Methods of producing an IL-1 RAP antibodies
The present invention provides an antibody or an antigen-binding fragment thereof with binding specificity for human interleukin-1 receptor accessory protein (IL1RAP) wherein the antibody or antigen-binding fragment is capable of inhibiting the binding of antibody ‘CAN04’ to human IL1RAP. The invention further provides the use of such antibodies or an antigen-binding fragments in the treatment and/or diagnosis of IL-1 associated diseases and conditions, including cancers such as acute myeloid leukemia and melanoma.
1. A method for producing an antibody or antigen-binding fragment, the method comprising culturing a host cell comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment comprising:
a. A heavy chain variable region (VH) comprising (i) heavy chain CDR1 comprising: GYAFTSS (amino acids 26-32 of SEQ ID NO:9); (ii) Heavy chain CDR2 comprising: YPGDGN (SEQ ID NO: 4); and (iii) Heavy chain CDR3 comprising: GYLDPMDY (SEQ ID NO: 5); and a light chain variable region (VL) comprising: (i) Light chain CDR 1 comprising: ASQGINNYLN (amino acids 25-34 of SEQ ID NO:16); (ii) Light chain CDR2 comprising: YTSGLHA (SEQ ID NO: 13); and (iii) Light chain CDR3 comprising: QQYSILPWT (SEQ ID NO: 14);
b. A heavy chain variable region (VH) comprising (i) heavy chain CDR1 comprising GYAFTSS (amino acids 26-32 of SEQ ID NO:9); (ii) heavy chain CDR2 comprising YPGDGN (SEQ ID NO: 4); and (iii) heavy chain CDR3 comprising GYLDPMDY (SEQ ID NO: 5); and a light chain variable region (VL) comprising (i) light chain CDR 1 comprising SASQGINNYLN (SEQ ID NO:12); light chain CDR2 comprising YTSGLHA (SEQ ID NO: 13); and (iii) light chain CDR3 comprising QQYSILPWT (SEQ ID NO: 14);
c. A heavy chain variable region (VH) comprising (i) heavy chain CDR1 comprising GYAFSSS (SEQ ID NO:3); (ii) heavy chain CDR2 comprising YPGDGN (SEQ ID NO: 4); and (iii) heavy chain CDR3 comprising GYLDPMDY (SEQ ID NO: 5); and a light chain variable region (VL) comprising (i) light chain CDR 1 comprising SASQGINNYLN (SEQ ID NO:12); light chain CDR2 comprising YTSGLHA (SEQ ID NO: 13); and (iii) light chain CDR3 comprising QQYSILPWT (SEQ ID NO: 14);
d. A heavy chain variable region (VH) comprising (i) heavy chain CDR1 comprising GYAFSSS (SEQ ID NO:3); (ii) heavy chain CDR2 comprising YPGDGN (SEQ ID NO: 4); and (iii) heavy chain CDR3 comprising GYLDPMDY (SEQ ID NO: 5); and a light chain variable region (VL) comprising (i) light chain CDR 1 comprising ASQGINNYLN (amino acids 25-34 of SEQ ID NO:16); light chain CDR2 comprising YTSGLHA (SEQ ID NO: 13); and (iii) light chain CDR3 comprising QQYSILPWT (SEQ ID NO: 14);
e. A heavy chain variable region (VH) comprising (i) heavy chain CDR1 comprising GYTFTSS (amino acids 26-32 of SEQ ID NO:10); (ii) heavy chain CDR2 comprising YPGDGQ (amino acids 52-57 of SEQ ID NO:10); and (iii) heavy chain CDR3 comprising GYLDPMDY (SEQ ID NO: 5); and a light chain variable region (VL) comprising (i) light chain CDR 1 comprising SASQGINNYLN (SEQ ID NO:12); light chain CDR2 comprising YTSGLHA (SEQ ID NO: 13); and (iii) light chain CDR3 comprising QQYSILPWT (SEQ ID NO: 14); or
f. A heavy chain variable region (VH) comprising (i) heavy chain CDR1 comprising GYTFTSS (amino acids 26-32 of SEQ ID NO:10); (ii) heavy chain CDR2 comprising YPGDGQ (amino acids 52-57 of SEQ ID NO:10); and (iii) heavy chain CDR3 comprising GYLDPMDY (SEQ ID NO: 5); and a light chain variable region (VL) comprising (i) light chain CDR 1 comprising ASQGINNYLN (amino acids 25-34 of SEQ ID NO:16); light chain CDR2 comprising YTSGLHA (SEQ ID NO: 13); and (iii) light chain CDR3 comprising QQYSILPWT (SEQ ID NO: 14);
under conditions that permit expression of the encoded antibody or antigen-binding fragment.
2. The method of claim 1 , wherein the antibody exhibits one or more of the following properties:
a) a binding affinity (KD) for human IL1RAP of 200 pM or greater;
b) cross-reactivity with IL1RAP from Macaca fascicularis;
c) an inhibitory action on IL1 signalling;
d) capability of inducing ADCC in one or more cancer cell lines;
e) capability of inducing ADCC of cells expressing IL1RAP; and
f) capability of internalisation upon binding to one or more cancer cell lines.
3. The method of claim 1 , wherein the antibody is an Fv fragment or an Fab fragment.
4. The method of claim 1 , wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acids of SEQ ID NO: 9.
5. The method of claim 1 , wherein the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of the amino acids of SEQ ID NO: 16.
6. The method of claim 1 , wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising or consisting of the amino acids of SEQ ID NO: 9, and a light chain variable region comprising or consisting of the amino acids of SEQ ID NOs: 16.
7. The method of claim 1 , wherein the antibody or antigen-binding fragment comprises:
a) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 15;
b) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 15;
c) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 15;
d) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 15;
e) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 16;
f) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 16;
g) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 16;
h) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 16;
i) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 17;
j) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 17;
k) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 17; or
l) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 17.
8. The method of claim 1 , wherein the antibody comprises:
a heavy and light chain constant region, or part thereof.
9. The method of claim 1 , wherein the antibody comprises an Fc region.
10. The method of claim 1 , wherein the antibody further comprises a cytotoxic moiety.
11. The method of claim 1 , wherein the antibody further comprises a detectable moiety.
12. The method of claim 1 , wherein the antibody is humanized.
13. The method of claim 1 , wherein the antibody or antigen-binding fragment is capable of inducing ADCC of cells expressing IL1RAP.
14. The method of claim 1 , wherein the host cell is a bacterial cell, mammalian cell, or a human cell.
15. The method of claim 9 , wherein the Fc region is artificial or engineered to alter the effector function of the Fc region.
16. The method of claim 15 , wherein the Fc region is engineered to comprise one or more of the mutations identified in Table 1.
17. The method of claim 15 , wherein the Fc region is engineered to lack or be low in fucose residues.
18. The method of claim 1 , wherein the method further comprises linking at least one moiety and/or a polypeptide for increasing the in vivo half-life of the antibody or antigen-binding fragment to the antibody or antigen-binding fragment.
19. The method of claim 18 , wherein the moiety is selected from a polyethylene glycol (PEG), fusion proteins, human serum albumin, glycosylation groups, fatty acids and dextran.
20. The method of claim 18 , wherein the method further comprises linking the antibody or antigen-binding fragment to a functional or therapeutic moiety.