CN117580855A - IL-2 based constructs - Google Patents

IL-2 based constructs Download PDF

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Publication number
CN117580855A
CN117580855A CN202280043198.4A CN202280043198A CN117580855A CN 117580855 A CN117580855 A CN 117580855A CN 202280043198 A CN202280043198 A CN 202280043198A CN 117580855 A CN117580855 A CN 117580855A
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Prior art keywords
chimeric protein
protein complex
cancer
modified
cell
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CN202280043198.4A
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Chinese (zh)
Inventor
N·克雷
E·德普拉
L·扎贝奥
A·考威尔
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Orenice Bioscience Pte Ltd
Orionis Biosciences Inc
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Orenice Bioscience Pte Ltd
Orionis Biosciences Inc
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Priority claimed from PCT/US2022/025143 external-priority patent/WO2022221746A1/en
Publication of CN117580855A publication Critical patent/CN117580855A/en
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Abstract

The present invention relates in part to chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprising interleukin 2 or a variant thereof, and their use as therapeutic agents.

Description

IL-2 based constructs
Technical Field
The present invention relates in part to chimeric proteins or chimeric protein complexes (including Fc-based chimeric protein complexes) comprising interleukin 2 (IL-2) or variants thereof, and their use as therapeutic agents.
Cross Reference to Related Applications
The present application claims the benefit of U.S. provisional patent application Ser. Nos. 63/175,827, filed on 16 months 4 of 2021, and 63/297,330, filed on 7 months 1 of 2022, the disclosures of which are hereby incorporated by reference in their entireties.
Sequence listing
The contents of the text file submitted electronically with the text are incorporated by reference in their entirety. A computer-readable format copy of the sequence listing (file name: ORN-080pc_st25.Txt, creation date: 2022, 4 months, 14 days; file size: 1,151,315 bytes).
Background
Interleukin 2 (IL-2) is a member of the cytokine family, each with a four alpha helix bundle. IL-2 signals through the IL-2 receptor, which is a complex consisting of two or three polypeptide chains (i.e., an alpha chain and/or a beta chain and a gamma chain).
IL-2 has been approved for cancer treatment in high dose regimens. Illustrative cancers treated with IL-2 include renal cell (kidney) cancer and melanoma (a skin cancer).
IL-2 stimulates immune cell proliferation and activation through receptor signaling complexes containing alpha (IL-2Rα, CD 25) and/or beta (IL-2Rβ, CD 122) and a common gamma receptor polypeptide chain (yc, CD 132), but its effects are pleiotropic and contextually. At high doses, IL-2 binds to the heterodimeric IL-2Rβγ receptor, resulting in tumor killing of CD8 + Desired expansion of effector cells, memory T cells and NK cells. IL-2 also binds with greater affinity to its heterotrimeric receptor IL-2Rαβγ, thereby amplifying immunosuppressive CD4 expressing high constitutive levels of IL-2Rα + CD25 + Regulatory T cells (tregs). Expansion of tregs represents an adverse effect of IL-2 on cancer immunotherapy. In additionActivation of IL-2rαβγ on endothelial cells can cause Vascular Leak Syndrome (VLS).
In contrast, CD8 + Stimulation of T cells represents a detrimental effect of IL-2 in autoimmune disease therapy, where stimulation of tregs by IL-2 is required to suppress autoreactive CD8 + T cells. Because of their overall pleiotropic effects on a variety of cell types, IL-2 therapy has been plagued by serious side effects and systemic toxicity, including Vascular Leak Syndrome (VLS), which limits the exploitation of its therapeutic potential.
Constructs for receptor-selective IL-2 (e.g., constructs that have defective or reduced IL-2Rα and IL-2Rβ interactions) have been prepared. However, these constructs still present side effects and toxicity problems. Some problems can be ameliorated by reducing the dose (e.g., by extending the half-life), but reducing the dose alone does not address the cell selectivity issue. Thus, there remains a need for IL-2-based agents with reduced cellular side effects (off-target effects) and lower systemic toxicity that can be used for IL-2rβγ targeting (e.g., CD8 for use in cancer treatment) + T cell activation, thelp cell activation for use in cancer therapy, and NK cell activation for use in cancer therapy) and IL-2rαβγ targeting (e.g., treg stimulation for use in autoimmune diseases).
Thus, there remains a need for safe and effective IL-2-based therapeutics with improved target selectivity, pharmacokinetic and therapeutic profiles, as well as minimal toxicity profiles.
Disclosure of Invention
Thus, in some aspects, the invention relates to chimeric proteins and chimeric protein complexes, including Fc-based chimeric protein complexes, comprising modified IL-2 as a signaling agent. In some embodiments, the modified IL-2 is a modified wild-type IL-2, wherein the wild-type IL-2 has the amino acid sequence of SEQ ID NO:1. in some embodiments, the modified IL-2 is a modified neoleukocyte, wherein the neoleukocyte has the amino acid sequence of SEQ ID NO:2.
In some embodiments, the modified IL-2 signaling agent comprises: (i) A mutation selected from the group consisting of D20E, D20F, D20G, D20H, D I, D20K, D L and D20V; or (ii) a mutation selected from the group consisting of N88G, N88E, N88K, N Q and N88V; or (iii) a mutation selected from the group consisting of Q126G, Q126A, Q E, Q F, Q126H, Q126I, Q126K, Q126L, Q N, Q126P, Q R, Q126S, Q T, Q V, Q W and Q126Y; or (iv) a mutation selected from (i) and (iii) or a mutation selected from (ii) and (iii); or (v) one or more mutations selected from the group consisting of: R38A/F42K/N88G/C125K/C125E/C125F/C125G/C125H/C125I/C125K/C125L/C125N/C125S/C125 20T/C125V/C125 88A/C125D/C125 88G/C125E/C125H/C125 88I/C125 88K/C125 88Q/C125R/C125T/C125 88V/C125E/R38A/F42K/C125V/R38A/F42K C125A/F42Y/E62A/C125A/F42Y/Y45A/E62Y/Y45A/L72A/F42Y/Y45A/E62A/C125Y/Y45A/L72G/C125A/F42K/N88A/F42K/Q126G/C125A/F42K/Q126I/C125A/F38A/F42K/Q126Y/C125A/F42K/E62A/N88G and R38A/F42K/E62A/N88G/C125A.
In some embodiments, the modified IL-2 signaling agent is a modified IL-2 signaling agent relative to SEQ ID NO:1 comprises one or more mutations at an amino acid residue selected from R38, F42, E62, C125, Y45, L72, D20, N88 and Q126. In such embodiments, the modified IL-2 signaling agent is relative to SEQ ID NO:1 comprises one or more mutations selected from R38 42 42 62 125 45 20 20 20 20 20 20 20 20 20 20 20 20 88 88 88 88 38A/F42Y/E62A/C125Y/Y45A/L72G/C125 42K/C125 38A/F42K/C125E/C125 20G/C125S/C125 20T/C125 20V/C125A/C125 88D/C125 88G/C125 88H/C125 88Q/C125 88T/C125 20E/R38A/F42K/C125 20V/R38A/F42K/C125 38A/F42K/N88G/C126 126 126W and Q126Y 126 126 126 126.
In various embodiments, the modified IL-2 signaling agent is specific for a peptide of seq id NO:1 comprises one or more mutations selected from the group consisting of D20 20 88K/C125E/C125 20G/C125 20S/C125T/C125 20V/C125A/C125 88D/C125 88G/C125 88H/C125 88Q/C125 88T/C125E/R38A/F42K/C125V/R38A/F42K/E62A/C125 38A/F42K/N88G/C125 38A/F42K/E62A/N88A/F42K/E126A/N126G 126 126 126W and Q126Y 126.
In certain embodiments, the modified IL-2 signaling agent is specific for the amino acid sequence of seq id NO:1 comprises one or more mutations selected from the group consisting of R38A/F42Y/E62A/C125A, F Y/Y45A/L72G/C125A, F K/C125A, D E/C125A, D G/C125A, D S/C125A, D20T/C125A, D V/C125A, N A/C125A, N88D/C125A, N G/C125A, N88H/C125A, N88Q/C125A, N88T/C125A, D20E/R38A/F42K/C125A, D V/R38A/F42K/C125A and R38A/F42K/N88G/C125A.
In some embodiments, the modified IL-2 signaling agent is a modified neoleukocyte comprising the amino acid sequence of SEQ ID NO:2. in such embodiments, the nucleic acid sequence comprising SEQ ID NO:2, the modified IL-2 signaling agent has one or more mutations at amino acid residue D15 and/or N40. For example, in some embodiments, the polypeptide comprising SEQ ID NO:2 comprises D15T, D15H, N40I, N G or N40R.
In some embodiments, the one or more mutations or modifications increase the safety profile of the modified IL-2 relative to, for example, wild-type IL-2. In some embodiments, the one or more mutations or modifications reduce the biological activity of the modified IL-2 relative to, for example, wild-type IL-2. For example, the one or more mutations or modifications can reduce the affinity of the modified IL-2 for the target receptor. In some embodiments, the target receptor is a heterotrimeric receptor IL-2Rαβγ, which consists of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits. In some embodiments, the target receptor is the heterodimeric receptor IL-2Rβγ, which consists of IL-2Rβ and IL-2Rγ subunits. In some embodiments, the modified IL-2 comprises one or more mutations or modifications that reduce its affinity for the IL-2Rα subunit. In another embodiment, the modified IL-2 comprises one or more mutations or modifications that reduce its affinity for IL-2Rβ. In yet another embodiment, the modified IL-2 comprises one or more mutations or modifications that reduce its affinity for IL-2Rgamma. In one embodiment, the modified IL-2 comprises one or more mutations or modifications that reduce its affinity for any combination of IL-2Rα, IL-2Rβ, and IL-2Rγ (e.g., one or more mutations or modifications that reduce the affinity of the modified IL-2 for IL-2Rα, IL-2Rβ, and IL-2Rγ or for IL-2Rα and IL-2Rβ or for IL-2Rγ and IL-2Rβ or for IL-2Rα and IL-2Rγ).
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rα and IL2rβ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of D20E/R38A/F42K, D E/R38A/F42K/C125A, D V/R38A/F42K, D V/R38A/F42K/C125A, D V/R38A/F42K/E62A, D20V/R38A/F42K/E62A/C125A, R a/F42K/N88G, R a/F42K/N88G/C125A, R a/F42K/E62A/N88G and R38A/F42K/E62A/N88G/C125A.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rα and IL-2rγ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of R38A/F42K/Q126G, R a/F42K/Q126I and R38A/F42K/Q126Y.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rβ, optionally wherein the modified IL-2 comprises one or more mutations selected from D20E, D20E, D20E, D20E, D20E, D20E, D20E, D20/20E, D20E, D20E, D88E/C125E, D20F/C125E, D G/C125E, D H/C125E, D I/C125E, D20L/C125E, D20K/C125E, D N/C125E, D S/C125E, D T/C125E, D V/C125E, D a/C125E, D88D/C125E, D88G/C125E, D E/C125E, D H/C125E, D I/C125E, D K88Q/C125E, D R/C125T/C125A and N/C88A.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rγ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q S, Q126T, Q126V, Q W and Q126Y.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2Rβγ, optionally wherein the modified IL-2 comprises a polypeptide selected from the group consisting of D20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126V/Q20V/Q126 20V/Q126 20V/Q E/Q126V/Q20V/Q126V/Q20V/Q126 20V/Q126 20V/Q126 20V/Q126V/Q20V/Q126V/Q20V/Q126 20V/Q126 20. In further embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rβγ and increased selectivity for activated CD8 cells.
In another aspect, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rβ and relative to a polypeptide having the amino acid sequence of SEQ ID NO:1 comprises a single mutation selected from the group consisting of D20E, D20F, D20G, D20H, D20I, D20K, D20L, D20N, D20V, N88/C125G, N88Q/C125A and N88/C125G, N E/C125G, N F/C125G, N G/C125G, N H/C125G, N I/C125G, N K/C125G, N L/C125G, N20N/C125G, N S/C125G, N T/C125G, N V/C125G, N88A/C125G, N88D/C125G, N88G/C125G, N H/C G, N88Q/C125A and N88T/C125A, and does not comprise a mutation that confers reduced affinity or biological activity to the IL-2rα subunit (IL-2rα). In further embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rβ and exhibits increased selectivity for activated CD8 cells and reduced selectivity for Treg.
In some embodiments, the target receptor is a heterotrimeric receptor IL-2Rαβγ, which consists of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits. In some embodiments, the target receptor is the heterodimeric receptor IL-2Rβγ, which consists of IL-2Rβ and IL-2Rγ subunits.
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce or eliminate the activity or affinity of the modified IL-2 for an IL-2R alpha chain and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R lacking an alpha chain. In some embodiments, the mutation or modification that reduces the activity or affinity of the modified IL-2 and/or reduces the modified IL-2 can be restored at the target cell or therapeutic site of action by linking to the targeting moiety of the modified IL-2.
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R lacking an alpha chain and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R comprising an alpha chain, such mutations or modifications being attenuating mutations whose activity or affinity can be restored by inducing access to a target or therapeutic action site (e.g. a cell or extracellular matrix) via a targeting moiety.
In various embodiments, the invention encompasses chimeric proteins, chimeric protein complexes, or Fc-based chimeric protein complexes comprising a modified IL-2 signaling agent that contains a glycosylation mutation. In some embodiments, the glycosylation mutation reduces or eliminates glycosylation of the modified IL-2 signaling agent. In some embodiments, the glycosylation mutation is a T3 substitution, wherein the mutation is one of T3A, T3F, T3H, T3L, T V and T3Y. In some embodiments, the glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2.
In some embodiments, the chimeric proteins comprise one or more additional signaling agents, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be modified.
In some embodiments, the chimeric proteins comprise one or more targeting moieties that allow for recovery of attenuated or attenuated activity described herein (e.g., activity on IL-2). In some embodiments, the chimeric proteins comprise one or more targeting moieties having a recognition domain (e.g., antigen recognition domain, including but not limited to various antibody formats, including single domain antibodies) that specifically bind to a target of interest (e.g., antigen, receptor). In various embodiments, the targeting moiety has a recognition domain that specifically binds to a target of interest (e.g., antigen, receptor), including targets found on one or more immune cells, which may include, but are not limited to, T cells, tregs, cytotoxic T lymphocytes, T helper cells, natural Killer (NK) cells, natural Killer T (NKT) cells, anti-tumor or tumor macrophages (e.g., M1 or M2 macrophages, respectively), B cells, and dendritic cells. In some embodiments, the immune cell is a T cell and the targeting moiety targets CD8. In various embodiments, the immune cell is a Treg and the targeting moiety targets CTLA4. In various embodiments, the immune cell is an NK cell and the targeting moiety is NKp46.
In some embodiments, the recognition domain specifically binds to a target of interest (e.g., antigen, receptor) and effectively recruits one or more immune cells. In some embodiments, the target of interest (e.g., antigen, receptor) is found on one or more tumor cells. In some embodiments, the chimeric proteins of the invention can recruit immune cells, such as immune cells that can kill and/or suppress tumor cells, directly or indirectly to a site of action (such as a tumor microenvironment, as a non-limiting example). In some embodiments, the recognition domain binds to a target of interest found in normal target tissue. In some embodiments, the chimeric proteins of the invention can bind to target cells to direct localization of the chimeric proteins to target tissue cells, while also being able to bind to a second, different cell type (e.g., immune cells) to confer a tissue localization biological effect mediated by modified IL-2. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., antigen, receptor) that is part of a non-cellular structure.
In various embodiments, the chimeric proteins of the present technology provide improved cellular target selectivity, safety, and/or therapeutic activity and/or pharmacokinetic characteristics (e.g., increased serum half-life) as compared to non-targeted modified IL-2.
In some embodiments, the chimeric proteins of the present technology are single chain polypeptides.
In some embodiments, the chimeric proteins of the present technology are protein complexes comprising two or more polypeptide chains. In some embodiments, the targeting moiety (or moieties) and the one or more signaling agents (e.g., modified IL-2) are located on the same polypeptide chain in a protein complex. In some embodiments, the targeting moiety (or moieties) and the one or more signaling agents (e.g., modified IL-2) are located on different polypeptide chains in a protein complex.
In various embodiments, the chimeric proteins of the invention are useful in the treatment of various diseases or disorders, such as cancer, infections, immune disorders, autoimmune diseases, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and disorders, and the invention encompasses various methods of treatment.
In another aspect, the invention relates to an Fc-based chimeric protein complex comprising a modified IL-2 as a signaling agent. In some embodiments, the Fc-based chimeric protein complexes of the invention comprise a polypeptide having a nucleotide sequence identical to SEQ ID NO: a polypeptide having 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 any one of 291-296, 298-335. In a particular embodiment, the Fc-based chimeric protein complex of the present invention comprises a polypeptide having a nucleotide sequence identical to SEQ ID NO: 292. 293, 294, 299, 301, 310, 311, 312, 315, 316, 319, 320, 325, 328, 332, 333, and 335, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical.
In some embodiments, the invention relates to a chimeric protein or an Fc-based chimeric protein complex comprising a polypeptide having a nucleotide sequence identical to SEQ ID NO: a polypeptide having an amino acid sequence of any one of 290-449, 478-495, or 501-531 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.
Drawings
Various non-limiting illustrative diagrams of Fc-based chimeric protein complexes of the invention are shown in fig. 1A-1F, 2A-2H, 3A-3H, 4A-4D, 5A-5F, 6A-6J, 7A-7D, 8A-8F, 9A-9J, 10A-10F, 11A-11L, 12A-12L, 13A-13F, 14A-14L, 15A-15L, 16A-16J, 17A-17J, 18A-18F, and 19A-19F. In various embodiments, each schematic is a composition of the present invention. Where applicable in the figures, "TM" refers to a "targeting moiety" as described herein, and "SA" refers to a "signaling agent" as described herein,is as described hereinTwo long parallel rectangles are human Fc domains as described herein, e.g., from IgG1, from IgG2, or from IgG4, and optionally with effector knockout and/or stabilizing mutations as also described herein, and the two long parallel rectangles (one with a protrusion and the other with a depression) are human Fc domains as described herein, e.g., from IgG1, from IgG2, or from IgG4, with knob entry hole and/or ion pair (aka charged pair, ionic bond, or charged residue pair) mutations as described herein, and optionally with effector knockout and/or stabilizing mutations as also described herein. At least one signaling agent is IL-2, or a modified IL-2, or a neoleukocyte, or a modified neoleukocyte.
FIGS. 1A-1F show illustrative homodimer 2-chain complexes. These figures show illustrative configurations of homodimer 2-chain complexes.
Fig. 2A-2H show illustrative homodimer 2-chain complexes having two Targeting Moieties (TM) (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In various embodiments, the constructs shown in the boxes (i.e., fig. 2G and 2H) have a Signaling Agent (SA) between TM1 and TM2 or between TM1 and Fc.
Figures 3A-3H show illustrative homodimer 2-chain complexes having two signaling agents (more signaling agents may be present in some embodiments as described herein). In various embodiments, the locations of SA1 and SA2 are interchangeable. In various embodiments, the constructs shown in the boxes (i.e., fig. 3G and 3H) have a TM between Fc and SA1 and/or SA2, between the N-terminus or C-terminus of the Fc chain.
Fig. 4A-4D show an illustrative heterodimer 2 chain complex with split TM chains and SA chains, i.e., TM on knob chain of Fc and SA on hole chain of Fc.
Fig. 5A-5F show an illustrative heterodimer 2-chain complex with split TM chains and SA chains, i.e., both TM on the knob chain of the Fc and SA on the hole chain of the Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be the same.
Fig. 6A-6J show that an illustrative heterodimer 2-chain complex, i.e., TM, has two signaling agents on the knob chain of the Fc (more signaling agents may be present in some embodiments as described herein). In these orientations and/or configurations, one SA is on the knob chain and one SA is on the hole chain. In various embodiments, the locations of SA1 and SA2 are interchangeable.
Fig. 7A-7D show an illustrative heterodimer 2 chain complex with split TM chains and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc.
Fig. 8A-8F show an illustrative heterodimer 2-chain complex with split TM chains and SA chains, i.e., SA on the knob chain of Fc, and both TM on the hole chain of Fc, with two targeting moieties (as described herein, more targeting moieties may be present in some embodiments). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be the same.
Fig. 9A-9J show an illustrative heterodimer 2-chain complex, i.e., TM on the hole chain of Fc, with two signaling agents (more signaling agents may be present in some embodiments as described herein). In these orientations and/or configurations, one SA is on the knob chain and one SA is on the hole chain. In various embodiments, the locations of SA1 and SA2 are interchangeable.
FIGS. 10A-10F show an illustrative heterodimer 2-chain complex in which TM and SA are on the same chain, i.e., SA and TM are both on the knob chain of Fc.
Fig. 11A-11L show an illustrative heterodimer 2-chain complex in which TM and SA are on the same chain, i.e., SA and TM are both on the knob chain of the Fc, with two targeting moieties (more targeting moieties may be present in some embodiments as described herein). In various embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be the same.
Fig. 12A-12L show an illustrative heterodimer 2-chain complex in which TM and SA are on the same chain, i.e., SA and TM are both on the knob chain of Fc, with two signaling agents (more signaling agents may be present in some embodiments as described herein). In various embodiments, the locations of SA1 and SA2 are interchangeable.
FIGS. 13A-13F show illustrative heterodimer 2-chain complexes in which TM and SA are on the same chain, i.e., SA and TM are both on the hole chain of Fc.
Fig. 14A-14L show an illustrative heterodimer 2-chain complex in which TM and SA are on the same chain, i.e., SA and TM are both on the hole chain of the Fc, with two targeting moieties (more targeting moieties are present in some embodiments as described herein). In various embodiments, the positions of TM1 and TM2 are interchangeable. In various embodiments, TM1 and TM2 may be the same.
Fig. 15A-15L show an illustrative heterodimer 2-chain complex in which TM and SA are on the same chain, i.e., SA and TM are both on the hole chain of Fc, with two signaling agents (more signaling agents may be present in some embodiments as described herein). In various embodiments, the locations of SA1 and SA2 are interchangeable.
Fig. 16A-16J show an illustrative heterodimer 2-chain complex having two targeting moieties (as described herein, more targeting moieties may be present in some embodiments), and wherein SA is on knob Fc and TM is on each chain. In various embodiments, TM1 and TM2 may be the same.
Fig. 17A-17J show an illustrative heterodimer 2-chain complex having two Targeting Moieties (TM) (as described herein, more targeting moieties may be present in some embodiments), and wherein SA is on the hole Fc and TM is on each chain. In various embodiments, TM1 and TM2 may be the same.
Fig. 18A-18F show an illustrative heterodimer 2-chain complex having two signaling agents (as described herein, more signaling agents may be present in some embodiments) and having split SA and TM chains: SA is on the knob and TM is on the hole Fc.
Fig. 19A-19F show an illustrative heterodimer 2-chain complex having two signaling agents (as described herein, more signaling agents may be present in some embodiments) and having split SA and TM chains: TM on the knob and SA on the hole Fc.
FIG. 20 depicts IL-2 and Fc-IL-2 driven STAT5 phosphorylation in PBMC lymphocyte populations.
Figures 21A-21G show the effect of CD25 knockout mutation and CD8 targeting on STAT5 phosphorylation in lymphocyte populations.
FIGS. 22A-22G depict sensorgrams of binding of IL-2 and Fc-IL-2 fusion proteins to biotinylated CD25 in a biological layer interferometry.
FIGS. 23A-23R depict a CD8 for + 、CD4 + CD25 - And CD4 + CD25 + Screening of D20 mutants by pSTAT5 in PMBC.
FIGS. 24A-24R depict a CD8 for + 、CD4 + CD25 - And CD4 + CD25 + Screening of N88 mutants by pSTAT5 in PMBC.
Figures 25A-25D show the effect of β mutation (here N88G) and CD8 targeting on STAT5 phosphorylation in a PBMC sub-population.
FIGS. 26A-26J show various combinations of CD25 and IL-2Rβ mutations and their effect on pSTAT5 in PMBC populations.
FIGS. 27A-27B depict binding of CD25 scFv Fc fusions to HEK-Blue IL-2 and HEK-Blue IL-1 cells in FACS.
FIG. 28 shows the effect of CD25 scFv Fc fusion on wild-type IL-2 signaling in HEK-Blue IL-2 cells.
Fig. 29A-29M depict the effect of CD25 targeting of ALN2 variants with CD25 or beta mutations on STAT5 phosphorylation in certain PBMC sub-populations.
FIGS. 30A-30H show STAT5 phosphorylation of IL-2 and Fc-IL-2 mimetic/neoleukocyte variants in a sub-population of PBMCs.
FIG. 31 depicts different bivalent (if VHH1 and VHH2 are the same) or bispecific (if VHH1 and VHH2 represent different targeting domains) knob hole ALN2 versions.
Fig. 32 depicts various ALN2 configurations.
Fig. 33A-33G depict STAT5 comparisons of STAT5 phosphorylation of divalent and bispecific CD8 VHH-targeted ALN2 compared to monovalent counterparts.
Fig. 34A-34E depict STAT5 phosphorylation of different types of ALN2 in cd8+ and Treg PBMC sub-populations.
Fig. 35A-35D show that the single peptide ALN2 induced pSTAT5 in cd8+ compared to Treg PBMC sub-populations.
FIGS. 36A-36M depict STAT5 phosphorylation of CD8 VHH-Fc-IL-2 and its T3O-glycosylated variants in CD8+ and CD8-PBMC lymphocyte populations.
Figures 37A-37D show mouse CD8 targeting α in cd8+ and Treg primary mouse spleen cells: STAT5 phosphorylation of beta ALN2 variants.
FIG. 38 depicts tumor growth (in mm) in animals treated with buffer, fc-ALN2 or CD8-Fc-ALN2 in MC38 and CT26 isogenic mouse colon cancer models 3 In units).
FIG. 39 shows the effect of CD8-Fc-ALN2 and anti-PD-1 co-therapy in MC38 and CT26 mouse models.
Figures 40A-40D show STAT5 phosphorylation of CD 8-targeted fcγ common Q126 mutants in CD 8-positive PBMCs.
FIGS. 41A-41S depict sensorgrams of binding of CD8-Fc-IL-2 and its Q126 mutant fusion proteins to biotinylated CD25 in BLI.
Fig. 42A-42D show α in cd8+ and Treg PBMC sub-populations of three different donors: pSTAT5 phosphorylation of gamma mutant.
Fig. 43A-43D depict β of a "resting" or transactant activated cd8+ or Treg PBMC sub-population: gamma ALN2 STAT5 phosphorylation.
FIG. 44 shows the effect of CD8-Fc-ALN2 (12.5. Mu.g), fc-ALN2 (10.7. Mu.g) or Fc-IL2 (10.7. Mu.g) on T cell activation 1 day after a single dose.
FIG. 45 depicts the effect of CD8-Fc-ALN2 (12.5 μg), fc-ALN2 (10.7 μg) or Fc-IL2 (10.7 μg) on T cell activation 7 days after two doses with or without co-treatment with anti-PD-1 Ab.
FIG. 46 shows the effect of CD8-Fc-ALN2 (25 μg), fc-ALN2 (21.4 μg) or Fc-IL2 (21.4 μg) on T cell activation 3 days after a single dose.
FIG. 47 depicts the effect of three injections of 12.5 μg CD8-Fc-ALN2 and anti-PD-1 Ab compared to wild-type Fc-IL2,1 in an A20 mouse model.
FIG. 48 shows the effect of three doses of 12.5 μg CD8-Fc-ALN2 compared to equimolar Fc-ALN2 treatment in a B16F10 mouse model.
FIG. 49 depicts the effect of two injections of 12.5 μg CD8-Fc-ALN2 compared to equimolar Fc-ALN2 treatment in the Panc02 mouse model.
FIG. 50 depicts STAT5 phosphorylation in spleen cells (single population) of mice comparing wild-type IL2, PD-L1 targeted and non-targeted ALN 2.
FIG. 51 shows the effect of three doses of 12.5 μg CD8-Fc-ALN2 and anti-PD-1 Ab compared to equimolar Fc-ALN2 treatment in MC38 mouse model.
FIG. 52 depicts the effect of three doses of 12.5 μg TNCA1-Fc-ALN2 or equivalent doses of non-targeted ALN2 in the MC38 mouse model without or with co-treatment with anti-PD-1 Ab.
Figure 53 shows that mouse CD8 or NKp46 or bispecific targeting α in CD8 and NK primary mouse splenocytes compared to non-targeted or wild type IL-2: STAT5 phosphorylation of beta ALN2 variants.
FIG. 54 depicts up-regulation of CD25 (left) and PD-1 (right) by ALN2 variants R38A_F42K_D20E (up) and R38A_F42 K_N8G (down) in three PBMC donors. Data are plotted as mean ± SEM.
FIG. 55 shows the effect of hCD8-Fc-ALN2 on human T-cell activation in Human Immune System (HIS) mice 45 minutes after 1 treatment (5. Mu.g).
Figure 56 depicts CD25 upregulation of mouse CD 8-targeted or non-targeted ALN2 variants with different loss-of-function β mutations in primary mouse spleen cells.
FIG. 57 shows the effect of treatment with 12.5 μg of N88G-based or D20E-based CD8-Fc-ALN2 or equimolar non-targeted ALN2 on days 6 and 9 in the MC38 mouse model.
Fig. 58 depicts STAT5 phosphorylation of ALN2 variants with different loss-of-function gamma mutations in primary mouse spleen cells.
FIG. 59 shows 12.5 or 4.17ug CD8-Fc-ALN 2. Alpha. For three doses in the MC38 mouse model: the effect of gamma mutation combinations compared to equimolar doses of non-targeted Fc-ALN.
FIG. 60 shows three doses of 4.17 or 1.4 μg CD8-Fc-ALN2 beta in the MC38 mouse model: the effect of gamma mutation combinations compared to equimolar doses of non-targeted Fc-ALN.
FIG. 61 depicts signaling of the D20 ALN2 variant in HEK-B1ue CD8 and HEK-Blue NKp46 cells.
FIG. 62 depicts signaling of the N88 ALN2 variant in HEK-Blue CD8 and HEK-Blue NKp46 cells.
Detailed Description
The inventive technique is based in part on the following findings: targeted chimeric proteins or protein complexes (including Fc-based chimeric protein complexes) comprising modified IL-2 exhibit beneficial therapeutic properties and reduced side effects. For example, chimeric proteins of the present technology have high activity and/or long-acting effects while causing minimal side effects. The present technology provides pharmaceutical compositions comprising chimeric proteins and the use of the pharmaceutical compositions in the treatment of various diseases.
In some embodiments, chimeric proteins or protein complexes with modified IL-2 may be advantageous for attenuating CD8 that predominantly expresses IL-2 receptors β and γ + Activation of T cells, thelp cells or NK cells (which may provide an anti-tumor effect) is not beneficial for T with IL-2 receptors α, β and γ reg (this may provide immunosuppression, oncologic effects). Furthermore, in some embodiments, preference for IL-2Rβ and/or IL-2Rγ over IL-2Rα avoidanceNumerous IL-2 side effects, such as pulmonary edema and/or Vascular Leak Syndrome (VLS), are observed. Moreover, IL-2-based chimeras are useful in the treatment of diseases (e.g., autoimmune diseases), such as when the modified IL-2 is antagonistic (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations at IL-2Rβ and/or IL-2Rγ, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference). For example, the constructs of the invention may be advantageous for attenuating CD8 with IL-2 receptors beta and gamma + Inhibition of T cells (and thus suppress immune responses) against T with IL-2 receptors α, β and γ reg . Alternatively, in some embodiments, chimeras carrying modified IL-2 favor T reg Is beneficial for the agonistic activation of (c) and thus for the immunosuppression, but not for CD8 + Activation of T cells. For example, these constructs may be used to treat diseases or diseases that would benefit from immunosuppression, such as autoimmune diseases.
In some embodiments, the chimeric protein has a binding to CD8 as described herein + A targeting moiety of a T cell, a modified IL-2 having reduced affinity and/or activity for IL-2rβ and/or IL-2rγ and/or substantially reduced or eliminated affinity and/or activity for IL-2rα. In some embodiments, these constructs provide targeted CD8 + T cell activity, and generally for T reg Cells are inactive (or have substantially reduced activity). In some embodiments, such constructs have enhanced immunostimulatory effects (e.g., without wishing to be bound by theory, by not stimulating Treg) compared to wild-type IL-2, while eliminating or reducing systemic side effects and/or toxicity associated with wild-type IL-2 and modified IL-2 protein constructs that preferentially target IL-2rβγ, but not in a cell-selective, targeted, and conditional manner, e.g., as described herein.
In some embodiments, the invention relates to a chimeric protein or an Fc-based chimeric protein complex comprising a polypeptide having a nucleotide sequence identical to SEQ ID NO: a polypeptide having an amino acid sequence of any one of 290-449, 478-495, or 501-531 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. In further embodiments, the chimeric protein or Fc-based chimeric protein complex comprises a polypeptide having the amino acid sequence constructed in one or more of examples 1-25.
Modified IL-2
In one aspect, the present technology provides a chimeric protein comprising modified IL-2. In various embodiments, the chimeric proteins of the present technology comprise modified IL-2 as a signaling agent. In various embodiments, the modified IL-2 signaling agent encompasses a functional derivative, analog, precursor, isoform, splice variant or fragment of the modified IL-2. In some embodiments, the modified IL-2 encompasses the amino acid sequence of SEQ ID NO:1, a mutation and/or modification of SEQ ID NO:1 is the amino acid sequence of wild-type IL-2. In some embodiments, the modified IL-2 has reduced binding affinity for its target receptor.
In some embodiments, the modified IL-2 signaling agent has reduced affinity and/or activity for IL-2Rα and/or IL-2Rβ and/or IL-2Rγ. In some embodiments, the modified IL-2 has reduced affinity and/or activity for IL-2Rβ and/or IL-2Rγ. In some embodiments, the modified IL-2 has substantially reduced or eliminated affinity and/or activity for IL-2Rα. Such embodiments may be of relevance in the treatment of cancer, for example, when the modified IL-2 has agonism at IL-2rβγ.
In various embodiments, the IL-2 has one or more mutations or modifications that distance it from an IL-2R having an alpha chain, and one or more attenuating mutations that reduce the activity or affinity of IL-2 at an IL-2R having only a beta gamma chain in a recoverable manner.
In various embodiments, the IL-2 has one or more mutations or modifications that distance it from IL-2R having only a βγ chain, and an attenuating mutation that reduces the activity or affinity of IL-2 at IL-2R having an α chain in a recoverable manner.
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce or eliminate the activity or affinity of the modified IL-2 for the IL-2rα chain and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2rβγ complex. In some embodiments, the reduced activity or affinity of the modified IL-2 for the IL-2rα chain can be restored by inducing proximity to a target (e.g., a cell) via the targeting moiety disclosed herein. In some embodiments, the mutation or modification that eliminates the activity or affinity of the modified IL-2 for the IL-2rα chain cannot be restored by inducing proximity to a target (e.g., a cell) via the targeting moiety. In some embodiments, the reduced activity or affinity of the modified IL-2 for the IL-2rβγ complex can be restored by inducing proximity to a target (e.g., a cell) via the targeting moiety disclosed herein. In some embodiments, the one or more mutations that reduce the activity or affinity of the modified IL-2 for the IL-2rα chain and IL-2rβγ complex, respectively, can be restored by inducing proximity to a target (e.g., a cell) via a targeting moiety disclosed herein.
In various embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that substantially eliminate the activity or affinity of modified IL-2 for IL-2R alpha chain and/or one or more mutations or modifications that reduce the activity or affinity of modified IL-2 for IL-2R lacking alpha chain, such reduced activity or affinity may be restored by inducing access to a target (e.g., a cell) via the targeting moiety, and such IL-2 is substantially inactive for IL-2R on a cell that does not express the target of the targeting moiety, or substantially inactive for IL-2R comprising alpha chain when contacted with a cell that expresses the target of the targeting moiety, and active for IL-2R lacking alpha chain and/or a cell that expresses the target of the targeting moiety, as compared to wild-type IL-2. In various embodiments, the activity or affinity from a mutation or modification is about 3-fold or about 5-fold, or about 10-fold, or about 30-fold, or about 100-fold, or about 300-fold, or about 500-fold, or about 1000-fold lower than the activity or affinity from a different mutation or modification.
In various embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rαβγ and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rα, such reduced activity or affinity being recoverable by induction of proximity to a target (e.g., a cell) via a targeting moiety.
In various embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rαβγ and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rβγ, such reduced activity or affinity may be restored by inducing proximity to a target (e.g., a cell) via a targeting moiety, and such IL-2 is substantially inactive for IL-2R on cells that do not express the target of the targeting moiety, or is substantially inactive for IL-2rαβγ when contacted with cells that express the target of the targeting moiety, and is active for IL-2rβγ and/or cells that express the target of the targeting moiety, as compared to wild-type IL-2.
In various embodiments, the IL-2 has a selective (e.g., biased and/or strongly biased) effect on IL-2Rβγ (rather than IL-2Rαβγ), but the activity on IL-2Rβγ is further attenuated by mutation or modification, thereby rendering the IL-2 safe for systemic use (e.g., IL-2 is active primarily on IL-2Rβγ at the site of therapeutic action, which is targeted by the targeting moiety).
In various embodiments, the IL-2 has a selective (e.g., biased and/or strongly favored) effect on NK cells and/or cd8+ cells, but is substantially non-selective for Treg cells, and the selective (e.g., biased and/or strongly favored) effect on NK cells and/or cd8+ cells is controlled by targeting, thereby reducing or eliminating systemic side effects. For example, in some embodiments, the chimeric proteins of the invention are directed not only to cells having an immunostimulatory effect, but also in a controlled manner that concentrates IL-2 action at a desired treatment site. As described herein, in various embodiments, such chimeric proteins can be used to safely stimulate the immune system to obtain an anticancer effect.
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R lacking an alpha chain and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for an IL-2R comprising an alpha chain, such mutations or modifications being attenuating mutations whose activity or affinity can be restored by inducing access to a target (e.g., a cell) via a targeting moiety.
In various embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of modified IL-2 for IL-2R lacking an alpha chain and/or one or more mutations or modifications that reduce the activity or affinity of modified IL-2 for IL-2R comprising an alpha chain, such reduced activity or affinity may be restored by inducing access to a target (e.g., a cell) via a targeting moiety, and such IL-2 is substantially inactive for IL-2R on a cell that does not express the target of the targeting moiety, or substantially inactive for IL-2R lacking an alpha chain when contacted with a cell that expresses the target of the targeting moiety, and active for IL-2R comprising an alpha chain and/or a cell that expresses the target of the targeting moiety, as compared to wild-type IL-2.
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rβγ and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rα, such mutations or modifications being attenuating mutations whose activity or affinity can be restored by inducing access to a target (e.g., a cell) via a targeting moiety.
In various embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rβγ and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rαβγ, such reduced activity or affinity being recoverable by induction of proximity to a target (e.g., a cell) via a targeting moiety.
In various embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rβγ and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2rβγ, such reduced activity or affinity may be restored by inducing proximity to a target (e.g., a cell) via a targeting moiety, and such IL-2 is substantially inactive for IL-2R on cells that do not express the target of the targeting moiety, or is substantially inactive for IL-2rβγ when contacted with cells that express the target of the targeting moiety, and is active for IL-2rββγ and/or cells that express the target of the targeting moiety, as compared to wild-type IL-2.
In various embodiments, the modified IL-2 has a selective (e.g., biased and/or strongly biased) effect on IL-2rαβγ (rather than IL-2rβγ), but the activity on IL-2rαβγ is further attenuated by mutation or modification, thereby rendering IL-2 safe for systemic use (e.g., IL-2 is active primarily on IL-2rβγ at the site of therapeutic action, targeted to that site by the targeting moiety).
In some embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2Rβγ chain and the normal activity or affinity for the IL-2Rα chain, wherein the reduced activity or affinity of the modified IL-2 for the IL-2Rβγ chain can be restored by inducing proximity to a target (e.g., a cell) via the targeting moiety.
In various embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for IL-2Rα and the normal activity or affinity for IL-2Rβγ chains, wherein the reduced activity or affinity of the modified IL-2 for IL-2Rα chains can be restored by inducing proximity to a target (e.g., a cell) via the targeting moiety.
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that eliminate the activity or affinity of modified IL-2 for IL-2rα chains and/or one or more mutations or modifications that reduce the activity or affinity of modified IL-2 for IL-2rβγ chains, wherein the reduced activity or affinity of modified IL-2 for IL-2rβγ chains can be restored by inducing proximity to a target (e.g., a cell) via a targeting moiety (e.g., a CD8, CD3, or PD-1 targeting moiety) and can be used to mediate a T cell-mediated immune response (e.g., a CD8T cell-mediated immune response).
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of modified IL-2 for IL-2rα chains and/or one or more mutations or modifications that reduce the activity or affinity of modified IL-2 for IL-2rβγ chains, wherein the reduced activity or affinity of modified IL-2 for IL-2rα and/or IL-2rβγ chains can be restored by inducing proximity to a target (e.g., a cell) via a targeting moiety (e.g., a CD8, CD3, or PD-1 targeting moiety) and can be used to mediate a T cell-mediated immune response (e.g., a CD8T cell-mediated immune response).
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2rα chain and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2rβγ chain, wherein the reduced activity or affinity of the modified IL-2 for the IL-2rα chain and/or IL-2rβγ chain can be restored by inducing proximity to a target (e.g., a cell) via a targeting moiety and can be used to mediate a Treg response.
In some embodiments, the IL-2 has one or more mutations or modifications: one or more mutations or modifications that eliminate the activity or affinity of the modified IL-2 for the IL-2rα chain and/or one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2rβγ chain, wherein the reduced activity or affinity of the modified IL-2 for the IL-2rβγ chain can be restored by inducing proximity to a target (e.g., a cell) via a targeting moiety and can be used to mediate a Treg response.
In some embodiments, the IL-2 has one or more mutations or modifications that reduce the activity or affinity of the modified IL-2 for the IL-2rβγ chain and the normal activity or affinity for the IL-2rα chain, wherein the reduced activity or affinity of the modified IL-2 for the IL-2rβγ chain can be restored by inducing proximity to a target (e.g., a cell) via the targeting moiety and can be used to mediate a Treg response.
In various embodiments, the IL-2 has a selective (e.g., biased and/or strongly biased) effect on Treg cells, but is substantially non-selective for NK cells and/or cd8+ cells, and the selective (e.g., biased and/or strongly biased) effect on Treg cells is controlled by targeting, thereby reducing or eliminating systemic side effects. For example, in some embodiments, the chimeric proteins of the invention are directed not only against cells having immunosuppressive effects, but also in a controlled manner that concentrates IL-2 action at the desired treatment site. As described herein, in various embodiments, such chimeric proteins can be used to safely stimulate the immune system to obtain an anti-autoimmune effect.
In some embodiments, the modified IL-2 signaling agent is a modified wild-type IL-2. Wild-type IL-2 has the following amino acid sequence:
In some embodiments, the modified IL-2 signaling agent comprises: (i) A mutation selected from the group consisting of D20E, D20F, D20G, D20H, D I, D20K, D L and D20V; or (ii) a mutation selected from the group consisting of N88G, N88E, N88K, N Q and N88V; or (iii) a mutation selected from the group consisting of Q126G, Q126A, Q E, Q F, Q126H, Q126I, Q126K, Q126L, Q N, Q126P, Q R, Q126S, Q T, Q V, Q W and Q126Y; or (iv) a mutation selected from (i) and (iii) or a mutation selected from (ii) and (iii); or (v) one or more mutations selected from the group consisting of: R38A/F42K/N88G/C125K/C125E/C125F/C125G/C125H/C125I/C125K/C125L/C125N/C125S/C125 20T/C125V/C125 88A/C125D/C125 88G/C125E/C125H/C125 88I/C125 88K/C125 88Q/C125R/C125T/C125 88V/C125E/R38A/F42K/C125V/R38A/F42K C125A/F42Y/E62A/C125A/F42Y/Y45A/E62Y/Y45A/L72A/F42Y/Y45A/E62A/C125Y/Y45A/L72G/C125A/F42K/N88A/F42K/Q126G/C125A/F42K/Q126I/C125A/F38A/F42K/Q126Y/C125A/F42K/E62A/N88G and R38A/F42K/E62A/N88G/C125A.
In some embodiments, the modified IL-2 signaling agent is a modified IL-2 signaling agent relative to SEQ ID NO:1 comprises one or more mutations at an amino acid residue selected from R38, F42, E62, C125, Y45, L72, D20, N88 and Q126. In such embodiments, the modified IL-2 signaling agent is relative to SEQ ID NO:1 comprises one or more mutations selected from R38 42 42 62 125 45 20 20 20 20 20 20 20 20 20 20 20 20 88 88 88 88 38A/F42Y/E62A/C125Y/Y45A/L72G/C125 42K/C125 38A/F42K/C125E/C125 20G/C125S/C125 20T/C125 20V/C125A/C125 88D/C125 88G/C125 88H/C125 88Q/C125 88T/C125 20E/R38A/F42K/C125 20V/R38A/F42K/C125 38A/F42K/N88G/C126 126 126W and Q126Y 126 126 126 126.
In various embodiments, the modified IL-2 signaling agent is relative to SEQ ID NO:1 comprises one or more mutations selected from the group consisting of D20 20 88K/C125E/C125 20G/C125 20S/C125T/C125 20V/C125A/C125 88D/C125 88G/C125 88H/C125 88Q/C125 88T/C125E/R38A/F42K/C125V/R38A/F42K/E62A/C125 38A/F42K/N88G/C125 38A/F42K/E62A/N88A/F42K/E126A/N126G 126 126 126W and Q126Y 126.
In certain embodiments, the modified IL-2 signaling agent is a modified IL-2 signaling agent relative to SEQ ID NO:1 comprises one or more mutations selected from the group consisting of R38A/F42Y/E62A/C125A, F Y/Y45A/L72G/C125A, F K/C125A, D E/C125A, D G/C125A, D S/C125A, D20T/C125A, D V/C125A, N A/C125A, N88D/C125A, N G/C125A, N88H/C125A, N88Q/C125A, N88T/C125A, D20E/R38A/F42K/C125A, D V/R38A/F42K/C125A and R38A/F42K/N88G/C125A.
In some embodiments, the modified IL-2 signaling agent comprises one or more of the combination of mutations selected from those listed in table 8.
Without wishing to be bound by theory, it is believed that the modified IL-2 agent has a reduced affinity for high affinity IL-2 receptors (i.e., IL-2rαβγ) and retains affinity for medium affinity IL-2 receptors (i.e., IL-2rβγ receptors) compared to wild-type IL-2.
Without wishing to be bound by theory, it is further believed that these modified IL-2 agents have a reduced affinity for IL-2 receptors (e.g., IL-2rβγ) compared to wild-type IL-2.
In some embodiments, the modified IL-2 signaling agent having mutations at R38, F42, Y45, and/or E62 is capable of inducing expansion of effector cells, including cd8+ T cells and NK cells, but not Treg cells and/or endothelial cells. In some embodiments, the modified IL-2 signaling agent having a mutation at R38, F42, Y45, and/or E62 is less toxic than the wild-type IL-2 agent. Chimeric proteins comprising such modified IL-2 agents having substantially reduced affinity and/or activity for IL-2 ra may be used, for example, in oncology.
In some embodiments, the modified IL-2 agent has a mutation at amino acid residue D20. For example, the modified IL-2 agent may comprise D20E, D20G, D20S, D20T or D20V. In some embodiments, the modified IL-2 agent having a mutation at D20 has reduced toxicity compared to the wild-type IL-2 agent. In some embodiments, the D20 mutation reduces toxicity by inhibiting endothelial cell binding and activation.
In some embodiments, the modified IL-2 agent has a mutation at amino acid C125. For example, the modified IL-2 agent may comprise mutation C125A. In some embodiments, modified IL-2 agents having a mutation at C125 optimize the preparation of such modified IL-2 agents. In some embodiments, the mutation at C125 promotes stability of such modified IL-2 agents by removing unpaired cysteine residues. In such embodiments, the modified IL-2 agent may have substantially reduced affinity and/or activity for IL-2rα, as described, for example, in carminate et al (2013) The Journal of Immunology,190:6230-6238, the entire disclosure of which is hereby incorporated by reference.
In other embodiments, the modified IL-2 agent may have substantially reduced affinity and/or activity for IL-2Rβ, as described, for example, in WO 2016/025385, the entire disclosure of which is hereby incorporated by reference. In such embodiments, the modified IL-2 agent may induce expansion of Treg cells rather than effector cells (such as cd8+ T cells and NK cells). Chimeric proteins comprising such modified IL-2 agents having substantially reduced affinity and/or activity for IL-2rβ may be used, for example, in the treatment of autoimmune diseases. In some embodiments, the modified IL-2 agent may comprise a mutation at amino acid residue N88. For example, the modified IL-2 agent may comprise N88A, N88D, N88G, N88H, N Q or N88T.
In some embodiments, the modified IL-2 signaling agent comprises SEQ ID NO:1, deletion of Ala at the N-terminus. In some embodiments, the modified IL-2 agent comprises SEQ ID NO:1 with serine or alanine substituted for cysteine at position 125. In some embodiments, the modified IL-2 agent comprises SEQ ID NO:1 and a serine or alanine substitution at position 125.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rα and IL2rβ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of D20E/R38A/F42K, D E/R38A/F42K/C125A, D V/R38A/F42K, D V/R38A/F42K/C125A, D V/R38A/F42K/E62A, D20V/R38A/F42K/E62A/C125A, R a/F42K/N88G, R a/F42K/N88G/C125A, R a/F42K/E62A/N88G and R38A/F42K/E62A/N88G/C125A.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rα and IL-2rγ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of R38A/F42K/Q126G, R a/F42K/Q126I and R38A/F42K/Q126Y.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rβ, optionally wherein the modified IL-2 comprises one or more mutations selected from D20E, D20E, D20E, D20E, D20E, D20E, D20E, D20/20E, D20E, D20E, D88E/C125E, D20F/C125E, D G/C125E, D H/C125E, D I/C125E, D20L/C125E, D20K/C125E, D N/C125E, D S/C125E, D T/C125E, D V/C125E, D a/C125E, D88D/C125E, D88G/C125E, D E/C125E, D H/C125E, D I/C125E, D K88Q/C125E, D R/C125T/C125A and N/C88A.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rγ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of Q126A, Q126E, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126N, Q126P, Q126R, Q S, Q126T, Q126V, Q W and Q126Y.
In some embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2Rβγ, optionally wherein the modified IL-2 comprises a polypeptide selected from the group consisting of D20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126V/Q20V/Q126 20V/Q126 20V/Q E/Q126V/Q20V/Q126V/Q20V/Q126 20V/Q126 20V/Q126 20V/Q126V/Q20V/Q126V/Q20V/Q126 20V/Q126 20. In further embodiments, the modified IL-2 exhibits reduced affinity and/or activity for IL-2rβγ and increased selectivity for activated CD8 cells.
Novel leukopenia
In some embodiments, the modified IL-2 signaling agent is a modified neoleukocyte, comprising the amino acid sequence:
in such embodiments, the nucleic acid sequence comprising SEQ ID NO:2, the modified IL-2 signaling agent has one or more mutations at amino acid residue D15 and/or N40. For example, in some embodiments, the polypeptide comprising SEQ ID NO:2 comprises D15T, D15H, N40I, N G or N40R.
In some embodiments, the modified IL-2 signaling agent comprises an amino acid sequence that hybridizes to SEQ ID NO:1 or 2 has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94% >. Or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).
In various embodiments, the modified IL-2 is modified to have one or more mutations. In some embodiments, the modified IL-2 is modified to have one or more mutations and one or more modifications (e.g., glycosylation). IL-2 contains a threonine O-glycosylation site at position 3 (T3). For example, in some embodiments, the modified IL-2 signaling agent comprises a T3O-glycosylation mutation, wherein the mutation is one of T3A, T3F, T3H, T3L, T V and T3Y. In some embodiments, the modified IL-2 signaling agent comprises a T3O-glycosylation deletion mutation, wherein the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2 sequence are deleted.
In some embodiments, the one or more amino acid mutations may be independently selected from the group consisting of substitutions, insertions, deletions, and truncations.
In some embodiments, the amino acid mutation is an amino acid substitution, and may include conservative and/or non-conservative substitutions.
"conservative substitutions" may be made, for example, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobicity: met, ala, val, leu, ile; (2) neutral hydrophilicity: cys, ser, thr, asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.
As used herein, "conservative substitutions" are defined as the exchange of one amino acid for another listed within the same set of six standard amino acid sets shown above. For example, asp is exchanged for Glu such that one negative charge is retained in the polypeptide so modified. In addition, glycine and proline may be substituted for each other based on their ability to disrupt the alpha helix.
As used herein, "non-conservative substitution" is defined as the exchange of one amino acid listed in a different one of the six standard amino acid groups (1) to (6) shown above for another amino acid.
In various embodiments, the substitutions may also include non-classical amino acids (such as selenocysteine, pyrrolysine, N-formylmethionine beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminocaproic acid, aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocysteine, sulfoalanine, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoroaminoacids, designer amino acids such as beta-methylaminoacids, Cα -methylaminoacids, Nα -methylaminoacids and amino acid analogs.
In some embodiments, the mutation allows the modified IL-2 to have one or more reduced activities, such as one or more of reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, relative to an unmutated IL-2, e.g., a wild-type form of IL-2 (e.g., SEQ ID NO: 1). For example, the one or more reduced activities, such as reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, can be at a receptor, such as IL-2rαβγ, relative to an unmutated IL-2, e.g., a wild-type form of IL-2 (e.g., SEQ ID NO: 1). As a result, in various embodiments, the mutations allow for reduced systemic toxicity, reduced side effects, and reduced off-target effects of the modified IL-2 relative to non-mutated IL-2 (e.g., wild-type forms of IL-2).
In some embodiments, the modified IL-2 is modified to have one or more mutations that reduce its binding affinity or activity at the therapeutic or target receptor. In some embodiments, the modified IL-2 provides an activity that is agonism at the therapeutic or target receptor (e.g., activating a cellular effect at the therapeutic site) or antagonism at the therapeutic or target receptor (e.g., reducing or eliminating a cellular effect at the therapeutic site). For example, the modified IL-2 may activate a therapeutic receptor or a target receptor. In such embodiments, the mutation results in reduced activation activity of the modified IL-2 at the therapeutic or target receptor.
In some embodiments, attachment to the targeting moiety restores reduced affinity or activity at the therapeutic or target receptor. In other embodiments, attachment to the targeting moiety does not substantially restore reduced affinity or activity at the therapeutic or target receptor. In various embodiments, the chimeric proteins of the present technology reduce off-target effects because the modified IL-2 has mutations that impair binding affinity or activity at the therapeutic or target receptor. In various embodiments, such a reduction in side effects is observed relative to, for example, wild-type IL-2. In various embodiments, the modified IL-2 is substantially inactive for the pathway of the therapeutically active site and its effect is substantially directed against the specifically targeted cell type, thereby greatly reducing unwanted side effects.
In various embodiments, the modified IL-2 has one or more mutations that result in the modified IL-2 having reduced or decreased affinity for one or more therapeutic receptors, e.g., binding (e.g., K D ) And/or activation (which may be measured as, for example, K A And/or EC(s) 50 ). In various embodiments, reduced affinity at the therapeutic receptor allows for attenuation of activity and/or signaling from the therapeutic receptor.
In various embodiments, the modified IL-2 has an affinity of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% -20%, about 20% -40%, about 50%, about 40% -60%, about 60% -80%, about 80% -100% relative to wild-type IL-2 for a therapeutic or target receptor (e.g., IL-2rαβγ or IL-2rβγ or any of their subunits IL-2rα, IL-2rβ, and/or IL-2rγ). In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower relative to the wild-type IL-2.
Receptor binding activity can be measured using methods known in the art. For example, the binding data may be analyzed by means of a Scatchard plot and computer fit (e.g., scatchard, the attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672, 1949) or by means of, for example, brecht et al Biosens Bioelectron 1993;8:387-392, the entire contents of which are hereby incorporated by reference, are hereby incorporated by reference in their entirety for evaluation of affinity and/or binding activity by performing reflectance interferometry under flow-through conditions. In some embodiments, receptor binding activity is measured by Biological Layer Interferometry (BLI).
In various embodiments, the reduced activity at the therapeutic or target receptor, reduced affinity at the therapeutic or target receptor, can be restored by attachment to a targeting moiety (e.g., an antibody or antibody form described herein) having high affinity for the antigen at the therapeutically active site. Targeting is achieved by linking the modified IL-2 to a targeting moiety. In one embodiment, the modified IL-2 is linked via its amino terminus to a targeting moiety. In another embodiment, the modified IL-2 has its carboxy terminus attached to a targeting moiety. In this way, in some embodiments, the chimeric proteins of the invention provide localized, mid-target, and controlled therapeutic effects at the therapeutic or target receptor.
In various embodiments, the activity of the modified IL-2 at the therapeutic or target receptor is enhanced by attachment to a targeting moiety (e.g., an antibody or antibody form described herein) having a high affinity for an antigen at the therapeutically active site. Targeting is achieved by linking the modified IL-2 to a targeting moiety. In one embodiment, the modified IL-2 is linked via its amino terminus to a targeting moiety. In another embodiment, the modified IL-2 has its carboxy terminus attached to a targeting moiety. In this way, in some embodiments, the chimeric proteins of the invention provide localized, mid-target, and controlled therapeutic effects at the therapeutic or target receptor.
Therapeutic agents comprising interleukins or variants thereof
In various embodiments, the present invention provides chimeric proteins or protein complexes comprising one or more targeting moieties that bind to one or more of the following targets: CD8, CTLA4, CD3, CD4, DNAM-1, nrp1 (neuropilin), TNFR1, TNFR2, GITR, ICOS, CD20, CD70, clec9a, NKp46, PD-1, PD-L2, SIRP1 alpha, FAP, XCR1, tenascin and ECM proteins.
Targeting partial cell recruitment
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, additionally comprise one or more targeting moieties having a recognition domain that specifically binds to a target of interest (e.g., antigen, receptor). In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may comprise two, three, four, five, six, seven, eight, nine, ten, or more targeting moieties. In illustrative embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, comprises two or more targeting moieties. In such embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can target two different cells (e.g., to form a synapse) or targeting portions of the same cell (e.g., to obtain a more concentrated signaling agent effect).
In some embodiments, the target of interest (e.g., antigen, receptor) is directed against immune cells and/or organ cells and/or tissue cells.
In various embodiments, the target of interest (e.g., antigen, receptor) can be found on one or more immune cells, which can include, but are not limited to, T cells, cytotoxic T lymphocytes, T helper cells, natural Killer (NK) cells, natural Killer T (NKT) cells, anti-tumor or tumor-associated macrophages (e.g., M1 or M2 macrophages), B cells, breg cells, dendritic cells, or a subset thereof. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., antigen, receptor) and is effective to recruit one or more immune cells, either directly or indirectly. In some embodiments, the target of interest (e.g., antigen, receptor) is found on one or more tumor cells. In some embodiments, the target of interest (e.g., antigen, receptor) is found on or in tumor neovasculature. In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can recruit immune cells directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells modulated for therapeutic effect). In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can recruit immune cells, e.g., immune cells that can kill and/or suppress tumor cells, directly or indirectly to a site of action (such as a tumor microenvironment, as a non-limiting example).
In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, presents a targeting moiety having a recognition domain that specifically binds to a target (e.g., antigen, receptor) that is part of a non-cellular structure. In some embodiments, the antigen or receptor is not an integral component of an intact cell or cell structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-protein, non-cellular marker, including but not limited to a nucleic acid, including DNA or RNA, such as, for example, DNA released from necrotic tumor cells or extracellular deposits such as cholesterol.
In some embodiments, the target of interest (e.g., antigen, receptor) is part of or a marker associated with a non-cellular component of the matrix or extracellular matrix (ECM). As used herein, a matrix refers to a connective and supportive framework of a tissue or organ. The matrix may include cells such as fibroblasts/myofibroblasts, glia, epithelium, fat, immunity, blood vessels, smooth muscle and immune cells, extracellular matrix (ECM) and a pool of extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is part of a matrix such as extracellular matrix and non-cellular components of extracellular molecules. As used herein, ECM refers to non-cellular components present within all tissues and organs. ECM is made up of a large collection of biochemically distinct components including, but not limited to, proteins, glycoproteins, proteoglycans, and polysaccharides. These components of the ECM are typically produced by adjacent cells and secreted into the ECM via exocytosis. Once secreted, ECM components tend to aggregate to form complex macromolecular networks. In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric proteins, comprise a targeting moiety that recognizes a target (e.g., an antigen or receptor or a non-protein molecule) located on any component of the ECM. Illustrative components of ECM include, but are not limited to, proteoglycans, non-proteoglycans, fibers, and other ECM proteins or ECM non-proteins, such as polysaccharides and/or lipids, or ECM related molecules (e.g., proteins or non-proteins, such as polysaccharides, nucleic acids, and/or lipids).
In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on ECM proteoglycans. Proteoglycans are glycosylated proteins. The basic proteoglycan unit includes a core protein with one or more covalently linked glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge, thereby attracting positively charged sodium ions (na+), which attract water molecules via osmosis, thereby keeping ECM and resident cells hydrated. Proteoglycans may also help capture and store growth factors within the ECM. Illustrative proteoglycans to which the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be targeted include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In one embodiment, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide such as hyaluronic acid.
In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on ECM fibers. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in the ECM. Collagen exists in the ECM in the form of fibrous proteins and provides structural support to reside cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagen present in the ECM, including, but not limited to, fibrillar collagen (type I, type II, type III, type V, type XI), inter-triple helix fibril-associated collagen (fast collogen) (type IX, type XII, type XIV), short chain collagen (type VIII, type X), basement membrane collagen (type IV), and/or type VI, type VII, or type XIII collagen. Elastin fibers provide elasticity to tissue, allowing them to stretch when needed and then return to their original state. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or elastin fiber.
In some embodiments, the targeting moiety recognizes one or more ECM proteins, including but not limited to tenascin, fibronectin, fibrin, laminin (laminin) or entactin (nidogen)/endonectin (entactin).
In one embodiment, the targeting moiety recognizes and binds to tenascin. Glycoproteins of the Tenascin (TN) family include at least four members, namely tenascin C, tenascin R, tenascin X, and tenascin W. The primary structure of tenascin includes several common motifs, which are in sequence in the same contiguous sequence: an amino-terminal heptavalent repeat, an Epidermal Growth Factor (EGF) -like repeat, a fibronectin type III domain repeat, and a carboxy-terminal fibrinogen-like globular domain. Each protein member is associated with typical variations in the number and nature of EGF-like repeats and fibronectin type III repeats. Isoform variants also exist specifically with respect to tenascin C. More than 27 splice variants and/or isoforms of tenascin C are known. In certain embodiments, the targeting moiety recognizes and binds to tenascin CA1. Similarly, tenascin R also has different splice variants and isoforms. Tenascin R is usually present in the form of dimers or trimers. Tenascin X is the largest member of the tenascin family and is known to exist in trimeric form. Tenascin W exists in the form of a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on tenascin. In some embodiments, the targeting moiety recognizes monomeric and/or dimeric and/or trimeric and/or hexameric forms of tenascin.
In one embodiment, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that links cells with collagen fibers in the ECM, allowing cells to migrate through the ECM. Upon binding to integrins, fibronectin unfolds to form functional dimers. In some embodiments, the targeting moiety recognizes monomeric and/or dimeric forms of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In an illustrative embodiment, the targeting moiety recognizes fibronectin Extracellular Domain A (EDA) or fibronectin Extracellular Domain B (EDB). Elevated EDA levels are associated with a variety of diseases and conditions including psoriasis, rheumatoid arthritis, diabetes and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing EDA isoforms and may be used to target the chimeric protein or chimeric protein complex, such as Fc-based chimeric protein complexes, to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing the EDB isoform. In various embodiments, such targeting moieties can be used to target the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, to tumor cells, including tumor neovasculature.
In one embodiment, the targeting moiety recognizes and binds to fibrin. Fibrin is another proteinaceous substance often found in the matrix network of the ECM. Fibrin is formed by the action of the protease thrombin on fibrinogen, which causes fibrin to polymerize. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes monomeric as well as polymeric forms of fibrin.
In one embodiment, the targeting moiety recognizes and binds to laminin. Laminin is the major component of the basal layer, which is the basis of the protein network of cells and organs. Laminin is a heterotrimeric protein containing an alpha chain, a beta chain and a gamma chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric forms of laminin.
In one embodiment, the targeting moiety recognizes and binds to nestin or integrin. Nestin/integrin is a highly conserved family of sulfated glycoproteins. They constitute the main structural components of the basement membrane and function to connect the laminin and collagen IV network in the basement membrane. Members of this family include nestin-1 and nestin-2. In various embodiments, the targeting moiety recognizes an epitope on nestin-1 and/or nestin-2.
In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any of the targets described herein. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on the protein. As used herein, a linear epitope refers to any contiguous amino acid sequence present on the protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on the protein. As used herein, conformational epitope refers to one or more amino acid segments (possibly discontinuous) that form a three-dimensional surface having features and/or shape and/or tertiary structure that are capable of being recognized by an antigen recognition domain.
In various embodiments, the targeting moiety may bind to the full length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or any other naturally occurring or synthetic analogs, variants or mutants of any of the targets described herein. In various embodiments, the targeting moiety can bind to any form of the proteins described herein, including monomeric, dimeric, trimeric, tetrameric, heterodimeric, multimeric, and associative forms. In various embodiments, the targeting moiety can bind to any translationally modified form of the proteins described herein, such as glycosylated and/or phosphorylated forms.
In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an extracellular molecule, such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In one embodiment, the DNA is shed from necrotic or apoptotic tumor cells or other diseased cells into the extracellular space.
In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known. Fibrolipid (fibrofat) plaques are characterized by accumulation of lipid-laden cells under the intima of the artery. Below the endothelium is a fibrous cap covering the atherogenic core of the plaque. The core includes lipid-laden cells (macrophages and smooth muscle cells), fibrin, proteoglycans, collagen, elastin, and cellular debris with elevated levels of tissue cholesterol and cholesterol esters. In advanced plaques, the central core of the plaque typically contains extracellular cholesterol deposits (released from dead cells) that form regions of cholesterol crystals with hollow needle-like slits. At the periphery of the plaque are younger foam cells and capillaries. Fibrous plaque is also localized under the intima, within the arterial wall, causing the wall to thicken and distend, and sometimes causing multiple plaque localized narrowing of the lumen and a degree of muscular layer atrophy. Fibrous plaques contain collagen fibers (eosinophilic), precipitating calcium (hematoxylin) and lipid-laden cells. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, cellular debris, and calcium or other inorganic deposits or precipitates. In some embodiments, the cellular debris is nucleic acid, such as DNA or RNA, released from dead cells.
In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures located in amyloid plaques found in the brain of an alzheimer's disease patient. For example, the targeting moiety may recognize and bind to peptide amyloid β, which is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures located in brain plaques found in huntington's disease patients. In various embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in plaques associated with other neurodegenerative or musculoskeletal diseases such as dementia with lewy bodies and inclusion body myositis.
In some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, may have two or more targeting moieties that bind to non-cellular structures. In some embodiments, there are two targeting moieties, one targeting moiety targeting a cell and the other targeting moiety targeting a non-cellular structure. In various embodiments, the targeting moiety can recruit cells, such as disease cells and/or effector cells, directly or indirectly. In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can or can be used in methods involving altering the balance of immune cells that are favorable to immune attack of a tumor. For example, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, can alter the immune cell ratio at a clinically significant site, thereby facilitating killing and/or suppressing cells of a tumor (e.g., T cells, cytotoxic T lymphocytes, T helper cells, natural Killer (NK) cells, natural Killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or a subset thereof), and combating tumor-protecting cells (e.g., bone marrow-derived suppressor cells (MDSCs), regulatory T cells (tregs), tumor-associated neutrophils (TAN), M2 macrophages, tumor-associated macrophages (TAMs), or a subset thereof). In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, are capable of increasing the ratio of effector T cells to regulatory T cells.
For example, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a T cell. In some embodiments, the recognition domain recruits T cells directly or indirectly. In one embodiment, the recognition domain specifically binds to effector T cells. In some embodiments, the recognition domain recruits effector T cells directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells modulated for therapeutic effect). Illustrative effector T cells include cytotoxic T cells (e.g., αβ TCR, CD3 + 、CD8 + 、CD45RO + )；CD4 + Effector T cells (e.g., αβ TCR, CD3 + 、CD4 + 、CCR7 + CD62L high, IL - 7R/CD127 + )；CD8 + Effector T cells (e.g., αβ TCR, CD3 + 、CD8 + 、CCR7 + CD62L high, IL - 7R/CD127 + ) The method comprises the steps of carrying out a first treatment on the surface of the Effector memory T cells (e.g., CD62L low, CD44 + 、TCR、CD3 + 、IL - 7R/CD127 + 、IL-15R + CCR7 low); central memory T cells (e.g. CCR7 + 、CD62L + 、CD27 + The method comprises the steps of carrying out a first treatment on the surface of the Or CCR7 high, CD44 + CD62L high, TCR, CD3 + 、IL-7R/CD127 + 、IL-15R + )；CD62L + Effector T cells; CD8 + Effector memory T cells (TEM), including early effector memory T cells (CD 27 + CD62L - ) And late effector memory T cells (CD 27) - CD62L - ) (TemE and TemL, respectively); CD 127% + ) CD25 (low/-) effector T cells; CD 127% - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44 (Low) CD62L (high) CD122 (high) sca ] + ) A) is provided; TH1 effector T cells (e.g. CXCR3 + 、CXCR6 + And CCR5 + The method comprises the steps of carrying out a first treatment on the surface of the Or alpha beta TCR, CD3 + 、CD4 + 、IL-12R + 、IFNγR + 、CXCR3 + ) TH2 effector T cells (e.g. CCR3 + 、CCR4 + And CCR8 + The method comprises the steps of carrying out a first treatment on the surface of the Or alpha beta TCR, CD3 + 、CD4 + 、IL-4R + 、IL-33R + 、CCR4 + 、IL-17RB + 、CRTH2 + ) The method comprises the steps of carrying out a first treatment on the surface of the TH9 effector T cells (e.g., αβTCR, CD3 + 、CD4 + ) The method comprises the steps of carrying out a first treatment on the surface of the TH17 effector T cells (e.g., ααβTCR, CD3 + 、CD4 + 、IL-23R + 、CCR6 + 、IL-1R + )；CD4 + CD45RO + CCR7 + Effector T cells, ICOS + Effector T cells; CD4 + CD45RO + CCR7( - ) Effector T cells; and effector T cells that secrete IL-2, IL-4 and/or IFN-gamma.
Illustrative T cell antigens of interest include, for example (and where appropriate, extracellular domains): CD8, CD3, SLAMF4, IL-2Rα, 4-1BB/TNFRSF9, IL-2R β, ALCAM, B7-1, IL-4R, B7-H3, BLAME/SLAMFS, CEACAM1, IL-6R, CCR3, IL-7R α, CCR4, CXCR1/IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-10R β, CCRS, IL-12R β1, CCR9, IL-12Rβ2, CD2, IL-13R α1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5 ase:Sub>A, luteglin (lutegrin) α4/CD49d, CDS, integrin αE/CD103, CD6, integrin αM/CD 11B, CDS, integrin αX/CD11c, integrin β2/CD1S, KIR/CD15S, CD/TNFRSF 7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, CD40 ligand/TNFSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD/SLAMF 3, NKG2D, CD F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, common gammase:Sub>A chain/IL-2R gammase:Sub>A, osteomodulin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX CR1, CX3CL1, L-selectin, CXCR3, SIRP betase:Sub>A 1, SIRP 4, SLAM 6, TCCR/WSX-1, DNAM-1, thymine, TIM-6, fasF-6, TNFX-6, fasF-3, TNFX-6, fasB-6, TNF3., fcgammaRIII/CD 16, TIM-6, TNFR1/TNFRSF1A, granulysin, TNF RIII/TNFRSF1B, TRAIL R1/TNFRSF1OA, ICAM-1/CD54, TRAIL R2/TNFRSF 10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN- γR1, TRAIL R4/INFRSF10D, IFN- γR2, TSLP, IL-1 R1 and TSLP R. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these illustrative T cell antigens.
As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, comprises a targeting moiety to one or more of PD-1, PD-L1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR for expression on T cells.
For example, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a B cell. In some embodiments, the recognition domain recruits B cells directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells modulated for therapeutic effect). Illustrative B cell antigens of interest include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/B, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, CDw150, CS1, and B Cell Maturation Antigen (BCMA). In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these illustrative B cell antigens.
As another example, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a natural killer cell. In some embodiments, the recognition domain recruits natural killer cells directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells modulated for therapeutic effect). Illustrative target natural killer cell antigens include, for example, TIGIT, 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, fc- εRII, LMIR6/CD300LE, fc- γR1/CD64, MICA, fc- γRIIB/CD32B, MICB, fc- γRIIB/CD32 c, MULT-1, fc- γRIIA/CD32 ase:Sub>A, binder-2/CD 112, fc- γRIII/CD16, NKG2A, fcRH/IRTA 5, NKG2C, fcRH/IRTA 4 NKG2D, fcRH4/IRTA1, NKp30, fcRH5/IRTA2, NKp44, fc receptor-like protein 3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, rae-1. Betase:Sub>A., rae-1. Deltase:Sub>A., H60, rae-1. Epsilon., ILT2/CD85j, rae-1. Gammase:Sub>A., ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85 ase:Sub>A, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d, and ULBP-3. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these illustrative NK cell antigens.
Moreover, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with macrophages/monocytes. In some embodiments, the recognition domain recruits macrophages/monocytes directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells modulated for therapeutic effect). Illustrative target macrophage/monocyte antigens include, for example, SIRP1a, B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, common beta chain, integrin alpha 4/CD49d, BLAME/SLAMF8, integrin alpha X/CD11C, CCL6/C10, integrin beta 2/CD18, CD155/PVR, integrin beta 3/CD61, CD31/PECAM-1, latex, CD36/SR-B3, leukotriene B4R 1, CD40/TNFRSF5, LIMPIIISR-B2, CD43, LMIR1/CD300A, CD, LMIR2/CD300C, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARC, CC/AMF 7, ECF 83-52, ECF 2, CD 83-CD 2 EMMPRIN/CD147, MGL2, endothelin/CD 105, ossein/GPNMB, fc-gamma RI/CD64, ossein, fc-gamma RIIB/CD32B, PD-L2, fc-gamma RIIC/CD32C, siglec-3/CD33, fc-gamma RIIA/CD32a, SIGNR1/CD209, fc-gamma RIII/CD16, SLAM, GM-CSF Ralpha, TCCR/WSX-1, ICAM-2/CD102, TLR3, TLR IFN- γR1, TLR4, IFN- γR2, TREM-1, IL-1 RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1, 2B4/SLAMF 4, IL-10Rα, ALCAM, IL-10R β, aminopeptidase N/ANPEP, ILT2/CD85j, common β chain, ILT3/CD85k, clq R1/CD93, ILT4/CD85d, CCR1, and, ILT5/CD85a, CCR2, integrin α4/CD49d, CCR5, integrin αM/CDll B, CCR8, integrin αX/CDllc, CD155/PVR, integrin β2/CD18, CD14, integrin β3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, LMIR3/CD300LF, coagulation factor III/tissue factor, LMIR5/CD300LB, CX3CR1, CX3CL1 LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, endothelin/CD 105, NCAM-L1, fc- γRI/CD64, PSGL-1, fc- γRIIICD 16, RP105, G-CSF R, L-selectin, GM-CSF Rα, siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6R, TREM-2, CXCR1/IL-8RA, TREM-3 and TREML1/TLT-1. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these illustrative macrophage/monocyte antigens.
Moreover, in some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a dendritic cell. In some embodiments, the recognition domain recruits dendritic cells directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells modulated for therapeutic effect). Illustrative target dendritic cell antigens include, for example, CLEC9A, XCR1, RANK, CD36/SRB3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-A1/MSR, CD5L, SREC-1, CL-P1/COLEC12, SREC-II, LIMPIIISRB, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand/TNFSF 9, IL-12/IL-23 p40, 4-amino-1, 8-naphthalenedicarboxamide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85D, 8D6A, ILT5/CD85a, A2B5, lutegline alpha 4/CD49D, aag, integrin beta 2/CD18, AMICA, langerhan protein (Larin) B7/CD 86, leukotriene B7/CD 7, 3H 7, 3-B7, 3B 7 LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, clq R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD/SLAMF 5, MMR, CD97, NCAML1, CD2F-10/SLAMF9, CD40/TNFRSF5, MAG/Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD/SLAMF 5 bone activin GPNMB, chern 23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8, siglec-2/CD22, CRACC/SLAMF7, siglec-3/CD, DC-SIGN, siglec-5, DC-SIGNR/CD299, siglec-6, DCAR, siglec-7, DCIR/CLEC4A, siglec-9, DEC-205, siglec-10, dectin-1/CLEC7A, siglec-F, dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC/CLEC4C, SLAM, EMMPRIN/CD147, TCCR/WSX-1, fc- γR1/CD64, TLR3, fc- γRIIB/CD32b, TREM-1, fc- γRIIC/CD32c, TREM-2, fc- γRIIA/CD32a, TREM-3, fc- γRIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and capsaicin R1. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these illustrative DC antigens.
In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) on an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, bone marrow cells, monocytes, eosinophils, or a subset thereof. In some embodiments, the recognition domain recruits megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, bone marrow cells, monocytes, eosinophils, or a subset thereof, directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a location with one or more disease cells or cells modulated for therapeutic effect). In some embodiments, the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, mast cells, monocytes, erythrocytes, bone marrow cells, bone marrow derived suppressor cells, NKT cells, and NK cells, or derivatives thereof. In some embodiments, the immune cell is a T cell and the targeting moiety targets CD8. In some embodiments, the immune cell is a Treg and the targeting moiety targets CTLA4. In some embodiments, the immune cell is an NK cell and the targeting moiety is NKp46.
In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with megakaryocytes and/or platelets. Illustrative megakaryocyte and/or platelet antigens of interest include, for example, GP IIb/IIIa, GPIb, vWF, PF4 and TSP. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these illustrative megakaryocyte and/or platelet antigens.
In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a red blood cell. Illustrative target erythrocyte antigens include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb/IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2 (MHC-II), and rhesus antigens. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these illustrative erythrocyte antigens.
In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a mast cell. Illustrative target mast cell antigens include, for example, SCFR/CD117, fcεRI, CD2, CD25, CD35, CD88, CD203C, C5R1, CMA1, FCER1A, FCER2, TPSAB1. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these mast cell antigens.
In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with basophils. Illustrative target basophil antigens include, for example, fceri, CD203c, CD123, CD13, CD107a, CD107b, and CD164. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these basophil antigens.
In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with neutrophils. Illustrative target neutrophil antigens include, for example, 7D5, CD10/CALLA, CD13, CD16 (FcRIII), CD18 protein (LFA-1, CR3 and p150, 95), CD45, CD67 and CD177. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these neutrophil antigens.
In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with eosinophils. Illustrative eosinophil antigens of interest include, for example, CD35, CD44, and CD69. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these eosinophil antigens.
In various embodiments, the recognition domain can bind to any suitable target, antigen, receptor, or cell surface marker known to those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue specific marker. Illustrative tissue-specific markers include, but are not limited to, endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAM1, PROCR, SELE, SELP, TEK, THBD, VCAM1, VWF; smooth muscle cell surface markers such as ACTA2, MYH1O, MYH11, MYH9, MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COL1A1, COL1A2, COL3A1, FAP, PH-4; epithelial cell surface markersA memory such as CD1D, K IRS2, KRT1O, KRT, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUC1, TACSTD1; neovascular markers, such as CD13, TFNA, alpha-vbeta-3 (alpha) V β 3 ) E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN. In various embodiments, the chimeric protein or chimeric protein complex, such as the targeting portion of an Fc-based chimeric protein complex, binds one or more of these antigens. In various embodiments, the chimeric protein or targeting portion of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds to one or more of the cells having these antigens.
In some embodiments, the recognition domain specifically binds to a target (e.g., antigen, receptor) associated with a tumor cell. In some embodiments, the recognition domain recruits tumor cells directly or indirectly. For example, in some embodiments, tumor cells are recruited directly or indirectly to one or more effector cells (e.g., immune cells described herein) that may kill and/or suppress the tumor cells.
Tumor cells or cancer cells refer to uncontrolled growth of cells or tissues and/or abnormal increases in cell survival time and/or inhibition of apoptosis that interfere with normal function of body organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasia, dormant tumors, or micrometastases. Illustrative tumor cells include, but are not limited to, the following: basal cell carcinoma, biliary tract carcinoma; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal carcinoma; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of urinary system; vulvar cancer; lymphomas, including hodgkin's and non-hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated split cell NHL, giant tumor NHL (bulky disease NHL), mantle cell lymphomas, AIDS-related lymphomas, and Waldenstrom's macroglobulinemia (Waldenstrom's Macroglobulinemia), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), and abnormal vascular hyperplasia associated with zematous misstructure tumors, oedema (e.g., brain tumor-associated oedema) and meissymondme's syndrome.
Tumor cells or cancer cells also include, but are not limited to, carcinoma such as various subtypes including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myelogenous, acute lymphoblastic, chronic myelogenous, chronic lymphocytic, and hair cells), lymphomas and myelomas (including, for example, hodgkin's and non-hodgkin's lymphomas, light chain, non-secretory, MGUS, and plasmacytoma), and central nervous system cancers (including, for example, brain (such as gliomas (such as astrocytomas, oligoglimas, and ependymomas), meningiomas, pituitary adenomas, and neuromas and spinal cord tumors (such as meningiomas and fibrogliomas).
Illustrative tumor antigens include, but are not limited to MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin B, colorectal associated antigen (CRC) -0017-1A/GA733, carcinoembryonic antigen (CEA) and immunogenic epitopes CAP-1 and CAP-2, etv, aml1, prostate Specific Antigen (PSA) and immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate Specific Membrane Antigen (PSMA), T cell receptor/CD 3-zeta chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5) MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, gnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-linked protein, beta-and gamma-linked proteins, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), cytosol, connexin 37, ig idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, smad family tumor antigens, 1mp-1, NA, EBV encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD47, CS1, CD38, ASGPR, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, ep-CAM, PD-L1, PMSA-2, and BCMA (tnsa 17). In various embodiments, the chimeric protein or targeting moiety of a chimeric protein complex, such as an Fc-based chimeric protein complex, binds one or more of these tumor antigens. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to HER2. In another embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, binds to PD-L2.
In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have one or more targeting moieties to immune cells selected from T cells, B cells, dendritic cells, neutrophils, bone marrow-derived suppressor cells, macrophages, NK cells, or a subset thereof, along with any of the signaling agents described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to T cells (including but not limited to effector T cells), along with any signaling agent described herein. In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to B cells, along with any signaling agent described herein. In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to dendritic cells, along with any signaling agent described herein. In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to macrophages, along with any signaling agent described herein. In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to NK cells, along with any of the signaling agents described herein.
As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety to a T cell mediated, for example, by targeting: CD8, SLAMF4, IL-2R α, 4-1BB/TNFRSF9, IL-2R β, ALCAM, B7-1, IL-4R, B7-H3, BLAME/SLAMFS, CEACAM1, IL-6R, CCR3, IL-7R α, CCR4, CXCR1/IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-10R β, CCRS, IL-12R β1, CCR9, IL-12R β2, CD2, IL-13R α1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5 ase:Sub>A, lutegrin (luterin) α4/CDS 49d, CDS, integrin αE/CD103, CD6, integrin αM/CD 11B, CDS, integrin αC 11/CD 35, CD 35/CD 35, SF3, CD3, CD4, ILT3/CDS 5j, ILT3/CDS 3, and CD4 KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, CD40 ligand/TNFSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2 3297, NKG2C, CD229/SLAMF3, NKG2D, CD F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, common gammase:Sub>A chain/IL-2R gammase:Sub>A, endostatin, huperzine CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX CR1, CX3CL1, L-selectin, CXCR3, SIRP betase:Sub>A 1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, thyminogen, EMMPRIN/CD147, TIM-1, ephB6, TIM-2, fas/TNFRSF6, TIM-3, fas ligand/TNFSF 6, TIM-4, fcgammaRIII/CD 16, fcgammaPrIN/CD 147, TIM-6, TNFR1/TNFRSF1A, granulysin, TNF RIII/TNFRSF1B, TRAIL R1/TNFRSF1OA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN- γR1, TRAIL R4/TNFRSF10D, IFN- γR2, TSLP, IL-1 R1 or TSLP R; along with any signaling agent described herein (e.g., modified IL-2).
As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a checkpoint marker expressed on T cells, e.g., one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR, along with any signaling agent described herein.
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have one or more targeting moieties to PD-1. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has one or more targeting moieties that selectively bind to a PD-1 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-1 polypeptide.
In one embodiment, the targeting moiety comprises the anti-PD-1 antibody, certolizumab (aka MK-3475, keyruda) or a fragment thereof. The pentraxin and other humanized anti-PD-1 antibodies are disclosed in Hamid et al (2013) New England Journal of Medicine 369 (2): 134-44, US 8,354,509 and WO 2009/114335, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the plagiozumab or antigen-binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:7 and/or a heavy chain comprising the amino acid sequence SEQ ID NO: 8.
In one embodiment, the targeting moiety comprises the anti-PD-1 antibody nivolumab (aka BMS-936558, MDX-1106, ONO-4538, OPDIVO) or fragments thereof. Nivolumab (clone 5C 4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US 8,008,449 and WO 2006/121168, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the nivolumab or antigen binding fragment thereof comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:9 and/or a heavy chain comprising the amino acid sequence SEQ ID NO: 10.
In one embodiment, the targeting moiety comprises the anti-PD-1 antibody Pidilizumab (also known as CT-011, hBAT or hBAT-1) or a fragment thereof. Pi Deli bead monoclonal antibodies and other humanized anti-PD-I monoclonal antibodies are disclosed in US 2008/0025980 and WO 2009/101611, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, an anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs of US 2008/0025980: 15-18: SEQ ID NO of US 2008/0025980: 15 (SEQ ID NO: 11); SEQ ID NO of US 2008/0025980: 16 (SEQ ID NO: 12); SEQ ID NO of US 2008/0025980: 17 (SEQ ID NO: 13); and SEQ ID No. of US 2008/0025980: 18 (SEQ ID NO: 14); and/or a heavy chain comprising a sequence selected from the group consisting of SEQ ID NOs of US 2008/0025980: 20-24: SEQ ID NO of US 2008/0025980: 20 (SEQ ID NO: 15); SEQ ID NO of US 2008/0025980: 21 (SEQ ID NO: 16); SEQ ID NO of US 2008/0025980: 22 (SEQ ID NO: 17); SEQ ID NO of US 2008/0025980: 23 (SEQ ID NO: 18); and SEQ ID No. of US 2008/0025980: 24 (SEQ ID NO: 19).
In one embodiment, the targeting moiety comprises the sequence of SEQ ID NO:18 A light chain of (SEQ ID NO: 14) and a light chain comprising SEQ ID NO of US 2008/0025980: 22 (SEQ ID NO: 17).
In one embodiment, the targeting moiety comprises AMP-514 (also known as MEDI-0680).
In one embodiment, the targeting moiety comprises the pd-12-Fc fusion protein AMP-224 disclosed in WO2010/027827 and WO 2011/066342, the entire disclosures of which are hereby incorporated by reference. In such embodiments, the targeting moiety may comprise a sequence comprising SEQ ID NO:4 (SEQ ID NO: 20) and/or a targeting domain comprising SEQ ID NO:83 (SEQ ID NO: 21) B7-DC fusion protein.
In one embodiment, the targeting moiety comprises a PD-L1-Fc fusion protein and/or a PD-1-Fc fusion protein, as described in WO 2019/191519.
In one embodiment, the targeting moiety comprises the peptide AUNP 12 or any other peptide disclosed in US 2011/0318373 or 8,907,053. For example, the targeting moiety may comprise AUNP 12 (i.e., compound 8 of US 2011/0318373 or SEQ ID NO: 49) having the sequence:
(SEQ ID NO：22)。
In one embodiment, the targeting moiety comprises an anti-PD-1 antibody 1E3, or fragment thereof, as disclosed in US 2014/0044738, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, 1E3 or antigen binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:23 and/or comprises the amino acid sequence SEQ ID NO: 24.
In one embodiment, the targeting moiety comprises an anti-PD-1 antibody 1E8, or fragment thereof, as disclosed in US 2014/0044738, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, 1E8 or antigen binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:25 and/or a heavy chain comprising the amino acid sequence SEQ ID NO: 26.
In one embodiment, the targeting moiety comprises an anti-PD-1 antibody 1H3, or fragment thereof, as disclosed in US 2014/0044738, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, 1H3 or antigen binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:27 and/or comprises the amino acid sequence SEQ ID NO: 28.
In one embodiment, the targeting moiety comprises a VHH for PD-1 as disclosed in, for example, US 8,907,065 and WO 2008/071447, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the VHH directed against PD-1 comprises the amino acid sequence of SEQ ID NO of US 8,907,065: 347-351 (SEQ ID NO:347 of US 8,907,065 (SEQ ID NO: 29), SEQ ID NO:348 of US 8,907,065 (SEQ ID NO: 30), SEQ ID NO:349 of US 8,907,065 (SEQ ID NO: 31), SEQ ID NO:350 of US 8,907,065 (SEQ ID NO: 32), and SEQ ID NO:351 of US 8,907,065 (SEQ ID NO: 351)).
In one embodiment, the targeting moiety comprises any of the anti-PD-1 antibodies or fragments thereof as disclosed in US2011/0271358 and WO2010/036959, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID NOs: 25-29 (SEQ ID NO:25 of US2011/0271358 (SEQ ID NO: 34), SEQ ID NO:26 of US2011/0271358 (SEQ ID NO: 35), SEQ ID NO:27 of US2011/0271358 (SEQ ID NO: 36), SEQ ID NO:28 of US2011/0271358 (SEQ ID NO: 37), and SEQ ID NO:29 of US2011/0271358 (SEQ ID NO: 38)); and/or a light chain comprising a sequence selected from SEQ ID NOs: 30-33 (SEQ ID NO:30 of US2011/0271358 (SEQ ID NO: 39), SEQ ID NO:31 of US2011/0271358 (SEQ ID NO: 40), SEQ ID NO:32 of US2011/0271358 (SEQ ID NO: 41), and SEQ ID NO:33 of US2011/0271358 (SEQ ID NO: 42)).
In various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, comprises one or more antibodies or antibody fragments thereof directed against PD-1 selected from TSR-042 (Tesaro, inc.), reg 2810 (Regeneron Pharmaceuticals, inc.), PDR001 (Novartis Pharmaceuticals), and BGB-a317 (BeiGene ltd.).
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have one or more targeting moieties to PD-L1. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has one or more targeting moieties that selectively bind to a PD-L1 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L1 polypeptide.
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab) or a fragment thereof. MEDI4736 is selective for PD-L1 and blocks binding of PD-L1 to PD-1 and CD80 receptors. MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprise heavy and light chains or heavy and light chain variable regions. The sequence of MEDI4736 is disclosed in WO/2016/06272, the entire content of which is hereby incorporated by reference. In an illustrative embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises an amino acid sequence comprising SEQ ID NO:43 and/or a heavy chain comprising the amino acid sequence SEQ ID NO: 44.
In an illustrative embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises the amino acid sequence of SEQ ID NO:4 (SEQ ID NO: 45) and/or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 (SEQ ID NO: 46).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody alt Zhu Shankang (atezolizumab) (also known as MPDL3280A, RG 7446) or a fragment thereof. In an illustrative embodiment, the alt Zhu Shan antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:47 and/or a heavy chain comprising the amino acid sequence SEQ ID NO: 48.
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody avermectin (avelumab) (also known as MSB 0010718C) or fragment thereof. In an illustrative embodiment, the avermectin or antigen binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:49 and/or a heavy chain comprising the amino acid sequence of SEQ ID NO: 50.
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105) or a fragment thereof as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, BMS-936559 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO: 51) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 52).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3G10, or fragment thereof, as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 3G10 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising an amino acid sequence (SEQ ID NO: 53) and/or a light chain variable region comprising an amino acid sequence (SEQ ID NO: 54).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 10A5, or fragment thereof, as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 10A5 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 55) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 56).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 5F8, or fragment thereof, as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 5F8 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 57) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 58).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 10H10, or fragment thereof, as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 10H10 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 59) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 60).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1B12 or a fragment thereof as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 1B12 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 61) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 62).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 7H1 or a fragment thereof as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 7H1 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 63) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 64).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 11E6 or a fragment thereof as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 11E6 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 65) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 66).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 12B7, or a fragment thereof, as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 12B7 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 67) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 68).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 13G4, or fragment thereof, as disclosed in US 2013/0309250 and WO2007/005874, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 13G4 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 69) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 70).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1E12, or fragment thereof, as disclosed in US 2014/0044738, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, 1E12 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 71) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 72).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1F4, or a fragment thereof, as disclosed in US 2014/0044738, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, 1F4 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 73) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 74).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2G11, or fragment thereof, as disclosed in US 2014/0044738, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, 2G11 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 75) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 76).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3B6, or fragment thereof, as disclosed in US 2014/0044738, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, 3B6 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 77) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 78).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3D10, or fragment thereof, as disclosed in US 2014/0044738 and WO2012/145493, the entire disclosure of which is hereby incorporated by reference. In illustrative embodiments, 3D10 or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence (SEQ ID NO: 79) and/or a light chain variable region comprising the amino acid sequence (SEQ ID NO: 80).
In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies as disclosed in US2011/0271358 and WO2010/036959, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID nos: 34-38 (SEQ ID No. 34 of US2011/0271358 (SEQ ID No. 81), SEQ ID No. 35 of US2011/0271358 (SEQ ID No. 82), SEQ ID No. 36 of US2011/0271358 (SEQ ID No. 83), SEQ ID No. 37 of US2011/0271358 (SEQ ID No. 84), and SEQ ID No. 38 of US2011/0271358 (SEQ ID No. 85)); and/or a light chain comprising an amino acid sequence selected from (SEQ ID No:39-42 of U.S. Pat. No. 2011/0271358; SEQ ID No:39 of U.S. Pat. No. 2011/0271358 (SEQ ID No: 86); SEQ ID No:40 of U.S. Pat. No. 2011/0271358 (SEQ ID No: 87); SEQ ID No:41 of U.S. Pat. No. 2011/0271358 (SEQ ID No: 88); and SEQ ID No:42 of U.S. Pat. No. 2011/0271358 (SEQ ID No: 89)).
In one embodiment, the targeting moiety comprises anti-PD-L1 antibody 2.7a4 or fragment thereof as disclosed in WO 2011/066389, US8,779,108 and US2014/0356353, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 2.7a4 or antigen binding fragment thereof for use in the methods provided herein comprises the amino acid sequence comprising SEQ ID No:2 (SEQ ID NO: 90) and/or a heavy chain variable region comprising the amino acid sequence of WO 2011/066389: 7 (SEQ ID NO: 91).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.9d10, or fragment thereof, as disclosed in WO 2011/066389, US8,779,108 and US2014/0356353, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the 2.9d10 or antigen binding fragment thereof used in the methods provided herein comprises the amino acid sequence of SEQ ID No:12 The heavy chain variable region of (SEQ ID NO: 92) and/or the SEQ ID NO:17 (SEQ ID NO: 93).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 2.14h9 or fragment thereof as disclosed in WO 2011/066389, US8,779,108 and US2014/0356353, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 2.14h9 or antigen binding fragment thereof for use in the methods provided herein comprises the amino acid sequence comprising SEQ ID No:22 The heavy chain variable region of (SEQ ID NO: 94) and/or the SEQ ID NO:27 (SEQ ID NO: 95).
In one embodiment, the targeting moiety comprises anti-PD-L1 antibody 2.20a8 or fragment thereof as disclosed in WO 2011/066389, US8,779,108 and US2014/0356353, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 2.20a8 or antigen binding fragment thereof for use in the methods provided herein comprises the amino acid sequence comprising SEQ ID No:32 (SEQ ID NO: 96) and/or SEQ ID No:37 (SEQ ID NO: 97).
In one embodiment, the targeting moiety comprises anti-PD-L1 antibody 3.15g8 or fragment thereof as disclosed in WO 2011/066389, US8,779,108 and US2014/0356353, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 3.15g8 or antigen binding fragment thereof for use in the methods provided herein comprises the amino acid sequence comprising SEQ ID No:42 (SEQ ID NO: 98) and/or a heavy chain variable region comprising an amino acid sequence, SEQ ID No:47 (SEQ ID NO: 99).
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 3.18g1 or fragment thereof as disclosed in WO 2011/066389, US8,779,108 and US2014/0356353, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, 3.18g1 or antigen binding fragment thereof for use in the methods provided herein comprises the amino acid sequence comprising SEQ ID No:52 The heavy chain variable region of (SEQ ID NO: 100) and/or the SEQ ID No:57 (SEQ ID NO: 101).
In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7a4opt, or fragment thereof, as disclosed in WO 2011/066389, US8,779,108 and US2014/0356353, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the 2.7a4opt, or antigen binding fragment thereof, used in the methods provided herein comprises the amino acid sequence comprising SEQ ID No:62 (SEQ ID NO: 102) and/or a heavy chain variable region comprising an amino acid sequence, SEQ ID No:67 (SEQ ID NO: 103) a light chain variable region.
In one embodiment, the targeting moiety comprises an anti-PD-L1 antibody 2.14h9opt, or fragment thereof, as disclosed in WO 2011/066389, US8,779,108 and US2014/0356353, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the 2.14h9opt or antigen binding fragment thereof used in the methods provided herein comprises the amino acid sequence of SEQ ID No:72 (SEQ ID NO: 104) and/or SEQ ID No:77 (SEQ ID NO: 105) a light chain variable region.
In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO2016/061142, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID nos: 18. 30, 38, 46, 50, 54, 62, 70 and 78 (SEQ ID No:18 (SEQ ID No: 106) of WO 2016/061142), SEQ ID No:30 (SEQ ID No: 107) of WO2016/061142, SEQ ID No:38 (SEQ ID No: 108) of WO2016/061142, SEQ ID No:46 (SEQ ID No: 109) of WO2016/061142, SEQ ID No:50 (SEQ ID No: 110) of WO2016/061142, SEQ ID No:54 (SEQ ID No: 111) of WO2016/061142, SEQ ID No:62 (SEQ ID No: 112) of WO2016/061142, SEQ ID No:70 (SEQ ID No: 113) of WO2016/061142 and SEQ ID No:78 (SEQ ID No: 114) of WO 2016/061142); and/or a light chain comprising a sequence selected from the group consisting of SEQ ID nos of WO 2016/061142: 22. 26, 34, 42, 58, 66, 74, 82 and 86 (SEQ ID No. 22 of WO2016/061142 (SEQ ID No. 115), SEQ ID No. 26 of WO2016/061142 (SEQ ID No. 116), SEQ ID No. 34 of WO2016/061142 (SEQ ID No. 117), SEQ ID No. 42 of WO2016/061142 (SEQ ID No. 118), SEQ ID No. 58 of WO2016/061142 (SEQ ID No. 119), SEQ ID No. 66 of WO2016/061142 (SEQ ID No. 120), SEQ ID No. 74 of WO2016/061142 (SEQ ID No. 121), SEQ ID No. 82 of WO2016/061142 (SEQ ID No. 122), and SEQ ID No. 86 of WO2016/061142 (SEQ ID No. 123)).
In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO2016/022630, the entire content of which is hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID nos: 2. 6, 10, 14, 18, 22, 26, 30, 34, 38, 42 and 46 (SEQ ID NO:2 of WO2016/022630 (SEQ ID NO: 124), SEQ ID NO:6 of WO2016/022630 (SEQ ID NO: 125), SEQ ID NO:10 of WO2016/022630 (SEQ ID NO: 126), SEQ ID NO:14 of WO2016/022630 (SEQ ID NO: 127), SEQ ID NO:18 of WO2016/022630 (SEQ ID NO: 128), SEQ ID NO:22 of WO2016/022630 (SEQ ID NO: 129), SEQ ID NO:26 of WO2016/022630 (SEQ ID NO: 130), SEQ ID NO:30 of WO2016/022630 (SEQ ID NO: 131), SEQ ID NO:34 of WO2016/022630 (SEQ ID NO: 132), SEQ ID NO:38 of WO2016/022630 (SEQ ID NO: 133), SEQ ID NO:42 of WO2016/022630 (SEQ ID NO: 134), and SEQ ID NO: 2016/022630 (SEQ ID NO: 135)); and/or a light chain comprising a sequence selected from the group consisting of SEQ ID nos: 4. 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 (SEQ ID NO:4 of WO2016/022630 (SEQ ID NO: 136), SEQ ID NO:8 of WO2016/022630 (SEQ ID NO: 137), SEQ ID NO:12 of WO2016/022630 (SEQ ID NO: 138), SEQ ID NO:16 of WO2016/022630 (SEQ ID NO: 139), SEQ ID NO:20 of WO2016/022630 (SEQ ID NO: 140), SEQ ID NO:24 of WO2016/022630 (SEQ ID NO: 141), SEQ ID NO:28 of WO2016/022630 (SEQ ID NO: 142), SEQ ID NO:32 of WO2016/022630 (SEQ ID NO: 143), SEQ ID NO:36 of WO2016/022630 (SEQ ID NO: 144), SEQ ID NO:40 of WO2016/022630 (SEQ ID NO: 145), SEQ ID NO:44 of WO2016/022630 (SEQ ID NO: 146), and SEQ ID NO:147 of WO 2016/022630).
In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO2015/112900, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID nos: 38. 50, 82 and 86 (SEQ ID No. 38 of WO2015/112900 (SEQ ID No. 148), SEQ ID No. 50 of WO2015/112900 (SEQ ID No. 149), SEQ ID No. 82 of WO2015/112900 (SEQ ID No. 150), and SEQ ID No. 86 of WO2015/112900 (SEQ ID No. 151)); and/or a light chain comprising a sequence selected from the group consisting of SEQ ID nos of WO 2015/112900: 42. 46, 54, 58, 62, 66, 70, 74 and 78 (SEQ ID No. 42 of WO2015/112900 (SEQ ID No. 152), SEQ ID No. 46 of WO2015/112900 (SEQ ID No. 153), SEQ ID No. 54 of WO2015/112900 (SEQ ID No. 154), SEQ ID No. 58 of WO2015/112900 (SEQ ID No. 155), SEQ ID No. 62 of WO2015/112900 (SEQ ID No. 156), SEQ ID No. 66 of WO2015/112900 (SEQ ID No. 157), SEQ ID No. 70 of WO2015/112900 (SEQ ID No. 158), SEQ ID No. 74 of WO2015/112900 (SEQ ID No. 159) and SEQ ID No. 78 of WO2015/112900 (SEQ ID No. 160)).
In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO 2010/077634 and US 8,217,149, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises the amino acid sequence of SEQ ID No:20 (SEQ ID NO: 161) and/or SEQ ID NO:21 (SEQ ID NO: 162).
In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from hybridomas obtainable under CNCM accession numbers CNCM I-4122, CNCM I-4080 and CNCM I-4081 as disclosed in US 20120039906, the entire disclosure of which is hereby incorporated by reference.
In one embodiment, the targeting moiety comprises a VHH for PD-L1 as disclosed in e.g. US 8,907,065 and WO 2008/071447, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the VHH directed against PD-L1 comprises SEQ ID No. of US 8,907,065: 394-399 (SEQ ID No. 394 of US 8,907,065 (SEQ ID No. 163), SEQ ID No. 395 of US 8,907,065 (SEQ ID No. 164), SEQ ID No. 396 of US 8,907,065 (SEQ ID No. 165), SEQ ID No. 397 of US 8,907,065 (SEQ ID No. 166), SEQ ID No. 398 of US 8,907,065 (SEQ ID No. 167), and SEQ ID No. 399 of US 8,907,065 (SEQ ID No. 168)).
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have one or more targeting moieties to PD-L2. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has one or more targeting moieties that selectively bind to a PD-L2 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L2 polypeptide.
In one embodiment, the targeting moiety comprises a VHH for PD-L2 as disclosed in, for example, US 8,907,065 and WO 2008/071447, the entire disclosures of which are hereby incorporated by reference. In an illustrative embodiment, the VHH directed against PD-L2 comprises SEQ ID No. of US 8,907,065: 449-455 (SEQ ID No. 449 of US 8,907,065 (SEQ ID No. 169), SEQ ID No. 450 of US 8,907,065 (SEQ ID No. 170), SEQ ID No. 451 of US 8,907,065 (SEQ ID No. 171), SEQ ID No. 452 of US 8,907,065 (SEQ ID No. 172), SEQ ID No. 453 of US 8,907,065 (SEQ ID No. 173), SEQ ID No. 454 of US 8,907,065 (SEQ ID No. 174), and SEQ ID No. 455 of US 8,907,065 (SEQ ID No. 175)).
In one embodiment, the targeting moiety comprises any of the anti-PD-L2 antibodies as disclosed in US2011/0271358 and WO2010/036959, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID nos: 43-47 (SEQ ID No. 43 of U.S. Pat. No. 2011/0271358 (SEQ ID No. 176), SEQ ID No. 44 of U.S. Pat. No. 2011/0271358 (SEQ ID No. 177), SEQ ID No. 45 of U.S. Pat. No. 2011/0271358 (SEQ ID No. 178), SEQ ID No. 46 of U.S. Pat. No. 201I/0271358 (SEQ ID No. 179), and SEQ ID No. 47 of U.S. Pat. No. 2011/0271358 (SEQ ID No. 180)); and/or a light chain comprising a sequence selected from SEQ ID nos: 48-51 (SEQ ID No. 48 of US2011/0271358 (SEQ ID No. 181), SEQ ID No. 49 of US2011/0271358 (SEQ ID No. 182), SEQ ID No. 50 of US2011/0271358 (SEQ ID No. 183), and SEQ ID No. 51 of US2011/0271358 (SEQ ID No. 184)).
In various embodiments, the targeting moiety of the invention may comprise a polypeptide having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity (e.g., a targeted PD-1 having about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% sequence identity with any of the sequences disclosed herein PD-L1 and/or PD-L2.
In various embodiments, the targeting moiety of the invention may comprise any combination of heavy, light, heavy chain variable, light chain variable, complementarity Determining Regions (CDRs) and framework region sequences that target PD-1, PD-L1 and/or PD-L2 as disclosed herein.
Other antibodies, antibody derivatives or forms, peptides or polypeptides or fusion proteins that selectively bind or target PD-1, PD-L1 and/or PD-L2 are disclosed in the following documents: WO 2011/066389, US 2008/0025980, US 2013/0034559, US 8,779,108, US 2014/0356353, US 8,609,089, US 2010/028330, US 2012/011020449, WO2010/027827, WO 2011,/066342, US 8,907,065, WO 2016/062722, WO 2009/101611, WO2010/027827, WO 2011/066342, WO 2007/005874, WO 2001/014556, US2011/0271358, WO 2010/036959, WO 2010/077634, US 8,217,149, US 2012/0039906, WO 2012/145493, US 2011/0318373, US 8,779,108, US 20140044738, WO 2009/089149, WO 2007/00587, WO 2016061142, WO 2016,02263, WO 2010/077634 and WO 2015/112900, the entire disclosures of which are hereby incorporated by reference.
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to T cells, e.g., mediated by targeting CD8, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to CD8 on T cells.
In one embodiment, the targeting moiety for CD8 comprises a VHH comprising the following amino acid sequence:
in one embodiment, the targeting moiety for CD8 comprises a VHH comprising the following amino acid sequence:
in one embodiment, the targeting moiety for CD8 comprises a VHH comprising the following amino acid sequence:
in various embodiments, the targeting moiety of the invention may comprise a polypeptide having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity (e.g., with any of the sequences disclosed herein about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98% > About 99% or about 100% sequence identity) of a CD 8-targeting sequence.
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to T cells, e.g., mediated by targeting CD4, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to CD4 on T cells.
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to T cells, for example, mediated by targeting CD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpoint markers, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to CD3 on T cells and a second targeting moiety to PD-L1 or PD-L2 on tumor cells.
In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have one or more targeting moieties to CD3 expressed on T cells. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has one or more targeting moieties that selectively bind to a CD3 polypeptide. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a CD3 polypeptide.
In one embodiment, the targeting moiety comprises the anti-CD 3 antibody moromonab (muromonab) -CD3 (also known as Orthoclone OKT 3) or a fragment thereof. Moromolizumab-CD 3 is disclosed in U.S. Pat. No. 4,361,549 and Wilde et al (1996) 51:865-894, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, the Moromorphab-CD 3 or antigen binding fragment thereof used in the methods provided herein comprises a heavy chain comprising the amino acid sequence (SEQ ID NO: 185) and/or a light chain comprising the amino acid sequence (SEQ ID NO: 186).
In one embodiment, the targeting moiety comprises the anti-CD 3 antibody oxlizumab (otelizumab) or a fragment thereof. Oxybutyrimab is disclosed in U.S. Pat. No. 20160000916 and Chatenoud et al (2012) 9:372-381, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, the oxybutynin or antigen binding fragment thereof used in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:187 and/or a heavy chain comprising the amino acid sequence SEQ ID NO: 188.
In one embodiment, the targeting moiety comprises the anti-CD 3 antibody telizumab (AKA MGA031 and hOKT3 γ1 (Ala-Ala)) or a fragment thereof. Tilicarbazemab is disclosed in Chatenoud et al (2012) 9:372-381, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, the telithromab or antigen-binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:189 and/or comprises the amino acid sequence SEQ ID NO: 190.
In one embodiment, the targeting moiety comprises the anti-CD 3 antibody, victimuzumab (also known as visilizumab)HuM 291) or a fragment thereof. Wiceizumab is disclosed in U.S.5,834,597 and WO2004052397 and Cole et al, transformation (1999) 68:563-571, the entire disclosure of which is hereby incorporated by reference. In an illustrative embodiment, the velutinab or antigen binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:191 and/or comprises the amino acid sequence SEQ ID NO:192, and a light chain variable region of 192.
In one embodiment, the targeting moiety comprises the anti-CD 3 antibody, foralumab (also known as NI-0401) or a fragment thereof. In various embodiments, the targeting moiety comprises any of the anti-CD 3 antibodies disclosed in US20140193399, US 7,728,114, US20100183554 and US 8,551,478, the entire disclosures of which are hereby incorporated by reference.
In an illustrative embodiment, the anti-CD 3 antibody or antigen binding fragment thereof for use in the methods provided herein comprises the amino acid sequence of SEQ ID No:2 and 6 (SEQ ID NO:2 of US 7,728,114 (SEQ ID NO: 193) and SEQ ID NO:6 of US 7,728,114 (SEQ ID NO: 194)) and/or a light chain variable region comprising the amino acid sequence SEQ ID NO 4 and 8 of US 7,728,114 (SEQ ID NO 4 of US 7,728,114 (SEQ ID NO: 195) and SEQ ID NO 8 of US 7,728,114 (SEQ ID NO: 196)).
In one embodiment, the targeting moiety comprises the amino acid sequence comprising SEQ ID NO:2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4. In one embodiment, the targeting moiety comprises any of the anti-CD 3 antibodies disclosed in US2016/0168247, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID nos: 6-9 (SEQ ID No. 6 of U.S. Pat. No. 2016/0168247 (SEQ ID No. 197), SEQ ID No. 7 of U.S. Pat. No. 2016/0168247 (SEQ ID No. 198), SEQ ID No. 8 of U.S. Pat. No. 2016/0168247 (SEQ ID No. 199), and SEQ ID No. 9 of U.S. Pat. No. 2016/0168247 (SEQ ID No. 200)); and/or a light chain comprising a sequence selected from the group consisting of SEQ ID nos: 10-12 (SEQ ID No. 10 of US2016/0168247 (SEQ ID No. 201), SEQ ID No. 11 of US2016/0168247 (SEQ ID No. 202), and SEQ ID No. 12 of US2016/0168247 (SEQ ID No. 203)).
In one embodiment, the targeting moiety comprises any of the anti-CD 3 antibodies disclosed in US2015/0175699, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising a sequence selected from the group consisting of SEQ ID nos: 9 (SEQ ID NO: 204); and/or comprising a sequence selected from the group consisting of SEQ ID No of US 2015/0175699: 10 (SEQ ID NO: 205) a light chain of the amino acid sequence.
In one embodiment, the targeting moiety comprises any of the anti-CD 3 antibodies disclosed in US 8,784,821, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID nos: 2. 18, 34, 50, 66, 82, 98 and 114 (SEQ ID No. 2 of US 8,784,821 (SEQ ID No. 206), SEQ ID No. 18 of US 8,784,821 (SEQ ID No. 207), SEQ ID No. 34 of US 8,784,821 (SEQ ID No. 208), SEQ ID No. 50 of US 8,784,821 (SEQ ID No. 209), SEQ ID No. 66 of US 8,784,821 (SEQ ID No. 210), SEQ ID No. 82 of US 8,784,821 (SEQ ID No. 211), SEQ ID No. 98 of US 8,784,821 (SEQ ID No. 212), and SEQ ID No. 114 of US 8,784,821 (SEQ ID No. 213)); and/or a light chain comprising a sequence selected from the group consisting of SEQ ID nos of US 8,784,821: 10. 26, 42, 58, 74, 90, 106 and 122 (SEQ ID No. 10 of US 8,784,821 (SEQ ID No. 214), SEQ ID No. 26 of US 8,784,821 (SEQ ID No. 215), SEQ ID No. 42 of US 8,784,821 (SEQ ID No. 216), SEQ ID No. 58 of US 8,784,821 (SEQ ID No. 217), SEQ ID No. 74 of US 8,784,821 (SEQ ID No. 218), SEQ ID No. 90 of US 8,784,821 (SEQ ID No. 219), SEQ ID No. 106 of US 8,784,821 (SEQ ID No. 220), and SEQ ID No. 122 of US 8,784,821 (SEQ ID No. 221)).
In one embodiment, the targeting moiety comprises any of the anti-CD 3 binding constructs disclosed in US20150118252, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen binding fragment thereof for use in the methods provided herein comprises a polypeptide comprising a sequence selected from the group consisting of SEQ ID nos: 6 and 86 (SEQ ID No. 6 of U.S. Pat. No. 20150118252 (SEQ ID No. 222) and SEQ ID No. 86 of U.S. Pat. No. 20150118252 (SEQ ID No. 223)). And/or comprising a sequence selected from the group consisting of SEQ ID No of US 2015/0175699: 3 (SEQ ID No: 224) of U.S. Pat. No. 3, 20150118252).
In one embodiment, the targeting moiety comprises any of the anti-CD 3 binding proteins disclosed in US2016/0039934, the entire contents of which are hereby incorporated by reference. In an illustrative embodiment, an antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID nos: 6-9 (SEQ ID No. 6 of U.S. Pat. No. 2016/0039934 (SEQ ID No. 225), SEQ ID No. 7 of U.S. Pat. No. 2016/0039934 (SEQ ID No. 226), SEQ ID No. 8 of U.S. Pat. No. 2016/0039934 (SEQ ID No. 227), and SEQ ID No. 9 of U.S. Pat. No. 2016/0039934 (SEQ ID No. 228)); and/or a light chain comprising a sequence selected from the group consisting of SEQ ID nos: 1-4 (SEQ ID No. 1 of U.S. Pat. No. 2016/0039934 (SEQ ID No. 229), SEQ ID No. 2 of U.S. Pat. No. 2016/0039934 (SEQ ID No. 230), SEQ ID No. 3 of U.S. Pat. No. 2016/0039934 (SEQ ID No. 231), and SEQ ID No. 4 of U.S. Pat. No. 2016/0039934 (SEQ ID No. 232)).
In various embodiments, the targeting moiety of the invention may comprise a polypeptide having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, with any of the sequences disclosed herein, about 99% or about 100% sequence identity) of a CD3 targeting sequence.
In various embodiments, the targeting moiety of the invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity Determining Region (CDR), and framework region sequences that target CD3 as disclosed herein. In various embodiments, the targeting moiety of the invention may comprise any heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity Determining Region (CDR), and framework region sequences of a CD3 specific antibody, including but not limited to X35-3, VIT3, BMA030 (BW 264/56), CLB-T3/3, CRIS7, YTH12.5, F1 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3B, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, WT31, and F101.01. These CD 3-specific antibodies are well known in the art and are described, inter alia, in tunepaliffe (1989), int.immunol.1, 546-550, the entire disclosure of which is hereby incorporated by reference.
Other antibodies, antibody derivatives or forms, peptides or polypeptides or fusion proteins that selectively bind or target CD3 are disclosed in U.S. patent publication nos. 2016/0000916, 4,361,549, 5,834,597, 6,491,916, 6,406,696, 6,143,297, 6,750,325, and international publication No. WO 2004/052397, the entire disclosures of which are hereby incorporated by reference.
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to T cells, mediated, for example, by targeting PD-1, along with any signaling agent described herein (e.g., modified IL-2).
As a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety to a B cell mediated, for example, by targeting: CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, or CDw150, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to CD 20.
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to B cells, e.g., mediated by targeting CD19, CD20, or CD70, along with any signaling agent described herein (e.g., modified IL-2).
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to B cells, e.g., mediated by targeting CD20, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to CD20 on B cells and a second targeting moiety to PD-L1 or PD-L2 on tumor cells. As an example, in some embodiments, the CD20 targeting moiety is a recombinant heavy chain-only antibody (VHH) having the sequence:
as a non-limiting example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, has a targeting moiety to NK cells mediated, for example, by targeting: 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, fc- εRII, LMIR6/CD300LE, fc- γR1/CD64, MICA, fc- γRIIB/CD32B, MICB, fc- γRIIC/CD32c, MULT-1, fc- γRIIA/CD32 ase:Sub>A, integrin-2/CD 112, fc- γRIII-CD16, NKG2A, fcRH/IRTA 5, NKG2C, fcRH/IRTA 4, NKG2D, fcRH/IRTA 1 NKp30, fcRH5/IRTA2, NKp44, fc receptor-like protein 3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, rae-1, rae-1α, rae-1β, rae-1δ, H60, rae-1ε, ILT2/CD85j, rae-1γ, ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85 ase:Sub>A, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DIA/CD158d, or ULBP-3; along with any signaling agent described herein (e.g., modified IL-2).
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to NK cells, e.g., mediated by targeting Kir1 a, DNAM-1 or CD64, along with any of the signaling agents described herein (e.g., modified IL-2).
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to NK cells, e.g., mediated by targeting TIGIT or KIR1, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to TIGIT on NK cells and a second targeting moiety to PD-L1 or PD-L2 on tumor cells.
As a non-limiting example, in various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have targeting moieties to dendritic cells mediated, for example, by targeting: CLEC-9A, XCR1, RANK, CD36/SRB3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-A1/MSR, CD5L, SREC-1, CL-P1/COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand/TNFSF 9, IL-12/IL-23P 40, 4-amino-1, 8-naphthalenedicarboxamide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85D, 8D6A, ILT5/CD85a, A2B5, rutegmedia alpha 4/CD49D, aa, integrin beta 2/CD18, langerhans protein (Langerhane), B7-2/CD86, leukoR 4/CD 7/CD 85, LMR 7/CD 3/CD300, LMR 3/CD300, LMIR2/CD 300; clq R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD/SLAMF 5, MMR, CD97, NCAML1, CD2F-10/SLAMF9, bone activin GPNMB, chern 23, PD-L2, CLEC-1, RP105, CLEC-2, siglec-2/CD22, CRACC/SLAMF7, siglec-3/CD33, DC-SIGN, siglec-5, DC-SIGNR/CD299, siglec-6, DCAR, siglec-7, DCIR/CLEC 4-9, DEC-205, de-10, CLEC-1, RP105, CLEC-2/CD 22, CRCC-3/CD 35, siglec-6, CLEC-977, CLEC-97/CD 9, DEC 7/CD 35, DEC 1/CD 35 SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, fc- γR1/CD64, TLR3, fc- γRIIB/CD32b, TREM-1, fc- γRIIC/CD32c, TREM-2, fc- γRIIA/CD32a, TREM-3, fc- γRIII/CD16, TREML1/TLT-1, ICAM-2/CD102 or capsaicin R1; along with any signaling agent described herein (e.g., modified IL-2).
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to dendritic cells, e.g., mediated by targeting CLEC-9A, DC-SIGN, CD64, CLEC4A or DEC205, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to CLEC9A on dendritic cells and a second targeting moiety to PD-L1 or PD-L2 on tumor cells.
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to dendritic cells, e.g., mediated by targeting CLEC9A, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to CLEC9A on dendritic cells and a second targeting moiety to PD-L1 or PD-L2 on tumor cells.
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to dendritic cells, e.g., mediated by targeting XCR1, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety directed against XCR1 on dendritic cells and a second targeting moiety directed against PD-L1 or PD-L2 on tumor cells.
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety directed against dendritic cells, e.g., mediated by targeting RANK, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to RANK on dendritic cells and a second targeting moiety to PD-L1 or PD-L2 on tumor cells.
As a non-limiting example, in various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have targeting moieties to monocytes/macrophages mediated, for example, by targeting: SIRP1a, B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, common beta chain, integrin alpha 4/CD49d, BLAME/SLAMF8, integrin alpha X/CDllc, CCL6/C10, integrin beta 2/CD18, CD155/PVR, integrin beta 3/CD61, CD31/PECAM-1, latex, CD36/SR-B3, leukotriene B4R 1, CD40/TNFRSF5, LIMPIIISR-B2, CD43, LMIR1/CD300A, CD, LMIR2/CD300C, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163, LRP-1, CD 2F-10/SLF 9, MARCO, CRACC/SLAMF7, MD-1, ECF-52, NMF-83, NMIN-147, EMML 2/CD 105, GPLI-CD 105, and GPLI-LI-2. Fc-gamma RI/CD64, ossein, fc-gamma RIIB/CD32B, PD-L2, fc-gamma RIIB/CD 32C, siglec-3/CD33, fc-gamma RIIA/CD32a, SIGNR1/CD209 Fc- γRIII-CD16, SLAM, GM-CSF Rα, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN- γR1, TLR4, IFN- γR2, TREM-1, IL-1 RII, TREM-2, fc-gamma RIII-CD16, SLAM, GM-CSF Rα, TCCR/WSX-1, ICAM-2/CD102, TLR3 IFN- γR1, TLR4, IFN- γR2, TREM-1, IL-1 RII, TREM-2, integrin αM/CDll B, CCR8, integrin αX/CDllc, CD155/PVR, integrin β2/CD18, CD14, integrin β3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, coagulation factor III/tissue factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4 LRP-1, CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, endothelin/CD 105, NCAM-L1, fc-gamma RI/CD64, PSGL-1, fc-gamma RIIICD 16, RP105, G-CSFR, L-selectin, GM-CSF Rα, siglec-3/CD33, HVEM/TNFRSF 14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6R, TREM-2, CXCR1/IL-8RA, TREM-3 or TRL 1/TLT-1; along with any signaling agent described herein (e.g., modified IL-2).
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to monocytes/macrophages mediated, for example, by targeting B7-H1, CD31/PECAM-1, CD163, CCR2, or macrophage mannose receptor CD206, along with any signaling agent described herein (e.g., modified IL-2).
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to monocytes/macrophages mediated, for example, by targeting SIRP1a, along with any signaling agent described herein (e.g., modified IL-2). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a targeting moiety to SIRP1a on macrophages and a second targeting moiety to PD-L1 or PD-L2 on tumor cells.
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have one or more targeting moieties for one or more of the checkpoint markers, e.g., PD-1/PD-L1 or PD-L2, CD28/CD80 or CD86, CTLA4/CD80 or CD86, ICOS/ICOSL or B7RP1, BTLA/HVEM, KIR, LAG3, CD137/CD137L, OX/OX 40L, CD27, CD40L, TIM3/Gal9, CD47, CD70, and A2aR, along with any signaling agent described herein (e.g., modified IL-2).
In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, comprise one or more targeting moieties to the same or different immune cells. In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have one or more targeting moieties to immune cells selected from T cells, B cells, dendritic cells, macrophages, NK cells, or a subset thereof, along with any of the signaling agents described herein (e.g., modified IL-2).
In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, comprise a targeting moiety to a tumor cell. In such embodiments, the targeting moiety may bind to any of the tumor antigens described herein.
In some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, comprises one or more targeting moieties having a recognition domain that binds to a target of interest (e.g., antigen, receptor) including a target found on one or more cells selected from the group consisting of: adipocytes (e.g., white adipocytes, brown adipocytes), hepatic adipocytes, hepatocytes, renal cells (e.g., renal wall cells, renal salivary glands, renal mammary glands, etc.), ductal cells (ductal cells of seminal vesicles, prostates, etc.), intestinal brush border cells (with microvilli), exocrine glandular ductal cells, gall bladder epithelial cells, output tubule ciliated cells, epididymal master cells, epididymal basal cells, endothelial cells, enamel cell epithelial cells (enamel secretion), semilunar squamous epithelial cells of the vestibular system of the ear (proteoglycan secretion), organs of Corti interproximal epithelial cells (secretion of the covering membrane of the hair cells), loose connective tissue fibroblasts, cornea fibroblasts (cornea cells), tendon fibroblasts, bone cells bone marrow reticulocytes, non-epithelial fibroblasts, pericytes, nucleus pulposus cells of the intervertebral disc, cementoblasts/cementoblasts (root bone-like ewan cell secretion), odontoblasts/dentin cells (odontoblast/odontocyte) (dentin secretion), hyaline chondrocytes, fibrochondrocytes, elastic chondrocytes, osteoblasts/bone cells (osteoblast/osteocyte), osteoprogenitors (stem cells of osteoblasts), vitreous cells of the vitreous of the eye, astrocytes of the perilymph space of the ear, hepatic stellate cells (Ito cells), pancreatic stellate cells, skeletal muscle cells, satellite cells, myocardial cells, smooth muscle cells, myoepithelial cells of the iris, myoepithelial cells of the exocrine glands, exocrine secretory epithelial cells (e.g., salivary gland cells, breast cells, lacrimal gland cells, sweat gland cells, sebaceous gland cells, prostate cells, gastric gland cells, pancreatic acinar cells, alveolar cells), hormone secretory cells (e.g., pituitary cells, nerve secretory cells, intestinal and respiratory tract cells, thyroid cells, parathyroid cells, adrenal cells, testicular interstitial cells, islet cells), keratinocytes, moisture-stratified barrier epithelial cells, neuronal cells (e.g., sensory transduction cells, autonomic neuronal cells, sensory organ and peripheral nerve support cells, and central nervous system neurons and glial cells such as interneurons, main cells, astrocytes, oligodendrocytes, and ependymal cells).
Targeting moiety forms
In various embodiments, the chimeric proteins or targeting moieties of chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, are protein-based agents, such as antibodies or derivatives thereof, capable of specific binding. In one embodiment, the targeting moiety comprises an antibody. In various embodiments, the antibody is a full length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises a variable region (e.g., V H ) And at least three constant regions (e.g., CH 1 、CH 2 And CH (CH) 3 ) And each light chain comprises a variable region (V L ) And a constant region (C L ). The variable region determines the specificity of the antibody. Each variable region comprises three hypervariable regions, also known as Complementarity Determining Regions (CDRs), flanked by four relatively conserved Framework Regions (FR). The three CDRs (termed CDR1, CDR2, and CDR 3) contribute to antibody binding specificity. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.
In some embodiments, the targeting moiety comprises an antibody derivative or antibody form. In some embodiments, the targeting moiety of the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, is a single domain antibody, heavy chain-only recombinant antibody (VHH), single chain antibody (scFv), heavy chain-only shark antibody (VNAR), micro protein (cysteine knot protein, knottin), DARPin; tetranectin (Tetranectin); affibody; trans bodies (trans bodies); anti-cargo proteins; adNectin; affilin; a microsome (Microbody); a peptide aptamer; altrex (alterase); a plastic antibody; fei Luoti (phylomer); stradobody (stradobody); giant body (maxibody); ivermectin (eviody); finobody (fynomer), armadillo-repeat, kunitzdomain, high affinity multimer (avimer), atrimer, pra Luo Ti (probody), immunobody (immunobody), qu Aoshan anti (triomab), troybody; pepbody (pepbody); vaccinal bodies (vaccinal bodies), monospecific bodies (UniBody); affimer, bispecific (DuoBody), fv, fab, fab ', F (ab') 2, peptide mimetic or synthetic molecules, as described in U.S. patent numbers or patent publications below: US 7,417,130, US 2004/132094, US 5,831,012, US 2004/02347, US 7,250,297, US 6,818,418, US 2004/209443, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794,144, US 2010/239633, US 7,803,907, US 2010/119446 and/or US 7,166,697, the contents of which are hereby incorporated by reference in their entirety. See also Storz MAbs.2011, 5 months-6 months; 3 (3): 310-317.
In one embodiment, the targeting moiety comprises a single domain antibody, such as a VHH from, for example, a VHH antibody-producing organism (such as camelid, shark), or a engineered VHH. VHHs are therapeutic proteins of antibody origin that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies from camels that lack light chains. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH 2 and CH 3). VHH are commercially available under the trademark NANOBODY or NANOBODY ies.
In one embodiment, the targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.
In some embodiments, the VHH comprises a fully human VH domain, such as, for example, temaboy (Crescendo Biologics, cambridge, UK). In some embodiments, the fully human VH domain (e.g., HUMABODY) is monovalent, bivalent, or trivalent. In some embodiments, the fully human VH domain (e.g., HUMABODY) is monospecific or multispecific, such as monospecific, bispecific, or trispecific. Illustrative fully human VH domains (e.g. HUMABODIES) are described, for example, in WO 2016/113555 and WO 2016/113557, the entire contents of which are incorporated by reference.
In various embodiments, the targeting moiety of the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, is a protein-based agent capable of specifically binding to a cellular receptor, such as a natural ligand of the cellular receptor. In various embodiments, the cellular receptor is found on one or more immune cells, which may include, but are not limited to, T cells, cytotoxic T lymphocytes, T helper cells, natural Killer (NK) cells, natural Killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or a subset thereof. In some embodiments, the cellular receptor is found on megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof.
In some embodiments, the targeting moiety is a natural ligand, such as a chemokine. Illustrative chemokines that may be included in a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CXCL3 1, HCC-4, and LDGF-PBP. In an illustrative embodiment, the targeting moiety may be XCL1, which is a chemokine that recognizes and binds to the dendritic cell receptor XCR 1. In another illustrative embodiment, the targeting moiety is CCL1, which is a chemokine that recognizes and binds to CCR 8. In another illustrative embodiment, the targeting moiety is CCL2, which is a chemokine that recognizes and binds to CCR2 or CCR 9. In another illustrative embodiment, the targeting moiety is CCL3, which is a chemokine that recognizes and binds to CCR1, CCR5, or CCR 9. In another illustrative embodiment, the targeting moiety is CCL4, which is a chemokine that recognizes and binds to CCR1 or CCR5 or CCR 9. In another illustrative embodiment, the targeting moiety is CCL5, which is a chemokine that recognizes and binds to CCR1 or CCR3 or CCR4 or CCR 5. In another illustrative embodiment, the targeting moiety is CCL6, which is a chemokine that recognizes and binds to CCR 1. In another illustrative embodiment, the targeting moiety is CCL7, which is a chemokine that recognizes and binds to CCR2 or CCR 9. In another illustrative embodiment, the targeting moiety is CCL8, which is a chemokine that recognizes and binds to CCR1 or CCR2B or CCR5 or CCR 9. In another illustrative embodiment, the targeting moiety is CCL9, which is a chemokine that recognizes and binds to CCR 1. In another illustrative embodiment, the targeting moiety is CCL10, which is a chemokine that recognizes and binds to CCR 1. In another illustrative embodiment, the targeting moiety is CCL11, which is a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR 9. In another illustrative embodiment, the targeting moiety is CCL13, which is a chemokine that recognizes and binds to CCR2 or CCR3 or CCR5 or CCR 9. In another illustrative embodiment, the targeting moiety is CCL14, which is a chemokine that recognizes and binds to CCR1 or CCR 9. In another illustrative embodiment, the targeting moiety is CCL15, which is a chemokine that recognizes and binds to CCR1 or CCR 3. In another illustrative embodiment, the targeting moiety is CCL16, which is a chemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR 8. In another illustrative embodiment, the targeting moiety is CCL17, which is a chemokine that recognizes and binds to CCR 4. In another illustrative embodiment, the targeting moiety is CCL19, which is a chemokine that recognizes and binds to CCR 7. In another illustrative embodiment, the targeting moiety is CCL20, which is a chemokine that recognizes and binds to CCR 6. In another illustrative embodiment, the targeting moiety is CCL21, which is a chemokine that recognizes and binds to CCR 7. In another illustrative embodiment, the targeting moiety is CCL22, which is a chemokine that recognizes and binds to CCR 4. In another illustrative embodiment, the targeting moiety is CCL23, which is a chemokine that recognizes and binds to CCR 1. In another illustrative embodiment, the targeting moiety is CCL24, which is a chemokine that recognizes and binds to CCR 3. In another illustrative embodiment, the targeting moiety is CCL25, which is a chemokine that recognizes and binds to CCR 9. In another illustrative embodiment, the targeting moiety is CCL26, which is a chemokine that recognizes and binds to CCR 3. In another illustrative embodiment, the targeting moiety is CCL27, which is a chemokine that recognizes and binds to CCR 10. In another illustrative embodiment, the targeting moiety is CCL28, which is a chemokine that recognizes and binds to CCR3 or CCR 10. In another illustrative embodiment, the targeting moiety is CXCL1, which is a chemokine that recognizes and binds to CXCR1 or CXCR 2. In another illustrative embodiment, the targeting moiety is CXCL2, which is a chemokine that recognizes and binds to CXCR 2. In another illustrative embodiment, the targeting moiety is CXCL3, which is a chemokine that recognizes and binds to CXCR 2. In another illustrative embodiment, the targeting moiety is CXCL4, which is a chemokine that recognizes and binds to CXCR 3B. In another illustrative embodiment, the targeting moiety is CXCL5, which is a chemokine that recognizes and binds to CXCR 2. In another illustrative embodiment, the targeting moiety is CXCL6, which is a chemokine that recognizes and binds to CXCR1 or CXCR 2. In another illustrative embodiment, the targeting moiety is CXCL8, which is a chemokine that recognizes and binds to CXCR1 or CXCR 2. In another illustrative embodiment, the targeting moiety is CXCL9, which is a chemokine that recognizes and binds to CXCR 3. In another illustrative embodiment, the targeting moiety is CXCL10, which is a chemokine that recognizes and binds to CXCR 3. In another illustrative embodiment, the targeting moiety is CXCL11, which is a chemokine that recognizes and binds to CXCR3 or CXCR 7. In another illustrative embodiment, the targeting moiety is CXCL12, which is a chemokine that recognizes and binds to CXCR4 or CXCR 7. In another illustrative embodiment, the targeting moiety is CXCL13, which is a chemokine that recognizes and binds to CXCR 5. In another illustrative embodiment, the targeting moiety is CXCL16, which is a chemokine that recognizes and binds to CXCR 6. In another illustrative embodiment, the targeting moiety is LDGF-PBP, which is a chemokine that recognizes and binds to CXCR 2. In another illustrative embodiment, the targeting moiety is XCL2, which is a chemokine that recognizes and binds to XCR 1. In another illustrative embodiment, the targeting moiety is CX3CL1, which is a chemokine that recognizes and binds to CX3CR 1.
In some embodiments, the targeting moiety is a natural ligand, such as an FMS-like tyrosine kinase 3 ligand (Flt 3L) or a truncated region thereof (e.g., that is capable of binding Flt 3). In some embodiments, the targeting moiety is the extracellular domain of Flt 3L. In some embodiments, the targeting moiety comprises a Flt3L domain, wherein the Flt3L domain is a single chain dimer, optionally wherein one FIt L domain is linked to another Flt3L domain via one or more linkers, wherein the linker is a flexible linker. In some embodiments, the targeting moiety of the invention comprises: a Flt3L domain, wherein said Flt3L domain is a single chain dimer; and an Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and/or stabilize a hinge region in the Fc domain. In some embodiments, the targeting moiety recognizes CD20. In some embodiments, the targeting moiety recognizes PD-L1. In some embodiments, the targeting moiety recognizes Clec9A.
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, comprise targeting moieties in various combinations. In one illustrative embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, may comprise two targeting moieties, wherein both targeting moieties are antibodies or derivatives thereof. In another illustrative embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, may comprise two targeting moieties, wherein both targeting moieties are natural ligands for a cellular receptor. In another illustrative embodiment, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, may comprise two targeting moieties, wherein one targeting moiety is an antibody or derivative thereof and the other targeting moiety is a natural ligand for a cellular receptor.
In various embodiments, the recognition domain of the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, functionally modulate (partially or fully neutralize, in a non-limiting manner) a target of interest (e.g., antigen, receptor), e.g., substantially inhibit, reduce, or neutralize, a biological effect that the antigen has. For example, each recognition domain can be directed against one or more tumor antigens that actively block or have the ability to block the immune system of a patient, e.g., a patient suffering from a tumor. For example, in some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, functionally modulates an immunosuppressive signal (e.g., a checkpoint inhibitor), e.g., one or more of TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, sirpa, ICOS, CD172a, and TMIGD 2. For example, in some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, is engineered to disrupt, block, reduce, and/or inhibit the transmission of immunosuppressive signals, as non-limiting examples, the binding of PD-1 to PD-L1 or PD-L2 and/or the binding of CTLA-4 to one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R 5A.
In various embodiments, the recognition domain of a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, binds to but does not functionally modulate a target of interest (e.g., antigen, receptor), e.g., the recognition domain is or resembles a binding antibody. For example, in various embodiments, the recognition domain targets only an antigen or receptor, but does not substantially inhibit, reduce, or functionally modulate the biological effect that the antigen or receptor has. For example, some of the smaller antibody formats described above (e.g., as compared to, e.g., an intact antibody) have the ability to target an epitope that is difficult to access and provide a greater range of specific binding sites. In various embodiments, the recognition domain binds to an epitope that is physically separated from an antigenic site (e.g., an active site of an antigen) that is important for the biological activity of the antigen or receptor.
Such non-neutralizing binding can be used in various embodiments of the invention, including methods of recruiting active immune cells to a desired site using a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, directly or indirectly via an effector antigen, such as any of the effector antigens described herein. For example, in various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, can be used in a method of reducing or eliminating a tumor to recruit cytotoxic T cells directly or indirectly to the tumor cells via CD8 (e.g., the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can comprise an anti-CD 8 recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to recruit cytotoxic T cells expressing CD8 directly or indirectly but not to functionally modulate the activity of the CD 8. In contrast, in these embodiments, CD8 signaling is an important part of tumor reduction or elimination. As another example, in various methods of reducing or eliminating a tumor, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, is used to recruit Dendritic Cells (DCs) directly or indirectly via CLEC9A (e.g., the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may comprise an anti-CLEC 9A recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to recruit DCs expressing CLEC9A directly or indirectly but not functionally modulate the activity of CLEC 9A. In contrast, CLEC9A signaling is an important part of tumor reduction or elimination in these embodiments.
In various embodiments, the recognition domain of a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, binds to XCR1 on, for example, dendritic cells. For example, in some embodiments, the recognition domain comprises all or part of XCL1 or a non-neutralizing anti-XCR 1 agent.
In various embodiments, the recognition domain of a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, binds to an immunomodulatory antigen (e.g., an immunostimulatory or immunosuppressive antigen). In various embodiments, the immunomodulatory antigen is one or more of the following: 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30, CD40, ICOS ligand; OX-40 ligand, LIGHT (CD 258), GITR ligand, CD70, B7-1, B7-2, CD30 ligand, CD40 ligand, ICOS ligand, CD137 ligand, and TL1A. In various embodiments, such immunostimulatory antigens are expressed on tumor cells. In various embodiments, the recognition domains of the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, bind to but do not functionally modulate such immunostimulatory antigens, thus allowing recruitment of cells expressing these antigens without reducing or losing their potential tumor reducing or eliminating capabilities.
In various embodiments, the recognition domain of a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, can be in the range of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprising two recognition domains with neutralizing activity, or comprising two recognition domains with non-neutralizing (e.g., binding) activity, or comprising one recognition domain with neutralizing activity and one recognition domain with non-neutralizing (e.g., binding) activity.
Fc domain
Fragment crystallizable domains (Fc domains) are tail regions in antibodies that interact with Fc receptors located on the cell surface of cells involved in the immune system (e.g., B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells). In IgG, igA and IgD antibody isotypes, the Fc domain consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody. In IgM and IgE antibody isotypes, the Fc domain comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
In some embodiments, the Fc-based chimeric protein complexes of the present technology comprise an Fc domain. In some embodiments, the Fc domain is selected from IgG, igA, igD, igM or IgE. In some embodiments, the Fc domain is selected from IgG1, igG2, igG3, or IgG4.
In some embodiments, the Fc domain is selected from human IgG, igA, igD, igM or IgE. In some embodiments, the Fc domain is selected from human IgG1, igG2, igG3, or IgG4.
In some embodiments, the Fc domain of the Fc-based chimeric protein complex comprises the CH2 and CH3 regions of IgG. In some embodiments, the IgG is human IgG. In some embodiments, the human IgG is selected from IgG1, igG2, igG3, or IgG4.
In some embodiments, the Fc domain comprises one or more mutations. In some embodiments, one or more mutations of the Fc domain reduce or eliminate effector function of the Fc domain. In some embodiments, the mutant Fc domain has reduced affinity or binding to a target receptor. As an example, in some embodiments, the mutation of the Fc domain reduces or eliminates binding of the Fc domain to fcγr. In some embodiments, fcγr is selected from fcγri; fcγriia, 131R/R; fcγriia, 131H/H, fc γriib; and fcγriii. In some embodiments, the mutation of the Fc domain reduces or eliminates binding to a complement protein (such as, for example, C1 q). In some embodiments, the mutation of the Fc domain reduces or eliminates binding to both fcγr and complement proteins (such as, for example, C1 q).
In some embodiments, the Fc domain comprises LALA mutations to reduce or eliminate effector function of the Fc domain. As an example, in some embodiments, LALA mutations include L234A and L235A substitutions in human IgG (e.g., igG 1) (where numbering is based on the common numbering of CH2 residues of human IgG1 according to the EU convention (PNAS, edelman et al, 1969;63 (1) 78-85).
In some embodiments, the Fc domain of the human IgG comprises a mutation at 46 to reduce or eliminate effector function of the Fc domain. As an example, in some embodiments, the mutation is selected from L234A, L234F, L235A, L235E, L235Q, K322A, K Q, D265A, P G, P329A, P331G and P331S.
In some embodiments, the Fc domain comprises a FALA mutation to reduce or eliminate effector function of the Fc domain. As an example, in some embodiments, the FALA mutation comprises F234A and L235A substitutions in human IgG 4.
In some embodiments, the Fc domain of human IgG4 comprises a mutation at one or more of F234, L235, K322, D265, and P329 to reduce or eliminate effector function of the Fc domain. As an example, in some embodiments, the mutation is selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G and P329A.
In some embodiments, the one or more mutations of the Fc domain stabilize the hinge region in the Fc domain. As an example, in some embodiments, the Fc domain comprises a mutation at S228 of a human IgG to stabilize the hinge region. In some embodiments, the mutation is S228P.
In some embodiments, one or more mutations of the Fc domain promote chain pairing in the Fc domain. In some embodiments, chain pairing is facilitated by ion pairing (also known as charged pairs, ionic bonds, or charged residue pairs).
In some embodiments, the Fc domain comprises mutations at one or more of the following amino acid residues of IgG to facilitate ion pairing: d356, E357, L368, K370, K392, D399 and K409.
As an example, in some embodiments, the human IgG Fc domain comprises one of the combinations of mutations in table 1 to facilitate ion pairing.
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In some embodiments, strand pairing is facilitated via knob hole mutation. In some embodiments, the Fc domain comprises one or more mutations to achieve knob-in-hole interactions in the Fc domain. In some embodiments, the first Fc strand is engineered to express a "knob" and the second Fc strand is engineered to express a complementary "hole". As an example, in some embodiments, the human IgG Fc domain comprises the mutations of table 2 to achieve knob hole interactions.
In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology comprises any combination of the mutations disclosed above. As an example, in some embodiments, the Fc domain comprises mutations that promote ion pairing and/or knob-hole interactions. As an example, in some embodiments, the Fc domain comprises a mutation having one or more of the following properties: facilitating ion pairing, inducing knob-hole interactions, reducing or eliminating effector functions of the Fc domain, and causing Fc stabilization (e.g., at the hinge).
As an example, in some embodiments, a human IgG Fc domain comprises mutations disclosed in table 3 that promote ion pairing in the Fc domain and/or promote knob-hole interactions.
As an example, in some embodiments, a human IgG Fc domain comprises mutations disclosed in table 4 that promote ion pairing in the Fc domain, promote knob-hole interactions, or a combination thereof. In various embodiments, "chain 1" and "chain 2" of table 4 may be interchanged (e.g., chain 1 may have Y407T and chain 2 may have T366Y).
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As an example, in some embodiments, a human IgG Fc domain comprises the mutations disclosed in table 5 that reduce or eliminate fcγr and/or complement binding in the Fc domain. In various embodiments, there are mutations in both strands of Table 5.
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In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a homodimer, i.e., the Fc region in the chimeric protein complex comprises two identical protein fragments.
In some embodiments, the Fc domain in the Fc-based chimeric protein complexes of the present technology is a heterodimer, i.e., the Fc domain comprises two different protein fragments.
In some embodiments, the heterodimeric Fc domain is engineered using ion pairing and/or knob hole mutation described herein. In some embodiments, the Fc-based chimeric protein complex of the heterodimer has a trans orientation/configuration. In the trans orientation/configuration, in various embodiments, the targeting moiety and the signaling agent (e.g., IL-2) are not found on the same polypeptide chain of the Fc-based chimeric protein complex of the invention.
In some embodiments, the Fc domain comprises or begins at the core hinge region of wild-type human IgG1, comprising the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 276). In some embodiments, the Fc domain further includes an upper hinge or portion thereof (e.g., DKTHTCPPC (SEQ ID NO: 451); see WO 2009053368), EPKSCDKTHTCPPC (SEQ ID NO: 452) or EPKSSDKTHTCPPC (SEQ ID NO: 453); see Lo et al, volume Protein Engineering, volume 11, 6, pages 495-500, 1998)).
Fc-based chimeric protein complexes
The Fc-based chimeric protein complexes of the present technology comprise at least one Fc domain disclosed herein, at least one Signaling Agent (SA) disclosed herein (e.g., IL-2), and at least one Targeting Moiety (TM) disclosed herein.
It is to be understood that the Fc-based chimeric protein complexes of the invention can encompass complexes of two fusion proteins, each fusion protein comprising an Fc domain.
In some embodiments, the Fc-based chimeric protein complex is heterodimeric. In some embodiments, the Fc-based chimeric protein complex of the heterodimer has a trans orientation/configuration. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a cis orientation/configuration.
In some embodiments, the heterodimeric Fc domain is engineered using ion pairing and/or knob hole mutation described herein. In some embodiments, the Fc-based chimeric protein complex of the heterodimer has a trans orientation.
In the trans orientation, in various embodiments, the targeting moiety and the signaling agent are not found on the same polypeptide chain of the Fc-based chimeric protein complex of the invention. In the cis orientation, in various embodiments, the targeting moiety and the signaling agent are found on separate polypeptide chains of the Fc-based chimeric protein complex. In the cis orientation, in various embodiments, the targeting moiety and the signaling agent are found on the same polypeptide chain of the Fc-based chimeric protein complex.
In some embodiments, when more than one targeting moiety is present in the heterodimeric protein complexes described herein, one targeting moiety may be in a trans orientation (relative to the signaling agent) and the other targeting moiety may be in a cis orientation (relative to the signaling agent). In some embodiments, the signaling agent and the target moiety are on the same end/side (N-terminal or C-terminal) of the Fc domain. In some embodiments, the signaling agent and the targeting moiety are on different sides/ends (N-terminus and C-terminus) of the Fc domain.
In some embodiments, where more than one targeting moiety is present in the heterodimeric protein complexes described herein, the targeting moieties can be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the targeting moieties are trans relative to each other due to being on different Fc chains). In some embodiments, where more than one targeting moiety is present on the same Fc chain, the targeting moieties may be on the same or different sides/ends (N-terminus or/and C-terminus) of the Fc chain.
In some embodiments, when more than one signaling agent is present in the heterodimeric protein complexes described herein, the signaling agents can be found on the same Fc chain or on two different Fc chains in the heterodimeric protein complex (in the latter case, the signaling agents are trans relative to each other due to being on different Fc chains). In some embodiments, where more than one signaling agent is present on the same Fc chain, the signaling agents may be on the same or different sides/ends (N-terminus or/and C-terminus) of the Fc chain.
In some embodiments, where more than one signaling agent is present in the heterodimeric protein complexes described herein, one signaling agent may be in a trans orientation (e.g., relative to the targeting moiety) and the other signaling agent may be in a cis orientation (e.g., relative to the targeting moiety).
In some embodiments, the heterodimeric Fc-based chimeric protein complex does not comprise a signaling agent (e.g., IL-2) and a targeting moiety on a single polypeptide.
In some embodiments, the Fc-based chimeric protein has an improved in vivo half-life relative to a chimeric protein lacking Fc or a chimeric protein that is not a heterodimeric complex. In some embodiments, the Fc-based chimeric proteins have improved solubility, stability, and other pharmacological properties relative to chimeric proteins lacking Fc or chimeric proteins that are not heterodimeric complexes.
The heterodimeric Fc-based chimeric protein complex consists of two different polypeptides. In the embodiments described herein, the targeting domain is located on a different polypeptide than the signaling agent (e.g., IL-2), so that the protein contains only one copy of the targeting domain, and only one copy of the signaling agent (e.g., IL-2) can be made (this provides a configuration in which potential interference with the desired property can be controlled). Furthermore, in various embodiments, only one targeting domain (e.g., VHH) can avoid cross-linking of antigens on the cell surface (which may trigger adverse effects in some cases). Furthermore, in various embodiments, a single signaling agent (e.g., IL-2) may mitigate molecular "crowding" and potential interference with affinity-mediated restoration of effector functions that rely on the targeting domain. Furthermore, in various embodiments, the heterodimeric Fc-based chimeric protein complex may have two targeting moieties, and these targeting moieties may be disposed on two different polypeptides. For example, in various embodiments, the C-termini of two targeting moieties (e.g., VHH) can be masked to avoid potential autoantibodies or pre-existing antibodies (e.g., VHH autoantibodies or pre-existing antibodies). Furthermore, in various embodiments, fc-based chimeric protein complexes, e.g., having a targeting domain on a different polypeptide than a heterodimer of a signaling agent (e.g., IL-2) (e.g., a wild-type signaling agent, e.g., IL-2), may be advantageous for "cross-linking" of two cell types (e.g., tumor cells and immune cells). Furthermore, in various embodiments, the heterodimeric Fc-based chimeric protein complex may have two signaling agents, each located on a different polypeptide to allow for more complex effector responses.
Furthermore, in various embodiments, fc-based chimeric protein complexes, e.g., heterodimers with a targeting domain on different polypeptides than a signaling agent (e.g., IL-2), provide combinatorial diversity of targeting moieties and signaling agents (e.g., IL-2) in a practical manner. For example, in various embodiments, a polypeptide having any of the targeting moieties described herein may be "off-the-shelf" combined with a polypeptide having any of the signaling agents described herein to allow for rapid production of various combinations of targeting moieties and signaling agents in a single Fc-based chimeric protein complex.
In some embodiments, the Fc-based chimeric protein complex comprises one or more linkers. In some embodiments, the Fc-based chimeric protein complex comprises a linker connecting an Fc domain, one or more signaling agents (e.g., IL-2), and one or more targeting moieties. In some embodiments, the Fc-based chimeric protein complex comprises a linker that connects each signaling agent (e.g., IL-2) to a targeting moiety (or connects a signaling agent (e.g., IL-2) to one of the targeting moieties when more than one targeting moiety is present). In some embodiments, the Fc-based chimeric protein complex comprises a linker that connects each signaling agent (e.g., IL-2) to an Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker that connects each targeting moiety to an Fc domain. In some embodiments, the Fc-based chimeric protein complex comprises a linker that connects a targeting moiety to another targeting moiety. In some embodiments, the Fc-based chimeric protein complex comprises a linker that connects a signaling agent (e.g., IL-2) to another signaling agent.
In some embodiments, the Fc-based chimeric protein complex comprises two or more targeting moieties. In such embodiments, the targeting moieties may be the same targeting moiety or they may be different targeting moieties.
In some embodiments, the Fc-based chimeric protein complex comprises two or more signaling agents. In such embodiments, the signaling agents may be the same targeting moiety or they may be different targeting moieties.
As an example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, at least two Signaling Agents (SA), and at least two Targeting Moieties (TM), wherein the Fc domain, signaling agent, and targeting moiety are selected from any of the Fc domains, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is homodimeric.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 1A-1F.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 2A-2H.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematics of fig. 3A-3H.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 4A-4D.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 5A-5F.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematics of fig. 6A-6J.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 7A-7D.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 8A-8F.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 9A-9J.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 10A-10F.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 11A-11L.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 12A-12L.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 13A-13F.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 14A-14L.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 15A-15L.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 16A-16J.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 17A-17J.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 18A-18F.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 19A-19F.
In various embodiments, the Fc-based chimeric protein complex takes the form of any one of the schematic diagrams of fig. 20A-20E.
In various embodiments, the Fc-based chimeric protein complex takes the form of the schematic of fig. 31.
In various embodiments, the Fc-based chimeric protein complex takes the form of the schematic of fig. 32.
In some embodiments, the invention encompasses Fc-based chimeric protein complexes comprising one or more variants of table 7 and/or one or more constructs listed within column "ALN2 variants" in table 7.
In some embodiments, the signaling agent is linked to the targeting moiety and the targeting moiety is linked to the same end of the Fc domain (see fig. 1A-1F). In some embodiments, the Fc domain is homodimeric.
In some embodiments, the signaling agent and the targeting moiety are linked to an Fc domain, wherein the targeting moiety and the signaling agent are linked at the same terminus (see fig. 1A-1F). In some embodiments, the Fc domain is homodimeric.
In some embodiments, the targeting moiety is linked to a signaling agent and the signaling agent is linked to the same end of the Fc domain (see fig. 1A-1F). In some embodiments, the Fc domain is homodimeric.
In some embodiments, the Fc-based chimeric protein complex of the homodimer has two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling agents, the targeting moieties are linked to the Fc domain, and the signaling agents are linked to the same end of the targeting moiety (see fig. 2A-2H). In some embodiments, the Fc domain is homodimeric. In some embodiments, in the presence of four targeting moieties and two signaling agents, two targeting moieties are linked to the Fc domain and two targeting moieties are linked to signaling agents that are linked to the same end of the Fc domain (see fig. 2A-2H). In some embodiments, the Fc domain is homodimeric. In some embodiments, in the presence of four targeting moieties and two signaling agents, the two targeting moieties are linked to each other and one targeting moiety in each pair is linked to the same end of the Fc domain and the signaling agent is linked to the same end of the Fc domain (see fig. 2A-2H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where there are four targeting moieties and two signaling agents, the two targeting moieties are linked to each other, wherein one targeting moiety in each pair is linked to a signaling agent (e.g., IL-2) and the other targeting moiety in the pair is linked to an Fc domain, wherein the targeting moieties linked to the Fc domain are linked on the same end (see fig. 2A-2H). In some embodiments, the Fc domain is homodimeric.
In some embodiments, the homodimer Fc-based chimeric protein complex has two or more signaling agents. In some embodiments, where four signaling agents and two targeting moieties are present, the two signaling agents are linked to each other, and one signaling agent in the pair is linked to the same end of the Fc domain, and the targeting moieties are linked to the same end of the Fc domain (see fig. 3A-3H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where four signaling agents and two targeting moieties are present, both signaling agents are attached to the same end of the Fc domain, and two of the signaling agents are each attached to a targeting moiety, wherein the targeting moieties are attached to the same end of the Fc domain (see fig. 3A-3H). In some embodiments, the Fc domain is homodimeric. In some embodiments, where four signaling agents and two targeting moieties are present, the two signaling agents are linked to each other, and one signaling agent in the pair is linked to the targeting moiety, and the targeting moiety is linked to the same end of the Fc domain (see fig. 3A-3H). In some embodiments, the Fc domain is homodimeric.
As an example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, wherein the Fc domain comprises one or more ion pairing mutations and/or one or more knob-in-hole mutations; at least one signaling agent (e.g., IL-2); and at least one targeting moiety, wherein the ion pairing motif and/or knob-in hole motif, signaling agent (e.g., IL-2), and targeting group are selected from any of the ion pairing groups and/or knob-in hole motifs, signaling agents, and targeting moieties disclosed herein. In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, the signaling agent (e.g., IL-2) is linked to the targeting moiety that is linked to the Fc domain (see fig. 10A-10F and fig. 13A-13F). In some embodiments, the targeting moiety is linked to the signaling agent (e.g., IL-2) that is linked to the Fc domain (see fig. 10A-10F and fig. 13A-13F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, the signaling agent (e.g., IL-2) and the targeting moiety are linked to the Fc domain (see fig. 4A-4D, fig. 7A-7D, fig. 10A-10F, and fig. 13A-13F). In some embodiments, the targeting moiety and the signaling agent (e.g., IL-2) are linked to the same end of different Fc chains (see fig. 4A-4D and fig. 7A-7D). In some embodiments, the targeting moiety and the signaling agent (e.g., IL-2) are linked to different ends of different Fc chains (see fig. 4A-4D and fig. 7A-7D). In some embodiments, the targeting moiety and the signaling agent (e.g., IL-2) are linked to the same Fc chain (see fig. 10A-10F and fig. 13A-13F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of one signaling agent (e.g., IL-2) and two targeting moieties, the signaling agent (e.g., IL-2) is linked to the Fc domain, and the two targeting moieties can: 1) Are linked to each other, wherein one targeting moiety is linked to the Fc domain; or 2) each linked to an Fc domain (see FIGS. 5A-5F, 8A-8F, 11A-11L, 14A-14L, 16A-16J, and 17A-17J). In some embodiments, the targeting moiety is attached to one Fc chain and the signaling agent (e.g., IL-2) is on another Fc chain (see FIGS. 5A-5F and 8A-8F). In some embodiments, the pair of targeting moiety and signaling agent (e.g., IL-2) are linked to the same Fc chain (see fig. 1 iA-11L and fig. 14A-14L). In some embodiments, one targeting moiety is linked to an Fc domain, the other targeting moiety is linked to a signaling agent (e.g., IL-2), and a pair of targeting moieties is linked to the Fc domain (see fig. 11A-11L, 14A-14L, 16A-16J, and 17A-17J). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked to the same Fc chain (see fig. 11A-11L and fig. 14A-14L). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked to different Fc chains (see fig. 16A-16J and 17A-17J). In some embodiments, the unpaired targeting moiety and the paired targeting moiety are linked at the same terminus (see fig. 16A-16J and 17A-17J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of one signaling agent (e.g., IL-2) and two targeting moieties, the targeting moiety is linked to a signaling agent (e.g., IL-2) that is linked to an Fc domain, and the unpaired targeting moiety is linked to the Fc domain (see fig. 11A-11L, 14A-14L, 16A-16J, and 17A-17J). In some embodiments, the paired signaling agent (e.g., IL-2) and the unpaired targeting moiety are linked to the same Fc chain (see fig. 11A-11L and fig. 14A-14L). In some embodiments, the paired signaling agent (e.g., IL-2) and the unpaired targeting moiety are linked to different Fc chains (see fig. 16A-16J and fig. 17A-17J). In some embodiments, the paired signaling agent (e.g., IL-2) and the unpaired targeting moiety are linked at the same terminus (see FIGS. 16A-16J and 17A-17J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of one signaling agent (e.g., IL-2) and two targeting moieties, the targeting moieties are linked together, and the signaling agent (e.g., IL-2) is linked to one of the pair of targeting moieties, wherein the targeting moiety not linked to the signaling agent (e.g., IL-2) is linked to the Fc domain (see fig. 11A-11L and fig. 14A-14L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of one signaling agent (e.g., IL-2) and two targeting moieties, the targeting moieties are linked together, and the signaling agent (e.g., IL-2) is linked to one of the pair of targeting moieties, wherein the signaling agent (e.g., IL-2) is linked to the Fc domain (see fig. 11A-11L and fig. 14A-14L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of one signaling agent (e.g., IL-2) and two targeting moieties, both targeting moieties are linked to the signaling agent (e.g., IL-2), wherein one of the targeting moieties is linked to an Fc domain (see fig. 11A-11L and fig. 14A-14L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of one signaling agent (e.g., IL-2) and two targeting moieties, the targeting moiety and the signaling agent (e.g., IL-2) are linked to the Fc domain (see fig. 16A-16J and 17A-17J). In some embodiments, the targeting moiety is attached to the terminus (see FIGS. 16A-16J and 17A-17J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, where two signaling agents and one targeting moiety are present, the signaling agents are attached to the same end of the Fc domain and the targeting moiety is attached to the Fc domain (see fig. 6A-6J and fig. 9A-9J). In some embodiments, the signaling agent is linked to an Fc domain on the same Fc chain, while the targeting moiety is linked to another Fc chain (see fig. 18A-18F and fig. 19A-19F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of two signaling agents and one targeting moiety, the signaling agent (e.g., IL-2) is linked to the targeting moiety, which is linked to the Fc domain, and the other signaling agent (e.g., IL-2) is linked to the Fc domain (see fig. 6A-6J, 9A-9J, 12A-12L, and 15A-15L). In some embodiments, the targeting moiety and unpaired signaling agent (e.g., IL-2) are linked to different Fc chains (see fig. 6A-6J and fig. 9A-9J). In some embodiments, the targeting moiety and unpaired signaling agent (e.g., IL-2) are linked to the same end of different Fc chains (see fig. 6A-6J and fig. 9A-9J). In some embodiments, the targeting moiety and unpaired signaling agent (e.g., IL-2) are linked to different ends of different Fc chains (see fig. 6A-6J and fig. 9A-9J). In some embodiments, the targeting moiety and unpaired signaling agent (e.g., IL-2) are linked to the same Fc chain (see fig. 12A-12L and fig. 15A-15L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, where two signaling agents and one targeting moiety are present, the targeting moiety is linked to a signaling agent (e.g., IL-2), the signaling agent is linked to an Fc domain, and the other signaling agent (e.g., IL-2) is linked to the Fc domain (see fig. 6A-6J and fig. 9A-9J). In some embodiments, a paired signaling agent (e.g., IL-2) and an unpaired signaling agent (e.g., IL-2) are linked to different Fc chains (see FIGS. 6A-6J and 9A-9J). In some embodiments, a paired signaling agent (e.g., IL-2) and an unpaired signaling agent (e.g., IL-2) are linked to the same end of different Fc chains (see FIGS. 6A-6J and 9A-9J). In some embodiments, a paired signaling agent (e.g., IL-2) and an unpaired signaling agent (e.g., IL-2) are linked to different ends of different Fc chains (see FIGS. 6A-6J and 9A-9J). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, where two signaling agents and one targeting moiety are present, the signaling agents are linked together and the targeting moiety is linked to one of the pairs of signaling agents, wherein the targeting moiety is linked to the Fc domain (see fig. 12A-12L and fig. 15A-15L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of two signaling agents and one targeting moiety, the signaling agents are linked together and one of the signaling agents is linked to the Fc domain and the targeting moiety is linked to the Fc domain (see fig. 12A-12L, 15A-15L, 18A-18F, and 19A-19F). In some embodiments, the paired signaling agent and targeting moiety are linked to the same Fc chain (see fig. 12A-12L and fig. 15A-15L). In some embodiments, the pairs of signaling agent and targeting moiety are linked to different Fc chains (see fig. 18A-18F and fig. 19A-19F). In some embodiments, the pairs of signaling agent and targeting moiety are linked to the same end of different Fc chains (see fig. 18A-18F and fig. 19A-19F). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, in the presence of two signaling agents and one targeting moiety, both signaling agents are linked to the targeting moiety, wherein one of the signaling agents is linked to the Fc domain (see fig. 12A-12L and fig. 15A-15L). In some embodiments, the Fc domain is heterodimeric. In some embodiments, the Fc domain comprises a mutation that reduces or eliminates its effector function.
In some embodiments, where two signaling agents and one targeting moiety are present, the signaling agents are linked together, and one of the signaling agents is linked to the targeting moiety, and the other signaling agent (e.g., IL-2) is linked to the Fc domain (see fig. 12A-12L and 15A-15L).
In some embodiments, where two signaling agents and one targeting moiety are present, each signaling agent (e.g., IL-2) is linked to an Fc domain, and the targeting moiety is linked to one of the signaling agents (see fig. 12A-12L and fig. 15A-15L). In some embodiments, the signaling agents are linked to the same Fc chain (see fig. 12A-12L and fig. 15A-15L).
In some embodiments, the targeting moiety or signaling agent (e.g., IL-2) is linked to an Fc domain comprising one or both of the CH2 and CH3 domains and optionally a hinge region. For example, vectors encoding targeting moieties linked as a single nucleotide sequence to an Fc domain, signaling agents (e.g., IL-2), or combinations thereof, can be used to prepare such polypeptides.
In some embodiments, the Fc-based chimeric protein complex comprises a polypeptide having a nucleotide sequence identical to SEQ ID NO: a polypeptide having an amino acid sequence of at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to any of 291-296, 298-335, 340-345, 347-359, 361-368, 371-375, 377-449.
In some embodiments, the Fc-based chimeric protein complex comprises a polypeptide having a nucleotide sequence identical to SEQ ID NO: a polypeptide having an amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical to any one of 291-296 and 298-335. In various embodiments, the Fc-based chimeric protein complex comprises a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 291-296, 298-335 and having fewer than 10 mutations relative to said amino acid sequence. In various embodiments, the Fc-based chimeric protein complex comprises a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 291-296, 298-335 and having fewer than 5 mutations relative to said amino acid sequence. In some embodiments, the Fc-based chimeric protein complex comprises a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 291-296, 298-335. In certain embodiments, the Fc-based chimeric protein complex comprises a polypeptide having a nucleotide sequence identical to SEQ ID NO: 292. 293, 294, 299, 301, 310, 311, 312, 315, 316, 319, 320, 325, 328, 332, 333, and 335, or a polypeptide having an amino acid sequence that is at least 95%, or at least 98%, or at least 99% identical. In certain embodiments, the Fc-based chimeric protein complex comprises a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 292. 293, 294, 299, 301, 310, 311, 312, 315, 316, 319, 320, 325, 328, 332, 333 and 335.
Additional signalling agents
In one aspect, the invention provides chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, that comprise one or more signaling agents (e.g., immunomodulators) in addition to the modified IL-2 described herein. In illustrative embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can comprise two, three, four, five, six, seven, eight, nine, ten, or more signaling agents in addition to the modified IL-2 described herein. In various embodiments, the additional signaling agent is modified so as to have reduced affinity or activity for one or more of its receptors, thereby allowing for attenuation of activity (including agonism or antagonism) and/or preventing non-specific signaling or undesired chelation of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex.
In various embodiments, the additional signaling agent is antagonistic in its wild-type form and carries one or more mutations that attenuate its antagonistic activity. In various embodiments, the additional signaling agent is antagonistic due to one or more mutations, e.g., an agonistic signaling agent is converted to an antagonistic signaling agent, and optionally, such a converted signaling agent also carries one or more mutations that attenuate its antagonistic activity (e.g., as described in WO 2015/007520, the entire contents of which are hereby incorporated by reference).
In various embodiments, the additional signaling agent is selected from the group consisting of cytokines, growth factors, and modified versions of hormones. Illustrative examples of such cytokines, growth factors, and hormones include, but are not limited to, lymphokines, monokines, traditional polypeptide hormones (such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone); parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; a relaxin source; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH); liver growth factors; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and tumor necrosis factor-beta; miaole tube inhibitors; a mouse gonadotrophin-related peptide; inhibin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet growth factors; transforming Growth Factors (TGFs) such as TGF- α and TGF- β; insulin-like growth factor-I and insulin-like growth factor-II; an osteoinductive factor; interferons such as, for example, interferon- α, interferon- β and interferon- γ (as well as type I, type II and type III interferons), colony Stimulating Factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as, for example, IL-1β, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15 and IL-18; tumor necrosis factors such as, for example, TNF- α or TNF- β; and other polypeptide factors including, for example, LIF and Kit Ligand (KL). As used herein, cytokines, growth factors, and hormones include proteins obtained from natural sources or produced by recombinant bacterial, eukaryotic, or mammalian cell culture systems, as well as biologically active equivalents of the native sequence cytokines.
In some embodiments, the additional signaling agent is a modified version of a growth factor selected from, but not limited to, transforming Growth Factors (TGFs) such as (TGF- α and TGF- β), epidermal Growth Factor (EGF), insulin-like growth factors such as insulin-like growth factor-I and insulin-like growth factor-II, fibroblast Growth Factor (FGF), genetic-regulatory protein (heregulin), platelet-derived growth factor (PDGF), vascular Endothelial Growth Factor (VEGF).
In one embodiment, the growth factor is a modified version of a Fibroblast Growth Factor (FGF). Illustrative FGF includes, but is not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, murine FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.
In one embodiment, the growth factor is a modified version of Vascular Endothelial Growth Factor (VEGF). Illustrative VEGF include, but are not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D and PGF and their isoforms, including the various isoforms of VEGF-A, such as VEGF 121 、VEGF 121 b、VEGF 145 、VEGF 165 、VEGF 165 b、VEGF 189 And VEGF (vascular endothelial growth factor) 206 。
In one embodiment, the growth factor is a modified version of a Transforming Growth Factor (TGF). Illustrative TGFs include, but are not limited to, TGF- α and TGF- β and subtypes thereof, including various subtypes of TGF- β, including tgfβ1, tgfβ2, and tgfβ3.
In some embodiments, the additional signaling agent is a modified version of a hormone selected from, but not limited to: human chorionic gonadotropin, gonadotrophin releasing hormone, androgen, estrogen, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin releasing hormone, growth hormone releasing hormone, adrenocorticotropic hormone releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoid, mineralocorticoid, epinephrine, norepinephrine, progesterone, insulin, ascending hormone, pullulan, calcitriol, calciferol, atrial natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptides Y, ghrelin, PYY3-36, insulin-like growth factor (IGF), leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen.
In some embodiments, the additional signaling agent is an immunomodulatory agent, such as one or more of an interleukin, an interferon, and tumor necrosis factor.
In some embodiments, the additional signaling agent is an interleukin, including, for example, IL-1 β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or a fragment, variant, analog or family member thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes and macrophages. Known functions include stimulation of proliferation of immune cells (e.g., T helper cells, B cells, eosinophils and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be measured using assays known in the art: matthews et al, lymphokines and Interferons: a Practical Approach, clemens et al, IRL Press, washington, D.C.1987, pages 221-225; and Orenecole and Dinarello (1989) Cytokine 1, 14-20.
In some embodiments, the signaling agent is a modified version of an interferon, such as a type I interferon, a type II interferon, and a type III interferon. Illustrative interferons include, for example, interferon-alpha-1, interferon-alpha-2, interferon-alpha-4, interferon-alpha-5, interferon-alpha-6, interferon-alpha-7, interferon-alpha-8, interferon-alpha-10, interferon-alpha-13, interferon-alpha-14, interferon-alpha-16, interferon-alpha-17 and interferon-alpha-21, interferon-beta, and interferon-gamma, interferon kappa, interferon epsilon, interferon tau, and interferon omega.
In various embodiments, the additional signaling agent is a type I interferon. In various embodiments, the type I interferon is selected from the group consisting of IFN alpha 2, IFN alpha 1, IFN beta, IFN gamma, consensus IFN, IFN epsilon, IFN kappa, IFN tau, IFN delta and IFN v.
In some embodiments, the additional signaling agent is Tumor Necrosis Factor (TNF) or a protein in the TNF family, including but not limited to TNF- α, TNF- β, LT- β, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and modified versions of TRAIL.
In various embodiments, the additional signaling agent is a modified (e.g., mutated) form of the signaling agent having one or more mutations. In various embodiments, the mutation allows the modified signaling agent to have one or more reduced activities, such as one or more of reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, relative to an unmodified or unmutated, i.e., wild-type form of the signaling agent (e.g., comparing wild-type form of the same signaling agent to a modified (e.g., mutated) form). In various embodiments, the mutation allows the modified signaling agent to have one or more reduced activities, such as one or more of reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, relative to the unmodified or unmutated signaling agent, i.e., unmutated IL-2. In some embodiments, mutations that reduce or decrease binding or affinity include those that substantially reduce or eliminate binding or activity. In some embodiments, mutations that reduce or decrease binding or affinity are different from those that substantially reduce or eliminate binding or activity. As a result, in various embodiments, the mutations allow for safer signaling agents relative to non-mutated, i.e., wild-type signaling agents, e.g., with reduced systemic toxicity, reduced side effects, and reduced off-target effects (e.g., comparing wild-type forms of the same signaling agent to modified (e.g., mutated) forms). In various embodiments, the mutation allows the signaling agent to be safer than an unmutated signaling agent, e.g., an unmutated sequence of IL-2, e.g., with reduced systemic toxicity, reduced side effects, and reduced off-target effects.
In various embodiments, the additional signaling agent is modified to have one or more mutations that reduce its binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate binding affinity or activity for the receptor. In some embodiments, the activity provided by the wild-type signaling agent is agonism at the receptor (e.g., activating a cellular effect at the treatment site). For example, the wild-type signaling agent may activate its receptor. In such embodiments, the mutation results in reduced or eliminated activation activity of the modified signaling agent at the receptor. For example, the mutation may cause the modified signaling agent to deliver a reduced activation signal to the target cell, or may eliminate the activation signal. In some embodiments, the activity provided by the wild-type signaling agent is antagonism at the receptor (e.g., blocking or suppressing a cellular effect at the treatment site). For example, the wild-type signaling agent may antagonize or inhibit the receptor. In these embodiments, the mutation results in a reduction or elimination of the antagonistic activity of the modified signaling agent at the receptor. For example, the mutation may cause the modified signaling agent to deliver a reduced inhibitory signal to the target cell, or may eliminate the inhibitory signal. In various embodiments, the signaling agent is antagonistic due to one or more mutations, e.g., converting an agonistic signaling agent into an antagonistic signaling agent (e.g., as described in WO 2015/007520, the entire contents of which are hereby incorporated by reference), and optionally, such converted signaling agent also carries one or more mutations that reduce or substantially reduce or eliminate binding affinity or activity to one or more of its receptors.
In some embodiments, reduced affinity or activity at the receptor may be induced or restored by attachment to one or more targeting moieties or upon inclusion in the Fc-based chimeric protein complexes disclosed herein. In other embodiments, the reduced affinity or activity at the receptor is not substantially induced or restored by the activity of one or more targeting moieties or after inclusion in the Fc-based chimeric protein complexes disclosed herein.
In various embodiments, the additional signaling agent is active on the target cell because the one or more targeting moieties compensate for the lack/deficiency of binding (e.g., without limitation, and/or avidity) required for substantial activation. In various embodiments, the modified signaling agent is substantially inactive for the pathway of the therapeutically active site and its effect is substantially directed against the specifically targeted cell type, thereby greatly reducing unwanted side effects.
In some embodiments, the additional signaling agent may include one or more mutations that reduce or decrease binding or affinity to one receptor (i.e., a therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or activity at a second receptor. In such embodiments, the mutations may be at the same or different positions (i.e., the same mutation or mutations). In some embodiments, one or more mutations that reduce binding and/or activity at one receptor are different from one or more mutations that substantially reduce or eliminate binding and/or activity at another receptor. In some embodiments, the one or more mutations that reduce binding and/or activity at one receptor are the same as the one or more mutations that substantially reduce or eliminate binding and/or activity at another receptor. In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, have a modified signaling agent that combines a mutation that reduces binding and/or activity at a therapeutic receptor and thus allows for a more controlled in-target therapeutic effect (e.g., relative to a wild-type signaling agent) with a mutation that substantially reduces or eliminates binding and/or activity at another receptor and thus reduces side effects (e.g., relative to a wild-type signaling agent).
In some embodiments, a substantial reduction or elimination of binding or activity is not substantially induced or restored by the targeting moiety or after inclusion in the Fc-based chimeric protein complexes disclosed herein. In some embodiments, substantial reduction or elimination of binding or activity may be induced or restored by the targeting moiety or after inclusion in the Fc-based chimeric protein complexes disclosed herein. In various embodiments, substantially reducing or eliminating binding or activity at the second receptor may also prevent adverse effects mediated by another receptor. Alternatively or additionally, substantially reducing or eliminating binding or activity at another receptor improves the therapeutic effect, as the reduced or eliminated chelation of the therapeutic chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is away from the site of therapeutic action. For example, in some embodiments, this circumvents the need for high doses of chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes that can compensate for losses at another receptor. This ability to reduce the dosage also provides a lower likelihood of side effects.
In various embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have a reduced, substantially reduced, level of one or more of its receptorsReduced or eliminated affinities, e.g. binding (e.g. K D ) And/or activation (e.g., when the modified signaling agent is an agonist of its receptor, it may be measured as, for example, K) A And/or EC(s) 50 ) And/or inhibition (e.g., when the modified signaling agent is an antagonist of its receptor, it may be measured as, for example, K I And/or IC 50 ). In various embodiments, reduced affinity at the signaling agent receptor allows for attenuation of activity (including agonism or antagonism). In such embodiments, the modified signaling agent has an affinity for the receptor of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% -20%, about 20% -40%, about 50%, about 40% -60%, about 60% -80%, about 80% -100% relative to the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower relative to the wild-type signaling agent (including, but not limited to non-mutated IL-2).
In embodiments where the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has a mutation that reduces binding at one receptor and substantially reduces or eliminates binding at a second receptor, the modified signaling agent reduces or reduces the binding affinity for one receptor to a lesser extent than the substantial reduction or elimination of affinity for the other receptor. In some embodiments, the decrease or decrease in binding affinity of the modified signaling agent for one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% less than the substantial decrease or elimination in affinity for the other receptor. In various embodiments, substantially reduced or eliminated refers to a greater reduction in binding affinity and/or activity than a decrease or decrease.
In various embodiments, the additional modified signaling agent comprises one or more mutations that reduce the endogenous activity of the signaling agent, e.g., to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, relative to the wild-type signaling agent (including, but not limited to, relative to unmutated IL-2).
In various embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and/or activity for a receptor of any of the cytokines, growth factors, and hormones described herein.
In some embodiments, the additional modified signaling agent comprises one or more mutations that result in the signaling agent having a reduced affinity for its receptor, lower than the binding affinity of the one or more targeting moieties to its receptor or receptors. In some embodiments, this difference in binding affinity exists between a signaling agent/receptor and a targeting moiety/receptor on the same cell. In some embodiments, this difference in binding affinity allows the signaling agent (e.g., a mutated signaling agent) to have a localized off-target effect and minimizes off-target effects that underlie the side effects observed with wild-type signaling agents. In some embodiments, such binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold, or at least about 25-fold, or at least about 50-fold, or at least about 100-fold, or at least about 150-fold lower.
Receptor binding activity can be measured using methods known in the art. Affinity and/or binding activity can be assessed, for example, by conducting a Scatchard plot analysis and computer fitting (e.g., scatchard, 1949) on the binding data or by conducting reflectance interferometry under flow conditions as described by Brecht et al (1993), the entire contents of all documents being incorporated herein by reference. In some embodiments, receptor binding activity is measured using Biological Layer Interferometry (BLI).
The amino acid sequences of the wild-type signaling agents described herein are well known in the art. Thus, in various embodiments, the first and second substrates, the additional modified signaling agent comprises a polypeptide having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89% >, a signaling agent with a known wild-type amino acid sequence of the signaling agent described herein; or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity).
In the context of the various embodiments of the present invention, the additional modified signaling agent comprises a signaling agent having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about, or about 98%, or about 99% sequence identity).
In various embodiments, the additional modified signaling agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations may be independently selected from the group consisting of substitutions, insertions, deletions, and truncations.
In some embodiments, the amino acid mutation is an amino acid substitution, and may include conservative and/or non-conservative substitutions as described herein.
As described herein, the additional modified signaling agent carries mutations that affect affinity and/or activity at one or more receptors. In various embodiments, there is a reduced affinity and/or activity for the therapeutic receptor, e.g., the receptor through which the desired therapeutic effect (agonism or antagonism) is mediated. In various embodiments, the modified signaling agent carries a mutation that substantially reduces or eliminates affinity and/or activity at the receptor, e.g., by which the desired therapeutic effect is not mediated (e.g., as a result of hybridization of binding). Receptors for any modified signaling agent, such as one of cytokines, growth factors, and hormones, as described herein are known in the art.
Illustrative mutations that provide reduced affinity and/or activity (e.g., agonism) at the receptor are found in WO 2013/107791 (e.g., for interferon), WO 2015/007542 (e.g., for interleukin), and WO 2015/007903 (e.g., for TNF), the entire contents of each of which are hereby incorporated by reference. Illustrative mutations that provide reduced affinity and/or activity (e.g., antagonism) at therapeutic receptors are found in WO 2015/007520, the entire contents of which are hereby incorporated by reference.
In some embodiments, the additional modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and/or activity for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a Tumor Necrosis Factor Receptor (TNFR) receptor in the superfamily, a TGF- β receptor, a receptor in the immunoglobulin (Ig) superfamily, and/or a receptor in the tyrosine kinase superfamily.
In various embodiments, the receptor of the additional signaling agent is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2 (beta subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and receptors for Thrombopoietin (TPO), prolactin, and growth hormone. Illustrative type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.
In various embodiments, the receptor of the additional signaling agent is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits and are receptors primarily for interferon. This family of receptors includes, but is not limited to, the receptors for interferon-alpha, interferon-beta and interferon-gamma, IL10, IL22 and tissue factor. Illustrative type II cytokine receptors include, but are not limited to, IFNaR1 and IFNAR2, IFN beta receptor, IFN gamma receptor (e.g., IFNGR1 and IFNGR 2) and type II IL receptor.
In various embodiments, the receptor of the additional signaling agent is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors that have seven transmembrane structures and are coupled to G proteins for signal transduction. Chemokine receptors include, but are not limited to, the CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor, and XC chemokine receptor (XCR 1). Illustrative chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3B, CXCR, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.
In various embodiments, the receptor of the additional signaling agent is a TNFR family member. Tumor Necrosis Factor Receptor (TNFR) family members share a cysteine-rich domain (CRD) formed by three disulfide bonds around the CXXCXXC core motif, forming an elongate molecule. Illustrative tumor necrosis factor receptor family members include: CD 120 a (TNFRSF 1A), CD 120B (TNFRSF 1B), lymphotoxin beta receptor (LTBR, TNFRSF 3), CD 134 (TNFRSF 4), CD40 (CD 40, TNFRSF 5), FAS (FAS, TNFRSF 6), TNFRSF6B (TNFRSF 6B), CD27 (CD 27, TNFRSF 7), CD30 (TNFRSF 8), CD137 (INFRSF 9), TNFRSF1OA (TNFRSF 1 OA), TNFRSF1OB (TNFRSF 1 OB), TNFRSF1OC (TNFRSF 1 OC), TNFRSF1OD (TNFRSF 1 OD), RANK (TNFRSF 1A), osteoprotegerin (TNFRSF 1 IB), TNFRSF12A (TNFRSF 12A), TNFRSF13B (TNFRSF 13B), TNFRSF13C (TNFRSF 13C), TNFRSF14 (TNFRSF 14), nerve growth factor receptor (NGFR, TNFRSF 16), 17 (21), and 19 (25 (TNFRSF).
In various embodiments, the receptor of the additional signaling agent is a TGF- β receptor. The TGF-beta receptor is a single pass serine/threonine kinase receptor. TGF-beta receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.
In various embodiments, the receptor of the additional signaling agent is an Ig superfamily receptor. Receptors in the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors in the Ig superfamily include, but are not limited to interleukin 1 receptors, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.
In various embodiments, the receptor of the additional signaling agent is a tyrosine kinase superfamily receptor. Receptors in the tyrosine kinase superfamily are well known in the art. There are approximately 58 known Receptor Tyrosine Kinases (RTKs) that fall into 20 subfamilies. Receptors in the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.
In one embodiment, the additional modified signaling agent is interferon alpha. In such embodiments, the modified ifnα agents have reduced affinity and/or activity for the IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified IFN- α agent has substantially reduced or eliminated affinity and/or activity for the IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chain.
Mutant forms of interferon alpha are known to those skilled in the art. In an illustrative embodiment, the modified signaling agent is a polypeptide having the amino acid sequence of SEQ ID NO:233 allelic form of ifnα2a.
In an illustrative embodiment, the modified signaling agent is a polypeptide having the amino acid sequence of SEQ ID NO:234 (which differs from ifnα2a at amino acid position 23).
In some embodiments, the IFN alpha 2 mutant (IFN alpha 2a or IFN alpha 2 b) at 144-154 in one or more amino acids, such as amino acid position 145, 148, 149 and/or 153 mutation. In some embodiments, the IFN alpha 2 mutant comprises selected from the group consisting of A145G, L153A, R A and M148A one or more mutations. Such mutants are described, for example, in WO2013/107791 and Piehler et al, (2000) J.biol. Chem,275:40425-33, the entire contents of all documents are hereby incorporated by reference.
In some embodiments, the IFN alpha 2 mutant has reduced affinity and/or activity for IFNAR 1. In some embodiments, the IFN alpha 2 mutant comprises selected from the group consisting of as described in WO 2010/030671F 64A, N65A, T69A, L80A, Y A and Y89A one or more mutations, the literature is incorporated by reference in its entirety.
In some embodiments, the IFN alpha 2 mutant comprises selected from the group consisting of WO2008/124086 as described in K133A, R144A, R A and L153A one or more mutations, the literature is incorporated by reference in its entirety.
In some embodiments, the IFN alpha 2 mutant comprises selected from the group consisting of WO2015/007520 and WO2010/030671 as described in R120E and R120E/K121E one or more mutations, the literature is incorporated by reference in its entirety. In such embodiments, the IFN alpha 2 mutant antagonizes wild type IFN alpha 2 activity. In such embodiments, the mutant IFN alpha 2 has reduced affinity and/or activity for IFNAR1, but retains the affinity and/or activity for IFNR 2.
In some embodiments, the human IFN alpha 2 mutant comprises (1) selected from the group consisting of R120E and R120E/K121E one or more mutations, without wishing to be bound by theory, the mutation can produce antagonistic effect; and (2) one or more mutations selected from K133A, R144A, R149A and L153A, which allow attenuation of the effect at IFNAR2, for example, without wishing to be bound by theory. In one embodiment, the human IFN alpha 2 mutant containing R120E and L153A.
In some embodiments, the human IFN alpha 2 mutant contains one or moreA plurality of mutations selected from L15A, A19W, R A, R23A, L A, F A, L30A, L30V, K31A, D A, R33K, R A, R33Q, H A, D40A, D114R, L117A, R120A, R134A, R144 145A, R145A, R148A, R149A, R152 a and N156A as disclosed in WO 2013/059885, the entire disclosure of which is hereby incorporated by reference. In some embodiments, the human IFN alpha 2 mutant comprises as disclosed in WO 2013/059885 in the mutant H57Y, E58N, Q S and/or L30A. In some embodiments, the human IFN- α2 mutant comprises the mutation H57Y, E58N, Q S and/or R33A as disclosed in WO 2013/059885. In some embodiments, the human IFN alpha 2 mutant comprises as disclosed in WO 2013/059885 mutations of H57Y, E58N, Q S and/or M148A. In some embodiments, the human IFN alpha 2 mutant comprises as disclosed in WO 2013/059885 in the mutant H57Y, E58N, Q S and/or L153A. In some embodiments, the human IFN alpha 2 mutant comprises as disclosed in WO 2013/059885 mutations of N65A, L80A, Y A and/or Y89A. In some embodiments, the human IFN alpha 2 mutant comprises as disclosed in WO 2013/059885 mutations of N65A, L80A, Y85A, Y A and/or D114A. In some embodiments, the human IFN alpha 2 mutant comprises selected from the group consisting of R144X 1 、A145X 2 And one or more mutations of R33A, wherein X 1 Selected from A, S, T, Y, L and I, and wherein X 2 Selected from G, H, Y, K and D. In some embodiments, the human ifnα2 mutant relative to the amino acid sequence of SEQ ID NO:233 or 234 comprises a member selected from R33A, T X 3 、R120E、R144X 1 A145X 2 One or more mutations of M148A, R149A and L153A, wherein X 1 Selected from A, S, T, Y, L and I, wherein X 2 Selected from G, H, Y, K and D, and wherein X 3 Selected from A and E.
In some embodiments, the additional modified signaling agent is ifnα1. In such embodiments, the modified ifnα1 signaling agent also has reduced affinity and/or activity for the IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified IFN alpha 1 agent on IFN alpha/beta receptor (IFNAR), i.e. IFNAR1 and/or IFNAR2 chain has substantially reduced or eliminated affinity and/or activity.
In some embodiments, the amino acid sequence relative to SEQ ID NO:450, the additional modified ifnα1 signaling agent is modified to have a mutation at one or more amino acids at positions L15, a19, R23, S25, L30, D32, R33, H34, Q40, D115, L118, K121, R126, E133, K134, K135, R145, a146, M149, R150, S153, L154, and N157. The mutation may optionally be a hydrophobic mutation and may for example be selected from alanine, valine, leucine and isoleucine. In some embodiments, the amino acid sequence relative to SEQ ID NO:450, said ifnα1 interferon is modified to have one or more mutations selected from the group consisting of: l15 19 23 25 30 33 33 33 34 40 118 121 121 121 121 126 126 145 145 145 145 145 145 145 145 145 145 145 145 145 145 145 145 145 145 145 145 145 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 149 149 153a and N157A. In some embodiments, the amino acid sequence relative to SEQ ID NO:450, the IFN alpha 1 mutant comprises one or more selected from the group consisting of: L30A/H58Y/E59N/Q62S, R A/H58Y/E59N/Q62S, M A/H58Y/E59N/Q62S, L A/H58Y/E59N/Q62S, R145A/H58Y/E59N/Q62S, D A/R121A, L A/R121A, L A/R121A/K122A, R A/K122A and R121E/K122E.
In one embodiment, the additional modified ifnα1 signaling agent or variant thereof is modified relative to SEQ ID NO:450 have one or more mutations at amino acid positions C1, C29, C86, C99 or C139. In this regard, beilharz et al Journal of interferon research 6.6.6 (1986): 677-685 (which is hereby incorporated by reference in its entirety) describe various ifnα1 mutations that can be used to introduce into the modified ifnα1 of the present invention. The mutation at position C86 may be, for example, C86S or C86A or C86Y. These C86 mutants of ifnα1 are referred to as reduced cysteine-based aggregation mutants. In some embodiments, the amino acid sequence relative to SEQ ID NO:450, IFN alpha 1 variants including the C1, C86 and C99 mutations.
In one embodiment, the additional modified signaling agent is interferon beta. In such embodiments, the modified interferon beta agent also has reduced affinity and/or activity for the IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified interferon beta agent has substantially reduced or eliminated affinity and/or activity for the IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chain.
In an illustrative embodiment, the modified additional signaling agent is IFNbeta. In various embodiments, the IFN beta comprises IFN beta functional derivatives, analogues, precursors, isoforms, splice variants or fragments. In various embodiments, the IFN beta from any species of IFN beta. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a modified version of mouse ifnβ. In another embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a modified version of human ifnβ. Human IFN beta is composed of 166 amino acid residues of molecular weight of about 22kDa polypeptide. The amino acid sequence of human IFN beta is SEQ ID NO:277.
in some embodiments, the human IFN beta is in the human IFN beta glycosylation form of IFN beta 1a. In some embodiments, the human IFN beta is in the human IFN beta non glycosylation form of IFN beta-1 b, it has Met-1 deletion and Cys-17 to Ser mutation.
In various embodiments, the modified IFN beta has one or more mutations, the one or more mutations reduce the IFN alpha 1 variants on IFNAR1 subunit binding or affinity. In one embodiment, the modified IFN beta at IFNAR1 with reduced affinity and/or activity. In various embodiments, the modified IFN beta is human IFN beta, and in the position of F67, R71, L88, Y92, I95, N96, K123 and R124 has one or more mutations. In some embodiments, the one or more mutations is a substitution selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y3592S, I95A, N G, K123G and R124G. In one embodiment, the modified IFN beta contains F67G mutation. In one embodiment, the modified IFN beta contains K123G mutation. In one embodiment, the modified IFN beta contains F67G and R71A mutation. In one embodiment, the modified IFN beta contains L88G and Y92G mutation. In one embodiment, the modified IFN beta contains Y92G, I A and N96G mutations. In one embodiment, the modified IFN beta contains K123G and R124G mutation. In one embodiment, the modified IFN beta contains F67G, L G and Y92G mutation. In one embodiment, the modified IFN beta contains F67S, L S and Y92S mutation.
In some embodiments, the modified IFN beta has one or more mutations, the one or more mutations reduce the IFN alpha 1 variants on IFNAR2 subunit binding or affinity. In one embodiment, the modified IFN beta at IFNAR2 with reduced affinity and/or activity. In various embodiments, the modified IFN beta is human IFN beta, and in the position of W22, R27, L32, R35, V148, L151, R152 and Y155 has one or more mutations. In some embodiments, the one or more mutations are substitutions selected from the group consisting of W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G and Y155G. In one embodiment, the modified IFN beta contains W22G mutation. In one embodiment, the modified IFN beta contains L32A mutation. In one embodiment, the modified IFN beta contains L32G mutation. In one embodiment, the modified IFN beta contains R35A mutation. In one embodiment, the modified IFN beta contains R35G mutation. In one embodiment, the modified IFN beta contains V148G mutation. In one embodiment, the modified IFN beta contains R152A mutation. In one embodiment, the modified IFN beta contains R152G mutation. In one embodiment, the modified IFN beta contains Y155G mutation. In one embodiment, the modified IFN beta contains W22G and R27G mutation. In one embodiment, the modified IFN beta contains L32A and R35A mutation. In one embodiment, the modified IFN beta contains L151G and R152A mutation. In one embodiment, the modified IFN beta contains V148G and R152A mutation.
In some embodiments, the modified IFN beta has one or more of the following mutations: R35A, R35T, E42K, M I, G78S, A Y, A142T, E149K and R152H. In some embodiments, the modified IFN beta has one or more of the following mutations: R35A, R35T, E42K, M I, G78S, A Y, A142T, E149K and R152H, in combination with C17S or C17A.
In some embodiments, the modified IFN beta has one or more of the following mutations: R35A, R35T, E42K, M I, G78S, A Y, A142T, E149K and R152H, in combination with any other ifnβ mutation described herein.
The crystal structure of human IFNbeta is known and described in Karpusas et al, (1998) PNAS,94 (22): 11813-11818. In particular, the structure of human IFNbeta has been shown to include five alpha helices (i.e., A, B, C, D and E) and four loop regions (i.e., AB, BC, CD and DE loops) connecting these helices. In various embodiments, the modified IFN beta in A, B, C, D, E spiral and/or AB, BC, CD and DE ring in one or more mutations, the one or more mutations reduce the modified IFN beta at the treatment receptor such as IFNAR binding affinity or activity. Illustrative mutations are described in WO 2000/023914 and US20150011732, the entire contents of which are hereby incorporated by reference. In illustrative embodiments, the modified IFN beta is in amino acid position 15, 16, 18, 19, 22 and/or 23 containing alanine substituted human IFN beta. In an illustrative embodiment, the modified IFN beta is in amino acid position 28-30, 32 and 33 containing alanine substituted human IFN beta. In one illustrative embodiment, the modified IFN beta is in amino acid position 36, 37, 39 and 42 containing alanine substituted human IFN beta. In an illustrative embodiment, the modified IFN beta is a human IFN beta, the human IFN beta at amino acid positions 64 and 67 containing alanine substitution and at position 68 containing serine substitution. In one illustrative embodiment, the modified IFN beta is in amino acid position 71-73 containing alanine substituted human IFN beta. In an illustrative embodiment, the modified IFN beta is in amino acid position 92, 96, 99 and 100 containing alanine substituted human IFN beta. In an illustrative embodiment, the modified IFN beta is a human IFN beta comprising alanine substitutions at amino acid positions 128, 130, 131 and 134. In an illustrative embodiment, the modified IFN beta is in amino acid position 149, 153, 156 and 159 containing alanine substituted human IFN beta. In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at W22, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at R27, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at W22 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R27 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at L32, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at R35, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at L32 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), isoleucine (I), methionine (M), and valine (V); and a mutation at R35, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at F67 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at R71 which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at F67 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R71 which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at L88, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at Y92, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at F67 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V); and a mutation at Y92 which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at L88, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V); and a mutation at Y92 which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at I95 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), methionine (M), and valine (V); and a mutation at Y92 which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at N96, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V); and a mutation at Y92 which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at Y92 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at I95 which is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), methionine (M) and valine (V); and a mutation at N96, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at K123 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at R124, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at K123 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R124, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at L151, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at R152 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at L151 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), isoleucine (I), methionine (M), and valine (V); and a mutation at R152 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at V148, said mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I) and methionine (M).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at V148 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at R152 that is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M), and valine (V).
In some embodiments, the mutant ifnβ comprises the amino acid sequence of SEQ ID NO:277 and a mutation at Y155, which mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).
In some embodiments, the invention relates to a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprising: (a) A modified ifnβ having the amino acid sequence of SEQ ID NO:277 and a mutation at position W22, wherein the mutation is an aliphatic hydrophobic residue; and (b) one or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest, the modified ifnβ and the one or more targeting moieties optionally being linked to one or more linkers. In various embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M and V. In various embodiments, the mutation at position W22 is G.
Other illustrative IFN beta mutants are provided in PCT/EP2017/061544, the entire disclosure of which is incorporated herein by reference.
In some embodiments, the modified additional signaling agent is interferon gamma. In such embodiments, the modified interferon gamma agent has reduced affinity and/or activity for the interferon gamma receptor (IFNGR), i.e., IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon gamma agent has substantially reduced or eliminated affinity and/or activity for an interferon gamma receptor (IFNGR), i.e., IFNGR1 and IFNGR2 chains.
In some embodiments, the modified additional signaling agent is consensus interferon. Consensus interferon can be generated by scanning the sequences of several human non-allelic ifnα subtypes and assigning the most frequently observed amino acids at each respective position. The consensus interferon differs from IFN alpha 2b by 20 out of 166 amino acids (88% homology), and shows more than 30% amino acid position identity compared to IFN beta. In various embodiments, the consensus interferon comprises the following amino acid sequence SEQ ID NO:278.
In some embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO:279, which amino acid sequence corresponds to the amino acid sequence SEQ ID NO:278 differ by one amino acid, i.e. SEQ ID NO:279 lacks SEQ ID NO: 278.
In various embodiments, the consensus interferon comprises a modified version of the consensus interferon, i.e., a consensus interferon variant, as a signaling agent. In various embodiments, the consensus interferon encompasses functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of the consensus interferon.
In one embodiment, the consensus interferon variant is selected from consensus interferon variants disclosed in U.S. Pat. nos. 4,695,623, 4,897,471, 5,541,293 and 8,496,921, the entire contents of all of which are hereby incorporated by reference. For example, the consensus interferon variant may comprise IFN-CON as disclosed in U.S. Pat. Nos. 4,695,623, 4,897,471 and 5,541,293 2 Or IFN-CON 3 Amino group of (2)Acid sequence. In one embodiment, the consensus interferon variant comprises IFN-CON 2 Amino acid sequence of (a): SEQ ID NO:280.
in one embodiment, the consensus interferon variant comprises IFN-CON 3 Amino acid sequence of (a): SEQ ID NO:281.
in one embodiment, the consensus interferon variant comprises the amino acid sequence of any of the variants disclosed in U.S. patent No. 8,496,921. For example, the consensus variant may comprise the amino acid sequence SEQ ID NO:282.
in another embodiment, the consensus interferon variant may comprise the amino acid sequence of SEQ ID NO:283.
in some embodiments, the consensus interferon variant may be pegylated, i.e., comprise a PEG moiety. In one embodiment, the consensus interferon variant may comprise a sequence linked to the sequence set forth in SEQ ID NO:283 at the S156C position.
In some embodiments, the engineered interferon is a variant of human IFN alpha 2a, insertion of Asp at about position 41 of the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQ ID NO: 284) results in Glu-Glu-Phe-Asp-Gly-Asn-Gln (SEQ ID NO: 285) (which results in renumbering of the sequence relative to the IFN alpha 2a sequence) and mutations as follows: arg23Lys, leu26Pro, glu53Gln, thr54Ala, pro56Ser, asp86Glu, ile104Thr, gly106Glu, thr110Glu, lys117Asn, arg125Lys and Lys136Thr. All embodiments described herein for consensus interferon apply equally to this engineered interferon.
In some embodiments, the additional modified signaling agent is Vascular Endothelial Growth Factor (VEGF). VEGF is a potent growth factor that plays a major role in physiological and pathological angiogenesis, regulates vascular permeability, and can act as a growth factor on cells expressing VEGF receptors. Additional functions include, among others, stimulation of cell migration in macrophage lineages and endothelial cells. There are several members of the VEGF growth factor family, and at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3). Members of the VEGF family may bind to and activate more than one VEGFR type. For example, VEGF-A binds to VEGFR-1 and VEGFR-2, while VEGF-C binds to VEGFR-2 and VEGFR-3.VEGFR-1 and VEGFR-2 activation regulate angiogenesis, whereas VEGFR-3 activation is associated with lymphangiogenesis. The primary pro-angiogenic signal is generated by VEGFR-2 activation. VEGFR-1 activation was reported to be likely associated with negative effects in angiogenesis. It has also been reported that VEGFR-1 signaling is important for tumor progression in vivo via bone marrow-derived VEGFR-1 positive cells (which contribute to the formation of a pre-metastatic niche in bone). Several therapeutic antibody therapies based on VEGF-Sub>A directed/neutralising have been developed, primarily for the treatment of various human tumors that rely on angiogenesis. These therapies are not without side effects. This may be surprising given that these therapies act as inhibitors of general non-cell/tissue specific VEGF/VEGFR interactions. Thus, it is desirable to limit VEGF (e.g., VEGF-Sub>A)/VEGFR-2 inhibition to specific target cells (e.g., tumor vascular endothelial cells).
In some embodiments, the VEGF is VEGF-A, VEGF-B, VEFG-C, VEGF-D or VEGF-E and isoforms thereof, including various isoforms of VEGF-A, such as VEGF 121 、VEGF 121 b、VEGF 145 、VEGF 165 、VEGF 165 b、VEGF 189 And VEGF (vascular endothelial growth factor) 206 . In some embodiments, the modified signaling agent has reduced affinity and/or activity for VEGFR-1 (Flt-1) and/or VEGFR-2 (KDR/Flk-1). In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-1 (Flt-1) and/or VEGFR-2 (KDR/Flk-1). In one embodiment, the modified signaling agent has reduced affinity and/or activity for VEGFR-2 (KDR/Flk-1) and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1 (Flt-1). Such embodiments may be used, for example, in wound healing methods or in the treatment of ischemia-related diseases (without wishing to be bound by theory, these diseases are mediated by the effects of VEGFR-2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1 (Flt-1) associated with cancer and pro-inflammatory activity is avoided. In various embodiments, VEGFR-1 (Flt-1) acts as a decoy receptor, thereby greatly reducing or eliminating affinity at the receptor, thereby avoiding chelation of the therapeutic agent. In one embodiment, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-1 (Flt-1) and/or substantially reduced or eliminated affinity and/or activity for VEGFR-2 (KDR/Flk-1). In some embodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signaling agent has reduced affinity and/or activity for VEGFR-3. Alternatively, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-3.
Pro-angiogenic therapies are also important in a variety of diseases (e.g., ischemic heart disease, hemorrhage, etc.), and include VEGF-based therapies. Activation of VEGFR-2 has a pro-angiogenic effect (on endothelial cells). Activation of VEFGR-1 stimulates migration of inflammatory cells (including, for example, macrophages) and leads to inflammation-associated high vascular permeability. Activation of VEFGR-1 can also promote bone marrow-related tumor niche formation. Thus, in such cases, it would be desirable to select a VEGF-based therapeutic for VEGFR-2 activation. In addition, cell-specific targeting of endothelial cells, for example, is desired.
In some embodiments, the additional modified signaling agent has reduced affinity and/or activity (e.g., antagonism) for VEGFR-2 and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1. For example, when tumor vascular endothelial cells are targeted via a targeting moiety that binds to a tumor endothelial cell marker (e.g., PSMA, etc.), such constructs will specifically inhibit VEGFR-2 activation on such marker positive cells without activating VEGFR-1 in transit and on the target cells (if the activity is eliminated), thereby eliminating induction of an inflammatory response. This will provide Sub>A more selective and safer anti-angiogenic therapy for many tumor types than VEGF-Sub>A neutralization therapies.
In some embodiments, the additional modified signaling agent has reduced affinity and/or activity (e.g., agonism) for VEGFR-2 and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1. In some embodiments, by targeting vascular endothelial cells, such constructs promote angiogenesis without causing induction of VEGFR-1 related inflammatory responses. Thus, such constructs will have a targeted pro-angiogenic effect and substantially reduce the risk of side effects caused by systemic activation of VEGFR-2 as well as VEGFR-1.
In an illustrative embodiment, the modified signaling agent is VEGF 165 (wild type) the VEGF 165 Has the amino acid sequence SEQ ID NO:235.
in another illustrative embodiment, the additional modified signaling agent is VEGF 165b (wild type) the VEGF 165b Has the amino acid sequence SEQ ID NO:236.
in these embodiments, the modified signaling agent has a mutation at amino acid I83 (e.g., a substitution mutation at I83, such as I83K, I83R or I83H). Without wishing to be bound by theory, it is believed that such mutations may result in reduced receptor binding affinity. See, for example, U.S. patent No. 9,078,860, the entire contents of which are hereby incorporated by reference.
In one embodiment, the additional modified signaling agent is tnfα. TNF is a pleiotropic cytokine that has a number of different functions including regulating cell growth, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and migration, inflammation, and septic shock. It binds to two unique membrane receptors on target cells: TNFR1 (p 55) and TNFR2 (p 75). TNFR1 exhibits a very broad expression pattern, whereas TNFR2 is preferentially expressed on certain populations of lymphocytes, tregs, endothelial cells, certain neurons, microglia, cardiomyocytes and mesenchymal stem cells. Very unique biological pathways are activated in response to receptor activation, but there is also some overlap. In general, without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), while TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFkB pathway). The administration of TNF is systemically toxic and this is mainly due to TNFR1 dosing. However, it should be noted that activation of TNFR2 is also associated with a variety of activities, and as with TNFR1, control of TNF targeting and activity is important in the context of developing TNF-based therapeutics.
In some embodiments, the additional modified signaling agent has reduced affinity and/or activity for TNFR1 and/or TNFR 2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TNFR1 and/or TNFR 2. TNFR1 is expressed in most tissues and is involved in cell death signaling, while TNFR2 is in contrast involved in cell survival signaling. Thus, in embodiments directed to methods of treating cancer, the modified signaling agent has reduced affinity and/or activity for TNFR1 and/or substantially reduced or eliminated affinity and/or activity for TNFR 2. In these embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can be targeted to cells in need of apoptosis, e.g., tumor cells or tumor vascular endothelial cells. In embodiments directed to methods of promoting cell survival, for example, in neurogenesis for treating neurodegenerative disorders, the modified signaling agent has reduced affinity and/or activity for TNFR2 and/or substantially reduced or eliminated affinity and/or activity for TNFR 1. In other words, in some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, comprise a modified TNF- α agent that allows for a beneficial death or survival signal.
In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has modified TNF having reduced affinity and/or activity for TNFR1 and/or substantially reduced or eliminated affinity and/or activity for TNFR 2. In some embodiments, promotion of T against wild-type TNF and/or carry-over aloneSuch chimeras are more potent apoptosis inducers than chimeras of one or more mutations that have reduced affinity and/or activity of NFR 1. In some embodiments, such chimeras are useful for inducing tumor cell death or tumor vascular endothelial cell death (e.g., for treating cancer). Furthermore, in some embodiments, for example, these chimeras avoid or reduce activation of T via TNFR2 reg Cells, thus further supporting TNFR 1-mediated antitumor activity in vivo.
In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, has modified TNF having reduced affinity and/or activity for TNFR2 and/or substantially reduced or eliminated affinity and/or activity for TNFR 1. In some embodiments, such chimeras are more potent cell survival activators in some cell types that may be the target of a particular treatment under stimulation of neurogenesis, including but not limited to various disease settings. In addition, the TNFR 2-preferred chimeras are also useful in the treatment of autoimmune diseases (e.g., crohn's disease, diabetes, MS, colitis, etc., and numerous other autoimmune diseases described herein). In some embodiments, the chimera targets autoreactive T cells. In some embodiments, the chimera promotes T reg Cell activation and indirect suppression of cytotoxic T cells.
In some embodiments, the chimera causes death of autoreactive T cells, for example, by activating TNFR2 and/or avoiding TNFR1 (e.g., modified TNF having reduced affinity and/or activity for TNFR2 and/or substantially reduced or eliminated affinity and/or activity for TNFR 1). Without wishing to be bound by theory, these autoreactive T cells have their apoptosis/survival signal altered, for example, by NFkB pathway activity/signaling changes.
In some embodiments, TNFR 2-based chimeras have additional therapeutic applications in a variety of diseases, including various autoimmune diseases, heart diseases, demyelinating and neurodegenerative disorders, and infectious diseases, among others.
In one embodiment, the wild-type tnfα has the amino acid sequence of SEQ ID NO:237.
in such embodiments, the modified tnfα agent has mutations at one or more amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, thereby producing a modified tnfα with reduced receptor binding affinity. See, for example, U.S. patent No. 7,993,636, the entire contents of which are hereby incorporated by reference.
In some embodiments, the modified human tnfα moiety has mutations at one or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, a109, P113, Y115, E127, N137, D143, and a145 as described, for example, in WO/2015/007903, the entire contents of which are hereby incorporated by reference (numbering according to the human TNF sequence, genbank accession No. BAG70306, version BAG70306.1, G1: 197692685). In some embodiments of the present invention, in some embodiments, the modified human tnfα moiety has a substitution mutation selected from R32G, N34G, Q3573G, L75G, L A, L75S, T77A, S86G, Y87Q, Y L, Y87 7991G, V91A, I97A, I97S, T105G, P/G, P115/G, P/143/G, P/145G and a 145T. In one embodiment, the human tnfα moiety has a mutation selected from Y87Q, Y87L, Y a and Y87F. In another embodiment, the human tnfα moiety has a mutation selected from the group consisting of I97A, I Q and I97S. In another embodiment, the human tnfα moiety has a mutation selected from Y115A and Y115G.
In some embodiments, the modified tnfα agent has one or more mutations selected from N39Y, S147Y and Y87H as described in WO2008/124086, the entire contents of which are hereby incorporated by reference.
In one embodiment, the additional modified signaling agent is tnfβ. TNF beta may form homotrimers or heterotrimers with LT-beta (LT- α1β2). In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TNFR1 and/or TNFR2 and/or herpes virus entry mediator (HEVM) and/or LT- βR.
In one embodiment, the wild-type tnfβ has the amino acid sequence of SEQ ID NO:238.
in such embodiments, the modified tnfβ agent may comprise a mutation at one or more amino acids at positions 106-113, thereby producing a modified tnfβ having reduced receptor binding affinity for TNFR 2. In one embodiment, the modified signaling agent has one or more substitution mutations at amino acid positions 106-113. In an illustrative embodiment, the substitution mutation is selected from the group consisting of Q107E, Q107D, S E, S D, Q107R, Q107N, Q E/S106E, Q107E/S106D, Q107D/S106E and Q107D/S106D. In another embodiment, the modified signaling agent has an insertion of about 1 to about 3 amino acids at positions 106-113.
In some embodiments, the additional modified agent is a TNF family member (e.g., TNF- α, TNF- β), which may be in the form of a single chain trimer as described in WO 2015/007903, the entire contents of which are incorporated by reference.
In some embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β) that has reduced affinity and/or activity, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations) at TNFR1, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference. In these embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β), which also optionally has substantially reduced or eliminated affinity and/or activity for TNFR 2. In some embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β) that has reduced affinity and/or activity, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations) at TNFR2, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference. In these embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β), which also optionally has substantially reduced or eliminated affinity and/or activity for TNFR 1. Constructs of such embodiments may be used, for example, in methods of suppressing TNF responses in a cell-specific manner. In some embodiments, the antagonistic TNF family member (e.g., TNF- α, TNF- β) is a single chain trimeric version as described in WO 2015/007903.
In one embodiment, the additional modified signaling agent is TRAIL. In some embodiments, the modified TRAIL agent has reduced affinity and/or activity for DR4 (TRAIL-RI) and/or DR5 (TRAIL-RII) and/or DcR1 and/or DcR 2. In some embodiments, the modified TRAIL agent has substantially reduced or eliminated affinity and/or activity for DR4 (TRAIL-RI) and/or DR5 (TRAIL-RII) and/or DcR1 and/or DcR 2.
In one embodiment, the wild-type TRAIL has the amino acid sequence of SEQ ID NO:239.
in such embodiments, the modified TRAIL agent may comprise mutations at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-1220, K224-A226, W231, E236-L239, E249-K251, T261-H264 and H270-E271 (numbering according to human sequence, genbank accession number NP-003801, version 10NP-003801.1, G1:4507593; see above).
In one embodiment, the additional modified signaling agent is tgfα. In such embodiments, the modified tgfα agents have reduced affinity and/or activity for Epidermal Growth Factor Receptor (EGFR). In some embodiments, the modified tgfα agents have substantially reduced or eliminated affinity and/or activity for Epidermal Growth Factor Receptor (EGFR).
In one embodiment, the additional modified signaling agent is tgfβ. In such embodiments, the modified signaling agent has reduced affinity and/or activity for TGFBR1 and/or TGFBR2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TGFBR1 and/or TGFBR2. In some embodiments, the modified signaling agent optionally has reduced or substantially reduced or eliminated affinity and/or activity for TGFBR3, which TGFBR3 may act as a reservoir for ligands of TGF- β receptors, without wishing to be bound by theory. In some embodiments, tgfβ may be more prone to TGFBR1 relative to TGFBR2, or tgfβ may be more prone to TGFBR2 relative to TGFBR 1. Similarly, without wishing to be bound by theory, LAP may act as a reservoir for ligands of TGF- β receptors. In some embodiments, the modified signaling agent has reduced affinity and/or activity for TGFBR1 and/or TGFBR2 and/or substantially reduced or eliminated affinity and/or activity for a potentially-related peptide (LAP). In some embodiments, such chimeras may be used in Kamurati-Engelmann disease or other diseases associated with inappropriate TGF beta signaling.
In some embodiments, the additional modified agent is a TGF family member (e.g., tgfα, tgfβ) that has reduced affinity and/or activity, i.e., antagonistic activity, at one or more of TGFBR1, TGFBR2, TGFBR3 (e.g., natural antagonistic activity or antagonistic activity resulting from one or more mutations, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference). In these embodiments, the modified agent is a tgffamily member (e.g., tgfα, tgfβ), which also optionally has substantially reduced or eliminated affinity and/or activity at one or more of TGFBR1, TGFBR2, TGFBR 3.
In some embodiments, the additional modified agent is a TGF family member (e.g., tgfα, tgfβ) that has reduced affinity and/or activity, i.e., antagonistic activity, at TGFBR1 and/or TGFBR2 (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference). In these embodiments, the modified agent is a TGF family member (e.g., tgfα, tgfβ), which also optionally has substantially reduced or eliminated affinity and/or activity at TGFBR 3.
In one embodiment, the additional modified signaling agent is IL-1. In one embodiment, the modified signaling agent is IL-1α or IL-1β. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-1R1 and/or IL-1 RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-1R1 and/or IL-1 RAcP. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-1R2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-1R2. For example, in some embodiments, the modified IL-1 agents of the invention avoid interactions at IL-1R2 and thus substantially reduce their function as attractants and/or recipients of therapeutic agents.
In one embodiment, the wild-type IL-1β has the amino acid sequence of SEQ ID NO:240.
IL-1β is a pro-inflammatory cytokine and an important modulator of the immune system. It is a potent activator of the CD 4T cell response, increasing the proportion of Th17 cells and expanding cells that produce IFNγ and IL-4. IL-1β is also a potent modulator of CD8+ T cells, enhancing antigen-specific CD8+ T cell expansion, differentiation, migration to the periphery and memory. IL-1. Beta. Receptors comprise IL-1R1 and IL-1R2. Binding to IL-1R1 and signaling via IL-1R1 constitute the mechanism by which IL-1 beta mediates a wide variety of its biological (and pathological) activities. IL1-R2 may act as a decoy receptor, thereby reducing the availability of IL-1β for interaction and signaling via IL-1R 1.
In some embodiments, the modified IL-1β has reduced affinity and/or activity (e.g., agonistic activity) for IL-1R 1. In some embodiments, the modified IL-1β has substantially reduced or eliminated affinity and/or activity for IL-1R 2. In such embodiments, there is inducible or recoverable IL-1β/IL-1R1 signaling, and loss of therapeutic chimera at IL-R2 is prevented, and thus the dose of IL-1 required is reduced (e.g., relative to wild-type or chimeras carrying only attenuating mutations to IL-R1). Such constructs may be used, for example, in methods of treating cancer, including, for example, stimulating the immune system to mount an anti-cancer response.
In some embodiments, the modified IL-1β has reduced affinity and/or activity (e.g., antagonistic activity, such as natural antagonistic activity or antagonistic activity due to one or more mutations) for IL-1R1, see, e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference. In some embodiments, the modified IL-1β has substantially reduced or eliminated affinity and/or activity for IL-1R 2. In such embodiments, there is non-inducible or non-recoverable IL-1β/IL-1r1 signaling and loss of therapeutic chimera at IL-R2 is prevented, and thus the dose of IL-1β required is reduced (e.g., relative to wild-type or chimeras carrying only attenuating mutations to IL-R1). Such constructs may be used, for example, in methods of treating autoimmune diseases, including, for example, suppressing the immune system.
In such embodiments, the modified signaling agent has a deletion of amino acids 52-54, which results in modified human IL-1 β having reduced binding affinity for type I IL-1R and reduced biological activity. See, for example, WO 1994/000491, the entire contents of which are hereby incorporated by reference. In some embodiments, the modified human IL-1β has one or more substitution mutations selected from the group consisting of A117G/P118G, R120G/L126G, R127G, R130G, R131G, R132G/Q138G, R145/146G, R145A/L147G, R148G/Q150G/D151G, R152/162A/Q164G, R166E/E167G, R169G/D170G, R172G, R174/D170G, R208G, R209G, R209A/K210G, R219/G, R221S/N224 52224S/K225G, R244G, R245Q (wherein X may be any amino acid change, e.g., non-conservative changes) to exhibit reduced binding to IL-1R, as described, for example, in WO2015/007542 and WO/2015/007536, the entire contents of which are hereby incorporated by reference (numbering based on the human IL-1β sequence, genbank accession No. np_000567, version NP-G, R, G1: G, R). In some embodiments, the modified human IL-1β may have one or more mutations selected from the group consisting of R120A, R120G, Q130 8235 146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209D, K219S, K219Q, E S and E221K. In one embodiment, the modified human IL-1β comprises mutations Q131G and Q148G. In one embodiment, the modified human IL-1 β comprises mutations Q148G and K208E. In one embodiment, the modified human IL-1β comprises mutations R120G and Q131G. In one embodiment, the modified human IL-1β comprises mutations R120G and H146G. In one embodiment, the modified human IL-1 β comprises mutations R120G and K208E. In one embodiment, the modified human IL-1 β comprises mutations R120G, F162A and Q164E.
In one embodiment, the additional modified signaling agent is IL-3. In some embodiments, the modified signaling agent has reduced affinity and/or activity for an IL-3 receptor that is a heterodimer of a unique alpha chain paired with a common beta (βc or CD 131) subunit. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for an IL-3 receptor that is a heterodimer with a unique alpha chain paired with a common beta (βc or CD 131) subunit.
In one embodiment, the additional modified signaling agent is IL-4. In such embodiments, the modified signaling agent has reduced affinity and/or activity for type 1 and/or type 2 IL-4 receptors. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for type 1 and/or type 2 IL-4 receptors. Type 1 IL-4 receptors are composed of IL-4Rα subunits with a common gamma chain and bind specifically to IL-4. Type 2 IL-4 receptors include IL-4Rα subunits that bind to a different subunit known as IL-13Rα 1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for a type 2 IL-4 receptor.
In one embodiment, the wild-type IL-4 has the amino acid sequence of SEQ ID NO:242.
in such embodiments, the modified IL-4 agent has one or more mutations at amino acids R121 (R121A, R121D, R121E, R121F, R121H, R I, R121K, R121N, R P, R121T, R W), E122 (E122F), Y124 (Y124A, Y124Q, Y124R, Y124S, Y T) and S125 (S125A). Without wishing to be bound by theory, it is believed that these modified IL-4 agents maintain type I receptor-mediated activity, but significantly reduce other receptor-mediated biological activity. See, for example, U.S. patent No. 6,433,157, the entire contents of which are hereby incorporated by reference.
In one embodiment, the additional modified signaling agent is IL-6.IL-6 signals through a cell surface type I cytokine receptor complex that includes a ligand binding IL-6R chain (CD 126) and a signal transduction component gp130.IL-6 may also bind to soluble forms of IL-6R (sIL-6R), the latter being the extracellular portion of IL-6R. The sIL-6R/IL-6 complex may be involved in neurite outgrowth and neuronal survival, and thus may be important in nerve regeneration by remyelination. Thus, in some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-6R/gp130 and/or sIL-6R. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-6R/gp130 and/or sIL-6R.
In one embodiment, the wild-type IL-6 has the amino acid sequence of SEQ ID NO:243.
in such embodiments, the modified signaling agent has one or more mutations at amino acids 58, 160, 163, 171, or 177. Without wishing to be bound by theory, it is believed that these modified IL-6 agents exhibit reduced binding affinity for IL-6rα and reduced biological activity. See, for example, WO 97/10338, the entire contents of which are hereby incorporated by reference.
In one embodiment, the additional modified signaling agent is IL-10. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-10 receptor 1 and IL-10 receptor 2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-10 receptor 1 and IL-10 receptor 2.
In one embodiment, the additional modified signaling agent is IL-11. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-11Rα and/or IL-11Rβ and/or gp 130. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-11Rα and/or IL-11Rβ and/or gp 130.
In one embodiment, the additional modified signaling agent is IL-12. In this embodiment, the modified signaling agent has reduced affinity and/or activity for IL-12Rβ1 and/or IL-12Rβ2. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-12Rβ1 and/or IL-12Rβ2.
In one embodiment, the additional modified signaling agent is IL-13. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-4 receptor (IL-4Rα) and IL-13Rα1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-4 receptor (IL-4Rα) or IL-13Rα1.
In one embodiment, the wild-type IL-13 has the amino acid sequence of SEQ ID NO:244.
in such embodiments, the modified IL-13 agent has one or more mutations at amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114. Without wishing to be bound by theory, it is believed that these modified IL-13 agents exhibit reduced biological activity. See, for example, WO 2002/018422, the entire contents of which are hereby incorporated by reference.
In one embodiment, the signaling agent is wild-type or modified IL-15. In various embodiments, the modified IL-15 has reduced affinity and/or activity for an interleukin 15 receptor.
In one embodiment, the wild-type IL-15 has the following amino acid sequence:
in such embodiments, the modified IL-15 agent is relative to SEQ ID NO:292 has one or more mutations at amino acids S7, D8, K10, K11, E46, L47, V49, I50, D61, N65, L66, I67, I68, L69, N72, Q108.
In one embodiment, the additional modified signaling agent is IL-18. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-18Rα and/or IL-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-18Rα and/or IL-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for type II IL-18rα, i.e., an IL-18rα isoform lacking the TIR domain required for signaling.
In one embodiment, the wild-type IL-18 has the amino acid sequence of SEQ ID NO:245.
in such embodiments, the modified IL-18 agent may comprise one or more mutations at an amino acid or amino acid region selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183, and Q190-N191 as described in WO/2015/007542, the entire contents of which are hereby incorporated by reference (numbering based on human IL-18 sequence, genbank accession number AAV38697, version AAV38697.1, G1: 54696650).
In one embodiment, the additional modified signaling agent is IL-33. In such embodiments, the modified signaling agent has reduced affinity and/or activity for ST-2 receptors and IL-1 RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for ST-2 receptor and IL-1 RAcP.
In one embodiment, the wild-type IL-33 has the amino acid sequence of SEQ ID NO:246.
in such embodiments, the modified IL-33 agent may comprise one or more mutations at an amino acid or amino acid region selected from the group consisting of I113-Y122, S127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227, and T260-E269 as described in WO/2015/007542, the entire contents of which are hereby incorporated by reference (numbering based on human sequences, genbank accession number NP-254274, version NP-254274.1, G1: 15559209).
In one embodiment, the modified signaling agent is Epidermal Growth Factor (EGF). EGF is a member of the family of potent growth factors. Members include EGF, HB-EGF, and other members such as TGF alpha, amphiregulin, neuregulin, epithelial regulatory protein, beta cell protein. EGF family receptors include EGFR (ErbB 1), erbB2, erbB3 and ErbB4. These receptors may act as homodimer and/or heterodimeric receptor subtypes. Different EGF family members exhibit different selectivities for various receptor subtypes. For example, EGF is associated with ErbB1/ErbB1, erbB1/ErbB2, erbB4/ErbB2 and some other heterodimeric subtypes. HB-EGF has a similar pattern, but it is also related to ErbB 4/4. Modulation of EGF (EGF-like) growth factor signaling, either positively or negatively, is of considerable therapeutic interest. For example, inhibition of EGFR signaling is of interest in the treatment of various cancers where EGFR signaling constitutes the primary growth-promoting signal. Alternatively, stimulation of EGFR signaling is of therapeutic interest, for example, in promoting wound healing (acute and chronic), oral mucositis (a major side effect of various cancer therapies including, but not limited to, radiation therapy).
In some embodiments, the additional modified signaling agent has reduced affinity and/or activity for ErbB1, erbB2, erbB3, and/or ErbB 4. Such embodiments may be used, for example, in methods of treating wounds. In some embodiments, the modified signaling agent binds to one or more of ErbB1, erbB2, erbB3, and ErbB4 and antagonizes the activity of these receptors. In such embodiments, the modified signaling agents have reduced affinity and/or activity for ErbB1, erbB2, erbB3, and/or ErbB4, which allows antagonizing the activity of these receptors in a attenuated manner. Such embodiments may be used, for example, in the treatment of cancer. In one embodiment, the modified signaling agent has reduced affinity and/or activity for ErbB 1. ErbB1 is a therapeutic target for kinase inhibitors-most of these kinase inhibitors have side effects because of their low selectivity (e.g., gefitinib, erlotinib, afatinib, bucatinib (brinatinib), and Ai Keti ni (icotinib)). In some embodiments, reduced antagonistic ErbB1 signaling is more targeted and has fewer side effects than other agents that target the EGF receptor.
In some embodiments, the additional modified signaling agent has reduced affinity and/or activity (e.g., antagonism, e.g., natural antagonism or antagonism due to one or more mutations, see e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference) for ErbB1 and/or substantially reduced or eliminated affinity and/or activity for ErbB4 or other subtypes with which it may interact. By specific targeting via the targeting moiety, cell selective suppression of ErbB1/ErbB1 receptor activation (antagonism, e.g., natural antagonism or antagonism due to one or more mutations, see e.g., WO 2015/007520, the entire contents of which are hereby incorporated by reference) will be achieved without involvement of other receptor subtypes that may be associated with inhibition-related side effects. Thus, such constructs will provide cell selective (e.g., tumor cells with activated EGFR signaling due to receptor expansion, overexpression, etc.) anti-EGFR (ErbB 1) drug effects with reduced side effects compared to EGFR kinase inhibitors that inhibit EGFR activity in all cell types in vivo.
In some embodiments, the additional modified signaling agent has reduced affinity and/or activity (e.g., agonism) for ErbB4 and/or other isoforms that may interact therewith. By targeting specific target cells via the targeting moiety, selective activation of ErbB1 signaling (e.g., epithelial cells) is achieved. In some embodiments, such constructs are useful for treating wounds (promoting healing) with reduced side effects, particularly for treating chronic conditions and applications other than topical application of therapeutic agents (e.g., systemic wound healing).
In one embodiment, the modified signaling agent is insulin or an insulin analog. In some embodiments, the modified insulin or insulin analog has reduced affinity and/or activity for insulin receptor and/or IGF1 or IGF2 receptor. In some embodiments, the modified insulin or insulin analog has substantially reduced or eliminated affinity and/or activity for insulin receptor and/or IGF1 or IGF2 receptor. The reduced response at the insulin receptor allows for control of diabetes, obesity, metabolic disorders, etc., while direct distance from IGF1 or IGF2 receptors avoids the effects of pre-cancers.
In one embodiment, the modified signaling agent is insulin-like growth factor I or insulin-like growth factor II (IGF-1 or IGF-2). In one embodiment, the modified signaling agent is IGF-1. In such embodiments, the modified signaling agent has reduced affinity and/or activity for insulin receptor and/or IGF1 receptor. In one embodiment, the modified signaling agent may bind to and antagonize the activity of IGF1 receptor. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IGF1 receptor, which allows antagonizing the activity of the receptor in a attenuated manner. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for insulin receptor and/or IGF1 receptor. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IGF2 receptor, which allows antagonizing the activity of the receptor in a attenuated manner. In one embodiment, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for insulin receptor and thus does not interfere with insulin signaling. In various embodiments, this applies to cancer treatment. In various embodiments, the agents of the invention may prevent IR isoform a from causing resistance to cancer treatment.
In one embodiment, the modified signaling agent is EPO. In various embodiments, the modified EPO agents have reduced affinity and/or activity for EPO receptor (EPOR) and/or ephrin receptor (EphR) relative to wild-type EPO or other EPO-based agents described herein. In some embodiments, the modified EPO agent has substantially reduced or eliminated affinity and/or activity for EPO receptor (EPOR) and/or Eph receptor (EphR). Illustrative EPO receptors include, but are not limited to, EPOR homodimers or EPOR/CD131 heterodimers. Also included are beta-common receptors (βcr) as EPO receptors. Illustrative Eph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6. In some embodiments, the modified EPO protein comprises one or more mutations that result in the EPO protein having reduced affinity for a receptor (e.g., heterodimer, heterotrimer, etc., including, but not limited to, EPOR-EPHB4, EPOR- βcR-EPOR) comprising one or more different EPO receptors or Eph receptors. Receptors for European patent publication No. 2492355, including but not limited to NEPOR, are also provided, the entire contents of which are hereby incorporated by reference.
In one embodiment, the human EPO has the amino acid sequence of SEQ ID NO:247 (the first 27 amino acids are signal peptides).
In one embodiment, the human EPO protein is a mature form of EPO (in which the signal peptide is cleaved) which is a 166 amino acid residue glycoprotein having the sequence of SEQ ID NO:248.
the structure of the human EPO protein is expected to contain four helical bundles including helix a, helix B, helix C and helix D. In various embodiments, the modified EPO protein comprises mutations located in four regions of the EPO protein important for biological activity, namely one or more of amino acid residues 10-20, 44-51, 96-108 and 142-156. In some embodiments, the one or more mutations are located at residues 11-15, 44-51, 100-108, and 147-151. These residues are limited to helix A (Val 11, arg14 and Tyr 15), helix C (Ser 100, arg103, ser104 and Leu 108), helix D (Asn 147, arg150, gly151 and Leu 155) and the A/B linker loop (residues 42-51). In some embodiments, the modified EPO protein comprises mutations between amino acids 41-52 and at residues of amino acids 147, 150, 151 and 155. Without wishing to be bound by theory, it is believed that mutation of these residues has a substantial effect on both receptor binding and in vitro biological activity. In some embodiments, the modified EPO protein comprises mutations at residues 11, 14, 15, 100, 103, 104 and 108. Without wishing to be bound by theory, it is believed that mutations at these residues have a moderate effect on receptor binding activity and a much greater effect on biological activity in vitro. Illustrative substitutions include, but are not limited to, one or more of the following: val11Ser, arg14Ala, arg14Gln, tyr15lle, pro42Asn, thr44lle, lys45Asp, val46Ala, tyr51Phe, ser100Glu, ser100Thr, arg103Ala, ser104lle, ser104Ala, leu108Lys, asn147Lys, arg150Ala, gly151ALa and Leu155Ala.
In some embodiments, the modified EPO protein comprises a mutation that affects biological activity without affecting binding, e.g., eliot et al Mapping of the Active Site of Recombinant Human Erythropoietin 1997, month 1 and 15; blood:89 Those mutations listed in (2), the entire contents of which are hereby incorporated by reference.
In some embodiments, the modified EPO protein comprises one or more mutations that involve surface residues in the EPO protein that are involved in receptor contact. Without wishing to be bound by theory, it is believed that mutations in these surface residues are unlikely to affect protein folding, thereby preserving some biological activity. Illustrative surface residues that may be mutated include, but are not limited to, residues 147 and 150. In an illustrative embodiment, the mutation is a substitution, including one or more of N147A, N147K, R a and R150E.
In some embodiments, the modified EPO protein comprises one or more mutations at residues N59, E62, L67 and L70 and one or more mutations that affect disulfide bond formation. Without wishing to be bound by theory, it is believed that these mutations affect folding and/or are expected to be at the embedded site and thus indirectly affect biological activity.
In one embodiment, the modified EPO protein comprises a K20E substitution that significantly reduces receptor binding. See, e.g., elliott et al, (1997) Blood,89:493-502, the entire contents of which are hereby incorporated by reference.
Other EPO mutations that can be incorporated into the chimeric EPO proteins of the invention are disclosed, for example, in the following documents: elliott et al, (1997) Blood,89:493-502, the entire contents of which are hereby incorporated by reference; and Taylor et al, (2010) PEDS,23 (4): 251-260, the entire contents of which are hereby incorporated by reference.
In various embodiments, the signaling agent is a toxin or a toxic enzyme. In some embodiments, the toxins or toxic enzymes are derived from plants and bacteria. Illustrative toxins or toxic enzymes include, but are not limited to, diphtheria toxin, pseudomonas toxin, anthrax toxin, ribosome Inactivating Proteins (RIP) such as ricin and saporin, curbitoxin, abrin, gelonin, and pokeweed antiviral protein. Additional toxins include Mathew et al, (2009) Cancer Sci 100 (8): 1359-65, the entire disclosure of which is hereby incorporated by reference. In such embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be used to induce cell death in a cell type specific manner. In such embodiments, the toxin may be modified, e.g., mutated, to reduce the affinity and/or activity of the toxin in order to attenuate effects, as described for other signaling agents herein.
Linker and functional group
In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, optionally comprise one or more linkers. In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, comprise a linker linking the targeting moiety and the signaling agent (e.g., modified IL-2). In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, comprise a linker-containing signaling agent (e.g., modified IL-2). In some embodiments, the linker may be used to link the various functional groups, residues, or moieties as described herein to a chimeric protein or chimeric protein complex such as an Fc-based chimeric protein complex. In some embodiments, the linker is a single amino acid or multiple amino acids that do not affect or reduce the stability, orientation, binding, neutralization, and/or clearance characteristics of the binding region and binding protein. In various embodiments, the linker is selected from a peptide, a protein, a sugar, or a nucleic acid.
In some embodiments, vectors encoding chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, linked to any of the linkers described herein in the form of a single nucleotide sequence are provided, and can be used to prepare such chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes. In various embodiments, the substituents of the Fc-based chimeric protein complex are expressed in the form of a nucleotide sequence in the vector.
In some embodiments, the linker length allows for efficient binding of the targeting moiety and signaling agent (e.g., modified IL-2) to its receptor. For example, in some embodiments, the length of the linker allows for effective binding of one of the targeting moieties and the signaling agent to a receptor on the same cell.
In some embodiments, the linker length is at least equal to the minimum distance between one of the targeting moieties and the binding site of the signaling agent to a receptor on the same cell. In some embodiments, the linker length is at least two times, or three times, or four times, or five times, or ten times, or twenty times, or 25 times, or 50 times, or one hundred times, or more the minimum distance between one of the targeting moieties and the binding site of the signaling agent to the receptor on the same cell.
As described herein, the length of the linker allows for efficient binding of one of the targeting moieties and the signaling agent to a receptor on the same cell, the binding being sequential, e.g., targeting moiety/receptor binding precedes signaling agent/receptor binding.
In some embodiments, there are two linkers in a single chimera, each flexible linker connecting the signaling agent to the targeting moiety. In various embodiments, the length of the linker allows for the formation of sites with disease cells and effector cells without steric hindrance that would prevent modulation of either cell.
The present invention contemplates the use of a variety of linker sequences. In various embodiments, the linker may be derived from a naturally occurring multidomain protein or an empirical linker as described, for example, in the following documents: chichili et al, (2013), protein Sci.22 (2): 153-167; chen et al, (2013), adv Drug Deliv rev.65 (10): 1357-1369, the entire contents of which are hereby incorporated by reference. In some embodiments, the joints may be designed using a joint design database and computer program, such as those described in the following documents: chen et al, (2013), adv Drug Deliv rev.65 (10): 1357-1369 and Crasto et al, (2000), protein eng.13 (5): 309-312, the entire contents of which are hereby incorporated by reference. In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve folding and/or stability of the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, improve expression, improve pharmacokinetics, and/or improve biological activity.
In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids long. For example, the linker can be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In some embodiments, the linker is a polypeptide. In some embodiments, the linker is more than about 100 amino acids long. For example, the linker can be more than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In some embodiments, the joint is flexible. In another embodiment, the joint is rigid.
In some embodiments involving chimeric proteins or chimeric protein complexes having two or more targeting moieties, such as Fc-based chimeric protein complexes, one linker connects two targeting moieties to each other and such linker has a shorter length, while one linker connects the targeting moiety and the signaling agent, such linker is longer than the linker connecting the two targeting moieties. For example, the difference in amino acid length between the linker connecting the two targeting moieties and the linker connecting the targeting moiety and the signaling agent can be about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids.
In various embodiments, the linker consists essentially of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). Lifting deviceFor example, in some embodiments, the linker is (Gly 4 Ser) n Wherein n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7 or 8 (SEQ ID NO:249-SEQ ID NO:256, respectively). For example, in some embodiments, the linker is (Gly 3 Ser) n Wherein n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7 or 8 (SEQ ID NO:457-SEQ ID NO:464, respectively). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 257). Other illustrative linkers include, but are not limited to, those having the sequences LE, GGGGS (SEQ ID NO: 249), (GGGGS) n (n＝1-4)(SEQ ID NO：249-SEQ ID NO：252)、(Gly) 8 (SEQ ID NO：258)、(Gly) 6 (SEQ ID NO：259)、(EAAAK) n (n＝1-3)(SEQ ID NO：260-SEQ ID NO：262)、A(EAAAK) n A(n＝2-5)(SEQ ID NO：263-SEQ ID NO：266)、AEAAAKEAAAKA(SEQ ID NO：263)、A(EAAAK) 4 ALEA(EAAAK) 4 A (SEQ ID NO: 267), PAAP (SEQ ID NO: 268), KESGSVSSEQLAQFRSLD (SEQ ID NO: 269), EGKSSGSGSESKST (SEQ ID NO: 270), GSAGSAAGSGEF (SEQ ID NO: 271) and (XP) n Wherein X represents any amino acid, such as Ala, lys or Glu. In various embodiments, the linker is GGS.
In some embodiments, the linker is one or more of GGGSE (SEQ ID NO: 272), GSESG (SEQ ID NO: 273), GSEGS (SEQ ID NO: 274), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 275), and a linker that places G, S and E randomly every 4 amino acid intervals.
In some embodiments, the linker is a hinge region of an antibody (e.g., igG, igA, igD and IgE, including subclasses (e.g., igG1, igG2, igG3, and IgG4, and IgA1 and IgA 2)). In various embodiments, the linker is a hinge region of an antibody (e.g., igG, igA, igD and IgE, including subclasses (e.g., igG1, igG2, igG3, and IgG4, and IgA1 and IgA 2)). The hinge region found in IgG, igA, igD and IgE class antibodies acts as a flexible spacer allowing the Fab portion to move freely in space. In contrast to the constant region, the hinge domains are structurally diverse, varying in both sequence and length by immunoglobulin class and subclass. For example, the length and flexibility of the hinge region varies from IgG class to IgG class. The hinge region of IgG1 encompasses amino acids 216-231 and because it is free and flexible, the Fab fragment can rotate about its axis of symmetry and move within a sphere centered on the first of the two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline duplex that is stabilized by an additional inter-heavy chain disulfide bridge. These properties constrain the flexibility of IgG2 molecules. IgG3 contains 62 amino acids (including 21 prolines and 11 cysteines) due to its unique extended hinge region (up to about four times the hinge of IgG 1), forming a non-flexible polyproline duplex unlike other subclasses. In IgG3, the Fab fragment is relatively far from the Fc fragment, thereby imparting greater flexibility to the molecule. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than IgG1 and its flexibility is between that of IgG1 and IgG2. The flexibility of the hinge region is reported in decreasing order as IgG3 > IgG1 > IgG4 > IgG2.
According to crystallographic studies, the immunoglobulin hinge region can be functionally further subdivided into three regions: an upper hinge region, a core region, and a lower hinge region. See Shin et al, 1992ImmunologicalR evieWs 130:87. the upper hinge region includes a hinge region of C H1 The first residue in the hinge that constrains movement is typically the amino acid of the first cysteine residue that forms an interchain disulfide bond between two heavy chains. The length of the upper hinge region is related to the segment flexibility of the antibody. The core hinge region contains a heavy interchain disulfide bridge, while the lower hinge region joins C H2 Amino-terminal to the domain and includes C H2 Residues in (a). As before. The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 276), which when dimerized by disulfide bond formation produces a cyclic octapeptide that is thought to act as a pivot, thus imparting flexibility. In various embodiments, the linker of the invention comprises any antibody (e.g., igG, igA, igD and IgE, including subclasses (e.g., igG1, igG2, igG3 and IgG4, and IgA1 and IgA 2), and one or both or three of the upper hinge region, the core region, and the lower hinge region. The hinge region may also contain one or more glycosylation sites, including numerous types of structurally unique sites for carbohydrate attachment. For example, igA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, which is considered to secrete an advantageous property of immunoglobulins. In various embodiments, the linker of the invention comprises one or more glycosylation sites. In various embodiments, the linker is a hinge-CH 2-CH3 domain of a human IgG4 antibody.
In some embodiments, the linker is a synthetic linker, such as PEG.
In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve folding and/or stability of the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, improve expression, improve pharmacokinetics, and/or improve biological activity. In another example, the linker can function to target the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, to a particular cell type or location.
In various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, may include one or more functional groups, residues, or moieties. In various embodiments, the one or more functional groups, residues, or moieties are linked or genetically fused to any of the signaling agents or targeting moieties described herein. In some embodiments, such functional groups, residues, or moieties impart one or more desired properties or functionalities to the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes. Examples of such functional groups and techniques for their incorporation into chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, are known in the art, for example, see Remington's Pharmaceutical Sciences, 16 th edition, mack Publishing co., easton, pa. (1980).
In various embodiments, each of the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, can be conjugated and/or fused with another agent to extend half-life or otherwise improve pharmacodynamics and pharmacokinetic properties. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can be fused or conjugated to one or more of PEG, XTEN (e.g., in rPEG form), polysialic acid (polysialic acid), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, and the like. In various embodiments, each of the individual chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, is combined with a biotugs (2015) 29:215-239, the entire contents of which are hereby incorporated by reference.
In some embodiments, the functional group, residue or moiety comprises a suitable pharmacologically acceptable polymer, such as poly (ethylene glycol) (PEG) or a derivative thereof (such as methoxypoly (ethylene glycol) or mPEG). In some embodiments, the attachment of the PEG moiety increases the half-life and/or reduces the immunogenicity of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex. In general, any suitable form of pegylation may be used, such as those used in the art for the pegylation of antibodies and antibody fragments (including but not limited to single domain antibodies, such as VHH); see, e.g., chapman, nat. Biotechnol, 54, 531-545 (2002); veronese and Harris, adv. Drug deliv. Rev.54, 453-456 (2003); harris and Chess, nat.rev. Drug.discover., 2, (2003) and WO04060965, the entire contents of which are hereby incorporated by reference. Various reagents for pegylation of proteins are also available commercially, for example, from Nektar Therapeutics in the united states. In some embodiments, site-directed pegylation is used, in particular via cysteine residues (see, e.g., yang et al, protein Engineering,16, 10, 761-770 (2003), the entire contents of which are hereby incorporated by reference). In some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, is modified to introduce one or more cysteine residues for attachment to PEG as appropriate, or an amino acid sequence comprising one or more cysteine residues for attachment to PEG, may be fused to the amino and/or carboxy terminus of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, using techniques known in the art.
In some embodiments, the functional group, residue or moiety comprises N-linked or O-linked glycosylation. In some embodiments, the N-linked or O-linked glycosylation is introduced as part of a co-translational and/or post-translational modification.
In some embodiments, the functional group, residue or moiety comprises one or more detectable labels or other signal generating groups or moieties. Suitable labels and techniques for attaching, using, and detecting them are known in the art and include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as Eu or other lanthanide metals), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminol, isoluminol, thermal acridinium ester (theromatic acridinium ester), imidazole, acridinium salt, oxalate, dioxetane or GFP and their analogs), radioisotopes, metals, metal chelates, or metal cations or other metals or metal cations particularly suitable for in vivo, in vitro, or in situ diagnostics and imaging, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, phosphotriose isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-glucosidase, beta-ribonuclease, acetyltransferase, glucose dehydrogenase, and phospholipase, and VI-amylase. Other suitable labels include moieties that can be detected using NMR or ESR spectroscopy. Such labeled VHH and polypeptides of the invention can be used, for example, in vitro, in vivo or in situ assays (including immunoassays known per se, such as ELISA, RIA, EIA and other "sandwich assays", etc.), as well as for diagnostic and imaging purposes in vivo, depending on the choice of the particular label.
In some embodiments, the functional group, residue or moiety comprises a tag attached or genetically fused to the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may include a single tag or multiple tags. For example, the tag is a peptide, sugar, or DNA molecule that does not inhibit or prevent binding of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, to its target or other antigen of interest, such as a tumor antigen. In various embodiments, the tag is at least about: three to five amino acids long, five to eight amino acids long, eight to twelve amino acids long, twelve to fifteen amino acids long, or fifteen to twenty amino acids long. Illustrative labels are described, for example, in U.S. patent publication No. US2013/0058962. In some embodiments, the tag is an affinity tag, such as glutathione-S-transferase (GST) and histidine (His) tag. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a His tag.
In some embodiments, the functional group, residue or moiety comprises a chelating group, e.g., to chelate one of a metal or metal cation. Suitable chelating groups include, for example, but are not limited to, diethylenetriamine pentaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA).
In some embodiments, the functional group, residue or moiety comprises a functional group that is part of a specific binding pair, such as a biotin- (streptavidin) binding pair. Such functional groups may be used to link a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, to another protein, polypeptide, or chemical compound that binds to the other half of the binding pair (i.e., by forming a binding pair). For example, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, can be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptavidin. For example, such conjugated chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, may be used as a reporter gene in, for example, diagnostic systems, wherein a detectable signal generator is conjugated to avidin or streptavidin. Such binding pairs may also be used, for example, to bind the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example is the liposomal formulation described by Cao and sursh, journal of Drug Targeting,8,4, 257 (2000). Such binding pairs can also be used to link a therapeutically active agent to a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex.
Preparation of chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes
Described herein are methods for preparing the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes. For example, DNA sequences encoding the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes (e.g., DNA sequences encoding a signaling agent (e.g., modified IL-2) and targeting moieties and linkers) can be chemically synthesized using methods known in the art. The synthetic DNA sequence may be linked to other suitable nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct encoding a desired chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex. Thus, in various embodiments, the invention provides isolated nucleic acids comprising a nucleotide sequence encoding a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex.
Nucleic acids encoding chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, may be incorporated (linked) into expression vectors that may be introduced into host cells by transfection, transformation or transduction techniques. For example, nucleic acids encoding chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be introduced into host cells by retroviral transduction. Illustrative host cells are E.coli cells, chinese Hamster Ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hep G2), insect Sf9 cells, and myeloma cells. The transformed host cells may be grown under conditions that allow the host cells to express genes encoding the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes. Thus, in various embodiments, the invention provides expression vectors comprising nucleic acids encoding the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes. In various embodiments, the invention additionally provides host cells comprising such expression vectors.
The specific expression and purification conditions will vary depending on the expression system employed. For example, if a gene is expressed in E.coli, it is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter such as Trp or Tac and a prokaryotic signal sequence. In another example, if an engineered gene is to be expressed in a eukaryotic host cell, such as a CHO cell, it is first inserted into an expression vector containing, for example, a suitable eukaryotic promoter, secretion signal, enhancer, and various introns. Transfection, transformation or transduction techniques may be used to introduce the genetic construct into a host cell.
The chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be produced by growing host cells transfected with expression vectors encoding the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, under conditions that allow expression of the proteins. After expression, the proteins may be collected and purified using techniques well known in the art, for example, affinity tags such as glutathione-S-transferase (GST) and histidine tags, or by chromatography.
Thus, in various embodiments, the invention provides a nucleic acid encoding a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex. In various embodiments, the invention provides a host cell comprising a nucleic acid encoding a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex.
In various embodiments, IL-2, variants thereof, or chimeric proteins or chimeric protein complexes comprising IL-2 or variants thereof, such as Fc-based chimeric protein complexes, may be expressed in vivo, e.g., in a patient. For example, in various embodiments, IL-2, a variant thereof, or a chimeric protein or chimeric protein complex comprising IL-2 or a variant thereof, such as an Fc-based chimeric protein complex, can be administered in the form of a nucleic acid encoding IL-2 or a variant thereof, or a chimeric protein or chimeric protein complex comprising IL-2 or a variant thereof, such as an Fc-based chimeric protein complex. In various embodiments, the nucleic acid is DNA or RNA. In some embodiments, the IL-2, variant thereof, or chimeric protein complex comprising IL-2 or variant thereof, such as an Fc-based chimeric protein complex, is encoded by a modified mRNA, i.e., an mRNA comprising one or more modified nucleotides. In some embodiments, the modified mRNA comprises one or more modifications found in U.S. patent No. 8,278,036, the entire contents of which are hereby incorporated by reference. In some embodiments, the modified mRNA comprises one or more of m5C, m5U, m A, s2U, ψ, and 2' -O-methyl-U. In some embodiments, the invention relates to the administration of modified mRNA encoding one or more chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes. In some embodiments, the invention relates to a gene therapy vector comprising the modified mRNA. In some embodiments, the invention relates to methods of gene therapy comprising the gene therapy vectors. In various embodiments, the nucleic acid is in the form of an oncolytic virus, such as adenovirus, reovirus, measles, herpes simplex, newcastle disease virus, or vaccinia.
In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a targeting moiety as a VHH. In various embodiments, the VHH is not limited to a particular biological source or a particular method of preparation. For example, the VHH can generally be obtained by: (1) By isolation of V from naturally occurring heavy chain antibodies H An H domain; (2) Encoding naturally occurring V by expression H Nucleotide sequence of H domain; (3) By naturally occurring V H "humanization" of H domains or by encoding such humanized V H Expression of nucleic acid of the H domain; (4) By "camelization" of naturally occurring VH domains from any animal species, such as from mammalian species, such as from humans, or by expression of nucleic acids encoding such camelized VH domains; (5) "camelization" by a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) The preparation of proteins, polypeptides or other amino acid sequences known in the art by using synthetic or semisynthetic techniques; (7) Preparing a nucleic acid encoding a VHH by using nucleic acid synthesis techniques known in the art, followed by expression of the nucleic acid so obtained; and/or (8) by any combination of one or more of the foregoing.
In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises V corresponding to a naturally occurring heavy chain antibody against a target of interest H VHH of the H domain. In some embodiments, such V may be generally produced or obtained by H H sequence: by suitably immunizing a camel (i.e. so as to generate an immune response and/or heavy chain antibodies against the target of interest) with molecules based on the target of interest (e.g. XCR1, clec9a, CD8, sirp1α, FAP, etc.); by obtaining a suitable biological sample (such as a blood sample or any B-fines) from the camelCell samples); and generating V against the target of interest by starting with the sample using any suitable known technique H H sequence. In some embodiments, the naturally occurring V against the target of interest H The H domain may be obtained from camel V H Natural libraries of H sequences, for example, such libraries are screened using the target of interest or at least a portion, fragment, epitope, or portion thereof, by using one or more screening techniques known in the art. For example, such libraries and techniques are described in WO 9937681, WO 0190190, WO 03025020 and WO 03035694, the entire contents of which are hereby incorporated by reference. In some embodiments, improved synthetic or semisynthetic libraries derived from natural VHH libraries may be used, such as VHH libraries obtained from natural VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as described for example in WO 0043507, the entire content of which is hereby incorporated by reference. In some embodiments, for obtaining V against a target of interest H Another technique for H sequences involves suitably immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., so as to generate an immune response and/or heavy chain antibodies to a target of interest), obtaining a suitable biological sample (such as a blood sample, or any B cell sample) from the transgenic mammal, and then using any suitable known technique starting with the sample to generate V against XCR1 H H sequence. For example, mice expressing heavy chain antibodies described in WO 02085945 and WO 04049794, as well as other methods and techniques (the entire contents of which are hereby incorporated by reference) can be used for this purpose.
In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a polypeptide that has been "humanized," i.e., by combining naturally occurring V H The replacement of one or more amino acid residues in the amino acid sequence of the H sequence (and in particular in the framework sequence) with a VHH from one or more amino acid residues present at corresponding positions in the VH domain of a conventional 4-chain antibody of human origin. This may be done using techniques known in the artIs performed by a humanization technique. In some embodiments, possible humanized substitutions or combinations of humanized substitutions may be determined by methods known in the art, for example, by comparing the sequence of a VHH to the sequence of a naturally occurring human VH domain. In some embodiments, the humanized substitutions are selected such that the resulting humanized VHH still retains advantageous functional properties. In general, as a result of humanization, the VHH of the invention compares to the corresponding naturally occurring V H The H domain may become more "human like" while still retaining advantageous properties, such as reduced immunogenicity. In various embodiments, the humanized VHH of the invention may be obtained in any suitable manner known in the art and is therefore not strictly limited to having been used to contain naturally occurring V H A polypeptide obtained from a polypeptide having an H domain as a starting material.
In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, comprises a V that has been "camelized", i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with a camelid heavy chain antibody H VHH of one or more amino acid residues present at corresponding positions in the H domain. In some embodiments, such "camelised" substitutions are inserted at amino acid positions formed and/or present at the VH-VL interface and/or at so-called camelidae marker residues (see for example WO9404678, the entire contents of which are hereby incorporated by reference). In some embodiments, the VH sequence used as a starting material or point for generating or designing a camelized VHH is a VH sequence from a mammal, e.g., a human VH sequence, such as a VH3 sequence. In various embodiments, the camelized VHH can be obtained in any suitable manner known in the art (i.e. as indicated in points (1) - (8) above), and is thus not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VH domain as starting material.
In various embodiments, "humanization" and "camelization" may be achieved byThe process is carried out: separately providing a coding for naturally occurring V H The nucleotide sequence of the H domain or VH domain is then altered in one or more codons in the nucleotide sequence in a manner known in the art in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. Such nucleic acids may then be expressed in a manner known in the art to provide the desired VHH of the invention. Or, respectively, based on naturally occurring V H The amino acid sequence of the H domain or VH domain, respectively, may be designed for the desired humanized or camelized VHH of the invention and then synthesized de novo using peptide synthesis techniques known in the art. And based on naturally occurring V respectively H The amino acid sequence or nucleotide sequence of the H domain or VH domain, respectively, may be designed to encode a desired humanized or camelized VHH, which is then synthesized de novo using nucleic acid synthesis techniques known in the art, after which the nucleic acid so obtained may be expressed in a manner known in the art to provide the desired VHH of the invention. In naturally occurring VH sequences or V H Other suitable methods and techniques for obtaining a VHH of the invention and/or nucleic acid encoding the VHH starting from an H sequence are known in the art and may for example comprise combining one or more parts of one or more naturally occurring VH sequences (such as one or more FR sequences and/or CDR sequences), one or more naturally occurring V sequences in a suitable manner H One or more portions of the H sequence (such as one or more FR sequences or CDR sequences) and/or one or more synthetic or semisynthetic sequences, in order to provide a VHH of the invention or a nucleotide sequence or nucleic acid encoding said VHH.
Pharmaceutically acceptable salts and excipients
The chimeric proteins or chimeric protein complexes described herein, such as Fc-based chimeric protein complexes, may have sufficiently basic functional groups that can react with inorganic or organic acids, or carboxyl groups that can react with inorganic or organic bases, to form pharmaceutically acceptable salts. As is well known in the art, pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids. Such salts include, for example, pharmaceutically acceptable salts listed in the following documents: journal of Pharmaceutical Science,66,2-19 (1977) and The Handbook of Pharmaceutical Salts; properties, selection, and use.P.H.Stahl and C.G.Wermuth (eds.), verlag, zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.
As non-limiting examples, pharmaceutically acceptable salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, methylbenzates, o-acetoxybenzoates, naphthalene-2-benzoates, isobutyrates, phenylbutyrate, alpha-hydroxybutyrates, butyne-1, 4-dicarboxylic acid salts, hexyne-1, 4-dicarboxylic acid salts, decanoates, octanoates, cinnamates, glycolates, heptanoates, hippurates, malates, hydroxymaleates, malonates, mandelates, methanesulfonates, nicotinates, phthalates, terephthalates, propiolates, propionates, phenylpropionates, sebacates, suberates, p-bromobenzenesulfonates, chlorobenzenesulfonates, ethanesulfonates, 2-isethionates, methylsulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, naphthalene-1, 5-sulfonates, xylenesulfonates and tartrates.
The term "pharmaceutically acceptable salt" also refers to salts of the compositions of the present invention having an acidic functional group, such as a carboxylic acid functional group, with a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia and organic amines such as unsubstituted or hydroxy-substituted mono-, di-or trialkylamines, dicyclohexylamines; tributylamine; pyridine; n-methyl, N-ethylamine; diethyl amine; triethylamine; mono (2-OH-lower alkylamine), bis (2-OH-lower alkylamine) or tris (2-OH-lower alkylamine) (such as mono (2-hydroxyethyl) amine, bis (2-hydroxyethyl) amine or tris (2-hydroxyethyl) amine), 2-hydroxy-tert-butylamine or tris (hydroxymethyl) methylamine, N-di-lower alkyl-N- (hydroxy-lower alkyl) -amine (such as N, N-dimethyl-N- (2-hydroxyethyl) amine) or tris (2-hydroxyethyl) amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.
In some embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.
Pharmaceutical composition and formulation
In various embodiments, the invention relates to pharmaceutical compositions comprising a chimeric protein or chimeric protein complex described herein, such as an Fc-based chimeric protein complex, and a pharmaceutically acceptable carrier or excipient. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally comprise an appropriate amount of pharmaceutically acceptable excipients in order to provide a form for proper administration.
In various embodiments, the pharmaceutical excipients may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipient may be, for example, physiological saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Physiological saline solution and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any of the agents described herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso r. Gennaro editions, 19 th edition 1995), which is incorporated herein by reference.
The invention includes various formulations of the described pharmaceutical compositions (and/or other therapeutic agents). Any of the inventive pharmaceutical compositions (and/or other therapeutic agents) described herein may be in the form of solutions, suspensions, emulsions, drops, tablets, pills, pellets, capsules, liquid-containing capsules, gelatin capsules, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, frozen suspensions, dried powders, or any other suitable form. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In another embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.
The pharmaceutical compositions (and/or other agents) of the present invention may also include a solubilizing agent, if desired. The agent may also be delivered with a suitable vehicle or delivery device as known in the art. The combination therapies outlined herein may be co-delivered in a single delivery vehicle or delivery device.
The formulations of the present invention comprising the pharmaceutical compositions (and/or other agents) of the present invention may suitably be presented in unit dosage form and may be prepared by any method well known in the pharmaceutical arts. Such methods generally include the step of associating the therapeutic agent with a carrier that constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing into association the therapeutic agent with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into dosage forms (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art) of the desired formulation.
In various embodiments, any of the pharmaceutical compositions (and/or other agents) described herein are formulated according to conventional procedures into compositions suitable for the mode of administration described herein.
Routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation or topical. Administration may be local or systemic. In some embodiments, the administration is achieved orally. In another embodiment, the administration is by parenteral injection. The mode of administration may be left to the discretion of the physician and depends in part on the site of the medical condition. In most cases, administration causes any of the agents described herein to be released into the blood stream.
In one embodiment, the chimeric proteins or chimeric protein complexes described herein, such as Fc-based chimeric protein complexes, are formulated according to conventional procedures into compositions suitable for oral administration. For example, compositions for oral delivery may be in the form of tablets, troches, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Compositions for oral administration may comprise one or more agents, for example a sweetener, such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen or cherry; a colorant; and a preservative to provide a pharmaceutically palatable preparation. Furthermore, when in tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. Any chimeric protein or chimeric protein complex driven by osmotic activity described herein, such as a permselective membrane around an Fc-based chimeric protein complex, is also suitable for use in orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is inhaled by the driving compound, which expands to displace the agent or agent composition through the orifice. These delivery platforms can provide essentially zero order delivery profiles, as opposed to spike profiles of immediate release formulations. Time delay materials such as glyceryl monostearate or glyceryl stearate are also useful. Oral compositions may include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. In one embodiment, the excipient is of pharmaceutical grade. Suspensions as well as the active compounds may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and the like, as well as mixtures thereof.
Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions) which may be dissolved or suspended in a sterile injectable medium immediately prior to use. They may contain, for example, suspending or dispersing agents known in the art. Formulation components suitable for parenteral administration include sterile diluents such as water for injection, physiological saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as EDTA; buffers such as acetate, citrate or phosphate; and tonicity adjusting agents such as sodium chloride or dextrose.
For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, cremophor (Cremophor) ELTM (BASF, parippany, NJ) or phosphate buffered physiological saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be preserved from microorganisms. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
The compositions provided herein may be formulated as aerosol formulations (i.e., "sprays") for administration via inhalation, alone or in combination with other suitable components. The aerosol formulation may be placed into a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.
Any of the inventive pharmaceutical compositions (and/or other agents) described herein may be administered by controlled release or sustained release means or by delivery devices well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5, 120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms may be used to provide controlled or sustained release of one or more active ingredients using, for example, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, other polymer matrices, gels, osmotic membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide a desired release profile at varying ratios. Suitable controlled release or sustained release formulations known to those skilled in the art, including those described herein, may be readily selected for use with the active ingredients of the agents described herein. The present invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, caplets and caplets suitable for controlled or sustained release.
The controlled or sustained release of the active ingredient may be stimulated by a variety of conditions including, but not limited to, a change in pH, a change in temperature, by the wavelength of appropriate light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
In another embodiment, the controlled release system may be placed in the vicinity of the target area to be treated, thus requiring only a portion of the systemic dose (see, e.g., goodson, medical Applications of Controlled Release, supra, volume 2, pages 115-138 (1984)). Langer,1990, science 249 can be used: 1527-1533) are described in the overview.
The pharmaceutical formulation is preferably sterile. Sterilization may be achieved, for example, by filtration through a sterile filtration membrane. In the case where the composition is lyophilized, filter sterilization may be performed before or after lyophilization and reconstitution.
Administration and dosage
It will be appreciated that the actual dosage of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, administered according to the present invention will vary depending on the particular dosage form and mode of administration. Many factors can alter the effect of a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex. Such as body weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combination, genetic predisposition, and sensitivity to response, can be within the purview of one skilled in the art. The administration may be performed continuously or in one or more discrete doses within the maximum tolerated dose. One skilled in the art can use conventional dosing tests to determine the optimal rate of administration for a given set of conditions.
In some embodiments, a suitable dose of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is in a range of about 0.01 μg/kg to about 100mg/kg subject body weight, about 0.01 μg/kg to about 10mg/kg subject body weight, or about 0.01 μg/kg to about 1mg/kg subject body weight, e.g., about 0.01 μg/kg, about 0.02 μg/kg, about 0.03 μg/kg, about 0.04 μg/kg, about 0.05 μg/kg, about 0.06 μg/kg, about 0.07 μg/kg, about 0.08 μg/kg, about 0.09 μg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 9mg, about 1.9mg, about 1.1mg, about 1.2mg, about 1.3mg, about 1mg, about 1.2mg, about 1.3mg, about 1.5mg, about 1, about 1.2mg, about 1, about 1.2mg and/kg.
The individual doses of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may be administered in a unit dosage form (e.g., tablet, capsule, or liquid formulation) containing, for example, from about 1 μg to about 100mg, from about 1 μg to about 90mg, from about 1 μg to about 80mg, from about 1 μg to about 70mg, from about 1 μg to about 60mg, from about 1 μg to about 50mg, from about 1 μg to about 40mg, from about 1 μg to about 30mg, from about 1 μg to about 20mg, from about 1 μg to about 10mg, from about 1 μg to about 5mg, from about 1 μg to about 3mg, from about 1 μg to about 1mg, or from about 1 μg to about 50 μg per unit dosage form. For example, the processing unit may be configured to, the unit dosage form may be about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about about 70 μg, about 80 μg, about 90 μg, about 0.1mg, about 0.2mg, about 0.3mg, about 0.4mg, about 0.5mg, about 0.6mg, about 0.7mg, about 0.8mg, about 0.9mg, about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg or about 100mg, including all values and ranges between these values.
In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is administered in an amount of about 1 μg to about 100mg per day, about 1 μg to about 90mg per day, about 1 μg to about 80mg per day, about 1 μg to about 70mg per day, about 1 μg to about 60mg per day, about 1 μg to about 50mg per day, about 1 μg to about 40mg per day, about 1 μg to about 30mg per day, about 1 μg to about 20mg per day, about 01 μg to about 10mg per day, about 1 μg to about 5mg per day, about 1 μg to about 3mg per day, or about 1 μg to about 1mg per day. In various embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is administered at the following daily doses: about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about about 80. Mu.g, about 90. Mu.g, about 0.1mg, about 0.2mg, about 0.3mg, about 0.4mg, about 0.5mg, about 0.6mg, about 0.7mg, about 0.8mg, about 0.9mg, about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg or about 100mg, including all values and ranges between these values.
According to certain embodiments of the invention, a pharmaceutical composition comprising the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may be administered, for example, more than once per day (e.g., about twice, about three times, about four times, about five times, about six times, about seven times, about eight times, about nine times, or about ten times per day), about once per day, about once every other day, about once every third day, about once a week, about once every two weeks, about once every month, about once every two months, about once every three months, about once every six months, or about once every year. In one embodiment, a pharmaceutical composition comprising a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is administered about three times per week.
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be administered chronically. For example, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can be administered as described herein for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, or at least about 12 weeks. For example, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can be administered for 12 weeks, 24 weeks, 36 weeks, or 48 weeks. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, can be administered for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.
Combination therapy and other therapeutic agents
In various embodiments, the pharmaceutical compositions of the present invention are co-administered in combination with one or more additional therapeutic agents. Co-administration may be performed simultaneously or sequentially.
In one embodiment, the additional therapeutic agent and the chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, are administered to a subject simultaneously. As used herein, the term "simultaneously" means that the other therapeutic agent and the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, are administered at a time interval of no more than about 60 minutes, such as no more than about 30 minutes, no more than about 20 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. Administration of the additional therapeutic agent and the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may be performed by simultaneous administration of a single formulation (e.g., a formulation comprising the additional therapeutic agent and the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex) or separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex).
Co-administration does not require simultaneous administration of each therapeutic agent, so long as the course of administration is such that the pharmacological activities of the other therapeutic agent and the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, overlap in time, thereby exerting a combined therapeutic effect. For example, the additional therapeutic agent and the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may be administered sequentially. As used herein, the term "sequentially" means that the other therapeutic agent and the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, are administered at intervals of greater than about 60 minutes. For example, the time between sequential administration of the other therapeutic agent and the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, may be more than about 60 minutes apart, more than about 2 hours apart, more than about 5 hours apart, more than about 10 hours apart, more than about 1 day apart, more than about 2 days apart, more than about 3 days apart, more than about 1 week apart, more than about 2 weeks apart, or more than about one month apart. The optimal time of administration will depend on the metabolic rate, excretion rate, and/or the pharmacokinetic activity of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, and other therapeutic agent being administered. The other therapeutic agent or the chimeric protein or chimeric protein complex such as Fc-based chimeric protein complex cells may be administered first.
Co-administration also does not require administration of the therapeutic agent to the subject by the same route of administration. In practice, each therapeutic agent may be administered by any suitable route, such as parenterally or parenterally.
In some embodiments, the chimeric proteins or chimeric protein complexes described herein, such as Fc-based chimeric protein complexes, act synergistically when co-administered with another therapeutic agent. In such embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes and other therapeutic agents, may be administered at lower doses than are employed when each agent is used in a monotherapy setting.
In some embodiments, the invention relates to chemotherapeutic agents as other therapeutic agents. For example, but not limited to, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, in combination with chemotherapeutic agents can be used to treat cancer, as described elsewhere herein. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclophosphamide; alkyl sulfonates such as busulfan (busulfan), imperoshu (imposulfan) and piposulfan (piposulfan); aziridines such as benzotepa (benzodopa), carboquinone (carboquone), mettussidine (meturedopa) and uratepa (uredopa); ethyleneimine and methyl melamines, including altretamine, triteramine, triethylphosphoramide, triethylthiophosphamide and trimethylol melamine; polyacetyl (acetogenin) (e.g., bullatacin and bullatacin); camptothecins (including the synthetic analog topotecan); bryostatin (bryostatin); sponge statin (call statin); CC-1065 (including adozelesin, carbozelesin, and bizelesin synthetic analogs thereof); candidiasis cyclic peptides (cryptophycins) (e.g., candidiasis cyclic peptide 1 and candidiasis cyclic peptide 8); dolastatin (dolastatin); duocarmycin (including synthetic analogs, KW-2189 and CB 1-TM 1); eosporin (eleutherobin); a podocarpine (pancratistatin); sarcandyl alcohol (sarcandylin); spongostatin (sponsin); nitrogen mustards such as chlorambucil (chloramabilin), napthalene mustards (chloraphanizine), cholesteryl phosphoramide (cholosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine, nitrogen mustards oxide hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), novobic (novembichin), bennethol (phenaestine), prednisone (prednisone), triamcinolone (trofosfamide), uratemustine (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouremycin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and Lei Mosi (ranimustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicin), especially calicheamicin # 1 and calicheamicin # 1 (see, e.g., agnew, chem. Intl. Ed. Engl.,33:183-186 (1994))); daptomycin (dyneimicin), including daptomycin a; bisphosphonates, such as clodronate (clodronate); epothilone (esperamicin); and novel carcinomatous chromophores (neocarzinostatin chromophore) and related pigment proteins enediyne antibiotic chromophores, aclacinomycin (acteosin), actinomycin (acteosin), amastatin (authamycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), karabin (carbicin), carminomycin (caminomycin), carcinophilin (carzinophilin), chromomycins (chromycins), actinomycin D (dactinomycin), daunorubicin, dithicin (deoxyubicin), 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (doxorubicin) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, ADRIAMYCIN) 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), elrubicin (esorcicin), idarubicin (idarubicin), maculomycin (marcelomycin), mitomycins (mitomycins) such as mitomycin C, mycofenac (mycophenolic acid), norgamycin (nogamycin), olivomycin (olivorin), pelomycin (peplomycin), prednisomycin (potfuromycin), puromycin (puromycin), quinamycin (queamycin), rodobucicin (rodobucicin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (zinostatin), fuzomycin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin (denopterin), methotrexate, pterin (pteroprerin), trimeoxate (trimetrexate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiamiprinne, thioguanine; pyrimidine analogs such as cyclocytidine (ancitabine), azacytidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine (dideoxyuridine), doxifluridine, enocitabine (enocitidine), fluorouridine (floxuridine); androgens such as carbosterone (calibretone), drotasone propionate (dromostanolone propionate), epithiostanol (epiostanol), melandrane (mepistostane), testosterone (testolactone); an anti-adrenal agent such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (aceglatone); aldehyde phosphoramidate glycoside (aldophosphamide glycoside); aminolevulinic acid (aminolevulinic acid); enuracil (eniluracil); amsacrine (amacrine); bei Sibu western (bestrebicil); bisantrene (bisantrene); edatraxate (edatraxate); colchicine (demecolcine); deaquinone (diaziquone); erlotinib (elformithin); ammonium elide (elliptiniurn acetate); epothilone (epothilone); etodolac (etoglucid); gallium nitrate; hydroxyurea; mushroom polysaccharide (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids) such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguazone); mitoxantrone (mitoxantrone); mo Pai darol (mopidanmol); diamine nitroacridine (nitroane); penstatin (pentastatin); egg ammonia nitrogen mustard (phenol); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophylloic acid (podophyllinic acid); 2-ethyl hydrazide; procarbazine (procarbazine); PSK polysaccharide complex (JHS Natural Products, eugene, oreg.); raschig (razoxane); rhizomycin (rhizoxin); dorzolopyran (sizofuran); germanium spiroamine (spirogmanium); tenozolic acid (tenuazonic acid); triiminoquinone (triaziquone); 2,2',2 "-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, myxomycin A (verracurin A), cyclosporin a (roridin a), and serpentine (anguidine)); uratam (urethan); vindesine (vindeline); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolaetol); pipobromine (pipobroman); gacetin (gacytosine); cytarabine (arabinoside) ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as TAXOL paclitaxel (paclitaxel) (Bristol-Myers SquibbOncology, priceton, n.j.), albumin engineered nanoparticle formulations of ABRAXANE paclitaxel without polyoxyethylated castor oil (Cremophor) (American Pharmaceutical Partners, schaumberg, 111.), and TAXOTERE docetaxel (Rhone-Poulenc rore, antny, france); chlorambucil; GEMZAR gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin (cispratin), oxaliplatin (oxaliplatin), and carboplatin (carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (vincristine); NAVELBINE vinorelbine (vinorelbine); norxiaoling (novantrone); teniposide (teniposide); edatraxate (edatrexate); daunorubicin (daunomycin); aminopterin (aminopterin); hilded (xeloda); ibandronate (ibandronate); irinotecan (irinotecan) (Camptosar, CPT-11) (including irinotecan with 5-FU treatment regimens and folinic acid (leucovorin)); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoid acid); capecitabine (capecitabine); combretastatin (combretastatin); folinic acid (LV); oxaliplatin, including oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-alphSub>A, raf, H-Ras, EGFR (e.g., erlotinib (TarcevSub>A)) and VEGF-A that reduce cell proliferation; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the method of treatment may further comprise the use of radiation. In addition, the method of treatment may further comprise using photodynamic therapy.
In some embodiments, including but not limited to infectious disease applications, the present invention relates to anti-infective agents as other therapeutic agents. In some embodiments, the anti-infective agent is an antiviral agent, including, but not limited to, abacavir (abaavir), acyclovir (Acyclovir), adefovir (Adefovir), amprenavir (Amprenavir), atazanavir (Atazanavir), cidofovir (Cidofovir), darunavir (Darunavir), delavirdine (Delavirdine), didanosine (Didanosine), doconazole Sha Nuo (doconol), efavirenz (Efavirenz), etidine (elvirigvir), emtricitabine (Emtricitabine), enfuvirtide (Enfuvirtide), etruvir, famciclovir (Famciclovir), and Foscarnet (foscanet). In some embodiments, the anti-infective agent is an antibacterial agent, including, but not limited to, cephalosporin antibiotics (cefalexin), cefuroxime (cefuroxime), cefadroxil (cefadroxil), cefazolin (cefazolin), cefalotin (cefalotin), cefaclor (cefaclor), cefamandole (cefamandole), cefoxitin (cefoxil), cefprozil, and ceftobiprole); fluoroquinolone antibiotics (fluoroquinolone antibiotics) (ciprofloxacin (ciprof), levofloxacin (Levaquin), ofloxacin (floxin), gatifloxacin (tequin), moxifloxacin (avelox), and norfloxacin (norflox)); tetracyclines (tetracyclines, minocycline (minocycline), hydroxytetracycline (oxytetracycline), and doxycycline (doxycycline)); penicillin antibiotics (amoxicillin (aroxillin), amibenicillin (ampicillin), penicillin V, dicloxacillin (dicarboxicillin), vancomycin (vancomycin), and methicillin (methicillin)); monoamide ring antibiotics (aztreonam); carbapenem antibiotics (ertapenem), doripenem (doripenem), imipenem (imipenem)/cilastatin (cilastatin) and meropenem). In some embodiments, anti-infective agents include antimalarial agents such as chloroquine, quinine, mefloquine, primary amine quinoline, doxycycline, artemether, lumefantrine, atovaquone, proguanil, and sulfadoxine, pyrimethamine, metronidazole, tinidazole, ivermectin, thiapyrimide, and albendazole.
In illustrative embodiments, the present invention relates to the use of hepatitis therapeutics as additional therapeutics. In various embodiments, hepatitis therapeutic agents include, but are not limited to, IFN- α (such as intra a) or pegylated IFN- α (such as Pegasys or PEG-intra), ribavirin (ribavirin), boceprevir (boceprevir), simeprevir (simeprevir), febuvir (sofosbuvir), cimeravir, dacarbatavir (daclatasvir), ledipasvir/febuvir (Harvoni), oxybitavir (ombasavir)/palitaconvir (ritonavir), oxybitaconvir/ritonavir (dacarbavir) (techonevir), oxybitaconvir/darinavir (dasativir), lamivudine (labelvudine), adefovir (efovir), aviconvalvir (ecaconvalvir), and any combination thereof. In one embodiment, the additional therapeutic agent is IFN- α (e.g., INTRON A) or pegylated IFN- α (e.g., pegasys or PEG-INTRON). In another embodiment, the additional therapeutic agent is ribavirin.
In some embodiments, the invention relates to combination therapies using the chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes and an immunosuppressive agent. In some embodiments, the invention relates to the administration of the Clec9A binding agent to a patient treated with an immunosuppressive agent.
In one embodiment, the immunosuppressive agent is TNF. In illustrative embodiments, the chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, act synergistically when co-administered with TNF. In illustrative embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, when co-administered with TNF for treating a tumor or cancer, act synergistically. For example, co-administration of a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, and TNF can act synergistically to reduce or eliminate the tumor or cancer, or to slow the growth and/or progression and/or metastasis of the tumor or cancer. In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, in combination with TNF can exhibit improved safety characteristics when compared to the agent alone in the case of monotherapy. In some embodiments, the chimeric protein or chimeric protein complex, such as Fc-based chimeric protein complex and TNF, may be administered at a lower dose than would be employed when each dose is used in a monotherapy setting.
In some embodiments, including but not limited to some embodiments of autoimmune applications, the other therapeutic agent is an immunosuppressant, which is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAID). Steroids, particularly adrenocorticosteroids and synthetic analogs thereof, are well known in the art. Examples of corticosteroids useful in the present invention include, but are not limited to, hydroxytriamcinolone (hydroxytriamcinolone), alpha-methyl dexamethasone (alpha-methyl outer peripheral), beta-methyl dexamethasone, beclomethasone dipropionate (beclomethasone dipropionate), betamethasone benzoate (betamethasone benzoate), betamethasone dipropionate, betamethasone valerate, clobetamethasone valerate (clobetasol valerate), desonide (desonide), desoxymethasone (desoxymethyl), dexamethasone (desoxymethyl), difluprethasone diacetate (diflorasone diacetate), diflupron (diflucortolone valerate), fludrolone (flucarbazone), fluclobetasone (fluclorolone acetonide), terfenasone (flumethasone pivalate), fluocinolone (fluosinolone acetonide), fluocinolone acetate (fluocinolone), fluocinolone (flucortine butylester), fludocetasone (fluocinolone), flubenzodiazide (desoxymethyl) and corresponding to the present invention, fludrolone (difluorosone diacetate), fludrolone (25), fludrolone (prednisone (5), fludrolone (54), fludrolone (25), fludrolone (54), fludrolone (5), fludrolone (54), fludrolone (35), fludroxide (prednisolide (and other than one Prednisone (chloroprednisone), clocortolone (closcortelone), clecinolone (clestinone), dichloropine (dichlorsoone), difluprednate (difluprednate), fluclonide (fludronide), flunisolide (flushinolide), fluorometholone (fludronate), fluprednisolone (fluprednisolone), hydrocortisone (hydrocortisone), methylprednisone (meprednisone), palatethasone (paramethasone), prednisone (prednisone), betamethasone dipropionate. (NSAIDs) that may be used in the present invention include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylamide, benzyl-2, 5-diacetoxybenzoic acid, ibuprofen (ibuprofen), sulindac (fulindac), naproxen (naproxen), ketoprofen (ketoprofen), etofenamate (etofenamate), phenylbutazone (phenylbutazone), and indomethacin (indomethacin). In some embodiments, the immunosuppressive agent may be a cytostatic agent, such as alkylating agents, antimetabolites (e.g., methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab), daclizumab (daclizumab) and moruzumab (muromonab)), anti-immunophilins (anti-immunophilins) (e.g., cyclosporin (cycloporine), tacrolimus (tacrolimus), sirolimus (sirolimus), interferons, opioids, TNF binding proteins, mycophenolic esters, and small biological agents (e.g., fingolimod, myriocin). Other anti-inflammatory agents are described, for example, in U.S. patent No. 4,537,776, the entire contents of which are hereby incorporated by reference.
In some embodiments, the invention relates to various agents for treating obesity as additional therapeutic agents. Illustrative agents for the treatment of obesity include, but are not limited to, orlistat (e.g., all, XENICAL), lorcaserin (locaserin) (e.g., BELVIQ), phentermine (phentermine) -topiramate (topiramate) (e.g., qsyia), sibutramine (sibutramine) (e.g., ductil or merjdii), rimonabant (ACOMPLLA), exenatide (exenatide) (e.g., BYETTA), pramlintide (pramlintide) (e.g., SYMLIN) phentermine, benzphetamine (benzphetamine), diethylpropion (diethyl propion), phendimetrazme, bupropion (bupropion), and metformin (metformin). Agents that interfere with the body's ability to absorb specific nutrients in food are among the additional agents, such as orlistat (e.g., ALU, XENICAL), glucomannan, and guar gum. Agents that suppress appetite are also among the additional agents, for example catecholamines and derivatives thereof (such as phentermine (phenteimine) and other amphetamine-based drugs), various antidepressants and mood stabilizers (such as bupropion and topiramate), anorectics (such as dexedrine, digoxin). Agents that increase bodily metabolism are also among the additional agents.
In some embodiments, the additional therapeutic agent may be selected from appetite suppressants, neurotransmitter re-uptake inhibitors, dopaminergic agonists, serotonergic agonists, gabaergic signaling modulators, anticonvulsants, antidepressants, monoamine oxidase inhibitors, substance P (NK 1) receptor antagonists, melanocortin receptor agonists and antagonists, lipase inhibitors, fat absorption inhibitors, energy intake or metabolism modulators, cannabinoid receptor modulators, agents for the treatment of addiction, agents for the treatment of metabolic syndrome, peroxisome proliferator-activated receptor (PPAR) modulators; dipeptidyl peptidase 4 (DPP-4) antagonists, agents for treating cardiovascular diseases, agents for treating elevated triglyceride levels, agents for treating low HDL, agents for treating hypercholesterolemia, and agents for treating hypertension. Some agents for cardiovascular disease include statins (e.g., lovastatin, atorvastatin, fluvastatin, rosuvastatin, simvastatin, and pravastatin) and omega-3 agents (e.g., LOVAZA, EPANQVA, VASCEPA, esterified omega-3, typically fish oil, krill oil, algae oil). In some embodiments, the additional agent may be selected from the group consisting of amphetamine, benzodiazepine, sulfonylurea, meglitinide (meglitinide), thiazolidinedione, biguanide, beta-blocker, XCE inhibitor, diuretic, nitrate, calcium channel blocker, phentermine, sibutramine, lorcaserin, cetiristat, rimonabant, tylonabant (taranabant), topiramate, gabapentin, valproate, vegabine, bupropion, tiagabine, sertraline, fluoxetine, trazodone, zonisamide, methylphenidate, valproine, diethylbenzene, lei Page, flunisolide, and meglitinide.
In some embodiments, the invention relates to agents useful as additional therapeutic agents for treating diabetes. Illustrative antidiabetic agents include, but are not limited to, sulfonylureas (e.g., dymelos (acetobutylurea), dibazene (chlorpropamide), ORINASE (methylsulfonylmethylurea) and TOLINASE (methylsulfonylmethylamine Zhuo Niao), glucotolol (glimepiride), glucotolal (prolonged release), dibeta (glibenclamide), MICRONASE (glibenclamide), glinasase prestarb (glibenclamide), and AMARYL (glimepiride)); biguanides (e.g., metformin (glucopyranose, glucopyranose XR, RIOMET, FORTAMET, and glucoetza)); thiazolidinediones (e.g. ACTOS (pioglitazone) and AVANDIA (rosiglitazone); alpha-glucosidase inhibitors (e.g., PRECOSE (acarbose) and GLYSET (miglitone); meglitinide (e.g., PRANDIN (repaglinide) and STARLIX (nateglinide)); an oral preparation of a dipeptidyl peptidase IV (DPP-IV) inhibitor (e.g., JANUVIA (sitagliptin), NESINA (alogliptin), ONGLYZA (saxagliptin) and TRADJEFTIN)), a sodium-glucose cotransporter 2 (SGLT 2) inhibitor (e.g., INVOKANA (Canagliflozin)), and a combination pill (e.g., GLUCOVANCE combining Globuzin (sulfonylurea) and metformin, metagliflozin (sulfonylurea) and Metagliflozin (Metagliflozin) in one pill and AVANDAMET, KAZANO (alogliptin and metformin) using metformin and rosiglitazone (AVANDIA), OSI (alogliptin), a biguanide, ACWES oral preparation, BYETTA subcutaneous preparation, JAVA oral preparation, lvUHOL oral preparation, NUMETIM oral preparation, methozimide oral preparation, gliptin, ZA-Methozimide preparation, UK-UK, ZUK-UK, ZUK, VIP, ZUK, VIP, and VIP, GLP, VIP, GLP-5, VIP-5, and oral preparation, VICTOZA 2-PAK subcutaneous agent, HUMALOG subcutaneous agent, STARLIX oral agent, FORTAMET oral agent, GLUCOVANCE oral agent, GLUCOPHAGE XR oral agent, NOVOLOG Mix 70-30FLEXPEN subcutaneous agent, GLYBURIDE-METRONMIN oral agent, acarbose oral agent, SYMLINPEN subcutaneous agent, GLUCOTRO1 XL oral agent, NOVOLIN R injection, GLUCOTROL oral agent, DUETACT oral agent, sitagliptin oral agent, SYMLINPEN subcutaneous agent, HUMALOG KWIKPEN subcutaneous agent JANUMET XR oral, GLIPIZIDE-METROMEIN oral, CYCLOSET oral, HUMALOG MIX 75-25 subcutaneous, nateglinide oral, HUMALOG Mix 75-25 KWIKWIKPEN subcutaneous, HUMULIN 70/30 subcutaneous, PRECASE oral, APIDRA subcutaneous, humulin R injection, jentadato oral, victorza 3-Pak subcutaneous, novolin 70/30 subcutaneous, NOVOLIN N subcutaneous, hyperinsulin subcutaneous, micronized glibenclamide oral GLYNASE oral agent, HUMULINN subcutaneous agent, insulin glargine subcutaneous agent, RIOMET oral agent, pioglitazone-METFORMIN oral agent, APIDRA SOLOSTAR subcutaneous agent, lispro insulin subcutaneous agent, GLYSET oral agent, HUMULIN 70/30 Pen subcutaneous agent, colesevelon oral agent, sitagliptin-METFORMIN oral agent, DIABETA oral agent, conventional human insulin injection, HUMULIN N Pen subcutaneous agent, exenatide subcutaneous agent, HUMALOG Mix 50-50 IKEN subcutaneous agent, liraglutide subcutaneous agent, KAZANO oral agent, repaglinide oral agent, chlorpropamide oral agent, insulin aspart subcutaneous agent, NOVOLOG Mix 70-30 subcutaneous agent, HUMALOG Mix 50-50 subcutaneous agent, saxagliptin oral agent, ACTOPS Met oral agent, miglitol oral agent, H recombinant human insulin, NPH 35H and conventional human insulin, mifepristone oral agent, insulin aspart protamine-insulin aspart subcutaneous agent, repaglinide-metformin oral agent, saxagliptin-metformin oral agent, linagliptin-metformin oral agent, nesia oral agent, OSENI oral agent, tolbutamide oral agent, insulin Lispro-protamine and insulin Lispro-subcutaneous agent, pranlukast Lin Taipi lower agent, insulin Lispro-subcutaneous agent, pioglitazone-glimepiride oral agent, PRANDIMET oral agent, NOVOLOG PenFill subcutaneous agent, linagliptin oral agent, exenatide microsphere subcutaneous agent, korllym oral agent, alogliptin-pioglitazone oral agent, alogliptin-metformin oral agent, canagliptin oral agent, insulin Lispro (humallog); insulin Aspart (NOVOLOG); insulin Glulisine (apidria); regular insulin (NOVOLIN R or HUMULIN R); NPH (NOVOLIN N or HUMULIN N); insulin glargine (LANTUS); insulin Detemir (level); HUMULIN or NOVOLIN 70/30; and NOVOLOG Mix 70/30 HUMALOG Mix 75/25 or 50/50.
In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, act synergistically when used in combination with Chimeric Antigen Receptor (CAR) T cell therapies. In an illustrative embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, synergistically acts when used in combination with CAR T cell therapy for treating a tumor or cancer. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex agent, synergistically acts when used in combination with CAR T cell therapy for treating a blood-based tumor. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, acts synergistically when used in combination with CAR T cell therapy to treat a solid tumor. For example, use of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex and CAR T cells, can act synergistically to reduce or eliminate the tumor or cancer, or to slow the growth and/or progression and/or metastasis of the tumor or cancer. In various embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, induces CAR T cell division. In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, induce CAR T cell proliferation. In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, prevent CART cell non-responsiveness.
In various embodiments, the CAR T cell therapy comprises T cells that target antigens (e.g., tumor antigens), such as, but not limited to, carbonic Anhydrase IX (CAIX), 5T4, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CS1, CD138, lewis-Y, L1-CAM, MUC16, ROR-1, IL13 ra 2, gp100, prostate Stem Cell Antigen (PSCA), prostate Specific Membrane Antigen (PSMA), B Cell Maturation Antigen (BCMA), human papilloma virus type 16 E6 (HPV-16 E6), CD171, folate receptor alpha (FR-a), GD2, human epidermal growth factor receptor 2 (HER 2), mesothelin, EGFRvIII, fibroblast Activation Protein (FAP), carcinoembryonic antigen (CEA) and vascular endothelial growth factor receptor 2 (VEGF-R2), as well as other tumor antigens well known in the art of CARs. Other illustrative tumor antigens include, but are not limited to MART-1/Melan-A, gp, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin B, colorectal associated antigen (CRC) -0017-1A/GA733, carcinoembryonic antigen (CEA) and immunogenic epitopes CAP-1 and CAP-2, etv, aml1, prostate Specific Antigen (PSA) and immunogenic epitopes PSA-1, PSA-2 and PSA-3, T cell receptor/CD 3-zeta chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A1, MAGE-B-C-A3, MAGE-A5, MAGE-A6, MAGE-C-A8, MAGE-A3, MAGE-C-A2, MAGE-A7, MAGE-C MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, gnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, alpha-fetoprotein, E-calpain, alpha-interlocking protein, beta-interlocking protein, and gamma-interlocking protein, p120ctn, gP100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), cytokinin, connexin 37, ig idiotypes, p15, gP75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, smad family tumor antigens, 1mp-1, NA, EBV encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, CD19, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, nmep-CAM, PD-L1 and PD-L2.
Illustrative CAR T cell therapies include, but are not limited to, JCAR014 (Juno Therapeutics), JCAR015 (Juno Therapeutics), JCAR017 (Juno Therapeutics), JCAR018 (Juno Therapeutics), JCAR020 (Juno Therapeutics), JCAR023 (Juno Therapeutics), JCAR024 (Juno Therapeutics), CTL019 (Novartis), KTE-C19 (kit Pharma), BPX-401 (Bellicum Pharmaceuticals), BPX-501 (Bellicum Pharmaceuticals), BPX-601 (Bellicum Pharmaceuticals), bb2121 (blubird Bio), CD-19 sleeping beauty cells (Ziopharm Oncology), UCART19 (Cellectis), UCART123 (Cellectis), UCART38 (Cellectis), uccs 1 (Cellectis), oxford biomedicia, MB-101 (Mustang Bio), and T cells developed by Innovative Cellular Therapeutics.
In some embodiments, chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, are used in methods of treating MS in combination with one or more Multiple Sclerosis (MS) therapeutic agents including, but not limited to, 3-interferon, glatiramer acetate, T-interferon, IFN-beta-2 (U.S. patent publication No. 2002/0025304), helical germanium (e.g., N- (3-dimethylaminopropyl) -2-aza-8, 8-dimethyl-8-germanium spiro [4:5] decane, N- (3-dimethylaminopropyl) -2-aza-8, 8-diethyl-8-germanium spiro [4:5] decane, N- (3-dimethylaminopropyl) -2-aza-8, 8-dipropyl-8-germanium spiro [4:5] decane, and N- (3-dimethylaminopropyl) -2-aza-8, 8-dibutyl-8-germanium spiro [4:5] decane), vitamin D analogs (e.g., 1, 25 (OH) 2D3 (see, e.g., U.S. Pat. No. 5,716,946)), prostaglandins (e.g., latanoprost, brimonidine, PGE1, PGE2, and PGE3 (see, e.g., U.S. Pat. publication No. 2002/0004525), tetracyclines, and derivatives (e.g., minocycline and doxycycline), see, e.g., U.S. Pat. publication No. 20020022608), VLA-4 binding antibodies (see, e.g., U.S. patent publication No. 2009/0202527), corticotropins, corticosteroids, prednisone (prednisone), methylprednisone (methylprednisone), 2-chlorodeoxyadenosine, mitoxantrone, sulfasalazine (sulfaphalazine), methotrexate, azathioprine, cyclophosphamide, cyclosporin, fumarates, anti-CD 20 antibodies (e.g., rituximab), and tebuconamidine hydrochloride (tizanidine hydrochloride).
In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is used in combination with one or more therapeutic agents that treat one or more symptoms or side effects of MS. Such agents include, but are not limited to, amantadine, baclofen, papaverine, meclozine, hydroxyzine, sulfamethoxazole, ciprofloxacin, docusate, pimeline, dantrolene, desmopressin, dexamethasone, tolterodine, phenytoin, oxybutynin, bisacodyl, venlafaxine, amitriptyline, hexamethylenetetramine, clonazepam, pantoprene, and the like isoniazid (isoniazid), vardenafil (vardenafil), nitrofurantoin (nitrofurantoin), psyllium mucilage (psyllium hydrophilic mucilloid), alprostadil (alprostadil), gabapentin (gabapentin), nortriptyline (nortriptyline), paroxetine (paroxetine), bromopropioline (propantheline bromide), modafinil (modafinil), fluoxetine (fluoxetine), phenazopyrine (phenazopyridine), methylprednisolone (methlyprednisolone), carbamazepine (carbamazepine), imipramine (imipramine), diazepine (buprofirine), bupropion (buprofirine) and sertraline (sertrack).
In some embodiments, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is used in a method of treating multiple sclerosis in combination with one or more Disease Modification Therapies (DMT) (e.g., an agent of table 6) described herein. In some embodiments, the invention provides improved therapeutic effects compared to the use of one or more DMTs described herein (e.g., the agents listed in table 6 below) without the use of one or more of the disclosed binding agents. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, in combination with the one or more DMT produces a synergistic therapeutic effect.
Illustrative disease modifying therapies include, but are not limited to:
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in some embodiments, the invention relates to combination therapies utilizing blood transfusion. For example, the compositions of the invention may be used as a supplement to blood transfusion. In some embodiments, the invention relates to combination therapies utilizing iron supplements.
In some embodiments, the invention relates to combination therapies utilizing one or more EPO-based agents. For example, the compositions of the present invention may be used as adjuvants for other EPO-based agents. In some embodiments, the compositions of the invention are used as maintenance therapies for other EPO-based agents. Other EPO-based agents include the following: epoetin α, including but not limited to DARBEPOETIN (ARANESP), EPOCEPT (LUPIN PHARMA), NANOKINE (NANOGEN PHARMACEUTICAL), EPOFIT (INTASPHARMA), EPOGEN (amben), EPOGIN, EPREX (JANSSEN-CILAG), bincrit 7 (SANDOZ), proclt; epoetin beta, including but not limited to NEORECORMON (HOFFMANN-LA ROCHE), RECOMON, monomethoxy polyethylene glycol-epoetin beta (MIRCERA, ROCHE); epoetin delta, including but not limited to DYNEPO (erythropoiesis stimulating protein, SHIRE PLC); epoetin omega, including but not limited to EPOMAX; epoetin ζ, including but not limited to SILAPO (STADA) and RETACRIT (HOSPIRA); and other EPO's including, but not limited to EPOCEPT (LUPIN PHARMACEUTICALS), EPOTRUST (PANACEA BIOTEC LTD), ercryro SAFE (BIOCON ltd.), repoittin (SERUM INSTITUTE OF INDIA LIMITED), VINTOR (EMCURE PHARMAC EUTICALS), EPOFIT (INTAS PHARMA), ERYKINE (INTAS BIO PHARMACEUTICA), WEPOX (WOCKHARDT BIOTECH), ESPO GEN (LG LIFE SCIENCES), RELIPOIETIN (RELIANCE LIFE SCI ENCES), SHANPOIETIN (SHANTHA BIOTECHNICSLTD), ZYR OP (CADILA HEALTHCARE ltd.), epaao (rhauepo) (SHENYA NG SUNSHINE PHARMACEUTICAL co.ltd), CINNAPOIETIN (CINNAGEN).
In some embodiments, the invention relates to combination therapies utilizing one or more immune modulating agents, such as, but not limited to, agents that modulate immune checkpoints. In various embodiments, the immunomodulatory agent targets one or more of PD-1, PD-L1, and PD-L2. In various embodiments, the immunomodulator is a PD-1 inhibitor. In various embodiments, the immunomodulatory agent is an antibody specific for one or more of PD-1, PD-Ll, and PD-L2. For example, in some embodiments, the immunomodulatory agent is an antibody, such as, but not limited to, nivolumab (ONO-4538/BMS-936558,MDX1106,OPDIVO,BRISTOL MYERS SQUIBB), pembrolizumab (keyruda, MERCK), pirimab (CT-011, cure TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL328OA (ROCHE). In some embodiments, the immunomodulatory agent targets one or more of CD137 or CD 137L. In various embodiments, the immunomodulatory agent is an antibody specific for one or more of CD137 or CD 137L. For example, in some embodiments, the immunomodulatory agent is an antibody, such as, but not limited to, wu Ruilu mab (urelumab) (also known as BMS-663513 and anti-4-1 BB antibodies). In some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, is combined with Wu Ruilu mab (optionally with one or more of nivolumab, li Lishan antibody (lirilumab) and Wu Ruilu mab) to treat solid tumors and/or B cell non-hodgkin's lymphoma and/or head and neck cancer and/or multiple myeloma. In some embodiments, the immunomodulatory agent is an agent that targets one or more of the following: CTLA-4, AP2M1, CD80, CD86, SHP-2 and PPP2R5A. In various embodiments, the immunomodulator is an antibody specific for one or more of the following: CTLA-4, AP2M1, CD80, CD86, SHP-2 and PPP2R5A. For example, in some embodiments, the immunomodulatory agent is an antibody, such as, but not limited to, ipilimumab (MDX-010, MDX-101, yervoy, bms) and/or tremelimumab (Pfizer). In some embodiments, a chimeric protein or chimeric protein complex of the invention, such as an Fc-based chimeric protein complex, is combined with ipilimumab (optionally with bavituximab) to treat one or more of melanoma, prostate cancer, and lung cancer. In various embodiments, the immunomodulator targets CD20. In various embodiments, the immunomodulator is an antibody specific for CD20. For example, in some embodiments, the immunomodulatory agent is an antibody, such as, but not limited to, ofatuzumab (GENMAB), obbintuzumab You Tuozhu mab (GAZYVA), AME-133v (APPLIEDMOLECULAR EVOLUTION), oreuzumab (GENENTECH), TRU-015 (TRU/EMERGENT), veltuzumab (veltuzumab) (IMMU-106).
In some embodiments, the present invention relates to combination therapies utilizing one or more of the intercalators described in WO 2013/10779, WO 2015/007536, WO 2015/007518, WO 2015/007542 and WO 2015/007903, the entire contents of which are hereby incorporated by reference in their entirety.
In some embodiments, the chimeric proteins or chimeric protein complexes described herein, such as Fc-based chimeric protein complexes, include modified derivatives, i.e., by covalently linking any type of molecule to the composition such that the covalent linkage does not interfere with the activity of the composition. By way of example and not limitation, derivatives include compositions modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications may be made using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
In other embodiments, the chimeric proteins or chimeric protein complexes described herein, such as Fc-based chimeric protein complexes, further comprise a cytotoxic agent, which in illustrative embodiments comprises a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. Such agents may be conjugated to the compositions described herein.
Thus, a chimeric protein or chimeric protein complex described herein, such as an Fc-based chimeric protein complex, may undergo post-translational modification to add effector moieties, such as chemical linkers; detectable moieties such as fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties; or functional moieties such as streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, and radioactive materials.
Illustrative cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopterin, 6-thioguanterin, cytarabine, 5-fluorouracil, dacarbazine; alkylating agents such as nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozocin, mitomycin C, cis-dichlorodiamide platinum (II) (DDP), cisplatin and carboplatin (berdine); anthracyclines including daunorubicin (previously daunorubicin), doxorubicin (doxorubicin), dithiino, carminomycin, idarubicin, epirubicin, mitoxantrone, and bisacodyl; antibiotics, including dactinomycin/actinomycin D, bleomycin, spinosyn, mithramycin and Aflatoxin (AMC); and antimitotic agents such as vinca alkaloids (vinca alkaloids), vincristine (vincristine), and vinblastine (vinblastine). Other cytotoxic agents include paclitaxel (paclitaxel), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetidine (emetine), etoposide, teniposide, colchicine, dihydroxyanthracenedione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O, P' - (DDD)), interferons, and mixtures of these cytotoxic agents.
Other cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, spinosad, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonist, EGFR antagonist, platinum, paclitaxel, irinotecan, 5-fluorouracil, gemcitabine, folinic acid, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine), moxetines, tyrosine kinase inhibitors, radiation therapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g., IL-12 or IL-2), IL-12R antagonists, toxin conjugated monoclonal antibodies, tumor antigen specific monoclonal antibodies, biturates (bituxins), alexanes (Avastins), oxacarrier (Abs), oxaden (Otuzumab (20), otuzumab (Pertuzumab), pertuzumab (20), and other anti-human antibodies,Or any combination thereof. Toxic enzymes from plants and bacteria, such as ricin, diphtheria toxin and pseudomonas toxin, can be conjugated to these therapeutic agents (e.g., antibodies) to produce cell type specific killing agents (you et al, proc. Nat 'l Acad. Sci. USA 77:5483 (1980); gilliland et al, proc. Nat' l) Acad. Sci. USA 77:4539 (1980); krolick et al, proc.nat' l acad.sci.usa 77:5419 (1980)).
Other cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. patent No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates in which the radionuclide emitting alpha or beta particles are stably coupled to a chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, with or without the use of a complex forming agent. Such radionuclides include beta-emitters such as phosphorus-32, scandium-47, copper-67, gallium-67, yttrium-88, yttrium-90, iodine-125, iodine-131, samarium-153, lutetium-177, rhenium-186, or rhenium-188; and an alpha emitter such as astatine-211, lead-212, bismuth-213, or actinium-225.
Illustrative detectable moieties also include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase, and luciferase. Other illustrative fluorescent materials include, but are not limited to, rhodamine, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Other illustrative chemiluminescent moieties include, but are not limited to, luminol. Other illustrative bioluminescent materials include, but are not limited to, fluorescein and aequorin. Other illustrative radioactive materials include, but are not limited to, iodine-125, carbon-14, sulfur-35, tritium, and phosphorus-32.
Therapeutic method
The methods and compositions described herein are applicable to the treatment of a variety of diseases and conditions, including, but not limited to, cancer, infection, immune conditions, anemia, autoimmune diseases, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and conditions.
In addition, any of the agents of the present invention may be used in the treatment of, or in the manufacture of a medicament for, a variety of diseases and conditions, including, but not limited to, cancer, infection, immune disorders, inflammatory diseases or conditions, and autoimmune diseases.
In some embodiments, the invention relates to the treatment of one or more of the following diseases or patients suffering from one or more of the following: chronic granulomatosis, osteosclerosis, idiopathic pulmonary fibrosis, friedreich's ataxia, atopic dermatitis, chagas Disease, cancer, heart failure, autoimmune diseases, sickle cell Disease, thalassemia, blood loss, transfusion reactions, diabetes, vitamin B12 deficiency, collagen vascular Disease, shu Wake Mannheim syndrome (Shwachman syndrome), thrombocytopenic purpura, celiac Disease, endocrine deficient conditions such as hypothyroidism or Edison's Disease, autoimmune diseases such as Crohn's Disease, systemic lupus erythematosus, rheumatoid arthritis or juvenile rheumatoid arthritis ulcerative colitis immune disorders such as eosinophilic fasciitis, hypoimmunoglobulin syndrome or thymoma/thymus cancer, graft versus host Disease, pre-leukemia, non-blood syndrome (e.g., down's), du Bowei z syndrome (Dubowwitz), seckel's), feerty syndrome (felter syndrome), hemolytic uremic syndrome, myelodysplastic syndrome, nocturnal paroxysmal hemoglobinuria, myelomas, whole blood cytopenia, pure red cell regeneration disorder, schoenlein-Henoch purura), malaria, protein starvation, menorrhagia, systemic sclerosis, cirrhosis, hypometabolic status and congestive heart failure.
In some embodiments, the invention relates to a method for treating cancer, the method comprising i) administering to a patient in need thereof an effective amount of a chimeric protein, chimeric protein complex, and/or Fc-based chimeric protein complex; ii) administering to a patient in need thereof an effective amount of a recombinant nucleic acid encoding a chimeric protein, chimeric protein complex and/or Fc-based chimeric protein complex; or iii) administering to a patient in need thereof an effective amount of a host cell comprising a recombinant nucleic acid encoding a chimeric protein, chimeric protein complex and/or Fc-based chimeric protein complex.
In some embodiments, the invention relates to the treatment of one or more of the following diseases or patients suffering from one or more of the following: chronic granulomatosis, osteosclerosis, idiopathic pulmonary fibrosis, friedel-crafts ataxia, atopic dermatitis, chagas disease, mycobacterial infection, cancer, scleroderma, hepatitis c, septic shock and rheumatoid arthritis.
In some embodiments, the invention relates to treating cancer or a patient having cancer. As used herein, cancer refers to any uncontrolled cell growth that may interfere with the normal function of body organs and systems, and includes both primary and metastatic tumors. The primary tumor or cancer migrates from its original location and the vital organs can eventually lead to death of the subject by hypofunction of the affected organ. Metastasis is a cancer cell or group of cancer cells that occurs at a location remote from the primary tumor due to the spread of the cancer cells from the primary tumor to other parts of the body. Metastasis may ultimately lead to death of the subject. For example, cancers may include benign and malignant cancers, polyps, hyperplasia, dormant tumors, or micrometastases.
Illustrative cancers that can be treated include, but are not limited to, carcinoma such as various subtypes including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myelogenous, acute lymphoblastic, chronic myelogenous, chronic lymphocytic, and hair cells), lymphomas and myelomas (including, for example, hodgkin's and non-hodgkin's lymphomas, light chain, non-secretory, MGUS, and plasmacytoma), and central nervous system cancers (including, for example, brain (such as gliomas (such as astrocytomas, oligoglioblastomas, and ependymomas), meningiomas, pituitary adenomas, and neuromas and spinal cord tumors (such as meningiomas and fibrogliomas).
Illustrative cancers that may be treated include, but are not limited to: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal carcinoma; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcomas (e.g., kaposi's sarcoma); skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of urinary system; vulvar cancer; lymphomas, including hodgkin's lymphoma and non-hodgkin's lymphoma, as well as B-cell lymphomas (including low grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated blastoid NHL, giant tumor NHL, mantle cell lymphoma, AIDS-related lymphoma, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal vascular hyperplasia associated with nevi, oedema (e.g., brain tumor-related oedema), and megers syndrome in one embodiment, the present invention relates to the treatment of leukemia, including capillary lymphoblastic leukemia, in another embodiment relates to the treatment of AIDS, the invention relates to the treatment of melanoma, in another embodiment relates to the treatment of AIDS, the invention relates to the treatment of the malignant sarcoma.
In some embodiments, the invention relates to the treatment of microbial and/or chronic infections or patients suffering from microbial and/or chronic infections. Illustrative infections include, but are not limited to, QIAGES disease, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, epstein-Barr virus (Epstein-Barr virus) or parvovirus, T-cell leukemia virus, bacterial overgrowth syndrome, fungal or parasitic infections.
In some embodiments, the invention relates to treating hepatitis. Illustrative hepatitis which may be treated include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, autoimmune hepatitis, alcoholic hepatitis, acute hepatitis and chronic hepatitis.
In an illustrative embodiment, the present invention relates to the treatment of chronic hepatitis c. In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be used to treat patients infected with any of the hepatitis c genotypes, including genotype 1 (e.g., 1a, 1 b), genotype 2 (e.g., 2a, 2b, 2c, and 2 d), genotype 3 (e.g., 3a, 3b, 3c, 3d, 3e, and 3 f), genotype 4 (e.g., 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, and 4 j), genotype 5a, and genotype 6a.
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be used to treat patients that do not respond well or respond well to standard-of-care antiviral therapies or otherwise are refractory to standard-of-care hepatitis c therapies. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is useful for treating patients exhibiting a low or no response to IL-2 therapy with or without ribavirin. In one embodiment, the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, is useful for treating a patient that is slow or non-responsive to a combination therapy of pegylated interferon and ribavirin. In one embodiment, the invention relates to the treatment of patients who are infected with hepatitis C genotype 1 or any other genotype but who are not responsive to prior IL-2 therapy. In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, are useful for treating patients with high baseline viral loads (e.g., greater than 800,000IU/mL). In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, are useful in treating patients suffering from severe liver injury, including those suffering from advanced liver fibrosis and/or cirrhosis.
In some embodiments, the invention relates to treating a patient who has not received antiviral therapy. In other embodiments, the invention relates to treating patients that are not responsive to prior antiviral therapies. In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, are useful for treating patients with relapse.
In some embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, are effective in treating hepatitis infections in all race including white, african americans, spanish and asians. In one embodiment, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, may be particularly effective in treating african americans that respond poorly to IL-2 therapy with or without ribavirin.
In various embodiments, the targeting chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, provide increased safety compared to, for example, non-targeting ifnα1 or unmodified wild-type IL-2 or modified IL-2. In illustrative embodiments, administration of the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, is associated with minimal side effects, such as those associated with the use of non-targeted IL-2 or unmodified wild-type IL-2 or modified IL-2 (e.g., influenza-like symptoms, myalgia, leukopenia, thrombocytopenia, neutropenia, depression, and weight loss).
In some embodiments, the targeted chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, exhibit increased therapeutic activity compared to, for example, non-targeted IL-2 or unmodified wild-type IL-2 or modified IL-2. In some embodiments, the targeted chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, exhibit improved pharmacokinetic characteristics (e.g., longer serum half-life and stability) compared to, for example, non-targeted IL-2 or unmodified wild-type IL-2 or modified IL-2.
Without wishing to be bound by theory, it is believed that due to such advantageous safety and pharmacokinetic and therapeutic profiles, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be used to treat patients at high doses and/or for prolonged periods of time. For example, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be used at high doses for initial induction therapy against chronic hepatitis c infection. In another example, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be used in long-term maintenance therapy to prevent disease recurrence.
In various embodiments, the compositions of the invention are used to treat or prevent one or more inflammatory diseases or conditions, such as inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, leigh Syndrome (Leigh syncrome), glycerol kinase deficiency, familial Eosinophilia (FE), autosomal recessive spasticity, laryngeal inflammatory disease; tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconiosis.
In various embodiments, the compositions of the invention are used to treat or prevent one or more autoimmune diseases or disorders, such as multiple sclerosis, diabetes, lupus, celiac disease, crohn's disease, ulcerative colitis, guillain-Barre syndrome (Guillain-Barre syndrome), scleroderma, goodpasture's syndrome, wegener's granulomatosis, autoimmune epilepsy, rassmassen's encephalitis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, ai Disen's disease (adison's disease), hashimoto's thyroiditis (Hashimoto's thyroiditis), fibromyalgia, menier's syndrome (Menier's syndrome); transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, reiter's syndrome, grave's disease, and other autoimmune diseases.
In various embodiments, the compositions of the invention are used to treat, control, or prevent cardiovascular diseases, such as diseases or conditions affecting the heart and blood vessels, including, but not limited to, coronary Heart Disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular disease (stroke), transient Ischemic Attacks (TIA), angina (stable and unstable), atrial fibrillation, cardiac arrhythmia, vascular disease, and/or congestive heart failure.
In various embodiments, the compositions of the invention are used to treat or prevent one or more metabolic-related disorders. In various embodiments, the invention is useful for treating, controlling or preventing diabetes, including type 1 diabetes and type 2 diabetes, as well as diabetes associated with obesity. The compositions and methods of the invention are useful for treating or preventing diabetes-related disorders including, but not limited to, diabetic nephropathy, hyperglycemia, impaired glucose tolerance, insulin resistance, obesity, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDL levels, atherosclerosis and its sequelae, vascular restenosis, irritable bowel syndrome, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), other inflammatory disorders, pancreatitis, abdominal obesity, neurodegenerative disorders, retinopathy, neoplastic disorders, adipose cell tumors, adipose cell cancers (such as liposarcoma), prostate cancer and other cancers (including gastric cancer, breast cancer, bladder cancer and colon cancer), angiogenesis, alzheimer's disease, psoriasis, hypertension, metabolic syndrome (e.g., the presence of three or more of abdominal obesity, hypertriglyceridemia, low fasting cholesterol, hypertension and high respiratory glucose), ovarian androgen-associated syndrome (multiple plasma) and other disorders such as sleep apnea of the ovarian component. The compositions and methods of the invention are useful for treating, controlling or preventing obesity (including genetic or environmental) and obesity-related disorders. The obesity-related disorders herein are associated with, caused by, or a result of obesity. Examples of obesity-related disorders include obesity, diabetes, overeating, binge eating and bulimia, hypertension, elevated plasma insulin concentrations and insulin resistance, dyslipidemia, hyperlipidemia, endometrial cancer, breast cancer, prostate cancer, renal and colon cancer, osteoarthritis, obstructive sleep apnea, gallstones, heart disease, abnormal heart rhythm and arrhythmia, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovary disease, craniopharyngeal neoplasia, prader-Willi Syndrome (Prader-Willi Syndrome), friehlich's Syndrome, GH deficient subjects, short stature, tenna's Syndrome, and other pathological conditions that exhibit reduced metabolic activity or reduced energy consumption at rest (percentage of total free fatty matter), such as children suffering from acute lymphoblastic leukemia. Other examples of obesity related disorders are metabolic syndrome, insulin resistance syndrome, abnormal genital hormones, sexual and reproductive dysfunction (such as impaired fertility, infertility, male hypogonadism and female hirsutism), fetal defects associated with maternal obesity, gastrointestinal motility disorders (such as obesity related gastroesophageal reflux), respiratory disorders such as obese pulmonary hypoventilation syndrome (Pexwell syndrome (Pickwickian syndrome)), shortness of breath, cardiovascular disorders, inflammation (such as systemic vasculitis), arteriosclerosis, hypercholesterolemia, lower back pain, gallbladder disease, hyperuricemia, gout and renal cancer, and increased risk of numbness. The compositions and methods of the invention are also useful for treating Alzheimer's disease.
In various embodiments, the compositions of the invention are used to treat or prevent one or more respiratory diseases, such as Idiopathic Pulmonary Fibrosis (IPF), asthma, chronic Obstructive Pulmonary Disease (COPD), bronchiectasis, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergy, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, hantavirus pulmonary syndrome (Hantavirus pulmonary syndrome, HPS), luffler's syndrome, goodpasture's syndrome, pleurisy, pneumonia, pulmonary edema, pulmonary fibrosis, sarcoidosis, complications associated with respiratory syncytial virus infection, and other respiratory diseases.
In some embodiments, the invention is used to treat or prevent one or more neurodegenerative diseases. Illustrative neurodegenerative diseases include, but are not limited to, friedreich's ataxia, multiple sclerosis (including, but not limited to, benign multiple sclerosis; relapsing-remitting multiple sclerosis (RRMS), secondary Progressive Multiple Sclerosis (SPMS), progressive Relapsing Multiple Sclerosis (PRMS), and Primary Progressive Multiple Sclerosis (PPMS)), alzheimer's Disease (including, but not limited to, early onset alzheimer's Disease, late onset alzheimer's Disease, and Familial Alzheimer's Disease (FAD), parkinson's Disease, and parkinson's Disease (including, but not limited to, idiopathic parkinson's Disease, vascular parkinson's Disease, drug-induced parkinson's Disease, lewy body dementia, hereditary parkinson's Disease, juvenile parkinson's Disease), huntington's Disease, amyotrophic lateral sclerosis (ALS, including, but not limited to, idiopathic ALS, familial ALS, western alien ALS, juvenile ALS, western malayan Disease (himalayadisease)).
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be used to treat wounds, e.g., non-healing wounds, ulcers, burns or frostbite, chronic or acute wounds, open or closed wounds, internal wounds or trauma (illustrative trauma is penetrating and non-penetrating).
In various embodiments, the chimeric proteins or chimeric protein complexes of the invention, such as Fc-based chimeric protein complexes, can be used to treat ischemia, as non-limiting examples, ischemia associated with acute coronary syndrome, acute Lung Injury (ALI), acute Myocardial Infarction (AMI), acute Respiratory Distress Syndrome (ARDS), arterial occlusive disease, arteriosclerosis, articular cartilage defect, aseptic systemic inflammation, atherosclerosis cardiovascular disease, autoimmune disease, bone fracture, cerebral edema, cerebral hypoperfusion, buerger's disease, burn, cancer, cardiovascular disease, cartilage injury, cerebral infarction, cerebral ischemia, stroke, cerebrovascular disease, chemotherapy-induced neuropathy, chronic infection, chronic mesenteric ischemia, lameness, congestive heart failure, connective tissue injury, contusion, coronary Artery Disease (CAD), cerebral ischemia, cerebral infarction, cerebral ischemia, cerebral stroke, cerebrovascular disease, chemotherapy-induced neuropathy, chronic infection, chronic mesenteric ischemia, lameness, congestive heart failure, connective tissue injury, contusion, coronary Artery Disease (CAD) Critical Limb Ischemia (CLI), crohn's disease, deep venous thrombosis, deep trauma, delayed ulcer healing, delayed wound healing, diabetes (type I and type II), diabetic neuropathy, diabetes-induced ischemia, disseminated Intravascular Coagulation (DIC), embolic cerebral ischemia, frostbite, graft versus host disease, hereditary hemorrhagic telangiectasia ischemic vascular disease, hyperoxia injury, hypoxia, inflammation, inflammatory bowel disease, inflammatory disease, tendon injury, intermittent claudication, intestinal ischemia, ischemic encephalopathy, ischemic heart disease, ischemic peripheral vascular disease, placental ischemia, ischemic kidney disease, ischemic vascular disease, inflammatory bowel disease, vascular graft versus host disease, vascular graft injury, intermittent claudication, ischemic heart disease, ischemic peripheral vascular disease, ischemic heart disease, and ischemic heart disease, ischemic reperfusion injury, laceration, left main coronary artery disease, limb ischemia, lower limb ischemia, myocardial infarction, myocardial ischemia, organ ischemia, osteoarthritis, osteoporosis, osteosarcoma, parkinson's disease, peripheral Arterial Disease (PAD), peripheral arterial disease, peripheral ischemia, peripheral neuropathy, peripheral vascular disease, precancerous, pulmonary edema, pulmonary embolism, remodeling disorders, renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers, solid organ transplantation, spinal cord injury, stroke, subchondral bone cyst, thrombosis, thrombotic cerebral ischemia, tissue ischemia, transient Ischemic Attacks (TIA), traumatic brain injury, ulcerative colitis, renal vascular disease, vascular inflammatory disorders, von Hippel-lindasyndrome, or tissue or organ trauma.
In various embodiments, the invention relates to the treatment of one or more anemias, including anemias caused by chronic kidney disease (e.g., by dialysis) and/or anticancer agents (e.g., chemotherapy and/or HIV treatment (e.g., zidovudine (zin) or Azidothymidine (AZT)), inflammatory bowel disease (e.g., crohn's disease and ulcerative colitis), anemias associated with inflammatory disorders (e.g., arthritis, lupus, IBD), anemias associated with diabetes, schizophrenia, cerebral malaria, aplastic anemia, and myelodysplastic syndromes resulting from cancer treatment (e.g., chemotherapy and/or radiation) and various myelodysplastic syndromes (e.g., sickle cell anemia, hemoglobinopathy C, alpha thalassemia and beta thalassemia, post-premature neonatal anemia, and corresponding disorders).
In some embodiments, the invention relates to treating anemia, i.e., a condition in which the number of red blood cells and/or the amount of hemoglobin found in red blood cells is below normal, or in patients suffering from anemia. In various embodiments, anemia may be acute or chronic. For example, the anemias of the invention include, but are not limited to, iron deficiency anemias, renal anemias, chronic disease/inflammatory anemias, pernicious anemias (such as megaloblastic, juvenile pernicious and congenital pernicious anemias), cancer-related anemias, anticancer-related anemias (e.g., chemotherapy-related anemias, radiotherapy-related anemias), simple red cell dysplasia, refractory anemias with excessive blasts, aplastic anemias, X-linked iron-particle-juvenile-cell anemias, hemolytic anemias, sickle-cell anemias, anemias caused by impaired ESA production, myelodysplastic syndrome, low-color anemias, microcytic anemias, iron-particle-juvenile-anemias, autoimmune hemolytic anemias, cooley's, thalassemias, wear-Buddha (Diamond Blackfan anemia), fanconi's anemia, and drug-induced immune hemolytic anemias. Anemia can lead to severe symptoms including hypoxia, chronic fatigue, inattention, pale skin, hypotension, dizziness and heart failure.
In some embodiments, the invention relates to treating anemia arising from chronic renal failure. In some embodiments, the invention relates to treating anemia arising from the use of one or more renal replacement therapies, including dialysis, hemodialysis, peritoneal dialysis, hemofiltration, hemodiafiltration, and renal transplantation.
In some embodiments, the invention relates to treating anemia in chronic kidney disease patients who have not undergone dialysis. For example, the invention relates to patients in stage 1 CKD, or stage 2 CKD, or stage 3 CKD, or stage 4 CKD, or stage 5 CKD. In some embodiments, the patient of the invention is stage 4 CKD or stage 5 CKD. In some embodiments, the patient of the invention has undergone kidney transplantation. In some embodiments, the invention relates to treating anemia in patients with Acute Kidney Injury (AKI).
In some embodiments, the anemia is induced by chemotherapy. For example, the chemotherapy may be any myelosuppressive chemotherapy. In some embodiments, the chemotherapy is one or more of Revlimid, thalomid, dexamethasone, doxorubicin (Adriamycin), and Doxil. In some embodiments, chemotherapy is one or more platinum-based drugs, including cisplatin (e.g., PLATINOL) and carboplatin (e.g., PARAPLATIN). In some embodiments, chemotherapy is any of the chemotherapeutic agents described herein. In some embodiments, the chemotherapy is Groopman et al J Natl Cancer Inst (1999) 91 (19): 1616-1634, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the compositions and methods of the invention are used to treat chemotherapy-related anemia in patients with advanced stage cancer (e.g., stage IV or stage III or stage II cancer). In some embodiments, the compositions and methods of the invention are used to treat chemotherapy-related anemia in cancer patients receiving dose-intensive chemotherapy or other invasive chemotherapy regimens.
In some embodiments, the invention relates to treating anemia in patients with one or more hematological-based cancers, such as leukemia, lymphoma, and multiple myeloma. Such cancers may directly affect bone marrow. Furthermore, the present invention relates to metastatic cancers that have spread to bone or bone marrow. In some embodiments, the invention relates to treating anemia in a patient undergoing radiation therapy. Such radiation therapy may damage the bone marrow, thereby reducing its ability to make red blood cells. In other embodiments, the invention relates to treating anemia in a patient with a decrease or deficiency in one or more of iron, vitamin B12, and folic acid. In other embodiments, the invention relates to treating anemia in patients with excessive blood loss (including but not limited to post-surgery or caused by tumors that cause internal bleeding). In other embodiments, the invention relates to treating anemia in a patient suffering from chronic anemia.
In some embodiments, the methods and compositions of the invention stimulate erythropoiesis. In some embodiments, the methods and compositions of the invention stimulate the division and differentiation of committed erythrocyte progenitors in the bone marrow.
Certain embodiments of the invention are particularly useful for treating chemotherapy-induced anemia in cancer patients. In some embodiments, the methods and compositions of the invention allow for continued administration of the chimeric protein or chimeric protein complex, such as an Fc-based chimeric protein complex, after the cancer patient has completed chemotherapy. In some embodiments, the methods and compositions of the invention allow for the treatment of cancer patients at doses that are not reduced relative to non-cancer patients. In some embodiments, the methods and compositions of the present invention allow for the treatment of cancer patients who are undergoing chemotherapy and are considered curable. In various embodiments, the cancer patient has one or more of a history of thrombosis, recent surgery, long-term bedridden or restricted activity, and is treated with a chemotherapeutic agent.
Medicine box
The invention also provides kits for administering any of the agents described herein (e.g., chimeric proteins or chimeric protein complexes such as Fc-based chimeric protein complexes with or without various other therapeutic agents). The kit is a combination of materials or components comprising at least one of the pharmaceutical compositions of the invention described herein. Thus, in some embodiments, the kit contains at least one pharmaceutical composition described herein.
The exact nature of the components disposed in the kit depends on their intended purpose. In one embodiment, the kit is configured for the purpose of treating a human subject.
Instructions for use may be included in the kit. Instructions for use typically include a tangible representation describing the techniques to be employed in achieving a desired result, such as treating cancer, using the components of the kit. Optionally, the kit also contains other useful components as would be readily understood by one skilled in the art, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials, or other useful attachments.
The materials and components assembled in the kit may be provided to the practitioner for storage in any convenient and suitable manner that preserves their operability and utility. For example, these components may be provided at room temperature, at refrigeration temperatures, or at refrigeration temperatures. These components are typically contained in a suitable packaging material. In various embodiments, the packaging material is constructed by well known methods, preferably to provide a sterile, non-contaminating environment. The packaging material may have an external label indicating the content and/or purpose of the kit and/or components thereof.
Definition of the definition
Throughout this application, the chimeric proteins or protein complexes of the invention may be represented by the terms "AcTaleukin-2" and/or "ALN 2".
As used herein, "a/an" or "the" may mean one or more than one.
As used herein, the term "or" is understood to be inclusive and to encompass both "or" and "unless specifically stated or apparent from the context.
Furthermore, the term "about" when used in connection with a numerical indication of a reference means that the numerical indication of the reference is plus or minus at most 10% of the numerical indication of the reference, e.g., within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the value. For example, the language "about 50" covers the range of 45 to 55.
An "effective amount" when used in conjunction with a medical use is an amount effective to provide a measurable treatment, prevention, or reduction in the incidence of a disease of interest.
As used herein, a property is "reduced" if a readout of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be appreciated by those of ordinary skill in the art, in some embodiments, activity decreases and some downstream reads will decrease but other downstream reads may increase.
Conversely, an activity is "increased" if the readout of the activity and/or effect is increased by a significant amount, e.g., by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus relative to the absence of such agent or stimulus.
As referred to herein, all compositional percentages are by weight of the total composition unless otherwise specified. As used herein, the word "comprise" and variations thereof are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the term "can/make" and variations thereof are intended to be non-limiting such that recitation of one embodiment as including certain elements or features does not exclude other embodiments of the present technology that do not include those elements or features.
Although the term "comprising" is used herein as a synonym for the term comprising, containing or having the meaning of describing and claiming the present invention, the present invention or embodiments thereof may alternatively be described using alternative terms such as "consisting of … …" or "consisting essentially of … …".
As used herein, the words "preferred" and "preferably" refer to implementations of the technology that provide certain benefits in certain circumstances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not mean that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
The amount of the compositions described herein required for achieving a therapeutic effect may be determined empirically according to routine procedures for a particular purpose. Generally, for administration of a therapeutic agent for therapeutic purposes, the therapeutic agent is administered in a pharmacologically effective dose. "pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount," or "effective amount" refers to an amount sufficient to produce the desired physiological effect or to achieve the desired result, particularly for the treatment of a condition or disease. As used herein, an effective amount will include an amount sufficient to, for example, delay the progression of symptoms of a disorder or disease, alter the course of symptoms of a disorder or disease (e.g., slow the progression of symptoms of a disease), reduce or eliminate one or more symptoms or manifestations of a disorder or disease, and reverse symptoms of a disorder or disease. Therapeutic benefits also include interrupting or slowing the progression of the underlying disease or condition, regardless of whether improvement is achieved.
Effective amounts, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage may vary depending upon the dosage form employed and the route of administration employed. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the LD50/ED50 ratio. In some embodiments, compositions and methods that exhibit a greater therapeutic index are preferred. The therapeutically effective dose can be estimated initially by in vitro assays, including, for example, cell culture assays. In addition, the dose may be formulated in animal models to achieve a circulating plasma concentration range that includes IC50 as determined in cell culture or in an appropriate animal model. The content of the described composition in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dose can be monitored by a suitable bioassay. The dosage may be determined by a physician and adjusted as necessary to accommodate the observed therapeutic effect.
In certain embodiments, the effect will cause a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will cause a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefits also include interrupting or slowing the progression of the underlying disease or condition, regardless of whether improvement is achieved.
As used herein, "method of treatment" is equally applicable to the use of the composition for treating a disease or disorder described herein and/or the use of the composition for the manufacture of a medicament for treating a disease or disorder described herein. The invention is further illustrated by the following non-limiting examples.
Examples
For the purposes of the following examples, the chimeric proteins or protein complexes of the invention may be referred to throughout the application by the terms "AcTaleukin-2" and/or "ALN 2".
Example 1: IL-2 and Fc-IL-2 drive STAT5 phosphorylation in Peripheral Blood Mononuclear Cells (PBMC)
This example describes how interleukin 2 (IL-2) is modified such that only target cells are activated, and cells with negative or adverse effects are no longer activated. In this example, the anti-tumor activity of CD8 expressing T cells (e.g., cytotoxic T cells or CTLs) is enhanced, while the immunosuppressive function of regulatory T cells (tregs) is limited. The uncoupling of this cellular function can be achieved in part by using the IL-2 receptor (IL-2R) in these cells: signaling in CTL is mediated by a medium affinity IL-2R complex consisting of IL-2rβ (CD 122) and gamma common (yc; CD 132) chains, whereas in Treg, the high affinity IL-2R complex also comprises IL-2rα (CD 25) chains. Strategies for developing CTL-specific IL-2 activity include: (i) CD8 targeting, (ii) elimination of CD25 binding, thereby eliminating the receptor from participating in signaling, and (iii) selection of loss-of-function receptor beta and gamma mutations that can be restored upon (CD 8) targeting.
First, the effect of Fc fusion on IL-2 activity was examined. For this, cytokines were cloned (in pcdna3.4 vector) through a flexible 20 x ggs linker to the C-terminus of the heterodimeric "knob-in-hole" human IgG1 Fc backbone. The Fc sequence contains either an L234A_L235A_K436Q effector mutation and a "pore" modification Y349C_T366S_L368A_Y407V (in sequence hFc 3) or a "knob" mutation S354C_T366W (in sequence hFc 4-hIL-2_C125A). For stability and manufacturability reasons, the free cysteine at position 125 in IL-2 was mutated to alanine (C125A).
To generate this "knob-in-hole" Fc-IL-2, a combination of "holes" and "knob" plasmids was transfected into ExpiCHO cells (ThermoFisher) according to the manufacturer's instructions. Seven days after transfection, recombinant proteins were purified using protein a rotor plate (thermo fisher), quantified, and purity tested using SDS-PAGE.
The resulting AcTaleukin-2 (ALN 2) was tested for STAT5 phosphorylation in the following cells: (i) CD8 positive T cells (cd8+); (ii) CD25 negative conventional T cells (CD 25-Tconv); (iii) CD25 positive conventional T cells (cd25+tconv); or (iv) regulatory T cells (Treg), defined as cd4+cd25+foxp3+. Briefly, PBMCs were isolated from buffy coats of healthy donors using density gradient centrifugation using Lymphoprep (StemCell technologies). Cells were stimulated with serial dilutions of wild-type recombinant IL-2 or Fc-IL-2 at 37℃for 30 min. After centrifugation, the cells were resuspended in lysis/fixation buffer (BD Biosciences) and incubated at 37 ℃ for an additional 10 minutes. Cells were washed and incubated with human FcR blocking reagent (Miltenyi Biotec) and stained with anti-CD 25 and anti-CD 8 for 30 min at room temperature. The cells were then permeabilized at 4℃for 30 min using permeabilization buffer III (BD Biosciences). Cells were finally stained with anti-CD 3, anti-CD 4, anti-FoxP 3 and anti-pSTAT 5 for 1 hour. Samples were collected on a MACQUANT X instrument (Miltenyi Biotec) and analyzed using Flowogic software (Miltenyi Biotec).
Figure 20 illustrates that (i) Treg cells are more sensitive to IL-2 than other cell types, and (ii) Fc fusions affect signaling in all test populations. For example, fc-IL-2 has a 20-fold lower activity on tregs than IL-2, whereas fusions have little activity on CD8 positive cells.
Example 2: CD25 knockout reduces Fc-IL-2 signaling in all cell types
This example examines the effect of five combinations of mutations described as reducing CD25 binding on Fc-IL-2 fusion behavior: (i) r38a_f42yy45a_e62a; (ii) f42y_y45 a_l7g; (iii) F42K; (iV) r38a_f42k; and (v) E61Q.
These residues in the hFc4-hIL-2_C125A construct were mutated (see below for the resulting sequences). These knob constructs were combined with the pore fusion hCD8 VHH-hFc3, wherein hCD8 VHH 1CDA65 sequence was fused to Fc with pore mutation via a 20 x ggs linker (sequence as follows), which allowed assessment of CD8 targeting efficiency. Plasmid combinations were transfected into expcho and purified as described above. The resulting proteins were tested for STAT5 phosphorylation in different lymphocyte populations (fig. 21A-21G). The data indicate that decreasing CD25 interactions (e.g., by the r38a_f42k mutation) affects signaling in all sub-populations, and that this effect is independent of the presence of CD25 (fig. 21A-21B). This may suggest that CD25 mutations not only directly affect CD25 binding, but also have some conformational effect. If CD8 VHH was used to target CD25 knockout mutants to CD8 expressing cells, signaling in these cells was significantly increased, while signaling in other cell types remained largely unchanged (fig. 21C-21G). The E61Q mutation was an exception (fig. 21G), and its attenuation was only modest compared to the other four variants.
The use of Biological Layer Interferometry (BLI) on the Octet RED96 instrument (ForteBio) also showed that CD25 mutations did reduce binding to CD 25. Briefly, recombinant CD25 (Acro Biosystems) was biotinylated using the Pierce IP antibody biotinylation kit and loaded onto a streptavidin sensor. The association and dissociation of seven concentrations of wild-type IL-2, fc-IL-2 or Fc-ALN2 with the mutant CD25 binding site (all CD 8-targeted) was monitored and used to calculate association and dissociation constants and thus affinity (fig. 22A-22G). Fusion to Fc did not appear to affect IL-2 binding to CD25 and indicated that the loss of activity observed in example 1 was due to lower binding efficiency to IL-2Rβ and/or IL-2Ryc chains. On the other hand, the combination mutation r38a_f42y_y45a_e62a; f42 y_y45a_l7g; F42K; r38a—f42k completely abrogated detectable CD25 binding (i.e., specific binding could not be measured). Furthermore, the effect of the mutation E61Q is only modest here and accounts for the more efficient STAT5 phosphorylation described previously.
In summary, CD25 knockout mutations attenuate IL-2 signaling (independent of CD25 expression) in all cell types, whereas CD8 targeting partially restores signaling in CD8 expressing cells. However, this ALN2 molecule with a mutated CD25 binding site still clearly signals in Treg cells, EC 50 50 to 250ng/ml.
Example 3: identification of mutated modified IL-2 with residues that remain IL-2Rβ binding
Previous experiments showed that mutation of the CD25 binding site resulted in reduced signaling, whereas CD8 targeting at least partially restored this loss of signaling. The resulting ALN2 variants are still capable of inducing STAT5 phosphorylation in hypersensitive Treg cells. To further reduce the latter signaling (and thus the CD8 selectivity of ALN2 activity), the option of recoverable (post-targeting) loss-of-function mutations of residues involved in IL-2rβ binding was examined.
The candidate residues D20 and N88 were mutated to any other amino acid (excluding C and M) in the hFc4-hIL-2_c125a construct (see below for sequences). The resulting construct was combined with CD8-hFc3 for expression in ExpiCHO. Recombinant proteins were purified from the supernatant using protein a rotor plate (ThermoFisher), quantified, and purity tested using SDS-PAGE.
The ALN2 variants obtained were tested at CD8 as follows + 、CD4 + CD25 - And CD4 + CD25 + STAT5 phosphorylation in PBMC population: PBMCs were blocked with human FcR blocking reagent (Miltenyi Biotec) and stained with fluorescently labeled CD4, CD8 and CD25 specific abs. After staining and washing, cells were stimulated with serial dilutions of ALN2 variants (as shown) for 30 minutes at 37 ℃. Cells were then fixed using lysis/fixation buffer (BD Biosciences) and permeabilized using permeabilization buffer III (BD Biosciences) according to the manufacturer's instructions. After overnight staining with pSTAT5 specific antibodies, samples were analyzed on a macquant X instrument (Miltenyi Biotec) and analyzed using FlowLogic software (Miltenyi Biotec).
FIGS. 23A-23R (screening of D20 mutants) and FIGS. 24A-24R (screening of N88 mutants) show that most mutations have a large effect on pSTAT5 results. Variants that retain the majority of activity (compared to wild type) are D20E, D20V, N88A, N88G (and to a lesser extent D20S, D20T, N88D, N88Q, N88H and N88T), EC 50 The value is between 3 and 18 ng/ml. The data also clearly indicate that even if the IL-2Rβ site is mutated and CD 8-targeted, it can be found in CD4 + CD25 + Clear pSTAT5 was detected at a concentration of 1 μg/ml in the population where the ultrasensitive Treg accounted for a small fraction.
Then, at CD8 + 、CD25 - Tconv、CD25 + The effect of the beta mutation (here exemplified by N88G) on STAT5 phosphorylation was evaluated in Tconv and Treg cells. Thus, wild-type IL-2 or IL-2_N88G (both having C125A) is expressed as a heterodimeric Fc fusion protein either as non-targeting (in combination with hFc 3) or CD8 targeting (in combination with CD8 VHH-hFc 3). pSTAT5 in PBMCs was prepared, purified and purified as described in example 1. The results shown in figures 25A-25D clearly demonstrate that the N88 mutation reduced signaling in all PBMC populations (figures 25A and 25C), and CD8 targeting increased signaling in antigen-expressing cells, while pSTAT5 in other sub-populations was essentially unaffected (fig. 25A and 25B and fig. 25C and 25D).
Example 4: combination of CD25 knockout and beta mutation
The purpose of this example is to combine a CD25 knockout mutation (e.g., r38a—f42k) with an IL-2rβ mutation that performs best (e.g., D20E, D20V, N88A and N88G). The resulting construct (see sequence below) was combined with a CD8 VHH-hFc3 partner and tested on CD8 as described in example 1 + 、CD25 - Tconv、CD25 + STAT5 phosphorylation in Tconv and Treg cells. The data in fig. 26A-26J and summary table 7 illustrate:
the cd25 knockout (e.g., r38a_k42k) mutation affects signaling in all sub-populations, independent of CD25 expression (fig. 26A and 26F).
il-2rβ mutations (e.g., D20E, D20V, N88A, N88G) also affect signaling in all sub-populations.
EC with mutant CD25 or variants with combined mutations at CD25 and beta sites on CD8 positive cells 50 The values were comparable, indicating that CD8 targeting compensated for signal transduction losses due to beta mutations (fig. 26B and 26G, fig. 26C and 26H, fig. 26D and 26I, and fig. 26E and 26J).
ALN2 variants with mutated CD25 or IL-2Rβ binding sites clearly signal in Treg cells despite reduced efficacy (FIG. 26B, FIG. 26C, FIG. 26D, FIG. 26E and FIG. 26F).
Combinations of CD25 and IL-2rβ mutations were required to almost completely eliminate signaling in hypersensitive Treg cells (fig. 26G-26J).
This combination of reduced CD25 binding and selected IL-2Rβ mutations and targeting to CD8 ultimately results in CD8 versus Treg + The specificity of (a) is up to 2000-fold (fig. 26F, 26G, 26I and 26J).
TABLE 7 EC of combinations of CD25 and IL-2Rβ mutations with reference to the groups depicted in FIGS. 26A-26J 50 (in pM; extrapolated if needed) and its effect on pSTAT5 in the PMBC population
And (3) a step of: failure to determine accurate EC 50
Overall, treg cells were 100,000 times more sensitive on average to wild-type IL-2 than cd8+ T cells. The combination of CD8 targeting, CD25 binding knockout and selected recoverable IL-2rβ mutation resulted in an ALN2 molecule with 1,000-fold specificity for cells that preferentially expressed CD8, and no detectable Treg activity at concentrations of 1,000 or even 10,000pm (equivalent to about 0.1-1 μg/ml). Notably, IL-2 molecules with only IL-2Ra mutations have shown maximum activation of tregs at 10,000pM (FIGS. 26A-26J).
Example 5: CD25 targeting of IL-2 activity
In the case of autoimmune diseases, it is thought that it may be desirable to specifically activate (e.g., by IL-2) Treg cells, while leaving other (IL-2) responsive cells unaffected. This example discusses the possibility of activating tregs with modified IL-2 molecules that target CD25 receptors that are highly expressed on these cells. scFv variants of two non-neutralizing anti-CD 25 antibodies (MA 251 and 7G7B 6) were selected as targeting domains. For this, VH and VL sequences were fused in two orientations (VH-VL and VL-VH) via 3 x ggggs linkers and ligated to an hFc3 sequence with effector mutations and pore mutations (see sequence below). The resulting construct was combined with a hFc4 expressing plasmid (to produce an unarmed knob-in-hole Fc construct) for preparation in expcho cells. The resulting protein was purified as described above and initially tested for (i) binding to cells expressing CD25, and (ii) neutralization of IL-2 signaling. For binding, two HEK-derived cell lines HEK-Blue-IL-2 (expressing functional IL-2R; invivoGen) and HEK-Blue-IL-1 (expressing functional IL-1R; invivoGen) were incubated with serial dilutions of MA251 and 7G7B6 scFv unarmed protein at 4℃for 2 hours. Binding was measured in FACS using fluorescence labelled anti-human Ab. Samples were collected on a MACQUANT X instrument (Miltenyi Biotec) and analyzed using Flowogic software (Miltenyi Biotec). The data in FIGS. 27A-27B clearly demonstrate that CD25 scFv targeting Fc fusions bind only to CD 25-expressing HEK-Blue-IL-2 cells, and not to the relevant HEK-Blue-IL-1 cells.
The effect on IL-2 signaling was measured in HEK-Blue IL-2 cells. Cells were not pre-incubated with or with excess (10 μg/ml) of the unarmed CD25 scFv Fc fusion prior to addition of serial dilutions of wild-type IL-2 (FIG. 28). After overnight stimulation, phospha-Light was used TM The SEAP reporter assay system (ThermoFisher) measures secreted phosphatase activity and plots the concentration function (fig. 28). This experiment shows that none of the proteins tested interfere with IL-2 signaling.
Then, the ability of CD25 targeting (here using the 7b6scfv_vh-VL targeting domain) to restore loss of biological activity of CD25 (here f38a_f42k) and IL2rβ mutations (here N88A and N88G) or combinations thereof (r38a_f42k_n88a and r38a_f38k_n88g) was studied. The hFc4-hIL-2 constructs with these mutations were combined with CD8 VHH-hFc3 or hFc3 constructs for preparation and purification in ExpiCHO cells. The resulting ALN2 variants were compared at CD25 as described previously - Tconv、CD25 + STAT5 phosphorylation in Tconv and Treg cells. The data in fig. 29A-29N illustrate:
CD25 targeting had no significant effect on wild-type Fc-IL-2 signaling (FIGS. 29B and 29C)
The cd25 or il2rβ mutations significantly affected signaling in all the tested sub-populations (fig. 29C and 29F, 29H, 29J, 29L and 29N). The combined CD25 and beta mutations (R38A_F42 K_N88G) completely abrogated STAT5 phosphorylation at concentrations up to 100nM (FIG. 29N)
Cd25 targeting significantly restored the activity of CD25 and IL2rβaln2 variants in CD25 expressing cells (fig. 29E and 29F and fig. 29G and 29H). This results in CD25 only + Medium conducting signals but no longer at CD25 - Variants of signaling in populations
The combined mutations (R38A_F42K_N88A and R38A_F42K_N88G) were only partially restored (FIGS. 29B and 29K and FIGS. 29B and 29M) compared to the wild-type Fc-IL-2 fusion
In summary, CD25 targeting (as shown by scFv versions of non-neutralizing anti-CD 25 antibodies herein) allows specific restoration of loss of function caused by CD25 and/or beta mutations in CD25 expressing cells versus CD25 negative cells.
Example 6: IL-2 mimetics (neoleukocins) and targeting of mutants thereof
Based on the crystal structure of IL-2, an IL-2/IL-15 mimetic was redesigned that lacks the CD25 binding site, but still is able to efficiently signal through the IL-2Rβ and yc receptors (see Silva et al, "De novo design of potent and selective mimics of IL-2 and IL-15," Nature, volume 565, pages 186-191, 2019). This 101 amino acid IL-2_mimetic triggers downstream cell signaling (e.g., STAT5 phosphorylation) independent of IL-2Rα and IL-15Rα and has therapeutic activity superior to IL-2 in melanoma and colon cancer mouse models.
In this example, this IL-2_mimetic sequence was fused to the C-terminus of the hig 1 Fc with effector mutations and knob mutations via a flexible linker 20 x ggs linker (hEc 4-IL-2_mimetic; see below for sequence). In this construct, residue D15 (corresponding to D20 in human IL-2) was mutated to T or H, and residue N40 (corresponding to N88 in human IL-2) was mutated to I, G and R in an attempt to attenuate binding to the IL-2Rβ chain. The resulting constructs (wild type or mutant) were combined with CD8 VHH-hFc3 for preparation in ExpiCHO cells. The resulting IL-2-mimetic (neoleukodin) variants were tested at CD8 as described in example 1 + 、CD25 - Tconv、CD25 + STAT5 phosphorylation in Tconv and Treg cells.
The data in fig. 30A-30H illustrate:
i. the sensitivity of the different cell populations to wild-type IL-2 was as follows: CD8+ < CD25-Tconv < CD25+Tconv < Treg (FIG. 30A).
Non-targeted IL-2_mimetic Fc variants have more or less the same activity on all PBMC subpopulations tested (e.g., EC50 values for cd8+, CD25-Tconv, treg and cd25+tconv are 6.1pM, 9.6pM, 2.3pM and 8.0pM cells, respectively) (fig. 30B).
CD8 targeting of wild-type IL-2_mimetic increased signaling in CD8 positive cells, but not in other cell types (fig. 30C), resulting in a selectivity index of about 10-fold.
Mutations D15T, D15H, N I and N40R almost completely abrogate IL-2_mimetic signaling in CD8 negative cells. Only at very high concentrations (100 nM) the pSTAT5 feature was observed in these cells (fig. 30D, 30E, 30F and 30H).
Cd8 targeting was able to restore (at least in part) signaling loss, resulting in an EC50 value of 38pM (D15T); 169pM (D15H); 53pM (N40I); and 12pM (N40R) (fig. 30D, fig. 30E, fig. 30F, and fig. 30H), and the selectivity index was increased by 10-100 fold compared to the targeted wild-type IL-2 mimetic in fig. 30C.
Loss of function of the N40G mutation was less severe on CD8 positive or CD8 negative cells (fig. 30G), but the selectivity index was increased by 10-100 fold compared to the targeted wild-type IL-2 mimetic in fig. 30C.
In summary, CD8 targeting of IL-2_mimetic and mutation of residues D15 and N40 significantly increased the selectivity of signaling in CD8 positive cells over CD8 negative cells. Targeting of wild-type IL-2-mimetics only resulted in a modest increase in selectivity.
Amino acid sequences of examples 1-6 above:
○hFc3(SEQ ID NO：290)
○hFc4-hIL-2_C125A(SEQ ID NO：291)
○hFc4-hIL-2_R38A_F42Y_Y45A_E62A_C125A(SEQ ID NO：292)
○hFc4-hIL-2_F42Y_Y45A_L72G_C125A(SEQ ID NO：293)
○hFc4-hIL-2_F42K_C125A(SEQ ID NO：294)
○hFc4-hIL-2_R38A_F42K_C125A(SEQ ID NO：295)
○hFc4-hIL-2_E61Q_C125A(SEQ ID NO：296)
○hCD8 VHH-hFc3(SEQ ID NO：297)
○hFc4-hIL-2_D20A_C125A(SEQ ID NO：298)
○hFc4-hIL-2_D20E_C125A(SEQ ID NO：299)
○hFc4-hIL-2_D20F_C125A(SEQ ID NO：300)
○hFc4-hIL-2_D20G_C125A(SEQ ID NO：301)
○hFc4-hIL-2_D20H_C125A(SEQ ID NO：302)
○hFc4-hIL-2_D20I_C125A(SEQ ID NO：303)
○hFc4-hIL-2_D20K_C125A(SEQ ID NO：304)
○hFc4-hIL-2_D20L_C125A(SEQ ID NO：305)
○hFc4-hIL-2_D20N_C125A(SEQ ID NO：306)
○hFc4-hIL-2_D20P_C125A(SEQ ID NO：307)
○hFc4-hIL-2_D20Q_C125A(SEQ ID NO：308)
○hFc4-hIL-2_D20R_C125A(SEQ ID NO：309)
○hFc4-hIL-2_D20S_C125A(SEQ ID NO：310)
○hFc4-hIL-2_D20T_C125A(SEQ ID NO：311)
○hFc4-hIL-2_D20V_C125A(SEQ ID NO：312)
○hFc4-hIL-2_D20W_C125A(SEQ ID NO：313)
○hFc4-hIL-2_D20Y_C125A(SEQ ID NO：314)
○hFc4-hIL-2_N88A_C125A(SEQ ID NO：315)
○hFc4-hIL-2_N88D_C125A(SEQ ID NO：316)
○hFc4-hIL-2_N88E_C125A(SEQ ID NO：317)
○hFc4-hIL-2_N88F_C125A(SEQ ID NO：318)
○hFc4-hIL-2_N88G_C125A(SEQ ID NO：319)
○hFc4-hIL-2_N88H_C125A(SEQ ID NO：320)
○hFc4-hIL-2_N88I_C125A(SEQ ID NO：321)
○hFc4-hIL-2_N88K_C125A(SEQ ID NO：322)
○hFc4-hIL-2_N88L_C125A(SEQ ID NO：323)
○hFc4-hIL-2_N88P_C125A(SEQ ID NO：324)
○hFc4-hIL-2_N88Q_C125A(SEQ ID NO：325)
○hFc4-hIL-2_N88R_C125A(SEQ ID NO：326)
○hFc4-hIL-2_N88S_C125A(SEQ ID NO：327)
○hFc4-hIL-2_N88T_C125A(SEQ ID NO：328)
○hFc4-hIL-2_N88V_C125A(SEQ ID NO：329)
○hFc4-hIL-2_N88W_C125A(SEQ ID NO：330)
○hFc4-hIL-2_N88Y_C125A(SEQ ID NO：331)
○hFc4-hIL-2_D20E_R38A_A42K_C125A(SEQ ID NO：332)
○hFc4-hIL-2_D20V_R38A_F42K_C125A(SEQ ID NO：333)
○hFc4-hIL-2_R38A_F42K_N88A_C125A(SEQ ID NO：334)
○hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：335)
oMA251 scFv_VH-VL-hFc3(SEQ ID NO：336)
○MA251 scFv_VL-VH-hFc3(SEQ ID NO：337)
○7G7B6 scFv_VH-VL-hFc3(SEQ ID NO：338)
○7G7B6 scFv_VL-VH-hFc3(SEQ ID NO：339)
O.hFc 4-IL-2-mimetic (SEQ ID NO: 340)
O.hFc4-IL-2_mimetic_D15T (SEQ ID NO: 341)
O.hFc4-IL-2_mimetic_D15H (SEQ ID NO: 342)
O.hFc4-IL-2_mimetic_N40I (SEQ ID NO: 343)
O.hFc4-IL-2_mimetic_N40G (SEQ ID NO: 344)
O.hFc 4-IL-2-mimetic-N40R (SEQ ID NO: 345)
Example 7: deletion of T3O-glycosylation in IL-2
IL-2 contains a threonine O-glycosylation site at position 3 (T3). In this example, the potential for removal of the site in the context of preparation, manufacturability and bioactivity was assessed. Two strategies were used: (i) A site-specific mutation, wherein T3 is mutated to A, F, H, L, V or Y, or (ii) a deletion mutagenesis, wherein the first 3, 4, 5, 6, 7, or 8 residues of the IL-2 sequence are deleted. Standard mutagenesis techniques with CD 8-Targeted ALN2 versionsMutation was performed. The IL-2 sequence (via a flexible 20 x ggs linker) was cloned into the C-terminus of an hIgG1 Fc sequence (termed hFc 4) with an l234a_l235a_k322Q effector mutation and an s357w "knob" mutation. IL-2 warhead contains the following mutations: (i) r38a_f42k blocks binding to CD25 or IL-2rα, (ii) N88G reduces interaction with IL-2rβ in a recoverable manner, and (iii) C125A removes free cysteine residues, resulting in superior manufacturability. The resulting construct was combined with a fusion of CD8 VHH 1CDA65 and hIG Fc (called hFc 3) with the l235a_k322Q effect mutation and the Y349c_t366s_l368a_y407V "pore" mutation. To generate this "knob-in-hole" Fc-ALN2, a combination of "holes" and "knob" plasmids was transfected into expiho cells (thermo fisher) according to the manufacturer's instructions. One week after transfection, the supernatant was collected and the cells were removed by centrifugation. Recombinant proteins were purified based on protein A binding properties (Hitrap MabSelect SuRe column, GE Healthcare) and by subsequent size exclusion chromatography (Superdex 200 increase HiScale 16/40 column, GE Healthcare), both in The purification was performed on a GE Healthcare (GE Healthcare). The concentration was measured using a spectrophotometer (NanoDrop instrument, thermo Scientific) and purity was estimated on SDS-PAGE.
The resulting ALN 2O-glycosylated variants were tested for STAT5 phosphorylation in the following cells: (i) CD8 positive T cells (CD 8) + ) The method comprises the steps of carrying out a first treatment on the surface of the (ii) CD25 negative conventional T cells (CD 25) - Tconv); (iii) CD25 positive conventional T cells (CD 25) + Tconv); or (iv) regulatory T cells (Treg), defined as CD4 + CD25 + FoxP3 + . Briefly, PBMCs were isolated from buffy coats of healthy donors using density gradient centrifugation using Lymphoprep (StemCell technologies). Cells were stimulated with serial dilutions of wild-type recombinant IL-2 or Fc-ALN2 at 37℃for 30 min. After centrifugation, the cells were resuspended in lysis/fixation buffer (BD Biosciences) and incubated at 37 ℃ for an additional 10 minutes. Cells were washed and incubated with human FcR blocking reagent (Miltenyi Biotec) and stained with anti-CD 25 and anti-CD 8 for 30 min at room temperature.The cells were then permeabilized at 4℃for 30 min using permeabilization buffer III (BD Biosciences). Cells were finally stained with anti-CD 3, anti-CD 4, anti-FoxP 3 and anti-pSTAT 5 for 1 hour. Samples were collected on a MACQUANT X instrument (Miltenyi Biotec) and analyzed using Flowogic software (Miltenyi Biotec).
To assess the stability of the O-glycosylated ALN2 variants, the purified protein was concentrated to 10mg/ml and subjected to five freeze (-20 ℃) to thaw cycles. After each cycle, the samples were centrifuged and the protein concentration was measured on a Nanodrop spectrophotometer. Before and after the freeze-thaw cycle, inSamples were analyzed on a purifier by size exclusion chromatography (Superdex 200 increase HiScale 16/40 column, GE Healthcare).
The following amino acid sequences were used in this example:
○hCD8 VHH-hFc3(SEQ ID NO：346)
○hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：347)
○hFc4-hIL-2_T3A_R38A_F42K_N88G_C125A(SEQ ID NO：348)
○hFc4-hIL-2_T3F_R38A_F42K_N88G_C125A(SEQ ID NO：349)
○hFc4-hIL-2_T3H_R38A_F42K_N88G_C125A(SEQ ID NO：350)
○hFc4-hIL-2_T3L_R38A_F42K_N88G_C125A(SEQ ID NO：351)
○hFc4-hIL-2_T3V_R38A_F42K_N88G_C125A(SEQ ID NO：352)
○hFc4-hIL-2_T3Y_R38A_F42K_N88G_C125A(SEQ ID NO：353)
○hFc4-hIL-2_R38A_F42K-N88G_C125A_del3(SEQ ID NO：354)
○hFc4-hIL-2_R38A_F42K_N88G_C125A_del4(SEQ ID NO：355)
○hFc4-hIL-2_R38A_F42K_N88G_C125A_del5(SEQ ID NO：356)
○hFc4-hIL-2_R38A_F42K_N88G_C125A_del6(SEQ ID NO：357)
○hFc4-hIL-2_R38A_F42K_N88G_C125A_del7(SEQ ID NO：358)
○hFc4-hIL-2_R38A_F42K_N88G_C125A_del8(SEQ ID NO：359)
example 8: bivalent and biparatopic targeting of IL-2 activity
In this example, the efficiency of IL-2 activity targeting CD8 positive cells was compared using a version with one or two VHHs. These VHHs may be identical (generating a bivalent configuration) or different (generating a so-called double paratope pattern). On the knob-in hole holder, VHH and IL-2 warheads can be cloned in different configurations, as outlined in fig. 31.
Specifically, seven different CD8 VHHs were cloned in vectors encoding ALN2 variants having configuration 1 depicted in fig. 31: 1CDA65, 2CDA5, 3CDA19, 2CDA47, 2CDA68, R2HCD26 and R3HCD129. IL-2 warheads contain R38A-F42K, N G and C125A mutations for CD25 knockout, recoverable beta attenuation, and manufacturability, respectively, as described in example 7. The resulting ALN2 pattern was generated by transient transfection of the expcho cells using the following plasmid combinations:
●hCD8 VHH 1CDA65-hFc3+hFc4-IL-2_R38A_F42K_N88G_C125A
●hCD8 VHH 1CDA65-hFc3+hCD8 VHH 1CDA65-hFc4-IL-2_R38A_F42K_N88G_C125A
●hCD8 VHH 1CDA65-hFc3+hCD8 VHH 2CDA5-hFc4-IL-2_R38A_F42K_N88G_C125A
●hCD8 VHH 1CDA65-hFc3+hCD8 VHH 3CDA19-hFc4-IL-2_R38A_F42K_N88G_C125A
●hCD8 VHH 1CDA65-hFc3+hCD8 VHH 2CDA47-hFc4-IL-2_R38A_F42K_N88G-C125A
●hCD8 VHH 1CDA65-hFc3+hCD8 VHH 2CDA68-hFc4-IL-2_R38A_F42K_N88G_C125A
●hCD8 VHH 1CDA65-hFc3+hCD8 VHH R2HCD26-hFc4-IL-2_R38A_F42K_N88G_C125A
●hCD8 VHH 1CDA65-hFc3+hCD8 VHH R3HCD 129-hFc4-IL-2_R38A_F42K_N88G_C125A
●hFc3+hCD8 VHH 1CDA65-hFc4-IL-2_R38A_F42K_N88G_C125A
●hFc3+hCD8 VHH 2CDA5-hFc4-IL-2_R38A_F42K_N88G_C125A
●hFc3+hCD8 VHH 3CDA19-hFc4-IL-2_R38A_F42K_N88G_C125A
●hFc3+hCD8 VHH 2CDA47-hFc4-IL-2_R38A_F42K_N88G_C125A
●hFc3+hCD8 VHH 2CDA68-hFc4-IL-2_R38A_F42K_N88G_C125A
●hFc3+hCD8 VHH R2HCD26-hFc4-IL-2_R38A_F42K_N88G_C125A
●hFc3+hCD8 VHH R3HCD 129-hFc4-IL-2_R38A_F42K_N88G_C125A
●hFc3+hFc4-IL-2_R38A_F42K_N88G_C125A
The resulting protein was purified from the supernatant as described previously and tested for CD8 + 、CD25-Tconv、CD25 + Tconv and CD4 + CD25 + FoxP3 + STAT5 phosphorylation in Treg cells.
The monospecific construct has configuration 4 of fig. 32, while VHH 1CDA65 is a construct with configuration 1 of fig. 32 and is produced as a monospecific construct. In FIGS. 33A-33G it is referred to as 1CDA-65-Fc-ALN2bis. The data in fig. 33A-33G illustrate that on CD8 cells:
i. construct 1CDA65-Fc-ALN2 and 1CDA65-Fc-ALN 2bis have similar efficacy;
divalent 1CDA65-1CDA65-Fc-ALN2 has similar activity to its monovalent counterpart 1CDA65-Fc-ALN2 or 1CDA65-Fc-ALN 2-bis; and is also provided with
An increase in biological activity was observed for the two-paratope 1CDA65-2CDA5-Fc-ALN2, 1CDA65-2CDA68-Fc-ALN2 and 1CDA65-R2HCD26-Fc-ALN 2. Notably, VHH 1CDA65 and 2CDA4774 belong to the same epitope bin, while VHH 2CDA5, 3CDA19, 2CDA68 and R2HCD26 belong to the second bin. Because of its lower affinity, the epitope bin of VHH R3HCD129 was not determined. Notably, the activity of an ALN2 variant with two targeting domains from different epitope bins was 10-fold higher on average than its monospecific counterpart.
The following amino acid sequences were used in this example:
●hCD8 VHH 1CDA65-hFc3(SEQ ID NO：360)
●hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：361)
●hCD8 VHH 1CDA65-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：362)
●hCD8 VHH 2CDA5-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：363)
●hCD8 VHH 3CDA19-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：364)
●hCD8 VHH 2CDA47-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：365)
●hCD8 VHH 2CDA68-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：366)
●hCD8 VHH R2HCD26-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：367)
●hCD8 VHH R3HCD129-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：368)
●hFc3(SEQ ID NO：369)
Example 9: evaluation of alternative ALN2 configuration
In this example, different CD 8-targeted ALN2 configurations were compared. Specifically, VHH and/or IL-2 warheads are cloned into the N-or C-terminus of the knob-in hole Fc scaffold, at the same or opposite sites of the molecule. The pattern may accommodate one or two warheads, cloned in tandem or on different Fc chains. The VHH and warhead can also be cloned in tandem, or the VHH can be cloned between two IL-2 moieties. All possible configurations are schematically outlined in fig. 32.
The following ALN2 plasmid combinations were transfected into ExpiCHO cells as described previously, purified from the supernatant, tested in CD8 + 、CD25 - Tconv、CD25 + Tconv and CD4 + CD25 + FoxP3 + pSTAT5 in Treg cells and screening for stability:
● hCD8 VHH 1CDA65-hFc3+ hFc4-IL-2_R38A_F42K_N88G_C125A (configuration 1 in FIG. 32)
● hCD8 VHH 1CDA65-hFc3+IL-2_R38A_F42K_N88G_C125A-hFc4-IL-2_R38A_F42K_N88G_C125A (configuration 9 in FIG. 32)
● hCD8 VHH 1CDA65-hFc3-hFc4-IL-2_R38A_F42K_N88G_C125A+hFc4-IL-2_R38A_F42K_N88G_C125A (configuration 13 in FIG. 32)
● hCD8 VHH 1CDA65-hFc3+hFc4-IL-2_R38A_F42K_N88G_C125A-IL-2_R38A_F42K_N88G_C125A (configuration 25 in FIG. 32)
● hCD8 VHH 1CDA65-hFc3+IL-2_R38A_A42K_N88G_C125A-IL-2_R38A_F42K_N88G_C125A-hFc4 (configuration 26 in FIG. 32)
In addition, the data in fig. 34A-34E demonstrate that all variants tested had well-defined pSTAT5 in cd8+ cells, but not in Treg cells, demonstrating their selectivity.
The following amino acid sequences were used in this example:
●hCD8 VHH 1CDA65-hFc3(SEQ ID NO：370)
●hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：371)
●IL-2_R38A_F42K_N88G_C125A-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：372)
●hCD8 VHH 1CDA65-hFc3-hFc4-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：373)
●hFc4-IL-2_R38A_F42K_N88G_C125A-IL-2_R38A_F42K_N88G_C125A(SEQ ID NO：374)
●IL-2_R38A_F42K_N88G_C125A-IL-2_R38A_F42K_N88G_C125A-hFc4(SEQ ID NO：375)
example 10: combination of beta and gamma mutations
The purpose of this example is to evaluate the loss-of-function mutation in the IL-2Rβ binding site (N88A, N88G, D E or D20V) and the blocking with gamma in IL-2R c The effect of the combination of mutations of the chain interactions. For the latter mutation, residue Q126 has been determined to be critical and is mutated.
Table 8 summarizes these mutations:
mutations or combinations thereof (as described in the examples above) were performed using standard mutagenesis techniques with CD 8-targeted ALN2 versions. Here, for manufacturability reasons, the IL-2 warhead contains only the C125A mutation. The resulting plasmid was combined with a CD8 VHH-hFc3 plasmid for transient transfection in ExpiCHO cells. Proteins were purified using protein a rotor plate (ThermoFisher), quantified, and purity tested using SDS-PAGE.
The ALN2 variants obtained were tested at CD8 as follows + 、CD4 + CD25 - And CD4 + CD25 + STAT5 phosphorylation in PBMC population: PBMCs were blocked with human FcR blocking reagent (Miltenyi Biotec) and stained with fluorescently labeled CD4, CD8 and CD25 specific abs. After staining and washing, cells were stimulated with serial dilutions of IL-2 or ALN2 variants for 30 min at 37 ℃. Cells were then fixed using lysis/fixation buffers (BDBiosciences) and permeabilized using permeabilization buffer III (BD Biosciences) according to the manufacturer's instructions. After overnight staining with pSTAT 5-specific antibodies, samples were analyzed on a macquant X instrument (Miltenyi Biotec) and using FloWLogic software (Miltenyi Biotec).
The following amino acid sequences were used in this example:
○hCD8 VHH-hFc3(SEQ ID NO：376)
○hFc4-hIL-2_C125A(SEQ ID NO：377)
○hFc4-hIL-2_C125A_D20E_Q126A(SEQ ID NO：378)
○hFc4-hIL-2_C125A_D20E_Q126D(SEQ ID NO：379)
○hFc4-hIL-2_C125A_D20E_Q126E(SEQ ID NO：380)
○hFc4-hIL-2_C125A_D20E_Q126F(SEQ ID NO：381)
○hFc4-hIL-2_C125A_D20E_Q126G(SEQ ID NO：382)
○hFc4-hIL-2_C125A_D20E_Q126H(SEQ ID NO：383)
○hFc4-hIL-2_C125A_D20E_Q126I(SEQ ID NO：384)
○hFc4-hIL-2_C125A_D20E_Q126K(SEQ ID NO：385)
○hFc4-hIL-2_C125A_D20E_Q126L(SEQ ID NO：386)
○hFc4-hIL-2_C125A_D20E_Q126M(SEQ ID NO：387)
○hFc4-hIL-2_C125A_D20E_Q126N(SEQ ID NO：388)
○hFc4-hIL-2_C125A_D20E_Q126P(SEQ ID NO：389)
○hFc4-hIL-2_C125A_D20E_Q126R(SEQ ID NO：390)
○hFc4-hIL-2_C125A_D20E_Q126S(SEQ ID NO：391)
○hFc4-hIL-2_C125A_D20E_Q126T(SEQ ID NO：392)
○hFc4-hIL-2_C125A_D20E_Q126V(SEQ ID NO：393)
○hFc4-hIL-2_C125A_D20E_Q126W(SEQ ID NO：394)
○hFc4-hIL-2_C125A_D20E_Q126Y(SEQ ID NO：395)
○hFc4-hIL-2_C125A_D20V_Q126A(SEQ ID NO：396)
○hFc4-hIL-2_C125A_D20V_Q126D(SEQ ID NO：397)
○hFc4-hIL-2_C125A_D20V_Q126E(SEQ ID NO：398)
○hFc4-hIL-2_C125A_D20V_Q126F(SEQ ID NO：399)
○hFc4_hIL-2_C125A_D20V_Q126G(SEQ ID NO：400)
○hFc4-hIL-2_C125A_D20V_Q126H(SEQ ID NO：401)
○hFc4-hIL-2_C125A_D20V_Q126I(SEQ ID NO：402)
○hFc4-hIL-2_C125A_D20V_Q126K(SEQ ID NO：403)
○hFc4-hIL-2_C125A_D20V_Q126L(SEQ ID NO：404)
○hFc4-hIL-2_C125A_D20V_Q126M(SEQ ID NO：405)
○hFc4-hIL-2_C125A_D20V_Q126N(SEQ ID NO：406)
○hFc4-hIL-2_C125A_D20V_Q126P(SEQ ID NO：407)
○hFc4-hIL-2_C125A_D20V_Q126R(SEQ ID NO：408)
○hFc4-hIL-2_C125A_D20V_Q126S(SEQ ID NO：409)
○hFc4-hIL-2_C125A_D20V_Q126T(SEQ ID NO：410)
○hFc4-hIL-2_C125A_D20V_Q126V(SEQ ID NO：411)
○hFc4-hIL-2_C125A_D20V_Q126W(SEQ ID NO：412)
○hFc4-hIL-2_C125A_D20V_Q126Y(SEQ ID NO：413)
○hFc4-hIL-2_C125A_N88A_Q126A(SEQ ID NO：414)
○hFc4-hIL-2_C125A_N88A_Q126D(SEQ ID NO：415)
○hFc4-hIL-2_C125A_N88A_Q126E(SEQ ID NO：416)
○hFc4-hIL-2_C125A_N88A_Q126F(SEQ ID NO：417)
○hFc4-hIL-2_C125A_N88A_Q126G(SEQ ID NO：418)
○hFc4-hIL-2_C125A_N88A_Q126H(SEQ ID NO：419)
○hFc4-hIL-2_C125A_N88A_Q126I(SEQ ID NO：420)
○hFc4-hIL-2_C125A_N88A_Q126K(SEQ ID NO：421)
○hFc4-hIL-2_C125A_N88A_Q126L(SEQ ID NO：422)
○hFc4-hIL-2_C125A_N88A_Q126M(SEQ ID NO：423)
○hFc4-hIL-2_C125A_N88A_Q126N(SEQ ID NO：424)
○hFc4-hIL-2_C125A_N88A_Q126P(SEQ ID NO：425)
○hFc4-hIL-2_C125A_N88A_Q126R(SEQ ID NO：426)
○hFc4-hIL-2_C125A_N88A_Q126S(SEQ ID NO：427)
○hFc4-hIL-2_C125A_N88A_Q126T(SEQ ID NO：428)
○hFc4-hIL-2_C125A_N88A_Q126V(SEQ ID NO：429)
○hFc4-hIL-2_C125A_N88A_Q126W(SEQ ID NO：430)
○hFc4-hIL-2_C125A_N88A_Q126Y(SEQ ID NO：431)
○hFc4-hIL-2_C125A_N88G_Q126A(SEQ ID NO：432)
○hFc4-hIL-2_C125A_N88G_Q126D(SEQ ID NO：433)
○hFc4-hIL-2_C125A_N88G_Q126E(SEQ ID NO：434)
○hFc4-hIL-2_C125A_N88G_Q126F(SEQ ID NO：435)
○hFc4-hIL-2_C125A_N88G_Q126G(SEQ ID NO：436)
○hFc4-hIL-2_C125A_N88G_Q126H(SEQ ID NO：437)
○hFc4-hIL-2_C125A_N88G_Q126I(SEQ ID NO：438)
○hFc4-hIL-2_C125A_N88G_Q126K(SEQ ID NO：439)
○hFc4-hIL-2_C125A_N88G_Q126L(SEQ ID NO：440)
○hFc4-hIL-2_C125A_N88G_Q126M(SEQ ID NO：441)
○hFc4-hIL-2_C125A_N88G_Q126N(SEQ ID NO：442)
○hFc4-hIL-2_C125A_N88G_Q126P(SEQ ID NO：443)
○hFc4-hIL-2_C125A_N88G_Q126R(SEQ ID NO：444)
○hFc4-hIL-2_C125A_N88G_Q126S(SEQ ID NO：445)
○hFc4-hIL-2_C125A_N88G_Q126T(SEQ ID NO：446)
○hFc4-hIL-2_C125A_N88G_Q126V(SEQ ID NO：447)
○hFc4-hIL-2_C125A_N88G_Q126W(SEQ ID NO：448)
○hFc4-hIL-2_C125A_N88G_Q126Y(SEQ ID NO：449)
example 11: single peptide ALN2 variants
This example shows the effect of a single peptide ALN2 variant. The alpha-free IL-2 variant (mutation R38A_F42K) was cloned into the single peptide A-Kine version. For this, the sequences encoding CD8 VHH 1CDA65 and IL-2 were fused via a flexible 20 x ggs linker in pcdna3.4 expression vectors for eukaryotic expression. A C-terminal 6 x his tag was added for purification purposes. The D20E, D20V, N a and N88G mutants were prepared using standard mutagenesis techniques and the resulting plasmids were transfected into expiho cells (thermosfisher) according to manufacturer's guidelines. One week after transfection, the supernatant was collected and the cells were removed by centrifugation. Recombinant proteins were purified based on His tag (HisTrap Excel column; cytiva) and by subsequent size exclusion chromatography (Superdex 200 increase HiScale 16/40 column, cytiva), both inThe purification was performed on a purifier (GEHealthcare). The concentration was measured using a spectrophotometer (NanoDrop instrument, thermo Scientific) and purity was estimated on SDS-PAGE.
The resulting proteins were tested for STAT5 phosphorylation in cd8+ and Treg cells as described previously. As shown in FIG. 35A-FIG. 3535D, all mutations except the mutation r38a_f42k_d20v allowed selective signaling of cd8+ over Treg cells over the range of concentrations tested. The N88 variant R38A_F42K_N88A (EC 50 :0.17 ng/nl) or R38A_F42K_N88G (EC) 50 : the 0.08ng/ml variant appeared to be more potent on CD8+ cells than the R38A_F42K_D20E mutant (EC 50 ：5.8ng/ml)。
The following amino acid sequences were used in this example:
○hCD8 VHH-hIL-2_D20E_R38A_F42K_C125A(SEQ ID NO：478)
○hCD8 VHH-hIL-2_D20V_R38A_F42K_C125A(SEQ ID NO：479)
○hCD8 VHH-hIL-2_R38A_F42K_N88A_C125A(SEQ ID NO：480)
○hCD8 VHH-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：481)
example 12: removal of T3O-glycosylation sites in IL-2 warheads
IL-2 contains a threonine O-glycosylation site at position 3 (T3). In this example, the potential for removal of the site in the context of preparation, manufacturability and bioactivity was assessed. Two strategies were used: (i) A site-specific mutation, wherein T3 is mutated to A, F, H, L, V or Y, or (ii) a deletion mutagenesis, wherein the first 3, 4, 5, 6, 7, or 8 residues of the IL-2 sequence are deleted. Mutations were performed using standard mutagenesis techniques with the CD 8-targeted ALN2 version. The IL-2 sequence (via a flexible 20 x ggs linker) was cloned into the C-terminus of an hIgG1 Fc sequence (termed hFc 4) with an l234a_l235a_k322Q effector mutation and an s357w "knob" mutation. IL-2 warhead contains the following mutations: (i) r38a_f42k blocks binding to CD25 or IL-2rα, (ii) N88G reduces interaction with IL-2rβ in a recoverable manner, and (iii) C125A removes free cysteine residues, resulting in superior manufacturability. The resulting construct was combined with a fusion of CD8 VHH 1CDA65 and hIG Fc (called hFc 3) with the l235a_k322Q effect mutation and the Y349c_t366s_l368a_y407V "pore" mutation. To generate this "knob-in-hole" Fc-ALN2, a combination of "holes" and "knob" plasmids was transfected into expiho cells (thermo fisher) according to the manufacturer's instructions. One week after transfection, the supernatant was collected and the cells were removed by centrifugation. Recombinant proteins were purified using protein a rotor plate (ThermoFisher), quantified, and purity tested using SDS-PAGE. The resulting variants were tested for STAT5 phosphorylation in CD8 positive (cd8+) and CD8 negative (CD 8-) PBMC populations as follows: PBMCs were blocked with human FcR blocking reagent (Miltenyi Biotec) and stained with fluorescently labeled CD8 specific abs. After staining and washing, cells were stimulated with serial dilutions of CD8 VHH-Fc-IL-2 variants (as shown) for 30 min at 37 ℃. Cells were then fixed using lysis/fixation buffer (BD Biosciences) and permeabilized using permeabilization buffer III (BD Biosciences) according to the manufacturer's instructions. After overnight staining with pSTAT5 specific antibodies, samples were analyzed on a macquant X instrument (Miltenyi Biotec) and analyzed using FlowLogic software (Miltenyi Biotec).
The data in FIGS. 36A-36M illustrate that substitution of T3 for H, I, L V, Y, or A, or deletion of up to 7 amino acids, has comparable activity on CD8+ cells, with EC50 values varying from 1 to 5 ng/ml. However, the IL-2_del8 variant was 10-fold less active than the wild-type IL-2. In contrast, the T3F (not shown) mutation no longer showed detectable activity. The effect on signaling in the CD8 population was shown to be very similar.
The following amino acid sequences were used in this example:
○hFc4-hIL-2_C125A(SEQ ID NO：482)
○hCD8 VHH-hFc3(SEQ ID NO：483)
○hFc4-hIL-2_T3A_C125A(SEQ ID NO：484)
○hFc4-hIL-2_T3H_C125A(SEQ ID NO：485)
○hFc4-hIL-2_T3L_C125A(SEQ ID NO：486)
○hFc4-hIL-2_T3V_C125A(SEQ ID NO：487)
○hFc4-hIL-2_T3Y_C125A(SEQ ID NO：488)
○hFc4-hIL-2_C125A_del3(SEQ ID NO：489)
○hFc4-hIL-2_C125A_del4(SEQ ID NO：490)
○hFc4-hIL-2_C125A_del5(SEQ ID NO：491)
○hFc4-hIL-2_C125A_del6(SEQ ID NO：492)
○hFc4-hIL-2_C125A_del7(SEQ ID NO：493)
○hFc4-hIL-2_C125A_del8(SEQ ID NO：494)
example 13: alpha: in vivo evaluation of beta combinations
Since human IL-2 is cross-reactive to the mouse IL-2 receptor, human α was tested: activity of beta ALN2 variants on mouse cells. For this purpose, in the VHH-Fc fusion with effector mutations and full mutations, the human CD 8-specific VHH (1 CDA 65) was replaced by the mouse CD 8-specific VHH R2CDE47. The resulting construct (mCD 8 VHH-hFc 3) was combined with knob Fc fused to an ALN2 bullet with one of the r38a_f42kα knockout mutation and the recoverable β mutation D20E, D20V, N a or N88G. The mouse CD 8-targeted ALN2 variant was expressed in expiho and purified as described above. Initially, these variants were tested for STAT5 phosphorylation in primary mouse spleen cells. Briefly, splenocytes from C57B1/6 mice (RBCs lysed with ACK buffer) were stained for cell surface markers (viability, CD3, CD4, CD 8a, CD8 β) and stimulated ex vivo with different mouse CD 8-targeted ALN2 variants for 30 min. Stimulation was stopped by adding 1X TFP fixation/permeation buffer (BD Pharmingen) and incubated at 4 ℃ for 50 minutes, followed by 2 wash steps with 1X TFP permeation/wash buffer (BD Pharmingen). The cell pellet was resuspended in permeabilization/washing buffer III (transcription factor phosphorylation buffer kit, BD Pharmingen) and incubated overnight at-20 ℃ followed by intracellular FoxP3 and pSTAT5 staining. Samples were collected on a MACSUAntl 6 flow cytometer and analyzed using FlowLogic 8.4 software (Miltenyi Biotec).
The D20E, N88A or N88G combinations were active on cd8+ cells quite (EC 50 Values were between 36ng/ml and 94 ng/ml), whereas signaling of these variants was only observed at very high concentrations in Treg cells (fig. 37A-37D). The D20V combination mutant appears inactive in both spleen cell subsets tested. Based on these data and preliminary preparation and stability observations, the mouse CD 8-targeted human r38a_f42k_n88g IL-2 mutant was used (in the remainder of the exampleReferred to as CD8-Fc-ALN 2) for further in vivo evaluation.
The anti-tumor efficacy of the mouse CD 8-targeted ALN2 variant (CD 8-Fc-ALN 2) with the combined mutation r38a_f42k_n88g was evaluated in two syngeneic mouse colon cancer models MC38 and CT26, and compared with the non-targeted ALN2 variant (Fc-ALN 2). Briefly, 6E+5 MC38 cells were injected subcutaneously (s.c.) in C57B1/6J mice (females, 9 weeks old) or 6E+5 CT26 cells were injected subcutaneously in Balb/C mice (females, 9 weeks old). After 7 days, the tumor volume reaches 20-90mm 3 (MC 38) or 40-120mm 3 (CT 26), beginning treatment: mice were intravenously (i.v.) received buffer or equimolar amounts of either non-targeted Fc-ALN2 (21 μg/mouse) or targeted CD8-Fc-ALN2 (25 μg/mouse) at day 7 and day 14 post tumor inoculation.
Tumor sizes (mean +/-SEM; n=5 per group) were measured daily and plotted in figure 38. The data clearly demonstrate that non-targeted ALN2 had no significant effect compared to the buffer treated animals, whereas CD8-Fc-ALN2 significantly and significantly inhibited tumor growth in both models. On day 21, even one animal treated with CD8-Fc-ALN2 was completely tumor-free in the MC38 model.
In a parallel experiment, the combined effect of CD8-Fc-ALN2 and anti-PD-1 Ab as a checkpoint inhibitor in MC38 and CT26 models was studied. For this purpose, tumor-bearing C57B1/6J (MC 38) or Balb/C mice (CT 26) were received intravenously with buffer or CD8-Fc-ALN2 (12.5. Mu.g/mouse) on days 7, 10, 14 and 17 after tumor inoculation. On the same day of intravenous treatment, the buffer or ALN2 treatment was supplemented with intraperitoneal (i.p.) anti-PD-1 Ab treatment (200 μg/mouse) for this purpose. The tumor sizes shown in fig. 39 are mean +/-SEM (n=5 per group). The number at the end of each curve represents the number of completely tumor-free mice at the end of the experiment (day 22). In the MC38 model, anti-PD-1 itself had no effect, CD8-Fc-ALN2 alone had moderate effect (2 out of 5 mice were completely tumor-free), but combined use resulted in complete tumor regression in all animals. Similar combined/additive effects were observed in the CT26 model: although there was no significant effect of both monotherapy (CD 8-Fc-ALN2 and anti-PD-1), the combination treatment slowed down tumor growth, even completely eradicated the tumor in one animal.
Notably, tumor-free MC38 mice subsequently received a re-challenge on the other side 56 days after the first MC38 tumor inoculation. All tumor-free and re-challenged mice remained completely tumor-free after 2 months, while control animals developed tumors 17 to 21 days after tumor inoculation, requiring euthanasia (up to 1500 mm) 3 )。
The following amino acid sequences were used in this example:
○mCD8 VHH-hFc3(SEQ ID NO：495)
○hFc4-hIL-2_D20E_R38A_F42K_C125A(SEQ ID NO：332)
○hFc4-hIL-2_D20V_R38A_F42K_C125A(SEQ ID NO：333)
○hFc4-hIL-2_R38A_F42K_N88A_C125A(SEQ ID NO：334)
○hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：335)
○hFc3(SEQ ID NO：369)
example 14: screening for gamma co-mutations
In the context of cancer, it may be desirable to selectively activate Cytotoxic T Lymphocytes (CTLs) with IL-2 while minimizing the impact on Treg cells that suppress these immune responses. One therapeutic strategy is to use these so-called alpha-free IL-2 variants, which no longer bind to the IL-2rα chain, which leads mainly to the hypersensitive of Treg cells to IL-2. As described above, CD 8-targeted afc-free variants are still capable of EC at 1 to 2ng/ml 50 Activating Treg cells. The combination with recoverable (after CD8 targeting) IL-2rβ mutations (D20E, D20V, N88A or N88G) further reduced Treg activation and increased selectivity for CD8 expressing cells (see example 4; fig. 26A-26J).
In this example, mutations at sites of interaction with gamma co-receptors were evaluated to assess the extent to which such mutations have the same beneficial effects as beta mutations. A possible candidate for the A-Kine mutation is a substitution of residue Q126, which is the core of the interaction between IL-2 and the gamma co-chain. Thus, this residue was mutated to any other (except cysteine) amino acid in the Fc4-IL-2_C125A chain and combined with CD8 VHH-hFc3 for preparation in ExpiCHO. One week after transfection, proteins were purified from the supernatant using protein a rotor plate (thermo fisher), quantified, and purity tested using SDS-PAGE. These mutations were compared to the effect of wild-type IL-2 (with the C125A mutation) on STAT5 phosphorylation in CD8 positive PBMCs, as described previously. Based on these responses, mutations were divided into four classes (fig. 40A-40D):
i. Null response mutation (0-10 times of activity loss)
Moderate effect mutation (10-50 times loss of activity)
Moderate effect mutation (50-500 times loss of activity)
Strong mutation (> 500-fold loss of activity) or almost complete loss of activity of Q126D
Subsequently, the Octet device was used to investigate how much the Q126 mutation affected the interaction/binding of IL-2 with the IL-2Rα chain. Briefly, the method comprises the following steps: biotinylated CD25 (IL-2 Rα) was loaded onto a streptavidin sensor, and the association and dissociation of two concentrations (50 and 10 nM) of CD8VHH-hFc3+ hFc4-IL-2 or its Q126 mutant were monitored and used to calculate association and dissociation constants and thus affinity. The data in fig. 41A-41S illustrate that most mutations do not affect this interaction, with two exceptions: mutations Q126F (reduced affinity by a factor of 10 due to lower association) and Q126P, which almost completely abrogate binding. The latter suggests that proline substitution leads to conformational transition, affecting other parts of the IL-2 molecule.
The following table provides association and dissociation constants and affinities:
KD(M) ka(1/Ms) kdis(1/s)
CD8 VHH-Fc-hIL-2_C125A 3，23E-08 2，36E+05 7，64E-03
CD8 VHH-Fc-hIL-2_C125A 2，61E-08 2，51E+05 6，56E-03
CD8 VHH-Fc-hIL-2_C125A_Q126A 1，91E-08 4，74E+05 9，06E-03
CD8 VHH-Fc-hIL-2_C125A_Q126A 1，50E-08 4，64E+05 6，95E-03
CD8 VHH-Fc-hIL-2_C125A_Q126D 2，08E-08 3，45E+05 7，16E-03
CD8 VHH-Fc-hIL-2_C125A_Q126E 1，35E-08 5，65E+05 7，61E-03
CD8 VHH-Fc-hIL-2_C125A_Q126F 1，02E-07 4，41E+04 4，49E-03
CD8 VHH-Fc-hIL-2_C125A_Q126G 2，20E-08 3，10E+05 6，82E-03
CD8 VHH-Fc-hIL-2_C125A_Q126H 1，78E-08 4，10E+05 7，31E-03
CD8 VHH-Fc-hIL-2_C125A_Q126I 2，68E-08 2，45E+05 6，55E-03
CD8 VHH-Fc-hIL-2_C125A_Q126K 1，36E-08 5，28E+05 7，21E-03
CD8 VHH-Fc-hIL-2_C125A_Q126L 3，19E-08 2，04E+05 6，49E-03
CD8 VHH-Fc-hIL-2_C125A_Q126M 2，54E-08 2，93E+05 7，46E-03
CD8 VHH-Fc-hIL-2_C125A_Q126N 1，60E-08 5，95E+05 9，52E-03
CD8 VHH-Fc-hIL-2_C125A_Q126P NA NA NA
CD8 VHH-Fc-hIL-2_C125A_Q126R 1，26E-08 5，42E+05 6，82E-03
CD8 VHH-Fc-hIL-2_C125A_Q126S 1，83E-08 4，82E+05 8，82E-03
CD8 VHH-Fc-hIL-2_C125A_Q126T 1，90E-08 4，76E+05 9，03E-03
CD8 VHH-Fc-hIL-2_C125A_Q126V 2，44E-08 3，29E+05 8，03E-03
CD8 VHH-Fc-hIL-2_C125A_Q126W 5，38E-08 1，14E+05 6，10E-03
CD8 VHH-Fc-hIL-2_C125A_Q126Y 2，83E-08 2，88E+05 8，14E-03
based on these two data sets, one mutation (moderate: Q126Y, moderate: Q126G, strong: Q126I) in each of the above categories was retained for further analysis in the following examples.
The following amino acid sequences were used in this example:
○hCD8 VHH-hFc3(SEQ ID NO：501)
○hFc4-hIL-2_C125A(SEQ ID NO：502)
○hFc4_C125A_Q126A(SEQ ID NO：503)
○hFc4_C125A_Q126D(SEQ ID NO：504)
○hFc4_C125A_Q126E(SEQ ID NO：505)
○hFc4_C125A_Q126F(SEQ ID NO：506)
○hFC4_C125A_Q126G(SEQ ID NO：507)
○hFc4_C125A_Q126H(SEQ ID NO：508)
○hFc4_C125A_Q126I(SEQ ID NO：509)
○hFc4_C125A_Q126K(SEQ ID NO：510)
○hFc4_C125A_Q126L(SEQ ID NO：511)
○hFc4_C125A_Q126M(SEQ ID NO：512)
○hFc4_C125A_Q126N(SEQ ID NO：513)
○hFc4_C125A_Q126P(SEQ ID NO：514)
○hFc4_C125A_Q126R(SEQ ID NO：515)
○hFc4_C125A_Q126S(SEQ ID NO：516)
○hFc4_C125A_Q126T(SEQ ID NO：517)
○hFc4_C125A_Q126V(SEQ ID NO：518)
○hFc4_C125A_Q126W(SEQ ID NO：519)
○hFc4_C125A_Q126Y(SEQ ID NO：520)
example 15: combination of alpha and gamma mutations
And alpha: beta combinations similarly, the alpha mutation (r38a—f42k) was combined with three selected gamma mutations (Q126G, Q I and Q126Y) in the deALN2 warhead. As described previously, three resulting ALN2 were produced, purified, and tested for STAT5 phosphorylation in cd8+ and Treg PBMC subsets of three different healthy donors. The results in fig. 42A-42D illustrate:
o as previously observed: treg sensitivity to IL-2 is 4 log higher than CD8+ cells
In contrast, cd8+ cell pairs have any combination α: gamma mutated ALN2 has approximately 3 log higher sensitivity than Treg.
The combination of the o with the strong Q126I mutation allowed clear signaling in cd8+ cells, whereas STAT5 phosphorylation was almost absent in Treg cells up to 10 μg/mL.
The following amino acid sequences were used in this example:
○hCD8 VHH-hFc3(SEQ ID NO：521)
○hFc4-IL-2_R38A_F42K_Q126G(SEQ ID NO：522)
○hFc4-IL-2_R38A_F42K_Q126I(SEQ ID NO：523)
○hFc4-IL-2_R38A_F42K_Q126Y(SEQ ID NO：524)
example 16: selectivity of combinations of beta and gamma mutations for activated CD8 cells
The first major disadvantage of using alpha-free IL-2 variants (see above) in cancer treatment is that these molecules are unable to compete with endogenously expressed IL-2 for binding to the IL-2 Ralpha chain. In fact, in the tumor environment, mainly CD4 + CD8 + Activation of the cells results in secretion of IL-2. This "newly secreted" IL-2 will preferentially activate cells expressing IL-2Rα, such as Tregs, thereby suppressing the anti-tumor response. The second disadvantage is that there is noThe alpha molecule cannot take advantage of the fact that activated CTLs have up-regulated IL-2rα levels, while IL-2rα remains the driving force for IL-2R activation. Based on these two concepts, the potential of combining the βA-Kine mutation (here D20E) with the γco A-Kine mutation (here Q126G or Q126Y) in the IL-2 warhead was evaluated.
To mimic the activation of cd8+ cells (and thus up-regulate CD 25), PBMCs of freshly isolated healthy donors were treated with tranact (nanomatrix conjugated with recombinant humanized CD3 and CD28 agonists; miltenyi Biotec) for two days, followed by stimulation with d20e_q168g or d20e_q16yaln2 variants with STAT5 phosphorylation as functional readout. As can be seen from fig. 43A-43D, these two β: gamma mutants were more active on tregs (approximately l 0-fold), but this sensitivity was completely reversed on activated PBMCs. Here, cd8+ cells vs β, probably due to up-regulation of IL-2rα: γaln2 variants become highly sensitive. These data demonstrate that these ALN2 variants are selective for activated CD8 positive cells.
The following amino acid sequences were used in this example:
○ hCD8 VHH-hFc3(SEQ ID NO：297)
○hFc4-IL-2_D20E_Q126G(SEQ ID NO：382)
○hFc4-IL-2_D20E_Q126Y(SEQ ID NO：395)
○hFc4-hIL-2_C125A(SEQ ID NO：482)
example 17:CD8-Fc-ALN2 is specific in activating and proliferating CD8 positive T cells and is superior to wild type FC-IL-2
C57B1/6J mice (female, 9 weeks old) were injected subcutaneously (s.c.) with 6e+5 MC38 cells. After 7 days, treatment was started: mice received intravenously (i.v.) either (1) buffer, (2) targeted CD8-Fc-ALN2 (12.5 μg/mouse), (3) equimolar doses of non-targeted Fc-ALN2 (10.7 μg/mouse) or (4) equimolar doses of wild-type Fc-IL2 (10.7 μg/mouse). After one day, spleens and tumors were removed and intracellular analyses of STAT5 phosphorylation (recording IL-2 receptor signaling) and membrane CD69 and CD25 expression (recording activation) were performed in CD8 or CD4 positive T cells. The results plotted in fig. 44 show that while non-targeted Fc-ALN2 did not induce pSTAT5, CD69 or CD25 in CD8 positive T cells, the induction of the CD8-Fc-ALN2 construct was overall very robust and surprising, even more effective than wild-type Fc-ALN 2. In contrast, wild-type Fc-IL2 very efficiently induced STAT5 phosphorylation in CD4 and FoxP3 positive regulatory T cells (Treg) as well as other (FoxP 3 negative) CD4 positive T cells. Importantly, CD8-Fc-ALN2 had no effect on FoxP3 positive regulatory T cells (tregs), as demonstrated by pSTAT5 data, even reduced activation of these cells.
To assess T cell proliferation, lymphocytes in the circulation (using Vetscan HMS, zoetis) as well as in the spleen and tumor (by flow cytometry) were counted 14 days after tumor inoculation (i.e., 1 week after initiation of treatment, i.e., 2 treatments on day 7 and day 10). The results plotted in fig. 45 show that targeting CD8-Fc-ALN2 is the only therapeutic method effective to cause lymphocyte proliferation in blood and spleen (as compared to non-targeted Fc-ALN2 and wild-type Fc-IL2 injected in equimolar amounts). Wild-type Fc-IL2 is able to increase the number of CD8 positive T cells in tumor tissue. The addition of anti-PD-1 Ab treatment further enhanced CD8-Fc-ALN2 induced circulation and lymphocyte proliferation in tumor tissue, but surprisingly Fc-IL2 did not.
Furthermore, in spleens collected 3 days after treatment, 25 μg of CD8-Fc-ALN2 was more able to enhance CD25 expression (and thus activation) in CD 8-positive T cells than equimolar 21.7 μg of wild-type Fc-IL2 (70% and 20%, respectively), whereas only wild-type Fc-IL2 induced CD25 expression in CD 4-positive T cells (see fig. 46).
The following amino acid sequences were used in this example:
○mCD8 VHH-hFc3(SEQ ID NO：495)
○hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：335)
○hFc3(SEQ ID NO：369)
○hFc4-hIL-2_C125A(SEQ ID NO：482)
example 18: efficacy of CD8-Fc-ALN2 in A20, B16 and Panc02 tumor models
Previously demonstrated, there is a combination of α: the anti-tumor efficacy of the mouse CD 8-targeted ALN2 variant (CD 8-Fc-ALN 2) with the β mutation r38a_f42 k_n8g was very successful in two isogenic mouse colon cancer models MC38 and CT 26. In addition, their antitumor efficacy was also analyzed in the other 3 tumor models: lymphoma a20, melanoma B16F10, and pancreatic adenocarcinoma Panc02. In this experiment, balb/C mice (female, 9 weeks old) were subcutaneously (s.c.) vaccinated with 6e+5 a20 cells and treatment was started on day 10, or C57B1/6J (female, 9 weeks old) mice were subcutaneously vaccinated with 6e+5B 16F10 or Panc02 cells and treatment was started on day 6.
First, at days 10, 14 and 17 after tumor inoculation, a20 tumor-bearing mice were treated intravenously (i.v.) as follows: (1) buffer, (2) targeted CD8-Fc-ALN2 (12.5. Mu.g/mouse) as monotherapy, (3) or combination of targeted CD8-Fc-ALN2 (12.5. Mu.g/mouse) with 200. Mu.g/mouse anti-PD-1 Ab, or (4) non-targeted wild-type Fc-IL2 (equimolar 10.7. Mu.g/mouse). Tumor sizes (mean +/-SEM; n=5 per group) are plotted in figure 47. FIG. 47 shows that both non-targeted wild-type Fc-IL-2 and targeted CD8-Fc-ALN2 significantly inhibited tumor growth, but wild-type Fc-IL-2 treated mice all died after the second treatment (indicated as "5/5+" or all animals died in FIG. 47). This result was in contrast to CD8-Fc-ALN2 treated mice, where the extreme sensitivity of Balb/c mice to wild type IL-2 toxic side effects was recorded, but not to CD8-Fc-ALN 2. At the end of the experiment, 4 out of 5 animals treated with CD8-Fc-ALN2 were completely tumor-free. Additional anti-PD-1 treatments were not initially able to enhance the therapeutic efficacy of CD8-Fc-ALN 2; furthermore, in this group, 4 out of 5 mice had no tumor, but this changed after two additional months of follow-up. At this later time point six mice (2 from the group treated with CD8-Fc-ALN2, 4 from the group treated with CD8-Fc-aln2+ anti-PD 1) remained tumor free and were re-challenged with a20 tumor cells on the other side. All 6 mice remained tumor-free, nor developed new tumors, compared to the initial mice vaccinated with the same a20 cells on the same day.
Second, on days 6, 9 and 13 after tumor inoculation, B16F10 tumor-bearing mice were treated intravenously with: (1) buffer, (2) targeted CD8-Fc-ALN2 (12.5. Mu.g/mouse) or (3) non-targeted Fc-ALN2 (equimolar 10.7. Mu.g/mouse). Tumor sizes (mean +/-SEM; n=4-5 per group) at day 16 post-inoculation are plotted in figure 48. CD8-Fc-ALN2 significantly slowed tumor growth, whereas non-targeted ALN2 had no effect.
Finally, at days 6 and 13 after tumor inoculation, panc02 tumor-bearing mice were treated intravenously with: (1) buffer, (2) targeted CD8-Fc-ALN2 (12.5. Mu.g/mouse) or (3) non-targeted Fc-ALN2 (equimolar 10.7. Mu.g/mouse). Tumor sizes (mean +/-SEM; n=5 per group) at day 16 post-inoculation are plotted in figure 49. CD8-Fc-ALN2 significantly slowed tumor growth, whereas non-targeted ALN2 did not.
The following amino acid sequences were used in this example:
○mCD8 VHH-hFc3(SEQ ID NO：495)
○hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：335)
○hFc3(SEQ ID NO：369)
example 19: tumor targeting of ALN2
In this example, the potential of using PD-L1 VHH to target ALN2 activity to tumors (or other immune cells) was assessed. The PD-L1-Fc-ALN2 protein was prepared by transfecting ExpiCHO cells with the following plasmid combination and purified as described previously:
●PD-L1-Fc-IL-2_R38A_F42K_N88G：mPD-L1vHH-hFc3+hFc4-IL-2_R38A_F42K_N88G
The potency and specificity of PD-L1ALN2 was verified on mouse spleen cells by analyzing phosphorylation of STAT5 as described in example 13. FIG. 50 shows that non-targeted Fc-ALN2 was unable to induce pSTAT5 in mouse spleen cells up to 10 μg/ml, whereas PD-L1 targeting was able to induce pSTAT5 signaling similar to WT IL-2.
To test the efficacy of the MC38 tumor model (PD-L1 expressing tumor), PD-L1 targeted ALN2 was selected. C57B1/6J mice (female, 9 weeks old) were subcutaneously injected with 6E+5 MC38 cells. Six days later, treatment was started: mice received (1) buffer intravenously (i.v.), either (2) targeted Fc-ALN2 (r38a_f42k_n88g with IL-2 mutation; 12.5 μg/mouse), or (3) non-targeted Fc-ALN2 three times (day 6, day 9 and day 13). Tumor sizes (mean +/-SEM; n=4-6 per group) at day 17 post-inoculation are plotted in figure 51, showing that only PD-L1 targeted ALN2 has specific tumor growth inhibition.
In addition, ALN2 (based on TenascinC-A1 specific VHH) targeting extracellular tumor stroma was generated in the case of cancer. The TNC-Fc-ALN2 protein was prepared by transfecting ExpiCHO cells with the following plasmid combination and purified as described above:
●TNC-Fc-IL-2_R38A_F42K_N88G：mTNC VHH-hFc3+hFc4-IL-2_R38A_F42K_N88G
to test efficacy in the MC38 tumor model, C57B1/6J mice (female, 9 weeks old) were subcutaneously injected with 6E+5 MC38 cells. Six days later, treatment was started: mice received (1) buffer, (2) non-targeted Fc-ALN2, or (3) TNCA 1-targeted Fc-ALN2 (r38a_f42k_n88g with IL-2 mutation; 12.5 μg/mouse) three times (day 6, day 9 and day 13). Tumor sizes were measured three times per week and mean +/-SEM (n=4-6) was plotted in figure 52. When mentioned, the ALN2 treatment was supplemented with intraperitoneal injection (i.p.) anti-PD-1 Ab treatment (200 μg/mouse) on the same day as intravenous treatment. It can be seen that TNC-targeted Fc-ALN2 therapy significantly and significantly inhibited tumor growth in combination with anti-PD-1 Ab therapy and compared to non-targeted Fc-ALN 2. Surprisingly, on day 21, some mice in which no tumor was detected were found only in the TNC-Fc-ALN2+PD-L1 treated group, i.e., three out of six animals.
Example 20: bispecific targeting of ALN2
FAP-targeted ALN2, NKp 46-targeted ALN2, and the following bispecific ALN2 were produced and purified from the expcho supernatant as described previously:
● NKp46-Fc-ALN2 (NKp 46 VHH-hFc3 (sequence below) +hfc4-hIL-2_r38a_f42k_n88 g). This construct targets ALN2 to NK cells.
● NKp46-CD8-Fc-ALN2 (combination of NKp46 VHH-hFc3+CD8VHH-hFc 4-hIL-2_R38A_F42K_N 88G; sequence as follows). This construct targets ALN2 to both NK cells and CD8 cells.
● FAP-Fc-ALN2 (FAP VHH-hFc3 (sequence below) +hfc4-hIL-2_r38a_f42k_n88 g). This construct targets ALN2 to tumor or cancer associated fibroblasts by FAP-specific VHH.
● FAP-CD8-Fc-ALN2 (FAP VHH-hFc3+CD8VHH-hFc 4-hIL-2_R38A_F42K_N88G in combination; sequence as follows). This construct targets ALN2 to tumor or cancer associated fibroblasts and CD8 cells by FAP-specific VHH.
The efficacy and specificity of NKp46-CD8-ALN2 was verified on mouse spleen cells by analyzing phosphorylation of STAT5 as described in example 13. FIG. 53 shows that non-targeted Fc-ALN2 did not induce pSTAT5 in CD8 cells at concentrations as high as 10. Mu.g/mL, whereas in NK cells only a slight effect was produced at an estimated EC50 value (extrapolated data since maximum signal was not reached at 10. Mu.g/mL) of about 5. Mu.g/mL. CD8 and NK cells can be stimulated simultaneously by bispecific constructs with EC50 of about 46 (relative to non-targeted > 200-fold window) and 2ng/ml (relative to non-targeted about 2500-fold window), respectively, which is at least similar to CD8 or NKp46 monospecific ALN 2.
As a follow-up to the above experiment, 2 additional bispecific constructs were generated and purified from the expcho supernatant as described previously:
● cd8_pd-1_fc-ALN2 (combination of PD-1 VHH-hfc3+cd8vhh-hfc4-hIL-2_r38a_f42k_n 88 g).
● CD8_PD-L1_Fc-ALN2 (combination of PD-L1VHH-hFc3+CD8VHH-hFc 4-hIL-2_R38A_F42K_N 88G)
These constructs were evaluated for their anti-tumor effect in MC38 tumor-bearing mice. These mice received one intravenous treatment at a tumor size of about 100-200mm3, or repeated treatments at 3-4 day intervals, with tumor sizes assessed every 2 to 3 days.
Sequences used in this experiment:
●mNKp46 VHH-hFc3(SEQ ID NO：525)
●mFAP VHH-hFc3(SEQ ID NO：526)
●mPD-L1 VHH-hFc3(SEQ ID NO：527)
●mTNC VHH-hFc3(SEQ ID NO：528)
●mCD8 VHH-hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：529)
●mPD-1VHH-hFc3(SEQ ID NO：530)
example 21: activation of CD8 cells with D20E and N88G CD8-Fc-ALN2 variants
Selective activation of human CD 8-targeted IL-2 variants with a combination of "alpha free" mutations (R38A_F42K) and recoverable loss of function beta mutations (hCD 8-Fc-ALN2_N88G or hCD 8-Fc-ALN2_D20E) that abrogate the activity on the IL-2Rα chain was assessed by analysis of cell surface expression of activation markers CD25 (IL-2Rα chain) and PD-1 in FACS. Expression in three PBMC donors was monitored five days later. The data (average of three donors) are plotted in fig. 54, clearly showing that CD8 targeting of these mutants allows selective upregulation (and thus activation) of these markers in CD8 positive cells relative to off-target cells.
Furthermore, in immunodeficient mice with human immune system (HIS mice) carrying MDA-MB-231 tumors, the same human CD 8-targeted ALN2 molecules induced STAT5 phosphorylation efficiently and specifically only in CD 8-positive T lymphocytes (analyzed 45 minutes after injection) (fig. 55). The humanized HIS mice are obtained by carrying out human umbilical cord blood CD34 positive stem cell irradiation and intrahepatic injection on 2-day-old NSG pups; tumor-bearing HIS mice were injected intravenously with hCD8-Fc-ALN2 at 6 months of age.
Similar to the human CD 8-targeted variants, the mouse CD 8-targeted Fc-ALN2 variants were generated by a combination of "no alpha" mutations with recoverable, loss-of-function β -chain mutations D20E or N88G (which have been used in the previous examples), as follows:
●mCD8-Fc-IL-2_R38A_F42K_D20E：mCD8 VHH-hFc3+hFc4-IL-2_R38A_F42K_D20E
●mCD8-Fc-IL-2_R38A_F42K_N88G：mCD8 VHH-hFc3+hFc4-IL-2_R38A_F42K_N88G
variants were expressed and purified as described above and activation of CD8 positive cells was tested on mouse spleen cells by measuring up-regulation of cell surface expression of CD25 after 6 days (fig. 56). Both the r38a_f42k_d20e and r38a_f42k_n88g variants significantly increased the expression of the activation marker in a selective and comparable manner, i.e. had a significant effect in CD8 positive cells but no effect in off-target (CD 8 negative) cells.
The following amino acid sequences were used in this example:
○mCD8 VHH-hFc3(SEQ ID NO：495)
○hCD8 VHH-hFc3(SEQ ID NO：483)
○hFc4-hIL-2_R38A_F42K_N88G_C125A(SEQ ID NO：335)
○hFc4-IL-2_R38A_F42K_D20E(SEQ ID NO：332)
Example 22: anti-tumor effects of D20E and N88G CD8-ALN2 variants
For evaluation in the MC38 tumor model, C57B1/6J mice (female, 9 weeks old) were subcutaneously injected with 6E+5 MC38 cells. Six days later, treatment was started: mice received either (1) buffer, (2) targeted CD8-Fc-ALN2 (12.5 μg/mouse), or (3) equimolar amounts of non-targeted Fc-ALN2 (10.7 μg/mouse) twice (day 6 and day 9).
●CD8-Fc-IL-2_R38A_F42K_D20E：mCD8 VHH-hFc3(SEQ ID NO：495)+hFc4-IL-2_R38A_F42K_D20E(SEQ ID NO：333)
●CD8-Fc-IL-2_R38A_F42K_N88G：mCD8 VHH-hFc3(SEQ ID NO：495)+hFc4-IL-2_R38A_F42K_N88G(SEQ ID NO：335)
●Fc-IL-2_R38A_F42K_N88G：VHH-hFc3(SEQ ID NO：369)+hFc4-IL-2_R38A_F42K_N88G(SEQ ID NO：335)
●Fc-IL-2_R38A_F42K_N88G：VHH-hFc3(SEQ ID NO：369)+hFc4-IL-2_R38A_F42K_D20E(SEQ ID NO：332)
Tumor size was measured three times per week and the average growth curve plotted in figure 57, showing that D20E-based CD8-Fc-ALN2 was at least as effective as N88G-based CD8-Fc-ALN2 in the absence of effect of both non-targeted counterparts.
Example 23: alpha in MC38 tumor model: in vivo evaluation of γALN2
In this example, the anti-tumor efficacy of a combination of "no alpha" mutation and recoverable loss-of-function gamma mutation was analyzed. The following constructs were expressed in primary spleen cells, purified and tested for STAT5 phosphorylation as described previously:
●CD8-Fc-IL-2_R38A_F42K_Q126G：mCD8 VHH-hFc3(SEQ ID NO.495)+hFc4-IL-2_R38A_F42K_Q126G(SEQ ID NO.522)
●CD8-Fc-IL-2_R38A_F42K_Q126I：mCD8 VHH-hFc3(SEQ ID NO.495)+hFc4-IL-2_R38A_F42K_Q126I(SEQ ID NO.523)
●CD8-Fc-IL-2_R38A_F42K_Q126Y：mCD8 VHH-hFc3(SEQ ID NO.495)+hFc4-IL-2_R38A_F42K_Q126Y(SEQ ID NO.524)
the data in fig. 58 shows that the combination of "no α" mutation with Q126G or Q126Y results in an ALN2 variant (EC 50 90 and 110ng/ml, respectively) and selectivity for CD8 positive mouse cells (little pSTAT5 in Treg cells). In the case of mice, the Q126I mutation was unable to recover at concentrations equal to or below 10 μg/mL, resulting in a variant with almost no activity (in contrast to the effect on human PBMC in the previous examples). The variant CD8-Fc-IL-2_R38A_F42K_Q126G was then evaluated in vivo.
For in vivo evaluation, C57B1/6J mice (females, 9 weeks old) were subcutaneously injected with 6E+5 MC38 cells. Six days later, treatment was started: mice received (1) buffer, (2) CD8-Fc-ALN2 (12.5 μg/mouse), (3) CD8-Fc-ALN2 (4.17 μg/mouse), (4) equivalent non-targeted ALN2 (12.5 μg/mouse), or (4) equivalent non-targeted ALN2 (3.57 μg/mouse) intravenously on days 6, 9, and 13. Tumor sizes on day 17 (i.e., 4 days after the last treatment) are plotted in figure 59 as mean +/-SEM (n=7). As can be seen in fig. 59, α: gamma ALN2 is capable of significantly delaying tumor growth in a dose-dependent manner, and CD 8-targeted ALN2 has excellent effects.
Example 24: beta: in vivo evaluation of gamma combinations
ALN2 variant CD8-Fc-IL-2_N88G_Q126G (mCD 8 VHH-hFc3 (SEQ ID NO: 495) +hFc 4-IL-2_N88G_Q126G) was expressed in ExpiCHO and purified from the supernatant as described previously:
○hFc4-IL-2_R38A_F42K_N88G_C125A_Q126G(SEQ ID NO：531)
for in vivo evaluation, C57B1/6J mice (females, 9 weeks old) were subcutaneously injected with 6E+5 MC38 cells. Six days later, treatment was started: mice received (1) buffer, (2) CD8-Fc-ALN2 (4.17 μg/mouse), (3) CD8-Fc-ALN2 (1.4 μg/mouse), (4) equivalent non-targeted ALN2 (3.57 μg/mouse), or (4) equivalent non-targeted ALN2 (1.19 μg/mouse) intravenously on days 6, 9, and 13. Tumor sizes on day 17 (i.e., 4 days after the last treatment) are plotted in figure 60 as mean +/-SEM (n=7). As can be seen in fig. 60, β: gamma ALN2 is capable of significantly delaying tumor growth in a dose-dependent manner, and CD 8-targeted ALN2 has excellent effects.
Example 25: screening of IL-2 variants specifically targeting cells expressing IL-2Rα chain
In the previous examples, β (D20 or N88) and γ (Q126) attenuating mutations were combined to form β with an intact IL-2rα binding site: γaln2 variants and CD8 targeting is used to increase selectivity for activated CD8 positive cells over, for example, treg. Unfortunately, expression of such ALN2 variants is poor. Thus, as an alternative to the combination of β and γ mutations, the β mutations (D20 and N88) that appeared unrecoverable in example 3 were rescreened. The initial screening of the beta mutants in example 3 was performed on "resting" or inactive CD8 positive PBMCs that were negative for IL-2rα expression. In this example, the expression of functional trimer α: beta: the D20 and N88 mutants were rescreened on cells of the gamma IL-2 receptor. For this purpose, signal transduction in HEK-Blue IL-2 cells (InvivoGen) stably expressing human CD8 (HEK-Blue CD 8) or human NKp46 (HEK-Blue NKp 46) was analyzed. D20 The data for the (fig. 61) and N88 (fig. 62) variants surprisingly demonstrate that for many mutants (e.g., D20F, D20G, D20H, D20I, D20K, D L, D20N, N88E, N I, N88K, N88Q, N88R and N88V), signaling is significantly attenuated (compared to CD8-Fc-IL 2) and that this loss of activity can be at least partially recovered after CD8 targeting (HEK-Blue CD8 and HEK-Blue NKp46 cells), whereas in functional dimer β: this is not the case for these mutants when screened on gamma IL-2 receptor.
Equivalent scheme
While the invention has been described in connection with specific embodiments thereof, it will be understood that, in general, further modifications can be made and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed by the scope of the following claims.
Incorporated by reference
All patents and publications cited herein are hereby incorporated by reference in their entirety.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention.
As used herein, all headings are for organization only and are not intended to limit the disclosure in any way. The contents of any individual section can be equally applicable to all sections.

Claims (162)

1. A chimeric protein or protein complex, comprising:
(a) A modified IL-2 signaling agent, wherein the modified IL-2 signaling agent is compared to a peptide having the amino acid sequence of SEQ ID NO:1, which one or more mutations or modifications confer to the modified IL-2 signaling agent increased safety and reduced affinity or biological activity for IL-2rαβγ and/or IL-2rβ and/or IL-2rγ and/or IL-2rα relative to the wild-type IL-2 signaling agent; and
(b) One or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest,
wherein the modified IL-2 and the one or more targeting moieties are optionally linked to one or more linkers,
wherein the modified IL-2 signaling agent comprises:
(i) A mutation selected from the group consisting of D20E, D20F, D20G, D20H, D I, D20K, D L and D20V; or (b)
(ii) A mutation selected from the group consisting of N88G, N88E, N88K, N88Q and N88V; or (b)
(iii) A mutation selected from Q126G, Q126A, Q126E, Q F, Q126H, Q I, Q K, Q126L, Q126N, Q P, Q126R, Q126S, Q126T, Q V, Q126W and Q126Y; or (b)
(iv) A mutation selected from (i) and (iii) or a mutation selected from (ii) and (iii); or (b)
(v) One or more mutations selected from the group consisting of:
R38A/F42K/N88G/C125A、
F42K/C125A、
D20E/C125A、
D20F/C125A、
D20G/C125A、
D20H/C125A、
D20I/C125A、
D20K/C125A、
D20L/C125A、
D20N/C125A、
D20S/C125A、
D20T/C125A、
D20V/C125A、
N88A/C125A、
N88D/C125A、
N88G/C125A、
N88E/C125A、
N88H/C125A、
N88I/C125A、
N88K/C125A、
N88Q/C125A、
N88R/C125A、
N88T/C125A、
N88V/C125A、
D20E/R38A/F42K、
D20E/R38A/F42K/C125A、
D20V/R38A/F42K、
D20V/R38A/F42K/C125A、
R38A/F42Y/E62A/C125A、
R38A/F42Y/Y45A/E62A、
F42Y/Y45A/L72G、
R38A/F42Y/Y45A/E62A/C125A、
F42Y/Y45A/L72G/C125A、
R38A/F42K/N88G、
R38A/F42K/Q126G、
R38A/F42K/Q126I、
R38A/F42K/Q126Y、
R38A/F42K/Q126G/C125A、
R38A/F42K/Q126I/C125A、
R38A/F42K/Q126Y/C125A、
R38A/F42K/E62A/N88G and
R38A/F42K/E62A/N88G/C125A。
2. the chimeric protein or protein complex of claim 1, wherein the modified IL-2 signaling agent further comprises a glycosylation mutation, wherein the mutation reduces or eliminates glycosylation of the modified IL-2 signaling agent.
3. The chimeric protein or protein complex of claim 2, wherein the glycosylation mutation is a T3 substitution, wherein the mutation is one of T3A, T3F, T3H, T3L, T3V and T3Y.
4. The chimeric protein or protein complex of claim 2, wherein the glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2.
5. A chimeric protein or protein complex, comprising:
(a) A modified IL-2 signaling agent, wherein the modified IL-2 signaling agent is compared to a peptide having the amino acid sequence of SEQ ID NO:1, which one or more mutations or modifications confer to the modified IL-2 signaling agent increased safety and reduced affinity or biological activity for IL-2rαβγ and/or IL-2rβ and/or IL-2rγ and/or IL-2rα relative to the wild-type IL-2 signaling agent; and
(b) One or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest,
wherein the modified IL-2 and the one or more targeting moieties are optionally linked to one or more linkers,
wherein the modified IL-2 signaling agent comprises a glycosylation mutation, wherein the mutation reduces or eliminates glycosylation of the modified IL-2 signaling agent.
6. The chimeric protein or protein complex of claim 5, wherein the glycosylation mutation is a T3 substitution, optionally wherein the mutation is one of T3A, T3F, T3H, T3L, T3V and T3Y.
7. The chimeric protein or protein complex of claim 5, wherein the glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2.
8. A chimeric protein or protein complex, comprising:
(a) A modified IL-2 signaling agent, wherein the modified IL-2 signaling agent is compared to a peptide having the amino acid sequence of SEQ ID NO:2, which one or more mutations or modifications confer to the modified IL-2 signaling agent increased safety and reduced affinity or biological activity for IL-2rαβγ and/or IL-2rβ and/or IL-2rγ and/or IL-2rα relative to the novel leukocyte signaling agent; and
(b) One or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest,
wherein the modified IL-2 signaling agent and the one or more targeting moieties are optionally linked to one or more linkers,
wherein the modified IL-2 signaling agent comprises one or more mutations at a position selected from D15 and N40, optionally wherein the mutation is selected from D15T, D15H, N40I, N G and N40R.
9. The chimeric protein or protein complex of any one of claims 1 to 8, wherein the modified IL-2 comprises one or more mutations that confer a reduced affinity for an IL-2 receptor chain.
10. The chimeric protein or protein complex of any one of claims 1 to 9, wherein the modified IL-2 exhibits reduced affinity for IL-2rαβγ.
11. The chimeric protein or protein complex of any one of claims 1 to 9, wherein the modified IL-2 exhibits reduced affinity for IL-2rβ, IL-2rγ, or a combination thereof.
12. The chimeric protein or protein complex of any one of claims 1 to 9, wherein the modified IL-2 exhibits an abrogated affinity for IL-2rα, IL-2rβ, IL2rγ, or a combination thereof.
13. The chimeric protein or protein complex of claim 10, wherein the modified IL-2 exhibits reduced affinity for:
IL-2Rα and IL2Rβ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of D20E/R38A/F42K, D E/R38A/F42K/C125A, D V/R38A/F42K, D V/R38A/F42K/C125A, D V/R38A/F42K/E62A, D20V/R38A/F42K/E62A/C125A, R A/F42K/N88G, R38A/F42K/N88G/C125A, R A/F42K/E62A/N88G and R38A/F42K/E62A/N88G/C125A; or (b)
IL-2rα and IL-2rγ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of R38A/F42K/Q126G, R a/F42K/Q126I and R38A/F42K/Q126Y; or (b)
IL-2Rβ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of D20E, D20E, D20E, D20E, D20E, D20E, D88/C125E, D E/C125E, D F/C125E, D G/C125E, D H/C125E, D I/C125E, D L/C125E, D K/C125E, D20N/C125E, D S/C125E, D T/C125E, D V/C125E, D A/C125E, D88D/C125E, D G/C125E, D E/C125E, D88H/C125 5288I/C125E, D K/C125Q/C125R/C88T/C125A and N88V/C125A; or (b)
IL-2rγ, optionally wherein the modified IL-2 comprises one or more mutations selected from Q126A, Q126E, Q126F, Q G, Q126H, Q126I, Q K, Q126L, Q126N, Q P, Q126R, Q S, Q T, Q126V, Q W and Q126Y, or a combination thereof.
14. The chimeric protein or protein complex according to any one of claim 1 to 9, wherein the modified IL-2 exhibits a reduced affinity for IL-2Rβγ, optionally wherein the modified IL-2 comprises a polypeptide selected from the group consisting of D20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126E/Q20E/Q126 20E/Q126V/Q126 20V/Q126V/Q20V/Q126V/Q20V/Q20E/Q126V 20V A/Q126 20V/Q126 20V/Q126 20V/Q126V/Q20V/Q, one or more mutations of N88G/Q126W and N88G/Q126Y.
15. The chimeric protein or protein complex of any one of claims 1 to 14, wherein the one or more mutations in the IL-2 signaling agent confer a reduced affinity or biological activity that can be recovered by ligation to one or more targeting moieties.
16. The chimeric protein or protein complex of any one of the above claims, wherein the one or more targeting moieties are directed against tumor cells, endothelial cells, epithelial cells, mesenchymal cells, tumor stroma or stromal cells and/or ECM.
17. The chimeric protein or protein complex of any one of the above claims, wherein the one or more targeting moieties are directed against immune cells and/or organ cells and/or tissue cells.
18. The chimeric protein or protein complex of claim 17, wherein the immune cell is selected from a T cell, a Treg, a cytotoxic T lymphocyte, a T helper cell, a B cell, a dendritic cell, an anti-tumor macrophage or tumor-associated macrophage (e.g., M1 or M2 macrophage), a neutrophil, a bone marrow-derived suppressor cell, a Natural Killer (NK) cell, and a Natural Killer T (NKT) cell, optionally the immune cell is a T cell and the targeting moiety targets CD8, or optionally wherein the immune cell is a Treg and the targeting moiety targets CTLA4, or optionally wherein the immune cell is a NK cell and the targeting moiety is NKp46.
19. The chimeric protein or protein complex according to any one of the preceding claims, wherein the targeting moiety comprises a recognition domain, the recognition domain is a full length antibody, a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv) heavy chain-only shark antibodies (VNAR), trace proteins (e.g., cysteine knot proteins, knottins), darpin, anti-carrier proteins,adnectin, aptamer, fv, fab, fab ', F (ab') 2 A peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.
20. The chimeric protein or protein complex of any one of the above claims, wherein the recognition domain is a single domain antibody.
21. The chimeric protein or protein complex of any one of the above claims, wherein the recognition domain is a VHH or a humanized VHH.
22. The chimeric protein or protein complex of any one of the above claims, wherein the recognition domain functionally modulates the antigen or receptor of interest.
23. The chimeric protein or protein complex of any one of the above claims, wherein the recognition domain binds to but does not functionally modulate the antigen or receptor of interest.
24. The chimeric protein or protein complex of any one of the above claims, comprising two or more targeting moieties.
25. The chimeric protein or protein complex of any one of the above claims, wherein the targeting moiety binds to one or more of the following targets: CD8, CTLA4, CD3, CD4, DNAM-1, nrp1 (neuropilin), TNFR1, TNFR2, GITR, ICOS, CD20, CD70, clec9a, NKp46, PD-1, PD-L2, SIRP1 alpha, FAP, XCR1, tenascin or ECM protein.
26. The chimeric protein or protein complex of any one of the above claims, further comprising one or more additional modified signaling agents.
27. The chimeric protein or protein complex of any one of the above claims, wherein the chimeric protein comprises two signaling agents and one targeting moiety or two targeting moieties and one signaling agent, or both.
28. The chimeric protein or protein complex of any one of the above claims, wherein the chimeric protein comprises three signaling agents or three targeting moieties, or both.
29. The chimeric protein or protein complex of claim 26, wherein the additional modified signaling agent comprises one or more mutations that confer reduced affinity or activity for a receptor relative to an unmutated signaling agent.
30. The chimeric protein or protein complex of claim 29, wherein the one or more mutations allow for attenuation of activity.
31. The chimeric protein or protein complex of claim 30, wherein agonistic or antagonistic activity is reduced.
32. The chimeric protein or protein complex of any one of the above claims, wherein the chimeric protein is suitable for use in a patient suffering from one or more of the following diseases: cancer, infection, immune disorders, autoimmune diseases, cardiovascular diseases, trauma, ischemia-related diseases, neurodegenerative diseases, and/or metabolic diseases.
33. A recombinant nucleic acid composition encoding one or more chimeric proteins or protein complexes according to any one of the preceding claims.
34. A host cell comprising the recombinant nucleic acid of claim 33.
35. A method for treating cancer, the method comprising administering to a patient in need thereof an effective amount of the chimeric protein or protein complex of any one of claims 1-32.
36. The method of claim 35, wherein the cancer is selected from one or more of the following: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal carcinoma; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcomas (e.g., kaposi's sarcoma); skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of urinary system; vulvar cancer; lymphomas, including hodgkin's and non-hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated schizophrenic NHL, giant tumor NHL, mantle cell lymphomas, AIDS-related lymphomas, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal vascular hyperplasia associated with nevus, oedema (e.g., brain tumor-related oedema), and meuges syndrome.
37. Use of the chimeric protein or protein complex of any one of claims 1-32 in the manufacture of a medicament.
38. Use of the chimeric protein or protein complex of any one of claims 1-31 in the treatment of cancer or an autoimmune disease in a subject in need thereof.
39. An Fc-based chimeric protein complex, comprising:
(a) A modified IL-2 signaling agent, wherein the modified IL-2 signaling agent is compared to a peptide having the amino acid sequence of SEQ ID NO:1, which one or more mutations or modifications confer to the modified IL-2 signaling agent increased safety and reduced affinity or biological activity for IL-2rαβγ and/or IL-2rβ and/or IL-2rγ and/or IL-2rα relative to the wild-type IL-2 signaling agent; and
(b) One or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest; and
(c) An Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and/or stabilize a hinge region in the Fc domain,
Wherein the modified IL-2 signaling agent comprises:
(i) A mutation selected from the group consisting of D20E, D20F, D20G, D20H, D I, D20K, D L and D20V; or (b)
(ii) A mutation selected from the group consisting of N88G, N88E, N88K, N88Q and N88V; or (b)
(iii) A mutation selected from Q126G, Q126A, Q126E, Q F, Q126H, Q I, Q K, Q126L, Q126N, Q P, Q126R, Q126S, Q126T, Q V, Q126W and Q126Y; or (b)
(iv) A mutation selected from (i) and (iii) or a mutation selected from (ii) and (iii); or (b)
(v) One or more mutations selected from the group consisting of:
R38A/F42K/N88G/C125A、
F42K/C125A、
D20E/C125A、
D20F/C125A、
D20G/C125A、
D20H/C125A、
D20I/C125A、
D20K/C125A、
D20L/C125A、
D20N/C125A、
D20S/C125A、
D20T/C125A、
D20V/C125A、
N88A/C125A、
N88D/C125A、
N88G/C125A、
N88E/C125A、
N88H/C125A、
N88I/C125A、
N88K/C125A、
N88Q/C125A、
N88R/C125A、
N88T/C125A、
N88V/C125A、
D20E/R38A/F42K、
D20E/R38A/F42K/C125A、
D20V/R38A/F42K、
D20V/R38A/F42K/C125A、
R38A/F42Y/E62A/C125A、
R38A/F42Y/Y45A/E62A、
F42Y/Y45A/L72G、
R38A/F42Y/Y45A/E62A/C125A、
F42Y/Y45A/L72G/C125A、
R38A/F42K/N88G、
R38A/F42K/Q126G、
R38A/F42K/Q126I、
R38A/F42K/Q126Y、
R38A/F42K/Q126G/C125A、
R38A/F42K/Q126I/C125A、
R38A/F42K/Q126Y/C125A、
R38A/F42K/E62A/N88G and
R38A/F42K/E62A/N88G/C125A。
40. the Fc-based chimeric protein complex of claim 39, wherein the modified IL-2 signaling agent further comprises a glycosylation mutation, wherein the mutation reduces or eliminates glycosylation of the modified IL-2 signaling agent.
41. The Fc-based chimeric protein complex of claim 40, wherein the glycosylation mutation is a T3 substitution, wherein the mutation is one of T3A, T3F, T3H, T3L, T3V and T3Y.
42. The Fc-based chimeric protein complex of claim 40, wherein said glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of said modified IL-2.
43. An Fc-based chimeric protein complex, comprising:
(a) A modified IL-2 signaling agent, wherein the modified IL-2 signaling agent is compared to a peptide having the amino acid sequence of SEQ ID NO:1, which one or more mutations or modifications confer to the modified IL-2 signaling agent increased safety and reduced affinity or biological activity for IL-2rαβγ and/or IL-2rβ and/or IL-2rγ and/or IL-2rα relative to the wild-type IL-2 signaling agent; and
(b) One or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest; and
(c) An Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and/or stabilize a hinge region in the Fc domain,
wherein the modified IL-2 signaling agent comprises a glycosylation mutation, wherein the mutation reduces or eliminates glycosylation of the modified IL-2 signaling agent.
44. The Fc-based chimeric protein complex of claim 43, wherein the glycosylation mutation is a T3 substitution, optionally wherein the mutation is one of T3A, T3F, T3H, T3L, T V and T3Y.
45. The Fc-based chimeric protein complex of claim 43, wherein said glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of said modified IL-2.
46. An Fc-based chimeric protein complex, comprising:
(a) A modified IL-2 signaling agent, wherein the modified IL-2 signaling agent is compared to a peptide having the amino acid sequence of SEQ ID NO:2, which one or more mutations or modifications confer to the modified IL-2 signaling agent increased safety and reduced affinity or biological activity for IL-2rαβγ and/or IL-2rβ and/or IL-2rγ and/or IL-2rα relative to the novel leukocyte signaling agent; and
(b) One or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest,
wherein the modified IL-2 signaling agent and the one or more targeting moieties are optionally linked to one or more linkers,
Wherein the modified IL-2 signaling agent comprises one or more mutations at a position selected from D15 and N40, optionally wherein the mutation is selected from D15T, D15H, N40I, N G and N40R.
47. The Fc-based chimeric protein complex of any one of claims 39-46, wherein the modified IL-2 comprises one or more mutations that confer reduced affinity for an IL-2 receptor.
48. The Fc-based chimeric protein complex of any one of claims 39-47, wherein the modified IL-2 exhibits reduced affinity for IL-2rαβγ.
49. The Fc-based chimeric protein complex of any one of claims 39-47, wherein the modified IL-2 exhibits reduced affinity for IL-2rβ, IL-2rγ, or a combination thereof.
50. The Fc-based chimeric protein complex of any one of claims 39-47, wherein the modified IL-2 exhibits an abrogated affinity for IL-2rα, IL-2rαβ, IL-2rγ, or a combination thereof.
51. The Fc-based chimeric protein complex of claim 48, wherein the modified IL-2 exhibits reduced affinity for:
IL-2Rα and IL2Rβ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of D20E/R38A/F42K, D E/R38A/F42K/C125A, D V/R38A/F42K, D V/R38A/F42K/C125A, D V/R38A/F42K/E62A, D20V/R38A/F42K/E62A/C125A, R A/F42K/N88G, R38A/F42K/N88G/C125A, R A/F42K/E62A/N88G and R38A/F42K/E62A/N88G/C125A; or (b)
IL-2rα and IL-2rγ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of R38A/F42K/Q126G, R a/F42K/Q126I and R38A/F42K/Q126Y; or (b)
IL-2Rβ, optionally wherein the modified IL-2 comprises one or more mutations selected from the group consisting of D20E, D20E, D20E, D20E, D20E, D20E, D88/C125E, D E/C125E, D F/C125E, D G/C125E, D H/C125E, D I/C125E, D L/C125E, D K/C125E, D20N/C125E, D S/C125E, D T/C125E, D V/C125E, D A/C125E, D88D/C125E, D G/C125E, D E/C125E, D88H/C125 5288I/C125E, D K/C125Q/C125R/C88T/C125A and N88V/C125A; or (b)
IL-2rγ, optionally wherein the modified IL-2 comprises one or more mutations selected from Q126A, Q126E, Q126F, Q G, Q126H, Q126I, Q K, Q126L, Q126N, Q P, Q126R, Q S, Q T, Q126V, Q W and Q126Y, or a combination thereof.
52. The Fc-based chimeric protein complex of any one of claims 39-47, wherein the modified IL-2 exhibits reduced affinity for IL-2Rβγ, optionally wherein the modified IL-2 comprises a polypeptide selected from the group consisting of D20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126 20E/Q126E/Q20E/Q126 20E/Q126V/Q126 20V/Q126V/Q20V/Q126V/Q20V/Q20E/Q126V 20V A/Q126 20V/Q126 20V/Q126 20V/Q126V/Q20V/Q, one or more mutations of N88G/Q126W and N88G/Q126Y.
53. The Fc-based chimeric protein complex of any one of claims 39-52, wherein the one or more mutations in the IL-2 signaling agent confer reduced affinity or biological activity that can be recovered by ligation to one or more targeting moieties or upon inclusion in the Fc-based chimeric protein complex.
54. The Fc-based chimeric protein complex of any one of claims 39-53, wherein the one or more targeting moiety is directed against a tumor cell, endothelial cell, epithelial cell, mesenchymal cell, tumor stroma or stromal cell and/or ECM.
55. The Fc-based chimeric protein complex of any one of claims 39-54, wherein the one or more targeting moieties are directed against immune cells and/or organ cells and/or tissue cells.
56. The Fc-based chimeric protein complex of claim 55, wherein the immune cell is selected from a T cell, B cell, dendritic cell, macrophage, neutrophil, mast cell, monocyte, erythrocyte, bone marrow cell, bone marrow derived suppressor cell, NKT cell, and NK cell or derivative thereof, optionally wherein the immune cell is a T cell and the targeting moiety targets CD8, or optionally wherein the immune cell is a Treg and the targeting moiety targets CTLA4, or optionally wherein the immune cell is an NK cell and the targeting moiety is NKp46.
57. The Fc-based chimeric protein complex of any one of claims 39-56, wherein the targeting moiety comprises a recognition domain that is a full-length antibody, a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a trace protein (e.g., a cysteine knot protein, a knottin), darpin, an anti-carrier protein, an adnectin, an aptamer, fv, fab, fab ', F (ab') 2 A peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.
58. The Fc-based chimeric protein complex of any one of claims 39-57, wherein the recognition domain is a single domain antibody.
59. As claimed inThe Fc-based chimeric protein complex of any one of claims 39-58, wherein said recognition domain is V HH Or humanized V HH 。
60. The Fc-based chimeric protein complex of any one of claims 39-59, wherein the recognition domain functionally modulates the antigen or receptor of interest.
61. The Fc-based chimeric protein complex of any one of claims 39-60, wherein the recognition domain binds to but does not functionally modulate the antigen or receptor of interest.
62. The Fc-based chimeric protein complex of any one of claims 39-61, comprising two or more targeting moieties.
63. The Fc-based chimeric protein complex of any one of claims 39-62, wherein the targeting moiety binds to one or more of the following targets: CD8, CTLA4, CD3, CD4, DNAM-1, nrp1 (neuropilin), TNFR1, TNFR2, GITR, ICOS, CD20, CD70, clec9a, NKp46, PD-1, PD-L2, SIRP1 alpha, FAP, XCR1, tenascin or ECM protein.
64. The Fc-based chimeric protein complex of any one of claims 39-63, further comprising one or more additional modified signaling agents.
65. The Fc-based chimeric protein complex of any one of claims 39-64, wherein the chimeric protein comprises two signaling agents or two targeting moieties, or both.
66. The Fc-based chimeric protein complex of any one of claims 39-65, wherein the chimeric protein comprises three signaling agents or three targeting moieties, or both.
67. The Fc-based chimeric protein complex of claim 64, wherein the additional modified signaling agent comprises one or more mutations that confer reduced affinity or activity for a receptor relative to an unmutated signaling agent.
68. The Fc-based chimeric protein complex of claim 67, wherein said one or more mutations allow for attenuation of activity.
69. The Fc-based chimeric protein complex of claim 68, wherein agonistic or antagonistic activity is reduced.
70. The Fc-based chimeric protein complex of any one of claims 39-69, wherein said chimeric protein is suitable for use in a patient suffering from one or more of the following diseases: cancer, infection, immune disorders, autoimmune diseases, cardiovascular diseases, trauma, ischemia-related diseases, neurodegenerative diseases, and/or metabolic diseases.
71. A recombinant nucleic acid composition encoding one or more of the Fc-based chimeric protein complexes or a constituent polypeptide thereof of any one of claims 39-70.
72. A host cell comprising the recombinant nucleic acid of claim 71.
73. A method for treating cancer, the method comprising: (i) Administering to a patient in need thereof an effective amount of the Fc-based chimeric protein complex of any one of claims 39-69; ii) administering to a patient in need thereof an effective amount of the recombinant nucleic acid of claim 71; or iii) administering to a patient in need thereof an effective amount of the host cell of claim 72.
74. The method of claim 73, wherein the cancer is selected from one or more of the following: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal carcinoma; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcomas (e.g., kaposi's sarcoma); skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of urinary system; vulvar cancer; lymphomas, including hodgkin's and non-hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated schizophrenic NHL, giant tumor NHL, mantle cell lymphomas, AIDS-related lymphomas, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal vascular hyperplasia associated with nevus, oedema (e.g., brain tumor-related oedema), and meuges syndrome.
75. Use of an Fc-based chimeric protein complex of any one of claims 39-70 in the manufacture of a medicament.
76. Use of the Fc-based chimeric protein complex of any one of claims 39-69 in the treatment of cancer or an autoimmune disease in a subject in need thereof.
77. The Fc-based chimeric protein complex of any one of claims 39-70, wherein the Fc domain is from IgG, igA, igD, igM or IgE.
78. The Fc-based chimeric protein complex of claim 77, wherein the IgG is selected from IgG1, igG2, igG3, or IgG4.
79. The Fc-based chimeric protein complex of any one of claims 39-70, wherein the Fc domain is from human IgG, igA, igD, igM or IgE.
80. The Fc-based chimeric protein complex of claim 79, wherein the human IgG is selected from human IgG1, igG2, igG3, or IgG4.
81. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-80, wherein the Fc chain pairing is facilitated by ion pairing and/or knob hole pairing.
82. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-81, wherein the one or more mutations in the Fc domain result in ion pairing between Fc chains in the Fc domain.
83. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-82, wherein the one or more mutations in the Fc domain result in knob-in hole pairing in the Fc domain.
84. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-83, wherein said one or more mutations in said Fc domain result in a reduction or elimination of effector function of said Fc domain.
85. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-84, wherein the Fc-based chimeric protein complex is a heterodimer and has a trans orientation/configuration with respect to any targeting moiety and signaling agent, or any targeting moiety with respect to each other, or any signaling agent with respect to each other.
86. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-85, wherein the Fc-based chimeric protein complex is a heterodimer and has a cis orientation with respect to any targeting moiety and signaling agent, or any signaling agent, with respect to each other.
87. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-86, wherein the Fc comprises L234A, L a and K322Q substitutions (numbering according to EU) in human IgG 1.
88. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-87, wherein said Fc is a human IgG1 and optionally comprises one or more mutations (numbering according to EU) of L234, L235, K322, D265, P329 and P331.
89. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-88, wherein the Fc-based chimeric protein complex has an orientation and/or configuration of any one of: 1A-1F, 2A-2H, 3A-3H, 4A-4D, 5A-5F, 6A-6J, 7A-7D, 8A-8F, 9A-9J, 10A-10F, 11A-11L, 12A-12L, 13A-13F, 14A-14L, 15A-15L, 16A-16J, 17A-17J, 18A-18F, 19A-19F, 31 and 32.
90. The Fc-based chimeric protein complex of any one of claims 39-70 and 77-89, wherein said Fc-based chimeric protein complex comprises a polypeptide having a sequence identical to SEQ ID NO: 292. 293, 294, 299, 301, 310, 311, 312, 315, 316, 319, 320, 325, 328, 332, 333, and 335, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical.
91. The chimeric protein of any one of claims 1-32 or the Fc-based chimeric protein complex of any one of claims 39-70 or 77-89, wherein the chimeric protein or Fc-based chimeric protein complex comprises a polypeptide having a nucleotide sequence identical to SEQ ID NO: a polypeptide having an amino acid sequence of any one of 290-449, 478-495, or 501-531 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.
92. A chimeric protein or protein complex, comprising:
(a) A modified IL-2 signaling agent, wherein the modified IL-2 signaling agent is compared to a peptide having the amino acid sequence of SEQ ID NO:1, which single mutation confers increased safety and reduced affinity or biological activity for IL-2rβ to the modified IL-2 signaling agent relative to the wild-type IL-2 signaling agent; and
(b) One or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest,
wherein the modified IL-2 and the one or more targeting moieties are optionally linked to one or more linkers,
Wherein the single mutation is selected from the group consisting of D20E, D20F, D20G, D H, D20I, D K, D L, D20N, D20V, N88G, N E, N88I, N88K, N Q, N R and N88V, and
wherein the modified IL-2 signaling agent does not comprise a mutation that confers reduced affinity or biological activity to IL-2rα.
93. The chimeric protein or protein complex of claim 92, wherein the modified IL-2 signaling agent further comprises a glycosylation mutation, wherein the mutation reduces or eliminates glycosylation of the modified IL-2 signaling agent.
94. The chimeric protein or protein complex of claim 93, wherein the glycosylation mutation is a T3 substitution, wherein the mutation is one of T3A, T3F, T3H, T3L, T3V and T3Y.
95. The chimeric protein or protein complex of claim 93, wherein the glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2.
96. The chimeric protein or protein complex of any one of claims 92 to 95, wherein the one or more mutations in the IL-2 signaling agent confer a reduced affinity or biological activity that can be recovered by ligation to one or more targeting moieties.
97. The chimeric protein or protein complex of any one of claims 92 to 96, wherein the one or more targeting moiety is directed against a tumor cell, an endothelial cell, an epithelial cell, a mesenchymal cell, a tumor stroma or stromal cell and/or ECM.
98. The chimeric protein or protein complex of any one of claims 92 to 97, wherein the one or more targeting moieties are directed against immune cells and/or organ cells and/or tissue cells.
99. The chimeric protein or protein complex of claim 98, wherein the immune cell is selected from a T cell, a Treg, a cytotoxic T lymphocyte, a T helper cell, a B cell, a dendritic cell, an anti-tumor macrophage or tumor-associated macrophage (e.g., M1 or M2 macrophage), a neutrophil, a bone marrow-derived suppressor cell, a Natural Killer (NK) cell, and a Natural Killer T (NKT) cell, optionally the immune cell is a T cell and the targeting moiety targets CD8, or optionally wherein the immune cell is a Treg and the targeting moiety targets CTLA4, or optionally wherein the immune cell is a NK cell and the targeting moiety is NKp46.
100. The chimeric protein or protein complex of any one of claims 92 to 99, wherein the targeting moiety comprises a recognition domain that is a full length antibody, a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a trace protein (e.g., a cysteine knot protein, a knottin), darpin, an anti-carrier protein, an adnectin, an aptamer, fv, fab, fab ', F (ab') 2 A peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.
101. The chimeric protein or protein complex of any one of claims 92 to 100, wherein the recognition domain is a single domain antibody.
102. The chimeric protein or protein complex of any one of claims 92 to 101, wherein the recognition domain is a VHH or a humanized VHH.
103. The chimeric protein or protein complex of any one of claims 92 to 102, wherein the recognition domain functionally modulates the antigen or receptor of interest.
104. The chimeric protein or protein complex of any one of claims 92 to 103, wherein the recognition domain binds to but does not functionally modulate the antigen or receptor of interest.
105. The chimeric protein or protein complex of any one of claims 92 to 104, comprising two or more targeting moieties.
106. The chimeric protein or protein complex of any one of claims 92 to 105, wherein the targeting moiety binds to one or more of the following targets: CD8, CTLA4, CD3, CD4, DNAM-1, nrp1 (neuropilin), TNFR1, TNFR2, GITR, ICOS, CD20, CD70, clec9a, NKp46, PD-1, PD-L2, SIRP1 alpha, FAP, XCR1, tenascin or ECM protein.
107. The chimeric protein or protein complex of any one of claims 92 to 106, further comprising one or more additional modified signaling agents.
108. The chimeric protein or protein complex of any one of claims 92 to 107, wherein the chimeric protein comprises two signaling agents and one targeting moiety or two targeting moieties and one signaling agent, or both.
109. The chimeric protein or protein complex of any one of claims 92 to 108, wherein the chimeric protein comprises three signaling agents or three targeting moieties, or both.
110. The chimeric protein or protein complex of claim 107, wherein the additional modified signaling agent comprises one or more mutations that confer reduced affinity or activity for a receptor relative to an unmutated signaling agent.
111. The chimeric protein or protein complex of claim 110, wherein the one or more mutations allow for attenuation of activity.
112. The chimeric protein or protein complex of claim 111, wherein agonistic or antagonistic activity is reduced.
113. The chimeric protein or protein complex of any one of claims 92 to 112, wherein the chimeric protein is suitable for use in a patient suffering from one or more of the following diseases: cancer, infection, immune disorders, autoimmune diseases, cardiovascular diseases, trauma, ischemia-related diseases, neurodegenerative diseases, and/or metabolic diseases.
114. A recombinant nucleic acid composition encoding one or more chimeric proteins or protein complexes of any one of claims 92 to 112.
115. A host cell comprising the recombinant nucleic acid of claim 114.
116. A method for treating cancer, the method comprising administering to a patient in need thereof an effective amount of the chimeric protein or protein complex of any one of claims 92 to 112.
117. The method of claim 116, wherein the cancer is selected from one or more of the following: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal carcinoma; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcomas (e.g., kaposi's sarcoma); skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of urinary system; vulvar cancer; lymphomas, including hodgkin's and non-hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated schizophrenic NHL, giant tumor NHL, mantle cell lymphomas, AIDS-related lymphomas, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal vascular hyperplasia associated with nevus, oedema (e.g., brain tumor-related oedema), and meuges syndrome.
118. Use of the chimeric protein or protein complex of any one of claims 92 to 112 in the manufacture of a medicament.
119. Use of the chimeric protein or protein complex of any one of claims 92 to 112 in the treatment of cancer or an autoimmune disease in a subject in need thereof.
120. An Fc-based chimeric protein complex, comprising:
(a) A modified IL-2 signaling agent, wherein the modified IL-2 signaling agent is compared to a peptide having the amino acid sequence of SEQ ID NO:1, which single mutation confers increased safety and reduced affinity or biological activity for IL-2rβ to the modified IL-2 signaling agent relative to the wild-type IL-2 signaling agent; and
(b) One or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest; and
(c) An Fc domain, optionally having one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pairing in the Fc domain, and/or stabilize a hinge region in the Fc domain,
Wherein the single mutation is selected from the group consisting of D20E, D20F, D20G, D H, D20I, D K, D L, D20N, D20V, N88G, N E, N88I, N88K, N Q, N R and N88V, and
wherein the modified IL-2 signaling agent does not comprise a mutation that confers reduced affinity or biological activity to IL-2rα.
121. The Fc-based chimeric protein complex of claim 120, wherein the modified IL-2 signaling agent further comprises a glycosylation mutation, wherein the mutation reduces or eliminates glycosylation of the modified IL-2 signaling agent.
122. The Fc-based chimeric protein complex of claim 121, wherein the glycosylation mutation is a T3 substitution, wherein the mutation is one of T3A, T3F, T3H, T3L, T3V and T3Y.
123. The Fc-based chimeric protein complex of claim 121, wherein the glycosylation mutation is a deletion of the first 3, 4, 5, 6, 7, or 8 residues of the modified IL-2.
124. The Fc-based chimeric protein complex of any one of claims 120 to 123, wherein the one or more mutations in the IL-2 signaling agent imparts a reduced affinity or biological activity that is capable of being recovered by ligation to one or more targeting moieties or upon inclusion in the Fc-based chimeric protein complex.
125. The Fc-based chimeric protein complex of any one of claims 120 to 124, wherein the one or more targeting moiety is directed against a tumor cell, endothelial cell, epithelial cell, mesenchymal cell, tumor stroma or stromal cell and/or ECM.
126. The Fc-based chimeric protein complex of any one of claims 120 to 125, wherein the one or more targeting moiety is directed against immune cells and/or organ cells and/or tissue cells.
127. The Fc-based chimeric protein complex of claim 126, wherein the immune cell is selected from a T cell, B cell, dendritic cell, macrophage, neutrophil, mast cell, monocyte, erythrocyte, bone marrow cell, bone marrow derived suppressor cell, NKT cell, and NK cell or derivative thereof, optionally wherein the immune cell is a T cell and the targeting moiety targets CD8, or optionally wherein the immune cell is a Treg and the targeting moiety targets CTLA4, or optionally wherein the immune cell is an NK cell and the targeting moiety is NKp46.
128. The Fc-based chimeric protein complex of any one of claims 120 to 127, wherein the targeting moiety comprises a recognition domain that is a full-length antibody, a single domain antibody, a heavy chain-only recombinant antibody (VHH), a single chain antibody (scFv), a heavy chain-only shark antibody (VNAR), a trace protein (e.g., a cysteine knot protein, a knottin), darpin, an anti-carrier protein, an adnectin, an aptamer, fv, fab, fab ', F (ab') 2 A peptidomimetic molecule, a natural ligand for a receptor, or a synthetic molecule.
129. The Fc-based chimeric protein complex of any one of claims 120 to 128, wherein the recognition domain is a single domain antibody.
130. The Fc-based chimeric protein complex of any one of claims 120 to 129, wherein theThe recognition domain is V HH Or humanized V HH 。
131. The Fc-based chimeric protein complex of any one of claims 120 to 130, wherein the recognition domain functionally modulates the antigen or receptor of interest.
132. The Fc-based chimeric protein complex of any one of claims 120 to 131, wherein the recognition domain binds to but does not functionally modulate the antigen or receptor of interest.
133. The Fc-based chimeric protein complex of any one of claims 120 to 132, comprising two or more targeting moieties.
134. The Fc-based chimeric protein complex of any one of claims 120 to 133, wherein the targeting moiety binds to one or more of the following targets: CD8, CTLA4, CD3, CD4, DNAM-1, nrp1 (neuropilin), TNFR1, TNFR2, GITR, ICOS, CD20, CD70, clec9a, NKp46, PD-1, PD-L2, SIRP1 alpha, FAP, XCR1, tenascin or ECM protein.
135. The Fc-based chimeric protein complex of any one of claims 120 to 134, further comprising one or more additional modified signaling agents.
136. The Fc-based chimeric protein complex of any one of claims 120 to 135, wherein the chimeric protein comprises two signaling agents or two targeting moieties, or both of the foregoing.
137. The Fc-based chimeric protein complex of any one of claims 120 to 136, wherein the chimeric protein comprises three signaling agents or three targeting moieties, or both.
138. The Fc-based chimeric protein complex of claim 135, wherein the additional modified signaling agent comprises one or more mutations that confer reduced affinity or activity for a receptor relative to an unmutated signaling agent.
139. The Fc-based chimeric protein complex of claim 138, wherein the one or more mutations allow for attenuation of activity.
140. The Fc-based chimeric protein complex of claim 139, wherein agonistic or antagonistic activity is reduced.
141. The Fc-based chimeric protein complex of any one of claims 120 to 140, wherein the chimeric protein is suitable for use in a patient suffering from one or more of the following diseases: cancer, infection, immune disorders, autoimmune diseases, cardiovascular diseases, trauma, ischemia-related diseases, neurodegenerative diseases, and/or metabolic diseases.
142. A recombinant nucleic acid composition encoding one or more of the Fc-based chimeric protein complexes or a constituent polypeptide thereof of any one of claims 120-140.
143. A host cell comprising the recombinant nucleic acid of claim 142.
144. A method for treating cancer, the method comprising: (i) Administering to a patient in need thereof an effective amount of the Fc-based chimeric protein complex of any one of claims 120 to 140; ii) administering to a patient in need thereof an effective amount of the recombinant nucleic acid of claim 142; or iii) administering to a patient in need thereof an effective amount of the host cell of claim 143.
145. The method of claim 144, wherein the cancer is selected from one or more of the following: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal carcinoma; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcomas (e.g., kaposi's sarcoma); skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of urinary system; vulvar cancer; lymphomas, including hodgkin's and non-hodgkin's lymphomas, and B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHL), small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated schizophrenic NHL, giant tumor NHL, mantle cell lymphomas, AIDS-related lymphomas, and waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal vascular hyperplasia associated with nevus, oedema (e.g., brain tumor-related oedema), and meuges syndrome.
146. Use of the Fc-based chimeric protein complex of any one of claims 120 to 140 in the manufacture of a medicament.
147. The use of the Fc-based chimeric protein complex of any one of claims 120 to 140 in the treatment of cancer or an autoimmune disease in a subject in need thereof.
148. The Fc-based chimeric protein complex of any one of claims 120 to 140, wherein the Fc domain is from IgG, igA, igD, igM or IgE.
149. The Fc-based chimeric protein complex of claim 148, wherein the IgG is selected from IgG1, igG2, igG3, or IgG4.
150. The Fc-based chimeric protein complex of any one of claims 120 to 140, wherein the Fc domain is from human IgG, igA, igD, igM or IgE.
151. The Fc-based chimeric protein complex of claim 150, wherein the human IgG is selected from human IgG1, igG2, igG3, or IgG4.
152. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 151, wherein the Fc chain pairing is facilitated by ion pairing and/or knob hole pairing.
153. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 152, wherein the one or more mutations in the Fc domain result in ion pairing between Fc chains in the Fc domain.
154. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 153, wherein the one or more mutations in the Fc domain result in knob-in hole pairing in the Fc domain.
155. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 154, wherein said one or more mutations in said Fc domain result in a reduction or elimination of effector function of said Fc domain.
156. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 155, wherein said Fc-based chimeric protein complex is a heterodimer and has a trans orientation/configuration with respect to any targeting moiety and signaling agent, or any targeting moiety with respect to each other, or any signaling agent with respect to each other.
157. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 156, wherein said Fc-based chimeric protein complex is a heterodimer and has a cis orientation with respect to any targeting moiety and signaling agent, or any signaling agent, with respect to each other.
158. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 157, wherein the Fc comprises L234A, L235A and K322Q substitutions (numbering according to EU) in human IgG 1.
159. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 158, wherein said Fc is a human IgG1 and optionally comprises one or more mutations (numbering according to EU) of L234, L235, K322, D265, P329 and P331.
160. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 159, wherein said Fc-based chimeric protein complex has an orientation and/or configuration of any one of: 1A-1F, 2A-2H, 3A-3H, 4A-4D, 5A-5F, 6A-6J, 7A-7D, 8A-8F, 9A-9J, 10A-10F, 11A-11L, 12A-12L, 13A-13F, 14A-14L, 15A-15L, 16A-16J, 17A-17J, 18A-18F, 19A-19F, 31 and 32.
161. The Fc-based chimeric protein complex of any one of claims 120 to 140 and 148 to 160, wherein said Fc-based chimeric protein complex comprises a polypeptide having a sequence identical to SEQ ID NO: 292. 293, 294, 299, 301, 310, 311, 312, 315, 316, 319, 320, 325, 328, 332, 333, and 335, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical.
162. The chimeric protein of any one of claims 92 to 112 or the Fc-based chimeric protein complex of any one of claims 120 to 140 or 148 to 160, wherein the chimeric protein or Fc-based chimeric protein complex comprises a polypeptide having a nucleotide sequence identical to SEQ ID NO: a polypeptide having an amino acid sequence of any one of 290-449, 478-495, or 501-531 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.
CN202280043198.4A 2021-04-16 2022-04-16 IL-2 based constructs Pending CN117580855A (en)

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