NZ736022B2

NZ736022B2 - De novo binding domain containing polypeptides and uses thereof

Info

Publication number: NZ736022B2
Application number: NZ736022A
Authority: NZ
Inventors: David M Hilbert; David William Lafleur
Original assignee: Arcellx Inc; Subdomain Llc
Filing date: 2016-04-04
Publication date: 2024-05-24

Abstract

Provided herein are de novo binding domain containing polypeptides (DBDpp) that specifically bind a target of interest. Nucleic acids encoding the DBDpp, and vectors and host cells containing the nucleic acids are also provided. Libraries of DBDpp, methods of producing and screening such libraries and the DBDpp identified from such libraries and screens are also encompassed. Methods of making and using the DBDpp are additionally provided. Such uses include, without limitation, affinity purification, and diagnostic and therapeutic applications. The DBDpp are antibody mimetics comprising three anti-parallel alpha helices joined by linker peptides.

Claims

WHAT IS CLAIMED IS:

1. A target-binding polypeptide comprising an amino acid sequence of MGSWX X FKX X LAX IKX X LEALGGSEAELAX FEX X IAX FEX X LQX Y 5 6 9 10 13 16 17 30 33 34 37 40 41 44 KGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2), MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX X EIX AFX X ELX AYKGKGNPE 32 33 36 39 40 43 VEALX X EAX AIX X ELX AYRHN (SEQ ID NO:3), 57 58 61 64 65 68 MGSWX EFX X RLX AIX X RLX ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEV 5 8 9 12 15 16 19 EX LRX X AAX IRX X LQAYRHN (SEQ ID NO:4), 55 58 59 62 65 66 MGSWX X FKX X LAX IKX X LEALGGSEAELAAFX X EIX AFX X ELX AYK 5 6 9 10 13 16 17 32 33 36 39 40 43 GKGNPEVEX LRX X AAX IRX X LQAYRHN (SEQ ID NO:5), 55 58 59 62 65 66 MGSWX EFX X RLX AIX X RLX ALGGSEAELAX FEX X IAX FEX X LQX Y 5 8 9 12 15 16 19 30 33 34 37 40 41 44 KGKGNPEVEALX X EAX AIX X ELX AYRHN (SEQ ID NO:6), 57 58 61 64 65 68 MGSWX X FKX X LAX IKX X LEALZ EAELAX FEX X IAX FEX X LQX YZ 5 6 9 10 13 16 17 1 28 31 32 35 38 39 42 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7), MGSWAEFKQRLAAIKTRLEALZ EAELAAFX X EIX AFX X ELX AYZ NPEVEALX 1 30 31 34 37 38 41 2 X EAX AIX X ELX AYRHN (SEQ ID NO:8), 52 53 56 59 60 63 MGSWX EFX X RLX AIX X RLX ALZ EAELAAFEKEIAAFESELQAYZ NPEVEX L 5 8 9 12 15 16 19 1 2 50 RX X AAX IRX X LQAYRHN (SEQ ID NO:9), 53 54 57 60 61 MGSWX X FKX X LAX IKX X LEALZ EAELAAFX X EIX AFX X ELX AYZ NP 5 6 9 10 13 16 17 1 30 31 34 37 38 41 2 EVEX LRX X AAX IRX X LQAYRHN (SEQ ID NO:10) or 50 53 54 57 60 61 MGSWX EFX X RLX AIX X RLX ALZ EAELAX FEX X IAX FEX X LQX YZ N 5 8 9 12 15 16 19 1 28 31 32 35 38 39 42 2 PEVEALX X EAX AIX X ELX AYRHN (SEQ ID NO:11), wherein 52 53 56 59 60 63 (a) X is a natural or non-natural amino acid; (b) Z and Z comprise between about 2 to about 30 natural or non-natural amino acids; (c) the target-binding polypeptide specifically binds a target of interest, wherein the specific binding of the target-binding polypeptide to the target of interest is greater than the binding of a reference polypeptide comprising the amino acid sequence of SEQ ID NO:1 to the target of interest; and (d) the target-binding polypeptide does not comprise the amino acid sequence of SEQ ID NO:50.

2. The target-binding polypeptide of claim 1 comprising an amino acid sequence of SEQ ID NO: 4.

3. The target-binding polypeptide of claim 1 or claim 2, wherein X is not a cysteine or a proline.

4. The target-binding polypeptide of any one of claim 1 to 3, wherein the target of interest is a cancer antigen, optionally wherein the cancer antigen is PD-L1, CD137, or CD123, and optionally wherein (a) the target of interest is PD-L1 and the target-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO: 43, and SEQ ID NO:44; (b) the target of interest is CD137 and the target-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; (c) the target of interest is CD123 and the target-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 92-126 and SEQ ID NO:127; or (d) the target of interest is CD123 and the target-binding polypeptide competes with the target-binding polypeptide of (c) for binding to CD123.

5. The target-binding polypeptide of any one of claims 1 to 3, that specifically binds to a protein comprising amino acids 19-305 of CD123 (SEQ ID NO: 187) or a protein that is at least 95% identical to CD123, optionally wherein the target-binding polypeptide is characterized by one or more of (a) the target-binding polypeptide binds to the protein comprising amino acids 19-305 of CD123 (SEQ ID NO: 187) or the protein that is at least 95% identical to CD123 with a dissociation constant (K ) -4 -12 between about 10 M and about 10 M, (b) the target-binding polypeptide comprises an amino acid sequence of MGSWX EFX X RLX AIX X RLX ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX 5 8 9 12 15 16 19 55 LRX X AAX IRX X LQAYRHN (SEQ ID NO:4), wherein X is a natural or non-natural amino 58 59 62 65 66 n acid, (c) the target-binding polypeptide comprises an amino acid sequence of MGSWX EFX X RLX AIX X RLX ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEX 5 8 9 12 15 16 19 55 LRX X AAX IRX X LQAYRHN (SEQ ID NO:4), wherein X is a natural or non-natural amino 58 59 62 65 66 n acid, and wherein X is not cysteine or proline, and (d) the target-binding polypeptide comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NO:60 – SEQ ID NO: 136, and (e) the target-binding polypeptide is capable of binding to a tumor.

6. A fusion protein comprising a first and second target-binding polypeptide according to claim 5 wherein the first and second target-binding polypeptide exhibits binding specificity for a tumor target, optionally wherein the first and second target-binding polypeptide exhibits binding specificity for different tumor targets.

7. The target-binding polypeptide of any one of claim 1 to 5, or the fusion protein of claim 6, wherein the target-binding polypeptide or fusion protein is labeled and optionally, wherein the label is a biotin moiety or is selected from the group consisting of an enzymatic label, a fluorescent label, a luminescent label, and a bioluminescent label.

8. The target-binding polypeptide of any one of claim 1 to 5 and 7, or the fusion protein of claim 6 or claim 7, wherein the target-binding polypeptideor fusion protein is conjugated to a therapeutic or cytotoxic agent.

9. A pharmaceutical composition comprising the target-binding polypeptide of any one of claim 1 to 5, 7 and 8, or the fusion protein of any one of claims 6 to 8 and further comprising a pharmaceutically acceptable carrier.

10. A kit comprising the target-binding polypeptide of any one of claim 1 to 5, 7 and 8, or the fusion protein of any one of claims 6 to 8.

11. An isolated nucleic acid molecule encoding the target-binding polypeptide of any one of claim 1 to 5, or the fusion protein of claim 6.

12. A vector comprising the isolated nucleic acid molecule of claim 11, and optionally further comprising a nucleotide sequence which regulates the expression of the target-binding polypeptide, DBDpp, or fusion protein encoded by the nucleic acid molecule.

13. An ex-vivo host cell comprising the nucleic acid molecule of claim 11 or the vector of Claim

14. An ex-vivo cell line engineered to express the target-binding polypeptide of any one of claim 1 to 5, or the fusion protein of claim 6.

15. A chimeric antigen receptor (CAR), wherein the CAR comprises (a) a targeting domain, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the targeting domain comprises the target-binding polypeptide of any one of claim 1 to 5, or the fusion protein of claim 6, optionally wherein the intracellular signaling domain is selected from the group consisting of a human CD3 zeta domain, 41BB domain, a CD28 domain and any combination thereof, optionally wherein the target-binding polypeptide or fusion protein binds to a tumor antigen associated with a hematologic malignancy or a solid tumor, and optionally wherein the tumor antigen is selected from the group consisting of CD137, PD-L1, CD123, CTLA4, CD47, KIR, DR5, TIM3, PD1, EGFR, TCR, CD19, CD20, CD22, ROR 1, mesothelin, CD33/lL3Ra, cMet, PSMA, Glycolipid F77, EGFRvIII, GD2, NY-ESO- 1, MAGE A3, and combinations thereof.

16. The CAR of claim 15, wherein the intracellular signaling domain comprises a costimulatory signaling region, optionally wherein the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-lBB, OX40, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

17. An isolated nucleic acid molecule comprising a sequence encoding the CAR of claim 15 or claim 16.

18. An ex-vivo cell comprising a nucleic acid molecule comprising a sequence encoding the CAR of claim 15 or claim 16, optionally wherein the cell is a T cell or a natural killer (NK) cell, and optionally wherein the cell exhibits an anti-tumor immunity when the target-binding polypeptide or fusion protein binds to its corresponding tumor antigen.

19. Use of an immune cell comprising the CAR of claim 15 or claim 16 in the manufacture of a medicament for the treatment of cancer.

20. The use of claim 19, wherein the CAR comprises: (a) a target binding domain comprising a target-binding polypeptide having an amino acid sequence selected from the group consisting of MGSWX X FKX X LAX IKX X LEALGGSEAELAX FEX X IAX FEX X LQX 5 6 9 10 13 16 17 30 33 34 37 40 41 4 YKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2), MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX X EIX AFX X ELX AYKGKGN 32 33 36 39 40 43 PEVEALX X EAX AIX X ELX AYRHN (SEQ ID NO:3), 57 58 61 64 65 68 MGSWX EFX X RLX AIX X RLX ALGGSEAELAAFEKEIAAFESELQAYKGKGNP 5 8 9 12 15 16 19 EVEX LRX X AAX IRX X LQAYRHN (SEQ ID NO:4), 55 58 59 62 65 66 MGSWX X FKX X LAX IKX X LEALGGSEAELAAFX X EIX AFX X ELX A 5 6 9 10 13 16 17 32 33 36 39 40 43 YKGKGNPEVEX LRX X AAX IRX X LQAYRHN (SEQ ID NO:5), and 55 58 59 62 65 66 MGSWX EFX X RLX AIX X RLX ALGGSEAELAX FEX X IAX FEX X LQX 5 8 9 12 15 16 19 30 33 34 37 40 41 4 YKGKGNPEVEALX X EAX AIX X ELX AYRHN (SEQ ID NO:6), wherein X is a 4 57 58 61 64 65 68 n natural or non-natural amino acid, wherein X is not a cysteine or a proline residue, wherein the target-binding polypeptide specifically binds a target of interest expressed by a cancer cell, and wherein the specific binding of the target-binding polypeptide to the target of interest is greater than binding of a polypeptide according to SEQ ID NO:1 to the target of interest, (b) a transmembrane domain selected from 41BB and CD28, and (c) an intracellular domain, wherein the intracellular domain comprises a signaling domain selected from an alpha, beta, or zeta chain of the T cell receptor, optionally wherein the immune cell is a T cell or a natural killer (NK) cell.

21. A method for transforming a reference polypeptide into a target-binding polypeptide capable of specifically binding to a target of interest, the method comprising: (a) modifying a plurality of amino acid residues from the reference polypeptide to generate a plurality of candidate target-binding polypeptides; wherein the candidate target-binding polypeptides comprise a variant of the amino acid sequence of SEQ ID NO: 1, wherein the candidate target-binding polypeptides comprise three anti-parallel alpha helices joined by linker peptides, wherein the amino acid residues modified are solvent accessible or solvent inaccessible, and wherein the modification comprises one or more conservative or non- conservative amino acid substitutions and does not include a substitution with a cysteine or a proline; (b) packaging the plurality of candidate target-binding polypeptides in a plurality of vectors to generate a candidate library; and (c) screening the candidate library for candidate target-binding polypeptides that exhibit specific binding to the target of interest; optionally further comprising identifying potentially immunogenic amino acid residues in a candidate target-binding polypeptide and modifying at least one of the potentially immunogenic amino acid residues in the candidate target-binding polypeptide, wherein the modification comprises an amino acid substitution, wherein the candidate target-binding polypeptides comprise an amino acid sequence selected from the group consisting of MGSWX X FKX X LAX IKX X LEALGGSEAELAX FEX X IAX FEX X LQX Y 5 6 9 10 13 16 17 30 33 34 37 40 41 44 KGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:2), MGSWAEFKQRLAAIKTRLEALGGSEAELAAFX X EIX AFX X ELX AYKGKGNPE 32 33 36 39 40 43 VEALX X EAX AIX X ELX AYRHN (SEQ ID NO:3), 57 58 61 64 65 68 MGSWX EFX X RLX AIX X RLX ALGGSEAELAAFEKEIAAFESELQAYKGKGNPEV 5 8 9 12 15 16 19 EX LRX X AAX IRX X LQAYRHN (SEQ ID NO:4), 55 58 59 62 65 66 MGSWX X FKX X LAX IKX X LEALGGSEAELAAFX X EIX AFX X ELX AYK 5 6 9 10 13 16 17 32 33 36 39 40 43 GKGNPEVEX LRX X AAX IRX X LQAYRHN (SEQ ID NO:5), 55 58 59 62 65 66 MGSWX EFX X RLX AIX X RLX ALGGSEAELAX FEX X IAX FEX X LQX Y 5 8 9 12 15 16 19 30 33 34 37 40 41 44 KGKGNPEVEALX X EAX AIX X ELX AYRHN (SEQ ID NO:6), 57 58 61 64 65 68 MGSWX X FKX X LAX IKX X LEALZ EAELAX FEX X IAX FEX X LQX YZ 5 6 9 10 13 16 17 1 28 31 32 35 38 39 42 2 NPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO:7), MGSWAEFKQRLAAIKTRLEALZ EAELAAFX X EIX AFX X ELX AYZ NPEVEALX 1 30 31 34 37 38 41 2 X EAX AIX X ELX AYRHN (SEQ ID NO:8), 52 53 56 59 60 63 MGSWX EFX X RLX AIX X RLX ALZ EAELAAFEKEIAAFESELQAYZ NPEVEX L 5 8 9 12 15 16 19 1 2 50 RX X AAX IRX X LQAYRHN (SEQ ID NO:9), 53 54 57 60 61 MGSWX X FKX X LAX IKX X LEALZ EAELAAFX X EIX AFX X ELX AYZ NP 5 6 9 10 13 16 17 1 30 31 34 37 38 41 2 EVEX LRX X AAX IRX X LQAYRHN (SEQ ID NO:10) and 50 53 54 57 60 61 MGSWX EFX X RLX AIX X RLX ALZ EAELAX FEX X IAX FEX X LQX YZ N 5 8 9 12 15 16 19 1 28 31 32 35 38 39 42 2 PEVEALX X EAX AIX X ELX AYRHN (SEQ ID NO:11), wherein X is a natural or 52 53 56 59 60 63 n non-natural amino acid and Z and Z comprise between about 2 to about 30 natural or non- natural amino acids.

22. Virus-like particles (VLPs) comprising a fusion polypeptide comprising the target-binding polypeptide of any one of claims 1 to 5, wherein the VLPs are suitable for use as immunogens for antibody generation, wherein said antibodies are directed against the target-binding polypeptide.

23. A target-binding polypeptide as claimed in any one of claims 1-5 and 7-8, substantially as described herein and with reference to any example thereof.

24. A fusion protein as claimed in any one of claims 6-8, substantially as described herein and with reference to any example thereof.

25. A pharmaceutical composition as claimed in claim 9, substantially as described herein and with reference to any example thereof.

26. A kit as claimed in claim 10, substantially as described herein and with reference to any example thereof.

27. An isolated nucleic acid as claimed in claim 11 or 17, or a vector as claimed in claim 12, sustantially as described herein and with reference to any example thereof.

28. A host cell as claimed in claim 13, a cell line as claimed in claim 14, or a cell as claimed in claim 18, substantially as described herein and with reference to any example thereof.

29. A CAR as claimed in claim 15 or 16, a use as claimed in claim 19 or 20, a method as claimed in claim 21, or VLPs as claimed in claim 22, substantially as described herein and with reference to any example thereof.