Patent Application
20240200031
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 12, 2022, is named MSKCC_053_WO1_ST25.txt and is 106,000 bytes in size.
For the purpose of only those jurisdictions that permit incorporation by reference, all of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention.
The activating receptor NKG2C present on certain immune cells binds to an HLA-E:peptide complex. HLA-E is a non-classical class I HLA molecule that presents a nine amino acid-long peptide, typically derived from the signal peptide sequence of HLA-A, -B, -C, or -G that is expressed in the same cell. In general, the absence of HLA-A, -B, -C, and -G expression (or some other specific proteins), HLA-E does not come to the cell surface. To force expression of HLA-E on the surface of feeder cells used for NKG2C-based activation and/or expansion of T cells or natural killer (“NK”) cells, while avoiding expression of other class I HLA molecules, a surrogate peptide needs to be provided for presentation on the HLA-E molecule.
The present invention involves recombinant HLA proteins. In particular, the present invention involves recombinant HLA proteins that are derived from HLA-E proteins but in which amino acids at the N-terminal end of the HLA-E protein are replaced with N-terminal amino acids derived from HLA-A, HLA-B, HLA-C or HLA-G proteins, such that the N-terminal leader peptide from HLA-A, HLA-B, HLA-C or HLA-G is present at the N-terminus of the HLA-E-derived recombinant HLA protein. Following proteolytic cleavage of the leader peptide and/or other intracellular processing, a 9 amino acid peptide (nonapeptide) from the leader peptide is available for loading into the HLA-E molecule, enabling expression of the HLA-E molecule on the cell surface and presentation of the loaded peptide to immune cells.
The present invention provides numerous of such novel recombinant HLA proteins, nucleic acid molecules encoding such recombinant HLA proteins, vectors comprising such nucleic acid molecules, host cells comprising such proteins, nucleic acid molecules, and/or vectors, and various compositions.
The present invention also provides various methods of use of such novel recombinant HLA proteins, nucleic acid molecules, vectors, host cells and compositions, including, but not limited to, methods in which host cells expressing the recombinant HLA proteins of the present invention are used to stimulate the expansion and or activation of immune cells such as T cells or NK cells.
These and other aspects of the present invention are described further in the Detailed Description, Drawings, Brief Description of the Drawings, Examples and Claims sections of this patent disclosure. Furthermore, one of skill in the art will recognize that the various embodiments of the present invention described throughout this patent disclosure can be combined in various different ways, and that such combinations are within the scope of the present invention.
In order that the present invention can be more readily understood, certain terms are defined below. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, The Dictionary of Cell and Molecular Biology (5th ed. J. M. Lackie ed., 2013), the Oxford Dictionary of Biochemistry and Molecular Biology (2d ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skill with general definitions of some terms used herein.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.
Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges provided herein are inclusive of the numbers defining the range.
Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are included.
Amino acids are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
The abbreviation “HLA” refers to human leukocyte antigen.
The term “vector” means a construct for delivery of a nucleic acid molecule to a cell. Examples of vectors include, but are not limited to, viruses, viral-derived vectors, naked DNA or RNA vectors, plasmid vectors, cosmid vectors, phage vectors, and the like. In some embodiments a vector may be an “expression vector” that is capable of delivering a nucleic acid molecule to a cell and that also contains elements required for expression of the nucleic acid molecule in the cell.
The term “nucleic acid molecule” as used herein includes both DNA molecules and RNA molecules. Similarly, the term “nucleic acid sequence” as used herein includes both DNA sequences and RNA sequences.
The terms “signal peptide,” “signal sequence,” “leader peptide,” and “leader sequence” are used interchangeably herein.
In each of the embodiments described herein, the recombinant HLA proteins and/or nucleic acid molecules and/or cells (or populations of cells) can optionally be in “isolated” form. An “isolated” protein or nucleic acid molecule or cell (or population of cells) is not within a living subject (or, in the case of proteins and/or nucleic acid molecules, is not within a cell) and is typically in a form not found in nature. In some embodiments an isolated protein or nucleic acid molecule or cell (or populations of cells) may be purified to a degree that it is not in a form in which it is found in nature. In some embodiments, an isolated protein or nucleic acid molecule or cell (or populations of cells) is substantially pure. In some embodiments, an isolated protein or nucleic acid molecule or cell (or populations of cells) has a purity of greater than 75%, or greater than 80%, or greater than 90%, or greater than 95% of the stated protein or nucleic acid molecule or cell (or populations of cells).
The terms “identical” or percent “identity” in the context of two or more amino acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.
Other terms are defined elsewhere in this patent disclosure, or else are used in accordance with their usual meaning in the art.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Ausubel et al. eds. (2015) Current Protocols in Molecular Biology (John Wiley and Sons); Greenfield, ed. (2013) Antibodies: A Laboratory Manual (2nd ed., Cold Spring Harbor Press); Green and Sambrook, eds. (2012), Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press); Krebs et al., eds. (2012) Lewin's Genes XI (11th ed., Jones & Bartlett Learning); Freshney (2010) Culture Of Animal Cells (6th ed., Wiley); Weir and Blackwell, eds., (1996) Handbook Of Experimental Immunology, Volumes I-IV (5th ed., Wiley-Blackwell); Borrebaeck, ed. (1995) Antibody Engineering (2nd ed., Oxford Univ. Press); Glover and Hames, eds., (1995) DNA Cloning: A Practical Approach, Volumes I and II (2nd ed., IRL Press); Rees et al., eds. (1993) Protein Engineering: A Practical Approach (1st ed., IRL Press); Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Nisonoff (1984) Introduction to Molecular Immunology (2nd ed., Sinauer Associates, Inc.); and Steward (1984) Antibodies: Their Structure and Function (1st ed., Springer Netherlands).
The present invention provides recombinant HLA proteins. In particular, the present invention provides recombinant HLA proteins that are modified HLA-E proteins in which amino acids of the leader peptide at the N-terminal end of the HLA-E protein are replaced with the amino acids of the N-terminal leader peptide from HLA-A, HLA-B, HLA-C or HLA-G proteins. In such proteins, the leader peptide from HLA-A, HLA-B, HLA-C or HLA-G is present at the N-terminus of the recombinant HLA protein and, after proteolytic cleavage of the signal peptide and/or other post-translational processing, a 9 amino acid peptide within the signal peptide is available for loading into the HLA-E molecule.
Accordingly, in some embodiments the present invention provides recombinant HLA proteins comprising, from N-terminal to C-terminal, a leader peptide from an HLA-A, HLA-B, HLA-C, or HLA-G molecule and an HLA-E protein, wherein the recombinant HLA protein does not comprise an HLA-E leader peptide. In some embodiments there are no intervening amino acids between the leader peptide and the HLA-E protein. In some embodiments there is no linker moiety between the leader peptide and the HLA-E protein.
In some embodiments the leader peptide from HLA-A, HLA-B, HLA-C, or HLA-G comprises amino acids 3-11 of any one of SEQ ID NOs. 33-47 (i.e., the 9 amino acid nonapeptide from SEQ ID NOs. 33-47 that can be loaded onto an HLA-E protein for presentation to an immune cell).
In some embodiments the leader peptide from HLA-A, HLA-B, HLA-C, or HLA-G comprises the amino acid sequence of any one of SEQ ID NOs. 33-47.
In some embodiments the HLA-E protein is derived from the amino acid sequence of SEQ ID NO. 31 or SEQ ID NO. 32.
In some embodiments the HLA-E protein comprises amino acids 22-358 of SEQ ID NO. 31 or SEQ ID NO. 32.
In some embodiments the recombinant HLA protein comprises any one of SEQ ID NOs. 1 to 30. For example, in one embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 1. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 2. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 3. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 4. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 5. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 6. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 7. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 8. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 9. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 10. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 11. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 12. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 13. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 14. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 15. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 16. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 17. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 18 In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 19. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 20. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 21. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 22. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 23. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 24. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 25. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 26. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 27. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 28. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 29. In another embodiment the present invention provides a recombinant HLA protein comprising SEQ ID NO. 30.
The recombinant HLA proteins described above are processed intracellularly leading to cell-surface HLA expression and peptide presentation. In some embodiments the present invention provides recombinant HLA proteins produced intracellularly from any one of the proteins described above, for example by proteolytic cleavage of its leader peptide.
The amino acid sequences of 30 examples of recombinant HLA proteins according to the present invention listed above, as identified by their SEQ ID NOs, are provided in Table A, below. In Table A, the amino acids of the leader sequence, which is derived from another HLA molecule (i.e., not from HLA-E), are bolded (i.e., the first 24 amino acids), and the portion of the leader peptide that constitutes the 9 amino acid peptide that is loaded into and displayed on the HLA-E-derived molecule is underlined (i.e., amino acids 3-11).
MV
VMAPRTLFL
LLSGALTLTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MA
VMAPRTLLL
LLSGALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MA
VMAPRTLVL
LLSGALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MA
VMAPRTLLL
LLLGALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MA
IMAPRTLVL
LLSGALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
ML
VMAPRTVLL
LLSAALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VTAPRTVLL
LLSGALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VTAPRTVLL
LLSAALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDD
MR
VTAPRTLLL
LLWGAVALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VTAPRTLLL
LLWGALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MRV
TAPRTVLL
LLWGAVALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VMAPRTLIL
LLSGALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDD
MR
VMAPRALLL
LLSGGLALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VMAPRTLLL
LLSGALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VMAPQALLL
LLSGALALIETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MV
VMAPRTLFL
LLSGALTLTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MA
VMAPRTLLL
LLSGALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MA
VMAPRTLLL
LLLGALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MA
IMAPRTLVL
LLSGALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
ML
VMAPRTVLL
LLSAALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VTAPRTVLL
LLSGALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VTAPRTVLL
LLSAALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDD
MR
VTAPRTLLL
LLWGAVALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VTAPRTLLL
LLWGALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VTAPRTVLL
LLWGAVALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VMAPRTLIL
LLSGALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDD
MR
VMAPRALLL
LLSGGLALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VMAPRTLLL
LLSGALALTETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
MR
VMAPQALLL
LLSGALALIETWAGSHSLKYFHTSVSRPGRGEPRFISVGYVD
The amino acid sequences of the HLA-E proteins from which the recombinant HLA proteins listed in Table A are derived are provided in Table B, below. In Table B, the amino acids of the leader sequence are bolded (i.e., the first 21 amino acids). These are the amino acids that are substituted with leader peptides from a different HLA molecule (HLA-A, HLA-B, HLA-C, or HLA-G) in the recombinant HLA proteins shown in Table A.
MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQF
MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQF
Amino acid sequences of leader peptides from various HLA-A, HLA-B, HLA-C, and HLA-G types, which can be used to replace the leader peptides of HLA-E molecules, are provided in Table C, below. Amino acid residues 3-11 of each of these leader peptides (underlined) constitutes a 9 amino acid peptide (nonapeptide) that can be loaded into an HLA-A molecule and presented on the cell surface of a host cell.
The full amino acid sequence of HLA-G01 (SEQ ID NO. 48) is illustrated in
In some embodiments the present invention provides variants of the recombinant HLA proteins described above or elsewhere herein. For example, in some embodiments the present 5 invention provides variants of the recombinant HLA proteins having the amino acid sequences of any of SEQ ID Nos 1-30. In some embodiments the present invention provides variants of the recombinant HLA proteins described above or elsewhere herein that comprise 1 or 2 or 3 or 4 or 5 or more amino acid point mutations (which can be insertions, deletions, or substitutions) as compared to the recombinant HLA proteins described above or elsewhere 10 herein, or have an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to that of any of the recombinant HLA proteins described above or elsewhere herein, provided that such variant recombinant HLA proteins can be expressed on the surface of a host cell and can be loaded with and present a nonapeptide derived from their leader peptide.
The present invention also provides nucleic acid molecules that encode any of the recombinant HLA proteins described herein. In some embodiments such nucleic acid molecules are DNA molecules. In some embodiments such nucleic acid molecules are RNA molecules.
For example, in one embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising any one of SEQ ID NOs. 1 to 30. In one embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 1. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 2. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 3. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 4. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 5. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 6. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 7. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 8. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 9. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 10. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 11. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO.12. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 13. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 14. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 15. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 16. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 17. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 18. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 19. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 20. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 21. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 22. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 23. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 24. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 25. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 26. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 27. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 28. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 29. In another embodiment the present invention provides a nucleic acid molecule that encodes a recombinant HLA protein comprising SEQ ID NO. 30.
In some embodiments the nucleic acid molecules are codon optimized for human expression.
In some embodiments the nucleic acid molecules are operatively linked to a promoter. In some such embodiments the promoter is the EF1α promoter.
The present invention also provides vectors that comprise any of the nucleic molecules described herein. In some of such embodiments the vectors are cloning vectors. In some of such embodiments the vectors are expression vectors. In some of such embodiments the vectors are viral vectors. In some of such embodiments the vectors are lentiviral vectors.
The present invention also provides cells that comprise a nucleic acid molecule that encodes a recombinant HLA protein as described herein, and/or that express a recombinant HLA protein encoded by such a nucleic acid molecule. Such cells are referred to herein as “host cells.” The host cells can be any cell type capable of expressing the recombinant HLA molecules of the present invention on their cell surface.
In some embodiments the host cells are mammalian cells. In some embodiments the host cells are human cells. In some embodiments the host cells are in vitro. In some embodiments the host cells are in vivo.
In some embodiments the host cells are K562 cells. See Lozzi et al., Blood, 1975 March; 45(3), pp. 321-34 for a description of K562 cells, which are well known and widely used in the art. In some embodiments the host cells are K562 cells modified to express 4-1BBL. In some embodiments the host cells are K526 cells modified to express membrane-bound IL-21 (mbIL-21). In some embodiments the host cells are K526 cells modified to express both 41BBL and mbIL-21. In some embodiments the host cells are the K562-derived cell line K562 c9.mbIL-21 (which is modified to modified to express CD64, CD86, CD137L, truncated CD19, eGFP, and membrane-bound IL-21, as described in the Examples section of this disclosure). See Singh et al. Cancer Res. 2011 May 15; 71(10), pp. 3516-27 for a description of modified K562 cells.
In some embodiments the host cells are the K562-derived cell line K562 c9.mbIL-21.E:G01 (described in the Examples section of this disclosure).
In some embodiments the host cells are 721.221 cells. See Shimizu et al., (1988). Proceedings of the National Academy of Sciences of the United States of America, 85(1), pp. 227-231 for a description of 721.221 cells.
In some embodiments the host cells are BAF3 cells. See Palacios et al., Nature. 1984 May 10-16; 309(5964), pp. 126-31 for a description of BAF3 cells.
In embodiments where the host cells are contacted in vitro (e.g., co-cultured) with immune cells, such as T cells or NK cells, the host cells may be referred to as “feeder” cells.
In some embodiments the host cells are isolated cells.
In some embodiments the present invention provides compositions that comprise a recombinant HLA protein as described herein, or a nucleic acid molecule encoding a recombinant HLA protein as described herein, or a host cell (or population of host cells) as described herein, and at least one additional component.
Examples of such additional components include diluents, saline solutions, cell culture media, buffers, carriers, stabilizers, dispersing agents, suspending agents, thickening agents, excipients, preservatives, and the like. In some embodiments the compositions are sterile.
In some embodiments the compositions are pharmaceutical compositions. The term “pharmaceutical composition” as used herein refers to a composition that is in such form as to permit the biological activity of the recombinant HLA protein, or nucleic acid molecule, or host cell (or population of host cells) therein, and which contains no components that are unacceptably toxic to cells or to living subjects.
The recombinant HLA proteins of the present invention can be produced by any of the methods known in the art for production of recombinant proteins. For example, in some embodiments the recombinant HLA proteins of the present invention are produced by expression in a host cell from a recombinant nucleic acid molecule that encodes the recombinant HLA protein. In some embodiments the recombinant nucleic acid molecule is a DNA molecule. In some embodiments the recombinant nucleic acid molecule is an RNA molecule.
Similarly, nucleic acid molecules encoding the recombinant HLA proteins of the present invention can be produced by any of the methods known in the art for production of nucleic acid molecules, such as standard methods of molecular biology and/or recombinant DNA technology.
The present invention also provides methods of use of the recombinant HLA proteins, nucleic acid molecules and host cells described herein.
In some embodiments the present invention provides methods for stimulating the expansion of immune cells. In some embodiments the present invention provides methods for modulating the activity of immune cells. In some embodiments the present invention provides methods for activating immune cells. In some embodiments the present invention provides methods for generating de novo populations of immune cells. In some embodiments the present invention provides methods of generating or preparing immune cells for use in adoptive cell therapy. In some embodiments the present invention provides methods of generating or preparing immune cells for administration to a subject, such as a human subject.
In some embodiments such methods involve contacting the immune cells with a recombinant HLA protein as described herein. In some embodiments such methods involve contacting the immune cells with a host cell expressing a recombinant HLA protein as described herein. In some embodiments such methods involve delivering a nucleic acid molecule encoding a recombinant HLA protein as described herein to a host cell. In some embodiments such methods involve delivering a vector comprising a nucleic acid molecule encoding a recombinant HLA protein as described herein to a host cell. In some embodiments such methods involve delivering a composition comprising a recombinant HLA protein, nucleic acid molecule or vector as described herein to a host cell.
In some embodiments, the immune cells are T cells. In some embodiments the T cells are CD8+ T cells. In some embodiments the T cells are NKG2C+CD8+ T cells. In some embodiments the T cells are NKG2C-CD8+ T cells. In some embodiments the T cells are CD8+ T cells in which the BCL11B gene has been knocked out (“BCL11B KO” cells).
In some embodiments the immune cells are NK cells.
For example, and as demonstrated experimentally in the Examples section of this patent disclosure, in some embodiments the present invention provides methods of activating NK cells by contacting the NK cells in vitro with a host cell expressing a recombinant HLA protein as described herein.
Similarly, and as also demonstrated experimentally in the Examples section of this patent disclosure, in some embodiments the present invention provides methods of activating T cells by contacting the T cells in vitro with a host cell expressing a recombinant HLA protein as described herein.
Similarly, in some embodiments the present invention provides methods of inducing de novo T cell populations by contacting T cells in vitro with a host cell expressing a recombinant HLA protein as described herein. For example, and as described in the Examples section of this patent disclosure, the present invention provides methods of inducing de novo NKG2C+ CD8+ T cell populations by contacting CD8+ T cells in which the BCL11B gene has been knocked out (“BCL11B KO” cells) in vitro with a host cell expressing a recombinant HLA protein as described herein.
In some embodiments the contacting is in vitro. In some embodiments the contacting is in vivo. Embodiments that involve contacting immune cells with a host cell expressing a recombinant HLA protein as described herein in vitro may be referred to herein as “feeder cell” methods or “co-culture methods” and the host cells used in such methods may be referred to as “feeder cells.”
The ability to stimulate the expansion of immune cells and/or modulate the activity of immune cells and/or induce a de novo immune cell population by contacting immune cells in vitro with a host cell expressing a recombinant HLA protein, as described herein, has several important clinical applications, including, but not limited to, in providing expanded and/or activated immune cells or de novo immune cell populations for use in adoptive cell therapy methods and/or other therapeutic methods in which immune cells (e.g., T cells or NK cells) are administered to subjects. Accordingly, in some embodiments, the methods described herein are used in conjunction with or as part of a method of generating or preparing immune cells for use in adoptive cell therapy. In some embodiments, the methods described herein are used in conjunction with or as part of a method of generating or preparing immune cells for administration to a subject, such as a human subject.
These and other embodiments of the invention are further described in the “Examples” section of this patent disclosure. All sections of this patent disclosure are intended to be read in conjunction with, and in the context of, all other sections of the present patent disclosure. Furthermore, one of skill in the art will recognize that the various embodiments of the present invention described herein can be combined in various ways, and that such combinations are within the scope of the present invention.
The invention is further described by, and understood with reference to, the following non-limiting Example(s).
The activating receptor NKG2C binds to HLA-E:peptide complexes. HLA-E is a non-classical class I HLA molecule that presents a nine amino acid-long peptide, typically derived from the signal peptide sequence of HLA-A, -B, -C, or -G that is expressed in the same cell. In general, in the absence of HLA-A, -B, -C, and -G expression (or some other specific proteins), HLA-E does not come to the cell surface. To force expression of HLA-E on the surface of cells (e.g, feeder cells used for activation of immune cells via NKG2C) while avoiding expression of other class I HLA molecules, a surrogate peptide (e.g., a nine amino acid peptide from the signal peptide sequence of HLA-A, -B, -C, or -G) needs to be provided alongside HLA-E.
The present invention provides recombinant HLA proteins that are chimeric molecules derived from HLA-E molecules but in which the HLA-E signal peptide (also referred to as leader peptide) sequence has been replaced with a signal peptide from an HLA-A, -B, -C, or G molecule.
K562 derivative cell lines are frequently used for expanding T cells and NK cells. In some experiments the chimeric proteins were expressed in K562s modified to express CD64, CD86, CD137L, truncated CD19, eGFP, and membrane-bound IL-21, referred to as “K562 c9.mbIL-21” cells.
Nucleotide sequences encoding the recombinant HLA proteins described herein were codon-optimized for human translation and synthesized in a pUC57 vector. Site-directed mutagenesis was used to convert HLA-E0101 sequences to HLA-E0103 sequences (i.e., to introduce an R128G mutation) using the Q5 Site-Directed Mutagenesis Kit [New England Biolabs (NEB)] with the forward primer 5′-GGGACCAGACGGGAGATTCCTG-3′ (SEQ ID NO. 49) and reverse primer 5′-AGCTCGCATCCGTGCATC-3′(SEQ ID NO. 49).
For expression, nucleotide sequences encoding the recombinant HLA proteins were cloned into the third-generation lentiviral expression vector, pERRL, such that their expression was under the control of an EF1α promoter. The pERRL vector was produced from the pRRLSIN.cPPT.PGK-GFP.WPRE vector (Addgene, #12252) by exchanging the human PGK promoter for the human EF1 promoter and inserting an XbaI restriction site in the 5′ multiple cloning site (MCS).
Lentivirus was produced as previously described. Briefly, 293 T cells (American Type Culture Collection) were transiently transfected with 15 μg of pERRL, 18 μg of pRSV-Rev (Addgene, #12253), 18 μg of pMDLg/pRRE (Addgene, #12251), and 7 μg of pCI-VSVG (Addgene, #1733) using Lipofectamine 2000 (Invitrogen). Supernatants were collected at 24 and 48 hours after transfection and concentrated by centrifugation at ≥30,000 g for ≥6 hours. Viral pellets were combined and resuspended in RPMI 1640 complete medium and used to transduce host cells.
Host cell lines used were K562 cells, modified K562 cells, 721.221 and BAF3 cells (K562 cells are frequently used for expanding T cells and NK cells). In some experiments modified K562 cells were used that were modified to express CD64, CD86, CD137L, truncated CD19, eGFP, and membrane-bound IL-21. These modified K562 cells referred to herein as “K562 c9.mbIL-21” cells or “K562 mod” cells.
Host cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
Host cells were transduced with the lentiviral vectors described above containing a nucleic acid sequence encoding the given recombinant HLA protein. A K62 cell line referred to herein as “K562 c9.mbIL-21.E:G01” was generated by transducing K562 c9.mbIL-21 cells with a lentiviral vector encoding HLA-E0103:G01 (SEQ ID NO. 1) and selecting a subclone with high surface expression of HLA-E (as determined by fluorescence activated cell sorting) by limiting
For experiments involving T cells certain T cell sub-populations were isolated by flow sorting. The sorted cells were rested overnight in complete media (RPMI with Penicillin/Streptomycin, L-Glutamine, and 10% FCS) with 100U/mL IL-2, at 1 million cells per mL in a humidified incubator at 37° C. and 5% CO2. The following day, an equal number of irradiated host cells were added to the T cell culture in an equal volume of complete media. Twice a week (e.g. Monday and Friday), the T cells were counted and re-stimulated with an equal number of irradiated host cells in fresh complete media containing 100U/mL IL-2 (1 million total cells per mL, 500,000 each of T cells and host cells).
For experiments involving NK cells NK cells were isolated by flow sorting, or isolated by magnetic cell separation (MACS, Miltenyi), or examined within the total peripheral blood mononuclear call (PBMC) population. NK cells were cultured in media containing 200U/mL of IL-2.
Unless stated otherwise, the above methods of variations of these methods were employed in the studies described in the following Examples.
Numerous recombinant HLA proteins were generated and expressed in multiple cell types. Fifteen of the recombinant HLA proteins listed in Table A were expressed in 721.221 host cells, twelve were expressed in K562 host cells, and seven were expressed in BAF3 host cells.
The upper panel of
Activity of Host Cells Expressing Recombinant HLA Proteins on NK Cells & T cells
Experiments were performed to investigate the activity of the recombinant HLA proteins of the present invention on NK cells.
Experiments were also performed to investigate the activity of recombinant HLA proteins on T cells.
Furthermore, additional studies showed that cells expressing the recombinant HLA proteins of the present invention could induce de novo T cell populations. Specifically, when CD8+ T cells in which the BCL11B gene had been knocked out (“BCL11B KO” cells) cells were exposed to K562 cells expressing HLA-E0103: G01 for 4 weeks a de novo NKG2C+CD8+ T cell population was induced. See
This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/173,966 filed on Apr. 12, 2021, the content of which is hereby incorporated by reference in its entirety.
This invention was made with government support under grant number AI150999 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/024435 | 4/12/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63173966 | Apr 2021 | US |
