WO2022148816A1 - Inhibition de l'activité des lymphocytes t - Google Patents

Inhibition de l'activité des lymphocytes t Download PDF

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Publication number
WO2022148816A1
WO2022148816A1 PCT/EP2022/050213 EP2022050213W WO2022148816A1 WO 2022148816 A1 WO2022148816 A1 WO 2022148816A1 EP 2022050213 W EP2022050213 W EP 2022050213W WO 2022148816 A1 WO2022148816 A1 WO 2022148816A1
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seq
amino acid
variant
cell
sequence
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PCT/EP2022/050213
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English (en)
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Alex BATEMAN
John Bradley
Jun Wang
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Cambridge Enterprise Limited
European Molecular Biology Laboratory (Embl)
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Publication of WO2022148816A1 publication Critical patent/WO2022148816A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to products and compositions for the inhibition of T cell activity and their use in therapy.
  • Background The immune system distinguishes self from non-self. It protects the host from infections by recognising pathogenic microbes as foreign and deploying mechanisms to attack and destroy them.
  • the immune system is also important for surveillance and removal of cancer cells. In some settings, the immune system results in harm. Autoimmunity results from a failure to distinguish self from non-self. In organ transplantation donor tissue is recognised as foreign and an alloimmune response provokes organ rejection. In graft versus host disease donor cells can attack the host’s tissues. In some infections the host’s immune response to the pathogen results in damage to host tissue.
  • Immunosuppressant drugs are limited by two broad categories of adverse events. Adverse events may relate to suppression of the immune system and may result in an increased risk of infection and increased susceptibility to cancer. Alternatively, adverse events may be unrelated to the immunosuppressant effect, for example through cytotoxicity to non-immune cells or adverse events unrelated to immunosuppression. There is an unmet need for immunosuppressive drugs that target specific immune cells or pathways, with reduced toxicity and other adverse effects.
  • a first aspect of the invention provides an isolated peptide comprising a fragment of SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof.
  • a peptide may comprise a fragment of SEQ ID NO: 15 or SEQ ID NO: 16 or a variant thereof.
  • Some preferred peptides may comprise a thrombin cleavage site of Factor V.
  • a peptide may comprise or consist of one or more of SEQ ID NOs: 3 to 11 or a variant thereof.
  • Other preferred peptides may comprise a fragment of SEQ ID NO: 14 or a variant thereof.
  • a peptide may comprise or consist of any one of SEQ ID NOs: 12 to 13 or a variant thereof.
  • a second aspect of the invention provides a pharmaceutical composition comprising one or more isolated peptides of the first aspect and a pharmaceutically acceptable excipient.
  • a third aspect of the invention provides a method of producing a pharmaceutical composition comprising admixing one or more isolated peptides of the first aspect with a pharmaceutically acceptable excipient.
  • a fourth aspect of the invention provides an isolated peptide of the first aspect for use in the treatment of the human or animal body.
  • a fifth aspect of the invention provides a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof and a pharmaceutically acceptable excipient.
  • a sixth aspect of the invention provides a method of producing a pharmaceutical composition comprising admixing an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof with a pharmaceutically acceptable excipient.
  • a seventh aspect of the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof for use in the treatment of the human or animal body.
  • An eighth aspect of the invention provides a method of treatment of a condition characterised by T cell mediated immune responses comprising administering to an individual in need thereof a peptidyl therapeutic agent that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a fragment of said sequence or a variant of said sequence or fragment.
  • a ninth aspect of the invention provides a peptidyl therapeutic agent that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a fragment of said sequence or a variant of said sequence or fragment for use in the treatment of a condition characterised by T cell mediated immune responses.
  • a tenth aspect of the invention provides the use of a peptidyl therapeutic agent that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a fragment of said sequence, or a variant of said sequence or fragment for the manufacture of a medicament for the treatment of a condition characterised by T cell mediated immune responses.
  • the peptidyl therapeutic agent of the eighth to the tenth aspects may be a peptide or polypeptide that comprises the amino acid sequence of any one of SEQ ID NOs: 3 to 13.
  • the peptidyl therapeutic agent of the eighth to the tenth aspects may be an isolated peptide of the first aspect.
  • Conditions characterised by T cell mediated immune responses in accordance with the eighth to the tenth aspects may include T cell malignancies, such as T cell lymphoma and T cell leukaemia; auto-immune conditions, such as inflammatory bowel disease, rheumatoid arthritis; type 1 diabetes; systemic lupus erythematosus; psoriasis; psoriatic arthritis; and vasculitis; and allo-immune conditions, such as Graft vs Host Disease (GvHD) or transplant rejection.
  • T cell malignancies such as T cell lymphoma and T cell leukaemia
  • auto-immune conditions such as inflammatory bowel disease, rheumatoid arthritis
  • type 1 diabetes systemic lupus erythematosus
  • psoriasis psoriatic arthritis
  • vasculitis vasculitis
  • allo-immune conditions such as Graft vs Host Disease (GvHD)
  • FIG. 2 shows the results of labelling CD4+T conventional cells (Tcon) with carboxyfluorescein diacetate succinimidyl ester (CFSE) (a), and stimulating them with Dynabeads T cell activator (b –i). ).
  • Cells proliferate in response to stimulation, so the dye is diluted and intensity of fluorescence decreases. Decreased fluorescence is therefore indicative of cell proliferation.
  • Medium concentration is close to the physiological plasma level at 20 nM, low concentration is 4 nM, high concentration is 100 nM.
  • Proliferation is shown to be inhibited in a concentration dependent manner by addition of native full length Factor V ( Figure 2 panels c – e), but not by activated factor V, Factor Va ( Figure 2 panels g – i).
  • Figure 3 shows the results of stimulating Tcon cells with Dynabeads T cell activator in the presence or absence of recombinant B domain (Construct 1; Factor V 738 - 1573), recombinant full length Factor V ( construct 2) and mutated Factor V (construct 3; Factor V R709A, R1018A, R1545A).
  • Tcon proliferation was not inhibited by construct 1, and thrombin and hirudin had no effect on their own and in combination with construct 1.
  • Figure 5 shows a diagram of Factor V showing basic region, acidic region, and regions of peptides.
  • Figure 6 shows the results of stimulating CD4 T cells with Dynabeads T cell activator in the presence or absence of peptides derived from Factor V. Peptide sequences are described in Table 1.
  • Figure 7 shows the results of stimulating T cells with Dynabeads T cell activator in the presence or absence of further peptides derived from Factor V. Peptide sequences are described in Table 1.
  • This invention relates to peptides derived from modified full-length factor V (FV) and the use of modified full- length factor V (FV) polypeptides and peptides derived therefrom to inhibit T cell proliferation and/or activation.
  • FV modified full-length factor V
  • FV modified full-length factor V
  • T cell proliferation and/or activation may be inhibited directly and not through the action of regulatory T cells (Tregs).
  • Tregs regulatory T cells
  • the FV polypeptides and peptides described herein may be useful, for example for the treatment of conditions characterised by T cell mediated immune responses.
  • Factor V is an essential cofactor of the blood coagulation cascade. FV circulates in plasma and is activated through proteolytic cleavage by thrombin into the active form (FVa).
  • Human Factor 5 (Gene ID: 2153) may have the amino acid sequence of NP_000121.2 and may be encoded by the nucleotide sequence of NM_000130.5.
  • An isolated peptide described herein may comprise the amino acid sequence of a fragment of a full-length Factor V sequence or a variant of such a fragment.
  • Full-length Factor V sequences may include the amino acid sequences of SEQ ID NOs: 1 and 2.
  • a fragment of a full-length Factor V sequence, such as SEQ ID NO: 1 or SEQ ID NO: 2 is a contiguous sequence of amino acids from the full-length protein sequence that consists of at least one fewer amino acid than the full-length protein sequence i.e. a fragment may contain fewer amino acids than the 2224 amino acids of the full-length Factor V sequence.
  • a fragment may lack a sequence of 10 or more, 20 or more, 50 or more of 100 or more amino acids relative to the full-length Factor V sequence.
  • Preferred fragments may comprise 100 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, or 20 or fewer amino acids.
  • Preferred fragments may comprise 6 or more, 8 or more, 10 or more, 15 or more, 20 or more, 30 or more or 40 or more amino acids.
  • a peptide described herein may comprise the amino acid sequence of a fragment of SEQ ID NO: 15 or 16.
  • a preferred fragment may comprise amino acids corresponding to one or more thrombin cleavage sites of Factor V.
  • a fragment may comprise one or more of positions 709, 1018 or 1545 in the full- length Factor V sequence.
  • the residue at the position in the fragment corresponding to positon 709, 1018 or 1545 is not R and the fragment is not susceptible to cleavage by thrombin.
  • a preferred fragment may comprise amino acids corresponding to the thrombin cleavage site at position 709 of Factor V and/or the thrombin cleavage site at position 1545 of Factor V.
  • a fragment described herein may comprise the amino acid sequence of residues 700 to 718 of human Factor V or a variant thereof.
  • an isolated peptide may comprise the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, a variant of said amino sequence, or a fragment of said amino acid sequence or variant.
  • NRLAAALGIRSFRNSSLNQ (SEQ ID NO: 3)
  • NRLAAALGIKSFRNSSLNQ (SEQ ID NO: 4)
  • a fragment described herein may comprise the amino acid sequence of residues 1009 to 1027 of human Factor V or a variant thereof.
  • an isolated peptide may comprise the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, a variant of said amino sequence, or a fragment of said amino acid sequence or variant.
  • a fragment described herein may comprise the amino acid sequence of residues 1536 to 1555 of human Factor V or a variant thereof.
  • an isolated peptide may comprise the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, a variant of said amino sequence, or a fragment of said amino acid sequence or variant.
  • a fragment described herein may comprise the amino acid sequence of residues 1300 to 1319 of human Factor V or a variant thereof.
  • an isolated peptide may comprise the amino acid sequence of SEQ ID NO: 12, a variant of said amino sequence, or a fragment of said amino acid sequence or variant.
  • GQMPISPDLSHTTLSPDLSH (SEQ ID NO: 12)
  • a fragment described herein may comprise the amino acid sequence of residues 1296 to 1330 of human Factor V or a variant thereof.
  • an isolated peptide may comprise the amino acid sequence of SEQ ID NO: 13, a variant of said amino sequence, or a fragment of said amino acid sequence or variant.
  • SPALGQMPISPDLSHTTLSPDLSHTTLSLDLSQTN SEQ ID NO: 13
  • An isolated peptide described herein may inhibit T cell proliferation and/or activation, such as CD4 T cell and CD8 T cell proliferation and/or activation.
  • the isolated peptide inhibits CD4 T cell proliferation and/or activation.
  • the isolated peptide does not inhibit B cell proliferation.
  • the isolated peptide is thrombin resistant.
  • the peptide may be devoid of thrombin cleavage sites, such that it is not susceptible to proteolytic cleavage by thrombin.
  • an amino acid residue in the peptide corresponding to R709 in human Factor V may be an amino acid residue other than R, preferably an amino acid residue other than R or Q.
  • the amino acid residue corresponding to R709 in human Factor V may be K or A.
  • amino acid residue in the peptide corresponding to R1018 in human Factor V may be an amino acid residue other than R, preferably an amino acid residue other than R or Q.
  • the amino acid residue corresponding to R1018 in human Factor V may be K or A.
  • An amino acid residue in the peptide corresponding to R1545 in human Factor V may be an amino acid residue other than R, preferably an amino acid residue other than R or Q.
  • amino acid residue corresponding to R1545 in human Factor V may be K or A.
  • an isolated peptide may comprise the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or a variant of any of these sequences.
  • NRLAAALGIX 1 SFRNSSLNQ (SEQ ID NO: 9)
  • HTHHAPLSPX 2 TFHPLRSEA (SEQ ID NO: 10)
  • the isolated peptide may also be resistant to proteolytic cleavage by Factor Xa.
  • An amino acid sequence described herein that is a variant of a reference sequence such as a peptide, polypeptide or protein sequence described herein, may have 1 or more amino acid residues altered relative to the reference sequence. For example, 50 or fewer amino acid residues may be altered relative to the reference sequence, preferably 45 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer or 3 or fewer, 2 or 1.
  • a variant described herein may comprise the sequence of a reference sequence with 50 or fewer, 45 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, 3 or fewer, 2 or 1 amino acid residues mutated.
  • An amino acid residue in the reference sequence may be altered or mutated by insertion, deletion or substitution, preferably substitution for a different amino acid residue.
  • Such alterations may be caused by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the encoding nucleic acid.
  • a peptide, polypeptide or protein as described herein that is a variant of a reference sequence, such as an amino acid sequence described above, may share at least 50% sequence identity with the reference amino acid sequence, at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • a variant of a protein described herein may comprise an amino acid sequence that has at least 50% sequence identity with the reference amino acid sequence, at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the reference amino acid sequence, for example one or more of SEQ ID NOS: 1 to 15.
  • GAP Garnier GAP (1990) J. Mol. Biol.215: 405-410
  • FASTA Pearson and Lipman (1988) PNAS USA 85: 2444-2448
  • Smith-Waterman algorithm Smith and Waterman (1981) J.
  • a peptide described herein may further comprise one or more heterologous amino acid sequences additional to the FV fragment.
  • the peptide may further comprise one or more additional domains, which improve stability, pharmacokinetics, targeting, affinity, purification and/or production properties.
  • a peptide described herein may be provided using synthetic or recombinant techniques which are standard in the art.
  • a peptide may be generated wholly or partly by chemical synthesis.
  • a peptide may be synthesised using liquid or solid-phase synthesis methods; in solution; or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • a peptide may be synthesised in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • peptides may be synthesized by Fmoc (N-(9-fluorenyl) methoxycarbonyl) chemistry as C-terminal amides on TentaGel R RAM resin in an automated synthesizer (e.g. Applied Biosystems Pioneer TM ).
  • a peptide described herein may be isolated, in the sense of being free from contaminants, such as other polypeptides and/or cellular components.
  • an isolated peptide described herein may be chemically modified, for example, by addition of one or more polyethylene glycol molecules, sugars, phosphates, and/or other such molecules, where the molecule or molecules are not naturally attached to wild-type FV. Suitable chemical modifications are well known to those of skill in the art.
  • the same type of modification may be present in the same or varying degree at several sites in the peptide.
  • a given peptide may contain multiple different modifications. Modifications can occur anywhere in the peptide sequence, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
  • Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a haem moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation,
  • a protecting group may be coupled to the N- and/or C-terminal end of a peptide to protect the peptide from enzymatic digestion. Suitable protecting groups are well-known in the art.
  • An isolated peptide as described herein may be structurally modified.
  • a structurally modified peptide is substantially similar in both three-dimensional shape and biological activity to a peptide described herein and preferably comprises a spatial arrangement of reactive chemical moieties that closely resembles the three- dimensional arrangement of active groups in the peptide sequence.
  • Examples of structurally modified peptides include pseudo-peptides, semi-peptides and peptoids.
  • An isolated peptide as described herein may be structurally modified to include one or more non-peptidyl bonds, for example pseudopeptide bonds.
  • suitable pseudopeptide bonds are known in the art, including retro-inverso pseudopeptide bonds ("Biologically active retroinverso analogues of thymopentin", Sisto A. et al in Rivier, J. E. and Marshall, G. R. (eds) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), pp.722-773) and Dalpozzo, et al. (1993), Int. J. Peptide Protein Res., 41:561-566), reduced isostere pseudopeptide bonds (Couder, et al. (1993), Int.
  • An isolated peptide comprising pseudopeptide bonds may have an identical amino acid sequence to the sequence described above, except that one or more of the peptide bonds are replaced by a pseudopeptide bond. In some embodiments, the most N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus. Further modifications also can be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
  • An isolated peptide as described herein may be structurally modified to eliminate peptide bonds. Suitable structurally modified peptides include peptoids (Simon, et al., 1992, Proc.
  • N-alkyl group of each glycine residue corresponds to the side chain of a natural amino acid.
  • Some or all of the amino acids of a peptide may be replaced with the N-substituted glycine corresponding to the replaced amino acid.
  • An isolated peptide as described herein may be structurally modified to comprise one or more D-amino acids.
  • a peptide may be an enantiomer in which one or more L-amino acid residues in the amino acid sequence of the peptide is replaced with the corresponding D-amino acid residue or a reverse-D peptide, which is a peptide consisting of D-amino acids arranged in a reverse order as compared to the L- amino acid sequence described above.
  • a reverse-D peptide which is a peptide consisting of D-amino acids arranged in a reverse order as compared to the L- amino acid sequence described above.
  • Glycosylated peptides may be produced for example by expression in recombinant eukaryotic cells
  • a nucleic acid encoding an isolated peptide described herein as described above and a vector comprising such a nucleic acid.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in mammalian cells.
  • a vector may also comprise sequences, such as origins of replication, promoter regions and selectable markers, which allow for its selection, expression and replication in bacterial hosts such as E. coli.
  • Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate.
  • plasmids viral e.g. phage, or phagemid
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds. John Wiley & Sons, 1992.
  • a nucleic acid or vector as described herein may be introduced into a host cell.
  • a recombinant cell comprising a nucleic acid or vector that expresses a peptide as described above.
  • a range of host cells suitable for the production of recombinant peptide are known in the art. Suitable host cells may include prokaryotic cells, in particular bacteria such as Escherichia coli and Lactococcus lactis and eukaryotic cells, including mammalian cells such as CHO and CHO-derived cell lines (Lec cells), HeLa, COS, HEK293 and HEK-EBNA cells, amphibian cells such as Xenopus oocytes, insect cells such as Trichoplusia ni, Sf9 and Sf21 and yeast cells, such as Pichia pastoris.
  • prokaryotic cells in particular bacteria such as Escherichia coli and Lactococcus lactis and eukaryotic cells
  • mammalian cells such as CHO and CHO-derived cell lines (Lec cells), HeLa, COS, HE
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. adenovirus, AAV, lentivirus or vaccinia.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well-known in the art.
  • the introduced nucleic acid may be on an extra-chromosomal vector within the cell or the nucleic acid may be integrated into the genome of the host cell. Integration may be promoted by inclusion of sequences within the nucleic acid or vector which promote recombination with the genome, in accordance with standard techniques.
  • the introduction may be followed by expression of the nucleic acid to produce the encoded peptide.
  • host cells which may include cells actually transformed although more likely the cells will be descendants of the transformed cells
  • expression may require the activation of the inducible promoter.
  • the expressed peptide may be isolated and/or purified, after production.
  • Another aspect of the invention provides a method of producing a peptide described herein comprising expressing a heterologous nucleic acid encoding the peptide in a host cell and optionally isolating and/or purifying the peptide thus produced. After production, the peptide may be investigated further, for example the pharmacological properties and/or activity may be determined. Methods and means of protein analysis are well-known in the art.
  • Heterologous refers to a polypeptide or nucleic acid that is foreign to a particular biological system, such as a host cell, and is not naturally occurring in that system.
  • a heterologous polypeptide or nucleic acid may be introduced to a biological system by artificial means, for example using recombinant techniques.
  • heterologous nucleic acid encoding a polypeptide may be inserted into a suitable expression construct which is in turn used to transform a host cell to produce the polypeptide.
  • a heterologous polypeptide or nucleic acid may be synthetic or artificial or may exist in a different biological system, such as a different species or cell type.
  • a recombinant polypeptide may be expressed from heterologous nucleic acid that has been introduced into a cell by artificial means, for example using recombinant techniques.
  • a recombinant polypeptide may be identical to a polypeptide that is naturally present in the cell or may be different from the polypeptides that are naturally present in that cell.
  • pharmaceutical compositions comprising one or more isolated peptides described herein.
  • a composition may comprise two or more, three or more, four or more or five or more different peptides as described herein.
  • a pharmaceutical composition may comprise, in addition to the peptide itself, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art.
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g., human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below. In some preferred embodiments, parenteral or oral routes of administration may be used.
  • the peptide may be provided in a lyophilised form for reconstitution prior to administration.
  • a lyophilised peptide may be re-constituted in sterile water and mixed with saline prior to administration to an individual.
  • Alternative solvents may be used for peptides that are not water soluble.
  • parenteral for example sub-cutaneous, intra-muscular or intra-venous administration, e.g. by injection, the pharmaceutical composition comprising a peptide described herein may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3’-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or
  • compositions and formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the isolated peptide described herein with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • a peptidyl therapeutic agent that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a fragment of said sequence or a variant of said sequence or fragment may be useful in methods for the treatment of a condition characterised by T cell mediated immune responses.
  • a method of treatment of a condition characterised by T cell proliferation may comprise administering a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a fragment of said sequence or a variant of said sequence or fragment to an individual in need thereof.
  • the peptidyl therapeutic agent may be a polypeptide that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof.
  • the peptidyl therapeutic agent may be an isolated peptide as described above that comprises a fragment of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or a variant thereof, preferably a fragment of the amino acid sequence of SEQ ID NO: 15 or 16, or a variant thereof.
  • the isolated peptide may comprise the amino acid sequence of any one of SEQ ID NOs: 3 to 13. Variants and fragments of reference sequences such as SEQ ID NO: 1 and SEQ ID NO: 2 are described above.
  • the peptidyl therapeutic agent may be a resistant to thrombin.
  • the peptidyl therapeutic agent may lack thrombin cleavage sites.
  • peptidyl therapeutic agent Whilst the peptidyl therapeutic agent may be administered alone, for example for the inhibition of T cell proliferation or the treatment of conditions characterised by T cell immune responses, it will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the peptidyl therapeutic agent. Suitable components are described above.
  • a condition characterised by T cell immune responses may be a disease, illness or medical condition characterised by undesirable, aberrant or pathological adaptive immune responses and/or excessive or aberrant proliferation or activation of CD4 and/or CD8 T cells relative to non-diseased controls.
  • Conditions characterised by T cell immune responses may include auto-immune conditions, such as inflammatory bowel disease, such as Crohn’s disease and ulcerative colitis, rheumatoid arthritis; type 1 diabetes; systemic lupus erythematosus; psoriasis; psoriatic arthritis; vasculitis; Sjögren’s syndrome; eosinophilic gastrointestinal disorders (EGIDs), such as eosinophilic esophagitis; systemic sclerosis; idiopathic pulmonary fibrosis; polymyositis; dermatomyositis; pemphigus; vitiligo; alopecia areata; lupus nephritis; ankylosing spondylitis; Wiskott Aldrich syndrome; and T cell malignancies, such as T cell leukaemia and T cell lymphoma.
  • EGIDs eosinophilic gastrointestinal disorders
  • Conditions characterised by T cell immune responses may also include allo-immune conditions, such as Graft vs Host Disease (GvHD) or transplant rejection. Conditions characterised by T cell immune responses may also include T cell malignancies, such as T cell lymphoma or T cell leukaemia.
  • Treatment may include therapeutic and prophylactic or preventative treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset).
  • the method of treatment may comprise administering a peptidyl therapeutic agent described herein to an individual in need thereof.
  • An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g.
  • a guinea pig, a hamster, a rat, a mouse canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang- utan, gibbon), or a human.
  • the individual is a human.
  • non-human mammals especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g.
  • murine, primate, porcine, canine, or rabbit animals may be employed.
  • Administration is normally in a "therapeutically effective amount” or “prophylactically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of pain in the patient.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners.
  • a composition may be administered alone or in combination with other treatments either simultaneously or sequentially dependent upon the circumstances of the individual to be treated.
  • Treatments that may be administered in combination include but are not limited to immunosuppressants, such as corticosteroids, anti-proliferative agents (e.g. mycophenolate, mofetil, azathioprine, cyclophosphamide), calcineurin inhibitors (e.g. ciclosporin, tacrolimus); mTor inhibitors (e.g. sirolimus), and biological agents targeting specific pathways or cellular components of the immune system.
  • immunosuppressants such as corticosteroids, anti-proliferative agents (e.g. mycophenolate, mofetil, azathioprine, cyclophosphamide), calcineurin inhibitors (e.g. ciclosporin, tacrolimus); mTor inhibitors (e.g. sirolimus), and biological agents targeting specific pathways or cellular components of the immune system.
  • prescription of treatment e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on
  • peptides and polypeptides are well known in the art (Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used.
  • a therapeutically effective amount or suitable dose of a peptide or polypeptide described herein may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known.
  • a peptidyl therapeutic agent described herein may also be useful in in vitro assays, for example to identify patients likely to respond to treatment with the agent.
  • a method may comprise; contacting a peptidyl therapeutic agent that comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a fragment of said sequence or a variant of said sequence or fragment with a T cell and determining the effect of the agent on the T cell.
  • the effect of the agent on the activity, function, marker expression, activation and/or proliferation of the T cell may be determined. Suitable techniques for determining effects on T cells are well known in the art.
  • the T cell may be contained in a sample obtained from an individual, such as a patient with a condition characterised by T cell immune responses. Inhibition of proliferation and/or activation of a T cell in the sample by the agent may be indicative that the individual is likely to respond to treatment.
  • Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term ”consisting essentially of”.
  • nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise. It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise. Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention. All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.
  • Proteinase inhibitor cocktail (Roche Diagnostics Ltd, West Wales, UK). Native human F5, F5a, Thrombin, Dynabeads Human T reg Expander, PCR kits, CYBRgreen kit and CFSE kits (Thermofisher Scientific, Loughborough, UK). CD4+CD25 + CD127 ⁇ T reg, CD8 T cell and B cell isolation kit (Miltenyi Biotec, Surrey, UK). Assay of FV plasma levels was performed using a human Factor V ELISA Kit (ab137976, Abcam, Cambridge, UK). Peptides were synthesised by Cambridge Research Biochemicals Limited, Cleveland, UK. > 90% purity was confirmed by HPLC / Mass Determination by Time of Flight.
  • Mouse monoclonal IgG1 anti-human Factor V light chain, mouse IgG anti-human Factor V heavy chain, and mouse IgG1 anti Factor V B domain were from Haematologic Technologies Inc., VT, USA. Unless otherwise indicated, all reagents were from Sigma-Aldrich Company Ltd (Dorset, UK). Healthy volunteers and patients; Healthy donor blood samples were obtained from the NIHR Cambridge BioResource and leukapheresis samples from the National Health Service Blood and Transfusion services (NHSBT, Cambridge) with written informed consent of donors and approval of the National Research Ethics Committee and Health Research Authority.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs Peripheral blood mononuclear cells
  • RPMI media supplemented with 10% human AB serum.
  • Cells were stained with a surface marker panel for 15 min at room temperature. Cells were then washed and fixed in BD CellFix and analysed promptly using a BD Fortessa flow cytometer (BD Biosciences, Oxford, UK). Neutrophils were isolated from blood by dextran sedimentation and discontinuous Percoll gradients.
  • RNA-Seq. CD4+CD25 + CD127 ⁇ Tregs were isolated using a T reg isolation kit and cell purity was checked with the panel of surface markers described above.
  • Total RNA was collected using RNeasy Plus Micro Kit (Qiagen, Manchester, UK).
  • Sample sequencing libraries were prepared from 250 ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina, Chesterford UK) according to the manufacturer’s instructions. Samples were individually indexed for pooling using a single index strategy.
  • Sequencing was performed using a high output flow cell with 2 ⁇ 75 cycles of sequencing providing 800 M paired end reads from 400 M unique clusters.
  • the number of reads that were generated and used at the starting point of the analysis ranged from 27.5 to 33.5 million for each sample, and the range of sequence length was from 35 to 76 for all the samples.
  • Reads were trimmed using TrimGalore v0.3.7 and mapped using STAR v2.4.0 h.
  • Ensembl Homo_sapiens.GRCh38.dna.primary_assembly reference genome file was used to do the mapping of reads, using the annotated transcripts from the Ensembl Homo_sapiens.GRCh38.80 GTF file.
  • T cell in vitro expansion assay T cons were isolated with the above-mentioned kit. Cells were stained with CFSE following the manufacturer’s instruction.5 ⁇ 104 labelled T cons were mixed with Dynabeads Human activator CD3/CD28 at cell-to-beads ratio of 2 to 1. Cells were collected and analysed using a BD Fortessa flow cytometer after 4 or 5 days.
  • Suppression index was calculated as the ratio between decreased percentage of proliferation and the total percentage of proliferation of the cells.
  • RT-PCR 1 ⁇ g total RNA was amplified with F5 forward (5′- ACCACAATCTACCATTTCAGGACTT -3′); F5 reverse (5′- CGCCTCTGCTCACGAGTTAT -3′) and Foxp3 forward (5′- GCTGCAGCTCTCAACGGT -3′); Foxp3 reverse (5′- GGCAAACATGCGTGTGAAC -3′) using RT-PCR system following manufacture’s instruction ; the PCR product was visualized by 1% low melting temperature agarose gel in Tris-Acetate- EDTA buffer. Immunoblotting.
  • T regs and the CD4 enriched fraction isolated from the kit described above were collected and lysates prepared for SDS polyacrylamide gel electrophoresis and immunoblotting as previously described (Wang et al, 2018. Signals were detected by enhanced chemiluminesence using ECL (Thermo Scienific, Paisley, UK). Images were collected and analysed using Image Lab (Bio-Rad, Hemel Hempsted, UK). Expression of Factor V constructs; Native and mutated Factor V proteins were produced by Peak Proteins, Macclesfield, UK.
  • FV B domain ((aa710-1545), (2) full length FV (aa 1 - 2224) and (3) [R709A, R1018A, R1545A]FV aa1-2224 were sub-cloned into a proprietary vector for the HEK293-6E system. All sequences contained a C-terminal 6His tag to facilitate purification.
  • Cells were transfected at a 500ml scale for each construct, media harvested 5-6 days after transfection and protein purified using a combination of Ni affinity and size exclusion chromatography and if required ion exchange. Purified proteins were analysed by reducing and non-reducing SDS-PAGE, A280 to determine concentration, size exclusion and mass spectrometry to confirm identity. Data analysis.
  • Tcon T conventional (Tcon) cells stimulated by anti-CD3 coated beads.
  • Tcon are incubated with a fluorescent dye ( Figure 2). This effect is highly reproducible and similar to that seen by incubating Tcon with Tregs. The effect is not seen if factor V is activated.
  • Thrombin did not increase proliferation in the absence of Factor V.
  • a cleavage resistant recombinant Factor V was found to be a potent inhibitor of CD4 cell proliferation.
  • recombinant full-length Factor V and a cleavage resistant recombinant Factor V were shown to suppress CD8 cell proliferation, but not B cell proliferation ( Figure 4).
  • Peptides spanning the cleavage sites of Factor V, and peptides derived from the repetitive sequences of the B domain were generated. The specific peptides are shown in Table 1.
  • Peptide 1 and Peptide 2 spanning the first cleavage site of Factor V suppressed T cell proliferation in three of three experiments, with Peptide 2 having a more marked effect in two of the experiments.
  • Peptides 4 and 6 also suppressed T cell proliferation in some experiments, but the effect was less marked.
  • Peptides 7 and 8 were derived from repetitive sequences in the B domain that are enriched with SPDLS and SPELS motifs. These peptides also suppressed T cell proliferation, with the longer peptide having a more marked effect ( Figure 7).
  • Peptide 1 (P1; SEQ ID NO: 3) – aa 700 to aa 718 NRLAAALGIRSFRNSSLNQ – sequence spanning Arg709 Peptide 2 (P2; SEQ ID NO: 4) – aa 700 to aa 718 NRLAAALGIKSFRNSSLNQ – Peptide 1 with Arg709 replaced by Lys Peptide 3 (P3; SEQ ID NO: 5) – aa 1009 to aa 1027 HTHHAPLSPRTFHPLRSEA – sequence spanning Arg1018 Peptide 4 (P4; SEQ ID NO: 6) – aa 1009 to aa 1027 HTHHAPLSPKTFHPLRSEA – Peptide 3 with Arg1018 replaced by Lys Peptide 5 (P5; SEQ ID NO: 7) – aa 1536 to aa 1555 PDNIAAWYLRSNNGNRRNY – sequence spanning Arg1545 Peptide 6 (P
  • SEQ ID NO: 10 PDNIAAWYLX 3 SNNGNRRNY where X 3 is any residue other than R, preferably A or K.
  • SEQ ID NO: 11 GQMPISPDLSHTTLSPDLSH
  • SEQ ID NO: 12 SPALGQMPISPDLSHTTLSPDLSHTTLSLDLSQTN SEQ ID NO: 13 SPELSEMLEYDRSHKSFPTDISQMSPSSEHEVWQTVISPDLSQVTLSPELSQTNLSPDLSHTTLSPELIQRNLS PALGQMPISPDLSHTTLSPDLSHTTLSLDLSQTNLSPELSQTNLSPALGQMPLSPDLSHTTLSLDFSQTNLSPE LSHMTLSPELSQTNLSPALGQMPISPDLSHTTLSLDFSQTNLSPELSQTNLSPALGQMPLSPDPSHTTLSLDLS QTNLSPELSQTNLSPDLSEMPLFADLSQIPLTPDLDQMTLSPDLGETDLSPNFGQMSLSPDLS SEQ ID NO:14 NRLAA

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Abstract

La présente invention concerne l'inhibition de la prolifération et/ou de l'activation de lymphocytes T par la protéine du facteur V (FV) pleine longueur et des peptides dérivés de la protéine FV pleine longueur. Ceci peut être utilisé pour exercer un effet immunosuppresseur en thérapie. L'Invention concerne des peptides et des protéines FV et leur utilisation dans le traitement de troubles auto-immuns et d'autres états caractérisés par des réponses immunitaires à médiation par les lymphocytes T.
PCT/EP2022/050213 2021-01-07 2022-01-06 Inhibition de l'activité des lymphocytes t WO2022148816A1 (fr)

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Citations (2)

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WO2016041616A1 (fr) * 2014-09-17 2016-03-24 Merck Patent Gmbh Méthode de traitement de maladies provoquées par des métastases osseuses, médicaments à cet effet, et méthode de prédiction du résultat clinique du traitement de maladies provoquées par des métastases osseuses
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