US2021369857A1PendingUtilityA1

Il2 and tnf mutant immunoconjugates

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Assignee: PHILOGEN SPAPriority: Nov 9, 2016Filed: Jul 23, 2021Published: Dec 2, 2021
Est. expiryNov 9, 2036(~10.3 yrs left)· nominal 20-yr term from priority
Inventors:Dario NeriRoberto De LucaSarah Wulhfard
A61K 47/6813A61K 47/6843A61K 38/191C07K 2317/622A61K 38/00C07K 14/525C07K 14/55A61P 35/00C07K 16/18C07K 2317/73A61K 47/6851A61K 38/2013C07K 2319/33C07K 2317/626C07K 2319/74
61
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Claims

Abstract

The present application relates to conjugates comprising interleukin 2 (IL2), and a mutant of tumour necrosis factor, such as tumour necrosis factor alpha (TNFα), and an antibody molecule. The antibody molecule preferably binds to an antigen associated with neoplastic growth and/or angiogenesis, such as the Extra-Domain A (EDA) or Extra-Domain B (EDB) of fibronectin. The conjugate may be used in the treatment of cancer.

Claims

exact text as granted — not AI-modified
1 . A conjugate comprising interleukin-2 (IL2), a tumour necrosis factor (TNF) mutant, and an antibody molecule which binds an antigen associated with neoplastic growth and/or angiogenesis, wherein the TNF mutant has reduced activity relative to the wild type TNF. 
     
     
         2 . The conjugate according to  claim 1  wherein the TNF mutant comprises the amino acid sequence of wild-type TNF with one or more amino acid substitutions, deletions or insertions which reduce the activity of the TNF mutant relative to the wild type TNF. 
     
     
         3 . The conjugate according to  claim 1  or  claim 2 , wherein the TNF is TNFα. 
     
     
         4 . The conjugate according to  claim 3  wherein the TNFα is human TNFα. 
     
     
         5 . The conjugate according to any one of the preceding claims wherein the TNF mutant has a mutation at position corresponding to R32 of SEQ ID NO: 15 or R52 of SEQ ID NO: 17. 
     
     
         6 . The conjugate according to  claim 5  wherein the R at said position is substituted for a non-polar amino acid. 
     
     
         7 . The conjugate according to  claim 6  wherein the TNF mutant has an R to A mutation at said position. 
     
     
         8 . The conjugate according to any one of the preceding claims wherein the TNF mutant comprises the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 39 or a variant thereof. 
     
     
         9 . The conjugate according to any one of  claims 1  to  8 , wherein the TNFα is linked to the antibody molecule via a peptide linker. 
     
     
         10 . The conjugate according to  claim 9 , wherein the peptide linker has the amino acid sequence of SEQ ID NO: 14. 
     
     
         11 . The conjugate according to any one of the preceding claims, wherein the IL2 is human IL2. 
     
     
         12 . The conjugate according to  claim 11 , wherein the IL2 comprises the sequence of SEQ ID NO: 12. 
     
     
         13 . The conjugate according to any one of  claims 1  to  12 , wherein the IL2 is linked to the antibody molecule by a peptide linker. 
     
     
         14 . The conjugate according to  claim 13  wherein the peptide linker has the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 49. 
     
     
         15 . The conjugate according to any one of  claims 1  to  14 , wherein the antibody molecule comprises a single chain Fv (scFv). 
     
     
         16 . The conjugate according to any one of  claims 1  to  14 , wherein the antibody molecule comprises a diabody. 
     
     
         17 . The conjugate according to any one of the preceding claims, wherein the antibody molecule binds fibronectin. 
     
     
         18 . The conjugate according to  claim 17 , wherein the antibody molecule binds the Extra Domain-A (ED-A) of fibronectin. 
     
     
         19 . The conjugate according to  claim 18 , wherein the antibody molecule comprises an antigen binding site having the complementarity determining regions (CDRs) of antibody F8 set forth in SEQ ID NOs 6-11. 
     
     
         20 . The conjugate according to  claim 19 , wherein the antibody molecule comprises the VH and VL domains of antibody F8 set forth in SEQ ID NOs 2 and 4 or variants thereof. 
     
     
         21 . The conjugate according to  claim 20 , wherein the antibody molecule is an scFv, and wherein the VH domain and the VL domain of the scFv are linked by a 10 to 20 amino acid linker. 
     
     
         22 . The conjugate according to  claim 17  to  21 , wherein the antibody molecule comprises the amino acid sequence of scFv F8 set forth in SEQ ID NO: 5 or a variant thereof. 
     
     
         23 . The conjugate according to  claim 17 , wherein the antibody molecule binds the Extra Domain-B (ED-B) of fibronectin. 
     
     
         24 . The conjugate according to  claim 23 , wherein the antibody molecule comprises an antigen binding site having the complementarity determining regions (CDRs) of antibody L19 set forth in SEQ ID NOs 18-23. 
     
     
         25 . The conjugate according to  claim 24 , wherein the antibody molecule comprises the VH and VL domains of antibody L19 set forth in SEQ ID NOs 24 and 25 or variants thereof. 
     
     
         26 . The conjugate according to  claim 25 , wherein the antibody molecule is an scFv, and wherein the VH domain and the VL domain of the scFv are linked by a 10 to 20 amino acid linker. 
     
     
         27 . The conjugate according to any one of  claims 23  to  26 , wherein the antibody molecule comprises the amino acid sequence of scFv L19 set forth in SEQ ID NO: 26 or a variant thereof. 
     
     
         28 . The conjugate according to any one of  claims 1  to  16 , wherein the antibody molecule binds the A1 Domain of Tenascin C. 
     
     
         29 . The conjugate according to  claim 28 , wherein the antibody molecule comprises an antigen binding site having the complementarity determining regions (CDRs) of antibody F16 set forth in SEQ ID NOs 27-32. 
     
     
         30 . The conjugate according to  claim 29 , wherein the antibody molecule comprises the VH and VL domains of antibody F16 set forth in SEQ ID NOs 33 and 34 or variants thereof. 
     
     
         31 . The conjugate according to  claim 30 , wherein the antibody molecule is an scFv, and wherein the VH domain and the VL domain of the scFv are linked by a 10 to 20 amino acid linker. 
     
     
         32 . The conjugate according to any one of  claims 28  to  31 , wherein the antibody molecule comprises the amino acid sequence of scFv F16 set forth in SEQ ID NO: 35 or SEQ ID NO: 46 or a variant thereof. 
     
     
         33 . The conjugate according to any one of  claims 1  to  32 , wherein the antibody molecule is, or comprises, a single chain Fv (scFv) and, wherein the IL2 is linked to the N-terminus of the VH domain of the scFv via a peptide linker and the TNF mutant is linked to the C-terminus of the VL domain of the scFv via a peptide linker. 
     
     
         34 . The conjugate according to any one of  claims 1  to  32 , wherein the antibody molecule is, or comprises, a single chain Fv (scFv) and, wherein the TNF mutant is linked to the N-terminus of the VH domain of the scFv via a peptide linker and the IL2 is linked to the C-terminus of the VL domain of the scFv via a peptide linker. 
     
     
         35 . The conjugate according to any one of  claims 1  to  32 , wherein the antibody molecule is, or comprises, a single chain Fv (scFv) and, wherein the IL2 and the TNF mutant are linked to C-terminus of the VL domain of the scFv via a peptide linker or the IL2 and the TNFα are linked to the N-terminus of the scFv via a peptide linker. 
     
     
         36 . The conjugate according to any one of  claims 33 ,  34  or  35 , wherein the peptide linker is 10 to 20 amino acids long. 
     
     
         37 . The conjugate according to any one of  claims 1  to  22 , wherein the conjugate comprises the amino acid sequence of SEQ ID NO: 1 with an R to A mutation at position 432 or the amino acid sequence of SEQ ID NO: 16 with an R to A mutation at position 452. 
     
     
         38 . The conjugate according to any one of  claims 1  to  22 , wherein the conjugate comprises the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 38 or a variant thereof. 
     
     
         39 . The conjugate according to any one of  claims 1  to  17  and  claims 23  to  27 , wherein the conjugate comprises the amino acid sequence of SEQ ID NO: 70 with an R to A mutation at position 430 or the amino acid sequence of SEQ ID NO: 71 with an R to A mutation at position 450. 
     
     
         40 . The conjugate according to any one of  claims 1  to  17  and  claims 23  to  27 , wherein the conjugate comprises the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or a variant thereof. 
     
     
         41 . The conjugate according to any one of  claims 1  to  16  and  claims 28  to  32 , wherein the conjugate comprises the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 48 or a variant thereof. 
     
     
         42 . A nucleic acid molecule encoding a conjugate according to any one of  claims 1  to  41 . 
     
     
         43 . An expression vector comprising the nucleic acid of  claim 42 . 
     
     
         44 . A host cell comprising the vector of  claim 43 . 
     
     
         45 . A method of producing a conjugate according to any one of  claims 1  to  41 , the method comprising culturing the host cell of  claim 44  under conditions for expression of the conjugate. 
     
     
         46 . The method of  claim 45  further comprising isolating and/or purifying the conjugate. 
     
     
         47 . The conjugate according to any one of  claims 1  to  41  for use in a method of treating cancer by targeting IL2 and TNF to the neovasculature in vivo. 
     
     
         48 . The conjugate according to any one of  claims 1  to  41  for use in a method of delivering IL2 and TNF to the tumour neovasculature in a patient. 
     
     
         49 . A method of treating cancer by targeting IL2 and TNF to the neovasculature in a patient, the method comprising administering a therapeutically effective amount of a conjugate according to any of  claims 1  to  41  to the patient. 
     
     
         50 . A method of delivering IL2 and TNF to the tumour neovasculature in a patient comprising administering to the patient a conjugate according to any one of  claims 1  to  41 . 
     
     
         51 . The conjugate for use according to  claim 47 , or the method according to  claim 49 , wherein the cancer is a melanoma, head and neck cancer, kidney cancer, or a sarcoma. 
     
     
         52 . The conjugate for use according to  claim 48 , or the method according to  claim 50 , wherein the tumour is the result of a melanoma, head and neck cancer, kidney cancer, or a sarcoma.

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