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BR-112018005573-B1 - CD3-BINDING POLYPEPTIDES

BR112018005573B1BR 112018005573 B1BR112018005573 B1BR 112018005573B1BR-112018005573-B1

Abstract

This disclosure relates to protein molecules that specifically bind to CD3, which may have at least one humanized CD3-binding domain. Such molecules are useful for cancer treatment. The CD3-binding protein molecule may have a second binding domain that binds to another target. In one embodiment, multispecific polypeptide molecules bind to both tumor antigen-expressing cells and the CD3 subunit of a T cell receptor complex on T cells to induce target-dependent T cell cytotoxicity, activation, and proliferation. The disclosure also provides pharmaceutical compositions comprising CD3-binding polypeptide molecules, nucleic acid molecules encoding these polypeptides, and methods for producing these molecules.

Inventors

  • Philip Tan
  • JOHN W. BLANKENSHIP

Assignees

  • APTEVO RESEARCH AND DEVELOPMENT LLC

Dates

Publication Date
20260317
Application Date
20160921
Priority Date
20150921

Claims (20)

  1. 1. CD3-binding polypeptide, characterized in that it comprises a CD3-binding domain that specifically binds to human CD3 and that it comprises a variable region of humanized immunoglobulin light chain and a variable region of humanized immunoglobulin heavy chain; wherein the variable region of the immunoglobulin light chain comprises an amino acid sequence of LCDR1 from SEQ ID NO: 94, an amino acid sequence of LCDR2 from SEQ ID NO: 95 and an amino acid sequence of LCDR3 from SEQ ID NO: 96 and wherein the variable region of the immunoglobulin heavy chain comprises an amino acid sequence of HCDR1 from SEQ ID NO: 91, an amino acid sequence of HCDR2 from SEQ ID NO: 92 and an amino acid sequence of HCDR3 from SEQ ID NO: 93; wherein the amino acid residue at position 9 according to the IMGT numbering system of the variable region of the immunoglobulin heavy chain is proline; where the amino acid residue at position 21 according to the IMGT numbering system of the variable region of the immunoglobulin light chain is methionine, and where the amino acid residue at position 87 according to the IMGT numbering system of the variable region of the immunoglobulin light chain is tyrosine.
  2. 2. CD3-binding polypeptide according to claim 1, characterized in that: (a) the variable region of the immunoglobulin light chain comprises SEQ ID NO: 88 and the variable region of the immunoglobulin heavy chain comprises SEQ ID NO: 86; or (b) the variable region of the immunoglobulin light chain comprises SEQ ID NO: 89 and the variable region of the immunoglobulin heavy chain comprises SEQ ID NO: 86.
  3. 3. CD3-binding polypeptide according to claim 1, characterized in that the amino acid residue at position 52, according to the IMGT numbering system, of the variable region of the immunoglobulin light chain is arginine and/or the amino acid residue at position 53, according to the IMGT numbering system, of the variable region of the immunoglobulin light chain is tryptophan.
  4. 4. CD3-binding polypeptide according to claim 1, characterized in that the amino acid residue at position 27, according to the IMGT numbering system, of the variable region of the immunoglobulin heavy chain is tyrosine.
  5. 5. CD3-binding polypeptide according to claim 1, characterized in that: the amino acid residue at position 53, according to the IMGT numbering system, of the variable region of the immunoglobulin heavy chain is isoleucine.
  6. 6. CD3-binding polypeptide according to claim 1, characterized in that the amino acid residue at position 86, according to the IMGT numbering system, of the variable region of the immunoglobulin light chain is aspartic acid.
  7. 7. CD3-binding polypeptide, according to any of the preceding claims, characterized in that the CD3-binding domain comprises SEQ ID NO:83 or SEQ ID NO:84.
  8. 8. CD3-binding polypeptide, according to any of the preceding claims, characterized in that the CD3-binding domain is a single-strand variable fragment (scFv).
  9. 9. CD3-binding polypeptide according to claim 8, characterized in that said scFv comprises a linker between the variable region of the heavy chain and the variable region of the light chain of said scFv and in that said linker comprises the amino acid sequence QRHNNSSLNTGTQMAGHSPNS (SEQ ID NO:148).
  10. 10. CD3-binding domain according to claim 8, characterized in that the variable region of the heavy chain of said scFv is amino-terminal to the variable region of the light chain of said scFv.
  11. 11. CD3-binding polypeptide, according to any of the preceding claims, characterized in that it further comprises a second binding domain, wherein the second binding domain is selected from PSMA-binding domain, CD37-binding domain, ROR1-binding domain and CD123-binding domain.
  12. 12. CD3-binding polypeptide according to claim 11, characterized in that said CD3-binding polypeptide comprises, from the amino-terminal to the carboxyl-terminal, (i) the second binding domain, (ii) a hinge region, (iii) an immunoglobulin constant region, (iv) a carboxyl-terminal linker, and (v) the CD3-binding domain.
  13. 13. CD3-binding polypeptide according to claim 12, characterized in that the constant region of the immunoglobulin comprises a human IgG1 CH2 domain comprising L234A, L235A, G237A and K322A substitutions, according to the EU numbering system.
  14. 14. CD3-binding polypeptide according to claim 11 or 13, characterized in that it comprises an amino acid sequence selected from SEQ ID NO: 62, 64, 66, 68, 72, 74, 76, 78, 80, 82, 100, 104, 108, 112, 116, 120, 197 and 198.
  15. 15. CD3-binding polypeptide, according to any one of claims 12 to 14, characterized in that the hinge region is derived from a hinge region of immunoglobulin.
  16. 16. CD3-binding polypeptide, according to any one of claims 12 to 15, characterized in that the carboxyl-terminal linker comprises or consists of SEQ ID NO:196.
  17. 17. CD3-binding polypeptide, according to any one of claims 12 to 16, characterized in that the constant region of the immunoglobulin comprises CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgD immunoglobulin.
  18. 18. CD3-binding polypeptide, according to any one of claims 11 to 17, characterized in that the CD3-binding polypeptide induces redirected T-cell cytotoxicity (RTCC).
  19. 19. CD3-binding polypeptide according to claim 18, characterized in that the CD3-binding polypeptide induces RTCC with an EC50 of 30 pM or less.
  20. 20. CD3-binding polypeptide according to any one of claims 11 to 19, characterized in that the second binding domain is a single-strand variable fragment (scFv).

Description

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 62/221,190, filed September 21, 2015. The contents of this application are incorporated by reference in their entirety. DESCRIPTION OF THE TEXT FILE SENT ELECTRONICALLY [0002] The contents of the attached text file are incorporated herein by reference in their entirety: A machine-readable copy of the Sequence Listing (file name: APVO_052_01WO_SeqList_ST25.txt, Record date: September 21, 2016, file size: 544 kilobytes). FIELD OF DISSEMINATION [0003] This disclosure relates to molecules that specifically bind to CD3, which may have at least one humanized CD3-binding domain. A therapeutic protein bound to CD3 may be a monospecific therapeutic protein or a multispecific therapeutic protein. A multispecific therapeutic protein may bind to both a tumor antigen and CD3 subunits of the T cell receptor complex on T cells to induce target-dependent T cell cytotoxicity, activation, and proliferation. Fundamentals of Disclosure [0004] Targeting the T-cell receptor complex (TCR) in human T cells with anti-CD3 antibodies has been proposed for the treatment of autoimmune diseases and related disorders, such as for the treatment of organ allograft rejection. In addition to monospecific therapeutics targeting CD3, multispecific polypeptides that selectively bind to T cells and tumor cells could offer a mechanism to redirect T-cell cytotoxicity to tumor cells. These multispecific polypeptides may be useful for cancer treatment. [0005] The clinical use of some anti-CD3 antibodies has been hampered by serious side effects. For example, OKT3, a mouse monoclonal antibody specific for human CD3, induced T cell proliferation and cytokine production in vitro and led to a large-scale release of cytokines in vivo (Hirsch et al. (1989) J. Immunol 142: 737-43). The release of cytokines (also referred to as a "cytokine storm") in turn led to a "flu-like" syndrome, characterized by fever, chills, headaches, nausea, vomiting, diarrhea, respiratory distress, septic meningitis, and hypotension (Chatenoud, (2003) Nature Reviews 3:123-132). [0006] There is a need for CD3-binding molecules that have improved thermal stability with a favorable manufacturing profile and reduced adverse effects. SUMMARY OF THE DISCLOSURE [0007] The disclosure encompasses CD3-binding domains and polypeptides that possess an advantageous manufacturing profile. The polypeptides comprising CD3-binding domains disclosed herein may be thermally stable. In some cases, a polypeptide has improved thermal stability compared to another CD3-binding polypeptide. The CD3-binding domains and polypeptides described herein may have reduced side effects (e.g., they may lead to the release of low levels of cytokines when administered to an individual). [0008] In certain embodiments, disclosure refers to a CD3-binding domain that specifically binds to human CD3 and that comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is (a) at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 88; or (b) at least about 94% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 89, wherein the variable region of the immunoglobulin heavy chain comprises an amino acid sequence that is at least approximately 82% identical, at least about 85% identical, at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 86. A CD3-binding domain may comprise an amino acid sequence that is at least about 87% identical, at least about 90% identical, at least about 92% identical, at least about 95% identical, at least approximately 97% identical, at least approximately 98% identical, or at least approximately 99% identical to the amino acid sequence of SEQ ID NO: 83 or SEQ ID NO: 84. A CD3-binding domain may comprise SEQ ID NO: 83 or SEQ ID NO: 84. [0009] In one embodiment, a CD3-binding domain comprises a variable immunoglobulin light chain region comprising an LCDR1 amino acid sequence from SEQ ID NO: 94, an LCDR2 amino acid sequence from SEQ ID NO: 95, and an LCDR3 amino acid sequence from SEQ ID NO: 96, and a variable immunoglobulin heavy chain region comprising an HCDR1 amino acid sequence from SEQ ID NO: 91, an HCDR2 amino acid sequence from SEQ ID NO: 92, and an HCDR3 amino acid sequence from SEQ ID NO: 93. In another embodiment, a CD3-binding domain comprises a variable immunoglobulin light chain region comprising an LCD