AU2020299636A1 - Compositions and methods of use - Google Patents
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- AU2020299636A1 AU2020299636A1 AU2020299636A AU2020299636A AU2020299636A1 AU 2020299636 A1 AU2020299636 A1 AU 2020299636A1 AU 2020299636 A AU2020299636 A AU 2020299636A AU 2020299636 A AU2020299636 A AU 2020299636A AU 2020299636 A1 AU2020299636 A1 AU 2020299636A1
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Abstract
The disclosure relates to peptides that can interact with RIP homotypic interaction motif (RHIM) proteins/peptides. These peptides also contain a RHIM motif which has an evolutionary conserved core tetrad sequence (V/I)-Q-(V/I/L/C)-G found in RIPK1, RIPK3 and their orthologues, such as DAI and TRIP. The peptide-peptide RHIM motif interaction causes multimerisation, amyloid formation and activation of the NLRP3 inflammasome. This in turn causes an artificial or temporary induction of inflammatory cell death, enhances the level of inflammatory mediators, such as IL-Ιβ, and results in an increase of inflammatory cell infiltrate, such as CD8+ T cells, into the tissue space. The therapeutic application is for tumour suppression, pathogenic regression and enhancing the immune response to vaccination.
Description
COMPOSITIONS AND METHODS OF USE
FIELD OF DISCLOSURE
This disclosure relates to the technical fields of molecular immune signalling platforms, inflammation immunity, cancer, cell death, molecular biology and medicine. In particular, the disclosure relates to agents and methods using agents which artificially or temporarily induce inflammatory cell death and enhance the level of inflammatory mediators, such as IL-Ib, in a subject sufficient to increases inflammatory cell infiltrate, such as CD8+ T cells, into the tissue space, which can be applied therapeutically where inflammatory-mediated tissue infiltrate could be of use, such as tumour suppression, pathogenic regression and vaccination.
BACKGROUND ART
The reference in this specification to any prior publication, or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Bibliographic details of documents referred to are listed at the end of the specification.
Hallmark inflammatory cell death pathways include necroptosis and pyroptosis, which have been predominantly investigated within the innate immunology field, where myeloid cells are the point of origin. Pro-inflammatory signaling cascades in macrophages can be propagated through supramolecular organizing centers (SMOCs) which are oligomeric protein complexes, usually activated downstream of pathways that sense pathogen, damage or homeostasis associated molecular patterns (PAMPs, DAMPs and HAMPs). SMOCs can recruit effector molecules and initiate inflammatory cell death. Some examples of SMOC scaffolds include the necrosome, and inflammasome. Interestingly, these scaffolds can be induced downstream of complexes containing a Rip homotypic interaction motif (RHIM), a motif that can form a fibrilar b sheet scaffold structure called an amyloid (Mompean, 2018 ;Lawlor, 2015);
The formation of amyloids intracellularly initiates a variety of biophysical phenomena leading to inflammation. Mechanistically this is thought to be mediated through the act of amyloid fibril formation, rather than the presence of the final amyloid scaffold itself (Nag, 2013; Last, 2011; Flach, 2012; Harper, 1997; Walsh, 1997; Winner, 2011; Abedini, 2016). In the context of pathological amyloid conditions it seems that native proteins required for cellular hemostasis (e.g. Tau, Amyloid-b, Islet amyloid
polypeptide (IAPP), etc) appear to be processed into a thermodynamically unfavorable unfolded intermediate. This exposes a sequence that induces protein nucleation to a more energetically favourable fibril which resembles the structural studies observed in RHIM formation (Harper, 1997; Walsh, 1997; Nag, 2013; Winner, 2011; Abedini, 2016.) Although this self-nucleation appears to be rapid, the oligomers or smaller soluble sub units appear to be responsible for disease progression and toxicity (Harper, 1997; Walsh, 1997; Nag, 2013; Winner, 2011; Abedini, 2016). Mechanistically, this could be a result of soluble fibril association with cellular membrane components, either the cell membrane itself or organelles such as mitochondria or lysosomes (Magzoub, 2012; Williamson, 2009; Patil, 2009; Hebda, 2009). Membrane association and fibril formation are reported to cause lipids to be ripped from the membrane surface during fibril polymerization, initiating pore-formation and ionic flux (Hebda, 2009; Last, 2011; Patel, 2015). This can trigger downstream inflammatory processes, including SMOCs such as the nod-like receptor protein 3 (NLRP3) inflammasome, which can be activated through the cellular efflux of cytoplasmic potassium ions (Masters, 2010; Niemi, 2011; Halle, 2008).
The NLRP3 inflammasome senses perturbances to cellular homeostasis, including cases where there is a deleterious accumulation of misfolded non-degradable proteins such as amyloids, or crystal structures (Hornung, 2008) or upon engagement of necroptotic (Conos, 2017; Gutierrez, 2017) and pyroptotic signalling (Kayagaki, 2015; Ding, 2016). IL-Ib release into the extracellular space is a hallmark of NLRP3 activation, facilitating immune cell infiltration into the tissue microenvironment. This cytokine is cleaved and released from cells by cytoplasmic NLRP3 binding to the adaptor protein ASC, and consequent ASC driven oligomerization and activation of Caspase-1 (CASP1). In the context of amyloids, including RHIM proteins, the release of IL-Ib downstream of NLRP3 activation has been clearly demonstrated (Masters, 2010; Halle, 2008; Niemi, 2011; Lawlor, 2015). Indeed it has been shown that the release of IL-Ib initiates can increase inflammatory disease progression.
RHIM containing proteins are known inducers of SMOCs, which contribute to amyloid scaffold formation (Mompean, 2018), cytokine production (Dondelinger, 2015) and inflammatory cell death. The most commonly studied RHIM containing proteins are the serine/threonine receptor interacting protein kinases (RIPK) 1 and 3. Upon activation, RIPK1 and RIPK3 independently or together co-polymerize into filamentous b-amyloid structures, through their consensus sequences IQIG and VQVG, with a unique Ser (RIPK1) and Cys (RIPK3) ladder described by Sun et al. JBC (2002) 227:9505-9511; Li et al. Cell (2012); 150(2):339-350 and Mompean, 2018. This b-sheet like scaffold
structure resembles traditional fibril amyloids, which confers insolubility and proteolytic resistance. Beyond the traditional and well-studied RIPK1 and RIPK3 RHIM domain proteins, mammalian TIR-domain-containing adaptor-inducing IFNp (TRIF), and mammalian DNA-induced activator of IFN (DAI, also known as DLM and ZBP1), as well as murine cytomegalovirus M45, and herpes simplex virus ICP6 and ICP10 have similar domain units comprised of about 60 amino acids with a core tetrad sequence (V/I)-Q-(V/I/L/C)-G. Interestingly, the stable formation of RHIM SMOC scaffold-like complexes (in any combination of the aforementioned proteins) are evolutionarily conserved and have been demonstrated both in mammals and in drosophila to trigger inflammatory cell death and cytokine production. Death and cytokine release is linked in drosophila to the IMD complex demonstrated by Kleino et al, Immunity 47, 635-647, 2017. In mammals the RHIM SMOC triggers i) the IKK complex, which induces NF-KB commencing inflammatory cytokine production (Dondelinger, 2015), ii) phosphorlyation of mixed lineage kinase domain like pseudokinase (MLKL) to cause necroptosis in mammals, and iii) caspase-8, resulting in NLRP3 activation of CASP1 (Lawlor, 2015).
RHIM containing proteins, such as RIPKl and RIPK3, activate the NLRP3 inflammasome to induce the cleavage and release of IL-Ib (Weng, 2014). In general, IL-Ib has been thought to play an important role in acute inflammation, pathogenic resolution and adaptive anti -tumour responses (Ben-Sasson, 2013). Indeed, intratumoral injection of IL-Ib in established malignancies can induce tumour regression, mediated by CD8 T-cell expansion (North, 1988), which follows similar pathogenic pathways. In fact, tumour regression by Thl cells in the context of solid tumours appears to require IL-Ib secretion (Haabeth, 2016). This may suggest that antigen presentation is required for T-cell infiltration, and that IL-Ib is required for antigen specific Thl cell expansion. Induction of inflammatory events (e.g. increased IL-Ib secretion) with checkpoint inhibition, vaccination, and/or engineered T-cells could induce significant benefit therapeutically broadly in tumour and pathogenic disorders.
There is provided new immuno-inflammatory agents for use in therapeutic and prophylactic applications.
SUMMARY OF THE DISCLOSURE
The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein, the term about, unless stated to the contrary, refers to +/- 10%, or +/- 5%, of the designated value.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
As used herein, the singular form "a", "an" and "the" include singular and plural references unless the context indicates otherwise.
In one aspect, the present application provides RIP homotypic interaction motifs (RHIM) interacting agents and methods using same that are able to effect in a subject an artificial lytic innate immune-inflammatory response (LIIIR) or a pro-inflammatory network sufficient, for example, to increases inflammatory cell infiltrate into tissues and to stimulate cytokine or growth factor sensitive tumour suppression in a triple negative breast cancer model. The RHIM interacting agents agonise or trigger an endogenous LIIIR pathway, or a number of different response pathways depending upon the cell contacted, sufficient to stimulate, for example, pro-inflammatory factor-sensitive tumour suppression in a triple negative breast cancer model. Accordingly, in one non-limiting embodiment, the RHIM interacting agents are particularly useful in treating subjects with a cancer or a hyperproliferative disease or the propensity to develop cancer or a hyperproliferative disease.
In one embodiment, the present application provides a method of inducing an artificial LIIIR, such as artificial inflammasome activation (e.g., NLRP3 inflammasome activation) in a cell or subject. In one embodiment, the method comprises administering to the cell or subject an effective amount of a composition comprising a RHIM- interacting agent, or an encoding nucleic acid, and optionally an agent that modulates cellular delivery. In one embodiment, the RHIM-interacting agent within one or more cells takes part in amyloid formation and induces a LIIIR.
The non-naturally occurring RHIM-interacting agents or compositions comprising same are modelled or selected to interact with the RHIM of endogenous RHIM containing polypeptides, including without limitation RIPK1, RIPK3, MCMV M45, DAI, dIMD, TRIF and homologues and orthologs thereof.
The term "RHIM" refers to a conserved region or domain of between 4 and 60 amino acids within RIPK1 and RIPK3 polypeptides that are important for mediating the binding between RIPK1 and RIPK3 and the formation of amyloid fibrils. The term RIP is an acronym for a receptor-interacting protein, now called RIPKl and RIPK3. A
consensus RHIM sequence determined from all murine containing RHIM sequences has the amino acid sequence TEIYNSSGVQIGNYNYM (SEQ ID NO: 39).
Thus, a functional RHIM is crucial for binding other RHIM domain containing proteins and inducing amyloid formation therewith. A sequence alignment of RIPK1 and RIPK3 orthologs identifies absolutely conserved core residues (IQIGXXN SEQ ID NO: 40) which have been shown to be necessary for amyloid formation and further conserved core-flanking residues (XXIXN(Xa)XXXEVQEVGXXNXM/L SEQ ID NO: 41) that are of secondary importance in RHIM-RHIM binding. The presence of all the conserved residues in a RHIM-interacting peptide is proposed to promote the potency of amyloid formation. Further flanking regions of the RHIM are proposed to mediate specificity of binding and thus may be employed where high specificity between the RHIM interacting agent and a specific RHIM containing polypeptide is desirable.
Peptide fragments of RIPK1 or RIPK3 comprising the core sequence of the RHIM, IQIG (SEQ ID NO: 42) or VQVG (SEQ ID NO: 43) are able to form amyloid in vitro while the longer evolutionarily conserved RHIM sequence has been described herein as XXIXN(Xa)XXXEVQEVGXXNXM/L (SEQ ID NO: 41) wherein Xa is present or absent and wherein X is an amino acid, or in one embodiment, an amino acid selected from the corresponding position in a RIPK1, RIPK3, DAI, TRIF or orthologous RHIM motif. Further flanking amino acids may be selected from RIPK1, RIPK3, DAI (encompassing DAI-1, 2, or 3 as appropriate) TRIF or orthologous motifs.
In one embodiment, the RHIM interacting agent is a peptide, typically a non- naturally occurring peptide, which within one or more cells takes part in amyloid formation and induces a LIIIR. In one embodiment, the peptide is bound or attached to a moiety that facilitates intracellular delivery. In other embodiments, the peptide is co administered with an agent that facilitates intracellular delivery. Similarly, when the peptide is administered as a nucleic acid for expression within a cell, the nucleic acid may be modified or introduced within a vector to facilitate intracellular delivery or co administered with an agent that facilitates intracellular delivery. Delivery specifically to target cells using targeting agents such as antibodies or antigen binding fragments is further contemplated using art recognised technologies.
In one embodiment, the RHIM-interacting agent interacts with a RHIM of one or more endogenous RHIM-containing proteins such as RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides, within one or more target cells, to induce a LIIIR comprising amyloid formation, cell death and inflammasome activation.
In one embodiment, the RHIM-interacting agent is a soluble peptide in monomeric form.
For illustrative purposes and without being bound by any particular mechanism of action it is proposed that the RHIM interacting agent forms amyloid with the one or more native intracellular proteins comprising a RHIM, such as RIPK1, and induces a form of necroptotic inflammatory cell death which may also facilitate active T-cell recruitment. Furthermore, within cells able to form inflammasomes, such as without limitation macrophages or certain tumour cells, amyloid formation induced by the RHIM interacting agents induces pyroptotic inflammatory cell death involving the release of inflammatory mediators such as cytokines including in particular, IL-Ib and IL-18.
The development of RHIM-interacting agents that can act intracellularly to form amyloid signalling complexes that artificially induce pyroptosis as well as necroptosis and act as therapeutic inflammatory mediators in cancer therapy and pathogenic disorders has wide ranging implications and applications in the field of medicine where the importance of being able to modulate or induce desirable immune responses, not limited to cancer treatment, is emerging.
The application discloses and enables the use a range of RHIM interacting agents based upon the initial finding that linear RHIM containing peptides comprising between about 15 and about 40 or 60 continuous amino acids within the approximately 60 amino acid RHIM can be transfected into cells in the presence of a transfection aid such as lipofectamine. Also, that a linear RHIM containing peptide conjugated to a cell penetrating peptide can also be taken up by cells and be effective in inducing amyloid formation by interacting with endogenous RHIM containing polypeptides and a LIIIR. A number of peptide modifications are known in the art to stabilise peptides against serum proteases and to enhance intracellular penetration and these are encompassed. Some such modified peptides can be expressed from nucleic acids in a cell, others are manufactured synthetically. Similarly, where it is desirable to target the RHIM interacting agent to one or more specific cell types, this may be achieved either by ex vivo manipulation of target cells, or incorporation of targeting moieties able to bind specifically to target cells, as known in the art.
In one embodiment, at least one cell is a myeloid cell of the innate immune system.
In one embodiment, the cell is a virally infected cell.
In one embodiment, administration of RHIM-interacting agents is systemic.
In one embodiment, the LIIIR comprises inflammasome activation and pro- inflammatory cytokine release.
In one embodiment, the LIIIR comprises NLRP3 inflammasome activation and IL-Ib release in the subject.
In one embodiment, the LIIIR facilitates reduced tumour growth, or reduced pathology in a tissue of the subject. In one embodiment, the LIIIR facilitates an enhanced immune response. In one embodiment the LIIIR facilitates enhanced vaccination efficiency in the subject when administered in conjunction with an antigen.
In one embodiment, the RHIM-interacting agent comprises a RHIM-containing peptide of 7-60 or 8-40 amino acids in length, comprising the conserved RHIM sequence XXIXN(Xa)XXXI/VQI/V GXXNXM/L (SEQ ID NO: 41) wherein Xa is present or absent and wherein X is an amino acid, or an amino acids selected from the corresponding position in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptide, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 conservative amino acid substitutions thereof. The non-X amino acid residues are essential or semi-essential amino acids whose presence either as naturally occurring or in unnatural forms is involved with RHIM binding and amyloid formation or changing the potency or specificity of the interaction. In one embodiment, for RIPK1 and RIPK3 based RHIM interacting peptides, one or more semi essential amino acid residues (non-X residues in XXIXN(Xa)XXXEVQEVGXXNXM/L(SEQ ID NO: 41)) may be absent provided the core residues (EVQEVGXXN(SEQ ID NO: 44)) are retained.
For example, the first X in human and mouse RIPK1 RHIM sequence comprising the above XXIXN(Xa)XXXEVQEVGXXNXM/L is Tyrosine (Tyr,Y) but Cysteine (Cys, C) is present in other orthologs. Thus, a conservative substitution of the RIPK1 based RHIM interacting agent at this position is an amino acid with a hydrophobic or polar/neutral side chain. A preferred substitution for Tyr is Phe. A preferred substation for Cys is Ser.
In one embodiment, the RHIM-interacting agent comprises a RHIM-containing peptide of 7-60 or 8-40 amino acids in length, comprising the conserved RHIM sequence XXIXN(Xa)XXXIQIGXXNXM/L wherein Xa is present or absent and wherein X is an amino acid, such as alanine, or an amino acids selected from the corresponding position in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptide, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, or 9 conservative amino acid substitutions thereof.
A "conservative amino acid substitution" is one in which the naturally or non- naturally occurring amino acid residue is replaced with a naturally or non-naturally occurring amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., Lys, Arg, His), acidic side chains (e.g., Asp, Glu), uncharged polar side chains (e.g., Gly, Asn, Gin, Ser, Thr, Tyr, Cys), nonpolar side chains (e.g., Ala, Val, Leu, He, Pro, Phe, Met, Trp), beta-branched side chains (e.g., Thr,
Val, lie) and aromatic side chains (e.g., Phe, Trp, His). Thus, a predicted nonessential amino acid residue in a RHIM, for example, may be replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine). A full amino acid sub-classification is set out in Table 4 and exemplary substitutions are set out in Table 5.
These adaptor proteins (intracellular proteins that organize downstream signalling cascades leading to a specific cellular response after exposure to a given pathogen) or their mimics in invading organisms are activated by the present RHIM interacting agents which are therefore agonists of adaptor protein activity. The administered RHIM interacting agents described herein are not full length RHIM containing proteins, that is they are based on the RHIM and its ability to induce amyloid formation by interacting with endogenous full length naturally occurring RHIM containing proteins. Specifically, for example RHIM interacting agents or constructs administered in accordance with the description do not comprise a death domain (RIP), or do not comprise a kinase (RIP), TRAF-binding (TRIF) or IAP (IMD) binding domain or Zbeta domain (DAI),
RHIM interacting peptides may comprise modifications known to modify the pharmacokinetic features of peptides, such as by increasing protease resistance in vivo and/or to increase cell permeability, e.g., stapled peptides, peptidomimetics.
In one embodiment, the peptide comprises one or more of a linker or spacer, a modified or non-natural or non-proteogenic amino acid, a modified side-chain, a modified backbone, terminal modified groups or comprises a modified spatial constraint or is a D-retro-inverso peptide.
In one embodiment, the peptide is a pseudopeptide, peptoid, azapeptide, cyclized, stapled, ether or lactam peptide or comprises a spatial constraint.
In one embodiment, the peptide is conjugated or otherwise attached/bound to a lipid, carbohydrate, polymer, protein, nanoparticle, peptide, antibody or fragment or antigen binding form thereof, aptamer, or nucleic acid.
In one embodiment, peptides include pharmaceutically acceptable salts, hydrates, sterioisomers, and pro-drugs.
In one embodiment, RHIM interacting agent comprises following structure I, N' to C:
ZlWaXl X2 X3 X4 X5 X6 X7 X8 X9 X10 XI 1 X12 X13 X14 X15 X16 X17
WbZ2
XI is present or absent and where present is any amino acid, or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position typically a residue with a hydrophobic or polar/neutral side chain
X2 is present or absent and where present is any amino acid or is an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position, typically polar or a hydrophobic residue
X3 is Isoleucine (I) or a hydrophobic residue such as Leucine or Norleucine X4 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position
X5 is Asparagine (N)
X6 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position
X7 is any amino acid
X8 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position, such as a Glycine (G) or Proline (P) or Alanine (A)
X9 is Isoleucine (I) or Valine (V)
XI 0 is Glutamine (Q)
XI I is Isoleucine (I) or Valine (V) or Leucine (L)
XI 2 is Glycine (G)
X13 is any amino acid or an amino acid corresponding to a RIPKl, RIPK3, DAI, TRIF or orthologous polypeptides at this position
X14 is any amino acid or an amino acid corresponding to a RIPKl, RIPK3, DAI, TRIF or orthologous polypeptides at this position
XI 5 is Asparagine (N)
XI 6 is present or absent and where present is any amino acid typically polar such as Tyrosine (Y)
XI 7 is present or absent and where present is a methionine (M) or Leucine (L) or Isoleucine (I);
Wa and Wb are present or absent and where present is each 5 to 20 contiguous amino acids that immediately flank XI to X17, where present, in a RIPKl, RIPK3, DAI, TRIF or orthologous polypeptides, or a conservative substitution thereof;
Z1 and Z2 are individually present or absent and where present are individually selected from one or more of a linker, stability enhancing, delivery enhancing or label moiety;
or a pharmaceutically acceptable salt, hydrate, tautomer, sterioisomer, pro-drug thereof.
In one embodiment, X is a "non-essential" amino acid residue meaning a residue that can be altered from the wild-type sequence of a polypeptide (e.g.,a RHIM of RIPK1) without abolishing or substantially altering its ability to bind to an endogenous RHIM containing polypeptide such as RIPK1 or RIPK3 and form amyloid.
In one embodiment, the RHIM interacting agent comprises or encodes a peptide comprising or consisting or consisting essentially of the amino acid sequence set out in SEQ ID NO: 1 to 5 (murine RHIM interacting peptide sequences) or 21 to 26 (human RHIM interacting peptide sequences). Illustrative peptides comprising a TAT cell pepntration sequence and a triglycine spacer have the sequences set forth in SEQ ID Nos: 7 to 11.
In one embodiment, the RHIM interacting agent comprises or encodes a peptide comprising or consisting or consisting essentially of the amino acid sequence set out in one of SEQ ID NO: 1 to 5 or 21 to 26 or 7 to 11 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. In one embodiment, the RHIM interacting agent having a small number of substituted or deleted residues retains the structure of structure I.
In one embodiment, the RHIM interacting agent comprises or encodes an amino acid sequence having 1, 2, 3, 4, 5 or 6 conservative or non-conservative amino acid substitution, deletion or addition to the sequence of structure I, provided the core conserved RHIM is maintained, XXIXN(Xa)XXXEVQEVGXXNXM/L wherein Xa is present or absent and wherein X is an amino acid.
In one embodiment, the RHIM interacting agent comprises a nucleic acid molecule from which the RHIM-interacting peptide is expressible.
Examples of suitable polynucleotide sequences are set forth in SEQ ID NO: 13 to 20 and 27 to 38. In certain embodiments, the present disclosure provides polynucleotides comprising nucleic acid sequences comprising a coding sequence for an encodable RHIM peptide or an encodable RHIM interacting agent. For example, in some embodiments, nucleic acids of the disclosure comprise, consists essentially of, or consists of a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 13 to 20 and 27 to 38.
In one embodiment, the nucleic acid molecule is an RNA or DNA or RNA:DNA or a chemically modified form thereof.
In one embodiment, a proportion of at least one type of nucleotide (e.g„ cysteine and/or uracil), is chemically modified to increase its stability in vivo.
In one embodiment, the nucleic acid is in the form of a viral or non-viral vector.
In one embodiment, the RHIM interacting agent is administered to cells ex vivo, such a transfection of cells with a RHIM-containing peptide with a lipofectamine or shuttle peptide or another transfection aid. The present invention encompasses the use of genetically modified cell depots (e.g. CAR T-cells, TCRs, genetically modified macrophage, etc).
In one embodiment, the RHIM interacting agent comprises an antibody or antibody fragment that targets the agent specifically to target cells, such as to myeloid cells such as macrophages, or, for example, targets the agent to tumor associated myeloid cells.
In one embodiment, the present application provides a pharmaceutical or physiological composition comprising a RHIM interacting agent as defined herein above.
In one embodiment, the present application provides a composition comprising a RHIM interacting agent as defined herein for use or when used in stimulating an artificial LIIIR, or specifically for stimulating NLRP inflammasome activation and release of IL- 1b, from a cell or in a subject.
The application enables a method of treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a composition comprising a RHIM interacting agent sufficient to induce intracellular amyloid formation and a LIIIR.
In one embodiment, the cancer is an IL-Ib sensitive cancer. In one embodiment, the RHIM interacting agent is administered systemically, or to the immune system as well as to the tumor.
In one embodiment a RHIM interacting agent composition is provided comprising, a peptide of less than 80 amino acids comprising a RHIM of an adaptor protein such as an RIPK1, RIPK3, DAI, TRIF or orthologous polypeptide or an encoding nucleic acid and optionally a heterologous delivery moiety such as a linker, cell penetrating or shuttle peptide or lipid nanoparticle or vector.
BRIEF DESCRIPTION OF THE FIGURES
The patent or application file contains at least one drawing executed in colour. Copies of this patent or patent application publication with colour drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.
The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention.
Figure 1 A-F RHIM peptide sequences form homo- and hetero- amyloid fibrils with endogenous counterparts. A. Evolutionarily conserved RHIM peptide sequences (mouse RIPK3 and RIPK1, were connected via a tri-lysine spacer to a Trans- Activator of Transcription (TAT) sequence from HIV. Consensus sequence determined from all murine containing RHIM sequences. An alanine substituted peptide was synthesized as a control. B. The two RIPK RHIM sequences were synthesized and analysed 24 hours post salt exposure via ThT fluorescence for homo- and hetero-amlyoid. C. RIPK1, RIPK3 and alanine substituted peptides were analysed for amyloid formation in a concentration and time dependent fashion. D. Confocal z-projection images of amyloid formation in BMDMs 30 minutes post-treatment with 25 mM RHIM peptide measured via ThT fluorescence, scale bar 28 pm. E. Fluorescence quantification of panel D via ImageJ. F. Amyloid formation measured via ThT fluorescence: RIPK1 peptide, RIPKl protein, RIPKl peptide incubated with a full length RIPKl protein, and saline. Data are representative of 3 independent experiments.
Figure 2 A-G RHIM eptides trigger the NLRP3 inflammasome. A. Immortalized BMDMs carrying an NF-KB reporter were incubated for 4 hours with 25 pM Rl, R3, A, Saline and 100 ng/mL LPS and NF-KB measured by flow cytometry (see methods). B. Secreted cell supernatant IL-6 was measured 24 hours from LPS, IL-4 and unprimed WT BMDMs, following incubation with 25 pM Rl, R3, A, or Saline. C. Secreted IL-Ib was measured from LPS primed WT BMDMs following a 24 hour incubation with various concentrations of Rl, R3, A, Saline. D. Secreted cell supernatant IL-Ib was measured from unprimed and LPS primed WT BMDMs following 24 hour incubation with various concentrations of Rl and Saline. E. Secreted IL-Ib was measured from LPS primed WT, Nlrp3-/-, Caspl-/- BMDMs following 24 hour incubation with 25 pM Rl, R3, A, Saline or 10 pM Nigericin. F. Secreted IL-Ib was measured from LPS primed WT BMDMs in media with and without IDUN. G. Secreted IL-Ib was measured from LPS primed WT BMDMs in media with and without 30 mM extracellular potassium, following a 24 hour incubation with 25 pM Rl, R3, Saline or 10 pM Nigericin. Data are representative of 3 independent experiments Means ± SD, replicate assays. * p<0.05, ** p<0.01, ***p<0.001, **** p< 0001. As determined by a two-way ANOVA.
Figure 3 A-J Lysosome rupture and pyroptosis both contribute to RHIM peptide induced cell death. A. WT BMDMs were incubated with various concentrations of Rl, R3 and A peptides. Incorporation of PI was analysed via flow cytometry. B. BMDMs
from WT and Nlrp3-/- mice were incubated for 24 hours with 25 uM Rl, R3, and A peptides. Incorporation of PI was analysed via flow cytometry C. BMDMs from WT and Caspl-/- mice were incubated for 24 hours with 25 uM Rl, R3, and A peptides. Incorporation of PI was analysed via flow cytometry D. BMDMs from WT and Gsdmd- /- mice were incubated for 24 hours with 25 uM Rl, R3, and A peptides. Incorporation of PI was analysed via flow cytometry E. Immortailized BMDMs from WT mice were incubated for 24 hours with 25 mM Rl, LPS, and extracellular potassium. Incorporation of PI was analysed via flow cytometry. F. Confocal images of WT BMDMs containing lysotracker following incubation with 25 pM RIPKl peptides. G. Quantification of confocal analysis performed in F at different time points. H. Immortalized BMDMs from WT, Ripkl-/- mice were incubated for 24 hours with and without 25 pM of Rl, R3, and A peptides. Incorporation of PI was analysed via flow cytometry I. IBMDMs from WT, Ripk3-/- mice were incubated for 24 hours with and without various concentrations of Rl, R3, and A peptides. Incorporation of PI was analysed via flow cytometry J. Analysis of Rl, R3 and A peptide induced lysis of human erythrocytes isolated from whole blood at various pHs. Data are representative of 3 independent experiments Means ± SD, replicate assays. * p<0.05, ** p<0.01, ***p<0.001, **** p< 0001. As determined by ANOVA.
Figure 4 A-E Systemic injection of RIPKl RHIM peptides induces IL-Ib production and reduces tumor growth. A. Systemic alternating injection schedule in orthotopic mammary tumor model (n=6, peptide injection 5 mg/kg). B. 4T1 Tumor measurements from alternating injection schedule (WxLxL/2, n=6). C. Systemic daily injection schedule in orthotopic mammary tumor model with and without IDUN (n=6, IDUN= 2.5 mg/kg, peptide= 3.5 mg/kg). D. 4T1 Tumor measurements (WxLxL/2) from daily injection (n=6 per treatment). E. IL-Ib staining and quantification in isolated tumor tissues from daily injection schedule (n=4 from n=6 animals). Data are representative of 2 independent experiments (1st experiment n=3 per treatment and 2nd experiment n=6 per experiment, data represented in figure is the collected data from 2nd experiment n=6 per treatment). Means ± SD. * p<0.05, ** p<0.01, ***p<0.001, **** p< 0001, as determined by two-way ANOVA.
Figure 5 graphically represents the results of in vitro ThT fluorescence assays of RHIM peptides. RHIM peptides, IAPP (positive control) and Bax BH3 (negative control) were dissolved in 0.01M acetic acid, diluted to 10 mM in PBS with or without 10 pM ThT, fluorescent readings were taken at 15 min intervals for 15 hours (a) IAPP (b) Bax BH3 (c) rRIPl (d)rRIP3 (e) mutRIPl (f) mutRIP3 (g) rTRIF (h) rMCMV (i) rDAI (j) rlMD. Data is representative of three independent experiments.
Figure 6 graphically represents the results of assays showing RHIM peptides trigger Il-ΐb secretion. BMDMs were primed with LPS (100 ng/mL, 1 h), transfected with RHIM peptides or IAPP (10 mM, Lipofectamine transfection,‘Lipo’) or treated with CpA (500 nM) and incubated (15 h). Supernatants were analysed for IL-Ib and TNF via ELISA (a) IL-Ib (b) TNF. *P < 0.01 and **P < 0.001 (two-tailed unpaired t-test). Data are representative of three independent experiments (mean and s.d.).
Figure 7 graphically represents the results of assays showing RHIM peptide activation of IL-Ib is mediated by the NLRP3 inflammasome. WT and NLRP3-/- BMDMs were primed with LPS (100 ng/mL, 1 h), transfected with RHIM peptides or IAPP (10 mM, Lipofectamine transfection) or treated with CpA (500 nM) and incubated (15 h). Supernatants were analysed for IL-Ib via ELISA. *P < 0.01 (two-tailed unpaired t-test). Data are representative of three independent experiments (mean and s.d).
Figure 8 A- D Pro-inflammatory cell infiltration into the tumor following systemic peptide injection. Systemic injection of RIPKl RHIM peptides reduces alters T-cell and macrophage composition following alternating daily injection (schedule outlined Figure 4A). A. Isolated macrophage percentages (MerTK/CD64+ cells) at time of sacrifice from digested tumors. B. Percentage CD86+, CD64/MerTK+ macrophages at time of sacrifice from digested tumors. C. Percentage CD206+, CD64/MerTK+ macrophages at time of sacrifice from digested tumors. D. Isolated CD3/CD8+ T-cell counts following tumor digestion. Data are representative of 2 independent experiments (n=3 and represented within figure n=6 animals). Means ± SD. * p<0.05, ** p<0.01, ***p<0.001, **** p< 0001, as determined by two-way ANOVA.
Figure 9 A-D Potency of amyloid formation of RHIM peptides influences tumor outcome. A. 4T1 Tumor measurements (WxLxL/2) following systemic injection of RHIM peptides (n=3). B. Serum IL-Ib levels at time of sacrifice. C. Isolated CD8+ T- cell counts per million cells from digested tumors at time of sacrifice. D. Isolated CD4+ T-cell counts per million cells from digested tumors at time of sacrifice. (n=3, schedule outlined figure 4A). Means ± SD. * p<0.05, ** p<0.01, ***p<0.001, **** p< 0001, as determined by two-way ANOVA.
Figure 10 A-D Peptides induce cell death in multiple cell lines. A) RIPK peptides induce cell death in human monocytic cell lines. THPl cells were exposed to RIPK RHIM peptides for 24 hours. Incorporation of PI was analysed via flow cytometry. B: 4T1 cells were exposed to RIPK RHIM peptides for 24 hours, results from an MTS assay where 100% viability was determined from untreated cells. C: Untreated and LPS primed 4T1 cells were exposed to 25 mM RIPK RHIM peptide and RIPK RHIM peptides and IDUN-5665 duel treatment for 24 hours. Incorporation of PI was analysed via flow
cytometry. D: WT and Ripkl-/- MDA-MB-231 cells were exposed to 25 mM R1 and 50 mM R3 RHIM peptide treatment for 24 hours. Incorporation of PI was analysed via flow cytometry. Data are representative of 2 independent experiments. Means ± SD, replicate assays. * p<0.05, ** p<0.01, ***p<0.001, **** p< 0001. As determined by a two-way ANOVA.
Figure 11 A-B RHIM peptide sequences form homo- and hetero- amyloid fibrils in a sequence, time, and concentration dependent manner both alone and with native protein. Peptide sequences, Trans- Activator of Transcription (TAT) sequence from HIV for solubility and cellular internalization, connected with a lysine spacer to evolutionarily conserved RHIM sequences (RIPK3, RIPKl, TRIF and DAI). The four mouse RIPK RHIM sequences were synthesized and analysed 24 hours post salt exposure via ThT fluorescence for homo-amyloid.
Figure 12 Deletion of proteins necessary for the progression of necroptosis, have limited to no impact on cell death, however when caspases are eliminated death proceeds through necroptosis. Top: BMDMs from WT, Mlkl-/- mice were incubated for 24 hours with and without various concentrations of Rl, R3, and A peptides, positive: 20 ng/mL TNF, 20mM zVAD, I mM 911. Bottom: In the presence of 20 mM zVAD cell death increases in WT macrophages, in Mlkl-/- death remains constant. Suggesting that blockage of caspases drives necroptosis dependent on MLKL in the presence of 25 mM of Rl peptide. Incorporation of PI was analysed via flow cytometry. Data are representative of 2 independent experiments.
Figure 13 Synthesized peptide characterization. Evolutionarily conserved RHIM peptide sequences (mouse RIPK3 and RIPKl, were connected via a tri -lysine spacer to a Trans- Activator of Transcription (TAT) sequence from HIV. An alanine substituted peptide was synthesized as a control (sequences can be located in Figure 1 of the manuscript). Peptides were synthesized by solid phase peptide synthesis by ELIM Biopharmaceuticals. Peptides were purified by high performance liquid chromatography (HPLC, right) to greater than 95% purity and verified to be the correct molecular weight by electrospray mass spectrometry (left). Al/2) Alanine substituted peptide HPLC and Mass Spec data (molecular weight -3640). Bl/2) RIPK3 peptide HPLC and Mass Spec data (molecular weight -3839) Cl/2) RIPKl peptide HPLC and Mass Spec data (molecular weight -3885).
Figure 14 Bone marrow macrophages were incubated with wild type and flagellin knockout luciferase containing L. pneumophila bacteria for 96 hours. TOP: Macrophages infected with wild type L. pneumophila bacteria (Lp02) did not produce significant infection rates. BOTTOM: However when macrophages were incubated with
flagelllin deleted (Af aA) bacteria macrophages were unable to contain infection unless they were co-incubated with 25mM R1 (RIPK1 peptide) or R3 (RIPK3 peptide). 25mM A (Alanine peptide) did not produce observations that differed from control.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.
Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1- 3, ed. Sambrook et ah, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (2001); and Current Protocols in Molecular Biology, ed. Ausubel et ah, Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates). Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Current Protocols in Immunology, ed. Coligan et ah, Greene Publishing and Wiley-Interscience, New York, (1992) (with periodic updates); Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, Mass., (2007); Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, FI, (2006); Medical Immunology, 6th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, (2007); and Harlow and Lane ANTIBODIES: A Laboratory Manual, Second edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014). Conventional methods of gene transfer and gene therapy may also be adapted for use in the present invention. See, e.g., Gene Therapy: Principles and Applications, ed. T. Blankenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press, 1997; Viral Vectors for Gene Therapy: Methods and Protocols, ed. Otto-Wilhelm Merten and Mohammed Al-Rubeai, Humana Press, 2011; and Nonviral Vectors for Gene Therapy: Methods and Protocols, ed. Mark A. Findeis, Humana Press, 2010. Amino Acids. 2018 Jan; 50(l):39-68. doi: 10.1007/s00726-017-2516-0. Epub 2017 Nov 28.
The present application enables an agent utilizing the RHIM consensus sequence as a way to induce inflammatory events through amyloid formation. In one embodiment, the agent would first act as a monomeric unit that could, upon cellular internalization or expression in a concentration dependent manner, polymerize into an amyloid fibril both independently and in combination with native RHIM containing proteins. These induced
fibrils could mimic the intracellular scaffold formations that are responsible both for concurrent intracellular cytokine production and lytic cell death. The present application discloses the use of the present agents in an in-vivo context to support the formation of a pro-inflammatory microenvironment that could be harnessed to induce disease regression where pro-inflammatory cues are shown to be beneficial, such as cancer, and pathogenic disorders.
The present disclosure is predicated, in part, on the development of a peptide that upon administration to a receptive cell or to a subject stimulates an immune- inflammatory cascade that is able to reduce tumour growth in a triple negative breast cancer model. This development has many applications where an immune-inflammatory stimulus can help the body to overcome or prevent cancer or other pathologies or prevent the establishment of infections such as by enhancing responses to vaccination. While a number of particular peptides and peptide constructs have been developed, the skilled person will appreciate that the invention encompasses routine modifications in peptide sequence or chemistry, to facilitate stability and delivery, as known in the art and described herein. Further, the peptides can be delivered in the form of a nucleic acid molecule or vector able to induce intracellular production of the peptide. In one embodiment, the peptide is connected to a cell penetrating peptide. In another embodiment, the peptide is transfected into cells as a lipid particle or micelle or together with a transfection aid in vivo or ex vivo.
Accordingly, in one aspect the disclosure provides methods for artificially or transiently inducing an artificial lytic immuno-inflammatory or pro-inflammatory response (LIIIR) in a subject. In one embodiment the methods are sufficient to increase the effectiveness of an immune response, to maintain or improve health. These methods generally comprise contacting a cell or a subject with an LIIIR-inducing amount of a RHIM-interacting agent or an encoding nucleic acid molecule. As determined herein a peptide construct comprising a RHIM-interacting portion and an intracellular delivery portion (such as cell penetrating peptide) was able to enter RHIM interactive cells and there promote an LIIIR. Specifically, for example, the RHIM-interacting portion induced amyloid formation and release of pro-inflammatory cytokines such as at least IL-Ib. Non-limiting examples of RHIM interacting agents include RHIM-containing peptides of Structure 1.
Death can also happen in tumor cells and other cell types to a lesser extent— however these cells do not express IL-1. A peptide can interact with RIPK1 (as demonstrated in the Figures/examples) which would suggest that any cell that has high
expression of this protein would be responsive. However the strongest responses have been observed have been from myeloid cells that express inflammasome components.
As disclosed in this application, RHIM-interacting agents induce in immune cells or cells able to form inflammasomes:
i. amyloid formation between intracellular RHIM containing molecules ii. intracellular membrane lysis leading to lytic cell death
iii. pro-inflammatory cytokine production and release from lysed cells
iv. recruitment of immune cells into the tissue microenvironment,
v. Caspase-1 and NLRP3 activation leading to cleavage of pro-IL-Ib and release of IL-Ib.
In one embodiment, the specificity of binding between a RHIM interacting agent and an endogenous RHIM-containing molecule can be reflected in a dissociation constant (KD) that is at least in the micromolar range. In one embodiment, the RHIM interacting agent forms homotypic or heterotpic interations with other exogenously administered RHIM interacting agent and the specificity of binding between RHIM interacting agents can be reflected in a dissociation constant (KD) that is at least in the micromolar range.
As disclosed in this application, the agents induce in receptive cells comprising RIPK1/RIPK3 cell signalling platforms able to interact with RHIM interacting agents: i. cell death - any cell type that has a high concentration of RIPK1/RIPK3 would likely be responsive (eg. GI track, skin). Ideal targets include any disease state where disease resolution is associated with increased inflammatory activity, such as cancer, pathogenic disorders. In these cases, a cell type of myeloid lineage is likely the target (i.e. tumor associated macrophages in solid tumors, and macrophages that have engulfed pathogenic materials.
Any organ that has a sufficient concentration of RIPK1 protein would likely be responsive (e.g. the GI track, skin, etc). As determined herein MLKL and RIPK3 dependence is promoted in the absence of caspases (shown in the presence of zVAD or IDUN). Ideal targets include any disease state where disease resolution is associated with increased inflammatory activity— such as cancer and pathogenic disorders. In these cases, a cell type of myeloid lineage is likely the target (i.e. tumor associated macrophages in solid tumors, and macrophages that have engulfed pathogenic materials. However, cell death is not specific to the myeloid lineage, and any cell type comprising RIPK1/RIPK3 etc can die through the use of the herein disclosed RHIM interacting agent/peptide. Notwithstanding the above, myeloid cells as determined herein are able to
produce abundant pro-inflmmatory cytokine with the presence of the priming RHIM- interacting agent/peptide.
As disclosed in this application, RHIM interacting agents induce in IL-Ib or other pro-inflammatory cytokine sensitive tumours:
i. immune cell recruitment and/or activation in tumour stroma
ii. altered macrophage composition e.g. decreased M2 and increased Ml iii. elevated extracellular circulating and intratumoral IL-Ib secretion
iv. reduction in tumour growth
In one embodiment, the RHIM-interacting agent, binds to RHIM containing molecules such as RIPK1, RIPK3, DAI, TRIF and other orthologs. In one embodiment, the RHIM-interacting agent binds to and agonises the activity of intracellular endogenous RHIM containing molecules to form amyloid. In one embodiment, the RHIM interacting agent comprises a RHIM-containing peptide and forms homodimers or heterodimers with endogenous RIPK1, RIPK3, or RIPK1 and RIPK3. In one embodiment, RHIM- interacting agents, including RHIM interacting or containing peptides penetrate reactive cells and stimulate the formation of amyloid therein.
While the activity of the RHIM interacting agents described here has been illustrated by demonstrating their effects in suppressing tumour growth, the disclosure is not limited to this particular application. Accordingly, in other aspects in the instant methods are used to enhance the effectiveness of the immune response such as by serving as an immune agonist or adjuvant in vaccines or to reduce pathogenesis.
In another aspect, this disclosure is directed to an agent or composition comprising a RHIM-interacting peptide as described herein.
Compositions include physiologically or pharmacologically or pharmaceutically acceptable vehicles that are not biologically or otherwise undesirable. Pharmacologically acceptable salts, esters, pro-drugs, or derivatives of a compound described here is a salt, ester, pro-drug, or derivative that is not biologically or otherwise undesirable.
In some embodiments, the peptide agent is modified. Peptide and agent activity are tolerant to additional moieties, flanking residues and substitutions within the defined boundaries. Similarly backbone modifications and replacements, side-chain modifications and N and C-terminal modifications are conventional in the art. Generally, the modification is to enhance stability or pharmacological profile, intracellular delivery such as to the nucleus or mitochondria and/or cell targeting/delivery. For example, peptide cyclisation or stapling is conventional for enhancing peptide stability and intracellular delivery. In another embodiment, peptides or agents are in the form of micro
or nano-particles or bubbles, liposomes, conjugates or fusion proteins including for example cell penetrating or endo-porter peptides or mixtures thereof comprising moieties adapted for stability, intracellular delivery and/or cell targeting/delivery.
In one embodiment, agents or their encoding nucleic acids where appropriate are assembled in liposomes, hydrogels, emulsions, viral vectors, viral-like particles or virosomes.
In one embodiment, specific binding moieties such as antibody or antibody fragments or mimics are used to target agents to an immune cell.
In one embodiment, peptide agents are delivered through biological synthesis in vivo such as via delivery of mRNA, gene editing such as CRISPR components, or bacteria or cells.
Compositions generally comprise a RHIM-interacting peptide, peptidomimetic or an encoding nucleic acid where appropriate, and a pharmaceutically acceptable carrier and/or diluent.
In one embodiment, the RHIM-interacting agents of the present disclosure are not naturally occurring molecules, but instead are modified forms of naturally occurring molecules which do not possess certain features or functions of the naturally occurring full length molecules.
RIPK1 or RIPK3 orthologous RHIM sequences are for example those listed in Cell (2012) 150 , 339-350.
Zi and Z2 are individually present or absent and where present is selected from one or more of a linker, a stability enhancing, delivery enhancing or label moiety. In one embodiment, the moiety may be an amino acid or non-amino acid moiety.
In one embodiment, Z\ and or Z2 comprises a linker or spacer.
Illustrative linkers include hydrophilic linkers such as a di or polylysine, a di or polyglycine, or a di or polyarginine or mixed (lysine/arginine) linker.
In one embodiment, Zi and or Z2 comprises a cell penetrating peptide or an antibody fragment.
In one embodiment, Zi and or Z2 comprises a label. In one embodiment the label is an affinity tag or detectable label, such as a fluorescent label.
Sequence alignment of RIPK1 and RIPK3 orthologs shows conservative residues within the RHIM domain as illustrated in Cell 750:339-350, 2012.
In one embodiment the RHIM-interacting agent comprises the human (17 residues X-X) or murine (residues X to X) of RIPK1 amino acid sequence as set forth in SEQ ID NO: 1 or 2.
In one embodiment the RHIM-interacting agent comprises the human (17 residues X-X) or murine (residues X to X) RIPK3 amino acid sequence set forth in SEQ ID NO: 3 or 4.
In one embodiment, the RHIM- interacting agent of Structure 1 or derivative comprises 8 to 60 amino acids, or 10 to 35 or 7 to 40 amino acids residues not including any intracellular targeting or cell targeting components.
In one embodiment, the RHIM- interacting agent of Structure 1 or derivative has 4 to 17 amino acids, or 4 to 40 or 7 to 17 or 4 to 100 amino acids residues including any intracellular targeting components.
For the avoidance of doubt, the RHIM-interacting agent of structure I or derivative does not comprise a death domain or a kinase domain.
In one embodiment, the RHIM-interacting peptide of structure I comprises at least X3 to Xi5, optionally a hydrophilic linker and optionally a cell penetrating peptide.
In one embodiment, amino acids X3 to X15 of structure I provide a peptide having the corresponding sequence from a RIPK1 or RIPK3 or DAI or MCMV M45 peptide or a sequence having at least one or more stabilizing amino acids such as D-amino acids, sugar amino acids, Beta-amino acids or N-methylated amino acid or wherein the peptide is cyclized or stapled peptide, or an ether peptide or lactam peptide etc.
In another embodiment, the RHIM-interacting peptide is constrained by means of a linker which is covalently bound to at least two amino acids in the peptide. Various cyclisation strategies are known in the art to increase stability and cellular permeability.
In some embodiments, the RHIM interacting agent is delivered in the form of nucleic acid molecules encoding same or pro-drugs thereof or vectors comprising nucleic acid molecules encoding same or pro-drugs thereof. In one embodiment, the nucleic acid is mRNA.
In another embodiment the RHIM interacting agent is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregates as micelles, insoluble monolayers, liquid crystals or lamellar layers in aqueous solution.
In one embodiment, the disclosure enables a composition comprising a RHIM- interacting agent as described herein which interacts with endogenous RHIM proteins to form amyloid or use as a medicament or for use in therapy.
In one embodiment, the composition stimulates an immune response in immune cells sufficient to reduce tumour development in a subject.
In one embodiment, the immune response includes primary responses by cells of the innate immune system such as monocytes and macrophages, and secondary responses by cells of adaptive immune system such as T-cells.
In one embodiment, the immune response results in elevated levels of serum inflammatory cytokines.
In another aspect, the present disclosure enables a composition for stimulating an immune response in immune cells in a subject in need thereof, the composition comprising a RHIM-interacting peptide or a nucleic acid molecule from which the peptide is expressible.
In one embodiment, the subject composition is co-administered with a second therapeutic or prophylactic agent, or a vaccine.
In another aspect, the present disclosure provides for the use of the RHIM interacting agent in the manufacture of a medicament for stimulating an immune response or for preventing or treating cancer.
In one embodiment, the application provides screening assays for RHIM- interacting agents as described herein, comprising assessing the ability of potential agents to induce intracellular amyloid formation or amyloid formation and Il-ΐb release from immune cells, such as primary BMDMs or transformed cells, such as 4T1 cells.
Peptides and modifications
Peptide-based therapeutics provide useful molecules because they are known to be potent and selective against biological targets that are otherwise difficult to manipulate with small molecules. To improve the pharmacokinetic properties of linear peptides, modified peptides have been successfully developed and used to target intracellular proteins.
The peptides of the present disclosure comprise amino acids. Reference to "amino acid" includes naturally occurring amino acids or non-naturally occurring amino acids.
Peptide compounds are generally and conventionally modifiable by addition of moieties, flanking peptide residues, and substitutions within understood parameters. Peptides can furthermore comprise routine modified backbones, side chains, peptide bond replacements, and terminal modifications using standard peptide chemistries.
The amino acids incorporated into the amino acid sequence described herein may be L-amino acids, D-amino acids, L- b -homo amino acids, D- b -homo amino acids or N-methylated amino acids, sugar amino acids, and/or mixtures thereof. Non-natural amino acids may not be recognised by proteases and may therefore alter the half-life. In one embodiment, the D-retro inversion sequence is employed.
Non-naturally occurring amino acids include chemical analogues of a corresponding naturally occurring amino acid. Examples of unnatural amino acids and derivatives include, but are not limited to, 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, nor leucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids.
In one embodiment, peptides are modified to enhance their pharmacodynamics properties using art recognised modifications. Peptides may be substituted, such as alanine substituted, or substituted with cross linkable moieties and/or linked. Suitable residues may comprise additional alpha-carbon substitutions selected from hetero- lower alkyl, hetero- methyl, ethyl, propyl and butyl. Peptide bond replacements such as trifluoroethylamines are used to produce more stable and active peptidomimetics.
Accordingly, cyclic or stapled peptides, peptoids, peptomers, and peptidomimetic forms of peptides are encompassed.
Backbone constrained peptidomimetics and cyclic peptides are protected against exopeptidases. Peptides can be cyclised coupling N- to C-terminus after cleavage. This can be achieved by direct coupling or by introduction of specific functional groups that permit defined cyclization by a biorthogonal reaction. Illustrative modifications Cys-Cys disulphide bridges, inclusion of sidechain modifications to include linkers forming macro lactam peptides, thio ether peptides or stapled peptides etc. Click variants are particularly useful for peptide cyclization. Another approach uses 2-amino-d, 1-dodecanoic acid (Laa) couples to the N-terminus and by replacing Asn with the lipoamine.
A more defined structure can be obtained by use of a more rigid back bone with heterocycles, N-methylated amine bonds or methylated alpha-carbon atoms.
Among the techniques used for peptide stapling, the two-component double Cu- catalysed azide-alkyne cycloaddition (CuAAC) strategy constrains the peptides in the bioactive conformation and simultaneously improves pharmacokinetic properties. Moreover, this strategy uses unnatural azido amino acids that can be easily synthesised and facilitates the functionalisation of the staple with cell-perm eabili sing motifs, fluorescent-labelled tags and photo-switchable linkers. The independent functionalisation of the staple can be particularly useful as the complex functionality is added to the staple rather than the N- or C-terminus of the peptide. In addition, this approach only requires one linear peptide to generate a variety of functionalised stapled peptides, facilitating the exploration of various functionalities on the linker and thus properties of the overall peptide.
Azapeptides are peptide analogs in which one or more of the amino residues is replaced by a semicarbazide. This substitution of a nitrogen for the a-carbon center results in conformational restrictions, which bend the peptide about the aza-amino acid residue away from a linear geometry. The resulting azapeptide turn conformations have been observed by x-ray crystallography and spectroscopy, as well as predicted based on computational models. In biologically active peptide analogs, the aza-substitution has led to enhanced activity and selectivity as well as improved properties, such as prolonged duration of action and metabolic stability.
Half-life may also be increased by acylating or amidating ends. Peptoids are produced with N-alklyated oligoglycines side chains. In some embodiments, peptides may be acetylated, acylated (e.g., lipopeptides), formylated, amidated, phosphorylated (on Ser,Thr and/or Tyr) , sulphated or glycosylated.
The term "macrocyclization reagent" or "macrocycle-forming reagent" as used herein refers to any reagent which may be used to prepare a peptidomimetic macrocycle by mediating the reaction between two reactive groups. Reactive groups may be, for example, an azide and alkyne, in which case macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, Cul or CuOTf, as well as Cu(II) salts such as Cu(CO.sub.2CH.sub.3).sub.2, CuSO.sub.4, and CuCl.sub.2 that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate. Macrocyclization reagents may additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh.sub.3).sub.2, [Cp*RuCl].sub.4 or other Ru reagents which may provide a reactive Ru(II) species. In other cases, the reactive groups are terminal olefins. In such embodiments, the macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbine complex catalysts such as Group VIII transition metal carbene catalysts. For example, such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and pentacoordinated. Additional catalysts are disclosed in Grubbs et ah, "Ring Closing Metathesis and Related Processes in Organic Synthesis" Acc. Chem. Res. 1995, 28, 446-452, and U.S. Pat. No. 5,811,515. In yet other cases, the reactive groups are thiol groups. In such embodiments, the macrocyclization reagent is, for example, a linker functionalized with two thiol -reactive groups such as halogen groups.
In one embodiment, a peptidomimetic macrocycle exhibits improved biological properties such as increased structural stability, increased affinity for a target, increased resistance to proteolytic degradation and/or increased cell penetrability when compared to a corresponding non-macrocyclic polypeptide. In another embodiment, a
peptidomimetic macrocycle comprises one or more .alpha.-helices in aqueous solutions and/or exhibits an increased degree of .alpha.-helicity in comparison to a corresponding non-macrocyclic polypeptide. In some embodiments, a macrocycle-forming linker increases cell permeability of the peptidomimetic macrocycle. Without wishing to be bound by theory, it is hypothesized that the macrocycle-forming linker may increase the overall hydrophobicity of the peptidomimetic macrocycle relative to a corresponding non-macrocyclicpolypeptide.
For example, the sequence of the peptide can be analyzed and azide-containing and alkyne-containing amino acid analogs of the invention can be substituted at the appropriate positions. The appropriate positions are determined by ascertaining which molecular surface(s) of the secondary structure is (are) required for biological activity and, therefore, across which other surface(s) the macrocycle forming linkers of the invention can form a macrocycle without sterically blocking the surface(s) required for biological activity. Such determinations are made using methods such as X-ray crystallography of complexes between the secondary structure and a natural binding partner to visualize residues (and surfaces) critical for activity; by sequential mutagenesis of residues in the secondary structure to functionally identify residues (and surfaces) critical for activity; or by other methods. By such determinations, the appropriate amino acids are substituted with the amino acids analogs and macrocycle forming linkers of the invention. For example, for a helical secondary structure, one surface of the helix (e.g., a molecular surface extending longitudinally along the axis of the helix and radially 45- 135. degree about the axis of the helix) may be required to make contact with another biomolecule in vivo or in vitro for biological activity. In such a case, a macrocycle-forming linker is designed to link two carbons of the helix while extending longitudinally along the surface of the helix in the portion of that surface not directly required for activity.
The peptidomimetic macrocycle resulting from a method of the invention may comprise an helix in aqueous solution. For example, the peptidomimetic macrocycle may exhibit increased helical structure in aqueous solution compared to a corresponding non- macrocyclic polypeptide. In some embodiments, the peptidomimetic macrocycle exhibits increased thermal stability compared to a corresponding non-macrocyclic polypeptide. In other embodiments, the peptidomimetic macrocycle exhibits increased biological activity compared to a corresponding non-macrocyclic polypeptide. In still other embodiments, the peptidomimetic macrocycle exhibits increased resistance to proteolytic degradation compared to a corresponding non-macrocyclic polypeptide. In
yet other embodiments, the peptidomimetic macrocycle exhibits increased ability to penetrate living cells compared to a corresponding non-macrocyclic polypeptide.
The term "amino acid analog" refers to a molecule which is structurally similar to a naturally occurring amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, without limitation, compounds which are structurally identical to an amino acid, as defined herein, except for the inclusion of one or more additional methylene groups between the amino and carboxyl group (e.g. .alpha.-amino .beta.-carboxy acids), or for the substitution of the amino or carboxy group by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution or the carboxy group with an ester).
The peptide may comprise an N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl, 2-furosyl and or a C-terminal hydroxyl, amide, ester or thioester group. In one embodiment, the peptide is acetylated at the N-terminus and amidated at the C- terminus. In one embodiment, chelators are introduced for example DOTA, DPTA. Peptides may be modified by, for example pegylation, lipidation, xtenylation, pasylation and other approaches to extend the half-life of the peptide in vivo or in vitro. In one embodiment, pegylation is used to increase peptide solubility and bioavailability. Various forms of peg are known in the art and include HiPeg, branched and forked Peg, releasable Peg, heterobifunctional Peg with end group NHS esters, malaimeide, vinyl sulphone, pyridyl disulphide, amines and carboxylic acids. Examples of therapeutic pegylated peptides include pegfilagrastin (Neulasta) made Amgen.
Linkers or spacers may be amino acids or nucleic acids or other atomic structures known in the art, typically between 2 and 10 amino acids or nucleotides in length. Spacers should be flexible enough to allow correct orientation of RHIM-interacting constructs as described herein, such as those including nanoparticles, antibody fragments, liposomes, cell penetrating and/or intracellular delivery moieties. One form of spacer is the hinge region from IgG suitable for use when the construct comprises an antigen binding moiety for cellular targeting.
Antigen-binding molecules include for example extracellular receptors, antibodies or antibody fragments (including molecules such as an ScFv). Signal peptides may be present at the N-terminal end. Bispecific antibodies capable of selectively binding to two or more epitopes are known in the art and could be used in the present RHIM interacting agents.
In one embodiment, the peptide is conjugated or otherwise associated (covalent or non-covalent attachment) with a delivery agent. In one embodiment the delivery agent
delivers the peptide to a target cell or cell population. In one embodiment, the delivery agent facilitates intracellular delivery of the peptide. In another embodiment, the delivery agent facilitates intracellular delivery to a cell compartment such as the nucleus, mitochondria. In one embodiment, delivery is to an immune cell or or a non-immune cells, or an immune cell and a tumor cell in vivo. In one embodiment delivery is ex vivo.
Derivatives of RHIM interacting agents of Structure 1 includes biologically active fragments thereof as described therein comprising the structure XXIXN(Xa)XXXI/VQI/VGXXNXM/L wherein Xa is present or absent and wherein X is an amino acid, or an amino acids selected from the corresponding position in a RIPK1, RIPK3, DAI or MCMC M45 ortholog. In one embodiment, a biologically active fragment comprises at least X9 to X12 and has the functional ability of the parent construct to form amyloid and induce cell death and cytokine secretion in one or two or more cell types. Biologically active fragments may contain for example 7 to 30 amino acids. Systematic shortening or alanine scanning or modelling around the conserved motif can be routinely conducted to identify minimal peptides with amyloid forming and cytokine secreting effects.
Derivatives also include molecules having a percent amino acid or polynucleotide sequence identity over a window of comparison after optimal alignment. In one embodiment the percentage identity is at least 80%-99% including any number in between 80 and 99.
Suitable assays for the biological activity of peptides or agents are known to the skilled addressee and are described in the examples and Figures.
In some embodiment, alternative or additional markers of peptide activity include: inflammasome activation; mitochondrial and lysosomal disruption; potassium influx into cells; cytokine production and secretion or loss from lysed cells selected from IL-Ib, IL- 18, TNF-alpha, IL-6, IL-8, IL-12, IFN-gamma., IFN-alpha, MCP-1, MIP-lalpha., MIP- lPeta., iNOS, IL-17, IL-23; cell penetration, cell targeting, decreased tumour growth. The effect of the RHIM interacting peptide to form amyloid with endogenous RHIM containing proteins can be confirmed using the methods described in Example 1. The effect of the peptide to induce the production of cytokines and IL-Ib production down stream of NLRP3 activation and the role of potassium flux to activate NLRP3 can be confirmed by, for example, the method described in Example 2. The effect of the peptide to induce intracellular membrane lysis can be confirmed by, for example, the method described in Example 3. The effect of the peptide of the present invention to activate inflammasomes can be confirmed by, for example, the method described in Examples 5 and 6, etc. The adjuvant or immune agonist effect of the peptide or agent of the present
invention can be confirmed by, for example, the above-mentioned methods for confirming the immunostimulatory effect (Examples 2 to 6), the methods described in Examples 7 and 8, etc.
In one embodiment, the RHIM-interacting agent is modified with a moiety which is not a naturally occurring amino acid residue. The moiety may be selected from the group consisting of a detectable label, a non-naturally occurring amino acid as described herein, a reactive group, a fatty acid, cholesterol, a lipid, a bioactive carbohydrate, a nanoparticle, a small molecule drug, and a polynucleotide. In one particular embodiment, the moiety is a detectable tag label. In one example the detectable label is selected from the group consisting of a fluorophore, a fluorogenic substrate, a luminogenic substrate, and a biotin. Art recognised tags or labels include affinity agents and moieties for detection include fluorescent and luminescent compounds, metals, dyes. Other useful moieties include affinity tags, biotin, lectins, chelators, lanthanides, fluorescent dyes, FRET acceptor/donors.
In one embodiment, the RHIM-interacting agent which may comprise a detectable label, is accompanied in a kit with a modified control version of the agent wherein the conserved residues of the RHIM as shown in Structure 1 are substituted with for example alanine. Kits comprising the agents are proposed for sale and may be used for screening purposes.
Peptides of this type may be obtained through the application of recombinant nucleic acid techniques as, for example, described in Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al. CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
Alternatively, peptides of this type may be synthesised using conventional liquid or increasingly solid phase synthesis techniques. For example, initial reference may be made to solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS : A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989), see particularly Chapter 9, or by Roberge et al. (1995 Science 269: 202).
Azapeptide synthesis was previously hampered by tedious solution-phase synthetic routes for selective hydrazine functionalization. Recently, the submonomer procedure for azapeptide synthesis, has enabled addition of diverse side chains onto a common semicarbazone intermediate, providing a means to construct azapeptide
libraries by solution- and solid-phase chemistry. In brief, aza residues are introduced into the peptide chain using the submonomer strategy by semicarbazone incorporation, deprotonation, N-alkylation, and orthogonal deprotection. Amino acylation of the resulting semicarbazide and elongation gives the desired azapeptide. Furthermore, a number of chemical transformations have taken advantage of the orthogonal chemistry of semicarbazone residues (e.g., Michael additions and N-arylations). In addition, oxidation of aza-glycine residues has afforded azopeptides that react in pericyclic reactions (e.g., Diels-Alder and Alder-ene chemistry). The bulk of these transformations of aza-glycine residues have been developed by the Lubell laboratory, which has applied such chemistry in the synthesis of ligands with promising biological activity for treating diseases such as cancer and age-related macular degeneration. Azapeptide analogues of growth hormone-releasing peptide-6 (His-d-Trp-Ala-Trp-d-Phe-Lys-NH2, GHRP-6) have for example been pursued as ligands of the cluster of differentiation 36 receptor (CD36) and show promising activity for the development of treatments for angiogenesis- related diseases, such as age-related macular degeneration, as well as for atherosclerosis. Azapeptides have also been employed to make a series of conformationally constrained second mitochondria-derived activator of caspase (Smac) mimetics that exhibit promising apoptosis-inducing activity in cancer cells. The synthesis of cyclic azapeptide derivatives was used to make an aza scan to study the conformation-activity relationships of the anticancer agent cilengitide, cyclo(RGDf-N(Me)V), and its parent counterpart cyclo(RGDfV), which exhibit potency against human tumor metastasis and tumor- induced angiogenesis. Innovations in the synthesis and application of azapeptides are described in Acc Chem Res. 2017 Jul 18;50(7): 1541-1556.
Alternatively, peptides can be produced by digestion of an adaptor polypeptide with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8- protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Measures that may be taken to optimize pharmacodynamics parameters of peptides and peptide analogs are described by Werle M. et al (2006) Strategies to improve plasma half-life time of peptide and protein drugs amino Acids 30(4):351-367; and Di L (2014) Strategic approaches to optimising peptide ADME properties AAPS J 1-10.
Antibody drug conjugate (ADC) may be obtained by binding an antibody or antibody fragment/derivative such as an ScFv recognizing a cell to the present peptide using an appropriate linker. The antibody portion of an antibody drug conjugate binds to a target molecule on the surface of a target cell or the ADC infiltrates into the cell. In some embodiments, the linker of the antibody drug conjugate is cleaved in the cell or in
the environment of the target cell and the peptide exerts its effect in the cell or local environment.
The peptide may be stabilised for example via nanoparticles, liposomes, micelles or for example PEG as known in the art. Methods to form liposomes are described in: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., the contents of which is incorporated herein by reference. Polymer nanoparticles ideally use surfactants that are not toxic or physically adsorbed to the nnoparticle. In one aspect, biodegradable surfmers are used. For example, biodegradable, polyethylene glycol) (PEG)ylated N-(2-hydroxypropyl) methacrylamide (HPMA) based surfmers are synthesized and used to stabilize lipophilic NPs. In particular, the NP core is made from a macromonomer comprising a poly(lactic acid) (PLA) chain functionalized with HPMA double bond. The nanoparticle forming polymer chains are then constituted by a uniform poly(HPMA) backbone that is biocompatible and water soluble and hydrolysable PEG and PLA pendants assuring the complete degradability of the polymer. The stability provided by the synthesized surfmers is studied in the cases of both emulsion free radical polymerization and solution free radical polymerization followed by the flash nanoprecipitation of the obtained amphiphilic copolymers.
Other stabilising or heterologous moieties include NMEG, albumin, albumin binding proteins, immunoglobulin Fc domain.
Traditional Fc fusion proteins and antibodies are examples of unguided interaction pairs, whereas a variety of engineered Fc domains have been designed as asymmetric interaction pairs as described by Spiess et al (2015) Molecular Immunology 67(2A): 95-106. Fc conjugates may comprise an amino acid sequence that is derived from an Fc domain of an IgG (IgGl, IgG2, IgG3, or IgG4), IgA (IgAl or IgA2), IgE, or IgM immunoglobulin. Such immunoglobulin domains may comprise one or more amino acid modifications (e.g., deletions, additions, and/or substitutions) that promote hetero or homo dimeric or multimeric amyloid formation within the host cell.
In some embodiments, nanoparticles comprising the RHIM-interacting agents of Structure 1 can be further modified by the conjugation of receptive cell type specific binding agents, antibodies or fragments thereof known in the art.
Although the peptide molecules may inherently permeate biological membranes, membrane permeation may further be increased by the conjugation of a membrane permeating moiety to the peptide. Accordingly, in one embodiment the peptide comprises at least one membrane permeating moiety. The membrane permeating moiety may be conjugated to the peptide or added to the composition comprising the peptide or
RHIM interacting agent. Suitable membrane permeating moieties include lipid moieties, cholesterol and proteins, such as cell-penetrating peptides and polycationic peptides.
One platform for the discovery of functional cell penetrating peptides for efficient intracellular delivery is described by Hoffmann et al. (2018) Scientific Reports 8:12538. One desirable feature of functional cell penetrating peptides for use herein is their capacity to evade endosomal trapping. Another useful feature, as discussed in relation to RHIM-interacting peptides is their modification to contain residues that are not susceptible to protease digestion in vivo or in vitro. One example is described for illustrative purposes in Wang et al. Oncogene, 2018 (doi.org/10.1038/s41388-018-0421- y). Illustrative CPPs are described in WO2019/018898, WO2014/25518, and WO2012/159164 to Phylogica.
Cell penetrating peptides known in the art include the peptides described in, for example, US 20090047272, US 20150266935 and US 20130136742. Accordingly, suitable cell penetrating peptides may include, but are not limited to, basic poly(Arg) and poly(Lys) peptides and basic poly(Arg) and poly(Lys) peptides containing non-natural analogues of Arg and Lys residues: such as: HIV TAT (47-57), W/R, AlkCWKis, KisWCCWKis (Di-CWKi8), Transportan, DipaLytic, Ki6RGD, Ki6GGCMFGCGG(Pl), KiJCRRARGD PDDRCT (P2), KKWKMRRNQFWVKVQRbAK (B) bA (P3), P3a, P9.3, Pep-1, Plae, Kplae, cKplae, GALFLGFLGGA AGS TMGAW S QPK SKRK V (MGP), WEAK(LAKA)2- LAKH(LAKA)2LKAC (HA2), (LARL)6NHCH3 (LARL46), KLLKLLLKLWLLKLLL (Hel-11-7), (KKKK)2GGC (KK), (KWKK)2GCC (KWK), (RWRR)2GGC (RWR), PKKKRKV (SV40 LS7), PEVKKKRKPEYP (NLS12), TPPKKKRKVEDP ( LS12a), GGGGPKKKRK V GG (SV40 LSI 3), GGGF S T SLRARK A (AV NLS13), CKKKKKK SEDE YP YVPN (AV RME NLS17), CKKKKKKK SEDEYP YVP F ST SLRARK A (AV FP NLS28), L VRKKRKTEEE SPLKDKD AKK SKQE (SV40 N1 LS24), and Loligomer; HSV-1 tegument protein VP22; HSV-1 tegument protein VP22r fused with nuclear export signal (NES); mutant B-subunit of Escherichia coli enterotoxin EtxB (H57S); detoxified exotoxin A (ETA); the protein transduction domain of the HIV-1 Tat protein, GRKKRRQRRRPPQ; the Drosophila melanogaster Antennapedia domain Antp (amino acids 43-58), RQIKIWFQ RRMKWKK; Buforin II, TRS SRAGLQFP V GRVHRLLRK; hClock-(amino acids 35-47) (human Clock protein DNA-binding peptide), KRVSR KSEKKRR; MAP (model amphipathic peptide), KLALKLALKALKAALKLA; K-FGF, AAVALLPAVLLALLAP; Ku70-derived peptide, comprising a peptide selected from the group comprising VPMLKE, VPMLK, PMLKE or PMLK; Prion, Mouse Pipe (amino acids 1 -28), MA LGYWLLALFVTMWTDVGLCKKRPKP; pVEC,
LLIILRRRIRKQAHAHSK; Pep-I, KETWWET WWTEW S QPKKKRK V; SynBl, RGGRLSYSRRRFSTSTGR; GWTLN S AGYLLGKINLKAL AALAKKIL;
Transportan-10, A G Y L L G K I N L K A L A A L A K K I L ; CADY, Ac- GLWR ALWRLLRSL WRLLWR A-cy steami de; Pep-7, SDLWEMMMV SLACQ Y ; HN-1, TSPLNIHNGQKL; VT5, DPKGDPKGVT VT VT VT VT GKGDPKPD ; or pISL, RVIRVWF QNKRCKDKK.
As described in ETnited States patent no. 9,982,267 rationally designed synthetic peptide shuttle agents are known for delivering peptide from an extracellular space to the cytosol and/or nucleus of a target cell.
Typical shuttle peptides comprise an endosome leakage domain (ELD) operably linked to a cell penetrating domain (CPD), and optionally one or more histidine-rich domains, to increase the transduction efficiency of an to a peptide in eukaryotic cells, such that the peptide gains access to the cytosol/nuclear compartment
In some embodiments, linkers may be formed by adding sequences of small hydrophobic amino acids without rotatory potential (such as glycine) and polar serine residues that confer stability and flexibility. Linkers may be soft and allow the domains of the shuttle agents to move. In some embodiments, prolines may be avoided since they can add significant conformational rigidity. In some embodiments, the linkers may be serine/glycine-rich linkers (e.g., GGS, GGSGGGS, GGSGGGSGGGS, or the like).
ELD characteristically having endosome escape activity and/or pH-dependent membrane disrupting activity. Non-limiting examples of endosome leakage domains are described in US 9,982,267 and include CM18KWKLFKKIGAVLKVLTTG (SEQ ID NO: 98), Diphtheria VGS SL S CINLD WD VIRDKTKTKIE SL( SEQ ID NO: 99),
T domain KEHGPIKNKMSESPNKTV SEEKAKQ (SEQ ID NO:), (DT)
YLEEFHQT ALEHPEL SELKT VT GTNP (SEQ ID NO: 100), PEA
VLAGNPAKHDLDIKPTVISHRLHFPE (SEQ ID NO: 101), LAH4
KKALLALALHHLAHLALHLALALK (SEQ ID NO: 102), HGP
LLGRRGWEVLKYWWNLLQYW SQEL (SEQ ID NO: 103), H5WYG
GLFHAIAHFIHGGWHGLIHGW Y G (SEQ ID NO: 104),
HA2 GLF GAIAGFIENGWEGMIDGWY G(SEQ ID NO: 105),
EB1 LIRLW SHLIHIWF QNRRLKWKKK(SEQ ID NO: 106),
VSVG KFTIVFPHNQKGNWKNVPSNYHYCP(SEQ ID NO: 107),
KALA WEAKLAKALAKALAKHLAKALAKA (SEQ ID NO: 108),
JST-1 GLFEALLELLESLWELLLEA(SEQ ID NO: 109).
Examples of cell-penetrating peptides include SP AAVALLPAVLLALLAP (SEQ ID NO: 45), TAT Y GRKKRRQRRR (SEQ ID NO: 46), Penetratin
RQIKIWF QNRRMKWKK (SEQ ID NO: 47), pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO: 48); M918 M VT VLFRRLRIRRAC GPPRVR V (SEQ ID NO: 49); Pep-1 KETWWETWWTEW SQPKKKRKV (SEQ ID NO: 50); Pep-2
KETWFETWFTEW SQPKKKRKV LCLRPVG (SEQ ID NO: 51) Arginine stretch RRRRRRRRR (SEQ ID NO: 52) Transportan
WTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 53) SynB l
RGGRLSYSRRRFSTSTGR (SEQ ID NO: 54) SynB3 RRLSYSRRRF (SEQ ID NO: 55) and PTD4 YARAAARQARA(SEQ ID NO: 56).
Examples of nuclear localization signals include Ela KRPRP (SEQ ID NO: 57) SV40 T-Ag PKKKRK V ( SEQ ID NO: 58) c-myc PAAKRVKLD(SEQ ID NO: 59) Op-T-NLS S SDDE AT AD S QH AAPPKKKRK V (SEQ ID NO: 60) Vp3 KKKRK(SEQ ID NO: 61) Nucleoplasm in KRPAATKKAGQAKKKK (SEQ ID NO: 62) Histone 2B DGKKRKRSRK (SEQ ID NO: 63) Xenopus N1
VRKKRKTEEE SPLKDKD AKK SKQE(SEQ ID NO: 64) PARP
KRKGDEVDGVDECAKKSKK (SEQ ID NO: 65) PDX-1 RRMKWKK (SEQ ID NO: 66) QKI-5 RVHPYQR (SEQ ID NO: 67) HCDA
KRP AC TLKPEC VQ QLL VC S QE AKK (SEQ ID NO: 68) H2B GKKRSKA (SEQ ID NO: 69) v-Rel KAKRQR(SEQ ID NO: 70) Amida RKRRR(SEQ ID NO: 71) RanBP3 PPVKRERTS (SEQ ID NO: 72) Pho4p PYLNKRKGKP (SEQ ID NO: 73); LEF-1 KKKKRKREK (SEQ ID NO: 74) TCF-1 KKKRRSREK (SEQ ID NO: 75) BDV-P PRPRKIPR (SEQ ID NO: 76) TR2 KDC VINKHHRNRCQ Y CRLQR (SEQ ID NO:
77) SOX9 PRRRK(SEQ ID NO: 78), and Max PQSRKKLR(SEQ ID NO: 79)
Subcellular localization signals include: mitochondrial signal NLVERCFTD (SEQ ID NO: 80), MLSLRQSIRFFK (SEQ ID NO: 81) cytochrome c oxidase subunit IV Mitochondrial signal MLISRCKW SRFPGNQR (SEQ ID NO: 82), peroxisome signal sequence - SKL, PTS1 nucleolar signal sequence MQRKPTIRRKNLRLRRK (SEQ ID NO: 83), and nucleolar signal sequence KQAWKQKWRKK (SEQ ID NO: 84).
In one embodiment the RHIM-interacting agent is encapsulated in PLGA (polylactide-co-glycolic-acid) nanoparticles, modified with, for example, 7 aminoacid gly copeptide (g7) ligand or PEG or Diptheria Toxin for crossing the blood brain barrier.
In one embodiment, the membrane permeating moiety is a lipid moiety, such as a Cio-C20 fatty acyl group, especially octadecanoyl (stearoyl; Ci8), hexadecanoyl (palmitoyl; Ci6) or tetradecanoyl (myristoyl; Ci4); most especially tetradecanoyl. In one embodiment, the membrane permeating moiety is conjugated to the N- or C-
terminal amino acid residue or through the amine of a lysine or other hydrophillic side- chain of the peptide containing molecule, especially the N-terminal amino acid residue. In one embodiment, the membrane permeating moiety is conjugated through the amine of the N-terminal amino acid residue.
In one embodiment the cell penetrating peptide is derived from HIV TAT peptides, such as Y GRKKRPQRRR (SEQ ID NO: 110) (HIV TAT47-57),
GRKKRRQRRRPPQ (SEQ ID NO: 111), GY GRKKRRQRRR(SEQ ID NO: 112), or RKKRRQRRR(SEQ ID NO: 113).
In one embodiment Z 1 and or Z2 comprise cell targeting or delivery moieties such as without limitation, RGD, NDG, NGR, CREKA, LyP-1, F3, SMS, IF7, and H2009 and antigens on or near the surface of target immune or tumor cells.
In one embodiment Z1 or Z2 comprise an aptamers (DNA or RNA).
Other suitable binding agents are known in the art and include antigen binding constructs such as affimers, aptamers, or suitable ligands (receptors) or parts thereof.
Antibodies, such as monoclonal antibodies, or derivatives or analogs thereof, include without limitation: Fv fragments; single chain Fv (scFv) fragments; Fab' fragments; F(ab')2 fragments; humanized antibodies and antibody fragments; camelized antibodies and antibody fragments, and multivalent versions of the foregoing. Multivalent binding reagents also may be used, as appropriate, including without limitation: monospecific or bispecific antibodies; such as disulfide stabilized Fv fragments, scFv tandems (scFv) fragments, diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e. leucine zipper or helix stabilized) scFv fragments.
The term "antibody fragments", as used herein, include any portion of an antibody that retains the ability to bind to the epitope recognized by the full length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), disulfide-linked Fvs (dsFv), and fragments comprising either a VL or VH region. Antigen-binding fragments of antibodies can comprise the variable region(s) alone or in combination with a portion of the hinge region, CHI, CH2, CH3, or a combination thereof. Preferably, the antibody fragments contain all six CDRs of the whole antibody, although fragments containing fewer than all six CDRs may also be functional.
"Single-chain FVs" ("scFvs") are antigen-binding fragments that contain the heavy chain variable region (VH) of an antibody linked to the light chain variable region (VL) of the antibody in a single polypeptide, but lack some or all of the constant domains of the antibody. The linkage between the VH and VL can be achieved through a short,
flexible peptide selected to assure that the proper three-dimensional folding of the VL and VH regions occurs to maintain the target molecule binding-specificity of the whole antibody from which the scFv is derived. scFvs lack some or all of the constant domains of antibodies.
Methods of making antigen-specific binding agents, including antibodies and their derivatives and analogs and aptamers, are well-known in the art. Polyclonal antibodies can be generated by immunization of an animal. Monoclonal antibodies can be prepared according to standard (hybridoma) methodology. Antibody derivatives and analogs, including humanized antibodies can be prepared recombinantly by isolating a DNA fragment from DNA encoding a monoclonal antibody and subcloning the appropriate V regions into an appropriate expression vector according to standard methods. Phage display and aptamer technology is described in the literature and permit in vitro clonal amplification of antigen-specific binding reagents with very affinity low cross-reactivity. Phage display reagents and systems are available commercially, and include the Recombinant Phage Antibody System (RPAS), commercially available from Amersham Pharmacia Biotech, Inc. of Piscataway, New Jersey and the pSKAN Phagemid Display System, commercially available from MoBiTec, LLC of Marco Island, Florida. Aptamer technology is described for example and without limitation in US Patent Nos. 5,270,163; 5,475,096; 5,840,867 and 6,544,776.
Optionally, one or more modified amino acid residues are selected from the group consisting of: a glycosylated amino acid, a PEGylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino acid, and an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent. RHIM interacting peptides may comprise at least one N-linked sugar, and may include two, three or more N-linked sugars. Peptides may also comprise O-linked sugars. RHIM interacting peptides or agents may be produced in a variety of cell lines that glycosylate the protein in a manner that is suitable for patient use, including engineered insect or yeast cells, and mammalian cells such as COS cells, CHO cells, HEK cells and NSO cells. In some embodiments the RHIM peptide is glycosylated and has a glycosylation pattern obtainable from a Chinese hamster ovary cell line. In most embodiments the RHIM interacting agent is synthesised and component parts added using techniques known in the art.
In some embodiments, the subject RHIM interacting agents have a half-life of about 1, 2, 3, 4, 6, 12, 24, 36, 48, or 72 hours in a mammal (e.g., a mouse or a human). Alternatively, they may exhibit a half-life of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 20, 25, or 30 days in a mammal (e.g., a mouse or a human) depending upon conjugate features
and the mode of administration. For example, peptides modified to bind to serum proteins typically display longer half lives. One strategy is to introduce peptide based ligand with high affinity for human albumin, in addition a fatty acid linked peptide may be employed.
The size of peptide may be modified to alter its hydrodynamic radium and renal clearance. PEGylation and lipidation often with linkers are established modifications to increase serum half life of agents by reducing clearance and protection from proteases. Second-generation PEGylation processes introduced the use of branched structures as well as alternative chemistries for PEG attachment. In particular, PEGs with cysteine reactive groups such as maleimide or iodoacetamide allow the targeting of the PEGylation to a single residue within a peptide reducing the heterogeneity of the final product. Furthermore, biodegradable hydrophilic amino acid polymers that are functional analogs of PEG have been developed, including XTEN (see US 20190083577) and PAS that are homogeneous and readily produced. Chemical linkage of antibody to peptide as developed by ConX illustrate illustrate a range of hybrid peptide half life extension methods that promise to overcome may of the disadvantages of earlier methods.
Oral and injectable solution solubilizing excipients include water-soluble organic solvents (polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerin, N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide), non ionic surfactants (Cremophor EL, Cremophor RH 40, Cremophor RH 60, d-a-tocopherol polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, Solutol HS 15, sorbitan monooleate, poloxamer 407, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire 44/14, Softigen 767, and mono- and di-fatty acid esters of PEG 300, 400, or 1750), water-insoluble lipids (castor oil, com oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil and palm seed oil), organic liquids/semi-solids (beeswax, d-a-tocopherol, oleic acid, medium-chain mono- and diglycerides), various cyclodextrins (a-cyclodextrin, b-cyclodextrin, hydroxypropyl-P-cyclodextrin, and sul fobuty 1 ether-P-cy cl odextri n ), and phospholipids (hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, 1-a- dimyristoylphosphatidylcholine, 1-a-dimyristoylphosphatidylglycerol). The chemical techniques to solubilize agents for oral and injection administration include pH adjustment, cosolvents, complexation, microemulsions, self-emulsifying drug delivery systems, micelles, liposomes, and emulsions.
Expression Constructs/Vectors
A construct or vector for expressing a RHIM-interacting peptide of Structure 1 in a recipient cell can comprise one or more DNA regions comprising a promoter operably linked to a nucleotide sequence encoding the peptide. The promoter can be inducible or constitutive. Examples of suitable constitutive promoters include, e.g., an immediate early cytomegalovirus (CMV) promoter, an Elongation Growth Factor - la (EF-la) gene promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, aMoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
The expression constructs may be generated by any suitable method including recombinant or synthetic techniques, utilizing a range of vectors known and available in the art such as plasmids, bacteriophage, baculovirus, mammalian virus, artificial chromosomes, among others. The expression constructs can be circular or linear, and should be suitable for replication and integration into eukaryotes. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses and lentiviruses. A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject stem cells. A number of retroviral systems are known in the art.
In a specific embodiment of the present invention, where the peptide is provided as a nucleic acid encoding the peptide, the nucleic acid may be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular (e.g., by use of a retroviral vector, by direct injection, by use of microparticle bombardment, by coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide or other intracellular targeting moiety. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression.
A nucleic acid molecule as described herein may in any form such as DNA or RNA, including in vitro transcribed RNA or synthetic RNA. Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules and modified forms thereof. A nucleic acid molecule may be single stranded or double stranded and linear or closed covalently to form a circle. The RNA may be modified by stabilizing sequences, capping, and polyadenylation. RNA or DNA and may be delivered as plasmids to express the peptide. RNA-based approaches are routinely available.
The term "RNA" relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a b- D-ribofuranosyl group. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non- naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
An optimised mRNA based composition could comprise a 5' and 3’ non translated region (5'-UTR, 3’-UTR) that optimises translation efficiency and intracellular stability as known in the art. An open reading frame encoding the RHIM-interating peptide of Structure 1. In one embodiment, removal of uncapped 5 '-triphosphates can be achieved by treating RNA with a phosphatase. RNA may have modified ribonucleotides in order to increase its stability and/or decrease cytotoxicity. For example, in one embodiment, in the RNA, 5-methylcytidine is substituted partially or completely, for cytidine. In one embodiment, the term "modification" relates to providing an RNA with a 5'-cap or 5'- cap analog. The term "5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'- cap, preferably to the 7-methylguanosine cap. The term "5'-cap" includes a 5'-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and/or enhance translation of RNA. Providing an RNA with a 5'-cap or 5'-cap
analog may be achieved by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'-cap analog, wherein said 5'-cap is co- transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5'-cap may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
A further modification of RNA may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5'- or 3 '-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA, for example, the exchange of the existing 3'-UTR with or the insertion of one or more, preferably two copies of a 3 '-UTR derived from a globin gene, such as alpha2-globin, alphal-globin, beta-globin. RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence. In order to increase stability and/or expression of the RNA it may be modified so as to be present in conjunction with a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues. In order to increase expression of the RNA it may be modified within the coding region so as to increase the GC-content to increase mRNA stability and to perform a codon optimization and, thus, enhance translation in cells. Modified mRNA may be synthesised enzymatically and packaged into nanoparticles such as lipid nanoparticles and administered, for example intramuscularly.
The nucleic acid molecule can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are known in the art and disclosed in Remington, the Science and Practice of Pharmacy, 20th Edition, Remington, T, ed. (2000).
Treatment and Administration
In accordance with this disclosure, the RHIM-interacting agent disclosed herein can be administered to patients with a cancer, such as a solid tumor. As determined herein intracellular amyloid formation through the action of the RHIM-interacting agent within receptive cells (comprising RHIM-containing polypeptides) leads to rapid internal membrane lysis and cell death. Additional activity of the RHIM-interacting agent is to engender cell death and activation and release of proinflammatory cytokines including IL-Ib within in extra-tumor, intratumor or intrastroma immune cells, or at one or two or more such locations sufficient to induce reduced tumor growth.
This ability of the present RHIM-interacting agents to induce in a time limited fashion an artificial inflammatory response will also find application in the use of the agents as adjuvants/ immunostimulators in vaccine compositions or in conjunction with vaccine compositions.
Furthermore, RHIM-interacting agents are for use in limiting pathogenesis in a subject.
Thus, compositions comprising RHIM-interacting agents of Structure 1 or derivatives or analogs thereof as described herein are for use in prophylaxis or therapy.
In one embodiment, the RHIM interacting agent comprises following structure I, N' to C:
ZiWaXi x2 x3 x4 Xs Xe Xv Xs X9 Xio Xu X12 X13 XM X15 Xie Xn WbZ2
Xi is present or absent and where present is any amino acid, or an amino acid corresponding to the amino acid in a RJPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position typically a residue with a hydrophobic or polar/neutral side chain
X2 is present or absent and where present is any amino acid or is an amino acid corresponding to the amino acid in a RJPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position, typically polar or a hydrophobic residue
X3 is Isoleucine (I) or a hydrophobic residue such as Leucine or Norleucine
X4 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1,
RIPK3, DAI, TRIF or orthologous polypeptides at this position
X5 is Asparagine (N)
X6 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position
X7 is any amino acid
X8 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position, such as a Glycine (G) or Proline (P) or Alanine (A)
X9 is Isoleucine (I) or Valine (V)
Xio is Glutamine (Q)
X11 is Isoleucine (I) or Valine (V) or Leucine (L)
Xi2 is Glycine (G)
Xi3 is any amino acid or an amino acid corresponding to a RIPKl, RIPK3, DAI, TRIF or orthologous polypeptides at this position
Xi4 is any amino acid or an amino acid corresponding to a RIPKl, RIPK3, DAI, TRIF or orthologous polypeptides at this position
Xi5 is Asparagine (N)
Xi6 is present or absent and where present is any amino acid typically polar such as Tyrosine (Y)
Xi7 is present or absent and where present is a methionine (M) or Leucine (L) or Isoleucine (I);
Wa and Wb are present or absent and where present is each 5 to 20 contiguous amino acids that immediately flank Xi to Xn, where present, in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides, or a conservative substitution thereof;
Zi and Z2 are individually present or absent and where present are individually selected from one or more of a linker, stability enhancing, delivery enhancing or label moiety; or a pharmaceutically acceptable salt, hydrate, tautomer, sterioisomer, pro-drug thereof.
In one embodiment, the ortholog or sequence employ the human sequence.
Treatment or therapy refers to effective inhibition (i.e., slowing down or elimination) of the growth or metastasis of the cancer.
In one embodiment, the cancer involves a solid tumour.
In one embodiment, the cancer is an epithelial cell or endothelial cells cancer.
In one embodiment, the cancer is a cytokine sensitive cancer such as an IL-IB sensitive cancer.
In one embodiment the cancer may be tested for sensitivity to the RHIM interacting agent and is selected from the group consisting of ABL1 protooncogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, brain stem glioma, brain and CNS tumors, breast cancer, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, cutaneous T-cell lymphoma, dermatofi brosarcoma-protuberans, desmoplastic-small-round-cell-tumor, ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconi anaemia, fibrosarcoma, gall bladder cancer, gastric cancer, genitourinary cancers, germ cell tumours, gestational-trophoblastic-disease, glioma, gynaecological cancers, hematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer,
intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhan's- cell-histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, li-fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-hodgkin's lymphoma, male breast cancer, malignant-rhabdoid-tumor-of- kidney, medulloblastoma, melanoma, merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(NSCLC), ocular cancers, oesophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral-neuroectodermal-tumors, pituitary cancer, polycythemia vera, prostate cancer, rare-cancers-and-associated-disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, rothmund-thomson syndrome, salivary gland cancer, sarcoma, schwannoma, sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell- cancer- (renal-pelvis-/- ureter), trophoblastic cancer, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom's-macroglobulinemia and Wilms' tumor.
Thus biopsy samples may be tested in vitro for direct sensitivity to RHIM interacting agents or sensitivity to inflammasome activated immune cells.
The treatments disclosed herein may be combined with other treatments for the specific cancer, pathology, or therapeutic or prophylactic needs identified. For such combination therapies, each component of the combination therapy may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect. Alternatively, the components may be together in a single dosage unit as a combination product. When administered separately, it may be preferred for the components to be administered by the same or different routes of administration.
The compositions may be delivered by injection, by topical or mucosal application, by inhalation or via oral route including modified release modes, over periods of time and in amounts which are effective to enhance inflammatory cytokine levels in a subject sufficient to increase the effectiveness of the immune response, to maintain or improve health. Administration may be systemic (e.g., parenteral via for
example intravenous, intraperitoneal, intradermal, sub cutaneous or intramuscular routes) or targeted.
The amount of the agent to be administered may be determined by standard clinical techniques by those of average skill within the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the nature of the agent and other clinical factors (such as the condition of the subject their weight, the route of administration and type of composition). The precise dosage to be therapeutically or prophylactically effective and non-detrimental can be determined by those skilled in the art. Pharmaceutical compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington, the Science and Practice of Pharmacy, 20th Edition, Remington, T, ed. (2000) and later editions.
However, suitable dosage ranges for intravenous administration of the peptide of the present invention are generally about 1.25 - 5 micrograms of active compound per kilogram (Kg) body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral compositions preferably contain 10% to 95% active ingredient.
By "derivative" is meant a peptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to a parent sequence including additions, or deletions that provide for functionally equivalent molecules.
By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state.
The term "subject," includes patient, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, most particularly a human, for whom prophylaxis or therapy is desired. The subject may be in need of prophylaxis or treatment for a cancer or other pathology, however, it will be understood that the aforementioned terms do not imply that symptoms are present.
The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotides in length.
The term sequence "identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base {e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Amino acid sequence identity may also be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is accessible at the website located at www.ebi.ac.uk/Tools/emboss/align/. This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are utilized which include Gap Open: 10.0 and Gap Extend 0.5. The default matrix "Blosum62" is utilized for amino acid sequences and the default matrix.
The term sequence "similarity" refer to the percentage number of amino acids that are identical or constitute conservative amino acid substitutions as defined in Table 1 below. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984 Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
The present description is further illustrated by the following examples, which should not be construed as limiting in any way.
EXAMPLES
EXAMPLE 1 - RIIIM consensus sequence peptides form intracellular amyloid fibrils in macrophages
It was proposed that a soluble cell penetrating monomeric unit would upon cellular internalization in a concentration dependent manner, polymerize into an amyloid fibril both independently and in combination with native RHIM containing proteins mimicking the intracellular scaffold formations that are responsible both for concurrent intracellular cytokine production and lytic cell death.
Peptides were created utilizing the evolutionarily conserved RHIM consensus sequences within RIPK1 and RIPK3 native proteins to induce artificial inflammatory events (RHIM consensus sequence XXIXNXXXIQIGXXNXM). The cell penetrating peptide, Trans- Activator of Transcription (TAT) sequence was added from human immunodeficiency virus (HIV), to both increase solubility and promote intracellular delivery (Figure 1A).
Several sequences were tested (TAT-RIPK1 -
Y GRKKRRQRRRKKKYTIFN S SGIQIGNHNYM (SEQ ID NO: 85) and TAT-RIPK3 - Y GRKKRRQRRRKKKL VFN C SE V QIGNY SL (SEQ ID NO: 86) to ensure stability and solubility of the monomeric unit, a 17 amino acid truncated version of the evolutionary conserved 34 amino acid consensus sequence of both RIPKl and RIPK3 were synthesized via solid state peptide synthesis, purified via HPLC and characterized by mass spectrometry
As a control, alanine amino acids were inserted into key amino acids identified to play an important role in RHIM amyloid self-assembly. These pure compounds were then subjected to a thioflavin t assay to assess relative homo- and hetero- amyloid formation both in a concentration (Figure 1b) and time dependent fashion (Figure 1C).
It became apparent that the kinetics (Figure 1C) and strength (Figure IB) of amyloid formation was sequence dependent with RIPKl enhancing overall ThT fluorescence.
Bone marrow derived macrophages (BMDMs) were cultured in the presence of RIPKl (Rl), RIPK3 (R3) and Alanine (A) containing peptide units. The intracellular kinetic and strength of amyloid formation was similar to that found initial ThT buffer screening assays. Consistent with previous observations RIPKl performed better in overall intracellular amyloid formation (Figure IE). The possibility of amyloid assembly with the native RIPKl protein was also investigated and indeed amyloid formation was enhance in the presence of native RIPKl (Figure IF) suggesting that not only are
amyloids form through peptide-peptide interactions but also through protein-peptide interactions.
Methods:
Peptide synthesis and characterization: A series of peptides were initially synthesized using solid phase peptide synthesis on a Liberty Blue Microwave synthesizer. Following synthesis peptides were cleaved utilizing reagent K and purified via high performance liquid chromatography, peptides were characterized for purity and accuracy of sequence via Matrix- Assisted Laser desorption/ionization time of flight mass spectrometry. Truncated constructs were subjected to amyloid, cell death and cytokine assays to validate solubility and efficacy. Following a series of validations, constructs were purchased through ELIM by Biopharmaceuticals, all assays represented in the patent were done using these synthesized constructs. HPLC traces and Electrospray Mass Spectrometry Characterization provided by ELIM can be found in Figure 13.
Animal Experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Washington and the animal ethics committee at the Walter and Eliza Hall Medical Institute. All studies performed under guidelines of veterinarians at the respective institutes in accordance with use and regulations. Bone marrow derived macrophages were obtained from the C57BL/6 background and were obtained from the Walter and Eliza Hall Parkville, mice used in culture were of a variety of ages and sexes, all experiments (except when noted) were performed utilizing three different mice often from different breading colonies and may not be littermate controls.
Soluble and cellular amyloid assays: The water soluble Thioflavin T (ThT) exhibits an excitation maximum shift and increased fluorescence intensity upon binding to amyloid fibrils from 385-450 to 445-482 nm, we utilized this molecule as a molecular readout for peptide amyloid formation both in a soluble assay at physiological salt and pH conditions and intracellularly through in-vitro cell culture assays. A 25 mM final concentration of Thioflavin T (ThT) in PBS at pH 7.4 was used in all assays (cellular and plate reader), to identify the relative fluorescence intensity of the formed amyloid fibril. For soluble assays run on the plate reader in 96 well plates, peptides were diluted in 100 uL to the final concentration noted within the figure legend, and 100 uL 50 pM of ThT was added prior to fluorescence intensity measurements, kinetic measurements were measured sequentially on the same samples. Measurements were run on a variety of different plate readers in two different countries and at least three different laboratories which had variability in fluorescence intensity, while all measurements were similar in output,
ranges of intensity varied between instruments. Experiments were performed in each location to ensure stability and reproducibility of the peptide amyloid formation; reported figures are a representative image from sum total of these experiments. Studies that were performed to analyze the relative ThT staining within bone marrow derived macrophages were performed similarly, however upon ThT analysis images from different microscopes were analyzed separately utilizing relative fluorescence intensity measurements in image J and normalized to the relative number of nuclei in each image. Briefly, BMDMs were plated and cultured on collagen coated glass coverslips, incubated with peptides at 25 mM concentration for 30 minutes, and 2 hour time points. Cells were fixed in 4% PFA at room temp for 15 minutes, washed 3x in PBS, blocked for 1 hour in a 5% BSA, .01% tween, 10% donkey serum solution and stained overnight in blocking solution with no serum containing 25 mM solution of ThT and DAPI (1 :5000).
EXAMPLE 2 - RIIIM based peptides induce cytokine secretion
Amyloid-inducing RHIM peptides activate the NLRP3 inflammasome.
RHIM domain containing scaffolds can activate NF-kB (Dondelinger, 2015). An immortalized BMDM NF-KB reporter cell line was used, and it was observed that the RIPKl RHIM peptide mediated enhanced NF-KB activation (Figure 2A). This was associated with increased production of IL-6, although not to the level of IL-6 induced by LPS (Figure 2B). As it was our initial hypothesis that these peptides could induce activation of the NLRP3 inflammasome downstream of amyloid formation, we sought to quantify the secretion of IL-Ib. Indeed, this was produced in a concentration dependent manner by the RHIM peptides (Figure 2C), and was greatly enhanced by pre-stimulating macrophages with LPS to first enhance NLRP3 and precursor IL-Ib levels (known as inflammasome priming) (Figure 2D). BMDMs deficient for either caspase-1 or NLRP3 ablated RHIM peptide-induced IL-Ib secretion (Figure 2E) demonstrating that, akin to other amyloid-forming proteins, the RIPK RHIM amyloid engages the NLRP3 inflammasome. The pan-caspase inhibitor IDUN-5556 (IDHN), currently in clinical trials, also prevented IL-Ib production by the RHIM peptides (Figure 2F). Caspase-8 deletion reduced both nigericin, a canonical NLRP3 activator, and RHIM peptide mediated IL-Ib secretion (Fig. S5), which most likely reflects the reported role for caspase-8 in inflammasome priming (Allam, 2014). Consistent with amyloid forming proteins triggering potassium ion efflux to activate NLRP3 (Masters, 2010) the inhibition of this event by incubating cells in high concentrations of extracellular potassium chloride also ablated RHIM peptide-induced IL-Ib secretion (Figure 2G). Overall, these
data suggest that amyloid forming RHIM peptides trigger the NLRP3 inflammasome to cause IL-Ib secretion in vitro.
Methods
Animal Experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Washington and the animal ethics committee at the Walter and Eliza Hall Medical Institute. All studies performed under guidelines of veterinarians at the respective institutes in accordance with use and regulations. A variety of knockout models (WT, Mlkl-/- (Murphy, 20130, Nlrp3-/- (Martinon, 2006), Caspl-/-(Kuida, 1995), Ripk3-/ (Newton, 2004)-, Gsdmd-/-(Shi, 2015; Kayagaki, 2015 ) on the C57BL/6 for in-vitro bone marrow macrophage cell culture were obtained from the Walter and Eliza Hall Parkville, mice used in culture were of a variety of ages and sexes, all experiments (except when noted) were performed utilizing three different mice often from different breading colonies and may not be littermate controls.
Peptide Macrophage Characteristics In Vitro. Bone marrow macrophages were generated from freshly isolated bone marrow flushed from the femurs of mice. Bone marrow was differentiated in DMEM containing 10% FBS, and 20% L929 supplemented for 7 days on non-TC treated plastic and scraped and plated. Macrophages and tumor cells were plated at 100,000 cells per well in a 96 well plate. Cells were assayed for confocal microscopy, flow cytometry, the supernatant of the cells was assayed for cytokine levels. Cytokine expression was analyzed following 4 hours of incubation with peptides, utilizing IL-Ib and IL-6 Duo mouse cytokine set from Biolegend or R&D Systems IL-lbeta Duo mouse kit (DY-401).
Example 3 -RHIM based peptides induce cell death and intracellular membrane lysis Not all pro-inflammatory cytokines are membrane permeable, and accordingly lytic mechanisms including those that induce cell death can be essential to induce cytokine release. As the present peptides had the potential to release multiple cytokines into the extracellular space, the inventors sought to investigate if this might be mediated through lytic cell death.
Amyloid forming RHIM peptides induce a lytic cell death
Consistent with amyloid being a potent cell death trigger, a concentration and sequence dependent cell death was observed on multiple cell lines treated with RHIM peptides (Figure 3 A, Figure 10). Genetically, a decrease in cell death was induced by RIPK RHIM
peptides at 25 mM by deletion of NLRP3, Caspase-1 and GSDMD (Figure 3B, C and D), consistent with the ability of RHIM peptide treatment to cause GSDMD processing to its active pore-forming p30 fragment. We had previously observed that NLRP3 activation downstream of RHIM peptide treatment was dependent on potassium efflux (Figure 2), and the presence of increased extracellular potassium also inhibited RIPK RHIM peptide-induced cell death (Figure 3E). Potassium efflux resulting from intracellular amyloid has been reported to be triggered as a consequence of lysosomal rupture (Masters, 2010; Niemi, 2011;Halle, 2008) and as early as 30 minutes following RHIM peptide exposure a significant decline in lysosomal function was observed, as reflected by the loss in lysotracker fluorescence (Figure 3F), with further decreases observed over the course of two hours (Figure 3G). Importantly, RHIM peptide induced cell death was significantly decreased by the genetic deletion of RIPK1, but not RIPK3 (Figure 3H and I), consistent with the present data suggesting that RHIM peptides can engage homo- and hetero-amyloid structures with endogenous full-length counterparts (Figure 1). Therefore, endogenous RIPK1 is likely to be the most proximal intracellular RHIM nucleating event leading to SMOC formation induced at relatively low concentrations of cellular RHIM interacting peptides.
Notably, the loss of the pyroptotic (i.e. NLRP3, CASP1, GSDMD) and necroptotic (RIPKl, RIPK3, MLKL) machinery at higher concentrations of RHIM interacting peptide failed to protect from RHIM peptide induced cell death, suggesting that RHIM peptides can also directly trigger cell lysis. Biophysical literature suggests that amyloids, when in their soluble protein form, can associate with cellular membranes and that subsequent polymerization of these proteins can lead to organelle rupture through physical self-assembly events (Nag, 2013; Last, 2011; Flach, 2012). Although we have been unable to observe such rupture in traditional membrane lysis models at physiological pH ~7.5 we did observe membrane lysis at pH 5.5 and 6.0, consistent with literature that has reported amyloid-induced lysis at acidic pH (Figure 3J) (Gustavsson, 1991 ; Colon, 1992). Therefore, it was conclude that these peptides induce lytic cell death likely mediated through combined biophysical and genetic mechanisms.
The inventors have shown that the present RHIM interacting agents induce robust lytic cell death. Accordingly, in one embodiment, reduced tumor progression is proposed through immune activation and lytic cell death mechanism, rather than through administration to the tumor cell alone.
Animal Experiments. (As above)
Peptide Macrophage Characteristics In Vitro. Bone marrow macrophages were generated from freshly isolated bone marrow flushed from the femurs of mice. Bone marrow was differentiated in DMEM containing 10% FBS, and 20% L929 supplemented for 7 days on non-TC treated plastic and scraped and plated. Macrophages and tumor cells were plated at 100,000 cells per well in a 96 well plate. Cells were assayed for confocal microscopy, flow cytometry, the supernatant of the cells was assayed for cytokine levels. For flow cytometry, after 24 hours of incubation with RHIM peptides, cells were removed from the wells following a wash with PBS, by incubation in 5mM EDTA/PBS solution on ice. During this process, cells were exposed to repeated pipetting or mechanical disruption to obtain a single cell suspension. Cells were then stained with PI (10pg/mL) and cell death quantified by flow cytometry. For confocal microscopy, cells were plated directly on collagen coated glass coverslips and imaged utilizing a Leica SP8X and images were quantified and analyzed utilizing ImageJ.
Biophysical lysis studies. Peptides were incubated at 37 degrees for 30 mins to 2 hours with human erythrocytes isolated from whole blood. For erythrocytes, hemolysis was assessed at a variety of buffered pHs ranging from 5.5-8, % hemolysis determined as a percentage of control (Triton-x at relative pH), absorbance of collected supernatants were measured at 541nm.
EXAMPLE 4 - RHIM peptides induce artificial inflammation events in-vivo to treat a triple negative breast cancer model (including cytokine secretion and modulation of pro-inflammatory cells (CD 8+ T-cells and macrophages))
RIPK1 RHIM peptide induces tumor regression andIL-Ib production in vivo.
Given increased interest in the use of inflammatory mediators together with agents that have an anti -turn or effect, the RIPKl RHIM peptide was used experimentally to treat a triple negative mammary cancer model in vivo (Figure 4A). Indeed, a decrease in tumor growth was observed following intravenous injection of RIPKl RHIM peptide (Figures 4B). Tumor growth inhibition correlated with increased IL-Ib serum levels (pilot data not shown), and both cytokine and tumor reduction was independent of traditional amyloid exposure (Figure 4B). This suggests that intracellular uptake and potentially peptide-protein interactions of the RIPKl peptide is essential for its efficacy. A modest increase in CD3/CD8+ cells was observed, and myeloid cells within all amyloid treated models, consistent with the formation of an inflammatory anti-tumor microenvironment (Figure 8). Given the dramatic increase in IL-Ib in peptide treated animals, the inventors sought to investigate if reduced tumor growth was dependent on
IL-Ib secretion. We have previously illustrated that IL-Ib secretion could be ablated through removal of caspase-1 both genetically and therapeutically with the clinical caspase inhibitor IDUN (Figure 2F). The inventors hypothesized that co-delivery of IDUN and RHIM interacting peptides simultaneously could rescue delayed tumor growth, through reduced IL-Ib secretion. Indeed, co-delivery of both RIPK1 RHIM peptide with IDUN returned tumor growth rate to the same level as saline treated conditions (Figure 4E), and also inhibited intratumoral IL-Ib production, suggesting that cytokine secretion is required for tumor suppression (Figure 4E and Figure 9). It appears that cytokine secretion needs to be present both within the tumor and systemically, as when peptides are injected intratumorally we observe no increase in systemic IL-Ib and no overall effect on tumor growth (Figure 9). In summary, this work documents the generation of a cell penetrant RHIM peptide that triggers cell death and systemic inflammasome activation for anti-tumor effects in vivo.
Animal Experiments. (As above)
4T1 Animal Model: 4T1 cells (500,000) in a 50:50 mixture of Matrigel (Fisher) were injected into the 4th nipple. Successful injection was confirmed via calipers. Tumors developed to a size ~ 50 mm3 within 5 days. Animals were injected retro-orbitally with 5 mg/kg (every other day) or 3.5 mg/kg (daily) peptide (Rl, R3, A, LAPP) in PBS or PBS alone (as noted in the figure legend) post-injection of cells. In some cases, animals were also simultaneously injected with IDUN-5556 at 2.5 mg/kg intraperitoneally, whenever possible IP injections were performed prior to retro-orbital (RO) injections to ensure complete caspase inhibition prior to peptide injection. IDUN-5556 and unmodified peptide constructs have very short half-lives (> 30 minutes) in-vivo (Brumatti, 2016; Brumatti, 2016; Mathur, 2016).
4T1 Animal Staining: Animals were sacrificed, perfused, and tumors were extracted and fixed at 4% PFA overnight at 4 degrees, transferred to 30% sucrose solution for 24 hours at 4 °C and flash frozen in Tissue-Tek OCT in preparation for immunofluorescence (IF) and cryosectioned at 20 pm. Sections were thawed, fixed in 4% PFA for 10 minutes at room temperature, washed 3x with PBS, and then incubated with blocking solution (5% BSA, .01% Tween, PBS, 10% Donkey Serum) for 60 minutes at room temperature. Tissue sections were then washed 3x with PBS and stained with primary anti-GSDMD and anti-IL-Ib antibodies (1 : 100, anti-rabbit, Abeam) overnight 4 degrees, followed by washing and secondary antibody incubation (1 :500, anti-Rabbit Alexa-647, 1 :5000 DAPI). Relative staining was analyzed from 3 random regions in 4 mice per group, each
mouse and parameters were quantified by manual measurements or by utilizing the analyze particle function in image J to measure the absolute number, or surface area. Absolute number measurements and area measurements required uniform threshold adjustments across all images, to ensure no overlap of“cells” within measured outputs; a cell was defined as having a single nucleus and analyzed statistically utilizing Prism. Tumor Digestions and Flow Cytometry Quantification. Freshly perfused tumors were placed into Miltenyi C-Tubes and enzymatically digested utilizing 5 mL RPMI with Collagenase type l(Sigma), Dispase (Sigma), and DNase-1 (Sigma). Briefly, tumors were placed into the enzymatic cocktail on ice and mechanically disrupted utilizing a gentleMACS Dissociator. Tumors were incubated utilizing agitation at 37 °C for 40 mins, and disrupted once more. Tumor lysates were spun down and applied to a 70 pm cell strainer. The subsequent cell suspension was counted and 200,000 cells were either freshly stained utilizing mouse FC Block, anti-mouse MerTK, CD64, CD206, CD86, CD3, or CD8 (Biolegend). Cell suspensions were run on a Miltenyi MACSQuant Analyzer and data was analyzed utilizing FlowJo and statistical analysis was run in Prism.
EXAMPLE 5
Illustrative peptide sequences
Note, the sample sequences above do not entail all DNA and RNA sequences. Additional RNA and DNA sequences can be obtained; codons can be replaced by utilizing the RHIM versions of the peptide sequence(s) (stated below and Figure 11) and the two codon tables below.
DNA:
**Note codons can be replaced, please see replacement table for substitution of codons a.
Replacement sequences:
RNA:
**Note codons can be replaced, please see replacement table for substitution of codons
Replacement codons:
Second Letter
3rd
tetter
Illustrative peptide modifications to improve stability:
1. Cyclization, examples
a. Disulfide bridges
b. Connection of phenolic side chains
c.ring-closing methatesis of dienes
d. Bisaryl ether bonds
2. N terminal modification, examples
a.Acetylation
b. Biotinylation
3. Non-natural amino acid substitution, examples:
a.D-amino acids
i. e.g. Octreotide
b. Alkylation of amino acids
c.p- and a- amino acids
4. Pseudopeptide replacements, examples:
a.Replacement of a labile peptide bond with a isoster b. Incorporation of reduced peptide bonds (CH2-NH) c. Retro-inversion
d. Incorporation of Azapeptides
5. Peptide-conjugation/prodrug, examples
a. Polymer (e.g. PEG, HPMA, etc)
i. Linear
1. e.g. Neulasta
2. e.g. Pegasys
ii. Micelle formulation
iii. Peptide conjugation
1. RGD
2. NDG
b. Protein
i. Albumin
ii. Fc
iii. Monocloncal and polyclonal antibodies iv. Peptides
v. Aptamers (DNA and RNA)
6. Peptide-formulation, examples:
a. Excipient
i. Cremophor
ii. PEG
b. Lipid membrane
i. liposomal
ii. Polymer
iii. micelle
7. Peptide-binding, examples
a. Albumin binding domain
b. Targeting peptide
i. RGD
Methods (for Figure 11):
Peptide synthesis and characterization: A series of peptides were initially synthesized using solid phase peptide synthesis on a Liberty Blue Microwave synthesizer. Following synthesis peptides were cleaved utilizing reagent K and purified via high performance liquid chromatography, peptides were characterized for purity and accuracy of sequence via Matrix- Assisted Laser desorption/ionization time of flight mass spectrometry. Truncated constructs were subjected to amyloid, cell death and cytokine assays to validate solubility and efficacy.
Soluble and cellular amyloid assays: The water soluble Thioflavin T (ThT) exhibits an excitation maximum shift and increased fluorescence intensity upon binding to amyloid fibrils from 385-450 to 445-482 nm, we utilized this molecule as a molecular readout for peptide amyloid formation both in a soluble assay at physiological salt and pH conditions and intracellularly through in-vitro cell culture assays. A 25 mM final concentration of Thioflavin T (ThT) in PBS at pH 7.4 was used in all assays (cellular and plate reader), to identify the relative fluorescence intensity of the formed amyloid fibril. For soluble assays run on the plate reader in 96 well plates, peptides were diluted in 100 uL to the final concentration noted within the figure legend, and 100 uL 50 mM of ThT was added prior to fluorescence intensity measurements, kinetic measurements were measured sequentially on the same samples. Measurements were run on a variety of different plate readers in two different countries and at least three different laboratories which had variability in fluorescence intensity, while all measurements were similar in output, ranges of intensity varied between instruments. Experiments were performed in each location to ensure stability and reproducibility of the peptide amyloid formation; reported figures are a representative image from sum total of these experiments.
EXAMPLE 6 - unmodified peptide assays showing RHIM interacting peptides are amyloidogenic
Peptide design and preparation
Synthetic peptides were purchased spanning the RHIM of RIP1, RIP3, TRIF, DAI, and MCMV-M45 (all murine sequences) and drosophila HMD, henceforth, rRIPl, rRIP3, rTRIF, rDAI, rMCMV, rlMD. IAPP peptide was purchased as an amyloidogenic positive control. Mutant forms of RIP 1 and RIP3 were designed using the aggregation prediction algorithm PASTA in an attempt to reduce amyloid propensity (see Table 1 for sequences). All peptides were received at >95% purity, dissolved in hexafluoroisopropanol, and stored at -80° in lyophilised aliquots. The Bax-BH3 peptide was the kind gift of Dr Dana Westphal. Peptides were resuspended in 0.1M acetic acid
before being diluted in MTPBS to a 500 mM stock, sonicated to eliminate aggregation and used within 30 min.
Peptide transfection
Mature, differentiated BMDMs, from WT and NLRP3 mice were harvested and plated at lxl05/well in 96-well plates and incubated overnight. Cells were primed with LPS (100 ng/ml, 1-2 h) and either untreated or treated with ThT (10 mM) before peptide transfection using lipofectamine as per manufacturers’ protocol (Synvolux Therapeutics), (final peptide concentration, 10 mM). Supernatants were collected after overnight incubation and cytokine release was assayed via ELISA (IL-Ib, R & D, eBioScience; TNF, eBioScience) according to manufacturers’ protocols. Transfection for microscopy was carried out in parallel.
Results
ThT bound to IAPP shows strong fluorescence (Figure 5a), whilst ThT fluorescence bound to Bax-BH3 is negligibly above background (Figure 5b). The RHIM peptides rRIP3 (Figure 6d) and rTRIF (Figure 5g) cause a strong fluorescent signal which increases slightly with time, whereas rRIPl (Figure 5c), rMCMV (Figure 5h), rIMD (Figure 5i) and rDAI (Figure 5j) elicit strong fluorescence which increases significantly over 5-10 hours and either stabilises or decreases slightly. In contrast, mutRIPl (Figure 5e) exhibits very low and fairly steady ThT fluorescence, while mutRIP3 (Figure 5f) has initially strong ThT fluorescence which rapidly decays to baseline.
These results show that wild-type RHIM peptides are amyloidogenic according to the criteria of ThT fluorescence. The engineered mutant peptides do not form intracellular amyloid. Furthermore, the engineered mutant peptides do not form intracellular amyloid when transfected into cells (results not shown).
EXAMPLE 7- RHIM peptides trigger IL-Ib secretion
It was investigated whether transfection of BMDMs with RHIM peptides could promote NLRP3 inflammasome activation of IL-Ib. The IAP antagonist, CpA was used as a positive control. This activates the ripoptosome and RIP3 to mediate NLRP3 activation of IL-Ib. Release of the pro-inflammatory cytokine TNF into the cell supernatant is used as a marker of normal TLR signalling (inflammasome‘priming’) and cytokine secretion, as TNF release is not dependent on inflammasome activation, so is a good indicator of inflammasome specificity when examining the effects of different stimuli or inhibitors on inflammasome function. Specifically, TNF release should not be altered by inflammsome inhibition, whereas release of IL-Ib will be reduced.
As expected TLR priming and IAP inhibition using CpA to activate the ripoptosome caused IL-I b release into the cell supernatant (Figure 6a). Similarly, TLR priming followed by transfection of WT BMDMs with rRIPl and rRIP3 alone resulted in the secretion of IL-Ib, which was significantly increased when compared to transfection of mutRIPl and mutRIP3 (/ O.O l and /K0.001 respectively) (Figure 6a). TLR priming and rTRIF and rIMD transfection resulted in less IL-Ib release, whilst rMCMV and rDAI resulted in intermediate IL-Ib secretion. None of the peptides, nor the transfection agent alone, affected TNF secretion (Figure 6b).
Example 8- RIIIM peptide activation of IL-Ib is mediated by the NLRP3 inflammasome
It was next examined whether the NLRP3 inflammasome could detect the RHIM peptides to modulate IL-Ib secretion. Transfection of TLR-primed NIrp3 BMDMs with RHIM peptides rRIPl, rRIP3, rIMD or IAPP showed a significant (/;<0.05) reduction in IL-Ib secretion when compared to WT macrophages (Figure 7). Similarly, the other RHIM peptides, rMCMV, rTRIF and rDAI showed a trend towards reduced IL- 1b secretion in Nlrp3 ! BMDMs, although this failed to reach significance. No reduction in IL-Ib activation was seen between WT and Nlrp3 ! when treated with mutRIPl or mutRIP3.
Together, these data indicate that IAP inhibition and RHIM peptides both activate IL-1 b through an NLRP3 -dependent mechanism, as shown by genetic deletion of NLRP3 inflammasome causing reduction of IL-Ib activation. Further, mutating residues of RIPl and RIP3 in order to reduce their amyloidigenicity reduces their ability to activate the NLRP3 inflammasome and cause IL-Ib secretion.
Example 9 - Peptides reduce pathogenic infections
Bone marrow macrophages were incubated with wild type and flagellin knockout luciferase containing L. pneumophila bacteria (Lp02). Macrophages recognize the flagellin on wild type (Lp02) L. pneumophila bacteria and control infection rates, when the flagellin has been knockout of the bacteria (AflaA) the macrophages can no longer control infection. The results demonstrate that if bone marrow macrophages were infected with Lp02 at multiplicity of infection of 1 for 96 hours, macrophages contained infection (Figure 14, top). However, when macrophages were incubated with Aria A bacteria (Figure 14, bottom), macrophages were unable to contain infection. When macrophages were co-incubated with R1 (RIPKl peptide), R3 (RIPK3 peptide) and A
(Alanine peptide), it was observed that RHIM peptides helped to facilitate decreased infection rates determined via luciferase measurements.
Animal Experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Washington and the animal ethics committee at the Walter and Eliza Hall Medical Institute. All studies performed under guidelines of veterinarians at the respective institutes in accordance with use and regulations. C57BL/6 for in-vitro bone marrow macrophage cell culture were performed utilizing 6-10 week-old female B ALB/c mice from Charles River Laboratories, it is unknown if mice were littermates.
Peptide Macrophage Characteristics In Vitro. Bone marrow macrophages were generated from freshly isolated bone marrow flushed from the femurs of mice. Bone marrow was differentiated in DMEM containing 10% FBS, and 20% L929 supplemented for 7 days on non-TC treated plastic and scraped and plated. Macrophages and tumor cells were plated at 100,000 cells per well in a 96 well plate.
L. pneumophila bacterial culture.
Lp02 and AflaA were streaked on BCYE with thymidine 0.1 mg/ml and incubated at 37 degrees C in 5% C02 for 4 days. Bacteria was harvested using a PBS rinse, pelleted and washed 3x with PBS. Dilution was determined to OD540 0.2-0.25 (2xl08 CFU/mL). Stocks were diluted to 5xl04 CFU/mL, vortexed and serial diluted and cultured 0.1 aliquotes on BCYE with thymidine.
Lp02 and AflaA Infection of macrophages
Lux Lp02 and Aria A were added at 5 xlO4 CFU/well (10 ul dilution or MOI of 1), centrifuged to ensure infection. Plates were incubated at 37 degrees for 2 hours, washed 3x with HBSS and read in luminometer. Plates were returned to the incubator and read at 24, 28, 72 and 96 hours.
Example 10- Peptides drive necroptosis when co-delivered with caspase inhibitors
It was next investigated if RHIM peptide sequences could be used for the induction of necroptosis. The data showed limited to no impact on cell death following the deletion of proteins necessary for the progression of necroptosis, such as MLKL (Figure 12) and RIPK3 (Figure 3). However, when caspases are simultaneously inhibited,
such as in co-treatment with zVAD (pan-caspase inhibitor), the peptides are able to drive necroptosis. This is demonstrated via the deletion of MLKL in Figure 12.
Animal Experiments. (As above) A variety of knockout models (WT, Mlkl-/-{Murphy, 2013)) on the C57BL/6 for in-vitro bone marrow macrophage cell culture were obtained from the Walter and Eliza Hall Parkville, mice used in culture were of a variety of ages and sexes, all experiments (except when noted) were performed utilizing three different mice often from different breading colonies and may not be littermate controls.
Peptide Macrophage Characteristics In Vitro. Bone marrow macrophages were generated from freshly isolated bone marrow flushed from the femurs of mice. Bone marrow was differentiated in DMEM containing 10% FBS, and 20% L929 supplemented for 7 days on non-TC treated plastic and scraped and plated. Macrophages and tumor cells were plated at 100,000 cells per well in a 96 well plate. For flow cytometry, after 24 hours of incubation with RHIM peptides and/or 20 mM zVAD, cells were removed from the wells following a wash with PBS, by incubation in 5mM EDTA/PBS solution on ice. During this process, cells were exposed to repeated pipetting or mechanical disruption to obtain a single cell suspension. Cells were then stained with PI (10pg/mL) and cell death quantified by flow cytometry. For confocal microscopy, cells were plated directly on collagen coated glass coverslips and imaged utilizing a Leica SP8X and images were quantified and analyzed utilizing ImageJ.
The RHIM interacting agent of the present invention has immune-inflammatory effect and therefore can be used as an immunostimulant. In one embodiment the agent is for use or when used as a cancer treatment or prophylaxis or a vaccine adjuvant. A vaccine composition containing or encoding the RHIM interacting agent of the present invention enables more effective therapy or prophylaxis. In one embodiment the agent is for use or when used to promote inflammasome, such as NLRP3, activation in a subject. Delivery may be to the immune system such as to particular immune cells or the lymph nodes or systemically or at site distant from determined pathology. Targeted delivery of agents to particular cell subsets can enhance the therapeutic index. Antibody targeted RHIM interacting agents that bind to cells comprising an antigen recognized by the antibody or binding fragments thereof. This include for example maleimide functionalized PEG-PLGA polymeric nanoparticules, or simply combining the RHIM interacting peptide in a composition comprising a delivery moiety or shuttle agent. Ex vivo approaches contemplate the administration of gene editing such as CRISPR components to modify cells to contain or express a RHIM interacting agent.
Table 1 shows unmodified RHIM interacting peptides (except for tat-RIP3 which is modified) and control peptides used in transfection, microscopy and ThT fluorescence studies described in Example 6.
Table 1
All peptides except IAPP and BH3 were purchased from GL Biochem, Shanghai, at >95% purity. IAPP was supplied by Calbiochem, and BH3 was the kind gift of Dana Westphal. MW, molecular weight. PASTA, an amyloidigenicity index, as determined by
PASTA accessed 3rd May 2013. Cross-beta fibrilliar amyloid structures usually have PASTA values < -4.0. Italic type indicates a change in residue from the WT peptide, tat prefix underlined aa, amino acid. PASTA is an amyloid prediction algorithm; i.e., generates an amyl oidigeni city index (Trovato A. et al. (2007) Protein engineering, design & selection : PEDS. 20(10):521-523).
Table 2 - Modified RHIM interacting peptide sequences
**A11 sequences include a TAT cell penetration motif with a tri-lysine spacer to increase cell penetration and solubility (YGRKKRRQRRRKKK)
**Note codons can be replaced, please see replacement table for substitution of codons
**Note codons can be replaced, please see replacement table for substitution of codons
Table 3. Human RHIM sequences
**Note codons can be replaced, please see replacement table for substitution of codons
**Note codons can be replaced, please see replacement table for substitution of codons
Table 4
Abbreviations
Table 5
Amino acid sub-classification
Table 6
Exemplary and Preferred Amino Acid Substitutions
KEY TO SEQUENCE LISTING
All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety
Those of skill in the art will appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.
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Claims (19)
1. A method for inducing an artificial lytic innate immune-inflammatory response (LIIIR) in a cell or subject, the method comprising administering to the cell or subject an effective amount of a composition comprising a RHIM-interacting agent, or an RHIM- interacting agent encoding nucleic acid, and optionally an agent that enhances cellular delivery, wherein the RHIM-interacting agent within one or more cells partakes in amyloid formation and induces a LIIIR.
2. The method of claim 1, wherein the RHIM-interacting agent interacts with one or more RHIM-containing proteins such as RIPK1, RIPK3, DAI, TRIF or an orthologous polypeptide within one or more target cells to induce amyloid formation, cell death and inflammasome activation.
3. The method of claim 1 or 2, wherein at least one cell is a myeloid cell of the innate immune system.
4. The method of any one of claims 1 to 3, wherein the LIIIR comprises NLRP3 inflammasome activation and IL-Ib release.
4. The method of any one of claims 1 to 4, wherein the LIIIR facilitates reduced tumor growth or reduced pathology in a tissue of the subject.
5. The method of any one of claims 1 to 3, wherein the LIIIR facilitates an enhanced immune response, or facilitates enhanced vaccination efficiency in the subject when administered in conjunction with an antigen.
6. The method of any one of claims 1 to 5, wherein the RHIM-interacting agent comprises a RHIM-containing peptide of 7-60 or 8-40 amino acids in length, comprising the conserved RHIM sequence XXIXN(Xa)XXXI/VQI/VGXXNXM/L wherein Xa is present or absent and wherein X is an amino acid, or an amino acid selected from the corresponding position in RIPKl, RIPK3, DAI, TRIF or orthologous polypeptides.
7. The method of claim 6, wherein the peptide comprises one or more of a linker, a modified or non-natural or non-proteogenic amino acid, a modified side-chain, a
modified backbone, terminal modified groups or comprises a modified spatial constrained or is a retro-inverso peptide.
8. The method of claim 6 or 7 wherein the peptide is a pseudopeptide, peptoid, azapeptide, cyclized, stapled, ether or lactam peptide or comprises a spatially constrained modification.
9. The method of any one of claims 6 to 8 wherein the peptide is conjugated or otherwise attached/bound to a lipid, carbohydrate, polymer, protein, nanoparticle, peptide, antibody or fragment thereof, aptamer, or nucleic acid.
10. The method of any one of claims 1 to 9 wherein the RHIM interacting agent comprises following structure I, N' to C:
ZiWaXi x2 x3 x4 Xs Xe Xv Xs X9 Xio Xu X12 X13 XM X15 Xie Xn WbZ2
Xi is present or absent and where present is any amino acid, or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptide at this position typically a residue with a hydrophobic or polar/neutral side chain
X2 is present or absent and where present is any amino acid or is an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptide at this position, typically polar or a hydrophobic residue
X3 is Isoleucine (I) or a hydrophobic residue such as Leucine or Norleucine
X4 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1,
RIPK3, DAI, TRIF or orthologous polypeptide at this position
X5 is Asparagine (N)
X6 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptide at this position
X7 is any amino acid
X8 is any amino acid or an amino acid corresponding to the amino acid in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position, such as a Glycine (G) or Proline (P) or Alanine (A)
X9 is Isoleucine (I) or Valine (V)
Xio is Glutamine (Q)
X11 is Isoleucine (I) or Valine (V) or Leucine (L)
Xi2 is Glycine (G)
Xi3 is any amino acid or an amino acid corresponding to a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position
Xi4 is any amino acid or an amino acid corresponding to a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides at this position
Xi5 is Asparagine (N)
Xi6 is present or absent and where present is any amino acid typically polar such as Tyrosine (Y)
Xi7 is present or absent and where present is a methionine (M) or Leucine (L) or Isoleucine (I);
Wa and Wb are present or absent and where present is each 5 to 20 contiguous amino acids that immediately flank Xi to Xn, where present, in a RIPK1, RIPK3, DAI, TRIF or orthologous polypeptides, or a conservative substitution thereof;
Zi and Z2 are individually present or absent and where present are individually selected from one or more of a linker, stability enhancing, delivery enhancing or label moiety; or a pharmaceutically acceptable salt, hydrate, tautomer, sterioisomer, pro-drug thereof.
11. The method of any one of claims 1 to 10, wherein the RHIM interacting agent comprises or encodes a peptide comprising or consisting the amino acid sequence set out in SEQ ID NO: 1 to 20.
12. The method of claim 1, wherein the RHIM interacting agent comprises a nucleic acid molecule that expresses the RHIM-interacting peptide.
13. The method of claim 12 wherein the nucleic acid molecule is an RNA or DNA or RNA:DNA or a chemically modified form thereof.
14. The method of claim 13, wherein a proportion of at least one type of nucleotide (e.g„ cysteine and/or uracil), is chemically modified to increase its stability in vivo.
15. The method of claim 12 wherein the nucleic acid is in the form of a viral or non- viral vector.
16. The method of any one of claims 1 to 15 where the RHIM interacting agent is administered to cells ex vivo, such a transfection of cells with a RHIM-containing peptide with lipofectamine or a similar transfection aid.
17. The method of any one of claims 1 to 15 wherein the RHIM interacting agent comprises an antibody or antibody fragment that targets the agent to myeloid cells such as macrophages, or targets the agent to tumor associated myeloid cells.
17. A composition comprising a RHIM interacting agent as defined in any one of claims 1 to 15.
18. A composition comprising a RHIM interacting peptide wherein the peptide comprises 7-60 or 10-40 amino acids in length, and comprises a core RHIM binding sequence selected from IQIG, VQVG or VQIG and comprising flanking amino acids following the conserved RHIM sequence XXIXN(Xa)XXXI/VQI/VGXXNXM/L wherein Xa is present or absent and X is an amino acid, or an amino acid derived from a RIPKl, RIPK3, DAI, TRIF or orthologous polypeptides.
19. A RHIM interacting agent as defined in any one of claims 1 to 15 for use in or for use in the manufacture of a medicament for use in stimulating an artificial lytic innate immune-inflammatory response (LIIIR), or for stimulating NLRP3 inflammasome activation and release of pro-inflammatory cytokine, in a cell or subject.
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