HUMANIZED ANTIBODIES

• US 20180222999A1
• Filed: 11/01/2017
• Published: 08/09/2018
• Est. Priority Date: 01/09/2012
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: SMIDER, Vaughn, BAZIRGAN, Omar A., MAO, Hongyuan Helen
• Granted Patent: US 10,562,980 B2
• Time in Patent Office: Days
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/5421 IL-8
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/1081 Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 2317/20 characterized by taxonomic origin
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2318/10 Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof

Abstract

The present disclosure provides humanized antibodies, including antibodies comprising an ultralong CDR3 and uses thereof.

Claims

1. A humanized antibody or binding fragment thereof comprising an ultralong CDR3.

2. The humanized antibody or binding fragment thereof of claim 1, wherein the ultralong CDR3 is 35 amino acids in length or longer, 40 amino acids in length or longer, 45 amino acids in length or longer, 50 amino acids in length or longer, 55 amino acids in length or longer, or 60 amino acids in length or longer.

4. The humanized antibody or binding fragment thereof of claim 1, wherein the ultralong CDR3 comprises 3 or more cysteine residues, 4 or more cysteine residues, 5 or more cysteine residues, 6 or more cysteine residues, 7 or more cysteine residues, 8 or more cysteine residues, 9 or more cysteine residues, 10 or more cysteine residues, 11 or more cysteine residues, or 12 or more cysteine residues.

6. The humanized antibody or binding fragment thereof of claim 1, wherein the antibodies or binding fragments thereof comprise a cysteine motif.

7. The humanized antibody or binding fragment thereof of claim 6, wherein the cysteine motif is selected from the group consisting of:

CX₁₀CX₅CX₅CXCX₇C (SEQ ID NO;

     41), CX₁₀CX₆CX₅CXCX₁₅C (SEQ ID NO;

     42), CX₁₁CXCX₅C (SEQ ID NO;

     43), CX₁₁CX₅CX₅CXCX₇C (SEQ ID NO;

     44), CX₁₀CX₆CX₅CXCX₁₃C (SEQ ID NO;

     45), CX₁₀CX₅CXCX₄CX₈C (SEQ ID NO;

     46), CX₁₀CX₆CX₆CXCX₇C (SEQ ID NO;

     47), CX₁₀CX₄CX₇CXCX₈C (SEQ ID NO;

     48), CX₁₀CX₄CX₇CXCX₇C (SEQ ID NO;

     49), CX₁₀CX₈CX₈C (SEQ ID NO;

     50), CX₁₀CX₆CX₅CXCX₇C (SEQ ID NO;

     51), CX₁₀CX₅CX₅C (SEQ ID NO;

     52), CX₁₀CX₅CX₆CXCX₇C (SEQ ID NO;

     53), CX₁₀CX₆CX₅CX₇CX₉C (SEQ ID NO;

     54), CX₉CX₇CX₅CXCX₇C (SEQ ID NO;

     55), CX₁₀CX₆CX₅CXCX₉C (SEQ ID NO;

     56), CX₁₀CXCX₄CX₅CX₁₁C (SEQ ID NO;

     57), CX₇CX₃CX₆CX₅CXCX₅CX₁₀C (SEQ ID NO;

     58), CX₁₀CXCX₄CX₅CXCX₂CX₃C (SEQ ID NO;

     59), CX₁₆CX₅CXC (SEQ ID NO;

     60), CX₆CX₄CXCX₄CX₅C (SEQ ID NO;

     61), CX₁₁CX₄CX₅CX₆X₃C (SEQ ID NO;

     62), CX₈CX₂CX₆CX₅C (SEQ ID NO;

     63), CX₁₀CX₅CX₅CXCX₁₀C (SEQ ID NO;

     64), CX₁₀CXCX₆CX₄CXC (SEQ ID NO;

     65), CX₁₀CX₅CX₅CXCX₂C (SEQ ID NO;

     66), CX₁₄CX₂CX₃CXCXC (SEQ ID NO;

     67), CX₁₅CX₅CXC (SEQ ID NO;

     68), CX₄CX₆CX₉CX₂CX₁₁C (SEQ ID NO;

     69), CX₆CX₄CX₅CX₅CX₁₂C (SEQ ID NO;

     70), CX₇CX₃CXCXCX₄CX₅CX₉C (SEQ ID NO;

     71), CX₁₀CX₆CX₅C (SEQ ID NO;

     72), CX₇CX₃CX₅CX₅CX₉C (SEQ ID NO;

     73), CX₇CX₅CXCX₂C (SEQ ID NO;

     74), CX₁₀CXCX₆C (SEQ ID NO;

     75), CX₁₀CX₃CX₃CX₅CX₇CXCX₆C (SEQ ID NO;

     76), CX₁₀CX₄CX₅CX₁₂CX₂C (SEQ ID NO;

     77), CX₁₂CX₄CX₅CXCXCX₉CX₃C (SEQ ID NO;

     78), CX₁₂CX₄CX₅CX₁₂CX₂C (SEQ ID NO;

     79), CX₁₀CX₆CX₅CXCX₁₁C (SEQ ID NO;

     80), CX₁₆CX₅CXCXCX₁₄C (SEQ ID NO;

     81), CX₁₀CX₅CXCX₈CX₆C (SEQ ID NO;

     82), CX₁₂CX₄CX₅CX₁₂CX₂C (SEQ ID NO;

     83), CX₁₂CX₅CX₅CXCX₈C (SEQ ID NO;

     84), CX₁₀CX₆CX₅CXCX₄CXCX₉C (SEQ ID NO;

     85), CX₁₁CX₄CX₅CX₈CX₂C (SEQ ID NO;

     86), CX₁₀CX₆CX₅CX₈CX₂C (SEQ ID NO;

     87), CX₁₀CX₆CX₅CXCX₈C (SEQ ID NO;

     88), CX₁₀CX₆CX₅CXCX₃CX₈X₂C (SEQ ID NO;

     89), CX₁₀CX₆CX₅CX₃CX₈C (SEQ ID NO;

     90), CX₁₀CX₆CX₅CXCX₂CX₆CX₅C (SEQ ID NO;

     91), CX₇CX₆CX₃CX₃CX₉C (SEQ ID NO;

     92), CX₉CXCX₈CX₅CX₆CX₅C (SEQ ID NO;

     93), CX₁₀CX₂CX₂CX₇CXCX₁₁CX₅C (SEQ ID NO;

     94), CX₁₀CX₆CX₅CX₂CX₈CX₄C (SEQ ID NO;

     95), CCX₃CXCX₃CX₂CCXCX₅CX₉CX₅CXC (SEQ ID NO;

     96), CX₆CX₂CX₅CX₄CCXCX₄CX₆CXC (SEQ ID NO;

     97), CX₇CXCX₅CX₄CCCX₄CX₆CXC (SEQ ID NO;

     98), CX₉CX₃CXCX₂CXCCCX₆CX₄C (SEQ ID NO;

     99), CX₅CX₃CXCX₄CX₄CCX₁₀CX₂CC (SEQ ID NO;

     100), CX₅CXCX₁CXCX₃CCX₃CX₄CX₁₀C (SEQ ID NO;

     101), CX₉CCCX₃CX₄CCCX₅CX₆C (SEQ ID NO;

     102), CCX₈CX₅CX₄CX₃CX₄CCXCX₁C (SEQ ID NO;

     103), CCX₆CCX₅CCCX₄CX₄CX₁₂C (SEQ ID NO;

     104), CX₆CX₂CX₃CCCX₄CX₅CX₃CX₃C (SEQ ID NO;

     105), CX₃CX₅CX₆CX₄CCXCX₅CX₄CXC (SEQ ID NO;

     106), CX₄CX₄CCX₄CX₄CXCX₁₁CX₂CXC (SEQ ID NO;

     107), CX₅CX₂CCX₅CX₄CCX₃CCX₇C (SEQ ID NO;

     108), CX₅CX₅CX₃CX₂CXCCX₄CX₇CXC (SEQ ID NO;

     109), CX₃CX₇X₃CX₄CCXCX₂CX₅CX₂C (SEQ ID NO;

     110), CX₉CX₃CXCX₄CCX₅CCCX₆C (SEQ ID NO;

     111), CX₉CX₃CXCX₂CXCCX₆CX₃CX₃C (SEQ ID NO;

     112), CX₈CCXCX₃CCX₃CXCX₃CX₄C (SEQ ID NO;

     113), CX₉CCX₄CX₂CXCCXCX₄CX₃C (SEQ ID NO;

     114), CX₁₀CSCS₃CX₂CXCCX₄CX₅CXC (SEQ ID NO;

     115), CXCXCX₃CX₂CXCCX₄CX₅CXC (SEQ ID NO;

     116), CX₆CCXCX₅CX₄CCXCX₅CX₂C (SEQ ID NO;

     117), CX₆CCXCX₃CXCCX₃CX₄CC (SEQ ID NO;

     118), CX₆CCXCX₃CXCX₂CXCX₄CX₈C (SEQ ID NO;

     119), CX₄CX₂CCX₃CXCX₄CCX₂CX₃C (SEQ ID NO;

     120) CX₃CX₅CX₃CCCX₄CX₉C (SEQ ID NO;

     121), CCX₉CX₃CXCCX₃CX₅C (SEQ ID NO;

     122), CX₉CX₂CX₃CX₄CCX₄CX₅C (SEQ ID NO;

     123), CX₉CX₇CX₄CCXCX₇CX₃C (SEQ ID NO;

     124), CX₉CX₃CCCX₁₀CX₂CX₃C (SEQ ID NO;

     125), CX₃CX₅CX₅CX₄CCX₁₀CX₆C (SEQ ID NO;

     126), CX₉CX₅CX₄CCXCX₅CX₄C (SEQ ID NO;

     127), CX₇CXCX₆CX₄CCCX₁₀C (SEQ ID NO;

     128), CX₈CX₂CX₄CCX₄CX₃CX₃C (SEQ ID NO;

     129), CX₇CX₅CXCX₄CCX₇CX₄C (SEQ ID NO;

     130), CX₁₁CX₃CX₄CCCX₈CX₂C (SEQ ID NO;

     131), CX₂CX₃CX₄CCX₄CX₅CX₁₅C (SEQ ID NO;

     132), CX₉CX₅CX₄CCX₇C (SEQ ID NO;

     133), CX₉CX₇CX₃CX₂CX₆C (SEQ ID NO;

     134), CX₉CX₅CX₄CCX₁₄C (SEQ ID NO;

     135), CX₉CX₅CX₄CCX₈C (SEQ ID NO;

     136), CX₉CX₆CX₄CCXC (SEQ ID NO;

     137), CX₅CCX₇CX₄CX₁₂ (SEQ ID NO;

     138), CX₁₀CX₃CX₄CCX₄C (SEQ ID NO;

     139), CX₉CX₄CCX₅CX₄C (SEQ ID NO;

     140), CX₁₀CX₃CX₄CX₇CXC (SEQ ID NO;

     141), CX₇CX₇CX₂CX₂CX₃C (SEQ ID NO;

     142), CX₉CX₄CX₄CCX₆C (SEQ ID NO;

     143), CX₇CXCX₃CXCX₆C (SEQ ID NO;

     144), CX₇CXCX₄CXCX₄C (SEQ ID NO;

     145), CX₉CX₅CX₄C (SEQ ID NO;

     146), CX₃CX₆CX₈C (SEQ ID NO;

     147), CX₁₀CXCX₄C (SEQ ID NO;

     148), CX₁₀CCX₄C (SEQ ID NO;

     149), CX₁₅C (SEQ ID NO;

     150), CX₁₀C (SEQ ID NO;

     151), and CX₉C (SEQ ID NO;

     152).

12. The humanized antibody or binding fragment thereof of claim 1, wherein the ultralong CDR3 comprises a non-human DH or a derivative thereof.

14. The humanized antibody or binding fragment thereof of claim 1, wherein the ultralong CDR3 comprises a JH sequence or a derivative thereof.

16. The humanized antibody or binding fragment thereof of claim 1, wherein the ultralong CDR3 comprises:

a non-human VH sequence or a derivative thereof;

a non-human DH sequence or a derivative thereof; and

/or

a JH sequence or derivative thereof.

19. The humanized antibody or binding fragment thereof of claim 1, wherein the ultralong CDR3 comprises a sequence derived from or based on SEQ ID NO:

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, or SEQ ID NO;

28.

20. The humanized antibody or binding fragment thereof of claim 1, wherein the ultralong CDR3 comprises a non-bovine sequence or a non-antibody sequence.

26. The humanized antibody or binding fragment thereof of claim 1, wherein the ultralong CDR3 comprises a X¹X²X³X⁴X⁵ motif, a CX¹X²X³X₄X₅ motif, a (X^aX^b)_z motif, a X¹X²X³X⁴X⁵X_n motif, a X_n(X^aX^b)_z motif, or a X¹X²X³X⁴X⁵X_n(X^aX^b)_z motif.

43. The humanized antibody or binding fragment thereof of claim 1, wherein the humanized antibody or binding fragment thereof comprises a human heavy chain germline sequence that is a VH4 family member gemline sequence.

49. The humanized antibody or binding fragment thereof of claim 1, wherein the humanized antibody or binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:

440, 441, 442, 443, or 444, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 470.

65. A polynucleotide encoding the humanized antibody or binding fragment thereof of claim 1.

68. A vector comprising the polynucleotide of claim 65.

69. A host cell comprising the vector of claim 68.

70. A nucleic acid library comprising a plurality of polynucleotides comprising sequences coding for humanized antibodies or binding fragments thereof of claim 1.

3. (canceled)

5. (canceled)

8-11. -11. (canceled)

13. (canceled)

15. (canceled)

17-18. -18. (canceled)

21-25. -25. (canceled)

27-42. -42. (canceled)

44-48. -48. (canceled)

50-64. -64. (canceled)

66-67. -67. (canceled)

71. A library of humanized antibodies or binding fragments thereof, wherein the antibodies or binding fragments thereof comprise an ultralong CDR3.

72-141. -141. (canceled)

142. A CDR3 scaffold comprising a X¹X²X³X⁴X⁵ motif,

wherein X¹ is threonine (T), glycine (G), alanine (A), serine (S), or valine (V), wherein X² is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein X³ is valine (V), alanine (A), threonine (T), or aspartic acid (D), wherein X⁴ is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and wherein X₅ is glutamine (Q).

175. A library comprising the CDR3 scaffold of claim 142.

143-179. -179. (canceled)

176-179. -179. (canceled)