to 34, wherein the cell binding agent is an anti-CanAg antibody, an anti-CD-19 antibody, an anti-CD-33 antibody, an anti-CALLA antibody, an anti-EGFR antibody, an anti-CD-56 antibody, an anti-IGF-IR antibody, or an anti-Her 2 antibody.

56. The method of any one of items 1 to 34, wherein the cell binding agent is the surface reconstituted antibody My9-6, KS77, or N901.

57. The method of any one of items 1 to 34, wherein the cell binding agent is trastuzumab, a B4 antibody, or a huC242 antibody.

58. The method of any one of items 1 to 34, wherein the cell-binding agent is a huC242 antibody.

59. The method of any one of items 1 to 34, wherein the cell binding agent is trastuzumab.

60. The method of item 1, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is less toxic than a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

61. The method of item 1, wherein the plasma clearance of the cell-binding agent maytansinoid conjugate is about the same as the antibody alone.

62. The method of item 1, wherein the maximum tolerated dose of the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is higher compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

63. The method of item 1, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has a higher persistence of biological activity compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

64. The method of item 1, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has lower activity against antigen-negative cells as compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

65. The method of item 1, wherein the cell-binding agent maytansinoid conjugate exhibits minimal bystander effect.

66. A method of clearing a cell, the method comprising: contacting the cell with a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are covalently linked to the cell-binding agent by a non-cleavable linker, and the cell-binding agent binds to the cell.

67. The method of item 66, wherein the non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, or disulfide bond cleavage.

68. The method of clause 66, wherein the non-cleavable linker does not carry a sulfur atom.

69. The method of clause 68, wherein the linker is derived from a dicarboxylic acid-based moiety.

70. The method of item 69, wherein the linker is derived from a moiety based on an α, ω -dicarboxylic acid, wherein the α, ω -dicarboxylic acid is of the general formula HOOC-X_l-Y_n-Z_m-COOH, wherein X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, Z is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group, wherein the heteroatom is selected from N, O or S, and wherein each of l, m and n is 0 or 1, with the proviso that they are not both 0 at the same time.

71. The method of clause 70, wherein the α, ω -dicarboxylic acid is adipic acid, glutaric acid, pimelic acid, hexene-1, 6-diacid, pentene-1, 5-diacid, cyclohexane-diacid, or cyclohexene-diacid.

72. The method of clause 66, wherein the non-cleavable linker bears a sulfur atom.

73. The method of clause 72, wherein the non-cleavable linker is derived from a maleimide-based moiety.

74. The method of clause 73, wherein the non-cleavable linker is derived from a maleimide-based moiety selected from the group consisting of: n-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl 4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate) (LC-SMCC), N-succinimidyl kappa-maleimidoundecanoate (KMUA), N-succinimidyl gamma-maleimidobutyrate (GMBS), N-hydroxysuccinimidyl epsilon-maleimidohexanoate (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N- (. alpha. -maleimidoacetate) -succinimidyl ester (AMAS), succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI).

75. The method of clause 74, wherein the non-cleavable linker is derived from SMCC.

76. The method of clause 72, wherein the non-cleavable linker is derived from a haloacetyl-based moiety.

77. The method of clause 76, wherein the non-cleavable linker is derived from a haloacetyl-based moiety selected from the group consisting of N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl Bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido) propionate (SBAP).

78. The method of clause 77, wherein the non-cleavable linker is derived from SIAB.

79. The method of item 66, wherein the linker is located at any one of the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl, or C-20 demethylation of at least one maytansinoid.

80. The method of clause 66, wherein the at least one maytansinoid is an N-methyl-alanine-containing ester of maytansinol.

81. The method of clause 66, wherein the at least one maytansinoid is an N-methyl-cysteine-containing ester of maytansinol.

82. The method of item 66, wherein the at least one maytansinoid is represented by formula (II ' -L), (II ' -D), or (II ' -D, L):

wherein:

Y₁' is representative of

Wherein:

83. The method of clause 82, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

84. The method of clause 82, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

85. The method of clause 66, wherein the at least one maytansinoid is represented by formula (II-L), (II-D), or (II-D, L):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

86. The method of clause 85, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

87. The method of clause 85, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

88. The method of clause 66, wherein the at least one maytansinoid is represented by formula 4₁’：

Wherein:

Y₁' is representative of

Wherein:

89. The method of clause 88, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

90. The method of clause 89, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

91. The method of clause 66, wherein the at least one maytansinoid is represented as 41:

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

92. The method of clause 91, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

93. The method of clause 91, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

94. The method of clause 66, wherein the at least one maytansinoid is DM 1.

95. The method of clause 66, wherein the at least one maytansinoid is DM 3.

96. The method of clause 66, wherein the at least one maytansinoid is DM 4.

97. The method of clause 66, wherein the maytansinoid is modified with a sulfur-free crosslinker to yield a compound of formula 5:

98. The method of clause 66, wherein the maytansinoid is modified with a sulfur-free crosslinker to yield a compound of formula 6:

99. The method of clause 66, wherein the maytansinoid is modified with a sulfur-free crosslinker to yield a compound of formula 7:

wherein R is H or SO₃ ^-Na⁺. The method of item 63, wherein the cell-binding agent binds to a tumor cell; a virally infected cell, a microbially infected cell, a parasite infected cell, an autoimmune cell, an activated cell, a bone marrow cell, an activated T cell, a B cell, or a melanocyte; cells expressing CD33, CD19, CanAg, cala, or Her-2 antigen; or express insulin growth factor receptor, epidermal growth factor receptor or folate receptorThe cell of (1).

100. The method of item 66, wherein the cell-binding agent binds to a cell selected from the group consisting of: breast cancer cells, prostate cancer cells, ovarian cancer cells, rectal cancer cells, stomach cancer cells, squamous cancer cells, small cell lung cancer cells, testicular cancer cells, and neuroblastoma cells.

101. The method of item 66, wherein the cell-binding agent is an antibody, single chain antibody, antibody fragment that specifically binds to a target cell, a monoclonal antibody, single chain monoclonal antibody, monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, chimeric antibody fragment that specifically binds to a target cell, a structural region antibody, structural region antibody fragment that specifically binds to a target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient transport molecule.

102. The method of item 66, wherein the cell binding agent is interferon, IL2, IL3, IL4, IL6, insulin, thyroid stimulating hormone releasing hormone, melanocyte stimulating hormone, steroid hormones, growth hormone release inhibiting factor, EGF, TGF- α, FGF, G-CSF, VEGF, MCSF, GM-CSF, folate, transferrin, estrogen analogs, androgens, or androgen analogs.

103. The method of item 66, wherein the cell-binding agent is an antibody, single chain antibody, or antibody fragment that specifically binds to a target cell.

104. The method of clause 66, wherein the cell-binding agent is a resurfaced antibody, a resurfaced single chain antibody, or a resurfaced antibody fragment that specifically binds to the target cell.

105. The method of item 66, 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.

106. The method of item 66, wherein the cell binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell.

107. The method of item 66, 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.

108. The method of item 66, 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.

109. The method of item 66, wherein the cell-binding agent is a chimeric antibody, a chimeric antibody fragment, a structural region antibody, or a structural region antibody fragment that specifically binds to a target cell.

110. The method of item 66, wherein the cell binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to tumor cells.

111. The method of item 66, 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.

112. The method of item 66, 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.

113. The method of item 66, wherein the cell-binding agent is a chimeric antibody, a chimeric antibody fragment, a structural region antibody, or a structural region antibody fragment that specifically binds to a tumor cell.

114. The method of item 66, wherein the cell binding agent is a monoclonal antibody, single chain monoclonal antibody, or monoclonal antibody fragment that specifically binds to a rectal cancer cell or a breast cancer cell.

115. The method of item 66, 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 rectal cancer cells or breast cancer cells.

116. The method of item 66, 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 rectal cancer cell or a breast cancer cell.

117. The method of item 66, 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.

118. The method of item 66, 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.

119. The method of any one of items 66 to 99, wherein the cell binding agent is an anti-CanAg antibody, an anti-CD-19 antibody, an anti-CD-33 antibody, an anti-CALLA antibody, an anti-EGFR antibody, an anti-CD-56 antibody, an anti-IGF-IR antibody, or an anti-Her 2 antibody.

120. The method of any one of claims 66 to 99, wherein the cell binding agent is the surface reconstituted antibody My9-6, KS77, or N901.

121. The method of any one of items 66 to 99, wherein the cell binding agent is trastuzumab, a B4 antibody, or a huC242 antibody.

122. The method of any one of items 66 to 99, wherein the cell-binding agent is a huC242 antibody.

123. The method of any one of items 66 to 99, wherein the cell binding agent is a resurfaced C242 antibody.

124. The method of clause 66, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is less toxic than a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

125. The method of item 66, wherein the plasma clearance of the cell-binding agent maytansinoid conjugate is about the same as the antibody alone.

126. The method of clause 66, wherein the maximum tolerated dose of the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is higher compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

127. The method of clause 66, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has a higher persistence of biological activity as compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

128. The method of clause 66, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has lower activity against antigen-negative cells as compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

129. The method of clause 66, wherein the cell-binding agent maytansinoid conjugate exhibits minimal bystander effect.

130. A method of treating an affliction selected from the group consisting of a tumor, an autoimmune disease, transplant rejection, graft-versus-host disease, a viral infection, and a parasitic infection, comprising: administering to a subject in need of treatment an effective amount of a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are covalently linked to the cell-binding agent by a non-cleavable linker, and the cell-binding agent binds to diseased or infected cells of the disease.

131. The method of item 130, wherein the tumor is selected from the group consisting of: lung, breast, colon, prostate, kidney, pancreas, ovary and lymphatic organ cancers.

132. The method of item 130, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.

133. The method of item 130, wherein the transplant rejection is selected from the group consisting of: renal transplant rejection, heart transplant rejection, and bone marrow transplant rejection.

134. The method of item 130, wherein the viral infection is selected from the group consisting of CMV, HIV, and AIDS.

135. The method of clause 130, wherein the parasitic infection is selected from the group consisting of giardiasis, amebiasis, and schistosomiasis.

136. The method of any one of items 130 to 135, wherein the non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, or disulfide bond cleavage.

137. The method of any one of items 130 to 135, wherein the non-cleavable linker does not carry a sulfur atom.

138. The method of item 137, wherein the linker is derived from a dicarboxylic acid-based moiety.

139. The method of clause 138, wherein the linker is derived from a moiety based on an α, ω -dicarboxylic acid, wherein the α, ω -dicarboxylic acid is of the formula HOOC-X_l-Y_n-Z_m-COOH, wherein X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, Z is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group, wherein the heteroatom is selected from N, O or S, and each of l, m and n is 0 or 1, provided that they are not both 0 at the same time.

140. The method of item 139, wherein the α, ω -dicarboxylic acid is adipic acid, glutaric acid, pimelic acid, hexene-1, 6-diacid, pentene-1, 5-diacid, cyclohexane-diacid, or cyclohexene-diacid.

141. The method of any of clauses 130 to 135, wherein the non-cleavable linker bears a sulfur atom.

142. The method of clause 141, wherein the non-cleavable linker is derived from a maleimide-based moiety.

143. The method of item 142, wherein the non-cleavable linker is derived from a maleimide-based moiety selected from the group consisting of: n-succinimidyl-4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate) (LC-SMCC), N-succinimidyl kappa-maleimidoundecanoate (KMUA), N-succinimidyl gamma-maleimidobutyrate (GMBS), N-hydroxysuccinimidyl epsilon-maleimidohexanoate (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N- (. alpha. -maleimidoacetate) -succinimidyl ester (AMAS), succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI).

144. The method of item 143, wherein the non-cleavable linker is derived from SMCC.

145. The method of clause 141, wherein the non-cleavable linker is derived from a haloacetyl-based moiety.

146. The method of clause 145, wherein the non-cleavable linker is derived from a haloacetyl-based moiety selected from the group consisting of N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl Bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido) propionate (SBAP).

147. The method of clause 146, wherein the non-cleavable linker is derived from SIAB.

148. The method of any one of items 130 to 135, wherein the linker is located at any one of the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl, or C-20 demethylation of at least one maytansinoid.

149. The process of any one of items 130 to 135, wherein the at least one maytansinoid is an N-methyl-alanine-containing ester of maytansinol.

150. The process of any one of items 130 to 135, wherein the at least one maytansinoid is an N-methyl-cysteine-containing ester of maytansinol.

151. The process of any one of items 130 to 135, wherein the at least one maytansinoid is represented by formula (II ' -L), (II ' -D), or (II ' -D, L):

wherein:

Y₁' is representative of

Wherein:

152. The method of item 151, wherein R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

153. The method of item 151, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

154. The process of any one of items 130 to 135, wherein the at least one maytansinoid is represented by formula (II-L), (II-D), or (II-D, L):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

155. The method of clause 154, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

156. The method of clause 154, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

157. The process of any one of items 130 to 135, wherein the at least one maytansinoid is represented by formula 41':

wherein:

Y₁' is representative of

Wherein:

158. The method of item 157, wherein said R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

159. The method of item 157, wherein said R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

160. The method of any one of items 130 to 135, wherein the at least one maytansinoid is represented as 41:

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

161. The method of clause 160, wherein said R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

162. The method of clause 160, wherein said R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

163. The process of any one of clauses 130 to 135, wherein the at least one maytansinoid is DM 1.

164. The process of any one of clauses 130 to 135, wherein the at least one maytansinoid is DM 3.

165. The process of any one of clauses 130 to 135, wherein the at least one maytansinoid is DM 4.

166. The process of any of clauses 130 to 135, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to yield a compound of formula 5:

167. The process of any of clauses 130 to 135, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to yield a compound of formula 6:

168. The process of any of clauses 130 to 135, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to yield a compound of formula 7:

wherein R is H or SO₃ ^-Na⁺。

169. The method of any one of items 130 to 135, wherein the cell-binding agent is an antibody, single chain antibody or antibody fragment that specifically binds to the diseased or infected cells that are afflicted.

170. The method of any one of items 130 to 135, wherein the cell-binding agent is a resurfaced antibody, a resurfaced single chain antibody, or a resurfaced antibody fragment that specifically binds to a diseased or infected cell that is afflicted.

171. The method of any one of items 130 to 135, wherein the cell-binding agent is a humanized antibody, a humanized single chain antibody, or a humanized antibody fragment that specifically binds to the diseased or infected cells of the disease.

172. The method of any one of items 130 to 135, wherein the cell binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a diseased or infected cell that is afflicted.

173. The method of any one of items 130 to 135, 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 diseased or infected cells that are afflicted.

174. The method of any one of items 130 to 135, 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 diseased or infected cells that are afflicted.

175. The method of clause 130, wherein the cell binding agent is an anti-CanAg antibody, an anti-CD-19 antibody, an anti-CD-33 antibody, an anti-cala antibody, an anti-EGFR antibody, an anti-CD-56 antibody, an anti-IGF-IR antibody, or an anti-Her 2 antibody.

176. The method of item 130, wherein the cell binding agent is the resurfaced antibody My9-6, KS77, or N901.

177. The method of clause 130, wherein the cell binding agent is trastuzumab, a B4 antibody, or a huC242 antibody.

178. The method of item 130, wherein the cell binding agent is a huC242 antibody.

179. The method of clause 130, wherein the cell binding agent is a resurfaced C242 antibody.

180. A method for in vitro application to treat: treating autologous bone marrow cells, which are performed before they are implanted into the same patient, in order to eliminate diseased or tumor cells; treating bone marrow cells or other tissues, which is performed prior to their transplantation, in order to eliminate viable T cells (component T cells) and prevent Graft Versus Host Disease (GVHD); treating the cell culture in order to eliminate all cells that do not express the desired variant of the target antigen; or treating the cell culture for the purpose of eliminating variant cells expressing the undesired antigen; the methods comprise treating the cells with an effective amount of a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are covalently linked to the cell-binding agent by a non-cleavable linker, and the cell-binding agent binds to the cells to be eliminated.

181. The method of clause 180, wherein the non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, or disulfide bond cleavage.

182. The method of clause 180, wherein the non-cleavable linker does not carry a sulfur atom.

183. The method of clause 182, wherein the linker is derived from a dicarboxylic acid-based moiety.

184. The method of item 183, wherein the linker is derived from a moiety based on an α, ω -dicarboxylic acid, wherein the α, ω -dicarboxylic acid is of the general formula HOOC-X_l-Y_n-Z_m-COOH, wherein X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, Z is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group, wherein the heteroatom is selected from N, O or S, and each of l, m and n is 0 or 1, provided that they are not both 0 at the same time.

185. The method of clause 184, wherein the α, ω -dicarboxylic acid is adipic acid, glutaric acid, pimelic acid, hexene-1, 6-diacid, pentene-1, 5-diacid, cyclohexane-diacid, or cyclohexene-diacid.

186. The method of clause 180, wherein the non-cleavable linker carries a sulfur atom.

187. The method of item 186, wherein the non-cleavable linker is derived from a maleimide-based moiety.

188. The method of item 187, wherein the non-cleavable linker is derived from a maleimide-based moiety selected from the group consisting of: n-succinimidyl-4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate) (LC-SMCC), N-succinimidyl kappa-maleimidoundecanoate (KMUA), N-succinimidyl gamma-maleimidobutyrate (GMBS), N-hydroxysuccinimidyl epsilon-maleimidohexanoate (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N- (. alpha. -maleimidoacetate) -succinimidyl ester (AMAS), succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI).

189. The method of clause 180, wherein the non-cleavable linker is derived from SMCC.

190. The method of clause 186, wherein the non-cleavable linker is derived from a haloacetyl-based moiety.

191. The method of item 190, wherein the non-cleavable linker is derived from a haloacetyl-based moiety selected from the group consisting of N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl Bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido) propionate (SBAP).

192. The method of item 191, wherein the non-cleavable linker is derived from a SIAB.

193. The method of item 180, wherein the linker is located at any one of the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl, or C-20 demethylation of at least one maytansinoid.

194. The method of clause 180, wherein the at least one maytansinoid is an N-methyl-alanine-containing ester of maytansinol.

195. The method of clause 180, wherein the at least one maytansinoid is an N-methyl-cysteine-containing ester of maytansinol.

196. The method of clause 180, wherein the at least one maytansinoid is represented by formula (II ' -L), (II ' -D), or (II ' -D, L):

wherein:

Y₁' is representative of

Wherein:

197The method of clause 196, wherein the R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

198. The method of item 196, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

199. The method of clause 180, wherein the at least one maytansinoid is represented by formula (II-L), (II-D), or (II-D, L):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

200. The method of clause 199, wherein the R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

201. The method of clause 199, wherein the R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

202. The method of clause 180, wherein the at least one maytansinoid is represented by formula 41':

wherein:

Y₁' is representative of

Wherein:

203. The method of item 202, wherein R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

204. The method of item 202, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

205. The method of clause 180, wherein the at least one maytansinoid is represented as 41:

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

206. The method of item 205, wherein said R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

207. The method of item 205, wherein said R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

208. The method of clause 180, wherein the at least one maytansinoid is DM 1.

209. The method of clause 180, wherein the at least one maytansinoid is DM 3.

210. The method of clause 180, wherein the at least one maytansinoid is DM 4.

211. The method of clause 180, wherein the maytansinoid is modified with a sulfur-free crosslinker to yield a compound of formula 5:

212. The method of clause 180, wherein the maytansinoid is modified with a sulfur-free crosslinker to yield a compound of formula 6:

213. The method of clause 180, wherein the maytansinoid is modified with a sulfur-free crosslinker to yield a compound of formula 7:

wherein R is H or SO₃ ^-Na⁺。

214. The method of item 180, wherein the cell-binding agent is an antibody, single chain antibody, or antibody fragment that specifically binds to the cells to be cleared.

215. The method of item 180, wherein the cell-binding agent is a resurfaced antibody, a resurfaced single chain antibody, or a resurfaced antibody fragment that specifically binds to the cells to be cleared.

216. The method of item 180, wherein the cell binding agent is a humanized antibody, a humanized single chain antibody, or a humanized antibody fragment that specifically binds to the cells to be cleared.

217. The method of item 180, wherein the cell binding agent is a monoclonal antibody, single chain monoclonal antibody, or monoclonal antibody fragment that specifically binds to the cells to be cleared.

218. The method of item 180, 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 the cells to be cleared.

219. The method of item 180, 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 the cells to be cleared.

220. The method of item 180, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is less toxic than a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

221. The method of item 180, wherein the plasma clearance of the cell-binding agent maytansinoid conjugate is about the same as the antibody alone.

222. The method of clause 180, wherein the maximum tolerated dose of the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is higher compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

223. The method of item 180, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has a higher persistence of biological activity as compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

224. The method of item 180, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has lower activity against antigen-negative cells as compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

225. The method of clause 180, wherein the cell-binding agent maytansinoid conjugate exhibits minimal bystander effect.

226. A cell-binding agent maytansinoid conjugate having at least one maytansinoid linked to a cell-binding agent via a non-cleavable linker, with the proviso that when the cell-binding agent is an antibody, the linker is not derived from a cross-linking agent selected from the group consisting of: succinimidyl 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC), thio-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), thio-MBS and N-succinimidyl-iodoacetate (SIA).

227. A cell-binding agent maytansinoid conjugate having at least one maytansinoid linked to a cell-binding agent via a non-cleavable linker, with the proviso that the cell-binding agent is not an antibody.

228. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, or disulfide bond cleavage.

229. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the non-cleavable linker does not carry a sulfur atom.

230. The cell-binding agent maytansinoid conjugate of item 229, wherein the linker is derived from a dicarboxylic acid-based moiety.

231. The cell binding agent maytansinoid conjugate of item 230, wherein the linker is derived from a moiety based on an α, ω -dicarboxylic acid, wherein the α, ω -dicarboxylic acid is of the general formula HOOC-X_l-Y_n-Z_m-COOH, wherein X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, Z is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group, wherein the heteroatom is selected from N, O or S, and wherein each of l, m and n is 0 or 1, with the proviso that they are not both 0 at the same time.

232. The cell-binding agent maytansinoid conjugate of item 231, wherein the α, ω -dicarboxylic acid is adipic acid, glutaric acid, pimelic acid, hexene-1, 6-diacid, pentene-1, 5-diacid, cyclohexane-diacid, or cyclohexene-diacid.

233. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the non-cleavable linker carries a sulfur atom.

234. The cell-binding agent maytansinoid conjugate of item 233, wherein the non-cleavable linker is derived from a maleimide-based moiety.

235. The cell-binding agent maytansinoid conjugate of item 234, wherein the non-cleavable linker is derived from a maleimide-based moiety selected from the group consisting of: n-succinimidyl-4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate) (LC-SMCC), N-succinimidyl kappa-maleimidoundecanoate (KMUA), N-succinimidyl gamma-maleimidobutyrate (GMBS), N-hydroxysuccinimidyl epsilon-maleimidohexanoate (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N- (. alpha. -maleimidoacetate) -succinimidyl ester (AMAS), succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI).

236. The cell-binding agent maytansinoid conjugate of item 235, wherein the non-cleavable linker is SMCC.

237. The cell-binding agent maytansinoid conjugate of item 233, wherein the non-cleavable linker is derived from a haloacetyl-based moiety.

238. The cell-binding agent maytansinoid conjugate of item 237, wherein the non-cleavable linker is derived from a haloacetyl-based moiety selected from the group consisting of N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl Bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido) propionate (SBAP).

239. The cell-binding agent maytansinoid conjugate of item 238, wherein the non-cleavable linker is derived from SIAB.

240. The cell-binding agent maytansinoid conjugate of any one of items 226 or 227, wherein the linker is located at any one of the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl, or C-20 demethylation of at least one maytansinoid.

241. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the at least one maytansinoid is an N-methyl-alanine-containing ester of maytansinol.

242. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the at least one maytansinoid is an N-methyl-alanine-containing ester of maytansinol.

243. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the at least one maytansinoid is represented by formula (II ' -L), (II ' -D), or (II ' -D, L):

wherein:

Y₁' is representative of

Wherein:

244. The cell-binding agent maytansinoid conjugate of item 243, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

245. The cell-binding agent maytansinoid conjugate of item 243Wherein said R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

246. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the at least one maytansinoid is represented by formula (II-L), (II-D), or (II-D, L):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

247. The cell-binding agent maytansinoid conjugate of item 246, wherein R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

248. The cell-binding agent maytansinoid conjugate of item 246, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0;or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

249. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the at least one maytansinoid is represented by formula 41':

wherein:

Y₁' is representative of

Wherein:

250. The cell-binding agent maytansinoid conjugate of item 249, wherein R is₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

251. The cell-binding agent maytansinoid conjugate of item 249, wherein R is₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

252. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the at least one maytansinoid is represented by 41:

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

253. The cell-binding agent maytansinoid conjugate of item 252, wherein R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

254. The cell-binding agent maytansinoid conjugate of item 252, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

255. The cell-binding agent maytansinoid conjugate of clauses 226 or 227, wherein the at least one maytansinoid is DM 1.

256. The cell-binding agent maytansinoid conjugate of clauses 226 or 227, wherein the at least one maytansinoid is DM 3.

257. The cell-binding agent maytansinoid conjugate of clauses 226 or 227, wherein the at least one maytansinoid is DM 4.

258. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to give a compound of formula 5:

259. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to give a compound of formula 6:

260. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the maytansinoid is modified with a sulfur-free cross-linking agent to give a compound of formula 7:

wherein R is H or SO₃ ^-Na⁺。

261. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the cell-binding agent binds to a tumor cell; a virally infected cell, a microbially infected cell, a parasite infected cell, an autoimmune cell, an activated cell, a bone marrow cell, an activated T cell, a B cell, or a melanocyte; cells expressing CD33, CD19, CanAg, cala, or Her-2 antigen; or cells expressing insulin growth factor receptor, epidermal growth factor receptor or folate receptor.

262. The cell-binding agent maytansinoid conjugate of item 261, wherein the cell-binding agent binds to a cell selected from the group consisting of: breast cancer cells, prostate cancer cells, ovarian cancer cells, rectal cancer cells, stomach cancer cells, squamous cancer cells, small cell lung cancer cells, testicular cancer cells, and neuroblastoma cells.

263. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is an antibody, a single chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody fragment that specifically binds to a target cell, a structural region antibody fragment that specifically binds to a target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient-transporting molecule.

264. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the cell-binding agent is a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient transport molecule

265. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the cell-binding agent is an interferon, IL2, IL3, IL4, IL6, insulin, thyroid-stimulating hormone releasing hormone, melanocyte stimulating hormone, steroid hormone, growth hormone release inhibiting factor, EGF, TGF- α, FGF, G-CSF, VEGF, MCSF, GM-CSF, folic acid, transferrin, estrogen analog, androgen, or androgen analog.

266. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is an antibody, single chain antibody, or antibody fragment that specifically binds to a target cell.

267. The cell-binding agent maytansinoid conjugate of item 226, 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.

268. The cell-binding agent maytansinoid conjugate of item 226, 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.

269. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell.

270. The cell-binding agent maytansinoid conjugate of item 226, 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.

271. The cell-binding agent maytansinoid conjugate of item 226, 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.

272. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is a chimeric antibody, a chimeric antibody fragment, a domain antibody, or a domain antibody fragment that specifically binds to a target cell.

273. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is a monoclonal antibody, a single-chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to tumor cells.

274. The cell-binding agent maytansinoid conjugate of item 226, 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.

275. The cell-binding agent maytansinoid conjugate of item 226, 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.

276. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is a chimeric antibody, a chimeric antibody fragment, a structural region antibody, or a structural region antibody fragment that specifically binds to a tumor cell.

277. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is a monoclonal antibody, single-chain monoclonal antibody, or monoclonal antibody fragment that specifically binds to rectal or breast cancer cells.

278. The cell-binding agent maytansinoid conjugate of item 226, 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 rectal cancer cells or breast cancer cells.

279. The cell-binding agent maytansinoid conjugate of item 226, 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 rectal cancer cells or breast cancer cells.

280. The cell-binding agent maytansinoid conjugate of item 226, 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 rectal cancer cells or breast cancer cells.

281. The cell-binding agent maytansinoid conjugate of item 226, 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.

282. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is an anti-CanAg antibody, an anti-CD 19 antibody, an anti-CD 33 antibody, an anti-CALLA antibody, an anti-EGFR antibody, an anti-CD 56 antibody, an anti-IGF-IR antibody, or an anti-Her 2 antibody.

283. The cell-binding agent maytansinoid conjugate of item 282, wherein the maytansinoid is DM1, DM2, or DM 3.

284. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is the resurfaced antibody My9-6, KS77, or N901.

285. The cell-binding agent maytansinoid conjugate of item 284, wherein the maytansinoid is DM1, DM2, or DM 3.

286. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is trastuzumab, a B4 antibody, or a huC242 antibody.

287. The cell-binding agent maytansinoid conjugate of item 286, wherein the maytansinoid is DM1, DM2, or DM 3.

288. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is a huC242 antibody.

289. The cell-binding agent maytansinoid conjugate of item 226, wherein the cell-binding agent is a resurfaced C242 antibody.

290. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is less toxic than a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

291. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the plasma clearance of the cell-binding agent maytansinoid conjugate is about the same as an antibody alone.

292. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has a higher maximum tolerated dose than a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

293. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has a higher persistence of biological activity compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

294. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has lower activity against antigen-negative cells compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

295. The cell-binding agent maytansinoid conjugate of item 226 or 227, wherein the cell-binding agent maytansinoid conjugate exhibits minimal bystander effect.

296. A composition comprising the cell-binding agent maytansinoid conjugate of item 226 and a carrier.

297. A composition comprising the cell-binding agent maytansinoid conjugate of item 227 and a carrier.

298. The composition of clauses 296 or 297, wherein the non-cleavable linker is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, or disulfide bond cleavage.

299. The composition of clauses 296 or 297, wherein the non-cleavable linker does not carry a sulfur atom.

300. The composition of item 299, wherein the linker is derived from a dicarboxylic acid-based moiety.

301. The composition of item 300, wherein the linker is derived from a moiety based on an α, ω -dicarboxylic acid, wherein the α, ω -dicarboxylic acid is of the general formula HOOC-X_l-Y_n-Z_m-COOH, wherein X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, Z is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group, wherein the heteroatom is selected from N, O or S, and wherein each of l, m and n is 0 or 1, with the proviso that they are not both 0 at the same time.

302. The composition of item 301, wherein the α, ω -dicarboxylic acid is adipic acid, glutaric acid, pimelic acid, hexene-1, 6-diacid, pentene-1, 5-diacid, cyclohexane-diacid, or cyclohexene-diacid.

303. The composition of clauses 296 or 297, wherein the non-cleavable linker carries a sulfur atom.

304. The composition of clause 303, wherein the non-cleavable linker is derived from a maleimide-based moiety.

305. The composition of clause 304, wherein the non-cleavable linker is derived from a maleimide-based moiety selected from the group consisting of: n-succinimidyl-4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate) (LC-SMCC), N-succinimidyl kappa-maleimidoundecanoate (KMUA), N-succinimidyl gamma-maleimidobutyrate (GMBS), N-hydroxysuccinimidyl epsilon-maleimidohexanoate (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N- (. alpha. -maleimidoacetate) -succinimidyl ester (AMAS), succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI).

306. The composition of clause 305, wherein the non-cleavable linker is SMCC.

307. The composition of clause 303, wherein the non-cleavable linker is derived from a haloacetyl-based moiety.

308. The composition of item 307, wherein the non-cleavable linker is derived from a haloacetyl-based moiety selected from the group consisting of N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl Bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido) propionate (SBAP).

309. The composition of clause 308, wherein the non-cleavable linker is derived from SIAB.

310. The composition of clauses 296 or 297, wherein the linker is located at any one of the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl, or C-20 demethylation of at least one maytansinoid.

311. The composition of clauses 296 or 297, wherein the at least one maytansinoid is an N-methyl-alanine-containing ester of maytansinol.

312. The composition of clauses 296 or 297, wherein the at least one maytansinoid is an N-methyl-cysteine-containing ester of maytansinol.

313. The composition of clauses 296 or 297, wherein the at least one maytansinoid is represented by formula (II ' -L), (II ' -D), or (II ' -D, L):

wherein:

Y₁' is representative of

Wherein:

314. The composition of item 313 wherein R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

315. The composition of item 313 wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or itIn R₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

316. The composition of clauses 296 or 299, wherein the at least one maytansinoid is represented by formula (II-L), (II-D), or (II-D, L):

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

317. The composition of item 316, wherein R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

318. The composition of item 316, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

319. The composition of clauses 296 or 297, wherein the at least one maytansinoid is represented by formula 41':

wherein:

Y₁' is representative of

Wherein:

320. The composition of clause 319, wherein the R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

321. The composition of clause 319, wherein the R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

322. The composition of clauses 296 or 297, wherein the at least one maytansinoid is represented as 41:

wherein:

Y₁represents (CR)₇CR₈)_l(CR₅CR₆)_m(CR₃CR₄)_nCR₁R₂S-, wherein:

323. The composition of item 323, wherein R₁Is methyl and R₂Is H, or R₁And R₂Is methyl.

324. The composition of item 323, wherein R₁Is methyl, R₂Is H, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; or wherein R is₁And R₂Is methyl, R₅、R₆、R₇、R₈Is H, l and m are 1, and n is 0.

325. The composition of clauses 296 or 297, wherein the at least one maytansinoid is DM 1.

326. The composition of clauses 296 or 297, wherein the at least one maytansinoid is DM 3.

327. The composition of clauses 296 or 297, wherein the at least one maytansinoid is DM 4.

328. The composition of clauses 296 or 297, wherein the maytansinoid is modified with a sulfur-free crosslinker to provide a compound of formula 5:

329. The composition of clauses 296 or 297, wherein the maytansinoid is modified with a sulfur-free crosslinker to provide a compound of formula 6:

330. The composition of clauses 296 or 297, wherein the maytansinoid is modified with a sulfur-free crosslinker to provide a compound of formula 7:

wherein R is H or SO₃ ^-Na⁺。

331. The composition of clauses 296 or 297, wherein the cell-binding agent binds to a tumor cell; a virally infected cell, a microbially infected cell, a parasite infected cell, an autoimmune cell, an activated cell, a bone marrow cell, an activated T cell, a B cell, or a melanocyte; cells expressing CD33, CD19, CanAg, cala, or Her-2 antigen; or cells expressing insulin growth factor receptor, epidermal growth factor receptor or folate receptor.

332. The composition of item 331, wherein the cell-binding agent binds to a cell selected from the group consisting of: breast cancer cells, prostate cancer cells, ovarian cancer cells, rectal cancer cells, stomach cancer cells, squamous cancer cells, small cell lung cancer cells, testicular cancer cells, and neuroblastoma cells.

333. The composition of item 296, wherein the cell-binding agent is an antibody, single chain antibody, antibody fragment that specifically binds to a target cell, a monoclonal antibody, single chain monoclonal antibody, monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, chimeric antibody fragment that specifically binds to a target cell, a structural region antibody, structural region antibody fragment that specifically binds to a target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient transport molecule.

334. The composition of clauses 296 or 297, wherein the cell binding agent is a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient transport molecule.

335. The composition of item 296 or 297, wherein the cell binding agent is an interferon, IL2, IL3, IL4, IL6, insulin, thyroid stimulating hormone releasing hormone, melanocyte stimulating hormone, steroid hormone, growth hormone release inhibiting factor, EGF, TGF- α, FGF, G-CSF, VEGF, MCSF, GM-CSF, folate, transferrin, estrogen, an estrogen analog, an androgen, or an androgen analog.

336. The composition of item 296, wherein the cell-binding agent is an antibody, single chain antibody, or antibody fragment that specifically binds to a target cell.

337. The composition of clause 296, wherein the cell-binding agent is a resurfaced antibody, a resurfaced single chain antibody, or a resurfaced antibody fragment that specifically binds to target cells.

338. The composition of item 296, 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.

339. The composition of item 296, wherein the cell binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell.

340. The composition of item 296, 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.

341. The composition of item 296, 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.

342. The composition of item 296, wherein the cell-binding agent is a chimeric antibody, a chimeric antibody fragment, a structural region antibody, or a structural region antibody fragment that specifically binds to a target cell.

343. The composition of item 296, wherein the cell binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to tumor cells.

344. The composition of item 296, 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.

345. The composition of item 296, 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.

346. The composition of item 296, wherein the cell-binding agent is a chimeric antibody, a chimeric antibody fragment, a structural region antibody, or a structural region antibody fragment that specifically binds to a tumor cell.

347. The composition of item 296, wherein the cell binding agent is a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a rectal cancer cell or a breast cancer cell.

348. The composition of item 296, 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 rectal cancer cells or breast cancer cells.

349. The composition of item 296, 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 rectal cancer cell or a breast cancer cell.

350. The composition of item 296, 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 rectal cancer cells or breast cancer cells.

351. The composition of item 296, 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.

352. The composition of item 296, wherein the cell binding agent is an anti-CanAg antibody, an anti-CD-19 antibody, an anti-CD-33 antibody, an anti-CALLA antibody, an anti-EGFR antibody, an anti-CD-56 antibody, an anti-IGF-IR antibody, or an anti-Her 2 antibody.

353. The composition of clause 352, wherein the maytansinoid is DM1, DM2, or DM 3.

354. The composition of item 296, wherein the cell binding agent is a resurfaced antibody My9-6, KS77, or N901.

355. The composition of clause 354, wherein the maytansinoid is DM1, DM2, or DM 3.

356. The composition of item 296, wherein the cell binding agent is trastuzumab, a B4 antibody, or a huC242 antibody.

357. The composition of clause 356, wherein the maytansinoid is DM1, DM2, or DM 3.

358. The composition of item 296, wherein the cell binding agent is a huC242 antibody.

359. The composition of clause 296, wherein the cell binding agent is a resurfaced C242 antibody.

360. The composition of clauses 296 or 297, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is less toxic than a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

361. The composition of clauses 296 or 297, wherein the plasma clearance of the cell-binding agent maytansinoid conjugate is about the same as the antibody alone.

362. The composition of clauses 296 or 297, wherein the maximum tolerated dose of the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker is higher compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

363. The composition of clauses 296 or 297, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has a higher persistence of biological activity as compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

364. The composition of clauses 296 or 297, wherein the cell-binding agent maytansinoid conjugate comprising a non-cleavable linker has lower activity against antigen-negative cells as compared to a cell-binding agent maytansinoid conjugate comprising at least one maytansinoid linked to a cell-binding agent via a cleavable linker.

365. The composition of clauses 296 or 297, wherein the cell-binding agent maytansinoid conjugate exhibits minimal bystander effect.

366. A process for preparing the cell-binding agent maytansinoid conjugate of item 226 or 227, the process comprising:

(a) providing a cell binding agent

(b) Modifying the cell-binding agent with a cross-linking agent, and

(c) linking the modified cell-binding agent and the maytansinoid or the thiol-containing maytansinoid, thereby providing a non-cleavable linker between the cell-binding agent and the maytansinoid or the thiol-containing maytansinoid, to produce the conjugate.

367. A process for preparing the cell-binding agent maytansinoid conjugate of item 226 or 227, the process comprising:

(a) providing maytansinoids or maytansinoids containing thiol,

(b) modifying the maytansinoid or the thiol-containing maytansinoid with a crosslinking agent to form a non-cleavable linker, and

(c) linking the modified maytansinoid or thiol-containing maytansinoid and the cell-binding agent, thereby providing a non-cleavable linker between the cell-binding agent and the maytansinoid or thiol-containing maytansinoid, to form the conjugate.

368. A process for preparing the cell-binding agent maytansinoid conjugate of item 226 or 227, the process comprising:

(a) to provide a maytansinoid which is,

(b) modifying the maytansinoid with a sulfur-free cross-linking agent to produce an ester of the maytansinoid, and

(c) linking the ester of the maytansinoid and the cell-binding agent, thereby providing a non-cleavable linker between the cell-binding agent and the maytansinoid, resulting in a conjugate.

369. A compound of formula 5:

370. A compound of formula 6:

371. A compound of formula 7:

wherein R is H or SO₃ ^-Na⁺。

372. A cell-binding agent maytansinoid conjugate of the formula:

trastuzumab-SMCC-maytansinoids.

373. A cell-binding agent maytansinoid conjugate of the formula:

trastuzumab-SMCC-DM 1.

374. A cell-binding agent maytansinoid conjugate of the formula:

huC 242-SMCC-maytansinoid.

375. A cell-binding agent maytansinoid conjugate of the formula:

huC242-SMCC-DM1。

brief Description of Drawings

Fig. 1 shows the structure of SMCC.

Figure 2 shows the structure of DM 1.

FIG. 3 shows graphically the results of a FACS binding assay (FACS binding assay) comparing huC242 antibody and the antibody-maytansinoid conjugate huC242-SMCC-DM 1.

FIG. 4 shows the cytotoxicity of huC242-SMCC-DM1 in a graphical manner.

FIG. 5 shows size exclusion chromatography (size exclusion chromatography) of huC242-SMCC-DM 1.

FIGS. 6A-C and 7 graphically show the cytotoxicity of huC242-SMCC-DM1, as compared to conjugates prepared with disulfide-containing linkers.

Figures 8A-D graphically show the cytotoxicity of SMCC-DM1 conjugates linked to various cell binding agents.

FIG. 9 graphically illustrates the cytotoxicity of the antibody-maytansinoid conjugate huC242-SIAB-DM 1.

FIG. 10A graphically shows the antitumor activity of huC242-SMCC-DM1 against COLO205 human colon cancer xenografts (xenografts) in SCID mice.

FIG. 10B graphically shows the anti-tumor activity of huC242-SMCC-DM1 against SUN16 human gastric tumor xenografts in SCID mice.

Figure 10C graphically shows the anti-tumor effect of trastuzumab-SMCC-DM 1(trastuzumab-SMCC-DM1) against human MCF7 tumor xenografts in SCID mice.

FIG. 11 graphically shows the plasma clearance of huC242-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.

FIGS. 12A-D graphically show the results of acute toxicity testing (acute toxicity tests) of huC242-SMCC-DM1, compared to conjugates prepared with disulfide-containing linkers.

FIG. 13 shows the persistence of cell cycle arrest (cell cycle arrest) and cell destruction activity exhibited by huC242-SMCC-DM1, compared to conjugates prepared with disulfide-containing linkers.

FIGS. 14A-C show the minimal bystander effect (bystander effect) activity of huC242-SMCC-DM1 compared to conjugates prepared with disulfide-containing linkers.

FIG. 15 shows a representative structure of a maleimide (maleimido) based cross-linking agent (cross-linking agent).

Fig. 16 shows a representative structure of a haloacetyl (haloacetyl) based cross-linker.

Figure 17 shows the structure of the antibody-SMCC-DM 1 conjugate.

FIG. 18 shows the structure of the antibody-SIAB-DM 1 conjugate.

Figure 19 shows the structure of the antibody-SMCC-DM 4 conjugate.

FIG. 20 shows the structure of an antibody-SIAB-DM 4 conjugate.

Figure 21 shows the synthesis of maytansinoid cell-binding agent conjugates linked via a non-cleavable linker without S.

FIG. 22 shows the cytotoxicity of huC 242-S-free non-cleavable linker-DM 1 in a graphical manner.

FIG. 23 shows graphically the results of FACS binding assays for huC 242-non-cleavable linker without S-DM 1.

Figure 24 graphically shows the results of a HER2ECD plate binding assay comparing trastuzumab to the antibody-maytansinoid conjugate trastuzumab-SMCC-DM 1.

Figure 25 graphically shows the cytotoxicity and specificity of trastuzumab-SMCC-DM 1.

Figure 26 shows size exclusion chromatography of trastuzumab-SMCC-DM 1.

Figure 27 shows graphically the results of the HER2ECD plate binding assay comparing trastuzumab to the antibody-maytansinoid conjugate trastuzumab-SIAB-DM 1.

FIG. 28 graphically illustrates the cytotoxicity and specificity of trastuzumab-SIAB-DM 1.

FIG. 29 shows size exclusion chromatography of trastuzumab-SIAB-DM 1.

Detailed description of representative embodiments of the invention

The art reveals that it is extremely difficult to modify existing drugs without reducing their cytotoxic potential. However, U.S. Pat. nos. 6,441,163Bl, 6,333,410Bl, 5,416,064 and 5,208,020 demonstrate that maytansinoids can be linked to suitable cell-binding agents via cleavable linkers, particularly those containing disulfide groups, to produce potent cytotoxic agents. The cell-binding agent maytansinoid conjugate allows the cytotoxic effects of maytansinoids to be fully applied in a targeted manner, only against unwanted cells, thereby avoiding side effects due to damage to non-targeted healthy cells.

The present inventors have unexpectedly found that maytansinoids attached to cell-binding agents via non-cleavable linkers are superior to maytansinoids attached via cleavable linkers in several important respects. In particular, the conjugates with non-cleavable linkers show equivalent in vitro and in vivo anti-tumor activity when compared to the conjugates containing cleavable linkers, whereas the conjugates with non-cleavable linkers show a significant reduction in plasma clearance and toxicity.

Thus, the present invention provides improved methods for targeting cells, particularly cells to be destroyed such as tumor cells (particularly solid tumor cells), virally infected cells, microbially infected cells, parasitically infected cells, autoimmune cells (autoantibody producing cells), activated cells (those involved in graft rejection or graft versus host disease), or any other form of diseased or abnormal cells, while exhibiting minimal side effects.

The conjugates used in the methods of the invention carry one or more maytansinoids linked to a cell-binding agent by a non-cleavable linker. In one method of making the conjugate, a cell-binding agent, e.g., an antibody, is first modified with a cross-linking agent, such as SMCC. In a second step, a reactive maytansinoid bearing a thiol group, such as DM1, is reacted with a modified antibody to produce an antibody-maytansinoid conjugate. Alternatively, the maytansinoid may be modified with a cross-linking agent, followed by reacting the maytansinoid with a cell-binding agent. See, for example, U.S. Pat. No. 6,441,163B 1.

Suitable maytansinoids

Maytansinoids suitable for use in the present invention are known in the art and may be isolated from natural sources according to known methods, produced by genetic engineering techniques (see Yu et al, 99PNAS7968-7973(2002)), or prepared synthetically according to known methods.

Examples of suitable maytansinoids include maytansinol and maytansinol analogs (analogues). Examples of suitable maytansinol analogues include those having modified aromatic rings and those having modifications at other positions.

Specific examples of suitable maytansinol analogues having a modified aromatic ring include:

(1) c-19-dechlorinated (U.S. Pat. No. 4,256,746) (prepared by LAH reduction of ansamitocin P2);

(2) c-20-hydroxy (or C-20-demethyl) +/-C-19-dechlorinated (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation with Streptomyces (Streptomyces) or Actinomycetes (Actinomyces), or by dechlorination with LAH); and

(3) c-20-demethoxy group, C-20-acyloxy group (-OCOR), +/-dechlorination (U.S. Pat. No. 4,294,757) (prepared by acylation with acid chloride).

Specific examples of suitable maytansinol analogues with modifications at other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (by reaction of maytansinol with H₂S or P₂S₅Prepared by reaction);

(2) C-14-Alkoxymethyl (demethoxy/CH)₂OR) (U.S. patent No. 4,331,598);

(3) c-14-hydroxymethyl or acyloxymethyl (CH)₂OH or CH₂OAc) (U.S. patent No. 4,450,254) (prepared from Nocardia (Nocardia);

(4) c-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by transformation of Streptomyces with maytansinol);

(5) c-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from the peach tree (Trewia nudiflora));

(6) C-18-N-demethylation (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces; and

(7)4, 5-deoxy (U.S. Pat. No. 4,371,533) (prepared by trichloro-peptide/LAH reduction of maytansinol).

Many sites on maytansinol are known to serve as attachment sites, depending on the type of attachment. For example, for the formation of ester bonds, a C-3 site with a hydroxyl group, a C-14 site modified with a hydroxymethyl group, a C-15 site modified with a hydroxyl group, and a C-20 site with a hydroxyl group are all suitable. However, the C-3 site is preferred, and the C-3 site of maytansinol is particularly preferred.

According to the invention, preferred maytansinoids have a free thiol group. Particularly preferred maytansinoids comprising a free thiol group include the N-methyl-alanine-containing esters of maytansinol, which are C-3 esters of maytansinol and its analogs. Preferred esters include the N-methyl-alanine-containing esters and the N-methyl-cysteine-containing esters of maytansinol. Synthetic methods for maytansinol esters with thiol groups have been previously described, for example, in U.S. Pat. No. 5,208,020, Chari et al, 52Cancer Res., 127-. Furthermore, U.S. Pat. No. 6,333,410B1, the entire disclosure of which is incorporated herein by reference, provides methods for the preparation and purification of thiol-containing maytansinoids suitable for attachment to cell-binding agents.

Many of the conjugates of the invention exemplified below employ the thiol-containing maytansinoid DM1, formally named N^2’-removing of ethylacyl-N^2’- (3-mercapto-1-oxopropyl) -maytansine. DM1 is represented by the following structural formula:

the synthesis of the thiol-containing maytansinoid DM1 has been previously described (U.S. Pat. No. 5,208,020).

U.S. patent application 10/849,136, the entire disclosure of which is incorporated herein by reference, describes sterically hindered (sterically hindered) thiol-containing maytansinoids having one or two alkyl substituents on the α -carbon bearing a thiol functional group. In addition, the acyl group of the acylated amino acid side chain of the sulfhydryl-bearing maytansinoid has a linear chain length of at least 3 carbon atoms between the carbonyl and sulfur atoms of the amide. These novel maytansinoids are suitable for use in the present invention.

The synthesis of maytansinoids having a sterically hindered thiol group can be described with reference to U.S. patent application 10/849,136, particularly to FIG. 3 thereof.

In one aspect of the invention, the maytansinoid contains a sterically hindered thiol group and is represented by formula (II ' -L), (II ' -D) or (II ' -D, L):

in the formula (II'), the reaction mixture is,

Y₁' is representative of

(CR₇R₈)_l(CR₉＝CR₁₀)_p(C≡C)_qA_o(CR₅R₆)_mD_u(CR₁₁＝CR₁₂)_r(C≡C)_sB_t(CR₃R₄)_nCR₁R₂SH。

A. B and D, each independently, is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, a simple or substituted aryl group, or a heterocyclic aryl or heterocycloalkyl group.

R₁To R₁₂Each independently is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl group, and further, R is₂To R₁₂May be H.

May represents a maytansinoid bearing a side chain at the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethyl (desmethyl).

Another maytansinoid useful in the present invention is represented by formula (II-L), (II-D) or (II-D, L):

in the formula (II), the compound represented by the formula (II),

Y₁represents (CR)₇R₈)_l(CR₅R₆)_m(CR₃R₄)_nCR₁R₂SH。

R₁To R₈Each independently is a linear alkyl or alkenyl group having 1 to 10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl group, and further, R is₂To R₈May be H.

May represents maytansinol with a side chain at the C-3 hydroxyl, C-14 hydroxymethyl, C-15 hydroxyl or C-20 demethyl (desmethyl).

Another useful maytansinoid is represented by formula 4₁’：

Wherein the substituents are as defined above for formula (II').

Another useful maytansinoid is represented by formula 4₁：

Wherein the substituents are as defined above for formula (II).

Preferred are any of the above compounds, wherein R₁Is H and R₂Is methyl, or R₁And R₂Is methyl.

Particularly preferred are any of the above compounds, wherein R₁Is H, R₂Is methyl, R₅、R₆、R₇And R₈Is H, each of l and m is 1, and n is 0; and wherein R₁And R₂Is methyl, R₅、R₆、R₇And R₈Are each H, l and m are 1, and n is 0.

Further, the L-aminoacyl stereoisomer is preferable.

Examples of linear alkyl or alkenyl groups having 1 to 10 carbon atoms include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, propenyl, butenyl, and hexenyl.

Examples of branched alkyl or alkenyl groups having 3 to 10 carbon atoms include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 1-ethylpropyl, isobutenyl, and isopentenyl.

Examples of the cyclic alkyl or alkenyl group having 3 to 10 carbon atoms include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl and cyclohexenyl.

Simple aryl groups include aryl groups having 6 to 10 carbon atoms, and substituted aryl groups include aryl groups having 6 to 10 carbon atoms bearing at least one alkyl substituent having 1 to 4 carbon atoms, or alkoxy substituents such as methoxy, ethoxy, or halogen substituents or nitro substituents.

Examples of simple aryl groups containing 6 to 10 carbon atoms include, but are not limited to, phenyl and naphthyl.

Examples of substituted aryl groups include, but are not limited to, nitrophenyl, dinitrophenyl.

Heterocyclic aryl groups include groups having 3 to 10 membered rings containing one or two heteroatoms selected from N, O or S.

Examples of heterocyclic aryl groups include, but are not limited to, pyridyl, nitropyridyl, pyrrolyl (pyrolyl), oxazolyl, thienyl, thiazolyl, and furyl.

Heterocycloalkyl includes cyclic compounds comprising a3 to 10 membered ring system, containing one or two heteroatoms, selected from N, O or S.

Examples of heterocycloalkyl groups include, but are not limited to, dihydrofuran, tetrahydrofuran, pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl (morpholino).

A particularly preferred maytansinoid comprising a side chain containing a sterically hindered thiol linkage is maytansinoid N^2’-deacetyl-N^2’- (4-mercapto-1-oxopentyl) -maytansine (known as DM3) and N^2’-deacetyl-N^2’- (4-methyl-4-mercapto-1-oxopentyl) -maytansine (known as DM 4). DM3 and DM4 are represented by the following structural formulae:

cell binding agents

The effectiveness of the compounds of the invention as therapeutic agents depends on the careful selection of an appropriate cell-binding agent. Cell binding agents may be of any kind currently known, or becoming known, including peptidic and non-peptidic substances. Generally, they may be antibodies (particularly monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules (such as transferrin), or any other cell-binding molecule or substance that specifically binds to a target.

More specific examples of cell-binding agents that may be used include:

polyclonal and monoclonal antibodies; including fully human antibodies;

single chain antibodies (polyclonal and monoclonal);

antibody fragments (polyclonal and monoclonal antibodies) such as Fab, Fab ', F (ab')₂And Fv (Parham,131J. Immunol.2895-2902 (1983); Spring et al, 113J. Immunol.470-478 (1974); Nisonoff et al, 89Arch. biochem. Biophys.230-244 (1960));

chimeric antibodies and antigen binding fragments thereof;

domain antibodies (dAbs) and antigen-binding fragments thereof, including camelid antibodies (Desmyter et al, 3Nature struct. biol,752,1996);

shark antibodies, termed new antigen receptors (IgNAR) (Greenberg et al, 374Nature,168,1995; Stanfield et al 305Science 1770-;

interferons (e.g., α, β, γ);

lymphokines such as IL-2, IL-3, IL-4, IL-6;

hormones such as insulin, TRH (thyroid stimulating hormone releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones such as androgen and estrogen;

growth factors and colony stimulating factors such as EGF, TGF-. alpha.FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess,5Immunology Today 155-158 (1984));

transferrin (O' Keefe et al, 260J.biol.chem.932-937 (1985)); and

vitamins, such as folate.

Monoclonal antibody technology allows the production of extremely specific cell-binding agents in the form of specific monoclonal antibodies. Techniques for making monoclonal antibodies, such as whole target cells, antigens isolated from target cells, whole viruses, attenuated whole viruses, and viral proteins such as viral capsid proteins, are particularly well known in the art, and are produced by immunizing a mouse, rat, hamster, or any other mammal with an antigen of interest. Sensitized human cells may also be used. Another method of making monoclonal antibodies is the use of scFv (single chain variable region) phage libraries, particularly human scFv phage libraries (see, e.g., Griffiths et al, U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al, WO 92/01047; limig et al, WO 99/06587). In addition, resurfaced antibodies disclosed in U.S. Pat. No. 5,639,641 may also be used, as may humanized antibodies.

The choice of a suitable cell-binding agent depends on the particular cell population to be targeted, but generally, if a suitable human monoclonal antibody is available, a human monoclonal antibody is preferred.

For example, monoclonal antibody J5 is a murine IgG2a antibody which is specific for the general type of acute lymphoblastic leukemia antigen (CALLA) (Ritz et al, 283Nature 583-585(1980)), which can be used if the target cell expresses CALLA, such as in acute lymphoblastic leukemia.

Monoclonal antibody MY9 is murine IgG₁Antibodies which specifically bind to the CD33 antigen (j.d. griffin et al 8 leukamia res.,521(1984)), may be used if the target cell expresses CD33, for example in Acute Myeloid Leukemia (AML).

Similarly, monoclonal antibody anti-B4, also interchangeably referred to as B4, is a murine IgG₁Which binds to the CD19 antigen on B cells (Nadler et al, 131J. Immunol.244-250(1983)), which antibodies can be used if the target cell is a B cell expressing this antigen or a diseased cell, for example in non-Hodgkin's lymphoma or chronic lymphocytic leukemia.

In addition, monoclonal antibody C242, which binds to the CanAg antigen (U.S. patent No. 5,552,293), can be used to treat CanAg-expressing tumors, such as rectal cancer, pancreatic cancer, non-small cell lung cancer, and gastric cancer. HuC242 is a humanized form of monoclonal antibody C242 and is described in U.S. patent No. 5,552,293, which hybridoma is deposited with the european collection of biologies (ECACC) under accession number 90012601. Humanized forms can be prepared by applying CDR-grafting methodology (U.S. Pat. Nos. 5,585,089; 5,693,761 and 5,693,762) or resurfacing methodology (U.S. Pat. No. 5,639,641). HuC242 may also be used to treat CanAg-expressing tumors, such as rectal cancer, pancreatic cancer, non-small cell lung cancer, and gastric cancer.

Further, the antibody trastuzumab can be used to treat breast cancer and other cancers, such as Her2 antigen expressing prostate and ovarian cancer.

anti-IGF-IR antibodies that bind to the insulin growth factor receptor are also useful.

Ovarian and prostate cancers, respectively, can be successfully targeted using, for example, anti-MUCI antibodies, such as anti-HMFG-2 (Taylor-Papadimitiou et al, 28.int.J.cancer 17-21,1981) or hCT 01(56Cancer Res.5179-5185,1996) and anti-PSMA (prostate specific membrane antigen) such as J591(Liu et al 57Cancer Res.3629-3634,1997).

Specific cell populations can be targeted with non-antibody molecules. For example, GM-CSF, which binds to myeloid cells, can be used as a cell-binding agent to target diseased cells from acute myeloid leukemia. In addition, IL-2 binding to activated T cells can be used to prevent transplant rejection, to treat and prevent graft versus host disease, and to treat acute T cell leukemia. Melanoma may be treated with MSH that binds to melanocytes. Folate can be used to target folate receptors expressed on ovarian cancer and other tumors. Epidermal Growth Factor (EGF) can be used to target squamous cancers such as lung and head and neck squamous cancers. Neuroblastoma and other tumor types can be targeted with somatostatin. Estrogens (or estrogen analogs) or androgens (or androgen analogs) can be used as cell-binding agents, respectively, to successfully target breast and testicular cancer.

Cross-linking agent (cross-linking reagent)

The maytansinoid is linked to the cell-binding agent by means of a cross-linking agent which, when reacted, forms a non-cleavable linker between the maytansinoid and the cell-binding agent.

As used herein, a "linker" is a cell-binding agentCovalently attached to any chemical moiety of the maytansinoid. In some cases, a portion of the linker is provided by a maytansinoid. For example, DM1, which is a thiol-containing maytansinoid (fig. 2), is a derivative of the natural maytansinoid, maytansine, and provides a portion of the linker. The side chain at the C-3 hydroxyl group of maytansine terminates in-CO-CH₃The side chain of DM1 terminates in-CO-CH₂-CH₂-SH. Thus, the final linker is composed of two parts, the cross-linker is introduced into the cell-binding agent and the side chain from DM 1.

A cleavable linker is a linker that can be cleaved under moderate conditions, i.e., under conditions in which the activity of the maytansinoid drug is unaffected. Many known linkers fall within this range, as described below.

Disulfide-containing linkers are linkers that can be cleaved by disulfide exchange, which can occur under physiological conditions.

An acid labile linker is a linker that is cleavable at acidic pH. For example, some intracellular compartments such as endosomes and lysosomes have acidic pH values (pH 4-5) providing conditions suitable for cleavage of acid-labile linkers.

The photolabile linker is useful on the body surface and in many body cavities where light can be received. In addition, infrared light can penetrate tissue.

Some linkers can be cleaved by peptidases. Only some peptides are easily cleaved either intracellularly or extracellularly. See, Trouet et al, 79Proc. Natl.Acad.Sci.USA, 626-. In addition, peptides are composed of α -amino acids and peptide bonds, which are chemically amide bonds between the carboxylate of one amino acid and the α -amino group of a second amino acid. Other amide bonds, such as the bond between the carboxylate ester of lysine and the epsilon-amino group, are understood not to be peptide bonds and are considered non-cleavable.

Some linkers can be cleaved by esterases. Likewise, only some of the esters may be cleaved by esterases present both intracellularly and extracellularly. Esters are formed by the condensation of carboxylic acids and alcohols. Simple esters are esters of simple alcohols, such as aliphatic alcohols, as well as small cyclic alcohols and small aromatic alcohols. For example, the inventors of the present invention did not find esterases that cleave esters at maytansine C-3, because the alcohol component of the ester, maytansinol, is very large and very complex.

A non-cleavable linker is any chemical moiety that is capable of linking the maytansinoid to the cell-binding agent in a stable covalent manner and is not included in the scope of cleavable linkers as listed above. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.

By "substantially resistant" (to cleavage) is meant that in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the population of cell-binding agent maytansinoid conjugates, the chemical bond in or attached to the linker remains uncleaved by acid, photolabile cleaving agents, peptidases, esterases, or chemical or physiological compounds that cleave the chemical bond (e.g., disulfide bond) in the cleavable linker for a period of time ranging from several hours to several days following treatment with any of the above agents.

Furthermore, "non-cleavable" (non-cleavable) refers to the ability of a chemical bond in or attached to a linker to resist cleavage induced by acids, photolabile cleaving agents, peptidases, esterases or chemical or biochemical compounds that cleave disulfide bonds without loss of activity of the maytansinoid or cell-binding agent.

One of ordinary skill in the art will readily distinguish between non-cleavable linkers and cleavable linkers.

An example of a suitable control for testing whether a linker is substantially resistant to cleavage is a linker with a chemical bond, such as a disulfide bond, which is sensitive to cleavage by any of the agents described above. Whether the linker is substantially resistant to cleavage can be tested by measuring the stability of the conjugate by ELISA, HPLC or other suitable method over a period of time ranging from a few hours to a few days, typically 4 hours to 5 days. Plasma concentration levels of the stable conjugate can be determined using ELISA assays.

Non-cleavable linkers are also characterized in that the half-life in vivo of a conjugate containing a non-cleavable linker is typically about 20% longer than the half-life of a conjugate containing a cleavable linker. In mice, the in vivo half-life of the IgG-maytansinoid conjugate linked via a non-cleavable linker is at least 4 days.

Suitable cross-linkers that form a non-cleavable linker between the maytansinoid and the cell-binding agent are known in the art, and may form non-cleavable linkers that contain a sulfur atom (e.g., SMCC) or do not contain a sulfur atom.

Preferred cross-linkers that form a non-cleavable linker between the maytansinoid and the cell-binding agent include maleimide or haloacetyl-based moieties. According to the invention, such non-cleavable linkers are expressed as originating from maleimide or haloacetyl based moieties. Crosslinkers that include maleimide-based moieties include N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate), which is a "long chain" analog of SMCC (LC-SMCC), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), gamma-maleimidobutanoic acid N-succinimidyl ester (GMBS), epsilon-maleimidohexanoic acid N-hydroxysuccinimidyl Ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), N- (. alpha. -maleimidoacetoxy) -succinimidyl ester [ AMAS ],(s), Succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI) (see FIG. 15 for representative structures of maleimide-based crosslinkers). These cross-linkers form non-cleavable linkers derived from maleimide-based moieties.

Crosslinkers that include haloacetyl-based moieties include N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl Bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido) propionate (SBAP) (see fig. 16 for representative structures of haloacetyl-based crosslinkers). These crosslinkers form non-cleavable linkers derived from haloacetyl-based moieties.

Although the active esters depicted in FIGS. 15 and 16 are composed of N-succinimidyl and thiosuccinimidyl esters, other active esters may be employed, such as N-hydroxyphthalimido esters, N-hydroxythiophthalimidyl esters, o-nitrophenyl esters, p-nitrophenyl esters, 2, 4-dinitrophenyl esters, 3-sulfonyl-4-nitrophenyl esters, 3-carboxy-4-nitrophenyl esters, pentafluorophenyl esters, and sulfonyltetrafluorophenyl esters.

Particularly preferred crosslinkers form non-cleavable linkers that do not contain a sulfur atom. FIG. 21 shows maytansinoid molecules derivatized with a crosslinking agent derived from an alpha, omega-dicarboxylic acid (alkane or alkene diacid where the alkane or alkene has 3 to 24 carbon atoms). When reacted with a cell binding agent, the cross-linker will form a non-cleavable linker that is free of sulfur (a non-cleavable linker that is free of S).

The maytansinoid molecule of FIG. 21 was prepared as follows. First, a monoester of adipic acid (also known as adipic acid or 1, 6-hexanedicarboxylic acid) is prepared by treatment with one equivalent of 2-trimethylsilylethanol (2-trimethylsilylethanol) in the presence of dicyclohexylcarbodiimide. Activation of the remaining carboxylic acid groups with isobutyl chloroformate followed by reaction with N-methyl-L-alanine gives the acylated N-methyl-L-alanine. Reaction with maytansinol in the presence of dicyclohexylcarbodiimide and zinc chloride, followed by removal of the trimethylsilyl protecting group with tetrabutylammonium chloride, produces the ester of maytansinoid with a free carboxyl group. Reacting with thio-N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide to esterify the carboxyl group to form an active ester of maytansinol, which can be reacted with a cell-binding agent to give a non-cleavable conjugate which does not contain a sulphur atom.

Using the above methods, non-cleavable linkers that do not contain a sulfur atom can also be derived from moieties based on other dicarboxylic acids. Suitable moieties based on other dicarboxylic acids include, but are not limited to, α, ω -dicarboxylic acids of formula (IV):

HOOC-X_l-Y_n-Z_m-COOH

(IV)

in formula (IV), X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, Z is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group, wherein the heteroatom is selected from N, O or S, and wherein each of l, m and n is 0 or 1, provided that they are not both 0 at the same time.

Maytansinoids can be represented by formula 5, which are derivatized to contain an active ester that can react directly with a cell-binding agent to form a conjugate with a non-cleavable linker that does not contain S:

wherein X, Y, Z, l, m and N are as defined above for formula (IV), further wherein E and carbonyl together form an active ester such as N-hydroxysuccinimide and thiosuccinimide esters, N-hydroxyphthalimidyl esters, N-hydroxythiophthalimidyl esters o-nitrophenyl esters, p-nitrophenyl esters, 2, 4-dinitrophenyl esters, 3-sulfonyl-4-nitrophenyl esters, 3-carboxy-4-nitrophenyl esters, pentafluorophenyl esters, and sulfonyl tetrafluorophenyl esters.

Preferred are derivatized maytansinoids, represented by formula 6:

wherein n represents an integer from 3 to 24, and E is as defined for the maytansinoid of formula 5.

A more preferred embodiment is a derivatized maytansinoid represented by formula 7:

wherein R is H or SO₃ ^-Na⁺。

The compounds of formulae 5,6 and 7 are novel maytansinoids.

Examples of linear alkyl, alkenyl or alkynyl groups having 2 to 20 carbon atoms include, but are not limited to, ethyl, propyl, butyl, pentyl, hexyl, propenyl, butenyl and hexenyl.

Examples of branched alkyl, alkenyl, or alkynyl groups having 2 to 20 carbon atoms include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 1-ethylpropyl, isobutenyl, isopentenyl, ethynyl, propynyl (propargyl), 1-butynyl, 2-butynyl, and 1-hexynyl.

Examples of cycloalkyl or cycloalkenyl groups having 3 to 10 carbon atoms include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl and cycloheptadienyl.

Examples of aryl groups having 6 to 10 carbon atoms include, but are not limited to, phenyl and naphthyl.

Examples of substituted aryl groups include, but are not limited to, nitrophenyl and dinitrophenyl.

Heterocyclic aryl groups include, but are not limited to, groups having 3 to 10 membered rings containing one or two heteroatoms selected from N, O or S.

Examples of substituted and unsubstituted heterocyclic aryl groups include, but are not limited to, pyridyl, nitropyridyl, pyrrolyl, oxazolyl, thienyl, thiazolyl, and furyl.

Heterocycloalkyl includes, but is not limited to, cyclic compounds including 3 to 10 membered ring systems containing one or two heteroatoms selected from N, O or S.

Examples of heterocycloalkyl groups include, but are not limited to, dihydrofuran, tetrahydrofuran, tetrahydropyrrolyl, piperidinyl, piperazinyl, and morpholinyl.

General formula HOOC-X_l-Y_n-Z_mExamples of α, ω -dicarboxylic acids of-COOH include, but are not limited to, adipic acid, glutaric acid, pimelic acid, hexene-1, 6-diacid, pentene-1, 5-diacid, cyclohexane-diacid, and cyclohexene-diacid.

Synthesis of cytotoxic conjugates

Conjugates of the cell-binding agent and the maytansinoid may be formed by any now known or later developed technique.

The process of linking the cell-binding agent and the maytansinoid typically involves two reaction steps. In one approach, which is described in U.S. Pat. No. 5,208,020, a cell-binding agent, such as an antibody, can be modified with a crosslinking agent to introduce one or more reactive groups, typically 1-10. The modified cell-binding agent is then reacted with one or more thiol-containing maytansinoids to form a conjugate.

Alternatively, as disclosed in U.S. Pat. No. 6,441,163B1, a thiol-containing maytansinoid may be first modified with a crosslinking agent, followed by reacting the modified maytansinoid with a cell-binding agent. For example, a thiol-containing maytansinoid can be reacted with a maleimide compound as depicted in FIG. 15 or a haloacetyl compound as depicted in FIG. 16 to provide a maytansinoid thioether with an active succinimidyl ester or thiosuccinimidyl ester. The reaction of these maytansinoids, which contain an active linker moiety, with a cell-binding agent provides an alternative method for producing non-cleavable cell-binding agent maytansinoid conjugates.

As disclosed above, in another aspect of the invention, a maytansinoid that is free of sulfur atoms can be first derivatized with a dicarboxylic acid-based crosslinking agent, and subsequently reacted with a cell-binding agent to form a conjugate, wherein the maytansinoid is attached to the cell-binding agent by a non-cleavable linker that is free of S.

Typically, an average of 1-10 maytansinoids are attached per antibody. The conjugate can be purified by Sephadex G-25 column.

The entire disclosures of U.S. Pat. Nos. 5,208,020 and 6,441,163B1 are specifically incorporated herein by reference.

Representative conjugates of the invention are antibody-maytansinoid derivatives, antibody fragment-maytansinoid derivatives, growth factor-maytansinoid conjugates such as Epidermal Growth Factor (EGF) -maytansinoid derivatives, hormone-maytansinoid conjugates such as Melanocyte Stimulating Hormone (MSH) -maytansinoid derivatives, Thyroid Stimulating Hormone (TSH) -maytansinoid derivatives, estrogen analogs-maytansinoid derivatives, androgen analogs-maytansinoid derivatives, and vitamin-maytansinoid conjugates such as folic acid maytansinoid.

Maytansinoid conjugates of antibodies, antibody fragments, protein hormones, protein growth factors and other proteins are prepared in the same manner. For example, peptides and antibodies may be modified with the non-cleavable crosslinkers mentioned above. Solutions of antibodies in aqueous buffers may be incubated with a molar excess of an antibody-modifying cross-linking agent such as succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate (SMCC), thio-SMCC, -maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), thio-MBS, succinimidyl-iodoacetyl ester, or N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate), which is a long chain analog of SMCC (LC-SMCC), thio-LC-SMCC, kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), thio-KMUA, gamma-maleimidobutanoic acid N-succinimidyl ester (GMBS), thio-GMBS, epsilon-maleimidohexanoic acid N-hydroxysuccinimide Ester (EMCS), thio-EMCS, N- (. alpha. -maleimidoacetoxy) -succinimidyl ester (AMAS), thio-AMAS, succinimidyl-6- (. beta. -maleimidopropionamido) hexanoate (SMPH), thio-SMPH, N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), thio-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, 101Eur.J.biochem.395-399 (1979); hashida et al, J.applied biochem.56-63 (1984); and Liu et al, 18690-697 (1979); uto et al, 138J.Immunol.meth.87-94 (1991); rich et al 18J.Med.chem.1004-1010 (1975); kitagawa and Aikawa,79J. biochem. (Tohyo) 233-; tanimori et al, 62J.Immunol.meth.123-128 (1983); hashida et al, 6J.appl.biochem.56-63 (1984); thorpe et al, 140Eur.J.biochem.63-71(1984), Chrisey et al, 24 Nucl.acids Res.3031-3039(1996), Annunziato et al, 4Bioconjugate chem.212-218(1993), Rector et al, 24J.Immunol.meth.321-336(1978), and Inman et al, 2bioconjugate.chem.458-463 (1991).

The modified antibody (1.25 molar equivalents per maleimide group or iodoacetyl group) was then treated with a thiol-containing maytansinoid to produce a conjugate. The mixture was incubated at about 4 ℃ overnight. The antibody-maytansinoid conjugate was purified by gel filtration through a Sephadex G-25 column. Spectrophotometric determination of the ratio of absorbance at 252nm and 280nm determined the number of maytansinoid molecules bound per antibody molecule. Typically, an average of 1-10 maytansinoids are bound per antibody.

In a preferred method, the antibody is modified with succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), a maleimido group is introduced, and the modified antibody is subsequently reacted with a thiol-containing maytansinoid to give a thioether-linked conjugate. Conjugates with 1 to 10 drug molecules per antibody molecule were also obtained. Examples of antibody-maytansinoid conjugates are shown in FIGS. 17-20.

Similarly, the C-17 hydroxyl groups of estrogen and androgen cell binding agents such as estradiol and androgens may be esterified, for example, by reaction with an appropriately protected thiol-containing chlorinated carboxylic acid (carboxylic acid chloride) such as 3-S-acetyl propionyl chloride. Other esterification methods may also be employed, as described in the literature (Haslam,36Tetrahedron 2400-. The protected or free thiol-containing androgen or estrogen can then be reacted with a thiol-containing maytansinoid to form a conjugate. The conjugate can be purified by column chromatography on silica gel, or by HPLC.

One particularly preferred method is to modify the maytansinoid with a cross-linking agent that results in a linkage that does not contain any sulfur atom, and subsequently reacting the modified maytansinoid with an antibody to form a conjugate.

Therapeutic Effect of the cytotoxic conjugates of the invention

The cell-binding agent maytansinoid conjugates of the invention can be evaluated for their ability to inhibit proliferation of a variety of cell lines in vitro. For example, cell lines such as human colon cancer cell line COLO205, human melanoma cell line A-375, human myeloid leukemia cell line HL 60, human breast cancer cell line SKBR3, or human epidermoid cancer cell line KB can be used to assess the cytotoxicity of these conjugates. Cells to be evaluated may be exposed to the compound for 24 hours and assayed for survival fraction by direct analysis by known methods. (see, e.g., Goldmacher et al, 135J.Immunol.3648-3651(1985), and Goldmacher et al, 102J.cell biol.1312-1319 (1986)). Then, from the analysis result, IC can be calculated₅₀The value is obtained.

High cytotoxicity is defined as showing IC₅₀The value (inhibitory concentration of toxic substance, the permissible survival score of said toxic substance being 0.5) is about 10^-8M or less, as determined by exposure of SKBR3 to the drug in vitro for 24 hours.

Figure 4 shows the in vitro potency and target specificity of the antibody-maytansinoid conjugates of the invention. The conjugate of huC242 and DM1 with the crosslinker SMCC was highly effective at destroying antigen-positive SKBR3 cells, IC₅₀The value is 3.5x 10^-12And M. In contrast, antigen-negative A-375 cells were approximately 800-fold less sensitive, indicating that the maytansinoid conjugates of the invention are highly potent and highly specific.

The huC242-SMCC-DM1 conjugate has equal or greater potency than the conjugate prepared with the disulfide-containing linker in both the clone formation assay (clonal assay) (FIGS. 6A-C) and the indirect cytotoxicity assay (FIG. 7). These results were unexpected based on previously published data demonstrating that anti-Her 2 antibodies linked to maytansinoids by SMCC do not exhibit specific activity (Chari et al, 52Cancer Res.127-133 (1992)).

The activity of conjugates prepared with SMCC non-cleavable linkers is not limited to huC242 conjugates. Trastuzumab with anti-Her 2 antibody; anti-CD 33 antibody My 9-6; anti-EGFR antibody KS 77; specific activity was also observed in vitro with the SMCC-DM1 conjugate of anti-CD 56 antibody N901 (fig. 8A-D and 25).

Furthermore, conjugates with non-cleavable linkers that exhibit specific activity in vitro are not limited to SMCC linkers. The huC242 conjugate of DM1 synthesized with a non-cleavable linker SIAB in an in vitro colony formation assay showed potency and antigen-specific cytotoxicity (figure 9). Further, trastuzumab conjugates of DM1 synthesized with SIAB were also cytotoxic in the colony formation assay (fig. 28). Further, the huC 242-S-free non-cleavable linker-DM 1 conjugate also demonstrated potency and antigen-specific cytotoxicity (figure 22) in an in vitro clonogenic assay.

Conjugates of antibodies using SMCC linker and DM1 were shown to be antagonistic in mice

Effects of human tumor xenografts (FIGS. 10A-C). First, as shown in figure 10A, significant inhibition of tumor growth was observed following treatment of COLO205 colon tumor grafts with huC242-SMCC-DM 1. In this experiment, 1 group of 5 animals with established subcutaneous tumors was treated with huC242-SMCC-DM1 and the dose of conjugated DM1 was 150. mu.g/kg. Tumor volumes were measured periodically and plotted as tumor volume versus time after tumor inoculation. Although 3 animals relapsed at different time points thereafter, all 5 treated animals were completely remitted, while 2 animals remained tumor-free until the end of the experiment (fig. 10A). In addition, as shown in figure 10B, this anti-tumor activity was observed at conjugate doses that did not affect mouse body weight, a measure of drug toxicity. In this experiment, 3 groups of 5 animals, each bearing established subcutaneous SUN tumors, were treated with huC242-SMCC-DM1 at 15. mu.g/kg, 30. mu.g/kg and 60. mu.g/kg conjugated DM1, respectively. Tumor volumes were measured periodically and plotted as tumor volume versus time after tumor inoculation. huC242-SMCC-DM1 showed a dose-dependent anti-tumor effect. The results show that treatment of mice bearing COLO205 colon cancer tumor xenografts with huC242-SMCC-DM1 conjugate resulted in complete tumor regression, with some mice remaining free of detectable tumors during the 2 month post-treatment period (figure 10A). Again, this activity was obtained at conjugate concentrations that did not affect mouse body weight. trastuzumab-SMCC-DM1 conjugate also showed significant tumor regression in the mouse tumor xenograft model with MCF-7 breast cancer cell line (fig. 10C).

The plasma clearance (plasma clearance) of antibody-maytansinoid conjugates synthesized with the non-cleavable linker SMCC was very slow and comparable to that of antibody alone. This is in sharp contrast to plasma clearance of conjugates prepared with relatively labile disulfide bonds, such as huC242-SPP-DM 1. For example, the half-life of SMCC conjugate clearance is approximately 320 hours, while the half-life of SPP conjugates is in the 40-50 hour range (fig. 11). However, the clearance of the antibody component of each class of conjugate was the same, suggesting that, in the case of the SPP-DM1 conjugate, the difference in the clearance of the conjugates measured was due to the loss of maytansinoids from the antibody conjugate. The non-cleavable SMCC linker is thus more resistant to maytansinoid-linker cleavage activity present in vivo than the SPP-DM1 conjugate. Further, the decreased clearance of SMCC-linked conjugates compared to SPP-DM1 conjugate resulted in an almost 5-fold increase in total maytansinoid exposure in animals as measured by the area under the curve (AUC). In some cases, this increased exposure may have a substantial effect on the drug effect.

Maytansinoid conjugates prepared with non-cleavable linkers, such as SMCC, show unexpectedly increased tolerability (tolerability) in mice compared to conjugates prepared with cleavable disulfide linkers. Acute toxicity tests using a single intravenous dose were performed in female CD-1 mice. The tolerability of the huC242-SMCC-DM1 conjugate (non-cleavable) and the huC242 conjugate prepared with a linker containing a cleavable disulfide bond was compared over 4 escalating series of dose ranges for each conjugate by monitoring mouse death (fig. 12A and B) and signs of toxicity (fig. 12C and D). The Maximum Tolerated Dose (MTD) of the SMCC-DM1 conjugate was higher than the highest dose tested (150mg/kg), while the MTD range for the disulfide-linked conjugate SPP-DM1 was 45-90 mg/kg. At 150mg/kg, SMCC-DM1

All mice in the treated group survived, whereas lethal toxicity (lethal toxicity) was observed in all mice in the SPP-DM1 treated group 96 hours after treatment.

Maytansinoid conjugates are believed to exert their cell-destroying activity by inhibiting the polymerization of tubulin. This inhibition of tubulin polymerization leads to cell cycle arrest, mainly in the G2/M phase. The cell antigen-dependent arrest caused by the antibody-maytansinoid conjugate was in the G2/M phase and could be monitored by flow cytometry analysis (fig. 13). Treatment of COLO205 cells with huC242-SPP-DM1 or huC242-SMCC-DM1 conjugates resulted in complete G2/M arrest at 6-10 hours. However, at 30 hours post-treatment, some cells arrested by treatment with the disulfide-linked huC242-SPP-DM1 conjugate escaped cell cycle arrest and resumed cell division. Surprisingly, at this later time point, cells treated with the non-cleavable conjugate did not escape cell cycle arrest. The difference in persistence of the activity of the two conjugates was also reflected in the percentage of dead cells at the 30 hour time point, as judged by the dye exclusion test (dye exclusion assay) performed using trypan blue. These results demonstrate the unexpected persistence of the molecular events induced by treatment with uncleavable SMCC linker conjugates.

Another aspect of conjugates prepared with a non-cleavable linker as compared to conjugates with a cleavable disulfide linker is that when they are in close proximity to antigen-positive cells, there is no activity against antigen-negative cells, which is referred to herein as a bystander effect. That is, conjugates prepared with non-cleavable linkers have minimal bystander activity. When cultured separately, both the huC242-SPP-DM1 (cleavable) and huC242-SMCC (non-cleavable) conjugates showed cell-damaging activity against the antigen-positive COLO205 cell line and were not active against the antigen-negative cell line Namalwa (FIGS. 14A-C). However, treatment of cocultures of COLO205 and Namalwa with huC242-SPP-DM1 revealed that the conjugates even displayed significant cell-destroying activity against antigen-negative Namalwa cells. In contrast, the huC242-SMCC-DM1 conjugate did not exhibit any such bystander activity in these cases. Even when Namalwa cells were co-cultured with antigen-positive COLO205 cells, no cell-damaging activity was observed with the huC242-SMCC-DM1 conjugate on Namalwa cells. This minimal bystander activity of the non-cleavable conjugate, as determined by such in vitro assays, may contribute to the increased tolerance of the conjugate with the non-cleavable linker observed in acute toxicity assays.

The above experimental results demonstrate that the maytansinoid conjugates of the invention with a non-cleavable linker have greatly improved anti-tumor activity compared to the previously described cell-binding agent maytansinoid conjugates.

Application method

A method in which the above conjugate can be used to target a maytansinoid 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 the one or more maytansinoids are covalently linked to the cell-binding agent by a non-cleavable linker and the cell-binding agent binds to cells in the selected cell population.

The above conjugates may also be used in a method of disrupting cells, the method comprising: contacting the cell with a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are covalently linked to the cell-binding agent by a non-cleavable linker, and the cell-binding agent binds to the cell.

The conjugates described above may also be used in a method of treating afflictions, including, but not limited to, malignancies, autoimmune diseases, transplant rejection, graft-versus-host disease, viral infections, microbial infections, and parasitic infections, comprising: administering to a subject in need of treatment an effective amount of a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are covalently linked to the cell-binding agent by a non-cleavable linker and the cell-binding agent binds to diseased or infected cells of the disease.

Examples of medical conditions that can be treated according to the methods of the present invention include, but are not limited to, any type of malignancy, including, for example, lung, breast, colon, prostate, kidney, pancreas, ovary, and lymphoid cancers; autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis; transplant rejection such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection; graft versus host disease; viral infections such as CMV infection, HIV infection, AIDS, and the like; and parasitic infections such as giardiasis, amebiasis, schistosomiasis, and other diseases as determined by one of ordinary skill in the art.

The method may be carried out in vitro or in vivo.

The above-described conjugates can be used in methods for in vitro applications to treat, for example, autologous bone marrow cells, which are performed before they are implanted in the same patient, in order to destroy diseased or malignant cells; treating bone marrow cells or other tissues prior to their transplantation for the purpose of destroying T cells and other lymphoid cells (lymphoid cells) and preventing Graft Versus Host Disease (GVHD); treating the cell culture in order to destroy all cells except those which do not express the desired variants of the target antigen (variants); or treating the cell culture for the purpose of destroying variant cells expressing the undesired antigen; the methods comprise treating the cells with an effective amount of a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids are covalently linked to the cell-binding agent by a non-cleavable linker, and the cell-binding agent binds to the cells to be destroyed.

One of ordinary skill in the art can readily determine the clinical and non-clinical in vitro use cases.

For example, the processing may be performed as follows. Bone marrow may be harvested from a patient or other individual and then incubated in serum-containing medium to which the cytotoxic agent of the present invention is added at a concentration ranging from about 10pM to 1nM for about 30 minutes to about 48 minutes at about 37 ℃. The exact concentration conditions and incubation time, i.e. dosage, can be readily determined by one of ordinary skill in the art. After incubation, the bone marrow cells are washed with serum-containing medium and returned intravenously to the patient according to known methods. In the case of patients undergoing other treatments such as ablative chemotherapy procedures or systemic radiotherapy procedures between the time of bone marrow harvest and reinfusion of the treated cells, the treated bone marrow cells can be cryopreserved in liquid nitrogen using standard medical equipment.

For clinical in vivo use, the cytotoxic agent may be supplied as a solution or lyophilized powder which is tested for sterility and endotoxin levels. The following are examples of suitable protocols for administering the conjugate. The conjugate may be administered weekly for 4 weeks in the form of an intravenous bolus (intravenous bolus). Bolus doses (Bolus doses) may be administered in 50 to 500ml of standard physiological saline to which 5 to 10ml of human serum albumin may be added. The dose will be, for each administration, 10mg to 2000mg, given intravenously (in the range 100ng to 20mg/kg daily). After 4 weeks of treatment, patients may continue to receive treatment on a weekly basis (on weekly basis).

The specific in vivo clinical protocol for the route of administration, excipients, diluents, dosage, time, etc., can be determined by one of ordinary skill in the art as the clinical situation permits.

If desired, other active agents such as other antineoplastic agents may be administered with the conjugate.

Novel conjugates, compositions and methods of making conjugates

While some conjugates of antibodies and maytansinoids linked via a non-cleavable linker are known, others are novel. Accordingly, there is provided a cell-binding agent maytansinoid conjugate having at least one maytansinoid linked to a cell-binding agent by a non-cleavable linker which does not include a group derived from a cross-linking agent selected from the group consisting of: succinimidyl 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC), thio-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), thio-MBS and succinimidyl-iodoacetate.

Novel conjugates can be prepared and used as described above.

The compositions include a cell-binding agent maytansinoid conjugate and a carrier.

The carrier may be a pharmaceutically acceptable carrier, diluent or excipient.

Suitable pharmaceutically acceptable carriers, diluents or excipients are well known and can be determined by one of ordinary skill in the art as clinical conditions permit.

Examples of suitable carriers, diluents and/or excipients include: (1) dulbecco phosphate buffered saline, pH about 7.4, with or without about 1mg/ml to 25mg/ml human serum albumin, (2) 0.9% normal saline (0.9% w/v NaCl), and (3) 5% (w/v) glucose; and may also contain antioxidants such as tryptamine and stabilizers such as tween 20.

Synthetic methods are also provided for these novel conjugates.

One of the methods for preparing cell-binding agent maytansinoid conjugates comprises:

(a) providing a cell binding agent

(b) Modifying the cell-binding agent with a cross-linking agent, and

Another method of making a cell-binding agent maytansinoid conjugate comprises:

(a) providing maytansinoids or maytansinoids containing thiol,

(b) modifying maytansinoids or thiol-containing maytansinoids with a crosslinking agent to form a non-cleavable linker, and

Additional methods of making cell-binding agent maytansinoid conjugates include:

(a) to provide a maytansinoid which is,

(b) modifying the maytansinoid to provide a sulfur-free maytansinol with an active ester, and

(c) linking the modified maytansinoid and the cell-binding agent, thereby providing an S-free, non-cleavable linker between the cell-binding agent and the maytansinol, resulting in a conjugate. These methods are described in detail above and in U.S. patents cited herein and specifically incorporated herein by reference.

Examples

The invention will now be illustrated with reference to non-limiting examples. All percentages, ratios, parts, etc., are by weight unless otherwise stated.

The buffers used for the following experiments were: 50mM potassium phosphate (KPi)/50mM sodium chloride (NaCl)/2mM ethylenediaminetetraacetic acid (EDTA), pH 6.5 (buffer A); l x Phosphate Buffered Saline (PBS), pH 6.5 (buffer B); and 0.1M KPi buffer/2 mM EDTA, pH 7.5 (assay buffer).

SMCC (product No. 22360, M.W.334.33g/mole) and SIAB (product No. 22329, M.W.402.15g/mole) were purchased from Pierce. The huC242 antibody is a humanized form of monoclonal antibody C242, described in U.S. patent No. 5,552,293, which hybridoma is deposited as ECACC accession Number 90012601. Trastuzumab was obtained from Genentech. DM1 (free thiol form; M.W.737.5g/mole) was prepared as previously described in U.S. Pat. Nos. 5,208,020 and 6,333,410B 1.

Chromatography was performed using chromatography Columns purchased from Amersham Biosciences (Sephadex G25NAP-25 pre-packed column (Amersham 17-0852-02); HiPrep26/10 Desalting column (desaling Columns), Sephadex G25 quality resin (fine resin), 3in series (3connected in series) (Amersham 17-5087-01)). A TSK-GEL G3000SWXL chromatography Column (TOSOH Bioscience,08541) with TSK Column Guard SWxl (TOSOH Bioscience08543) was also used.

The solvents used for the following experiments were dimethyl sulfoxide (DMSO), Dimethylacetamide (DMA), ethanol (EtOH) and 100mM Ellman's Reagent (DTNB, available from cayman chemical) dissolved in DMSO.

Example 1A

Preparation of huC242-SMCC-DM1 conjugate

a. Preparation and assay of huC242 antibodies

With an extinction coefficient at 280nm of 1.48(mg/mL)^-1And a molecular weight of 147,000g/mole, and the antibody concentration was determined.

b. Preparation and assay of SMCC stock solutions

A20 mM solution of SMCC (6.69mg/mL) was prepared in dimethylsulfoxide. Solution 1/40 was diluted in Assay Buffer (Assay Buffer) and the absorbance of the sample was measured at 302 nm. Using extinction coefficient 602M^-1cm^-1The concentration of the stock solution was calculated.

c. Preparation and determination of DM1 stock solution

A10 mM solution of DM1 (free thiol form) was prepared in Dimethylacetamide (DMA) (7.37mg/mL) (FIG. 2). The absorbance of the stock solution in a dilution of ethanol (EtOH) was measured at 280 nm. With an extinction coefficient at 280nm of 5700M^-1The concentration of stored DM1 was calculated. The concentration of free thiol or thiol groups (-SH) in the stored DM1 preparation was determined using Ellman's reagent (DTNB). Dilutions of the stock solutions were prepared in assay buffer prepared as 3% (v/v) DMA followed by the addition of 100mM DTNB (1/100th volume) in DMSO. Determining the increase in absorbance at 412nm over the blank with an extinction coefficient 14150^-1cm^-1The concentration was calculated. the-SH concentration obtained by Ellman's analysis represents the calculated DM1 stock concentration under ligation conditions.

d. Modification of huC242 with SMCC crosslinker

Dividing the antibody into 2 samples; one modified with a 7.5-fold molar excess of SMCC crosslinker and the other with an 8.5-fold molar excess of SMCC crosslinker. The samples were reacted at 8mg/mL antibody. The reaction was carried out in buffer A (95% v/v) containing DMSO (5% v/v) at room temperature for 2 hours with stirring.

G25 chromatography to remove excess SMCC

The column was pre-filled with Sephadex G25 resin equilibrated in buffer A at 1.5X 4.9cm, and the huC242-SMCC reaction mixture was gel filtered through the column. The loading and elution volumes were determined according to the manufacturer's instructions. The modified antibody eluate was analyzed with the above extinction coefficient to determine the antibody concentration. For a 7.5-fold molar excess of SMCC reaction, the yield of modified antibody was 74.6%; for the 8.5-fold molar excess SMCC reaction, the yield of modified antibody was 81.6%.

f. Connection of huC242-SMCC and DM1

The modified antibody samples were reacted with a 1.7 fold excess of DM1 over the linker (assuming 5 linkers per antibody). The reaction was carried out at a concentration of 2.5mg/mL antibody dissolved in buffer A (97% v/v) with DMA (3% v/v). After the addition of DM1, the reaction was incubated at room temperature for approximately 20 hours with stirring.

g. Purification of the conjugate by G25 chromatography

The ligation reaction mixture was subjected to gel filtration through a 1.5x 4.9cm SephadexG25 resin pre-packed column equilibrated in buffer B. The loading and elution volumes were determined according to the manufacturer's instructions. The number of DM1 molecules attached per mole of huC242 was determined by measuring the absorbance of the eluate at 252nm and 280 nm. The DM 1/antibody ratio was found to be 3.54 for the 7.5-fold molar excess SMCC sample and 3.65 for the DM 1/antibody ratio for the 8.5-fold molar excess SMCC sample. The yields of the ligation step were 83.7% and 75.4%, respectively. The two ligations were pooled together, sterile filtered and reanalyzed for drug and antibody concentrations. The pooled samples were designated Lot #1713-146C and analyzed for binding, cytotoxicity, specificity, degree of polymerization, and free drug content.

Characteristics of huC242-SMCC-DM1

Example 1B

In vitro assay for huC242-SMCC-DM1

a. Combination (Binding)

Binding affinity (binding affinity) of huC242 antibody and huC242-SMCC-DM1 was compared on COLO205 cells using an indirect method using 5X10 per well³Cells were incubated initially for 3 hours on ice. The results are shown in FIG. 3. Naked antibody (naked antibody) at 5.1X10^-1KD binding of M, conjugate at 5.52X 10^-10KD binding of M. Thus, attachment to DM1 did not appear to alter the binding affinity of huC 242.

b. Cytotoxicity and specificity

The in vitro cytotoxicity and specificity of the huC242-SMCC-DM1 conjugate was evaluated using a continuous exposure clonal assay (continuous exposure clonal assay). The results are shown in FIG. 4. huC242-SMCC-DM1 was effective in destroying antigen-positive SKBR3 cells (IC)₅₀＝3.5x10^-12M). Specificity was determined by comparing the IC of target SKBR3 cells₅₀IC of value and antigen negative cell line A375₅₀Value of, wherein the IC of the conjugate₅₀Value greater than 3.0x 10^-9M。

c. Size exclusion chromatography

The conjugates were analyzed using a TSK3000 size exclusion column (fig. 5). Peak 4 represents the monomeric portion of the conjugate, while the earlier peak represents the multimer and the later peak represents the fragment. The area under each curve divided by the total peak area represents the contribution of that peak to the sample. The conjugate sample was found to be 96.0% monomer.

d. Free drug

The percentage of free drug was determined by ELISA and found to be 4.4%.

Example 2A

Preparation of trastuzumab-SMCC-DM1 conjugate

The antibody trastuzumab was obtained from Genentech for ligation with the non-cleavable heterobifunctional (heterobifunctional) crosslinker SMCC and DM 1. The antibody was buffer exchanged from 50mM potassium phosphate/2 mM EDTA, pH 6.0, into 50mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5 (buffer A). The antibody was then reacted with a 7.5-fold molar excess of SMCC linker and purified on Sephadex G25 resin before being linked to DM 1. The final conjugate was purified again using Sephadex G25 resin. The resulting conjugate contained 3.1 moles of DM1 per mole of antibody.

a. Preparation and assay of antibody trastuzumab

Trastuzumab dissolved in 50mM potassium phosphate/2 mM EDTA buffer at pH 6.0 was passed through a Sephadex G25 column equilibrated with buffer A and eluted as buffer A. All buffers of this assay were endotoxin-free, using the chromogenic limulus amoebocyte Lysate Assay (LAL) method (Cambrex). With an extinction coefficient at 280nm of 1.45mL mg^-1cm^-1And a molecular weight of 145,423g, and the antibody concentration was measured.

b. Preparation and assay of SMCC stock solutions

A20 mM solution of SMCC (6.69mg/mL) was prepared in DMSO. The solution 1/40 was diluted in assay buffer and the absorbance of the sample was measured at 302 nm. Using a molar extinction coefficient of 602M^-1cm^-1The concentration of the stock solution was calculated.

c. Preparation and determination of DM1 stock solution

A10 mM DM1 solution (free thiol form) was prepared in DMA (7.37mg/mL) (FIG. 2). The absorbance of the stock solution in a dilution of ethanol (EtOH) was measured at 280 nm. With a molar extinction coefficient of 5700M at 280nm^-1The concentration of stored DM1 was calculated. The concentration of free-SH in the stored DM1 preparation was determined using Ellman's reagent (DTNB). Dilutions of the stock solutions were prepared in assay buffer, 3% (v/v) DMA, followed by the addition of 100mM DTNB (1/100) in DMSO^thVolume). Determining the increase in absorbance at 412nm over the blank with an extinction coefficient 14150M^-1cm^-1The concentration was calculated. the-SH concentration obtained by Ellman's analysis represents the calculated DM1 stock concentration under ligation conditions.

d. Modification of trastuzumab with SMCC crosslinker

At 20mg/mL antibody, the antibody was modified with a 7.5-fold molar excess of SMCC. The reaction was carried out in buffer A (95% v/v) with DMSO (5% v/v) at room temperature for 2 hours with stirring.

G25 chromatography to remove excess SMCC

The trastuzumab-SMCC reaction mixture was gel filtered through a 1.5x 4.9cm Sephadex G25 resin pre-packed column equilibrated in buffer a. The loading and elution volumes were determined according to the manufacturer's instructions (Amersham Biosciences). The concentration of the modified antibody solution was analyzed by the above extinction coefficient spectrophotometry. The yield of modified antibody was 88% based on protein concentration.

f. Ligation of trastuzumab-SMCC and DM1

The modified antibody was reacted with a 1.7 fold excess of DM1 over the linker (assuming 5 linkers per antibody). The reaction was carried out at a concentration of 10mg/mL antibody dissolved in buffer A (94% v/v) with DMA (6% v/v). After the addition of DM1, the reaction was incubated at room temperature for 16.5 hours with stirring.

g. Purification of the conjugate by G25 chromatography

The ligation reaction mixture was subjected to gel filtration through a 1.5x 4.9cm SephadexG25 resin pre-packed column equilibrated in buffer B. The loading and elution volumes were determined according to the manufacturer's instructions (Amersham Biosciences). The number of DM1 molecules attached per mole of trastuzumab was determined by measuring the absorbance of the eluate at 252nm and 280 nm. The DM 1/antibody ratio was found to be 3.13, and the yield of the ligation step was 95.7%. The overall yield of linked trastuzumab was 84% based on the starting antibody. The resulting conjugates were analyzed for binding, cytotoxicity, specificity, degree of polymerization, and free drug content.

TABLE II characterization of trastuzumab-SMCC-DM1

Example 2B

In vitro detection of trastuzumab-SMCC-DM1

Binding experiments showed that the attachment of antibody to DM1 did not affect the apparent K_D(ii) a Naked trastuzumab antibody and trastuzumab-SMCC-DM1 conjugate have the same binding affinity for ECD plates (5.5X 10)^-11M). In vitro cytotoxicity evaluation of the samples showed that the trastuzumab-SMCC-DM1 conjugate was both highly toxic (IC against antigen positive cell line)₅₀Is 3.6x 10^-12M) are also specific (IC against antigen-negative cell lines₅₀Greater than 3.0x 10^-9M)。

a. Bonding of

The binding affinities of trastuzumab and trastuzumab-SMCC-DM1 were compared using the HER2ECD plate binding assay provided by Genentech. The results are shown in FIG. 24. Naked antibody and conjugate were administered at 5.5X10^-11Apparent K of M_DAnd (4) combining. Thus, ligation to DM1 did not alter the binding affinity of trastuzumab.

b. Cytotoxicity and specificity

The trastuzumab-SMCC-DM1 conjugate was evaluated for cytotoxicity and specificity in vitro using a continuous exposure clonal assay (continuous exposure clonal assay). The results are shown in FIG. 25. trastuzumab-SMCC-DM1 was effective in destroying antigen-positive SKBR3 cells (IC)₅₀＝3.6x 10^-12M). Comparison of IC of target SKBR3 cells₅₀IC of value and antigen negative cell line A375₅₀In value, specificity is shown, in which IC of the conjugate₅₀Value greater than 3.0x 10^-9M。

c. Size exclusion chromatography

The conjugates were 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 the total peak area represents the contribution of that peak to the sample. The conjugate sample was found to be 95.3% monomer.

d. Free drug assay

The percentage of free drug was determined by ELISA and was found to be 3.4%.

e. Endotoxin levels

The conjugate was detected by chromatographic LAL assay (chromatographic LAL test) and found to contain 0.03 EU/mg.

Example 3A

Preparation of trastuzumab-SIAB-DM 1 conjugate

Trastuzumab antibodies were obtained from Genentech for ligation with non-cleavable heterobifunctional cross-linkers SIAB and DM 1. At pH 6.5, the antibody was reacted with a 7.0-fold molar excess of SIAB linker and purified on Sephadex G25F resin. Antibody-containing fractions were pooled and reacted with DM1 overnight at pH 6.5 and room temperature under standard ligation conditions, but in the dark. An aliquot (aliquot) was removed from the reaction vessel and analyzed to determine the incorporation of DM 1. After NAP 5 filtration, an aliquot was determined to be only 1.4 drug/Ab. An additional 8-fold excess of SIAB was added to the reaction for 2 hours, then the pH was increased to 8 before an additional 1.5-fold excess of DM1/SIAB was added. The reaction was allowed to proceed and purified on Sephadex G25F resin. The resulting conjugate contained 3.42 moles of DM1 per mole of antibody.

a. Determination of trastuzumab

With an extinction coefficient at 280nm of 1.45mL mg^-1cm^-1And a molecular weight of 145,423g, and the antibody concentration was measured.

b. Preparation and determination of SIAB stock solutions

A18 mM solution of SIAB (7.2mg/mL) was prepared in DMSO. Wavelength scans of solutions diluted into pH4 buffer were recorded for informational purposes only.

c. Preparation and determination of DM1 stock solution

An approximately 30mM DM1 solution (free thiol form) was prepared in DMA. The concentration of free-SH in the stored DM1 preparation was determined using Ellman's reagent (DTNB). Dilutions of the stock solutions were prepared in assay buffer prepared as 3% (v/v) DMA followed by the addition of 100mM DTNB (1/100 vol) in DMSO. Determining the increase in absorbance at 412nm over the blank with a molar extinction coefficient 14150M^-1cm^-1The concentration was calculated. the-SH concentration obtained by Ellman's analysis represents the DM1 stock concentration calculated as the ligation state.

d. Modification of trastuzumab with SIAB crosslinker

At 20mg/mL antibody, the antibody was modified with a 7.0-fold molar excess of SIAB. The reaction was carried out in buffer A (95% v/v) with DMSO (5% v/v) at room temperature for 2 hours with stirring.

G25 chromatography to remove excess SIAB

The trastuzumab-SIAB reaction mixture was gel filtered through HiPrep26/10 Desalting Columns (desaling Columns) equilibrated in buffer A. At 280nm, there appears to be interference from the SIAB reagent, thus assuming that the yield of modified antibody is 100% and assuming that 5 linker/antibody modifications were used to determine the amount of DM1 in the ligation reaction.

f. Ligation of trastuzumab-SIAB and DM1

The modified antibody was reacted with a 1.7 fold excess of DM1 over the linker, as described above, assuming 100% yield and 5 crosslinkers/antibody. The antibody concentration in the reaction was estimated to be 12.5mg/ml, and the reaction was carried out in buffer A (97% v/v) with DMA (3% v/v). After addition of DM1, the reaction was incubated in the dark at room temperature for 16.5 hours with stirring.

g. Ligation reagent analysis

A 0.25mL aliquot of the reaction mixture was removed and gel filtered through a pre-filled Sephadex G25 column equilibrated in buffer B. The number of DM1 molecules attached per mole of trastuzumab was determined by measuring the absorbance of the eluate at 252nm and 280 nm. The DM 1/antibody ratio was only 1.4.

h. Additional modification/ligation reactions

An additional 8-fold molar excess of SIAB was added and allowed to incubate for 2 hours at room temperature. DM1 was added in a 1.5-fold molar excess over SIAB and 1N NaOH was added to increase the pH of the reaction to 8. The reaction was incubated at room temperature in the dark and gel filtered through a G25F resin column equilibrated in buffer B. i. Combining and characterizing conjugates

The fractions containing the protein were pooled, filtered and the absorbance measured at 252nm as well as 280 nm. Samples of the conjugates were tested for endotoxin levels, binding, specific and non-specific cytotoxicity, percent monomer and free drug levels.

TABLE III characterization of trastuzumab-SIAB-DM 1

Example 3B

In vitro detection of trastuzumab-SIAB-DM 1

Binding experiments showed that the attachment of antibody to DM1 did not affect the apparent K_D(ii) a Naked trastuzumab and trastuzumab-SIAB-DM 1 have similar binding affinities (apparent K of Ab)_DIs 1.2x 10^-10M, apparent K of conjugate_DIs 1.9x 10^-10M). In vitro toxicity evaluation of the samples showed that the trastuzumab-SIAB-DM 1 conjugate was both highly toxic (IC against the antigen positive cell line SKBR 3)₅₀Is 5x10^-12M) are also specific (IC for antigen-negative cell line A375₅₀Greater than 3.0x 10^- ⁹M)。

a. Bonding of

Binding affinities of trastuzumab and trastuzumab-SIAB-DM 1 were compared using the HER2ECD plate binding assay provided by Genentech. The results are shown in FIG. 27. Naked trastuzumab and trastuzumab-SIAB-DM 1 have similar binding affinities (apparent K of antibody)_DIs 1.2x 10^-10M, apparent K of conjugate_DIs 1.9x 10^-10M)。

b. Cytotoxicity and specificity

In vitro cytotoxicity evaluation of the samples showed that the trastuzumab-SIAB-DM 1 conjugate was both highly toxic (IC against the antigen positive cell line SKBR 3)₅₀＝5x 10^-12M) are also specific (IC for antigen-negative cell line A375₅₀Greater than 3.0x 10^-9M). See fig. 28.

c. Size exclusion chromatography

The conjugates were 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 the total peak area represents the contribution of that peak to the sample. A sample of the conjugate was found to be 96.4% monomer.

d. Free drug

The percentage of free drug was determined by ELISA and was found to be 0.35%.

e. Endotoxin levels

The conjugate was detected by the chromatographic LAL test and was found to contain < 0.04 EU/mg.

Example 4

Linking of huC242 to crosslinker to form S-free non-cleavable linker

a. Synthesis of

Preparation of a stock solution of the crosslinker in DMA (see FIG. 21 for its structure), removal (spin out) of the insoluble precipitate, using ε²⁸⁰＝5700M^-1cm^-1The extinction coefficient of DM1 at this wavelength determines the concentration of the residual solution. This is only an estimate of the concentration, since the true extinction coefficient of the substance was not determined. Note that ε of DM1²⁵²/ε²⁸⁰Is 4.7 (in ETOH), and epsilon of this crosslinker solution²⁵²/ε²⁸⁰It was determined to be 1.42 (in pH 7.5 buffer) indicating either a different extinction value (extininion) or the presence of impurities.

The ligation reaction was performed at a level of 2mg with 2.8mg/ml huC242 antibody dissolved in 16% DMA in buffer E, pH 7.5 (buffer E ═ 50mM sodium phosphate, 150mM naci, 10mM EDTA). Based on the estimated crosslinker concentration in the stock solution, 30 equivalents of crosslinker/antibody were used (earlier experiments with 10 equivalents of crosslinker/antibody produced conjugates with only 0.9 DMl/antibody). The reaction was allowed to proceed for 3 hours, and the conjugate was then purified over a Nap 10(G25) column. After filtration (Millex GV filter, 0.2 μm pore size), the conjugate had 2.56DM 1/antibody (Lot #1749 ═ 78 a, antibody recovery). Free DM1 was determined in aliquots of the conjugates by HPLC (HiPrep column) and a considerable DM1 peak was observed at 12.09'. The sample was thus dialyzed against buffer B to remove this peak, and then re-analyzed. The final conjugate sample (Lot #1749-124A) was analyzed by HPLC for no free DM1 and 1.84DM 1/antibody. The conjugate was subjected to SECHPC, which showed 97% monomeric antibody.

b. Cytotoxicity and binding

The inventors performed binding and cytotoxicity studies on huC 242-S-free non-cleavable linker-DM 1 conjugate. First, the binding affinities of huC242 antibody, huC242-SMNP-DM3, and huC 242-S-free non-cleavable linker-DM 1 were compared using an indirect method on COLO205 cells. Using 5X10 per hole³Cells were incubated initially for 3 hours on ice. The results are shown in figure 23, which shows that the huC 242-S-free non-cleavable linker-DM 1 conjugate is approximately 2-fold higher than the apparent dissociation constant (apparent dissociation constant) of the free antibody (see figure 23). Furthermore, the in vitro cytotoxicity of the huC 242-S-free non-cleavable linker-DM 1 conjugate was comparable to that of huC242-SMNP-DM3 (IC of the S-free non-cleavable linker conjugate)₅₀＝7.0x 10^-12M) (see fig. 22).

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

All patents, publications, and other references cited herein are specifically incorporated by reference in their entirety.

Claims

1. A cell-binding agent maytansinoid conjugate, wherein the cell-binding agent maytansinoid conjugate has the formula:

wherein Ab is trastuzumab.

2. Use of a cell binding agent maytansinoid conjugate of claim 1 in the manufacture of a medicament for the treatment of cancer.