ANTIBODY-DRUG CONJUGATES AND IMMUNOTOXINS

Abstract

The present invention relates to conjugates, in particular antibody-drug conjugates and immunotoxins, having the formula I: A-(L-D)p (I) or a pharmaceutically acceptable salts or solvates thereof, wherein: A is an antibody that selectively binds Endoglin; L is a linker; D is a drug comprising a cytolysin or a Nigrin-b A-chain; and p is 1 to 10, and to use of such conjugates in the therapeutic treatment of tumors. Methods of producing such conjugates and components for use in such methods are disclosed.

Claims

1. A conjugate having the formula I:
A-(L-D).sub.p(I) or a pharmaceutically acceptable salt or solvate thereof, wherein: A is an antibody that selectively binds Endoglin; L is a linker; D is a drug comprising a cytolysin or a Nigrin-b A-chain; and p is 1 to 10.

2. The conjugate of claim 1, wherein A is a monoclonal antibody or binding fragment thereof that selectively binds to an extracellular region of human Endoglin.

3. The conjugate of claim 2, wherein A comprises heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having the following amino acid sequences: (i) CDRH1: SEQ ID NO: 7 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 7; (ii) CDRH2: SEQ ID NO: 8 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 8; (iii) CDRH3: SEQ ID NO: 9 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 9; (iv) CDRL1: SEQ ID NO: 10 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 10; (v) CDRL2: SEQ ID NO: 11 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 11; and (vi) CDRL3: SEQ ID NO: 12 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 12.

4. The conjugate of claim 3, wherein CDRH1-3 comprise the amino acid sequences of SEQ ID NOS: 7-9, respectively and wherein CDRL1-3 comprise the amino acid sequences of SEQ ID NOS: 10-12, respectively.

5. The conjugate of any one of the preceding claims, wherein A comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 5.

6. The conjugate of claim 5, wherein A comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5.

7. The conjugate of any one of the preceding claims, wherein A comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 6.

8. The conjugate of claim 7, wherein A comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 6.

9. The conjugate of any one of the preceding claims, wherein A comprises a heavy chain comprising an amino acid sequence having at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 3.

10. The conjugate of claim 9, wherein A comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3.

11. The conjugate of any one of the preceding claims, wherein A comprises a light chain comprising an amino acid sequence having at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 4.

12. The conjugate of claim 11, wherein A comprises a light chain comprising the amino acid sequence of SEQ ID NO: 4.

13. The conjugate of claim 1 or claim 2, wherein A competes with the anti-human Endoglin IgG1 antibody having the heavy chain amino acid sequence of SEQ ID NO: 3 and the light chain amino acid sequence of SEQ ID NO: 4 for binding to immobilized recombinant human Endoglin.

14. The conjugate of claim 1, wherein A is a monoclonal antibody or binding fragment thereof that selectively binds to an extracellular region of murine Endoglin.

15. The conjugate of claim 14, wherein A comprises heavy chain complementarity determining regions 1-3 (CDRH1-3) and light chain complementarity determining regions 1-3 (CDRL1-3) having the following amino acid sequences: (i) CDRH1: SEQ ID NO: 19 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 19; (ii) CDRH2: SEQ ID NO: 20 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 20; (iii) CDRH3: SEQ ID NO: 21 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 21; (iv) CDRL1: SEQ ID NO: 22 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 22; (v) CDRL2: SEQ ID NO: 23 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 23; and (vi) CDRL3: SEQ ID NO: 24 or a variant thereof having up to 1 or 2 amino acid substitutions compared with the sequence of SEQ ID NO: 24.

16. The conjugate of claim 15, wherein CDRH1-3 comprise the amino acid sequences of SEQ ID NOS: 19-21, respectively and wherein CDRL1-3 comprise the amino acid sequences of SEQ ID NOS: 22-24, respectively.

17. The conjugate of any one of claims 14 to 16, wherein A comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 17.

18. The conjugate of claim 17, wherein A comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17.

19. The conjugate of any one of claims 14 to 18, wherein A comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 18.

20. The conjugate of claim 19, wherein A comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18.

21. The conjugate of any one of claims 14 to 20, wherein A comprises a heavy chain comprising an amino acid sequence having at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 15.

22. The conjugate of claim 21, wherein A comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15.

23. The conjugate of any one of claims 14 to 22, wherein A comprises a light chain comprising an amino acid sequence having at least 90%, 95% or 99% sequence identity with the full-length sequence of SEQ ID NO: 16.

24. The conjugate of claim 23, wherein A comprises a light chain comprising the amino acid sequence of SEQ ID NO: 16.

25. The conjugate of claim 1 or claim 14, wherein A competes with the anti-murine Endoglin IgG1 antibody having the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16 for binding to immobilized recombinant murine Endoglin.

26. The conjugate of any one of the preceding claims, wherein D is a cytolysin of formula IV: ##STR00035## wherein: R.sup.2 (i) is directly or indirectly attached to linker L or (ii) is H or C.sub.1-C.sub.4 alkyl; R.sup.6 is C.sub.1-C.sub.6 alkyl; R.sup.7 is C.sub.1-C.sub.6 alkyl, CH.sub.2OR.sup.19 or CH.sub.2OCOR.sup.20, wherein R.sup.19 is alkyl, R.sup.20 is C.sub.2-C.sub.6-alkenyl, phenyl, or CH.sub.2-phenyl; R.sup.9 is C.sub.1-C.sub.6 alkyl; R.sup.10 is H, OH, O-alkyl or O-acetyl; f is 1 or 2; R.sup.11 has the following structure: ##STR00036## wherein R.sup.21 is H, OH, halogen, NH.sub.2, alkyloxy, phenyl, alkyl amino or dialkyl amino; R.sup.16 is H or a C.sub.1-C.sub.6-alkyl group; R.sup.17 (i) is directly or indirectly attached to linker L or (ii) is CO.sub.2H, CO.sub.2R.sup.18, CONHNH.sub.2, OH, NH.sub.2, SH or a optionally substituted alkyl, cycloalkyl, heteroalkyl or heterocycloalkyl group, wherein R.sup.18 is an optionally substituted alkyl, heteroalkyl or hetercycloalkyl group; and q is 0, 1, 2 or 3; and wherein the term optionally substituted relates to groups, wherein one or several H atoms can be replaced by F, Cl, Br or I or OH, SH, NH.sub.2, or NO.sub.2; the term optionally substituted further relates to groups, which can be exclusively or additionally substituted with unsubstituted C.sub.1-C.sub.6 alkyl, C.sub.2C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 heteroalkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.2-C.sub.9 heterocycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.1-C.sub.9 heteroaryl, C.sub.7-C.sub.12 aralkyl or C.sub.2-C.sub.11 heteroaralkyl groups.

27. The conjugate of claim 26, wherein R.sup.2 is a bond to linker L.

28. The conjugate of claim 26, wherein R.sup.17 is C(O)X, CONHNHX, OX, NHX or SX, wherein X is a bond to linker L.

29. The conjugate of any one of the preceding claims, wherein linker L further comprises a spacer.

30. The conjugate of claim 29, wherein the spacer has a chain length of 2 to 30 atoms.

31. The conjugate of claim 29 or claim 30, wherein the spacer comprises or consists of an alkylene (i.e. divalent alkyl) or heteroalkylene (i.e. divalent heteroalkyl) group.

32. The conjugate of any one of claims 29 to 31, wherein the spacer comprises or consists of an alkylene or oxyalkylene group.

33. The conjugate of claim 32, wherein the spacer comprises or consists of a group (CH.sub.2).sub.n or (OCH.sub.2CH.sub.2).sub.n, wherein n1.

34. The conjugate of claim 33, wherein the spacer comprises or consists of a group (OCH.sub.2CH.sub.2).sub.n, wherein n1.

35. The conjugate of claim 33 or claim 34, wherein n=1 to 15, 1 to 10, 1 to 6, or 2 to 5.

36. The conjugate of claim 35, wherein n=3 or 4.

37. The conjugate of any one of claims 29 to 36, wherein the spacer is directly attached to group R.sup.17, or is attached to group R.sup.17 via a bridging group.

38. The conjugate of claim 37, wherein the spacer is attached to group R.sup.17 via a C(O)X bridging group, wherein X is a bond to R.sup.17.

39. The conjugate of claim 38, wherein R.sup.17 is CONHNHX and the spacer is attached to group R.sup.17 via a C(O)X bridging group, wherein X represents the bond between the spacer and R.sup.17.

40. The conjugate of claim 39, wherein R.sup.17 is CONHNHX and the spacer is a (OCH.sub.2CH.sub.2).sub.n attached to R.sup.17 via a C(O)X bridging group, wherein n=2, 3 or 4.

41. The conjugate of claim 26, wherein D comprises a cytolysin having the following structure: ##STR00037##

42. The conjugate of claim 26, wherein D comprises a cytolysin having the following structure: ##STR00038##

43. The conjugate of any one of claims 26 to 42, wherein L comprises an attachment group for attachment to A and protease cleavable portion.

44. The conjugate of claim 43, wherein L comprises a valine-citrulline unit.

45. The conjugate of claim 44, wherein L comprises maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate.

46. The conjugate of claim 45, wherein the double bond of the maleimide is reacted with a thiol group of a cysteine residue of the antibody A to form a sulphur-carbon bond in order to effect linkage of the linker L to the antibody A.

47. The conjugate of any one of the preceding claims, wherein -L-D has a structure selected from the group consisting of: ##STR00039## ##STR00040##

48. The conjugate of claim 47, wherein -L-D has the following structure: ##STR00041##

49. The conjugate of claim 26, wherein -L-D has the following structure: ##STR00042##

50. The conjugate of claim 26, wherein -L-D has the following structure: ##STR00043##

51. The conjugate of any one of the preceding claims, wherein p is 1, 2, 3, 4 or 5.

52. The conjugate of any one of claims 1 to 25, wherein D is a Nigrin-b A-chain in the absence of a Nigrin-b B-chain.

53. The conjugate of claim 52, wherein the amino acid sequence of the Nigrin-b A-chain comprises or consists of the sequence of SEQ ID NO: 25.

54. The conjugate of claim 52 or claim 53, wherein the Nigrin-b A-chain is recombinantly-produced in a bacterial host cell.

55. The conjugate of any one of claims 52 to 54, wherein L comprises or is a disulphide bond.

56. The conjugate of any one of claims 52 to 55, wherein p is 1 to 5.

57. A conjugate as defined in any one of the preceding claims for use in medicine.

58. A conjugate as defined in any one of the preceding claims for use in a method of treatment of a tumor in a mammalian subject.

59. The conjugate for use according to claim 58, wherein said conjugate is for simultaneous, sequential or separate administration with one or more other antitumor drugs.

60. The conjugate for use according to claim 59, wherein said one or more other antitumor drugs comprise a cytotoxic chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent.

61. The conjugate for use according to claim 60, wherein said one or more other antitumor drugs comprise Gemcitabine, Abraxane bevacizumab, itraconazole, carboxyamidotriazole, an anti-PD-1 molecule or an anti-PD-L1 molecule.

62. The conjugate for use according to claim 61, wherein said anti-PD-1 molecule or anti-PD-L1 molecule comprises nivolumab or pembrolizumab.

63. The conjugate for use according to any one of claims 58 to 62, wherein the tumor is a blood neoplasm.

64. The conjugate for use according to any one of claims 58 to 62, wherein the tumor is a solid tumor.

65. The conjugate for use according to claim 64, wherein the treatment is of pancreatic cancer, Ewing sarcoma, breast cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer or colon cancer.

66. A method of treating a tumor in a mammalian subject, comprising administering a therapeutically effective amount of a conjugate as defined in any one of claims 1 to 56 to the subject in need thereof.

67. The method of claim 66, wherein said conjugate is administered simultaneously, sequentially or separately with one or more other antitumor drugs.

68. The method of claim 67, wherein said one or more other antitumor drugs comprise a cytotoxic chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent.

69. The method of claim 68, wherein said one or more other antitumor drugs comprise Gemcitabine, Abraxane bevacizumab, itraconazole, or carboxyamidotriazole, an anti-PD-1 molecule or an anti-PD-L1 molecule.

70. The method of claim 69, wherein said anti-PD-1 molecule or anti-PD-L1 molecule comprises nivolumab or pembrolizumab.

71. The method of any one of claims 66 to 70, wherein the tumor is a blood neoplasm.

72. The method of any one of claims 66 to 70, wherein the tumor is a solid tumor.

73. The method of claim 72, wherein the tumor is of pancreatic cancer, Ewing sarcoma, breast cancer, melanoma, lung cancer, head and neck cancer, ovarian cancer, bladder cancer or colon cancer.

74. Use of a cytolysin as defined in any one of claims 26 to 51 in the preparation of an antibody-drug conjugate that comprises an Endoglin-specific antibody.

75. Use according to claim 74, wherein the Endoglin-specific antibody is as defined in any one of claims 2 to 25.

76. An isolated Nigrin-b A-chain in the absence of the Nigrin-b B-chain.

77. The isolated Nigrin-b A-chain of claim 76, wherein the amino acid sequence of the Nigrin-b A-chain comprises or consists of the sequence of SEQ ID NO: 25.

78. Use of an isolated Nigrin-b A-chain according to claim 76 or claim 77 in the preparation of an immunotoxin.

79. Use according to claim 78, wherein the immunotoxin comprises an Endoglin-specific antibody.

80. Use according to claim 79, wherein the Endoglin-specific antibody is as defined in any one of claims 2 to 25.

81. A human monoclonal antibody: (i) that selectively binds human Endoglin and which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4; or (ii) that selectively binds murine Endoglin and which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO: 16.

82. The antibody of claim 81 for use in medicine.

83. The conjugate of any one of claims 1 to 56 or the antibody of claim 81 for use in a method of treating an inflammatory condition or an eye disease.

84. A method of treating an inflammatory condition or an eye disease in a mammalian subject, comprising administering a therapeutically effective amount of the conjugate as defined in any one of claims 1 to 56 or the antibody as defined in claim 81 to the subject in need thereof.

85. The conjugate or antibody for use according to claim 83 or the method according to claim 84, wherein: said inflammatory condition is rheumatoid arthritis; and/or said eye disease is diabetic retinopathy or macular degeneration.

86. Use of a monoclonal antibody as defined in claim 81 in the preparation of an antibody-drug conjugate or an immunotoxin.

87. A host cell comprising a vector comprising a polynucleotide that encodes at least one polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NOS: 1-6, 13-18 and 25.

88. The host cell of claim 87, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 26.

89. A process for the production of a conjugate as defined in any one of claims 52-56, comprising: (a) derivatising the antibody that selectively binds Endoglin to introduce at least one sulphydryl group; and (b) reacting the derivatised antibody and Nigrin-b A-chain under conditions which permit the formation of a disulphide bond linkage between the antibody and the Nigrin-b A-chain thereby producing the conjugate; and (c) optionally, purifying and/or isolating the conjugate.

90. The process of claim 89, wherein step (a) comprises reacting the antibody with 4-succynimidyloxycarbonyl--methyl--(2-pyridyl-dithio)toluene (SMPT), N-succynimidyl 3-(2-pyridyl-dithiopropionate) (SPDP) or methyl 4-mercaptobutyrimidate.

91. A process for the production of a conjugate as defined in any one of claims 26-51, comprising: (a) linking the antibody that selectively binds Endoglin to the linker via a thiol group; (b) linking the cytolysin to the linker; and (c) optionally, purifying and/or isolating the conjugate, wherein steps (a) and (b) can be performed in any order.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0087] FIG. 1 shows ELISA results of mE12-IgG for binding to recombinant mouse ENG (aa 27-581). Coated BSA was included as negative control. A concentration-dependent binding to mouse ENG was observed;

[0088] FIG. 2 shows flow cytometry analysis of binding of mE12-IgG to a) B16 cells using 50 g/ml antibody, b) B16 cells using 5 g/ml antibody, and c) HT1080 cells included as negative control. Shaded area: cells alone, unshaded area: cells incubated with antibody;

[0089] FIG. 3 shows a) ELISA of binding of A5 IgG1 to immobilized recombinant human ENG. b) Flow cytometry analysis of binding of A5-IgG to HT1080 cells;

[0090] FIG. 4 shows MALDI-Tof profile of recombinant nigrin-b A-chain. Observed mass (Da): 28546.55; Expected mass (Da): 28546.09; Mass deviation: 0.5; Mass Accuracy: 16 ppm.

[0091] FIG. 5 shows ribosome inactivating protein (RIP) activity of recombinant Nigrin-b A-chain (recNgA) tested in rabbit reticulocyte cell-free lysates (RRL) versus native (WT) Nigrin-b (3a, 3b, 6c, 9c) represent different formulations of recNgA

[0092] FIG. 6 shows cytotoxicity of recNgA tested on HT1080-FAP cell line through crystal violet viability assay (native Nigrindiamonds; recombinant Nigrin-b A-chainsquares)

[0093] FIG. 7 shows RIP activity of recNgA-conjugates in an RRL assay Native nigrin b: diamonds (.diamond-solid.); recNgA: squares (.square-solid.); recNgA-conjugates HPS-124-37-1 (triangles: .box-tangle-solidup.) HPS-124-37-2 (crosses: X)

[0094] FIG. 8 shows the general antibody conjugate structure for a cytolysin-conjugated antibody via a vcPABA linker. Attachment of the cytolysin may be via R.sub.1 or R.sub.4 (identified by arrows)

[0095] FIG. 9 shows analysis of A5 anti-huENG IgG1 internalization by discrimination of cells (n=10-30) showing only membrane staining (PM), PM and intracellular staining, or only intracellular staining.

[0096] FIG. 10 shows in vitro cytotoxic effect of cytolysin ADCs on (A) wild type (WT) and (B) antigen (AG)-expressing HT1080 cells. Cell proliferation arrest was evidenced through crystal violet staining after 72 h incubation of each compound at a concentration range from 10.sup.6 to 10.sup.12M. Parental TAM334 cytolysin was used as positive control for unspecific cytotoxicity.

[0097] FIG. 11 shows tumor growth inhibition effect of recNgA and cytolysin immunoconjugates. (A) recNgA conjugates, administrated as single agent (OMTX505) or in combination with Gemcitabine (OMTX505:GEM); (B) TAM471 (OMTX705-471) versus TAM551 (OMTX705-551) conjugates; (C) TAM471 (OMTX705-471) and TAM553 (OMTX705-553) versus TAM558 (OMTX705-558) conjugates. Vehicle and GEM (Gemcitabine): negative and positive control groups.

DETAILED DESCRIPTION OF THE INVENTION

[0098] In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

Endoglin

[0099] As used Endoglin may be an Endoglin protein of any mammalian species. In some cases Endoglin is human Endoglin (also known as CD105, ENG or END), the amino acid sequence of which is disclosed at UniProt accession No. P17813 (Version 154, dated 13 Nov. 2013) (SEQ ID NO: 27). In some cases, a molecule that binds Endoglin (e.g. an antibody molecule or a conjugate thereof) may bind to a region of the extracellular domain of Endoglin. The extracellular domain of human Endoglin comprises residues 26-561 of the full-length human Endoglin protein. In some cases Endoglin is murine Endoglin (also known as CD105, MJ7/18 antigen, ENG or END), the amino acid sequence of which is disclosed at UniProt accession No. Q63961 (Version 104, dated 13 Nov. 2013) (SEQ ID NO: 28). The extracellular domain of murine Endoglin comprises residues 27-581 of the full-length murine Endoglin protein.

Conjugate

[0100] As used herein conjugate includes the resultant structure formed by linking molecules and specifically includes antibody-drug conjugates (ADCs) and immunotoxins (ITs).

Selectively Binds

[0101] The terms selectively binds and selective binding refer to binding of an antibody, or binding fragment thereof, to a predetermined molecule (e.g. an antigen) in a specific manner. For example, the antibody, or binding fragment thereof, may bind to Endoglin, e.g. an extracellular portion thereof, with an affinity of at least about 110.sup.7M.sup.1, and may bind to the predetermined molecule with an affinity that is at least two-fold greater (e.g. five-fold or ten-fold greater) than its affinity for binding to a molecule other than the predetermined molecule.

Antibody Molecule

[0102] As used herein with reference to all aspects of the invention, the term antibody or antibody molecule includes any immunoglobulin whether natural or partly or wholly synthetically produced. The term antibody or antibody molecule includes monoclonal antibodies (mAb) and polyclonal antibodies (including polyclonal antisera). Antibodies may be intact or fragments derived from full antibodies (see below). Antibodies may be human antibodies, humanised antibodies or antibodies of non-human origin. Monoclonal antibodies are homogeneous, highly specific antibody populations directed against a single antigenic site or determinant of the target molecule. Polyclonal antibodies include heterogeneous antibody populations that are directed against different antigenic determinants of the target molecule. The term antiserum or antisera refers to blood serum containing antibodies obtained from immunized animals.

[0103] It has been shown that fragments of a whole antibody can perform the function of binding antigens. Thus reference to antibody herein, and with reference to the methods, arrays and kits of the invention, covers a full antibody and also covers any polypeptide or protein comprising an antibody binding fragment. Examples of binding fragments are (i) the Fab fragment consisting of V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; (ii) the Fd fragment consisting of the V.sub.H and C.sub.H1 domains; (iii) the Fv fragment consisting of the V.sub.L and V.sub.H domains of a single antibody; (iv) the dAb fragment which consists of a V.sub.H domain; (v) isolated CDR regions; (vi) F(ab).sub.2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a V.sub.H domain and a V.sub.L domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers (WO 93/11161) and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (WO94/13804; 58). Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains. Minibodies comprising a scFv joined to a CH3 domain may also be made.

[0104] In relation to a an antibody molecule, the term selectively binds may be used herein to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen-binding site is specific for a particular epitope that is carried by a number of antigens, in which case the specific binding member carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.

[0105] In some cases in accordance with the present invention the antibody may be a fully human antibody.

Cytotoxic Chemotherapeutic Agents

[0106] In some cases in accordance with any aspect of the present invention, the conjugate of the invention may administered with, or for administration with, (whether simultaneously, sequentially or separately) other antitumor drugs, including, but not limited to, a cytotoxic chemotherapeutic agent or an anti-angiogenic agent or an immunotherapeutic agent.

[0107] Cytotoxic chemotherapeutic agents are well known in the art and include anti-cancer agents such as:

Alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; 10 ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN-U) and streptozoein (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazolecarboxamide);
Antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2-deoxycofonnycin). Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorabicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin Q; enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes. Miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and antbracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o, p-DDD) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen. A further preferred cytotoxic agent is Gemcitabine (Gemzar). A further preferred cytotoxic agent is Paclitaxel bound to human serum albumin (Abraxane).

[0108] Anti-angiogenic agents are well known in the art and include anti-cancer agents such as bevacizumab, itraconazole, and carboxyamidotriazole.

[0109] Immunotherapeutic agents are known in the art and include, for example, anti-programmed cell death protein 1 (PD-1) antibodies and anti-programmed death-ligand 1 (PD-L1) antibodies, including Nivolumab (MDX1106) and Pembrolizumab (MK-3475).

Pharmaceutical Compositions

[0110] The conjugates of the present invention may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient.

[0111] A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the conjugate, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the conjugate.

[0112] In some embodiments, conjugates of the present invention may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised conjugates may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

[0113] Conjugates of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the conjugate. Thus pharmaceutical compositions may comprise, in addition to the conjugate, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the conjugate. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

[0114] For intra-venous administration, e.g. by injection, the pharmaceutical composition comprising the conjugate may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN, PLURONICS or polyethylene glycol (PEG).

Subject

[0115] The subject may be a human, a companion animal (e.g. a dog or cat), a laboratory animal (e.g. a mouse, rat, rabbit, pig or non-human primate), a domestic or farm animal (e.g. a pig, cow, horse or sheep). Preferably, the subject is a human. In some cases the subject may be a human diagnosed with or classified as being at risk of developing a cancer, e.g., an epithelial tumor, a solid tumor or a blood neoplasm. In certain cases the subject may be a laboratory animal, e.g., a mouse model of a cancer. In certain cases the subject may be a mammal (e.g. a human) that has been diagnosed with or classified as being at risk of developing an inflammatory condition, such as osteoarthritis or rheumatoid arthritis (RA). In particular, the subject may be a human having osteoarthritis or RA. In certain cases the subject may be a mammal (e.g. a human) that has been diagnosed with or classified as being at risk of developing an eye disease, such as diabetic retinopathy or macular degeneration.

Cancer

[0116] The anti-ENG conjugates described herein find use in the treatment of a tumor in a mammalian subject. The tumor may be a solid tumor. In particular, the tumor may be a pancreatic cancer, breast cancer, melanoma, Ewing sarcoma, lung cancer, head & neck cancer, ovarian cancer, bladder cancer or colon cancer.

Inflammatory Condition

[0117] In some cases in accordance with the present invention, the anti-ENG antibody or the antibody drug conjugate may be for use in the treatment of an inflammatory condition. ENG expression has been reported in osteoarthritis and rheumatoid arthritis. See, e.g., Szekanecz, Z. et al. Clinical Immunology and Immunopathology, 1995, 76, 187-194, and Leask A et al., Arthritis & Rheumatism, 2002, 46, 1857-1865. The present inventors believe that the anti-ENG antibodies described herein, and/or conjugates thereof described herein, are able to ameliorate osteoarthritis, rheumatoid arthritis and/or symptoms of osteoarthritis or rheumatoid arthritis.

Eye Disease

[0118] In some cases in accordance with the present invention, the anti-ENG antibody or the antibody drug conjugate may be for use in the treatment of an eye disease (e.g. diabetic retinopathy or macular degeneration, such as age related macular degeneration). ENG expression has been reported in certain eye conditions, including macular degeneration and retinopathy. See, e.g., Tsutomu Yasukawa et al., Curr Eye Res. 2000, 21, 952-961, and Abu El-Asrar A M et al., Mediators Inflamm. 2012, 2012:697489, and Malik R A et al., J Cell Mol Med. 2005, 9:692-7. The present inventors believe that the anti-ENG antibodies described herein, and/or conjugates thereof described herein, are able to ameliorate eye diseases and/or symptoms thereof (including diabetic retinopathy or macular degeneration, such as age related macular degeneration).

[0119] The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

EXAMPLES

Example 1

Production of Anti-ENG Antibodies

[0120] Anti-ENG scFvs isolated from synthetic antibody phage libraries have been described previously (29). One scFv, directed against the extracellular region of human ENG, known as A5 (29) and one scFv, directed against the extracellular region of murine ENG, known as mE12 (31) were converted into full-length IgG for subsequent characterisation studies and for generation of immunotoxins and ADCs. All scFvs were produced in E. coli and purified by IMAC, IgGs were produced in mammalian cells (CHO) using the Lonza GS expression vectors pEE6.4 and pEE14.4 developed for antibody production. Features of the scFv starting material are summarized in Table 1.

TABLE-US-00001 TABLE 1 antibodies, specificities, subclass, and vectors used as starting material V1 Plasmid Format Species Antigen Clone Subclass Vector DNA # scFv human human ENG A5 kappa pAB1 179 scFv human mouse ENG mE12 lambda pAB1 151.1

[0121] All scFvs were bacterially produced in E. coli TG1 and purified from the periplasmic extracts of 1 L cultures by IMAC.

[0122] Plasmids corresponding to full length IgG1 antibodies were generated and transfected into CHO cells for production of antibodies in Lonza's CHO expressing system with yields of approximately 1 mg/L of cell culture (lab scale). Antibodies were purified from cell culture supernatant by protein A chromatography. Purified proteins were characterized by SDS-PAGE and size exclusion chromatography. Bioactivity was analyzed by ELISA using recombinant ENG and detection of bound antibodies with HRP-conjugated anti-human IgG antibodies. Cell binding was analyzed by flow cytometry using ENG-expressing mouse B16 cell.

Results:

Plasmids Generated (and Sequenced):

[0123] A5 IgG1: pEE14.4 A5-IgG1 OCMTX003p (human anti-huENG IgG1)
mE12 IgG1: pEE14.4 mE12-IgG1 OCMTOO4p (human anti-muENG IgG1)

Example 2

Characterisation of Anti-ENG Antibodies

[0124] The amino acid sequences of anti-human ENG IgG1 A5 (A5-IgG1) heavy chain (HC) and light chain (LC), respectively are shown below:

TABLE-US-00002 Anti-humanEndoglinA5-IgG1-HC: (SEQIDNO:1) [00009] [00010] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN [00011] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aa 447 MWofprocessedHC 48,703 TheoreticalpI 8.36 Potentialglycosylationsite(doubleunderlined): N297 MutationsleadingtoADCCandCDCdeficiencyareshowninbold italics(seealsoWO99/58572) Signalsequenceisshownboxed VHdomainisunderlined;CDRH1-H3areshowninboldandcurved underlined. (SEQIDNO:2) [00012] [00013] [00014] NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC aa 214 MWofprocessedHC 23,113 theoreticalpI 7.76 signal sequenceisboxed VLdomainisunderlined;CDRL1-L3areshowninboldandcurved underlined. A5-IgG1-HC-withoutsignalsequence: (SEQIDNO:3) [00015] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN [00016] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A5-IgG1-LC-withoutsignalsequence: (SEQIDNO:4) [00017] [00018] NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC A5-VH: (SEQIDNO:5) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIYGSDGDTTYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARVFYTAGFDYWGQGTLVTVSS A5-VL: (SEQIDNO:6) DIELTQSPSSLSASVGDRVTITCRASQSISSSLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQAPAKPPTFGQGTKLEIKR A5-CDRH1: (SEQIDNO:7) SYAMS A5-CDRH2: (SEQIDNO:8) AIYGSDGDTTY A5-CDRH3: (SEQIDNO:9) VFYTAGFDY A5-CDRL1: (SEQIDNO:10) RASQSISSSLN A5-CDRL2: (SEQIDNO:11) AASSLQS A5-CDRL3: (SEQIDNO:12) QQAPAKPPT ParametersofthefullA5-IgGareasfollows: Totallengthoffull-lengthIgG(aa): 1,322 Calculatedmolecularmassoffull-lengthIgG: 143,577 Calculatedextinctioncoefficientoffull-lengthIgG: 195,440 Abs0.1%(= 1g/l) 1.361 theoreticalpI: 8.36 potentialglycosylationsite: N297 Anti-murineEndoglinmE12-IgG1-HC: (SEQIDNO:13) [00019] [00020] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH [00021] QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aa 446 MWofprocessedHC 48,574 CalculatedpI 8.88 Potentialglycosylationsite(doubleunderlined): N297 MutationsleadingtoADCCandCDCdeficiencyareshowninbold italics Signalsequenceisshownboxed VHdomainisunderlined Anti-murineEndoglinmE12-IgG1-LC: (SEQIDNO:14) [00022] [00023] SDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTECS aa 212 MW(processed) 22,516 CalculatedpI 6.69 Signalsequenceisshownboxed VLdomainisunderlined mE12-IgG1-HC-withoutsignalsequence: (SEQIDNO:15) [00024] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH [00025] QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK mE12-IgG1-LC-withoutsignalsequence: (SEQIDNO:16) [00026] SDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTECS mE12-VH: (SEQIDNO:17) EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLVWVSRINSDGSSTSYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARATGTWVMSWGQGTLVTVSS mE12-VL: (SEQIDNO:18) SSELIQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGN TASLTITGAQAEDEADYYCNSRDSSGTVFGGGTKLTVLG mE12-CDRH1: (SEQIDNO:19) SYGMH mE12-CDRH2: (SEQIDNO:20) RINSDGSSTSYADSVKG mE12-CDRH3: (SEQIDNO:21) ATGTWVMS mE12-CDRL1: (SEQIDNO:22) QGDSLRSYYAS mE12-CDRL2: (SEQIDNO:23) GKNNRPS mE12-CDRL3: (SEQIDNO:24) NSRDSSGTV

[0125] Purified anti-muENG mE12 and anti-huENG A5 antibodies were analyzed in ELISA for binding to recombinant ENG and using flow cytometry analysis (FACS). Affinity results are shown for mE12 anti-muENG (FIGS. 1 and 2) and A5 anti-huENG IgG1s (FIG. 3), and are summarized in Table 2. FIG. 1 shows concentration-dependent binding of mE12-IgG to recombinant mouse ENG (aa 27-581), but not to negative control (BSA). FIG. 2 shows flow cytometry analysis of binding of mE12-IgG to a) B16 cells using 50 g/ml antibody, b) B16 cells using 5 g/ml antibody, and c) HT1080 cells included as negative control. FIG. 3 demonstrates concentration-dependent binding of A5-IgG to human ENG by a) ELISA and b) flow cytometry analysis.

[0126] For scale-up the antibody constructs were cloned in GS double vectors (pEE14.4). The DNA plasmids were transformed, amplified, and transiently transfected into CHOK1SV cells for expression evaluation at a volume of 200 ml. In a second step the antibodies were transiently expressed in 5-10 L large scale cultures. Clarified culture supernatant was purified using one-step Protein A chromatography. Product quality analysis through SE-HPLC, SDS-PAGE and LAL was carried out using purified material at a concentration of 1 mg/ml, alongside an in-house human antibody as a control sample.

[0127] The purified protein samples were filtered through a 0.2 m filter and analysed by SE-HPLC chromatograms. The mE12-IgG was purified to >98.8%. The A5-IgG was purified to >90%. The endotoxin levels were <0.5 EU/mg.

[0128] All purified proteins were analyzed by SDS-PAGE in reducing and non-reducing conditions (data not shown).

[0129] Purified proteins A5-IgG and mE12-IgG were characterized by SDS-PAGE and size exclusion chromatography. Bioactivity was analyzed by ELISA, using recombinant mouse/human ENG and detection of bound antibodies with HRP-conjugated anti-human IgG antibodies. Cell binding was analyzed by flow cytometry, using ENG-positive HT1080 and ENG expressing mouse eEnd2. Melting points were determined by dynamic light scattering using a zetasizer nano. Affinities were determined by QCM using an Attana A100. Internalization study was performed by indirect immunofluorescence confocal microscopy on permeabilized cells, detecting bound and internalized antibodies with a FITC-labeled secondary antibody.

[0130] The full-length IgG1 purified antibodies were successfully produced at both lab scale and large scale, for the generation of immunoconjugates. A summary of antibody properties is shown in Table 2. The antibodies retained their specificity, as shown by ELISA and flow cytometry experiments. Affinities, as determined by QCM, were comparable with that of parental antibodies. QCM measurements indicated the contribution of avidity effects to high-affinity binding. Thermal stability differed between the different IgGs (64-72 C.).

[0131] Preliminary internalization studies indicate rapid cellular internalization for A5-IgG against cells expressing human ENG. Indeed within 30 min, almost 90% of the A5-IgG is found within HT1080 cells.

TABLE-US-00003 TABLE 2 Summary of antibody properties antibody A5-IgG1 mE12-IgG1 antigen human mouse endoglin endoglin isotype 1*/ 1*/ IgG type human human plasmid OCMTX003p OCMTX004p purity (SEC) minor aggregates Tm (DLS) 72 C. 64 C. EC.sub.50 ELISA 0.3 nM 60 nM (rhENG) (rmENG) EC.sub.50 FACS 0.4 nM 89 nM (HT1080) (eEnd2) binding rhENG: constants K.sub.D K.sub.D1 = 260 nM n.d. (QCM) K.sub.D2 = 2.5 nM internalization HT1080 n.d. 30-60 min y1* = deficient for ADCC and CDC (see Amour et al., 1999; Richter et al., 2013).

[0132] FIG. 9 shows internalization of anti-huENG A5 IgG1.

Example 3

Nigrin-b A-chain

[0133] In order to avoid side effects of free toxin that could be released in the bloodstream and to reduce potential immunogenicity of the RIP toxin, as extensively described with ricin, the enzymatic domain of Nigrin b, the A chain, was cloned and expressed in bacteria. The present inventors hypothesized that, if the A chain produced in bacteria was able to retain its activity, it would not be able to enter the cells, unless conjugated to a vehicle molecule, such as an antibody.

Production

[0134] Nigrin-b A-chain was synthetized taking into account codon optimization for bacterial expression and the synthetized gene was cloned in two different vectors, Nigrin pET30b-3 and Nigrin pET33b-1 (+/ His tag) for expression in two different E. coli strains, E. coli BLR(DE3) and E. coli HMS174(DE3). Different culture media were used to check different expression conditions. Process purification was established using Capto Q chromatography and SP Sepharose High Performance. Purified recombinant Nigrin-b A-chain (recNgA) was formulated at 5 mg/ml in PBS 1 pH7.4, DTT 0.5 mM, glycerol 10%. Endotoxin levels were <1 EU/mg of Nigrin and the purity >99% in monomeric form.

[0135] Eldman N-terminal sequencing revealed that N-terminal end of recNgA corresponded to the expected sequence.

TABLE-US-00004 RecombinantNigrin-bA-chainaminoacidsequence: (SEQIDNO:25) MIDYPSVSFNLDGAKSATYRDFLSNLRKTVATGTYEVNGLPVLRRESEVQ VKSRFVLVPLTNYNGNTVTLAVDVTNLYVVAFSGNANSYFFKDATEVQKS NLFVGTKQNTLSFTGNYDNLETAANTRRESIELGPSPLDGAITSLYHGDS VARSLLVVIQMVSEAARFRYIEQEVRRSLQQATSFTPNALMLSMENNWSS MSLEIQQAGNNVSPFFGTVQLLNYDHTHRLVDNFEELYKITGIAILLFRC SSPSND

[0136] The recombinant Nigrin-b A-chain has the following characteristics: Number of amino acids: 256

Molecular weight: 28546.0

Theoretical pI: 5.45

[0137] The nucleotide sequence encoding recombinant Nigrin-b A-chain is as follows:

TABLE-US-00005 (SEQIDNO:26) atagactatccctccgtctccttcaacttggatggagcca agtcggctacatacagggacttcctcagcaacctgcgaaa aacagtggcaactggcacctatgaagtaaacggtttacca gtactgaggcgcgaaagtgaagtacaggtcaagagtcggt tcgttctcgtccctctcaccaattacaatggaaacaccgt cacgttggcagtagatgtgaccaacctttacgtggtggct tttagtggaaatgcaaactcctactttttcaaggacgcta cggaagttcaaaagagtaatttattcgttggcaccaagca aaatacgttatccttcacgggtaattatgacaaccttgag actgcggcgaatactaggagggagtctatcgaactgggac ccagtccgctagatggagccattacaagtttgtatcatgg tgatagcgtagcccgatctctccttgtggtaattcagatg gtctcggaagcggcaaggttcagatacattgagcaagaag tgcgccgaagcctacagcaggctacaagcttcacaccaaa tgctttgatgctgagcatggagaacaactggtcgtctatg tccttggagatccagcaggcgggaaataatgtatcaccct tctttgggaccgttcagcttctaaattacgatcacactca ccgcctagttgacaactttgaggaactctataagattacg gggatagcaattcttctcttccgttgctcctcaccaagca atgat

Materials

[0138] Nigrin pET30b-3 genetic construct. [0139] Escherichia coli (Migula) Castellani and Palmers BLR(DE3) [0140] Culture media: auto induced medium (AIM) [0141] Extraction culture buffer: Glycine/NaOH 10 mM, Leupeptine 1 g/ml, Pepstatine 1 g/ml, pH 9.5. [0142] Extraction supernatant buffer Tris-HCl 50 mM, NaCl 200 mM, MgCl.sub.2 2 mM, leupeptine 1 gml.sup.1, pepstatine 1 gml.sup.1, lysozyme 0.1 mgml.sup.1, pH8.0. [0143] Dialysis solution: Citric acid/NaOH 25 mM pH5.0. -Capto Q FPLC: Equilibration buffer A: Glycine/NaOH 50 mM pH9.5. Elution buffer B: Glycine/NaOH 50 mM pH9.5, NaCl 1 M. [0144] Pooled fractions from Capto Q step (+80 ml extraction). [0145] SP Sepharose HP FPLC: Equilibration buffer A: Citric acid 25 mM pH4.0. Elution buffer B: Citric acid 25 mM pH4.0, NaCl 1 M.

Methods

[0146] E. coli BLR(DE3) holding expression Nigrin pET30b-3 cultivated in 1 L format of Auto Inducible Medium (AIM) with 30 gml.sup.1 Kanamycin. Protein expression was triggered by lactose activation and glucose depletion after about 3-4 hours of growth. Then, the temperature was lowered to 20 C. for an overnight duration.

[0147] For extraction, each cell pellet was initially resuspended in 80 ml of extraction buffer per liter of culture, and 3 cycles of 7 minutes disintegration at 1100-110 Bar were performed after 30 minutes of incubation at 8 C. under shaking. Then the extract underwent 60 minutes centrifugation at 15,900 g, 8 C. The supernatant was the purification's starting material.

[0148] Capto Q FPLC: 160 ml of extracted product from 81 culture were loaded into 160 ml Capto Q and equilibrated using 4 CV of equilibration buffer and washed with 15 CV of equilibration buffer. Elution was carried in three steps: 15 CV at 1.5 mS/cm (7.6% B); 20 CV at 23.8 mS/cm (18.9% B); 20 CV 100% B.

[0149] Dialysis was performed at the following conditions: 650 ml of the product were dialyzed in 45 L baths in citric acid/NaOH 25 mM pH5.0, cut-off 6-8000 Da. Dialysis factor 3500, <24 h. After dialysis, a 30 minutes centrifugation at 20,500 g and 8 C. allowed to separate soluble from insoluble fractions. SDS-PAGE was performed on the total and soluble fractions both pre and post dialysis (10 l loaded on SDS-PAGE). The eluent was dialysed into PBS pH7.4 and filtered =0.22 m using 220 cm.sup.2 EKV filters.

[0150] SP Sepharose HP: 610 ml of dialyzed pool of Capto Q in Citric acid 25 mM pH5.0 were loaded into 240 ml SP Sepharose High Performance with 4 CV of equilibration buffer and washed with 15 CV of equilibration buffer and eluted at 25 Cv gradient to 20% B; 4 CV step of 100% B.

[0151] Pooled fractions from SP Sepharose HP step were dialysed in PBS pH7.4, DTT 0.5 mM (54 L baths, pooled fractions of 950 mL at 0.97 mg/ml). Cut off was 6-8000 Da, dialysis factor was 3130, time >24 h. Afterwards a 30 min centrifugation at 20.55 g and 8 C. allowed to separate soluble from insoluble fractions. 10% glycerol was added afterwards.

[0152] Finally the eluent was dialysed into PBS pH7.4 (5 baths 3100) and filtered =0.2 m, then the recNg b A batch was snap frozen at 80 C. A SEC in Semi-Preparative S200 Superdex was later carried out.

[0153] Size exclusion chromatography and mass spectrometry analysis demonstrated monomeric and purification status of the obtained recombinant nigrin-b A-chain (recNgA) (FIG. 4).

[0154] Stability studies were performed to evaluate pH and temperature effect on nigrin-b A-chain protein itself and its activity. recNgA is stable at pH ranging from 5 to 9, and in presence or not of glycerol (from 10 to 45%) (data not shown).

Activity

[0155] The ribosome-inactivating protein (RIP) activity of recombinant Nigrin-b A-chain was tested in rabbit reticulocyte cell-free lysates: IC.sub.50 value obtained was similar to native nigrin-b and within 2.5 to 25 pM range (see FIG. 5). Thus, the A chain from Nigrin-b, expressed as a recombinant protein in bacteria, maintains its enzymatic activity, supporting that glycosylation is not required for RIP activity of Nigrin-b A-chain.

[0156] RecNgA retains its activity in rabbit reticulocyte cell-free lysates if stored frozen at (80 C.) and below 3 freeze-thaw cycles (not shown).

[0157] The cytotoxic activity of recNgA was tested on cell cultures through crystal violet-based viability assay. recNgA, lacking the B chain to translocate within cells, presents a 100 to 1000 less toxic activity than native Nigrin-b, as shown in FIG. 6. Native nigrin b showed an IC.sub.50210.sup.8M (similar to previous published data see 37), while recNgA showed an IC.sub.50210.sup.6M.

[0158] Previously published studies showed that native Nigrin b presents higher RIP activity than Ricin in RRL assay, while it is much less toxic (30-10,000 time, approximately) in cells or in vivo (see IC.sub.50 and LD.sub.50 values in Table 3).

[0159] Upon removing of B chain, Ricin A chain loses activity in both RRL assay and cytotoxicity assay. Unexpectedly, Nigrin b A chain, generated for the first time in this present invention, only loses activity in cell cytotoxicity assay, while it was even increased in RRL assay with respect to native Nigrin b. These data were suggesting that, in the case of Ricin, removing B chain was affecting not only binding and translocation of A chain, but also its RIP activity, while this was not the case for Nigrin b A chain that retains and even increases its RRL activity with respect to its native counterpart. As a result, Nigrin b A chain is 50 times more active than Ricin A chain in RRL.

[0160] Consequently, upon conjugation, Nigrin b A chain conjugates present higher cytotoxic activity (IC.sub.50 within pM range) than Ricin A chain conjugates (nM range)(not shown).

TABLE-US-00006 TABLE 3 In vitro and in vivo activity data for Ricin and Nigrin b (native and A chain). Rabbit Lysate HeLa Cells Mouse LD.sub.50 IC.sub.50 (pM) IC.sub.50 (pM) (gkg.sup.1) Nigrin b 30 27,600.00 (20- 12,000.00 2300 nM; dpt cell line) Nigrin b 6.5 750, 000.00 ND A chain (HT1080-FAP) 300, 000.00 (HT1080) Ricin 100 0.67 3.00 Ricin A 300 260,000.00 chain (T cells) ND (Inventors' own data Nigrin b A chain; see also Ferreras J. M. et al., Toxins, 3:420, 2011; Svinth M. et al., BBRC, 249: 637, 1998)

Example 4

Conjugation of Nigrin-b A-Chain to Anti-ENG Antibodies

[0161] For immunoconjugates containing RIPs to exhibit maximal cytotoxicity the RIP must be released from the targeting vehicle in fully active form, which requires avoiding steric hindrance (38). The disulfide bond is the only type of linkage that fit this criterium (39, 40). This bond allows conjugation using reagents for the introduction of free sulfhydryl groups such as N-succynimidyl 3(2-pyridyl-dithiopropionate) (SPDP) and 4-succynimidyloxycarbonyl--methyl- (2-pyridyl-dithio)toluene (SMPT). Immunotoxins consisting of mAbs covalently bound to toxins by hindered disulfide linkers, often labeled as second generation immunotoxins, are stable, long lived and display potent cytotoxicity to target cells (41).

[0162] SPDP has already been used in the making of immunotoxins (ITs) containing nigrin b (36, 42). Moreover SMPT protects the disulfide bond from attack by thiolate anions, improving in vivo stability of the linkage (43, 44).

Material

[0163] Recombinant nigrin b A chain in PBS, pH7.4, 10% glycerol, 0.5 mM DTT, 4.92 gl.sup.1, stored at 5 C. [0164] 5,5-dithio-bis-(2-nitrobenzoic acid) [0165] GE PD MiniTrap G-10 desalting columns. [0166] 0.2 m 28 mm sterile Minisart filters. [0167] Sciclone ALH 3000 workstation. [0168] Sarstedt Microtest Plate 96-Well Flat Bottom, ref no 82.1581.

Methods

[0169] Dithiothreitol (DTT, Cleland's reagent) is a redox agent that will be used to free the thiol groups present in the protein sample. Once said groups have been freed and so are available for reacting 5,5-dithio-bis-(2-nitrobenzoic acid) (Ellman reagent) will be added. Ellman reagent disulphide bridge will be cleaved and the 2 resulting thio-nitrobenzoate molecules (TNB) will attach to the protein at the thiol group sites. To titrate the TNBs absorbance values will be taken at =412 nm, a wavelength at which DTT is not absorbed, rendering the concentration of thiol groups. The proportion of these with the concentration of the protein taken from its absorbance at =280 will yield the number of free thiol groups per protein molecule.

[0170] Direct thiol titration was performed as follows:

[0171] 204 l recNg b A were dissolved in 796 l 20 mM phosphate 250 mM NaCl 1 mM EDTA pH 7.0 (assay buffer) (1.0033 gl.sup.1=final concentration). Ellman reagent was dissolved in phosphate 0.2 M at 3 gl.sup.1. For both buffers monobasic and dibasic sodium phosphate were added in a 1.61 to 1 mass proportion. PH was adjusted at room temperature and buffers were filtered. 100 ml Ellman buffer and 500 ml assay buffer were prepared. Ellman reagent was completely resuspended rather than weighed.

[0172] The recNgA sample was incubated in the presence of 4.8 mM DTT at room temperature for 30 min. The recNgbA sample was then purified in the column and the first 10 ml of the eluent aliquoted (V=0.5 ml).

[0173] The A.sub.280 of the aliquots was taken and the two most concentrated mixed. A.sub.280 was taken again. 10 l of 3 gl.sup.1 DTNB were added and A.sub.412 measured after 2 min (n=1), using Ellman diluted in assay buffer in the same concentration as a blank (n.sub.b=3). Readings belonged to the 0.1-3 AU linear range. Protein solutions were pipetted right beneath the meniscus after vortexing. 100 l were pipetted per well.

[0174] The results of this study show that the thiol group belonging to recNgA's single cysteine residue is free and available for reaction, not being blocked by its tertiary structure. This will allow recNgbA to be conjugated using a linker that requires a hindered inter-chain disulfide bond.

[0175] It is well established that immunoconjugates which contain ribosome-inactivating proteins exhibit maximal cytotoxicity only when the toxin molecule is released from the targeting vehicle in a fully active form. The separation of the RIP molecule from the carrier is required to avoid steric hindrance and to allow an effective translocation of the toxin into the cytoplasm (38). At present, the disulfide bond is the only type of linkage which appears to fit these criteria (40).

[0176] The coupling of two different protein macromolecules, that results in heterodimer formation, requires that each protein is modified prior to mixing them to react. In the case of the A chains of type 2 RIPs, the modification is limited to the reductive cleavage of the native cysteine residue that links the active (A) and the binding (B) chains of the molecule.

[0177] For IgG molecules, this is not possible because cysteine residues are involved in maintaining the tertiary and/or quaternary structure of the protein, so that it is not possible to reduce them without loss of the specific protein functions. Moreover, presumably some of the cysteine residues are not sterically accessible, as it was demonstrated by the 10 thiols groups per immunoglobulin that had to be generated for an optimal conjugation to an activated RIP (45).

[0178] For these reasons, in most IgG molecules, thiol groups are chemically inserted using hetero-bifunctional reagents, and several methods have been developed in order to generate hetero-conjugates avoiding or reducing to a minimum the formation of homopolymers. In most cases, the reagents used to introduce thiol groups react with amino groups, forming amide or amidine bonds. Amino groups are reactive, abundant and, in a limited way for most proteins, expendable. That is, a limited number of amino groups can be modified without diminishing the biological activity of the protein (40).

[0179] The most commonly used reagents for the introduction of free sulphydryl groups are N-succynimidyl 3-(2-pyridyl-dithiopropionate) (SPDP) and 4-succynimidyloxycarbonyl--methyl--(2-pyridyl-dithio)toluene (SMPT), that introduce 2-pyridyl disulphide groups into the protein by reacting with amino groups to form neutral amides, and methyl 4-mercaptobutyrimidate (2-iminothiolane.Traut's reagent) that introduces mercaptobutyrimidoyl groups, reacting to form charged amidines, thus preserving the positive charge of the derivatized amino acid (40;44).

[0180] SPDP and SMPT introduce hindered disulphide bond, while 2-iminothiolane SH must be protected by reacting it with 5,5-dithiobis-2-nitrobenzoic acid (Ellman's reagent).

[0181] The reaction with Ellman's reagent is also used for the quick measurement of protein sulphydryl groups (45, 46).

[0182] SMPT has a methyl group and a benzene ring attached to the carbon atom adjacent to disulphide bond that protects it from attack by thiolate anions, thus improving the in vivo stability of the linkage (43, 44).

[0183] Based on these data, IgG proteins can be modified with SMPT, which do not significantly affect the antigen binding property of the molecules in the following conditions, even if they change the charge of the protein in the reaction site.

[0184] In one study the present inventors investigated conjugating IgG1s with recNgA, using 2 different recNgA:mAb molar ratio of 2.5 and 3.5, after derivatization using an SMPT:mAb molar ratio of 6, following conjugation protocols (see 40). Purification was performed by Size Exclusion chromatography on Sephacryl S200 (see 41).

[0185] Under the described conditions, the immunotoxin is predominantly a mixture of antibody linked to one or two toxin molecules, with the presence of high molecular weight components (IgG linked to several RIP proteins), as well as free and polymeric RIPs (dimeric in the case of recNgA) and free antibody. Thus, a careful purification is thought to be desirable to obtain a pure product.

In Vitro Activity Testing

[0186] Activity testing on conjugates prepared as described above was performed though evaluation of RIP activity in rabbit reticulocyte cell-free lysate (RRL) assay. Results are presented in FIG. 7.

[0187] IC.sub.50 values obtained for the native Nigrin-b or recNgA were in the 2.5 pM range and those for conjugates were similar and within 1-0.5 pM range, even higher than native Nigrin-b positive control, showing that antibody conjugation did not affect the enzymatic activity of recNgA.

Example 5

Conjugation of Cytolysins to Anti-ENG Antibodies

[0188] The cytolysins employed for conjugation studies were chosen from the general structure shown above (formula IV). These structures exhibit activity against different cancer cell lines (nM to pM range).

[0189] Various linker systems can be used and attached to either R.sup.2 or R.sup.17 position of the molecule.

[0190] The general outline of the cytolysin conjugates, including the vcPABA linker and anti-ENG antibody, is shown in FIG. 8 (in the structure depicted in FIG. 8, the attachment site of the cytolysin to the vcPABA linker is at position R.sub.1 or R.sub.4the R.sub.1 and R.sub.4 numbering system used in FIG. 11 differs from the R group numbering system used, e.g., in the claims; it is intended that R.sub.1 of FIG. 11 corresponds to R.sup.2 in the claims and that R.sub.4 of FIG. 11 corresponds to R.sup.17 of the claims).

[0191] The vcPABA (valine-citrulline-PABC) protease-cleavable linker has been previously used in the ADC molecule Brentuximab Vedotine, developed by Seattle Genetics and Takeda, and recently approved by the FDA and EMEA as Adcetris (2011, and November 2012, respectively). In this ADC the vcPABA has been coupled at its free NH2 to maleimide caproyl for thiol-based conjugation on mAb (cAC10 anti-CD30 antibody). On the other side, vcPABA has been conjugated through its COOH to the Auristatin cytotoxic drug from Seattle Genetics (MMAE). (see 48)

[0192] The present inventors have used this linker (maleimide caproyl-vcPABA) to conjugate anti-ENG antibodies through thiol-based reaction with the maleimide caproyl, and on the other end, to the cytolysin cytotoxic molecules through its cyclic piperidine with vcPABA (R.sub.1 or R.sub.4 positions of the cytolysin shown in FIG. 8).

Synthesis of Maleimido-Val-Cit-PABOCO-Tubulysin/Cytolysin-TAM461:

[0193] ##STR00027##

TAM461 (Tubulysin/Cytolysin): 30.0 mg (0.041 mmol)

DMF: 3 mL

[0194] TAM465 (Linker): 35 mg (0.045 mmol)

HOBt: 1.4 mg

DIPEA: 10 L

[0195] TAM461 and TAM465 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18 h. The reaction mixture was concentrated and the resulting oil was purified by column chromatography using 2-6% methanol: DCM to give 35 mg (64%) of TAM467 as a white solid. ESI-MS: m/z=1371 [M+H].

Synthesis of Maleimido-Val-Cit-PABOCO-Tubulysin/Cytolysin-TAM470:

[0196] ##STR00028##

TAM470 (Tubulysin/Cytolysin): 0.07 mmol

DMF: 5 mL

[0197] TAM466 (Linker): 50 mg (0.065 mmol)

HOBt: 2.4 mg

DIPEA: 18 L

[0198] TAM470 and TAM466 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18 h and then analysed with TLC, indicating completion of reaction, The reaction mixture was concentrated and the resulting oil was purified with column chromatography using 4-12% methanol: DCM to give 56 mg of TAM471 (yield: 62%). ESI-MS: 1384.6 [M+1].

[0199] In vitro activity testing is performed. Functional activity is evaluated through microtubule inhibition assay, while cytotoxic activity is determined through crystal violet viability assay.

Generation of Cytolysin-Linker Derivatives

[0200] Different cytolysin-linker derivatives were synthesized according to the general structure presented in FIG. 11, where vcPABA linker was added either in position R1 (TAM467, TAM551) or R4 (TAM471, TAM553, TAM558), alone or with ethylene-glycol spacer (EG; n=1 to 3), or substituted by ethylene glycol groups (n=3) (TAM552). The respective chemical structures are presented in Table 4.

TABLE-US-00007 TABLE 4 Chemical structure of cytolysin-linker derivatives Product Code Mol. Wt. [00029] TAM467 1370.7 [00030] TAM551 1356.7 [00031] TAM471 1384.7 [00032] TAM552 1198.5 [00033] TAM553 1499.8 [00034] TAMS 58 1603.9

[0201] Microtubule inhibition activity and cytotoxic activity of each new derivative were evaluated through tubulin polymerization inhibition assay (TPI; Tubulin Polymerization assay kit; Cytoskeleton, Cat. #BKO11P), and cell proliferation arrest on HT1080 cells (CPA; crystal violet). IC50 were calculated and results are presented in Table 5.

TABLE-US-00008 TABLE 5 Microtubule inhibition activity and Cell Cytotoxicity activity of cytolysin-linker derivatives. IC50 (TPI IC50 (CPA Compound assay; M) assay; nM) TAM467 (Linker in R1) 150 230-420 TAM551 (Linker in R1) ND 90 TAM471 (Linker in R4; vcPABA) 14 17-42 TAM552 (Linker in R4; no 1.9 10 vcPABA; 3EG) TAM553 (Linker in R4; vcPABA; 6 98 1EG) TAM558 (Linker in R4; vcPABA; 1.9 98 3EG) TAM334 (parental cytolysin; no 2 0.3-0.6 linker) Tubulysin A ND 0.04-0.2 Tubulysin A + linker ND 5-20 MMAE (Seattle Genetics) ND 0.1-0.6 DM1-DM4 (Immunogen) ND 0.01-0.1 (ND: Not determined)

[0202] In vitro activity of parental cytolysin TAM334 is within the same range of other payloads currently used for the generation of antibody-drug conjugates such as auristatins (MMAE) or maytansinoids (DM1-DM4). As expected and previously described for other compounds from the Tubulysin A family, upon addition of linker, cell cytotoxic activity of cytolysins was decreased with respect to the parental compound TAM334. In addition, TAM467 derivative was presenting significantly lowest activity in both assays. All the derivatives were used in conjugation to generate ADC molecules and were evaluated comparatively both in vitro and in vivo to select the most active cytolysin-linker derivative.

Conjugation and Chemical Characterization of ADCs

[0203] Each of the newly generated derivatives was conjugated to monoclonal IgG1 human antibodies following a non-site-specific conjugation method on cysteine residues. To this aim, one batch of antibody was reduced and reacted with each of the derivatives. Different TCEP ratios were tested to reach optimal DAR of 3-4, less than 10% of free antibody and drug. Optimal conjugation conditions were as followed: TCEP=2.5 and 3.57 Thiol levels Ellmann's. Conjugates were then purified on G25 Sephadex and analysed through Size Exclusion Chromatography (SEC) to determine their purity, as well as Hydrophobic Interaction Chromatography (HIC) and Polymeric liquid reversed-phase chromatography (PLRP) to determine DAR, content of free antibody and distribution profile of different ADC species (0-8 drugs/mAb). Content of free drug was evaluated by UV detection method at 280 nm. Results of chemical analysis were determined (not shown) and biochemical characteristics of ADCs are shown in Table 6.

TABLE-US-00009 TABLE 6 Summary of chemical characteristics of the different ADC molecules HIC mAb free SEC purity Free Lot Drug Conc. mAb DAR 280 nm Drug Volume HPS157-039-001 TAM471 1.195 mg/mL 10.1% 3.38 92% 0% ~5.8 mL (6.931 mg) HPS157-039-002 TAM551 1.332 mg/mL 22.4% 3.08 74% 0% ~5.8 mL (7.726 mg) HPS157-039-003 TAM552 1.319 mg/mL 5.1% 3.84 97% 0% ~5.8 mL (7.650 mg) HPS157-039-004 TAM553 1.305 mg/mL 7.0% 4.10 84% 0% ~5.8 mL (7.569 mg) HPS157-039-005 TAM558 1.332 mg/mL 5.8% 3.92 93% 0% ~5.8 mL (7.726 mg)

[0204] The various drugs produced different levels of aggregation. Specifically ADC HPS157-039-002 (TAM551) showed highest level of aggregation already at DAR=3.08, leaving 22.4% of unconjugated antibody. A preliminary conjugation with TAM467 also showed high level of aggregation: at DAR 3.27, SEC purity was already only 67% with 16% of free drug (data not shown). These data were suggesting that vcPABA linker in position R1 was not optimal for this type of cytolysin molecules.

In Vitro Evaluation of Cytolysin Conjugates

[0205] Cytolysin ADC molecules were evaluated comparatively in vitro through proliferation arrest assay (crystal violet staining). Results are presented in FIG. 10 and IC.sub.50 values in Table 7.

TABLE-US-00010 TABLE 7 IC.sub.50 values obtained in Proliferation Arrest Assay (nM) Compound HT1080-WT HT1080-AG(+) TAM334 1.04 0.77 ADC-471 (HPS-157-039-001) 5.6 10.33 ADC-551 (HPS-157-039-002) 964 552 ADC-553 (HPS-157-039-004) 90 108 ADC-558 (HPS-157-039-005) 555 0.96

[0206] Location of vcPABA linker alone in R1 position (ADC-551) generated conjugates with much less cytotoxic activity in vitro with respect to R4 position (ADC-471) (FIG. 10; Table 7). In addition, increasing the number of ethylene-glycol groups as spacer to vcPABA linker in R4 position (ADC-471 (n=0) versus ADC-553 (n=1) and ADC-558 (n=3)) was shown to increase antigen-specific cytotoxic activity in vitro (FIG. 10). Indeed, while ADC-471 and ADC-553 showed low and no antigen-specific cytotoxic activity (10 nM and 100 nM IC.sub.50 range, respectively) with no difference between wild type (WT) and antigen (AG) expressing HT1080 cells, ADC-558 presented a 1 nM range specific cytotoxic activity with a specificity ratio of 500 between AG and WT HT1080 cells.

Example 6

Evaluation of In Vivo Anti-Tumoral Effect of Conjugates

[0207] Both types of immunoconjugates, recNgA- and cytolysin-conjugates, were evaluated for their anti-tumoral effect in vivo in a patient-derived xenograft mouse model for pancreas cancer (PAXF-736), previously selected for antigen expression.

[0208] Dose range studies were performed to define the maximum tolerated dose to be used in efficacy studies (not shown). For recNgA immunoconjugates, a highest tolerated dose of 0.5 mg/kg was found, while cytolysin conjugates, independently of the derivative used, were administrated at doses from 2.5 mg/kg up to 20 mg/kg, without any weight loss or toxic effect.

[0209] Immunoconjugates were then administrated once a week intraperitoneally over 5 weeks. Tumor volume and body weight were measured every 2-3 days. Vehicle-treated and Gemcitabine-treated (150 mg/kg) PDX mice were used as negative and positive control groups, respectively. Results are shown in FIG. 11.

[0210] The recNgA immunoconjugates (OMTX505) presented a high in vivo anti-tumoral efficacy (60%) at a dose of 0.5 mg/kg in PDX murine models of pancreas cancer (FIG. 11A). When combined with Gemcitabine, it even showed 100% tumor growth inhibition and tumor regression.

[0211] According to the in vitro results (see FIG. 10 & Table 7), location of vcPABA linker alone in R1 position (OMTX705-551) generated conjugates with no anti-tumoral activity in vivo (FIG. 11B).

[0212] Supporting the in vitro data, increasing the number of ethylene-glycol groups as spacer to vcPABA linker in R4 position (OMTX705-471 (n=0) versus OMTX705-553 (n=1) and OMTX705-558 (n=3)) was shown to increase anti-tumoral effect in vivo (FIG. 11C). OMTX705-471 and OMTX705-553 did not show any anti-tumoral effect in vivo, while OMTX705-558 presented a 40% tumor growth inhibition effect at 2.5 mg/kg dose in PDX mouse model for pancreas cancer.

[0213] From these data, recNgA and TAM558 molecules were selected as best payloads for anti-ENG conjugates.

Example 7

Ewing Sarcoma Models

[0214] Tumor cell plasticity enables certain types of highly malignant tumor cells to dedifferentiate and engage a plastic multipotent embryonic-like phenotype, which enables them to adapt during tumor progression and escape conventional therapeutic strategies. A recent study demonstrated that ENG expression correlates with tumor cell plasticity in Ewing sarcoma, and it is significantly associated with worse survival of Ewing sarcoma patients. Ewing sarcoma with reduced ENG levels showed reduced tumor growth in vivo. This study thus delineates an important role of ENG in tumor cell plasticity and progression of aggressive tumors (51).

[0215] The present inventors hypothesize the therapeutic potential of anti-ENG monoclonal antibodies, ITs and ADCs, in the treatment of Ewing Sarcoma.

[0216] 14 cell line models of Ewing sarcoma have been developed for in vitro studies, and their corresponding xenograft models, for the screening and characterization of therapeutic molecules for the treatment of Ewing Sarcoma. ENG expression has been confirmed in all the 14 cell lines, and all the human Ewing sarcoma patient samples (n=10) that have been examined.

[0217] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0218] The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

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