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1. WO2020181140 - CONSTRAINED CONDITIONALLY ACTIVATED BINDING PROTEINS

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[ EN ]

WHAT IS CLAIMED IS:

1. A fusion protein comprising, from N- to C-terminal:

a) a first sdABD-TTA;

b) a first domain linker;

c) a constrained Fv domain comprising:

i) a first variable heavy domain comprising a vhCDRl, vhCDR2 and vhCDR3;

ii) a constrained non-cleavable linker (CNCL); and

iii) a first variable light domain comprising vlCDRl, vlCDR2 and vlCDR3;

d) a second domain linker;

e) a second sdABD-TTA;

f) a cleavable linker (CL);

g) a constrained pseudo Fv domain comprising:

i) a first pseudo light variable domain;

ii) a non-cleavable linker (NCL); and

iii) a first pseudo heavy variable domain;

h) a third domain linker; and

i) a third sdABD that binds to human serum albumin;

wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; said first variable heavy domain and said first pseudo variable light domain intramolecularly associate to form an inactive Fv;

said first variable light domain and said first pseudo variable heavy domain intramolecularly associate to form an inactive Fv; and

wherein at least one of said sdABD-TTA is a sdABD-B7FI3 having a sequence selected from SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53 and SEQ ID NO:57.

2. A fusion protein according to claim 1 which is Pro664 and has SEQ ID NO:282.

3. A fusion protein comprising, from N- to C-terminal:

a) a first single domain antigen binding domain (sdABD) that binds to a human tumor target antigen (TTA) (sdABD-TTA);

b) a first domain linker;

c) a constrained Fv domain comprising:

i) a first variable heavy domain comprising a vhCDRl, vhCDR2 and vhCDR3;

ii) a constrained non-cleavable linker (CNCL); and

iii) a first variable light domain comprising vlCDRl, vlCDR2 and vlCDR3;

d) a second domain linker;

e) a second sdABD-TTA;

f) a cleavable linker (CL);

g) a constrained pseudo Fv domain comprising:

i) a first pseudo light variable domain;

ii) a non-cleavable linker (NCL); and

iii) a first pseudo heavy variable domain;

h) a third domain linker; and

i) a third sdABD that binds to human serum albumin;

wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; said first variable heavy domain and said first pseudo variable light domain intramolecularly associate to form an inactive Fv;

said first variable light domain and said first pseudo variable heavy domain intramolecularly associate to form an inactive Fv; and

wherein at least one of said sdABD-TTA is a sdABD-EpCAM having a sequence selected from SEQ ID NO:69 and SEQ ID NO:73.

4. A fusion protein comprising, from N- to C-terminal:

a) a first sdABD-TTA;

b) a first domain linker;

c) a constrained Fv domain comprising:

i) a first variable heavy domain comprising a vhCDRl, vhCDR2 and vhCDR3;

ii) a constrained non-cleavable linker (CNCL); and

iii) a first variable light domain comprising vlCDRl, vlCDR2 and vlCDR3;

d) a second domain linker;

e) a second sdABD-TTA;

f) a cleavable linker (CL);

g) a constrained pseudo Fv domain comprising:

i) a first pseudo light variable domain;

ii) a non-cleavable linker (NCL); and

iii) a first pseudo heavy variable domain;

h) a third domain linker; and

i) a third sdABD that binds to human serum albumin;

wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; said first variable heavy domain and said first pseudo variable light domain intramolecularly associate to form an inactive Fv;

said first variable light domain and said first pseudo variable heavy domain intramolecularly associate to form an inactive Fv; and

wherein at least one of said sdABD-TTA is a sdABD-Trop2 having a sequence selected from SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:93 and SEQ ID NO:97.

5. A fusion protein comprising, from N- to C-terminal:

a) a first sdABD-TTA;

b) a first domain linker;

c) a constrained Fv domain comprising:

i) a first variable heavy domain comprising a vhCDRl, vhCDR2 and vhCDR3;

ii) a constrained non-cleavable linker (CNCL); and

iii) a first variable light domain comprising vlCDRl, vlCDR2 and vlCDR3;

d) a second domain linker;

e) a second sdABD-TTA;

f) a cleavable linker (CL);

g) a constrained pseudo Fv domain comprising:

i) a first pseudo light variable domain;

ii) a non-cleavable linker (NCL); and

iii) a first pseudo heavy variable domain;

h) a third domain linker; and

i) a third sdABD that binds to human serum albumin;

wherein said first variable heavy domain and said first variable light domain are capable of binding human CD3 but said constrained Fv domain does not bind CD3; said first variable heavy domain and said first pseudo variable light domain intramolecularly associate to form an inactive Fv; and

said first variable light domain and said first pseudo variable heavy domain intramolecularly associate to form an inactive Fv.

wherein at least one of said sdABD-TTA is a sdABD-CA9 having a sequence selected from SEQ ID NO:101, SEQ ID NO:105, SEQ ID NO:109 and SEQ ID NO:113.

6. A fusion protein according to any of claims 1 and 3 to 5 wherein said first variable heavy domain is N-terminal to said first variable light domain and said pseudo light variable domain is N-terminal to said pseudo variable heavy domain.

7. A fusion protein according to any of claims 1 and 3 to 5 wherein said first variable heavy domain is N-terminal to said first variable light domain and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain.

8. A fusion protein according to any of claims 1 and 3 to 5 wherein said first variable light domain is N-terminal to said first variable heavy domain and said pseudo light variable domain is N-terminal to said pseudo variable heavy domain.

9. A fusion protein according to any of claims 1 and 3 to 5 wherein said first variable light domain is N-terminal to said first variable heavy domain and said pseudo variable heavy domain is N-terminal to said pseudo variable light domain.

10. A fusion protein according to any of claim 1 and 3 to 9 wherein said first and second TTA is the same.

11. A fusion protein according to any of claims 1 and 3 to 9 wherein said first and second TTA are different.

12. A fusion protein according to any of claims 1 and 3 to 11 wherein said half-life extension domain has SEQ ID NO: 117.

13. A fusion protein according to any of claims 1 and 3 to 12 having a sequence selected from the group consisting of Pro601 Pro602, V3 and V4, Pro665, Pro666, Pro667, Pro694, Pro695, Pro565, Pro566, Pro567, Pro727-731, Pro676-679, Pro808, Pro819, Pro621, Pro622, Pro640-643, Pro 744, Pro 746, Pro638, Pro639, Pro396, Pro476, Pro706, Pro709, Pro470, Pro471, Pro551, Pro552, Pro623, Pro624, Pro698, Pro655, Pro656, Pro657, Pro658, Pro516, Pro517, Pro518 and Pro519.

14. A nucleic acid encoding a fusion protein according to any of claims 1 to 13.

15. An expression vector comprising the nucleic acid of claim 14.

16. A host cell comprising the expression vector of claim 15.

17. A method of making a fusion protein comprising culturing the host cell of claim 16 under conditions wherein said protein is expressed and recovering said fusion protein.

18. A method of treating cancer comprising administering the protein of any of claims 1 to 13 to a patient.

19. A single domain antigen binding domain that binds human Trop2 having a sequence selected from SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:89 and SEQ ID NO: 93.

20. A single domain antigen binding domain that binds human B7H3 having a sequence selected from SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53 and SEQ ID NO:57.

21. A single domain antigen binding domain that binds human CA9 having a sequence selected from SEQ ID NO:101, SEQ ID NO:105, SEQ ID NO:109 and SEQ ID NO: 113.

22. A single domain antigen binding domain that binds human EpCAM having a sequence selected from SEQ ID NO:69 and SEQ ID NO:73.