KRAS EPITOPES AND ANTIBODIES

Abstract

The present invention relates to antibodies that bind to certain oncogenic mutant forms of KRAS. The invention also relates to certain epitopes of oncogenic mutant forms of KRAS. The invention also relates to immunoconjugates and compositions comprising such antibodies. The invention also provides methods of producing such antibodies. The invention further provides the use of such antibodies for therapeutic purposes, for example in the treatment of cancer.

Claims

1. An antibody which binds to an oncogenic mutant form of KRAS, said oncogenic mutant form of KRAS comprising an amino acid substitution at the position corresponding to position G13 or G12 of wild-type KRAS (SEQ ID NO:7), wherein said antibody binds to an epitope that is in the region of said oncogenic mutant form of KRAS that is defined by the amino acid residues that correspond to amino acid residues 10-21 of wild-type KRAS (SEQ ID NO:7), and wherein said antibody inhibits activity of said oncogenic mutant form of KRAS.

2. The antibody of claim 1, wherein said oncogenic mutant form of KRAS has the amino acid sequence of SEQ ID NO:14, 16, 13 or 15 and wherein said antibody binds to an epitope that is in the region of said oncogenic mutant form of KRAS that is defined by amino acid residues 10-21 of SEQ ID NO:14, 16, 13 or 15.

3. The antibody of claim 1 or claim 2, wherein said amino acid substitution at the position corresponding to position G13 or G12 of wild-type KRAS is a G13D or a G12D substitution.

4. The antibody of any one of claims 1 to 3, wherein said oncogenic mutant form of KRAS comprises an amino acid substitution at the position corresponding to position G13 of wild-type KRAS (SEQ ID NO:7) and said amino acid substitution is a G13D substitution.

5. The antibody of any one of claims 1 to 4, wherein said oncogenic mutant form of KRAS has the amino acid sequence of SEQ ID NO: 9, 12, 8 or 11 and wherein said antibody binds to an epitope that is in the region of said oncogenic mutant form of KRAS that is defined by amino acid residues 10-21 of SEQ ID NO: 9, 12, 8 or 11.

6. The antibody of any one of claims 1 to 5, wherein said oncogenic mutant form of KRAS has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 12 and wherein said antibody binds to an epitope that is in the region of said oncogenic mutant form of KRAS that is defined by amino acid residues 10-21 of SEQ ID NO:9 or SEQ ID NO:12.

7. The antibody of any one of claims 1 to 6, wherein said oncogenic mutant form of KRAS has the amino acid sequence of SEQ ID NO:8 or SEQ ID NO: 11 and wherein said antibody binds to an epitope that is in the region of said oncogenic mutant form of KRAS that is defined by amino acid residues 10-21 of SEQ ID NO:8 or SEQ ID NO:11.

8. The antibody of any one of claims 1 to 7, wherein said antibody binds to and inhibits at least one oncogenic mutant form of KRAS having an amino acid sequence of SEQ ID NO:8 or 11 and to at least one oncogenic mutant form of KRAS having an amino acid sequence of SEQ ID NO:9 or 12.

9. The antibody of any one of claims 1 to 8, wherein said antibody binds to an isolated peptide, said isolated peptide comprising (i) an amino acid sequence of SEQ ID NO:18 or SEQ ID NO:17; or (ii) an amino acid sequence substantially homologous to an amino acid sequence of (i), wherein said substantially homologous sequence has 1 or 2 amino acid substitutions or additions or deletions compared with said amino acid sequence and wherein in a sequence substantially homologous to SEQ ID NO:17 the X residue corresponding to position 3 of SEQ ID NO:17 is not altered and wherein in a sequence substantially homologous to SEQ ID NO:18 the X residue corresponding to position 4 of SEQ ID NO:18 is not altered.

10. The antibody of any one of claims 1 to 9, wherein said antibody binds to an isolated peptide, said isolated peptide comprising an amino acid sequence of SEQ ID NO:18 or SEQ ID NO:17.

11. The antibody of any one of claims 1 to 10, wherein said antibody binds to an isolated peptide, said isolated peptide comprising (i) an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1; or (ii) an amino acid sequence substantially homologous to an amino acid sequence of (i), wherein said substantially homologous sequence has 1 or 2 amino acid substitutions or additions or deletions compared with said amino acid sequence and wherein in a sequence substantially homologous to SEQ ID NO:1 the D residue corresponding to position 3 of SEQ ID NO:1 is not altered and wherein in a sequence substantially homologous to SEQ ID NO:2 the D residue corresponding to position 4 of SEQ ID NO:2 is not altered.

12. The antibody of any one of claims 1 to 11, wherein said antibody binds to an isolated peptide, said isolated peptide comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1.

13. The antibody of any one of claims 1 to 12, wherein said antibody binds to an isolated peptide, said isolated peptide comprising (i) an amino acid sequence of SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6; or (ii) an amino acid sequence substantially homologous to an amino acid sequence of (i), wherein said substantially homologous sequence has 1 or 2 amino acid substitutions or additions or deletions compared with said amino acid sequence and wherein in a sequence substantially homologous to SEQ ID NO:3 the D residue corresponding to position 3 of SEQ ID NO:3 is not altered and wherein in a sequence substantially homologous to SEQ ID NO:4 the D residue corresponding to position 4 of SEQ ID NO:4 is not altered and wherein in a sequence substantially homologous to SEQ ID NO:5 the D residue corresponding to position 5 of SEQ ID NO:5 is not altered and wherein in a sequence substantially homologous to SEQ ID NO:6 the D residue corresponding to position 4 of SEQ ID NO:6 is not altered.

14. The antibody of any one of claims 1 to 13, wherein said antibody binds to an isolated peptide, said isolated peptide comprising an amino acid sequence of SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6.

15. The antibody of any one of claims 1 to 14, wherein said antibody binds to an isolated peptide, said isolated peptide consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:6.

16. The antibody of any one of claims 1 to 15, wherein said antibody binds to an isolated peptide, said isolated peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:3.

17. The antibody of any one of claims 1 to 16, wherein said antibody binds to an isolated peptide, said isolated peptide consisting of an amino acid sequence of SEQ ID NO:5.

18. The antibody of any one of claims 1 to 17, wherein said antibody binds to a conjugate, said conjugate comprising an isolated peptide as defined in any one of claims 9 to 17 and a peptide carrier, preferably said peptide carrier is keyhole limpet hemocyanin (KLH).

19. The antibody of any one of claims 1 to 18, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:148 preferably SEQ ID NO: 149, and/or a VH CDR2 that has the amino acid sequence of SEQ ID NO:150 preferably SEQ ID NO: 151, and/or said light chain variable region comprises a VL CDR1 that has the amino acid sequence of SEQ ID NO:152 preferably SEQ ID NO: 153, and/or a VL CDR2 that has the amino acid sequence of SEQ ID NO:154 preferably SEQ ID NO:155.

20. The antibody of any one of claims 1 to 19, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises a variable light (VL) CDR3 that has the amino acid sequence of SEQ ID NO:37 or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequence.

21. The antibody of any one of claims 1 to 20, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR3 that has the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:74, or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequences.

22. The antibody of any one of claims 1 to 21, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:32, a VH CDR2 that has the amino acid sequence of SEQ ID NO:33 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:34, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:35, a VL CDR2 that has the amino acid sequence of SEQ ID NO:36 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:37, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequences.

23. The antibody of any one of claims 1 to 22, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:32, a VH CDR2 that has the amino acid sequence of SEQ ID NO:33 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:34 and wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:35, a VL CDR2 that has the amino acid sequence of SEQ ID NO:36 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:37.

24. The antibody of any one of claims 1 to 21, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:52, a VH CDR2 that has the amino acid sequence of SEQ ID NO:53 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:54, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:55, a VL CDR2 that has the amino acid sequence of SEQ ID NO:56 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:57, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequences.

25. The antibody of any one of claims 1 to 21 or claim 24, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:52, a VH CDR2 that has the amino acid sequence of SEQ ID NO:53 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:54 and wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:55, a VL CDR2 that has the amino acid sequence of SEQ ID NO:56 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:57.

26. The antibody of any one of claims 1 to 21, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:72, a VH CDR2 that has the amino acid sequence of SEQ ID NO:73 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:74, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:75, a VL CDR2 that has the amino acid sequence of SEQ ID NO:76 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:77, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequences.

27. The antibody of any one of claims 1 to 21 or claim 26, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:72, a VH CDR2 that has the amino acid sequence of SEQ ID NO:73 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:74 and wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:75, a VL CDR2 that has the amino acid sequence of SEQ ID NO:76 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:77.

28. The antibody of any one of claims 1 to 27, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein (i) the light chain variable region has the amino acid sequence of SEQ ID NO:31, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:30, or a sequence having at least 80% sequence identity thereto; (ii) the light chain variable region has the amino acid sequence of SEQ ID NO:51, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:50, or a sequence having at least 80% sequence identity thereto; or (iii) the light chain variable region has the amino acid sequence of SEQ ID NO:71, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:70, or a sequence having at least 80% sequence identity thereto.

29. The antibody of any one of claims 1 to 18, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:156 preferably SEQ ID NO: 157, and/or a VH CDR2 that has the amino acid sequence of SEQ ID NO:158 preferably SEQ ID NO: 159.

30. The antibody of any one of claims 1 to 18 or claim 29, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises: (a) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:156 or preferably SEQ ID NO:157, or a sequence substantially homologous thereto, (b) a VH CDR2 that has the amino acid sequence of SEQ ID NO:158 or preferably SEQ ID NO:159, or a sequence substantially homologous thereto, and (c) a VH CDR3 that has the amino acid sequence of SEQ ID NO:94 or SEQ ID NO:114 or SEQ ID NO:134, or a sequence substantially homologous thereto; and/or wherein said light chain variable region comprises: (d) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:95 or SEQ ID NO:115 or SEQ ID NO:135, or a sequence substantially homologous thereto, (e) a VL CDR2 that has the amino acid sequence of SEQ ID NO:96 or SEQ ID NO:116 or SEQ ID NO:136, or a sequence substantially homologous thereto, and (f) a VL CDR3 that has the amino acid sequence of SEQ ID NO:97, SEQ ID NO:117 or SEQ ID NO:137, or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequence.

31. The antibody of any one of claims 1 to 18, 29 or 30, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein light chain variable region comprises a variable light (VL) CDR2 that has the amino acid sequence of SEQ ID NO:96, or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequence.

32. The antibody of any one of claims 1 to 18, 29, 30 or 31, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:92, a VH CDR2 that has the amino acid sequence of SEQ ID NO:93 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:94, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:95, a VL CDR2 that has the amino acid sequence of SEQ ID NO:96 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:97, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequences.

33. The antibody of any one of claims 1 to 18, 29, 30, 31 or 32, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:92, a VH CDR2 that has the amino acid sequence of SEQ ID NO:93 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:94 and wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:95, a VL CDR2 that has the amino acid sequence of SEQ ID NO:96 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:97.

34. The antibody of any one of claims 1 to 18, 29, 30 or 31, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:112, a VH CDR2 that has the amino acid sequence of SEQ ID NO:113 and a VH CDR3 that has the amino acid sequence of SEQ ID NO: 114, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:115, a VL CDR2 that has the amino acid sequence of SEQ ID NO:116 and a VL CDR3 that has the amino acid sequence of SEQ ID NO: 117, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequences.

35. The antibody of any one of claims 1 to 18, 29, 30, 31 or 34, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:112, a VH CDR2 that has the amino acid sequence of SEQ ID NO:113 and a VH CDR3 that has the amino acid sequence of SEQ ID NO: 114 and wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO: 115, a VL CDR2 that has the amino acid sequence of SEQ ID NO: 116 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:117.

36. The antibody of any one of claims 1 to 18, 29 or 30, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:132, a VH CDR2 that has the amino acid sequence of SEQ ID NO:133 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:134, or sequences substantially homologous thereto, and/or wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:135, a VL CDR2 that has the amino acid sequence of SEQ ID NO:136 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:137, or sequences substantially homologous thereto, wherein said substantially homologous sequences are sequences containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequences, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequences.

37. The antibody of any one of claims 1 to 18, 29, 30 or 36, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:132, a VH CDR2 that has the amino acid sequence of SEQ ID NO:133 and a VH CDR3 that has the amino acid sequence of SEQ ID NO:134 and wherein said light chain variable region comprises a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:135, a VL CDR2 that has the amino acid sequence of SEQ ID NO:136 and a VL CDR3 that has the amino acid sequence of SEQ ID NO:137.

38. The antibody of claim 29, wherein said antibody comprises a VL CDR1, VL CDR2, a VL CDR3 and a VH CDR3 that have amino acid sequences as defined together in any one claims 30 or 32 to 37.

39. The antibody of any one of claims 1 to 18 or 29 to 38, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein (i) the light chain variable region has the amino acid sequence of SEQ ID NO:91, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:90, or a sequence having at least 80% sequence identity thereto; (ii) the light chain variable region has the amino acid sequence of SEQ ID NO: 111, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:110, or a sequence having at least 80% sequence identity thereto; or (iii) the light chain variable region has the amino acid sequence of SEQ ID NO:131, or a sequence having at least 80% sequence identity thereto and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:130, or a sequence having at least 80% sequence identity thereto.

40. The antibody of any one of claims 1 to 39, wherein said antibody is a monoclonal antibody or a polyclonal antibody.

41. The antibody of any one of claims 1 to 40, wherein said antibody is a monoclonal antibody.

42. The antibody of any one of claims 1 to 41, wherein said antibody is a whole antibody comprising an antibody constant region.

43. The antibody of any one of claims 1 to 42, wherein said antibody is an IgG antibody.

44. The antibody of any one of claims 1 to 41, wherein said antibody is an antigen binding fragment of an antibody.

45. The antibody of any one of claims 1 to 44, wherein said inhibition of activity is inhibition of GTPase activity and/or said inhibition of activity is characterised by the inhibition of ERK phosphorylation in cells that express said oncogenic mutant form of KRAS and/or said inhibition of activity is characterised by an increase in apoptosis in cells that express said oncogenic mutant form of KRAS and/or said inhibition of activity is characterised by an increase in necrosis in cells that express said oncogenic mutant form of KRAS.

46. A composition comprising an antibody of any one of claims 1 to 45 and a diluent, carrier or excipient, preferably a pharmaceutically acceptable diluent, carrier or excipient.

47. A nucleic acid molecule comprising a nucleotide sequence that encodes an antibody of any one of claims 1 to 45, or a set of nucleic acid molecules each comprising a nucleotide sequence, wherein said set of nucleic acid molecules together encode an antibody of any one of claims 1 to 45.

48. A method of producing an antibody according to any one of claims 1 to 45, comprising the steps of: (i) culturing a host cell comprising (i) one or more nucleic acid molecules encoding an antibody according to any one of claims 1 to 45 or (ii) a set of nucleic acid molecules each comprising a nucleotide sequence, wherein said set of nucleic acid molecules together encode an antibody of any one of claims 1 to 45, or (iii) one or more recombinant expression vectors comprising one or more of said nucleic acid molecules, under conditions suitable for the expression of the encoded antibody; and (ii) isolating or obtaining the antibody from the host cell or from the growth medium/supernatant.

49. An antibody as defined in any one of claims 1 to 45 for use in therapy.

50. An antibody as defined in any one of claims 1 to 45 for use in the treatment of cancer.

51. A method of treating cancer, said method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody as defined in any one of claims 1 to 45.

52. Use of an antibody as defined in any one of claims 1 to 45 in the manufacture of a medicament for use in therapy.

53. The use according to claim 52, wherein said therapy is the treatment of cancer.

54. An isolated peptide, wherein said isolated peptide is as defined in any one of claims 9 to 17.

55. A conjugate comprising an isolated peptide as defined in any one of claims 9 to 17 and a peptide carrier, preferably said peptide carrier is keyhole limpet hemocyanin (KLH).

Description

[0685] The invention will now be further described in the following non-limiting Examples with reference to the following drawings:

[0686] FIG. 1: Inhibition of phosphorylation of ERK in MDA-MB-231 (G13D) cells, induced by four anti-KRAS antibodies, G13D Ab1, G13D Ab2, G12D Ab1 and G12D Ab2. Antibodies were added to the cell medium (14-16 hours incubation) and intracellular phosphorylation of ERK was quantified using Perkin Elmer p-ERK 1/2 AlphaScreen Surefire assay kits. SAH-SOS1 (stabilized alpha helices of son of sevenless 1) was used as a positive control and rabbit IgG as a negative control (isotype control). Statistical significance is indicated as follows: *p<0.05, **p<0.01. Data is presented as mean±SEM. n=3 per treatment group except for G12D Ab2 27 nM and 67 nM where n=2.

[0687] FIG. 2: Determination of apoptosis in G12D mutated SK-LU-1 (lung adenocarcinoma) cells by four anti-KRAS antibodies, G13D Ab1, G13D Ab2, G12D Ab1 and G12D Ab2. Cells were treated with antibody or control and apoptosis was quantified in real time using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega). The isotype control was rabbit IgG. This graph represents the state of apoptosis at 52 hrs after addition of antibodies/controls to the cells. Luminescence is proportional to the level of apoptosis. Data was collected during a single day, n=4 per treatment. Statistical significance is indicated as follows: *p<0.05, **p<0.01, ****p<0.0001. Data is presented as mean±SEM.

[0688] FIG. 3: Determination of apoptosis in G12D mutated LS174T (colon adenocarcinoma) cells, induced by G12D Ab1, at 24, 48 and 72 hrs. Cells were treated with antibody or control and apoptosis was quantified by FACS, using fluorescence labelled Annexin V as the marker of apoptosis. Data is shown as % of control, where the control consisted of cells treated with vehicle (medium). n=1.

[0689] FIG. 4: Determination of apoptosis in G13D mutated HCT116 (colon carcinoma) cells, induced by four anti-KRAS antibodies, G13D Ab1, G13D Ab2, G12D Ab1 and G12D Ab2. Cells were treated with antibody or control and apoptosis was quantified in real time using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega). The isotype control was rabbit IgG. This graph represents the state of apoptosis at 52 hrs after addition of antibodies/controls to the cells. Luminescence is proportional to the level of apoptosis. Statistical significance is indicated as follows: **p<0.01. Data is presented as mean±SEM. n=3.

[0690] FIG. 5: Determination of apoptosis induced by antibodies G13D Ab1 and G12D Ab2 in (A) G13D mutated HCT116 (colon carcinoma) cells; (B) G12D mutated SK-LU-1 (lung adenocarcinoma) cells; and (C) non-cancerous wtKRAS CRL-1831 (colon epithelia) cells. Cells were treated with antibody or control and apoptosis was quantified in real time using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega). Vehicle is PBS/Tween. This graph represents the state of apoptosis at 52 hrs after addition of antibodies/control to the cells. For the HCT116 data, data was collected during 3 days with n=4 per day. For the SK-LU-1 data, data was collected during 2 days with n=4 per day. For the CRL-1831, data was collected during 1 day with n=4. Statistical significance is indicated as follows: **p<0.01, ****p<0.0001. Data is presented as mean±SEM.

[0691] FIG. 6: Determination of necrosis induced by antibodies G13D Ab1 and G12D Ab2 in (A) G13D mutated HCT116 (colon carcinoma) cells; (B) G12D mutated SK-LU-1 (lung adenocarcinoma) cells; and (C) non-cancerous wtKRAS CRL-1831 (colon epithelia) cells. Cells were treated with antibody or control and apoptosis was quantified in real time using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega). Vehicle is PBS/Tween. This graph represents the state of necrosis at 52 hrs after addition of antibodies/control to the cells. For the HCT116 data, data was collected during 3 days with n=4 per day. For the SK-LU-1 data, data was collected during 2 days with n=4 per day. For the CRL-1831, data was collected during 1 day with n=4. Statistical significance is indicated as follows: *p<0.05, ****p<0.0001. Data is presented as mean±SEM.

[0692] FIG. 7: Uptake of antibodies by cancer cells was quantified by FACS using fluorescence labelled antibody. G12D mutated LS174T (colon adenocarcinoma) cells and G13D mutated HCT116 (colon carcinoma) cells were treated with labelled antibody for 24 hrs and cell-associated fluorescence was quantified. FITC-A=fluorescence signal with emission measured at 488 nm. Area under the curve is proportional to the amount of cell-associated fluorescence. Fluorescence signal from non-antibody treated LS174T cells was used as control. n=1.

[0693] FIG. 8: Determination of apoptosis in wtKRAS NCI-H1975 (lung adenocarcinoma) cells by four anti-KRAS antibodies, G13D Ab1, G13D Ab2, G12D Ab1 and G12D Ab2. Cells were treated with antibody or vehicle. Apoptosis (A) and necrosis (B) were quantified in real time using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega). (A) represents the state of apoptosis at 10 hrs after addition of antibodies/vehicle to the cells. (B) represents the state of necrosis at 52 hrs after addition of antibodies/vehicle to the cells. Data was collected during a single day, n=4 per treatment. Data is presented as mean±SEM.

[0694] FIG. 9: Flow cytometry demonstrates uptake of antibodies G13D Ab1 and G12D Ab1 in mutated and wild-type KRAS cells. (A) Flow cytometry data showing antibody internalization at different time points after treatment with 220 nM ALEXA 488-conjugated G13D Ab1 antibody in a G13D-mutated KRAS cell line HCT-116, a G12D-mutated KRAS cell line SK-LU-1, and two wild-type KRAS cell lines A431, and NCI-H1975 (n=4). (B) Flow cytometry data showing antibody internalization at different time points after treatment with 220 nM ALEXA 488-conjugated G12D Ab1 antibody in a G13D-mutated KRAS cell line HCT-116, a G12D-mutated KRAS cell line SK-LU-1, and two wild-type KRAS cell lines A431 and, NCI-H1975 (n=4). Statistical significance was determined using a one-way ANOVA in combination with Dunnett's multiple comparisons test. Statistical significance is indicated as follows: **p<0.01, ***p<0.001, ****p<0.0001. Data is presented as mean±SEM.

[0695] FIG. 10: Evaluation of the apoptosis-inducing capacity of the mutation-selective G13D Ab1 and G12D Ab1 antibodies at 24 hrs in a panel of KRAS-mutated, wtKRAS, and non-cancerous cell lines. The different cell lines investigated include; G13D-mutated HCT116 (colon carcinoma) cells, G12D-mutated SK-LU-1 (lung adenocarcinoma) cells, G12V-mutated SW 620 (colorectal adenocarcinoma) cells, G12C-mutated MIA-PA-CA-2 (pancreatic carcinoma) cells, cancerous wtKRAS A431 (epidermoid carcinoma) cells, cancerous wtKRAS NCI-H1975 (non-small cell lung cancer) cells and non-cancerous wtKRAS CRL-1831 (colon epithelia) cells. Cells were treated with 220 nM antibody or vehicle and apoptosis was quantified in real time using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega). The graphs represent the level of apoptosis normalized to vehicle, at 24 hrs after addition of antibodies or vehicle to the cell cultures. For HCT116 cells n=10 (G13D Ab1) and 12 (vehicle and G12D Ab1). For SK-LU-1 cells n=8. For SW 620 cells n=8. For MIA-PA-CA-2 cells n=4. For A431 cells n=8. For NCI-H1975 cells n=4. For CRL-1831 cells n=6 (vehicle) and 8 (G13D Ab1 and G12D Ab1). Statistical significance was determined using one-way ANOVA in combination with Dunnett's multiple comparisons test except for SK-LU-1 cells where statistical significance was determined using a Kruskal-Wallis test in combination with Dunn's multiple comparisons test. **p<0.01, ***p<0.001, ****p<0.0001. Data is presented as mean±SEM.

[0696] FIG. 11: Evaluation of the apoptosis-inducing capacity of the mutation-selective G13D Ab1 and G12D Ab1 antibodies at 48 hrs in a panel of KRAS-mutated, wtKRAS, and non-cancerous cell lines. The different cell lines investigated include; G13D-mutated HCT116 (colon carcinoma) cells, G12D-mutated SK-LU-1 (lung adenocarcinoma) cells, G12V-mutated SW 620 (colorectal adenocarcinoma) cells, G12C-mutated MIA-PA-CA-2 (pancreatic carcinoma) cells, cancerous wtKRAS A431 (epidermoid carcinoma) cells, and non-cancerous wtKRAS CRL-1831 (colon epithelia) cells. Cells were treated with 220 nM antibody or vehicle and apoptosis was quantified in real time using RealTime-Go™ Annexin V Apoptosis and Necrosis Assay kit (Promega). Cancerous wtKRAS NCI-H1975 cells could not be evaluated at 48 hrs, using the above kit, due to a fast onset of apoptosis. The graphs represent the level of apoptosis normalized to vehicle, at 48 hrs after addition of antibodies or vehicle to the cell cultures. For HCT116 cells n=10 (G13D Ab1) and 12 (vehicle and G12D Ab1). For SK-LU-1 cells n=8. For SW 620 cells n=8. For MIA-PA-CA-2 cells n=4. For A431 cells n=8. For CRL-1831 cells n=6 (vehicle) and 8 (G13D Ab1 and G12D Ab1). Statistical significance was determined using one-way ANOVA in combination with Dunnett's multiple comparisons test except for CRL-183 and MIA-PA-CA-2 cells where statistical significance was determined using a Kruskal-Wallis test in combination with Dunn's multiple comparisons test. **p<0.01, ***p<0.001, ****p<0.0001. Data is presented as mean±SEM.

[0697] FIG. 12: Monoclonal antibody in hybridoma single cell clone supernatant was tested, using ELISA, for binding to a peptide having the amino acid sequence of SEQ ID NO:5 (which includes the G13D KRAS mutation). Clones 11D6-1, 4B81-1, 6B2-1, 7D11-1, and 7G2-1 were tested at various dilutions of peptide. Absorbance was measured at 405 nm. Higher absorbance indicates stronger binding.

EXAMPLE 1

Antibody Development

[0698] For each epitope (linear peptide) used for antibody generation, synthetic peptides including an additional cysteine residue, were synthesized and purified. These synthetic peptide sequences are set forth as SEQ ID NOs 3, 4, 5 and 6.

[0699] The peptides (epitopes) were linked by the terminal cysteine residue to keyhole limpet hemocyanin (KLH). The KLH-linked peptides (epitopes) were used to produce polyclonal antibodies by immunization of specific pathogen-free (SPF) rabbits following injection of the KLH linked peptides.

[0700] The peptides were produced by solid phase peptide synthesis (SPPS) with capping step. Linear peptides were conjugated to KLH by coupling SH-group on cysteine to NH2-group on KLH. Antibodies were purified using a Protein G column followed by affinity purification against the peptide.

[0701] The antibodies were affinity purified and subjected to ELISA tests. ELISA tests showed that the antibodies were able to bind to their respective peptides (i.e. the respective peptide used to immunize the rabbit to produce the relevant antibody). ELISA tests also showed that the antibodies were able to bind to the corresponding full-length oncogenic KRAS protein (isoform 2B versions).

[0702] Generation of both synthetic peptides and polyclonal antibodies were performed by Innovagen AB (Lund, Sweden).

[0703] The polyclonal antibody generated by immunization with the peptide of SEQ ID NO:3 is named G12D Ab1.

[0704] The polyclonal antibody generated by immunization with the peptide of SEQ ID NO:4 is named G12D Ab2.

[0705] The polyclonal antibody generated by immunization with the peptide of SEQ ID NO:5 is named G13D Ab1.

[0706] The polyclonal antibody generated by immunization with the peptide of SEQ ID NO:6 is named G13D Ab2.

Inhibition of KRAS Functional Activity

Inhibition of KRAS GTPase Activity

Materials and Methods

[0707] Recombinant wild-type, G12D and G13D forms of KRAS were produced by bacterial expression and purified. Purification tags were removed. KRAS was stored in 50 mM Tris, 100 mM NaCl, 10 mM MgCl.sub.2, 1 mM EDTA, 0.01% Triton-X-100, 1 mM DTT, pH7.5. Batch purity was assessed by gel filtration and SDS-PAGE.

[0708] KRAS activity was assessed using a microplate reader (Clariostar, BMG Labtech, Excitation/Emission=430/450). 1 g/L KRAS, 1 μM phosphate sensor (Thermo Fisher Scientific #PV4407), 6 μM GTP and antibody were prepared in buffer H (50 mM Tris, 100 mM NaCl, 10 mM MgCl.sub.2, 1 mM EDTA, 0.01% Triton X-100 and 1 mM DTT) and added to a 384-well plate and fluorescence was measured during 4 h. Fluorescence values after 4 h were compared between treatments.

[0709] The G13D Ab2, G12D Ab1 and G12D Ab2 antibodies were tested for their ability to inhibit GTP to GDP conversion (GTPase activity) by wild-type KRAS, G12D mutant KRAS and G13D mutant KRAS. The antibodies preferentially inhibited an oncogenic form of KRAS (G12D or G13D) as compared to wild-type KRAS. No significant inhibition of wild-type KRAS GTPase activity was observed.

Inhibition of ERK Phosphorylation

Materials and Methods

[0710] MDA-MB-231 cells (from American Type Culture Collection) were plated in 384 well plates at a density of 10000 cells/well and incubated overnight in a 5% CO.sub.2 incubator at 37° C. The next day, cells were treated with compounds (antibodies) or controls (rabbit IgG) in media containing serum and incubated for 14-16 hours in a 5% CO.sub.2 incubator at 37° C. The final working concentrations of compounds (antibodies) were 25 μg/ml (167 nM), 10 μg/ml (67 nM) and 4 μg/ml (27 nM). Rabbit IgG (−ve control) was added at a final concentration of 25 μg/ml (167 nM). The following day, media in each well was replaced with serum-free media containing compounds or controls. The K-Ras inhibitor SAH-SOS1 (stabilized alpha helices of son of sevenless 1, +ve control, 5 μM) was added to respective wells during this 4 h serum starvation period. After serum starvation, cells were treated with 10.9 ng/ml EGF (epidermal growth factor) for 10 min. After EGF treatment, cells were lysed for 10 min with lysis buffer provided in a kit (AlphaScreen® SureFire® p-ERK 1/2 (Thr202/Tyr204) Assay Kits, Perkin Elmer cat no. TGRESB500), which is a sandwich immunoassay for quantitative detection of phospho-ERK1/2 (phosphorylated on Thr202/Tyr204) in cellular lysates. ERK is Extracellular Signal-Regulated Kinase. The lysate was divided and used for measurement of total and phosphorylated ERK. Phosphorylated ERK was estimated following the AlphaScreen® SureFire® kit protocol (Perkin Elmer, cat no. TGRESB500). Total ERK was estimated following a kit protocol (AlphaScreen® SureFire® ERK 1/2 Total Assay Kit, Perkin Elmer cat. no. TGRTESB500), which is a sandwich immunoassay for quantitative detection of ERK (both phosphorylated and non-phosphorylated) in cellular lysates. Readings were taken on the Envision plate reader (Perkin Elmer) using standard AlphaScreen settings. AlphaScreen is a bead-based, non-radioactive Amplified Luminescent Proximity Homogeneous Assay. Statistical significance is indicated as follows: *p<0.05, **p<0.01. Data is presented as mean±SEM.

Results and Discussion

[0711] For antibodies to inhibit KRAS, they must enter the cells and be presented to the cytosol where KRAS resides. The level of phosphorylation of ERK in the MAPK pathway is in many cancer cells dependent on KRAS activity, where ERK is downstream to KRAS (MAPK/ERK pathway). ERK phosphorylation is often used as a measure of KRAS activity when testing anti-KRAS compounds, with inhibition of ERK phosphorylation a result of KRAS inhibition. We measured inhibition of ERK phosphorylation (as % inhibition of phospho-ERK) in the breast cancer cell line MDA-MB-231 by four anti-KRAS antibodies G13D Ab1, G13D Ab2, G12D Ab1 and G12D Ab2. As a positive control we used SAH-SOS1, a known polypeptide KRAS inhibitor that binds to KRAS, and as a negative control we used rabbit IgG which should not bind to KRAS. Antibodies were added to the medium outside the cells and, without wishing to be bound by theory, can enter the cells via the process of macropinocytosis. MDA-MB-231 cells bear a G13D KRAS mutation. We confirmed inhibition of ERK phosphorylation by the antibodies already at an extracellular concentration as low as 27 nM (FIG. 1).

Apoptosis

Materials and Methods

[0712] For experiments assessing apoptosis in HCT116 cells, SK-LU1 cells and CRL-1831 cells (i.e. the experiments that generated the data depicted in FIGS. 2, 4 and 5), the materials and methods are as follows:

[0713] The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega, cat no. JA1011) was used to measure apoptosis and necrosis in real time. This is a live-cell real-time (kinetic) assay that determines apoptosis by the exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane and determines necrosis by a cell-impermeant, profluorescent DNA dye that is able to enter cells during the necrotic process as the cell membrane disintegrate.

[0714] Cells were prepared as follows. HCT-116, SK-LU-1 and CRL-1831 cells (from American Type Culture Collection) were cultivated in T flasks. HCT-116 cells were grown in McCoy's 5a Medium Modified supplemented with 10% fetal bovine serum. SK-LU-1 cells were grown in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum. CRL-1831 cells were grown in DMEM:F12 Medium supplemented with 10% fetal bovine serum, 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocortisone and 20 ng/mL human recombinant EGF. Cells were harvested using pre-warmed trypsin solution (37° C.) and incubated for 15 minutes. Cells were transferred to L-15 assay medium, consisting of Leibovitz's L-15 Medium (ThermoFisher cat. no. 21083027), 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were counted and diluted to 200.000 cells/mL with L-15 assay medium. 50 μL of cell solution was added per well in 96 well plates (10.000 cells per well, Costar, white with clear opaque bottom, cat. no. 3903). For measurement of background, wells on the same plate were filled with 50 μL L-15 assay medium. Plates were sealed with adhesive film and incubated overnight at 37° C.

[0715] Antibodies were prepared as follows. Antibodies were first mixed into a total of 300 μL of phosphate-buffered saline containing 0.02% Tween (PBS/Tween). The final antibody concentration was 0.87 μM for all antibodies. Working solutions were prepared by mixing antibodies solutions with L-15 assay medium and kit detection reagent (210 μL assay medium+210 μL antibody solution+420 μL detection reagent). Detection reagent was prepared according to the kit instructions. One type of control was made where 210 μL antibody solution was replaced with 210 μL PBS/Tween (vehicle control). Another type of control was made where 210 μL antibody solution was replaced with 210 μL L-15 assay medium (medium control).

[0716] Assays were performed as follows. 200 μL of medium was removed from each plate well and replaced with 200 μL of working solution. The plates were the sealed with optic film and placed in a CLARIOstar® (BMG LABTECH) plate reader. Measurement of apoptosis was performed by reading luminescence signal and necrosis by fluorescence signal according to the kit instructions. Data analysis and interpretation was done according to the kit instructions.

[0717] Data is presented as mean±SEM. Statistical significance was determined using one-way ANOVA in combination with Dunnett's multiple comparisons test where each Ab (antibody) was compared to isotype control (rabbit IgG) or vehicle control. Statistical significance is indicated as follows: *p<0.05, **p<0.01, ****p<0.0001. Data is presented as mean±SEM.

For experiments assessing apoptosis in LS174T cells (i.e. the experiments that generated the data depicted in FIG. 3), the materials and methods are as follows:

[0718] LS174T cells were cultured to 70% confluence in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. Cells were treated with control (medium without antibody) or 3 ng/ml antibody in medium for 24, 48 or 72 hours. Cells were then washed and stained with fluorescence labelled Annexin V Pacific Blue (ThermoFisher, cat no. A35122) which binds to phosphatidylserine (PS) that becomes exposed on the outer leaflet of cells undergoing apoptosis. FACS was used for quantification of cell-associated fluorescence, and the level of apoptosis (% of control) was calculated as the ratio of fluorescence between antibody-treated cells and control cells.

Results and Discussion

[0719] In some experiments, apoptosis was quantified using a real-time apoptosis/necrosis kit from Promega, which measures the amount of phosphoserine lipid (PS) at the external side of the cellular membrane. PS amount increases during the apoptotic process. We have determined that the anti-KRAS antibodies here, G13D Ab1, G13D Ab2, G12D Ab1 and G12D Ab2, induce apoptosis in G12D mutated SK-LU-1 (lung adenocarcinoma) cells and/or G13D mutated HCT116 (colon carcinoma) cells (FIGS. 2, 4 and 5). Cells were treated with antibody by adding antibody to the cell medium. The data in FIG. 3 shows that the antibody G12D Ab1 is also able to induce apoptosis in LS174T (G12D) cells.

[0720] G13D Ab1 and G12D Ab1 caused high levels of apoptosis in both SK-LU-1 cells (FIG. 2) and HCT116 cells (FIG. 4) compared to isotype antibody control, or compared to vehicle PBS/Tween (FIG. 5A,B). This data indicates that both of these antibodies are active against both mutations (i.e. G12D and G13D mutant forms of KRAS). Without wishing to be bound by theory, it is believed that these antibodies may bind to the negatively charged aspartic acid (D) residue but that the antibodies can accommodate the D residue at either position 12 or 13 due to the small difference in distance. Some apoptosis was observed in CRL-1831 colon epithelia cells which have wild-type KRAS, although compared to cells harbouring mutated KRAS it was much less for both antibodies tested (FIG. 5C).

[0721] Apoptosis induced by KRAS antibodies of the present study shows that the synthetic antigens (which correspond essentially to amino acid residues 10-21 of oncogenic mutant forms of KRAS as described elsewhere herein) used for immunization and antibody production can be used to develop inhibitory KRAS antibodies. Furthermore, the data shows that antibodies can be generated which can inhibit both G12D and G13D mutant forms of KRAS.

Necrosis

Materials and Methods

[0722] Necrosis was recorded in parallel to apoptosis using the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit assay kit as described elsewhere.

Results and Discussion

[0723] It has been shown herein that, in addition to apoptosis, the anti-KRAS antibodies G13D Ab1 and G12D Ab1 caused significant necrosis in SK-LU-1 cells (FIG. 6A) and HCT116 cells (FIG. 6B) but not wild-type KRAS cells CRL-1831 (FIG. 6C).

Further Apoptosis and Necrosis Experiment

[0724] The effect of the antibodies G12D Ab1, G12D Ab2, G13D Ab1 and G13D Ab2 on apoptosis and necrosis of the lung adenocarcinoma cell line NCI-H1975 was tested. The NCI-H1975 cell line expresses wild-type KRAS. The materials and methods for this experiment are essentially as per the apoptosis and necrosis assays described above (the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega) was used).

[0725] In this experiment, the level of apoptosis and necrosis of NCI-H1975 cells treated with each of antibodies G12D Ab1, G12D Ab2, G13D Ab1 and G13D Ab2 was similar to, or less than, the level of apoptosis and necrosis observed with the vehicle-only control (FIG. 8).

Antibody Internalization

[0726] Materials and Methods Cells (LS174T cells or HCT116 cells) were cultured to 70% confluence in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. Cells were treated with 16 ng/ml rabbit anti-Goat IgG (H+L) Secondary Antibody, Alexa Fluor 488 (ThermoFisher, cat. no. A-11078) dissolved in the same medium for 24 hrs. Cells were washed and cell-associated fluorescence was quantified using FACS.

Results and Discussion

[0727] In order to assess whether cancer cells can internalize antibodies presented in the extracellular medium we used fluorescence-tagged antibodies and quantified uptake quantified by FACS. G12D mutated LS174T (colon adenocarcinoma) cells and G13D mutated HCT116 (colon carcinoma) cells were treated with labelled antibody for a duration, and cellular fluorescence was quantified. Based on the area under the curve of the accumulated florescence signal in treated cells, compared to the signal from untreated cells, we drew the conclusion that there exists a mechanism of uptake of antibody in these cancer cell types.

EXAMPLE 2

[0728] This Example shows that antibodies of the invention are internalized by cancer cells, including cancer cells expressing oncogenic mutant forms of KRAS. This Example also contains further data showing that antibodies of the invention cause apoptosis in cells expressing oncogenic mutant forms of KRAS.

Materials and Methods

Flow Cytometry Measurements of G13D Ab1 Antibody and G12D Ab1 Antibody Uptake

[0729] Antibody G13D Ab1 and antibody G12D Ab1 (of Example 1) were labelled with Alexa Fluor 488 using an Alexa Fluor 488 protein labeling kit (Cat # A10235, ThermoFisher Scientific). HCT 116, SK-LU-1, A431 and NCI-H1975 cells were cultured in 12-well microplates (Invitrogen). Cells were treated with 220 nM labelled G13D Ab1 antibody or G12D Ab1 antibody for 4 or 24 hours prior to measurements. Unstained cells were measured as a t(0) timepoint. Prior to measurements, cells were washed and harvested. Measurements were performed on a BD™ LSR II flow cytometer (BD Biosciences inc.). Analysis was performed in FlowJo v.10 (BD Biosciences). For each cell line and time-point n=4. n equals one measurement containing on average 4000 cells for HCT116, 8000 cells for SK-LU-1, 7000 cells for A431 and 4000 cells for NCI-H1975.

Antibody Induced Apoptosis of aKRA S Antibodies (G13D Ab1 Antibody and G12D Ab1 Antibody)

[0730] The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega, cat no. JA1011) was used to measure apoptosis in real time. Cultured cells were harvested using trypsin and transferred to L-15 assay medium, consisting of Leibovitz's L-15 Medium (ThermoFisher cat. no. 21083027), 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were counted and diluted to 200,000 cells/mL with L-15 assay medium. Cells were added in 96 well plates (10,000 cells per well, Costar, white with clear opaque bottom, cat. no. 3903) and incubated overnight.

[0731] Antibodies were diluted in L-15 assay medium and the detection reagent and added to the wells in a final concentration of 220 nM. The plates were the sealed with optic film and placed in a CLARIOstar® (BMG LABTECH) plate reader. Measurement of apoptosis was performed by reading of the luminescence signal.

2-3 biological replicates were performed for the following cell lines: HCT 116, SK LU 1, SW 620, CRL-1831 and A431. The NCI-H1975 and MIA-PA-CA-2 cell lines were tested once.

Results and Discussion

[0732] The in vitro anti-cancer efficacy of the G13D Ab1 and G12D Ab1 antibodies was determined by evaluating their capability to cause apoptosis in four different KRAS-mutated cell lines, i.e. G12D-mutated SK-LU-1 lung adenocarcinoma cells, G13D-mutated HCT116 colon carcinoma cells, G12V-mutated SW 620 colorectal adenocarcinoma cells, G12C-mutated MIA-PA-CA pancreatic carcinoma cells, as well as wild-type KRAS cell lines, i.e. A431 epidermoid carcinoma cells, and NCI-H1975 non-small cell lung cancer cells. A non-cancerous cell line CRL-1831 colon epithelial cells was also included as a control cell line in the apoptosis experiments. Although KRAS is an intracellular target, we exploited the fact that many cancer cell lines, including KRAS-mutated cells, have upregulated macropinocytosis. This uptake system has a well-documented capacity to internalize macromolecules, including proteins. We used flow cytometry and confocal microscopy experiments to evaluate internalization of Alexa Fluor 488-labelled G13D Ab1 and G12D Ab1 antibodies in the cell line bearing the G12D mutation (SK-LU-1) and the cell line bearing the G13D mutation (HCT116) as well as in the two wild-type KRAS cell lines A431, and NCI-H1975. The confocal microscopy images showed uptake of the G13D Ab1 and G12D Ab1 antibodies in all the different cell lines examined (data not shown). Flow cytometry experiments further confirmed these observations, and a statistically significant antibody uptake was observed in all tested KRAS-mutated and all tested wild-type KRAS cell lines as shown in FIG. 9. In the apoptosis experiments, the G13D Ab1 and G12D Ab1 antibodies were active only in G12D- and G13D-mutated cell lines after 24 hrs of treatment (FIG. 10). The antibodies did not cause apoptosis in any of the other cell lines tested which included G12C and G12V KRAS-mutated cells, wild type KRAS cancer cells and non-cancerous colon epithelial cells (FIG. 10). After 48 hrs of treatment (FIG. 11), we observed that the G13D Ab1 and G12D Ab1 antibodies elicited an increased level of apoptosis in G12D- and G13D-mutated cell lines, compared to the 24 h treatment. We also observed a small, but stastistically significant, increase of apoptosis in G12V-mutated cells after 48 h (FIG. 11). None of the other cell lines showed any increase in apoptosis after 48 h of antibody exposure (FIG. 11). Taken together, the data show that the apoptosis-inducing effect in G12D- and G13D-mutated cells compared to wild-type KRAS cells, is not a result of a quantitatively differential antibody uptake in favor of KRAS-mutated cells, but rather a result from a selective inhibition of G12D and G13D-mutated KRAS cells by the G13D Ab1 and G12D Ab1 antibodies.

[0733] The selective effects of the antibodies on G12D and G13D mutants but not on G12C and to a much lesser extent on G12V mutants may be explained by that the negatively charged aspartic acid residue (D) at the G12 and G13 position in the epitope region plays an important role in the binding of the antibodies. However, the allele location at either position 12 or 13, due to the small difference in distance, seems to be of minor importance for the binding interaction.

[0734] This study demonstrates two anti-KRAS antibodies that are capable of inhibiting G12D- and G13D-mutated KRAS with selectivity over other KRAS mutations (G12V and G12C) as well as over wild-type KRAS.

EXAMPLE 3

Materials and Methods

Production of Monoclonal Antibodies (Mouse IgG) Using the Hybridoma Technology

[0735] Mice were immunized with the linear peptide of SEQ ID NO:5 linked to keyhole limpet hemocyanin (the KLH linked peptide is as described above in Example 1). The “D” residue at residue number (position) 5 of SEQ ID NO:5 corresponds to the oncogenic G13D mutation of an oncogenic mutant G13D KRAS protein (e.g. an oncogenic mutant G13D KRAS protein of SEQ ID NO:9 or SEQ ID NO:12). Immune responses were evaluated with ELISA. After the immunization process mice were selected based on ELISA screening of serum, and spleen cells from mouse were extracted and fused with myeloma cells to produce hybridoma cells. Hybridomas were screened by ELISA to obtain positive clones (i.e. that produce antibody that binds to the target). After this screening, sub-cloning of the selected hybridomas was performed and a further round of screening was performed using ELISA. Sub-cloned hybridomas were then used to produce monoclonal antibodies. The binding properties of antibodies in the supernatant were tested using ELISA. Five monoclonal antibodies (hybridomas) were identified, 11D6-1, 4B8-1, 6B2-1, 7D11-1, 7G2-1. The peptide (immunogen) used to generate the 11D6-1, 4B8-1, 6B2-1, 7D11-1, 7G2-1 antibodies was the peptide of SEQ ID NO:5, and as mentioned above this was linked to keyhole limpet hemocyanin. The suffix “−1” in each of the antibody names is merely indicative that each of these antibodies is a daughter clone of a respective parental hybridoma (the parental hybridomas have the same names without the “−1” suffix, for example 11D6 is the parental hybridoma of the 11D6-1 antibody (daughter clone)).

Cloning and Sequencing of Mouse Hybridoma IgG

[0736] From mouse hybridomas (11D6-1, 4B8-1, 6B2-1, 7D11-1 and 7G2-1), RNA was prepared from which cDNA was synthesized. Variable Light (VL) and Variable Heavy (VH) regions of cDNA were amplified and cloned into standard cloning vector separately. Identification of positive clones was done by colony PCR followed by gel electrophoresis. VL and VH DNA and amino acid sequences were obtained from positive clones.

[0737] Sequencing of 6B2-1 identified one VH region (domain) sequence, but two different VL region (domain) sequences (i.e. two distinct VL region sequences were identified). This is discussed further below in the results section.

[0738] Sequencing of the cDNAs encoding the VH and VL domains of the other four antibodies (i.e. 11D6-1, 4B8-1, 7D11-1 and 7G2-1) identified one VH region (domain) sequence and one VL region (domain) sequence per antibody (i.e. per hybridoma).

Results and Discussion

[0739] Monoclonal antibody in hybridoma supernatant preparations from hybridoma clones 11D6-1, 4B8-1, 6B2-1, 7D11-1 and 7G2-1 were tested by ELISA for binding to various dilutions of the peptide having the amino acid sequence of SEQ ID NO:5 (which was peptide (immunogen) used to generate the 11D6-1, 4B8-1, 6B2-1, 7D11-1 and 7G2-1 antibodies). Quantitation of antibody bound to peptide was measured by absorbance at 405 nm. Absorbance is proportional to binding strength. All supernatants contained antibody that bound to the peptide of SEQ ID NO:5 (FIG. 12). A further ELISA performed using antibodies from clones 11D6-1, 4B8-1 and 7D11-1 and using a “wild-type” (WT) peptide (having the amino acid sequence CGAGGVGKSALTI (SEQ ID NO:160)) that has a G residue instead of a “mutant” D residue at position 5 (as compared to SEQ ID NO:5) showed that antibodies from clones 11D6-1, 4B8-1, and 7D11-1 clearly bound more strongly to the mutant G13D peptide (SEQ ID NO:5) than to the WT peptide (data not shown). If an antibody binds more strongly to a mutant-containing peptide than the WT peptide it is indicative that the antibody is selective for the mutant form of KRAS, that it has a preference for the mutated sequence. Without wishing to be bound by theory, such selective antibodies prefer to bind to a disease-causing mutant form of the KRAS protein. Such a preference may be important in a case when a cell or tissue is expressing not only the mutated form but also the wild-type form of the KRAS protein.

[0740] As mentioned above, sequencing of VH and VL regions (domains) of the 6B2-1 hybridoma identified one VH region (domain) sequence, but two different (i.e. distinct) VL region (domain) sequences. This could mean that hybridoma clone 6B2-1 has one heavy chain gene and two light chain genes that are producing antibodies. This phenomenon (i.e. of a hybridoma encoding two distinct VL domain sequences) is known to occur and has been discussed in the literature (e.g. Bradbury, A. R. M. et al. MAbs 10, 539-546 (2018)). A situation where there is one VH region (domain) sequence, but two different VL region (domain) sequences can result potentially in three different species of monoclonal antibodies ((i) a species that has two identical heavy chains and two identical light chains of sequence X; (ii) a species that has two identical heavy chains and two identical light chains of sequence Y; (iii) a species that has two identical heavy chains, one light chain of sequence X and one light chain of sequence Y). The supernatant (or purified antibody) from this hybridoma (6B2-1) might contain a mixture of one or more of these types of species. In normal circumstances only one heavy chain gene and one light chain gene are producing antibody. Hybridomas containing more than one light chain can arise during hybridoma generation and cultivation. There can be a number of causes. For example, a myeloma cell may fuse two B cells instead of only one. The tested 6B2-1 supernatant in this Example could contain one or more of the three potential species of antibody.

[0741] Elsewhere herein, the sequences of two potential monoclonal antibodies (two potential monoclonal antibody species) from the 6B2-1 hybridoma are set forth separately. These are (a) an antibody that has the identified VH domain sequence and only the first identified VL domain sequence, and (b) an antibody that has the identified VH domain sequence and only the second (or other) identified VL domain sequence. These distinct monoclonal antibodies (distinct monoclonal antibody species) have been given the names 6B2-1-1 and 6B2-1-2.

[0742] Sequences of the antibodies 11D6-1, 4B8-1, 7D11-1, 7G2-1, 6B2-1-1 and 6B2-1-2, are set out in Tables A-F herein.