Patent Application
20180133327
The present invention relates to the field of cell penetrating peptides, in particular as means for delivering a cargo into a cell, for example in biological and medical applications. The present invention also relates to complexes comprising the cell penetrating peptide and a cargo molecule and to the use of such complexes in medical applications such as the prevention and/or treatment of various human diseases, including infectious diseases and cancers.
The basic function of the cell membrane of eukaryotic cells is to protect the cell from its surroundings. Accordingly, the cell membrane tightly controls the movement of substances in and out from cells. Some very small compounds, such as certain molecules and ions, for example carbon dioxide (CO2) and oxygen (O2), can move across the cell membrane by diffusion. However, for other molecules and ions, the membrane acts as a barrier. If exogenously applied substances, such as drugs or nutrients, mediate their effects by or after cellular uptake, e.g., from the blood or from the gut, transport of those substances across the cell membrane must be ensured.
One example of such substances, for which cellular uptake is required, are antigens or antigenic epitopes administered in the context of immunotherapy. Immunotherapy is the prevention or treatment of a disease by inducing, enhancing or suppressing an immune response. Such a modification, in particular an induction or enhancement, of the immune response is typically mediated by cellular uptake of the administered antigen or antigenic epitope and presentation of said antigen or antigenic epitope at the surface of the cell in the context of major histocompatibility complex (MHC) class I and/or MHC class II. Thus, cell penetration is an essential prerequisite to immunotherapy.
Moreover, cell penetration is also an essential prerequisite for signal transduction therapy, for therapies influencing transcription and translation, e.g. tumor therapy based on modulation of transcription and translation, and, possibly, for gene therapy (cf. Reissmann S., 2015, Journal of peptide science, 2014, 20(10): 760-784).
Accordingly, the discovery of cell penetrating peptides (CPPs) by two laboratories in 1988 represented a major breakthrough for the transport of exogenous cargo molecules across the cell membrane. In addition to internalization and cargo transport into live cells, CPPs are also able to transport compounds through the skin, the blood-brain barrier, and through the conjunctiva of eyes. Because of their low cytotoxicity and final degradation to amino acids, CPPs are particularly favored in in vivo studies and for clinical applications.
In general, cell penetrating peptides are (short) peptides that are able to transport different types of cargo molecules across the cell membrane, and, thus, facilitate cellular uptake of various molecular cargoes (from nanosize particles to small chemical molecules and large fragments of DNA). Typically, the cargo is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions.
Cell penetrating peptides typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or have a sequence that contains an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. Cell-Penetrating peptides are of different sizes, amino acid sequences, and charges, but all CPPs have a common characteristic that is the ability to translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or to an organelle of a cell. At present, the theories of CPP translocation distinguish three main entry mechanisms: direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure (Jafari S, Solmaz M D, Khosro A, 2015, Bioimpacts 5(2): 103-111; Madani F, Lindberg S, Langel Ü, Futaki S, Gräslund A, 2011, J Biophys: 414729). CPP transduction is an area of ongoing research. Cell-penetrating peptides have found numerous applications in medicine as drug delivery agents in the treatment of different diseases including cancer and virus inhibitors, as well as contrast agents for cell labeling and imaging.
Frankel and Pabo simultaneously to Green and Lowenstein described the ability of the trans-activating transcriptional activator from the human immunodeficiency virus 1 (HIV-TAT) to penetrate into cells (Frankel, A. D. and C. O. Pabo, Cellular uptake of the tat protein from human immunodeficiency virus. Cell, 1988. 55(6): p. 1189-93). In 1991, transduction into neural cells of the Antennapedia homeodomain (DNA-binding domain) from Drosophila melanogaster was described (Joliot, A., et al., Antennapedia homeobox peptide regulates neural morphogenesis. Proc Natl Acad Sci U S A, 1991. 88(5): p. 1864-8). In 1994, the first 16-mer peptide CPP called Penetratin was characterized from the third helix of the homeodomain of Antennapedia (Derossi, D., et al., The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem, 1994. 269(14): p. 10444-50), followed in 1998 by the identification of the minimal domain of TAT required for protein transduction (Vives, E., P. Brodin, and B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem, 1997. 272(25): p. 16010-7). Over the past two decades, dozens of peptides were described from different origins including viral proteins, e.g. VP22 (Elliott, G. and P. O'Hare, Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell, 1997. 88(2): p. 223-33) and ZEBRA (Rothe, R., et al., Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator. J Biol Chem, 2010. 285(26): p. 20224-33), or from venoms, e.g. melittin (Dempsey, C.E., The actions of melittin on membranes. Biochim Biophys Acta, 1990. 1031(2): p. 143-61), mastoporan (Konno, K., et al., Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado). Toxicon, 2000. 38(11): p. 1505-15), maurocalcin (Esteve, E., et al., Transduction of the scorpion toxin maurocalcine into cells. Evidence that the toxin crosses the plasma membrane. J Biol Chem, 2005. 280(13): p. 12833-9), crotamine (Nascimento, F. D., et al., Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J Biol Chem, 2007. 282(29): p. 21349-60) or buforin (Kobayashi, S., et al., Membrane translocation mechanism of the antimicrobial peptide buforin 2. Biochemistry, 2004. 43(49): p. 15610-6).
Synthetic CPPs were also designed including the poly-arginine (R8, R9, R10 and R12) (Futaki, S., et al., Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem, 2001. 276(8): p. 5836-40) or transportan (Pooga, M., et al., Cell penetration by transportan. FASEB J, 1998. 12(1): p. 67-77).
However, many CPPs have drawbacks when used for delivering a cargo in vivo, such as in clinical applications. One of the biggest drawbacks results from the uptake of
CPPs and cargoes coupled to CPPs into intracellular endosomes. In order to release CPPs and cargoes coupled to CPPs from endosomes, destabilization by addition of auxiliary compounds or charged polymers such as PEIs is required. However, all these auxiliary compounds may be cytotoxic and, thus, cause severe side-effects in a clinical application. To avoid such endosomal trapping, it was searched for new CPPs with non-endosomal uptake mechanisms.
In 2010, Rothe et al. described that ZEBRA, a protein, which is responsible for the initiation of the Epstein-Barr virus lytic cycle and which belongs to the basic leucine zipper (bZIP) family of transcription factors, crosses the cell membrane in a pathway largely independent of endocytosis (Rothe, R., et al., Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator. J Biol Chem, 2010. 285(26): p. 20224-33). Moreover, Rothe et al. identified a minimal domain (MD) of ZEBRA, which is required for internalization. This minimal domain of the ZEBRA protein (245 amino acids in total) spans residues 170-220 of ZEBRA and was proposed as promising candidate CPP for therapeutic protein delivery applications (Rothe, R., et al., Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator. J Biol Chem, 2010. 285(26): p. 20224-33).
Based on this finding of ZEBRA's minimal domain, WO 2014/041505 A1 discloses improved cell penetrating peptides, which are derived from ZEBRA's minimal domain and which are 15 to 30 amino acids long. Thus, the cell penetrating peptides disclosed in WO 2014/041505 A1 are considerably shorter than ZEBRA's minimal domain. In particular, the fragments of ZEBRA's minimal domain, which 15 to 30 amino acids long as disclosed in WO 2014/041505 A1, provide a higher immunogenic potential as compared to shorter and longer fragments of ZEBRA's minimal domain.
However, there is still a need for providing improved cell penetrating peptides, which are in particular useful for clinical applications. In view of the above, it is the object of the present invention to overcome the drawbacks of current cell penetrating peptides outlined above and to provide an improved cell penetrating peptide and complexes comprising the cell penetrating peptide and a cargo molecule, which are in particular useful for clinical applications.
This object is achieved by means of the subject-matter set out below and in the appended claims.
Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Throughout this specification and the claims which follow, unless the context requires otherwise, the term “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step but not the exclusion of any other non-stated member, integer or step. The term “consist of” is a particular embodiment of the term “comprise”, wherein any other non-stated member, integer or step is excluded. In the context of the present invention, the term “comprise” encompasses the term “consist of”. The term “comprising” thus encompasses “including” as well as “consisting” e.g., a composition “comprising” X may consist exclusively of X or may include something additional e.g., X+Y.
The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
The word “substantially” does not exclude “completely” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
The term “about” in relation to a numerical value x means x±10%.
In the context of the present invention, i.e. throughout the present application, the terms “peptide”, “polypeptide”, “protein” and variations of these terms refer to peptide, oligopeptide, oligomer or protein including fusion protein, respectively, comprising at least two amino acids joined to each other preferably by a normal peptide bond, or, alternatively, by a modified peptide bond, such as for example in the cases of isosteric peptides. A peptide, polypeptide or protein can be composed of L-amino acids and/or D-amino acids. Preferably, a peptide, polypeptide or protein is either (entirely) composed of L-amino acids or (entirely) of D-amino acids, thereby forming “retro-inverso peptide sequences”. The term “retro-inverso (peptide) sequences” refers to an isomer of a linear peptide sequence in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted (see e.g. Jameson et al., Nature, 368,744-746 (1994); Brady et al., Nature, 368,692-693 (1994)). In particular, the terms “peptide”, “polypeptide”, “protein also include “peptidomimetics” which are defined as peptide analogs containing non-peptidic structural elements, which peptides are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. A peptidomimetic lacks classical peptide characteristics such as enzymatically scissile peptide bonds. In particular, a peptide, polypeptide or protein can comprise amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids, or it can be composed of amino acids other than the 20 amino acids defined by the genetic code. In particular, a peptide, polypeptide or protein in the context of the present invention can equally be composed of amino acids modified by natural processes, such as post-translational maturation processes or by chemical processes, which are well known to a person skilled in the art. Such modifications are fully detailed in the literature. These modifications can appear anywhere in the polypeptide: in the peptide skeleton, in the amino acid chain or even at the carboxy- or amino-terminal ends. In particular, a peptide or polypeptide can be branched following an ubiquitination or be cyclic with or without branching. This type of modification can be the result of natural or synthetic post-translational processes that are well known to a person skilled in the art. The terms “peptide”, “polypeptide”, “protein” in the context of the present invention in particular also include modified peptides, polypeptides and proteins. For example, peptide, polypeptide or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, amino acid addition such as arginylation or ubiquitination. Such modifications are fully detailed in the literature (Proteins Structure and Molecular Properties (1993) 2nd Ed., T. E. Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) B. C. Johnson, Ed., Academic Press, New York; Seifter et al. (1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 and Rattan et al., (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48-62). Accordingly, the terms “peptide”, “polypeptide”, “protein” preferably include for example lipopeptides, lipoproteins, glycopeptides, glycoproteins and the like.
However, in the context of the present invention “classical” peptides, polypeptides or proteins are preferred. A “classical” peptide, polypeptide or protein is typically composed of amino acids selected from the 20 amino acids defined by the genetic code, linked to each other by a normal peptide bond.
Cell Penetrating Peptide
In a first aspect the present invention provides a cell penetrating peptide (CPP) comprising an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 1, wherein the amino acid sequence sharing at least 80% sequence identity with SEQ ID NO:1:
Such a CPP allows for efficient delivery, i.e. transport and loading, in particular of at least one antigen or antigenic epitope, into cells, in particular into the antigen presenting cells (APCs), more preferably into dendritic cells (DCs) and, thus, to the dendritic cells' antigen processing machinery. The present inventors have surprisingly found that a CPP according to the present invention is unexpectedly superior to the cell penetrating peptides disclosed in WO 2014/041505 A1 as shown in the Examples of the present specification—in particular in in vivo settings, such as in tumor/cancer applications.
As used herein, the term “cell penetrating peptides” (“CPPs”) refers to (short) peptides that are able to transport different types of cargo molecules across plasma membrane, and, thus, facilitate cellular uptake of various molecular cargoes (from nanosize particles to small chemical molecules and large fragments of DNA). Typically, cell penetrating peptides (CPPs) are peptides of at least 8 amino acid residues that have the ability to cross the cell membrane and enter into most cell types. Alternatively, they are also called protein transduction domain (PTDs) reflecting their origin as occurring in natural proteins.
Cell penetrating functionality (or cellular internalization), of the cell penetrating peptide or complex comprising said cell penetrating peptide, according to the invention can be checked by standard methods known to one skilled in the art, including flow cytometry or fluorescence microscopy of live and fixed cells, immunocytochemistry of cells transduced with said peptide or complex, and Western blot. “Cellular internalization” of the cargo molecule linked to the cell penetrating peptide generally means transport of the cargo molecule across the plasma membrane and thus entry of the cargo molecule into the cell.
Depending on the particular case, the cargo molecule can, then, be released in the cytoplasm, directed to an intracellular organelle, or further presented at the cell surface.
According to the present invention, the cell penetrating peptide (CPP) comprises an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 1:
SEQ ID NO: 1 shows a peptide having a length of 42 amino acids, which corresponds to amino acids 178 to 219 of ZEBRA (SEQ ID NO: 2)—except that the cysteine (Cys) at position 189 in the ZEBRA amino acid sequence is substituted by a serine (Ser). This serine, which is located at position 12 of SEQ ID NO: 1, is shown underlined in SEQ ID NO: 1 above. The peptide according to SEQ ID NO: 1 has cell penetrating functionality as described herein, since it represents a large part of ZEBRA's minimal domain. The cell penetrating peptide according to SEQ ID NO: 1 is also referred to herein as “Z13”.
The term “ZEBRA” (also known as Zta, Z, EB1, or BZLF1) generally means the basic-leucine zipper (bZIP) transcriptional activator of the Epstein-Barr virus (EBV). The minimal domain of ZEBRA, which exhibits cell penetrating properties, has been identified as spanning from residue 170 to residue 220 of ZEBRA. The amino acid sequence of ZEBRA is disclosed under NCBI accession number YP_401673 and comprises 245 amino acids represented in SEQ ID NO: 2:
REVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF
Amino acids 178 to 219 of ZEBRA, which correspond to SEQ ID NO: 1—except that the cysteine (Cys) at position 189 in the ZEBRA amino acid sequence is substituted by a serine (Ser)—are shown underlined.
In the context of the present invention, i.e. throughout the present specification, an amino acid sequence “sharing a sequence identity” of at least, for example, 80% with a query amino acid sequence of the present invention, for example with SEQ ID NO: 1, is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence except that the subject amino acid sequence may include up to twenty (in the present example of 80%) amino acid mutations per each 100 amino acids of the query amino acid sequence. In other words, to obtain an amino acid sequence having a sequence of at least 80% identity to a query amino acid sequence, up to 20% (20 of 100) of the amino acid residues in the subject sequence may be deleted, inserted or substituted with another amino acid.
For amino acid sequences without exact correspondence, a “% identity” of a first sequence may be determined with respect to a second sequence. In general, to determine a “% identity” of two sequences to be compared, the sequences are aligned such that they give a maximum correlation between the sequences. This may include inserting “gaps” in either one or both sequences, to enhance the degree of alignment. A % identity may then be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
Methods for comparing the identity and homology of two or more sequences are well known in the art. The percentage to which two sequences are identical can, e.g., be determined using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877. Such an algorithm is integrated in the BLAST family of programs, e.g. BLAST or NBLAST program (see also Altschul et al., 1990, J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402), accessible through the home page of the NCBI at world wide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U. S. A 85, 2444-2448.). Sequences which are identical to other sequences to a certain extent can be identified by these programmes.
As used herein, the term “mutation” relates to a change in the nucleic acid sequence and/or in the amino acid sequence in comparison to a reference sequence, e.g. a corresponding naturally occurring sequence. A mutation, e.g. in comparison to a naturally occurring sequence, may be, for example, a spontaneous mutation, an induced mutation, e.g. induced by enzymes, chemicals or radiation, or a mutation obtained by site-directed mutagenesis (molecular biology methods for making specific and intentional changes in the nucleic acid sequence and/or in the amino acid sequence). Thus, the terms “mutation” or “mutating” shall be understood to also include physically making a mutation, e.g. in a nucleic acid sequence or in an amino acid sequence. A mutation includes substitution, deletion and insertion of one or more nucleotides or amino acids as well as inversion of several successive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, preferably a mutation may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide. A mutation may be achieved e.g., by altering, e.g., by site-directed mutagenesis, a codon of a nucleic acid molecule encoding one amino acid to result in a codon encoding a different amino acid, or by synthesizing a sequence variant, e.g., by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and by designing the synthesis of a nucleic acid molecule comprising a nucleotide sequence encoding a variant of the polypeptide without the need for mutating one or more nucleotides of a nucleic acid molecule.
Preferred mutations in the amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 1 in comparison to SEQ ID NO: 1 are substitution mutations. Generally, substitutions for one or more amino acids present in the referenced amino acid sequence should be made conservatively. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity properties, are well known (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1):105-132). Substitutions of one or more L-amino acids with one or more D-amino acids are to be considered as conservative substitutions in the context of the present invention. Exemplary conservative amino acid substitutions, which are preferred in the context of the present invention, are presented in Table 1 below:
In the context of the present invention, it is particularly preferred that in the amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 1 (i) either no amino acid mutation is present in comparison to SEQ ID NO: 1 (100% identity) or, (ii) at least one of the amino acids of SEQ ID NO: 1 selected from the group consisting of Y3, N5, Q18, L19, L20, Q21, H22, Y23, E25, S31, S32, E33, N34, and D35 is mutated (deleted, inserted or substituted; positions indicated in respect to SEQ ID NO: 1) but none of the other amino acids is mutated (deleted, inserted or substituted). Thereby, substitution mutations are preferred and conservative substitutions as described above are particularly preferred. Of note, according to the present invention the serine at position 12 of SEQ ID NO: 1 (S12) must not be mutated.
Since CPP according to the present invention comprises an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 1; and SEQ ID NO: 1 is 42 amino acids long, up to 8 amino acids may be mutated. However, the minimum length of the sequence sharing at least 80% sequence identity with SEQ ID NO: 1 is 36 amino acids, and, therefore, up to six amino acids may be deleted, but up to eight amino acids may be inserted or substituted. As described above, preferably an amino acid selected from the group consisting of Y3, N5, Q18, L19, L20, Q21, H22, Y23, E25, S31, S32, E33, N34, and D35 is mutated (deleted, inserted or substituted; positions indicated in respect to SEQ ID NO: 1), but none of the other amino acids of SEQ ID NO: 1 is mutated.
Preferably, the CPP according to the present invention comprises an amino acid sequence sharing a sequence identity of at least 85%, preferably a sequence identity of at least 90%, more preferably a sequence identity of at least 95% and most preferably a sequence identity of at least 98% with SEQ ID NO: 1 as described above. Thereby, it is understood that such a sequence provides cell penetrating functionality, has a serine at position 12, has a length of at least 36 amino acids in total and shares the preferred embodiments as described above, e.g. the type of preferred mutations and the selection of amino acids, which may be mutated in SEQ ID NO: 1.
In the present invention it is also preferred that the amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein, has a length of at least 38 amino acids in total. More preferably the amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein, has a length of at least 40 amino acids in total. Even more preferably the amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein, has a length of at least 42 amino acids in total.
It is also preferred that the amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein, has a length of no more than 50 amino acids in total. More preferably, the amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein, has a length of no more than 48 amino acids in total. Even more preferably, the amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein, has a length of no more than 45 amino acids in total. Most preferably, the amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein, has a length of no more than 43 amino acids in total.
Particularly preferably the amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein, has a length of 42 amino acids in total.
Preferably, the cell penetrating peptide according to the present invention has a length of no more than 100 amino acids in total, more preferably the cell penetrating peptide has a length of no more than 75 amino acids in total, even more preferably the cell penetrating peptide has a length of no more than 60 amino acids in total and most preferably the cell penetrating peptide has a length of no more than 50 amino acids in total.
Preferably, the CPP according to the present invention consists of an amino acid sequence sharing at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NO: 1, as described herein.
It is particularly preferred that the cell penetrating peptide according to the present invention consists of an amino acid sequence according to SEQ ID NO: 1.
It will be understood by one skilled in the art that the primary amino acid sequence of the cell penetrating peptide of the invention may further be post-translationally modified, such as by glycosylation or phosphorylation, without departing from the invention.
Moreover, the N- and/or the C-terminus of the CPP according to the present invention may be chemically modified, for example by addition of chemical groups. Preferred examples of such chemical groups, which can be added to the N- and/or the C-terminus of the cell penetrating peptide include for example a (G4S)n linker, wherein “G4S” refers to the amino acid sequence GGGGS (SEQ ID NO: 53), which may be present once or repeatedly, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, as indicated by the number “n”. Further preferred chemical groups, which can be added to the N- and/or the C-terminus of the cell penetrating peptide include the linkers, which are described with their amino acid sequences in Table I of Reddy Chichili V P, Kumar V, Sivaraman J. 2013. Linkers in the structural biology of protein-protein interactions. Protein Sci. 22:153-167.
Complex Comprising the Cell Penetrating Peptide and a Cargo Molecule
In a second aspect the present invention provides a complex comprising the cell penetrating peptide according to the present invention as described herein and a cargo molecule.
In the complex according to the present invention, the cargo molecule may be associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions. Preferably, the cell penetrating peptide according to the present invention as described herein and the cargo molecule are covalently linked in the complex according to the present invention.
A “covalent linkage” (also covalent bond), as used in the context of the present invention, refers to a chemical bond that involves the sharing of electron pairs between atoms. A “covalent linkage” (also covalent bond) in particular involves a stable balance of attractive and repulsive forces between atoms when they share electrons. For many molecules, the sharing of electrons allows each atom to attain the equivalent of a full outer shell, corresponding to a stable electronic configuration. Covalent bonding includes many kinds of interactions, including for example a-bonding, Tr-bonding, metal-to-metal bonding, agostic interactions, and three-center two-electron bonds.
The complex according to the present invention may comprise one single cell penetrating peptide or more than one cell penetrating peptides. Preferably, the complex according to the present invention comprises no more than five cell penetrating peptides, more preferably the complex according to the present invention comprises no more than four cell penetrating peptides, even more preferably the complex according to the present invention comprises no more than three cell penetrating peptides, particularly preferably the complex according to the present invention comprises no more than two cell penetrating peptides and most preferably the complex according to the present invention comprises one single cell penetrating peptide.
Preferably, in the complex according to the present invention, the cargo molecule is selected from the group consisting of: (i) a peptide, a polypeptide, or a protein; (ii) a polysaccharide; (iii) a lipid; (iv) a lipoprotein; (v) a glycolipid; (vi) a nucleic acid; (vii) a small molecule drug or toxin; and (viii) an imaging or contrast agent. Thus, the cargo molecule may be a peptide, a protein, a polysaccharide, a lipid, a combination thereof including lipoproteins and glycolipids, a nucleic acid (e.g. DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid, preferably siRNA or shRNA, more preferably siRNA), or a small molecule drug (e.g. cyclosporine A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid), or any combination thereof, in particular if more than one cargo molecule is comprised by the inventive complex.
It is understood that the cargo molecule can comprise for example at least one, i.e. one or more, peptides, polypeptides or proteins linked together and/or at least one, i.e. one or more, nucleic acids, e.g. wherein each one encodes one peptide or polypeptide. Also the at least one cargo molecule can be a combination of a protein, a lipid, and/or a polysaccharide including lipoproteins and glycolipids. Thus, in particular if the complex according to the present invention comprises more than one cargo molecule, it can comprise more than one peptide, polypeptide, or protein, more than one polysaccharide, more than one lipid, more than one lipoprotein, more than one glycolipid, more than one nucleic acid, more than one small molecule drug or toxin, or a combination thereof.
Preferably, the cargo molecule is a peptide, a polypeptide or a protein.
It is also preferred that the cargo molecule is at least one antigen or antigenic epitope.
As used herein, an “antigen” is any structural substance which serves as a target for the receptors of an adaptive immune response, in particular as a target for antibodies, T cell receptors, and/or B cell receptors. An “epitope”, also known as “antigenic determinant”, is the part (or fragment) of an antigen that is recognized by the immune system, in particular by antibodies, T cell receptors, and/or B cell receptors. Thus, one antigen has at least one epitope, i.e. a single antigen has one or more epitopes. In the context of the present invention, the term “epitope” is mainly used to designate T cell epitopes, which are presented on the surface of an antigen-presenting cell, where they are bound to Major Histocompatibility Complex (MHC). T cell epitopes presented by MHC class I molecules are typically, but not exclusively, peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, generally, but not exclusively, between 12 and 25 amino acids in length.
Preferably, the complex according to the invention comprises at least one antigen or antigenic epitope comprising or consisting of one or more epitope(s) from a tumor-associated antigen, a tumor-specific antigen, and/or an antigenic protein from a pathogen, including viral, bacterial, fungal, protozoal and multicellular parasitic antigenic protein.
More preferably, the at least one antigen or antigenic epitope comprises or consists of at least one pathogen epitope and/or at least one tumor epitope. Even more preferably, the at least one antigen or antigenic epitope comprises or consists of at least one tumor epitope.
It is particularly preferred that the complex according to the present invention comprises only such antigen(s) or antigenic epitope(s), which are tumor-associated antigen(s), tumor-specific antigen(s), and/or tumor epitope(s).
As used herein, “tumor epitope” means an epitope from a tumor-associated antigen or from a tumor-specific antigen. Such epitopes are typically specific (or associated) for a certain kind of tumor. For instance, tumor epitopes include glioma epitopes. Suitable tumor epitopes can be retrieved for example from tumor epitope databases, e.g. from van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer lmmun 2013; URL: http://www.cancerimmunity.org/peptide/, wherein human tumor antigens recognized by CD4+ or CD8+ T cells are classified into four major groups on the basis of their expression pattern, or from the database “Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed by Bioinformatics Core at Cancer Vaccine Center, Dana-Farber Cancer Institute; URL: http://cvc.dfci.harvard.edu/tadb/). Examples of tumor epitopes include e.g. TRP2-derived epitopes, glycoprotein 100 (gp100) melanoma antigen-derived epitopes, IEa epitopes, IL13rα2, Epha2 (ephrin type-A receptor 2), immunogenic fragments thereof, and fusions of such antigens and/or fragments.
Specific examples of tumor-related or tissue-specific antigens useful in a complex according to the present invention include, but are not limited to, the following antigens: Prostate: prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), PAP, PSCA (PNAS 95(4) 1735-1740 1998), prostate mucin antigen (PMA) (Beckett and Wright, 1995, Int. J. Cancer 62: 703-710), Prostase, Her-2neu, SPAS-1; Melanoma: TRP-2, tyrosinase, Melan A/Mart-1, gplOO, BAGE, GAGE, GM2 ganglioside; Breast: Her2-neu, kinesin 2, TATA element modulatory factor 1, tumor protein D52, MAGE D, ING2, HIP-55, TGF-1 anti-apoptotic factor, HOM-Mel-40/SSX2, epithelial antigen (LEA 135), DF31MUC1 antigen (Apostolopoulos et al., 1996 Immunol. Cell. Biol. 74: 457-464; Pandey et al., 1995, Cancer Res. 55: 4000-4003); Testis: MAGE-1, HOM-Mel-40/SSX2, NY-ESO-1; Colorectal: EGFR, CEA; Lung: MAGE D, EGFR Ovarian Her-2neu; Baldder: transitional cell carcinoma (TCC) (Jones et al., 1997, Anticancer Res. 17: 685-687), Several cancers: Epha2, Epha4, PCDGF, HAAH, Mesothelin; EPCAM; NY-ESO-1, glycoprotein MUC1 and NIUC10 mucins p5 (especially mutated versions), EGFR; Miscellaneous tumor: cancer-associated serum antigen (CASA) and cancer antigen 125 (CA 125) (Kierkegaard et al., 1995, Gynecol. Oncol. 59: 251-254), the epithelial glycoprotein 40 (EGP40) (Kievit et al., 1997, Int. J. Cancer 71: 237-245), squamous cell carcinoma antigen (SCC) (Lozza et al., 1997 Anticancer Res. 17: 525-529), cathepsin E (Mota et al., 1997, Am. J Pathol. 150: 1223-1229), tyrosinase in melanoma (Fishman et al., 1997 Cancer 79: 1461-1464), cell nuclear antigen (PCNA) of cerebral cavernomas (Notelet et al., 1997 Surg. Neurol. 47: 364-370), a 35 kD tumor-associated autoantigen in papillary thyroid carcinoma (Lucas et al., 1996 Anticancer Res. 16: 2493-2496), CDC27 (including the mutated form of the protein), antigens triosephosphate isomerase, 707-AP, A60 mycobacterial antigen (Macs et al., 1996, J. Cancer Res. Clin. Oncol. 122: 296-300), Annexin II, AFP, ART-4, BAGE, β-catenin/m, BCL-2, bcr-abl, bcr-abl p190, bcr-abl p210, BRCA-1, BRCA-2, CA 19-9 (Tolliver and O'Brien, 1997, South Med. J. 90: 89-90; Tsuruta at al., 1997 Urol. Int. 58: 20-24), CAMEL, CAP-1, CASP-8, CDC27/m, CDK-4/m, CEA (Huang et al., Exper Rev. Vaccines (2002)1:49-63), CT9, CT10, Cyp-B, Dek-cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1), EphA2 (Zantek et al., Cell Growth Differ. (1999) 10:629-38; Carles-Kinch et al., Cancer Res. (2002) 62:2840-7), EphA4 (Cheng at al., 2002, Cytokine Growth Factor Rev. 13:75-85), tumor associated Thomsen-Friedenreich antigen (Dahlenborg et al., 1997, Int. J Cancer 70: 63-71), ELF2M, ETV6-AML1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GnT-V, gp100 (Zajac et al., 1997, Int. J Cancer 71: 491-496), HAGE, HER2/neu, HLA-A*0201-R1701, HPV-E7, HSP70-2M, HST-2, hTERT, hTRT, iCE, inhibitors of apoptosis (e.g., survivin), KH-1 adenocarcinoma antigen (Deshpande and Danishefsky, 1997, Nature 387: 164-166), KIAA0205, K-ras, LACE, LAGE-1, LDLR/FUT, MAGE-1, MAGE-2, MAGE-3, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-B5, MAGE-B6, MAGE-C2, MAGE-C3, MAGE D, MART-1, MART-1/Melan-A (Kawakami and Rosenberg, 1997, Int. Rev. Immunol. 14: 173-192), MC1R, MDM-2, Myosin/m, MUC1, MUC2, MUM-1, MUM-2, MUM-3, neo-polyA polymerase, NA88-A, NY-ESO-1, NY-ESO-1a (CAG-3), PAGE-4, PAP, Proteinase 3 (Molldrem et al., Blood (1996) 88:2450-7; Molldrem et al., Blood (1997) 90:2529-34), P15, p190, Pm1/RARα, PRAME, PSA, PSM, PSMA, RAGE, RAS, RCAS1, RU1, RU2, SAGE, SART-1, SART-2, SART-3, SP17, SPAS-1, TEL/AML1, TPI/m, Tyrosinase, TARP, TRP-1 (gp75), TRP-2, TRP-2/INT2, WT-1, and alternatively translated NY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1 genes. Numerous other cancer antigens are well known in the art.
As used herein “pathogen epitope” means an epitope from an antigenic protein, an antigenic polysaccharide, an antigenic lipid, an antigenic lipoprotein or an antigenic glycolipid from a pathogen including viruses, bacteria, fungi, protozoa and multicellular parasites. Antigenic proteins, polysaccharides, lipids, lipoproteins or glycolipids from pathogens include, herewith, proteins, polysaccharides, lipids, lipoproteins and glycolipids, respectively, from pathogens responsible of diseases which can be a target for vaccination including, for instance, Amoebiasis, Anthrax, Buruli Ulcer (Mycobacterium ulcerans), Caliciviruses associated diarrhoea, Campylobacter diarrhoea, Cervical Cancer (Human papillomavirus), Chlamydia trachomatis associated genital diseases, Cholera, Crimean-Congo haemorrhagic fever, Dengue Fever, Diptheria, Ebola haemorrhagic fever, Enterotoxigenic Escherichia coli (ETEC) diarrhoea, Gastric Cancer (Helicobacter pylori), Gonorrhea, Group A Streptococcus associated diseases, Group B Streptococcus associated diseases, Haemophilus influenzae B pneumonia and invasive disease, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E diarrhoea, Herpes simplex type 2 genital ulcers, HIV/AIDS, Hookworm Disease, Influenza, Japanese encephalitis, Lassa Fever, Leishmaniasis, Leptospirosi, Liver cancer (Hepatitis B), Liver Cancer (Hepatitis C), Lyme Disease, Malaria, Marburg haemorrhagic fever, Measles, Mumps, Nasopharyngeal cancer (Epstein-Barr virus), Neisseria meningitidis Meningitis, Parainfluenza associated pneumonia, Pertussis, Plague, Poliomyelitis, Rabies, Respiratory syncytial virus (RSV) pneumonia, Rift Valley fever, Rotavirus diarrhoea, Rubella, Schistosomiasis, Severe Acute Respiratory Syndrome (SARS), Shigellosis, Smallpox, Staphylococcus aureus associated diseases, Stomach Cancer (Helicobacter pylori), Streptococcus pneumoniae and invasive disease, Tetanus, Tick-borne encephalitis, Trachoma, Tuberculosis, Tularaemia, Typhoid fever, West-Nile virus associated disease, Yellow fever.
Preferably, the at least one antigen or antigenic epitope will be presented at the cell surface in an MHC class I and/or MHC class II context and/or in a CD1 context, whereby presentation at the cell surface in an MHC class I and/or MHC class II context is preferred. The phrase “epitope presentation in the MHC class I context” refers in particular to a CD8+ epitope lying in the groove of a MHC class I molecule at the surface of a cell. The phrase “epitope presentation in the MHC class II context” refers in particular to a CD4+ epitope lying in the groove of a MHC class II molecule at the surface of a cell. The phrase “epitope presentation in the CD1 context” refers in particular to a lipidic epitope lying in the groove of a cluster of differentiation 1 molecule at the surface of a cell.
Preferably, in the complex according to the present invention, the cell penetrating peptide facilitates presentation of the at least one antigenic epitope at the cell surface in MHC class I and/or MHC class II context.
Advantageously, the complex according to the invention comprises a cell penetrating peptide and at least one antigen or antigenic epitope, and allows the transport and presentation of said epitopes at the cell surface of antigen presenting cells in an MHC class I and MHC class II context, and is, thus, useful in vaccination and immunotherapy. Therefore, it is particularly preferred in the complex according to the present invention that the cell penetrating peptide facilitates presentation of the at least one antigenic epitope at the cell surface in MHC class I and MHC class II context.
The present inventors assume that the two mechanisms of entry provided by ZEBRA-derived CPPs, namely (i) direct translocation and (ii) lipid raft-mediated endocytosis (Rothe R, Liguori L, Villegas-Mendez A, Marques B, Grunwald D, Drouet E, et al. Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator. The Journal of biological chemistry 2010;285(26):20224-33), should promote both MHC class I and II restricted presentation of cargo antigens to CD8+ and CD4+ T cells, respectively. Accordingly, such a CPP can deliver multi-epitopic peptides to dendritic cells (DCs), and subsequently to promote CTL and Th cell activation and anti-tumor function. Such a ZEBRA-derived CPP can thus efficiently deliver the complex according to the present invention to antigen presenting cells (APCs) and lead to multi-epitopic MHC class I and II restricted presentation.
In the context of the present invention, the term “MHC class I” designates one of the two primary classes of the Major Histocompatibility Complex molecules. The MHC class I (also noted “MHC I”) molecules are found on every nucleated cell of the body. The function of MHC class I is to display an epitope to cytotoxic cells (CTLs). In humans, MHC class I molecules consist of two polypeptide chains, α- and β2-microglobulin (b2m). Only the a chain is polymorphic and encoded by a HLA gene, while the b2m subunit is not polymorphic and encoded by the Beta-2 microglobulin gene. In the context of the present invention, the term “MHC class II” designates the other primary class of the Major Histocompatibility Complex molecules. The MHC class II (also noted “MHC II”) molecules are found only on a few specialized cell types, including macrophages, dendritic cells and B cells, all of which are dedicated antigen-presenting cells (APCs).
It is thus preferred that the complex according to the present invention comprises at least one antigen or antigenic epitope, which is at least one CD4+ epitope and/or at least one CD8+ epitope. More preferably, the complex according to the present invention comprises at least two antigenic epitopes, which are at least one CD4+ epitope and at least one CD8+ epitope.
The terms “CD4+ epitope” or “CD4+-restricted epitope”, as used herein, designate an epitope recognized by a CD4+ T cell, said epitope in particular consisting of an antigen fragment lying in the groove of a MHC class II molecule. A single CD4+ epitope comprised in the complex according to the present invention preferably consists of about 12-25 amino acids. It can also consist of, for example, about 8-25 amino acids or about 6-100 amino acids.
The terms “CD8+ epitope” or “CD8+-restricted epitope”, as used herein, designate an epitope recognized by a CD8+ T cell, said epitope in particular consisting of an antigen fragment lying in the groove of a MHC class I molecule. A single CD8+ epitope comprised in the complex according to the present invention preferably consists of about 8-11 amino acids. It can also consist of, for example, about 8-15 amino acids or about 6-100 amino acids.
Preferably, the at least one antigen can comprise or the at least one antigenic epitope can consist of a CD4+ epitope and/or a CD8+ epitope corresponding to antigenic determinant(s) of a tumor-associated antigen, a tumor-specific antigen, or an antigenic protein from a pathogen.
It is also preferred that the complex according to the present invention comprises at least two antigens or antigenic epitopes, wherein at least one antigen or antigenic epitope comprises or consists a CD4+ epitope and at least one antigen or antigenic epitope comprises or consists a CD8+ epitope. It is now established that Th cells (CD4+) play a central role in the anti-tumor immune response both in DC licensing and in the recruitment and maintenance of CTLs (CD8+) at the tumor site. Therefore, a complex according to the present invention comprising at least two antigens or antigenic epitopes, wherein at least one antigen or antigenic epitope comprises or consists of a CD4+ epitope and at least one antigen or antigenic epitope comprises or consists a CD8+ epitope, provides an integrated immune response allowing simultaneous priming of CTLs and Th cells and is thus preferable to immunity against only one CD8+ epitope or only one CD4+ epitope. For example, the complex according to the present invention may preferably comprise an Ealpha-CD430 epitope and a gp100-CD8+ epitope.
Preferably, the complex according to the present invention comprises or consists of more than one, i.e. at least two, antigens or antigenic epitopes, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens or antigenic epitopes. Accordingly, the complex according to the present invention may preferably comprise or consist of at least two, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, CD4+ epitopes and/or at least two, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, CD8+ epitopes. Thereby, the at least two antigens or antigenic epitopes are preferably different antigens or antigenic epitopes, more preferably the at least two antigens or antigenic epitopes are different from each other but relating to the same kind of tumor. A multi-antigenic vaccine will (i) avoid outgrowth of antigen-loss variants, (ii) target different tumor cells within a heterogeneous tumor mass and (iii) circumvent patient-to-patient tumor variability. Thus, the complex according to the present invention particularly preferably comprises at least four antigens or antigenic epitopes, in particular with at least two CD830 epitopes and at least two CD4+ epitopes. Such a complex according to the present invention induces multi-epitopic CD8 CTLs and CD4 Th cells to function synergistically to counter tumor cells and promote efficient anti-tumor immunity. Th cells are also involved in the maintenance of long-lasting cellular immunity that was monitored after vaccination. Such a complex according to the present invention induces polyclonal, multi-epitopic immune responses and poly-functional CD8+ and CD4+ T cells, and thus efficacious anti-tumor activity.
Preferably, the complex according to the present invention comprises at least two antigens or antigenic epitopes, more preferably the complex according to the present invention comprises at least three antigens or antigenic epitopes, even more preferably the complex according to the present invention comprises at least four antigens or antigenic epitopes, particularly preferably the complex according to the present invention comprises at least five antigens or antigenic epitopes and most preferably the complex according to the present invention comprises at least six antigens or antigenic epitopes. The antigens or antigenic epitopes comprised by the complex according to the present invention may be the same or different, preferably the antigens or antigenic epitopes comprised by the complex according to the present invention are different from each other. Preferably, the complex according to the present invention comprises at least one CD4+ epitope and at least one CD8+ epitope.
For example, the complex according to the present invention may preferably comprise a gp100-CD8+ epitope, an Ealpha-CD4+ epitope, and a further CD4+ epitope and a further CD8+ epitope. Even more preferably, the complex according to the present invention may comprise a polypeptide or protein comprising a gp100-CD8+ epitope and an Ealpha-CD4+ epitope. For example, such a polypeptide or protein comprised by the complex according to the present invention comprises or consists of an amino acid sequence according to SEQ ID NO: 3 or sequence variants thereof as defined above:
Accordingly, a particularly preferred complex according to the present invention comprises (i) the CPP according to SEQ ID NO: 1 and (ii) the cargo according to SEQ ID NO: 3. A preferred example of such a complex is according to SEQ ID NO: 4 shown below:
Preferably, the at least one antigen or antigenic epitope comprised by the complex according to the present invention is a peptide, polypeptide, or a protein. Examples of antigen or antigenic epitope of peptidic, polypeptidic, or proteic nature useful in the invention, include tumor antigens or antigenic epitopes thereof as described herein, allergy antigens or antigenic epitopes thereof, auto-immune self-antigens or antigenic epitopes thereof, pathogenic antigens or antigenic epitopes thereof as described herein, and antigens or antigenic epitopes thereof from viruses, preferably from cytomegalovirus (CMV), orthopox variola virus, orthopox alastrim virus, parapox ovis virus, molluscum contagiosum virus, herpes simplex virus 1, herpes simplex virus 2, herpes B virus, varicella zoster virus, pseudorabies virus, human cytomegaly virus, human herpes virus 6, human herpes virus 7, Epstein-Barr virus, human herpes virus 8, hepatitis B virus, chikungunya virus, O'nyong'nyong virus, rubivirus, hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellow fever virus, louping ill virus, St. Louis encephalitis virus, Japan B encephalitis virus, Powassan virus, FSME virus, SARS, SARS-associated corona virus, human corona virus 229E, human corona virus Oc43, Torovirus, human T cell lymphotropic virus type I, human T cell lymphotropic virus type II, HIV (AIDS), i.e. human immunodeficiency virus type 1 or human immunodeficiency virus type 2, influenza virus, Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Junin virus, Machupo virus, Borna disease virus, Bunyamwera virus, California encephalitis virus, Rift Valley fever virus, sand fly fever virus, Toscana virus, Crimean-Congo haemorrhagic fever virus, Hazara virus, Khasan virus, Hantaan virus, Seoul virus, Prospect Hill virus, Puumala virus, Dobrava Belgrade virus, Tula virus, sin nombre virus, Lake Victoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, Ivory Coast Ebola virus, influenza virus A, influenza virus B, influenza viruses C, parainfluenza virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus, human metapneumovirus, vesicular stomatitis Indiana virus, rabies virus, Mokola virus, Duvenhage virus, European bat lyssavirus 1+2, Australian bat lyssavirus, adenoviruses A-F, human papilloma viruses, condyloma virus 6, condyloma virus 11, polyoma viruses, adeno-associated virus 2, rotaviruses, orbiviruses, varicella including varizella zoster, etc., or antigens or antigenic epitopes from malaria parasite (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi), leishmania, typanosomes, amibes, bacteria, etc., or may be selected from epitopes or from variants of the above antigens or antigenic epitopes. Preferably, epitopes as well as variants of antigens as defined above exhibit an amino acid sequence identity of about 10%, in particular at least 10%, about 20%, in particular at least 20%, about 30%, in particular at least 30%, about 40%, in particular at least 40%, about 50%, in particular at least 50%, about 60%, in particular at least 60%, about 70%, in particular at least 70%, about 80%, in particular at least 80%, about 90% in particular at least 90%, at least 95% or at least 98% with one of the antigen or antigenic epitope sequences as shown or described above
Examples of antigens or antigenic epitopes in the category of peptide, polypeptide or protein include a combination of multiple glioma epitopes such as those described in Novellino et al (2005, Cancer Immunol Immunother, 54(3):187-207), Vigneron et al. (2013, Cancer Immun. 13:15). However, a single complex according to the present invention may also comprise only a subset, i.e. one or more of all of said glioma epitopes. In such a case preferably different complexes according to the present invention comprise different subsets of all of said glioma epitopes, so that for example a vaccine according to the present invention comprising such different complexes according to the present invention comprises all of said glioma epitopes but distributed in the different complexes.
Moreover, a complex according to the invention may also comprise at least one antigen or antigenic epitope, wherein said antigen or antigenic epitope is a polysaccharide, a lipid, a lipoprotein, and/or a glycolipid, in particular a polysaccharidic, lipidic, lipoproteic, and/or glycolipidic epitope, which can be, for example, pathogen epitopes as defined herewith.
In particular, the complex according to the invention may comprise at least one antigen or antigenic epitope, wherein said antigen or antigenic epitope is polysaccharidic, lipidic, lipoproteic, and/or glycolipidic, including viral, bacterial, fungal, protozoal and multicellular parasitic antigens or antigenic epitopes.
Preferably, said epitopes will be presented at the cell surface in an MHC class I and/or MHC class II context as described above.
Preferably, said lipidic epitopes will be presented at the cell surface in a CD1 (cluster of differentiation 1) context as described above.
The complex according to the present invention may also comprise at least one antigen or antigenic epitope, wherein said antigen or antigenic epitope is a small molecule drug or toxin.
Examples of cargo molecules within the category of small molecule drugs or toxins useful in the invention include cyclosporine A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid, diphtheria toxin, sunitinib and those molecules reviewed in De wit Amer (2010, Neuro Oncol, 12(3):304-16).
The complex according to the present invention comprises at least one antigen or antigenic epitope, preferably the complex according to the present invention comprises more than one antigen or antigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens or antigenic epitopes, more preferably the complex according to the present invention comprises (at least) two or three antigens or antigenic epitopes, even more preferably the complex according to the present invention comprises (at least) four or five antigens or antigenic epitopes.
If more than one antigen or antigenic epitope is comprised by the complex according to the present invention it is understood that said antigen or antigenic epitope is in particular also covalently linked in the complex according to the present invention, e.g. to another antigen or antigenic epitope and/or to another component of the complex according to the present invention, such as the cell penetrating peptide, and/or to another cargo molecule, e.g. a TLR peptide agonist.
The various antigens or antigenic epitopes comprised by the complex according to the present invention may be the same or different. Preferably, the various antigens or antigenic epitopes comprised by the complex according to the present invention are different from each other, thus providing a multi-antigenic and/or multi-epitopic complex.
Moreover, it is preferred that the more than one antigen or antigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens or antigenic epitopes, are positioned consecutively in the complex according to the present invention. This means in particular that all antigens and/or antigenic epitopes comprised by the complex are positioned in a stretch, which is neither interrupted by the cell penetrating peptide nor by another cargo molecule, such as a TLR peptide agonist. Rather, the CPP and, optionally, a further cargo molecule are positioned in the complex for example before or after such a stretch of all antigens and/or antigenic epitopes. However, the antigens and/or antigenic epitopes positioned consecutively in such a way may be linked to each other for example by a spacer or linker as described below, which is in particular neither a CPP nor another cargo molecule such as a TLR peptide agonist.
Alternatively, however, the various antigens and/or antigenic epitopes may also be positioned in any other way in the complex according to the present invention, for example with the CPP and, optionally, a further cargo molecule positioned in between two or more antigens and/or antigenic epitopes, i.e. with one or more antigens and/or antigenic epitopes positioned between the CPP and, optionally, a further cargo molecule (or vice versa) and, optionally, one or more antigens and/or antigenic epitopes positioned at the respective other end of the CPP and, optionally, a further cargo molecule.
It is understood that a number of different antigens or antigenic epitopes relating to the same kind of disease, in particular to the same kind of tumor, may be advantageously comprised by a single complex according to the present invention. Alternatively, a number of different antigens or antigenic epitopes relating to the same kind of disease, in particular to the same kind of tumor, may be distributed to subsets of different antigens or antigenic epitopes, in particular subsets complementing each other in the context of a certain kind of disease, e.g. tumor, which are comprised by different complexes according to the present invention, whereby such different complexes comprising different subsets may advantageously be administered simultaneously, e.g. in a single vaccine, to a subject in need thereof.
Preferably, the complex according to the present invention comprises a TLR peptide agonist. Thus, a TLR peptide agonist is a preferred peptidic cargo molecule in the complex according to the present invention.
Thereby, it is particularly preferred that the complex according to the present invention comprises—in addition to the CPP—at least one antigen or antigenic epitope as described above (i.e. one or more antigens or antigenic epitopes with the preferred embodiments as described above) and, furthermore, a TLR peptide agonist. Such a complex according to the present invention provides (i) stimulation of multi-epitopic cytotoxic T cell-mediated immunity, (ii) induction of Th cells and (iii) promotion of immunological memory. Thereby, a complex according to the present invention provides a potent vaccine, in particular having improved anti-tumor activity.
Recently, Toll Like Receptor (TLR) ligands are emerging as promising class of adjuvants (Baxevanis, C. N., I. F. Voutsas, and O. E. Tsitsilonis, Toll-like receptor agonists: current status and future perspective on their utility as adjuvants in improving anticancer vaccination strategies. Immunotherapy, 2013. 5(5): p. 497-511). A significant development of cancer vaccine studies was thus to include various TLR agonists to vaccine formulations, including TLR-3 (poly I:C), TLR-4 (monophosphoryl lipid A; MPL), TLR-5 (flagellin), TLR-7 (imiquimod), and TLR-9 (CpG) (Duthie M S, Windish H P, Fox C B, Reed S G. Use of defined TLR ligands as adjuvants within human vaccines. Immunol Rev. 2011; 239:178-196). The types of signaling and cytokines produced by immune cells after TLR stimulation control CD4+ T-cell differentiation into Th1, Th2, Th17, and Treg cells. Stimulation of immune cells such as dendritic cells (DCs) and T cells by most TLR-based adjuvants produces proinflammatory cytokines and promotes Th1 and CD8+ T responses (Manicassamy S, Pulendran B. Modulation of adaptive immunity with Toll-like receptors. Semin Immunol. 2009; 21:185-193).
In the complex according to the present invention, the TLR peptide agonist allows an increased targeting towards dendritic cells along with self-adjuvancity. Without being bound to any theory it is assumed that physical linkage of a TLR peptide agonist to the CPP and, optionally, to a further cargo molecule, such as at least one antigen or antigenic epitope as described herein in the complex according to the present invention provides an enhanced immune response by simultaneous stimulation of antigen presenting cells (APCs), in particular dendritic cells, that internalize, metabolize and display antigen(s).
As used in the context of the present invention, a “TLR peptide agonist” is an agonist of a Toll-like receptor (TLR), i.e. it binds to a TLR and activates the TLR, in particular to produce a biological response. Moreover, the TLR peptide agonist is a peptide, a polypeptide or a protein as defined above. Preferably, the TLR peptide agonist comprises from 10 to 150 amino acids, more preferably from 15 to 130 amino acids, even more preferably from 20 to 120 amino acids, particularly preferably from 25 to 110 amino acids, and most preferably from 30 to 100 amino acids.
Toll like receptors (TLRs) are transmembrane proteins that are characterized by extracellular, transmembrane, and cytosolic domains. The extracellular domains containing leucine-rich repeats (LRRs) with horseshoe-like shapes are involved in recognition of common molecular patterns derived from diverse microbes. Toll like receptors include TLRs1-10. Compounds capable of activating TLR receptors and modifications and derivatives thereof are well documented in the art. TLR1 may be activated by bacterial lipoproteins and acetylated forms thereof, TLR2 may in addition be activated by Gram positive bacterial glycolipids, LPS, LP A, LTA, fimbriae, outer membrane proteins, heat shock proteins from bacteria or from the host, and Mycobacterial lipoarabinomannans. TLR3 may be activated by dsRNA, in particular of viral origin, or by the chemical compound poly(LC). TLR4 may be activated by Gram negative LPS, LTA, Heat shock proteins from the host or from bacterial origin, viral coat or envelope proteins, taxol or derivatives thereof, hyaluronan containing oligosaccharides and fibronectins. TLR5 may be activated with bacterial flagellae or flagellin. TLR6 may be activated by mycobacterial lipoproteins and group B streptococcus heat labile soluble factor (GBS- F) or staphylococcus modulins. TLR7 may be activated by imidazoquinolines. TLR9 may be activated by unmethylated CpG DNA or chromatin—IgG complexes.
Preferably, the TLR peptide agonist comprised by the complex according to the present invention is an agonist of TLR1, 2, 4, 5, 6, and/or 10. TLRs are expressed either on the cell surface (TLR1, 2, 4, 5, 6, and 10) or on membranes of intracellular organelles, such as endosomes (TLR3, 4, 7, 8, and 9). The natural ligands for the endosomal receptors turned out to be nucleic acid-based molecules (except for TLR4). The cell surface-expressed TLR1, 2, 4, 5, 6, and 10 recognize molecular patterns of extracellular microbes (Monie, T. P., Bryant, C. E., et al. 2009: Activating immunity: Lessons from the TLRs and NLRs. Trends Biochem. Sci. 34(11), 553-561). TLRs are expressed on several cell types but virtually all TLRs are expressed on DCs allowing these specialized cells to sense all possible pathogens and danger signals.
However, TLR2, 4, and 5 are constitutively expressed at the surface of DCs. Accordingly, the TLR peptide agonist comprised by the complex according to the present invention is more preferably a peptide agonist of TLR2, TLR4 and/or TLR5. Even more preferably, the TLR peptide agonist is a TLR2 peptide agonist and/or a TLR4 peptide agonist. Particularly preferably, the TLR peptide agonist is a TLR4 peptide agonist. Most preferably, the TLR peptide agonist is one TLR peptide agonist, which is both, a TLR2 and a TLR4 agonist. TLR2 can detect a wide variety of ligands derived from bacteria, viruses, parasites, and fungi. The ligand specificity is often determined by the interaction of TLR2 with other TLRs, such as TLR1, 6, or 10, or non-TLR molecules, such as dectin-1, CD14, or CD36. The formation of a heterodimer with TLR1 enables TLR2 to identify triacyl lipoproteins or lipopeptides from (myco)bacterial origin, such as Pam3CSK4 and peptidoglycan (PGA; Gay, N. J., and Gangloff, M. (2007): Structure and function of Toll receptors and their ligands. Annu. Rev. Biochem. 76, 141-165; Spohn, R., Buwitt-Beckmann, U., et al. (2004): Synthetic lipopeptide adjuvants and Toll-like receptor 2—Structure-activity relationships. Vaccine 22(19), 2494-2499). Heterodimerization of TLR2 and 6 enables the detection of diacyl lipopeptides and zymosan. Lipopolysaccharide (LPS) and its derivatives are ligands for TLR4 and flagellin for TLR5 (Bryant, C. E., Spring, D. R., et al. (2010). The molecular basis of the host response to lipopolysaccharide. Nat. Rev. Microbiol. 8(1), 8-14).
TLR2 interacts with a broad and structurally diverse range of ligands, including molecules expressed by microbes and fungi. Multiple TLR2 agonists have been identified, including natural and synthetic lipopeptides (e.g. Mycoplasma fermentas macrophage-activating lipopeptide (MALP-2)), peptidoglycans (PG such as those from S. aureus), lipopolysaccharides from various bacterial strains (LPS), polysaccharides (e.g. zymosan), glycosylphosphatidyl-inositol-anchored structures from gram positive bacteria (e.g. lipoteichoic acid (LTA) and lipo-arabinomannan from mycobacteria and lipomannas from M. tuberculosis). Certain viral determinants may also trigger via TLR2 (Barbalat R, Lau L, Locksley R M, Barton G M. Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands. Nat Immunol. 2009: 10(11):1200-7). Bacterial lipopeptides are structural components of cell walls. They consist of an acylated s-glycerylcysteine moiety to which a peptide can be conjugated via the cysteine residue. Examples of TLR2 agonists, which are bacterial lipopeptides, include MALP-2 and it's synthetic analogue di-palmitoyl-S-glyceryl cysteine (Pam2Cys) or tri-palmitoyl-S-glyceryl cysteine (Pam3Cys).
A diversity of ligands interact with TLR4, including Monophosphoryl Lipid A from Salmonella minnesota R595 (MPLA), lipopolysaccharides (LPS), mannans (Candida albicans), glycoinositolphospholipids (Trypanosoma), viral envelope proteins (RSV and MMTV) and endogenous antigens including fibrinogen and heat-shock proteins. Such agonists of TLR4 are for example described in Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. Feb 24; 2006: 124(4):783-801 or in Kumar H, Kawai T, Akira S. Toll-like receptors and innate immunity. Biochem Biophys Res Commun. Oct. 30; 2009 388(4):621-5. LPS, which is found in the outer membrane of gram negative bacteria, is the most widely studied of the TLR4 ligands. Suitable LPS-derived TLR4 agonist peptides are described for example in WO 2013/120073 (A1).
TLR5 is triggered by a region of the flagellin molecule expressed by nearly all motile bacteria. Thus, flagellin, or peptides or proteins derived from flagellin and/or variants or fragments of flagellin are also suitable as TLR peptide agonists comprised by the complex according to the present invention.
Examples of suitable TLR peptide agonists thus include the TLR2 lipopeptide agonists MALP-2, Pam2Cys and Pam3Cys or modifications thereof, different forms of the TLR4 agonist LPS, e.g. N. meningitidis wild-type L3-LPS and mutant penta-acylated LpxL1-LPS, and the TLR5 agonist flagellin.
However, it is preferred that the TLR peptide agonist comprised by the complex according to the present invention is neither a lipopeptide nor a lipoprotein, neither a glycopeptide nor a glycoprotein. More preferably, the TLR peptide agonist comprised by the complex according to the present invention is a classical peptide, polypeptide or protein as defined herein.
A preferred TLR2 peptide agonist is annexin II or an immunomodulatory fragment thereof, which are described in detail in WO 2012/048190 A1, WO 2015/073632 A1 and U.S. patent application Ser. No. 13/0331546. In particular a TLR2 peptide agonist comprising an amino acid sequence according to SEQ ID NO: 4 or SEQ ID NO: 7 of WO 2012/048190 A1 or fragments or variants thereof are preferred. Particularly preferred TLR2 peptide agonists are disclosed in WO 2015/073632 A1, with the most preferred TLR2 peptide agonist having amino acid sequence according to SEQ ID NO: 1 of WO 2015/073632 A1.
Accordingly, a TLR2 peptide agonist comprising or consisting of an amino acid sequence according to SEQ ID NO: 5 or a sequence variant thereof is particularly preferred in the complex according to the present invention.
As used throughout the present specification, the term “sequence variant” refers to any alteration in a sequence as compared to a reference sequence. The term “sequence variant” includes nucleotide sequence variants and amino acid sequence variants. Preferably, a reference sequence is any of the sequences listed in the “Table of Sequences and SEQ ID Numbers” (Sequence listing), i.e. SEQ ID NO: 1 to SEQ ID NO: 52. Preferably, a sequence variant shares, in particular over the whole length of the sequence, at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, particularly preferably at least 95%, most preferably at least 98% sequence identity with a reference sequence, whereby sequence identity is calculated as described above. In particular, a sequence variant is a functional sequence variant, which means that the sequence variant preserves the specific function of the reference sequence (for example cell penetrating peptide functionality, antigenic epitope functionality, TLR agonist functionality etc., depending on the functionality of the reference sequence as disclosed herein). In particular, an amino acid sequence variant has an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the amino acid sequence variant has an amino acid sequence which is at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, particularly preferably at least 95%, most preferably at least 98% identical to the reference sequence. For example, variant sequences which are at least 90% identical have no more than 10 alterations, i.e. any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence.
Regarding TLR4, TLR peptides agonists are particularly preferred, which correspond to motifs that bind to TLR4, in particular (i) peptides mimicking the natural LPS ligand (RS01: Gln-Glu-lle-Asn-Ser-Ser- Tyr and RS09: Ala-Pro-Pro-His-Ala-Leu-Ser) and (ii) Fibronectin derived peptides. The cellular glycoprotein Fibronectin (FN) has multiple isoforms generated from a single gene by alternative splicing of three exons. One of these isoforms is the extra domain A (EDA), which interacts with TLR4.
Further suitable TLR peptide agonists thus comprise a fibronectin EDA domain or a fragment or variant thereof. Such suitable fibronectin EDA domains or a fragments or variants thereof are disclosed in EP 1 913 954 B1, EP 2 476 440 A1, US 2009/0220532 A1, and WO 2011/101332 A1.
Accordingly, a TLR4 peptide agonist comprising or consisting of an amino acid sequence according to SEQ ID NO: 6 or a sequence variant thereof is particularly preferred in the complex according to the present invention.
In addition, high-mobility group box 1 protein (HMGB1) and peptide fragments thereof are assumed to be TLR4 agonists. Such HMGB1-derived peptides are for example disclosed in US 2011/0236406 A1.
The complex according to the present invention preferably comprises one or more TLR peptide agonist, preferably the complex according to the present invention comprises exactly one TLR peptide agonist, however, the complex according to the present invention may also comprise more than one TLR peptide agonist, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TLR peptide agonists, for example the complex according to the present invention may comprise (at least) two or three TLR peptide agonists. If more than one TLR peptide agonist is comprised by the complex according to the present invention it is understood that said TLR peptide agonist is in particular also covalently linked in the complex according to the present invention, e.g. to another TLR peptide agonist and/or to the cell penetrating peptide and/or, optionally, to another cargo molecule, such as an antigen or antigenic epitope as described herein.
In a particularly preferred embodiment, the complex according to the present invention comprises one single TLR peptide agonist. In particularly, in this particularly preferred embodiment, the complex according to the present invention comprises one single TLR peptide agonist and no further component having TLR agonist properties except the one single TLR peptide agonist as described.
If the complex according to the present invention comprises more than one TLR peptide agonist, the various TLR peptide agonists comprised by the complex according to the present invention may be the same or different. Preferably, the various TLR peptide agonists comprised by the complex according to the present invention are different from each other. Moreover, it is preferred that more than one TLR peptide agonists, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 TLR agonists, are positioned consecutively in the complex according to the present invention. This means in particular that all TLR peptide agonists comprised by the complex are positioned in a stretch, which is neither interrupted by the cell penetrating peptide, nor by another cargo molecule, such as at least one antigen or antigenic epitope. Rather, the CPP and, optionally, another cargo molecule, such as at least one antigen or antigenic epitope are positioned in the complex for example before or after such a stretch of all TLR peptide agonists. However, the TLR peptide agonists positioned consecutively in such a way may be linked to each other for example by a spacer or linker as described below, which is in particular neither the CPP nor another cargo molecule, such as at least one antigen or antigenic epitope.
Alternatively, however, the various TLR peptide agonists may also be positioned in any other way in the complex according to the present invention, for example with the CPP and/or, optionally, another cargo molecule, such as at least one antigen or antigenic epitope positioned in between two or more TLR peptide agonists, i.e. with one or more TLR peptide agonist positioned between the CPP and, optionally, another cargo molecule, such as at least one antigen or antigenic epitope (or vice versa) and, optionally, one or more TLR peptide agonists positioned at the respective other end of the CPP and/or, optionally, another cargo molecule, such as at least one antigen or antigenic epitope.
It is understood that a number of different TLR peptide agonists activating the same or different TLR receptors may be advantageously comprised by a single complex according to the present invention. Alternatively, a number of different TLR peptide agonists activating the same or different TLR receptors may be distributed to subsets of different TLR peptide agonists activating the same or different TLR receptors, which are comprised by different complexes according to the present invention, whereby such different complexes comprising different subsets may advantageously be administered simultaneously, e.g. in a single vaccine, to a subject in need thereof.
Preferably, in the complex according to the present invention, the CPP and the one or more cargo molecules are covalently linked by chemical coupling in any suitable manner known in the art, such as cross-linking methods. However, attention is drawn to the fact that many known chemical cross-linking methods are non-specific, i.e., they do not direct the point of coupling to any particular site on the the CPP and the one or more cargo molecules. Thus, the use of non-specific cross-linking agents may attack functional sites or sterically block active sites, rendering the fused components of the complex according to the present invention biologically inactive. It is referred to the knowledge of the skilled artisan to block potentially reactive groups by using appropriate protecting groups. Alternatively, the use of the powerful and versatile oxime and hydrazone ligation techniques, which are chemo-selective entities that can be applied for the cross-linking of the CPP and the one or more cargo molecules may be employed. This linking technology is described e.g. by Rose et al. (1994), JACS 116, 30.
Coupling specificity can be increased by direct chemical coupling to a functional group found only once or a few times in the CPP and the one or more cargo molecules, which functional group is to be cross-linked to the another of the CPP and the one or more cargo molecules. As an example, the cystein thiol group may be used, if just one cystein residue is present in the CPP or in one or more cargo molecules of complex according to the present invention. Also, for example, if the CPP or one or more cargo molecules contains no lysine residues, a cross-linking reagent specific for primary amines will be selective for the amino terminus of the respective component. Alternatively, cross-linking may also be carried out via the side chain of a glutamic acid residue placed at the N-terminus of the peptide such that a amide bond can be generated through its side-chain. Therefore, it may be advantageous to link a glutamic acid residue to the N-terminus of the CPP or one or more cargo molecules. However, if a cysteine residue is to be introduced into the CPP or one or more cargo molecules, introduction at or near its N- or C-terminus is preferred. Conventional methods are available for such amino acid sequence alterations based on modifications of the CPP or the one or more cargo molecules by either adding one or more additional amino acids, e.g. inter alia an cystein residue, to the translocation sequence or by substituting at least one residue of the translocation sequence(s) being comprised in the respective component. In case a cystein side chain is used for coupling purposes, the CPP or one or more cargo molecules have preferably one cystein residue. Any second cystein residue should preferably be avoided and can, optionally, be replaced when they occur in the respective component comprised by the complex according to the present invention. When a cysteine residue is replaced in the original sequence of the CPP or of one or more cargo molecules, it is typically desirable to minimize resulting changes in the peptide folding of the respective component. Changes in folding are minimized when the replacement is chemically and sterically similar to cysteine. Therefore, serine is preferred as a replacement for cystein.
Coupling of the CPP and the one or more cargo molecules can be accomplished via a coupling or conjugating agent including standard peptide synthesis coupling reagents such as HOBt, HBTU, DICI, TBTU. There are several intermolecular cross-linking agents which can be utilized, see for example, Means and Feeney, Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43. Among these reagents are, for example, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-phenylene)bismaleimide; N,N′-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges; and 1,5-difluoro-2,4-dinitrobenzene. Other cross-linking agents useful for this purpose include: p,p′-difluoro-m,m′-dinitrodiphenylsulfone; dimethyl adipimidate; phenol-1,4-disulfonylchloride; hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate; glutaraldehyde and disdiazobenzidine. Cross-linking agents may be homobifunctional, i.e., having two functional groups that undergo the same reaction. A preferred homobifunctional cross-linking agent is bismaleimidohexane (BMH). BMH contains two maleimide functional groups, which react specifically with sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). The two maleimide groups are connected by a hydrocarbon chain. Therefore, BMH is useful for irreversible cross-linking of proteins (or polypeptides) that contain cysteine residues. Cross-linking agents may also be heterobifunctional. Heterobifunctional cross-linking agents have two different functional groups, for example an amine-reactive group and a thiol-reactive group, that will cross-link two proteins having free amines and thiols, respectively. Examples of heterobifunctional cross-linking agents are Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimide 4-(p-maleimidophenyl)butyrate (SMPB), an extended chain analog of MBS. The succinimidyl group of these cross-linkers reacts with a primary amine, and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine residue. Because cross-linking agents often have low solubility in water, a hydrophilic moiety, such as a sulfonate group, may be added to the cross-linking agent to improve its water solubility. Sulfo-MBS and sulfo-SMCC are examples of cross-linking agents modified for water solubility. Many cross-linking agents yield a conjugate that is essentially non-cleavable under cellular conditions. Therefore, some cross-linking agents contain a covalent bond, such as a disulfide, that is cleavable under cellular conditions. For example, Traut's reagent, dithiobis (succinimidylpropionate) (DSP), and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) are well-known cleavable cross-linkers. The use of a cleavable cross-linking agent permits the cell penetrating peptide and the one or more cargo molecules comprised by the complex according to the present invention to separate from each other after delivery into the target cell. For this purpose, direct disulfide linkage may also be useful. Chemical cross-linking may also include the use of spacer arms. Spacer arms provide intramolecular flexibility or adjust intramolecular distances between conjugated moieties and thereby may help preserve biological activity. A spacer arm may be in the form of a protein (or polypeptide) moiety that includes spacer amino acids, e.g. proline. Alternatively, a spacer arm may be part of the cross-linking agent, such as in “long-chain SPDP” (Pierce Chem. Co., Rockford, Ill., cat. No. 21651 H). Numerous cross-linking agents, including the ones discussed above, are commercially available. Detailed instructions for their use are readily available from the commercial suppliers. More detailed information on protein cross-linking and conjugate preparation, which is useful in the context of linkage of components a), b), and c) comprised by the complex according to the present invention can be retrieved from: Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991).
Cross-linking agents for peptide or protein crosslinking include for example (i) amine-to-amine crosslinkers, e.g. homobifunctional amine-specific protein crosslinking reagents based on NHS-ester and imidoester reactive groups for selective conjugation of primary amines; available in short, long, cleavable, irreversible, membrane permeable, and cell surface varieties; (ii) sulfhydryl-to-carbohydrate crosslinkers, e.g. crosslinking reagents based on maleimide and hydrazide reactive groups for conjugation and formation of covalent crosslinks; (iii) sulfhydryl-to-sulfhydryl crosslinkers, e.g. homobifunctional sulfhydryl-specific crosslinking reagents based on maleimide or pyridyldithiol reactive groups for selective covalent conjugation of protein and peptide thiols (reduced cysteines) to form stable thioether bonds; (iv) photoreactive crosslinkers, e.g. aryl azide, diazirine, and other photo-reactive (light-activated) chemical heterobifunctional crosslinking reagents to conjugate proteins, nucleic acids and other molecular structures involved in receptor-ligand interaction complexes via two-step activation; (v) amine-to-sulfhydryl crosslinkers, e.g. heterobifunctional protein crosslinking reagents for conjugation between primary amine (lysine) and sulfhydryl (cysteine) groups of proteins and other molecules; available with different lengths and types of spacer arms; and (vi) amine-to-amine crosslinkers, e.g. carboxyl-to-amine crosslinkers, e.g. Carbodiimide crosslinking reagents, DCC and EDC (EDAC), for conjugating carboxyl groups (glutamate, aspartate, C-termini) to primary amines (lysine, N-termini) and also N-hydroxysuccinimide (NHS) for stable activation of carboxylates for amine-conjugation.
Examples of crosslinkers in general, which can be used in the complex according to the present invention, include N-(α-Maleimidoacetoxy)-succinimide ester, N-5-Azido-2-nitrobenzyloxy-succinimide, 1,4-Bis-Maleimidobutane, 1,4-Bis-Maleimmidyl-2,3-dihydroxy-butane, Bis-Maleimidohexane, Bis-Maleimidoethane, N-(β-Maleimidopropionic acid)hydrazide*TFA, N-(β-Maleimidopropyloxy)succinimide ester, 1,8-Bis-Maleimidodiethylene-glycol, 1,11-Bis-Maleimidotriethyleneglycol, Bis (sulfosuccinimidyl)suberate, Bis (sulfosuccinimidyl)glutarate-d0, Bis (sulfosuccinimidyl)2,2,4,4-glutarate-d4, Bis (sulfosuccinimidyl)suberate-d0, Bis (sulfosuccinimidyl)2,2,7,7-suberate-d4, Bis (NHS)PEG5, Bis (NHS)PEG9, Bis (2-[succinimidoxycarbonyloxy]ethyl)sulfone, N,N-Dicyclohexylcarbodiimide, 1-5-Difluoro-2,4-dinitrobenzene, Dimethyl adipimidate*2HCl, Dimethyl pimelimidate*2HCl, Dimethyl suberimidate*2HCl, Disuccinimidyl glutarate, Dithiobis(succimidylpropionate) (Lomant's Reagent), Disuccinimidyl suberate, Disuccinimidyl tartarate, Dimethyl 3,3′-dithiobispropionimidate*2HCl, Dithiobis-maleimidoethane, 3,3′-Dithiobis (sulfosuccinimidylpropionate), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, Ethylene glycol bis (succinimidylsuccinate), N-ε-Maleimidocaproic acid, N-(ε-Maleimidocaproic acid)hydrazide, N-(ε-Maleimidocaproyloxy)succinimide ester, N-(γ-Maleimidobutyryloxy)succinimide ester, N-(κ-Maleimidoundecanoic acid)hydrazide, NHS-LC-Diazirine, Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate, Succinimidyl 6-(3′-[2-pyridyldithio] propionamido)hexanoate, L-Photo-Leucine, L-Photo-Methionine, m-Maleimidobenzoyl-N-hydroxysuccinimide ester, 4-(4-N-Maleimidophenyl)-butyric acid hydrazide*HCl, 2-[N2-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-L-lysinyl]ethylmethanethiosulfate, 2-{N2-[N6-(4-Azido-2,3,5,6-tetrafluorobenzoyI)-N6-(6-biotinamidocaproyl)-L-lysinyl]}ethylmethanethiosulfate, N-Hydroxysuccinimide, N-hydroxysuccinimide ester ethane azide, N-hydroxysuccinimide ester tetraoxapentadecane azide, N-hydroxysuccinimide ester dodecaoxanonatriacontane azide, NHS-Phosphine, 3-(2-Pyridyldithio)propionylhydrazide, 2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide, 2-pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide, N-(p-Maleimidophenyl)isocyanate, Succinimdyl 3-(bromoacetamido)propionate, NHS-Diazirine, NHS-SS-Diazirine, N-succinimidyl iodoacetate, N-Succinimidyl(4-iodoacetyl)aminobenzoate, Succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate, NHS-PEG2-Maliemide, NHS-PEG4-Maliemide, NHS-PEG6-Maleimide, NHS-PEG8-Maliemide, NHS-PEG12-Maliemide, NHS-PEG24-Maleimide, Succinimidyl 4-(p-maleimido-phenyl)butyrate, Succinimidyl-6-(β-maleimidopropionamido)hexanoate, 4-Succinimidyloxycarbonyl-methyl-α-(2-pyridyldithio)toluene, Succinimidyl-(4-psoralen-8-yloxy)butyrate, N-Succinimidyl 3-(2-pyridyldithio)propionate, Ethylene glycol bis (sulfo-succinimidyl succinate), N-(ε-Maleimidocaproyloxy)sulfosuccinimide ester, N-(γ-Maleimidobutryloxy)sulfosuccinimide ester, N-(κ-Maleimidoundecanoyloxy)sulfosuccinimide ester, Sulfo-NHS-LC-Diazirine, Sulfosuccinimidyl 6-(3′-[2-pyridyldithio]propionamido)hexanoate, m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-Hydroxysuccinimide, Sulfo-NHS-Phosphine, Sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylami no)hexanoate, Sulfo-NHS-(2-6-[Biotinamido]-2-(p-azidobezamido), Sulfo-NHS-Diazirine, Sulfo-NHS-SS-Diazirine, Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate, Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, Tris-(2-Maleimidoethyl)amine (Trifunctional), and Tris-(succimimidyl aminotricetate) (Trifunctional).
The linkage between the CPP and the one or more cargo molecules of the complex according to the present invention may be directly or indirectly, i.e. the CPP and the one or more cargo molecules directly adjoin or they may be linked by an additional component of the complex, e.g. a spacer or a linker.
A direct linkage may be realized preferably by an amide bridge, if the components to be linked have reactive amino or carboxy groups. More specifically, if the components to be linked are peptides, polypeptides or proteins, a peptide bond is preferred. Such a peptide bond may be formed using a chemical synthesis involving both components (an N-terminal end of one component and the C-terminal end of the other component) to be linked, or may be formed directly via a protein synthesis of the entire peptide sequence of both components, wherein both (protein or peptide) components are preferably synthesized in one step. Such protein synthesis methods include e.g., without being limited thereto, liquid phase peptide synthesis methods or solid peptide synthesis methods, e.g. solid peptide synthesis methods according to Merrifield, t-Boc solid-phase peptide synthesis, Fmoc solid-phase peptide synthesis, BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) based solid-phase peptide synthesis, etc. Alternatively, ester or ether linkages are preferred.
Moreover, in particular if the components to be linked are peptides, polypeptides or proteins, a linkage may occur via the side chains, e.g. by a disulfide bridge. Further components of other chemical nature, e.g. the at least one antigen or antigenic epitope if it is not of peptidic nature, may be likewise attached to the components of peptidic nature, e.g. the CPP and the one or more cargo molecules, if it is of peptidic nature. The linkage via a side chain will preferably be based on side chain amino, thiol or hydroxyl groups, e.g. via an amide or ester or ether linkage. A linkage of a peptidic main chain with a peptidic side chain of another component may also be via an isopeptide bond. An isopeptide bond is an amide bond that is not present on the main chain of a protein. The bond forms between the carboxyl terminus of one peptide or protein and the amino group of a lysine residue on another (target) peptide or protein.
The complex according to the present invention may optionally comprise a spacer or linker, which are non-immunologic moieties, which are preferably cleavable, and which link the CPP and the one or more cargo molecules, and/or optionally link one cargo molecule to another cargo molecule, and/or optionally link consecutive antigens or antigenic epitopes, and/or optionally link consecutive TLR peptide agonists, and/or which can be placed at the N- and/or C-terminal part of the components of the complex and/or at the N- and/or C-terminal part of the complex itself. A linker or spacer may preferably provide further functionalities in addition to linking of the components, and preferably being cleavable, more preferably naturally cleavable inside the target cell, e.g. by enzymatic cleavage. However, such further functionalities do in particular not include any immunological functionalities. Examples of further functionalities, in particular regarding linkers in fusion proteins, can be found in Chen X. et al., 2013: Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369, wherein for example also in vivo cleavable linkers are disclosed. Moreover, Chen X. et al., 2013: Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369 also discloses various linkers, e.g. flexible linkers and rigid linkers, and linker designing tools and databases, which can be useful in the complex according to the present invention or to design a linker to be used in the complex according to the present invention.
Said spacer (or linker) may be peptidic or non-peptidic, preferably the spacer is peptidic. Preferably, a peptidic spacer consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, more preferably of about 1, 2, 3, 4, or 5 amino acids. The amino acid sequence of the peptidic spacer may be identical to that of the N-terminal or C-terminal flanking region of any of the CPP or the one or more cargo molecules. For example, a preferred spacer is the natural flanking amino acid residues of the cargo sequence, for example the natural flanking amino acid residues of each antigenic epitope comprised by the complex according to the present invention. More preferably, the spacers/linkers used in the complex according to the present invention are the natural flanking four amino acid residues of each antigenic epitope comprised by the complex. Alternatively a peptidic spacer can consist of non-natural amino acid sequences such as an amino acid sequence resulting from conservative amino acid substitutions of said natural flanking regions or sequences of known cleavage sites for proteases such as an enterokinase target site (amino acid sequence: DDDK, SEQ ID NO: 7), factor Xa target site (amino acid sequence: IEDGR, SEQ ID NO: 8), thrombin target site (amino acid sequence: LVPRGS, SEQ ID NO: 9), protease TEV target site (amino acid sequence: ENLYFQG, SEQ ID NO: 10), PreScission protease target site (amino acid sequence LEVLFQGP, SEQ ID NO: 11), polycationic amino acids, e.g. poly K, furin target site (amino acid sequence RX(R/K)R, SEQ ID NO: 12). In a particular embodiment, the peptidic spacer does not contain any Cys (C) residues. In a preferred embodiment the linker sequence contains at least 20%, more preferably at least 40% and even more preferably at least 50% Gly or β-alanine residues, e.g. GlyGlyGly, CysGlyGly or GlyGlyCys, GlyGlyGlyGlyGly (SEQ ID NO: 13), GlyGlyGlyGly (SEQ ID NO: 14), and/or a (G4S)n linker as described herein wherein “G4S” refers to the amino acid sequence GGGGS (SEQ ID NO: 53) which may be present once or repeatedly, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times as indicated by the number “n”. Appropriate linker sequences can be easily selected and prepared by a person skilled in the art. They may be composed of D and/or L amino acids. Further examples of a peptidic spacer include the amino acid sequences EQLE (SEQ ID NO: 15) or TEWT (SEQ ID NO: 16) or any conservative substitutions thereof. Further preferred linkers/spacers include those, which are described with their amino acid sequences in Table I of Reddy Chichili V P, Kumar V, Sivaraman J. 2013. Linkers in the structural biology of protein-protein interactions. Protein Sci. 22:153-167.
A non-peptidic spacer can include or may be an ester, a thioester, and a di-sulfide.
In particular, the complex according to the invention may comprise a spacer or linker, in particular a peptidic spacer, placed between the CPP and the one or more cargo molecules. This peptidic spacer can be chosen by one skilled in the art so that it may be cut by the cell machinery once the complex comprising the cell penetrating peptide and the cargo molecule has been internalized.
When the complex comprises several antigens or antigenic epitopes or when the complex comprises several TLR peptide agonists, it will be clear for one skilled in the art that each of the antigens or antigenic epitopes and/or each of the TLR peptide agonists comprised in the complex of the invention can be either directly linked to each other or linked via spacers or linkers such as described herein, e.g., a peptidic spacer consisting of a few amino acids. Alternatively, when the complex according to the present invention comprises several antigens or antigenic epitopes or when the complex comprises several TLR peptide agonists, it is also possible that some antigens or antigenic epitopes and/or some TLR peptide agonists comprised by the complex of the invention are directly linked to each other and some other antigens or antigenic epitopes and/or some other TLR peptide agonists are linked via spacers or linkers such as described herein, e.g. a peptidic spacer consisting of a few amino acids.
For example, two successive antigens or antigenic epitopes or two successive TLR peptide agonists or in more general, any two successive cargo molecules or components comprised in the complex of the invention may be linked to each other by spacers consisting of the natural flanking regions of said antigens or antigenic epitopes or of said TLR peptide agonists, respectively. For example, such a spacer may consists of up to about 8 amino acids corresponding to up to about 4 amino acids of the N-terminal or C-terminal flanking region of the first component to be linked, followed by up to about 4 amino acids of the N-terminal or C-terminal flanking region of the second component to be linked.
In an illustration of the present invention, the spacer used to link a first antigen/antigenic epitope, a TLR peptide agonist, or in more general a first component of the complex (“antigen/epitope/TLR peptide agonist/component 1”) to a second antigen/antigenic epitope, a TLR peptide agonist, or in more general a second component of the complex (“antigen/epitope/TLR peptide agonist/component 2”) consists of about 8 amino acids corresponding to any possible combination ranging from: 0 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 8 flanking amino acids of antigen/epitope/TLR peptide agonist/component 2, to 8 flanking amino acids of antigen/epitope/TLR peptide agonist/component 1 and 0 flanking amino acid of antigen/epitope/TLR peptide agonist/component 2, i.e. including 1 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 7 flanking amino acids of antigen/epitope/TLR peptide agonist/component 2, 2 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 6 flanking amino acids of antigen/epitope/TLR peptide agonist/component 2, 3 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 5 flanking amino acids of antigen/epitope/TLR peptide agonist/component 2, 4 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 4 flanking amino acids of antigen/epitope/TLR peptide agonist/component 2, 5 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 3 flanking amino acids of antigen/epitope/TLR peptide agonist/component 2, 6 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 2 flanking amino acids of antigen/epitope/TLR peptide agonist/component 2, 7 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 1 flanking amino acid of antigen/epitope/TLR peptide agonist/component 2, 8 flanking amino acid of antigen/epitope/TLR peptide agonist/component 1 and 0 flanking amino acids of antigen/epitope/TLR peptide agonist/component 2. It will be understood that the total of 8 amino acids constituting a spacer linking two consecutive antigen/epitope/TLR peptide agonist/component is not an absolute value and the spacer could also be composed of a total of, for instance, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 9 amino acids or 10 amino acids. Similarly, equivalent combinations as mentioned above are also an illustration of the invention in the situation where a spacer has less or more than 8 amino acids.
In another particular illustration of the present invention, the spacer used to link a first antigen/antigenic epitope, a first TLR peptide agonist, or in more general a first component of the complex (“antigen/epitope/TLR peptide agonist/component 1”) to a second antigen/antigenic epitope, a first TLR peptide agonist, or in more general a second component of the complex (“antigen/epitope/TLR peptide agonist/component 2”) consists of e.g. 1, 2, 3, 4, or 5 amino acids. More particularly, said spacer's amino acid sequence can correspond to the 4 amino acids of the N-terminal or C-terminal flanking region of antigen/epitope/TLR peptide agonist/component 1 or antigen/epitope/TLR peptide agonist/component 2. A spacer as described above may also be placed at the C-terminal part of the last antigen/epitope/TLR peptide agonist/component comprised in the complex according to the present invention.
The technics for linking the CPP and, optionally, one or more cargo molecules are well documented in the literature and can depend on the nature of the cargo molecule, e.g. the at least one antigen or antigenic epitope. For instance, linkages between the CPP and, optionally, one or more cargo molecules can be achieved via cleavable disulphide linkages through total stepwise solid-phase synthesis or solution-phase or solid-phase fragment coupling, stable amide, thiazolidine, oxime and hydrazine linkage, disulphide linkage, stable thiomaleimide linkage, peptide bond (including peptide bonds between amino acids of a fusion protein), or electrostatic or hydrophobic interactions.
Preferably, the at least cargo molecule, e.g. the at least one antigen or antigenic epitope, comprised by the complex according to the present invention as well as any optional spacer or linker comprised by the complex according to the present invention are of peptidic nature. More preferably, all components of the complex according to the present invention, e.g. the cell penetrating peptide and the at least one cargo molecule, which is a peptide, polypeptide or protein and any optional peptidic linker or spacer are linked in the complex according to the present invention by a peptide bond. Most preferably, the complex according to the present invention is thus a peptide, a polypeptide or a protein, such as a fusion protein, e.g. a recombinant fusion protein.
Accordingly, it is preferred that the complex according to the present invention is a polypeptide or a protein, in particular a fusion protein or a fusion polypeptide. Of note, the complexes and the CPPs according to the present invention are typically recombinant peptides, polypeptides or proteins. The term “recombinant” as used herein means that it (here: the polypeptide or the protein) does not occur naturally. In this context it is noted that the CPP according to the invention has a serine (Ser) at position 12 (cf. SEQ ID NO: 1), which corresponds to position 189 of ZEBRA (SEQ ID NO: 2). Of note, naturally occurring ZEBRA (SEQ ID NO: 2) has a cysteine at the corresponding position 189. Accordingly, the CPP according to the present invention is a recombinant peptide. Therefore, the complex according to the present invention, which comprises the (recombinant) CPP according to the present invention is also recombinant. Preferably, the complex according to the present invention, comprises the CPP and one or more cargo molecules, wherein CPP and the cargo molecule are preferably of different origins, i.e. do not naturally occur in this combination. Thereby, it is preferred that the cargo molecule is not derived from EBV (Epstein-Barr virus), in particular the cargo molecule is preferably not a an EBV peptide or an EBV protein.
In this context, a complex comprising or consisting of an amino acid sequence according to SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, or of a sequence variant thereof as described herein or a complex comprising or consisting of an amino acid sequence sharing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with any of SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 is preferred; a complex comprising or consisting of an amino acid sequence according to SEQ ID NO: 18 or SEQ ID NO: 19, a sequence variant thereof as described herein or a complex comprising or consisting of an amino acid sequence sharing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with any of SEQ ID NO: 18 and SEQ ID NO: 19 is more preferred; a complex comprising or consisting of an amino acid sequence according to SEQ ID NO: 19 or a sequence variant thereof as described herein or a complex comprising or consisting of an amino acid sequence sharing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with SEQ ID NO: 19 is even more preferred.
Preferably the at least one antigen or antigenic epitope is positioned C-terminally of the cell penetrating peptide, whereby the cell penetrating peptide and the at least one antigen or antigenic epitope are optionally linked by a further component, e.g. a linker, a spacer, or by a TLR peptide agonist. Even more preferably, the at least one antigen or antigenic epitope is positioned C-terminally of the cell penetrating peptide, whereby the cell penetrating peptide and the at least one antigen or antigenic epitope are optionally linked by a further component, e.g. a linker, a spacer, but not by the at least one TLR peptide agonist.
Particularly preferably, the complex according to the present invention is a (recombinant) polypeptide or a (recombinant) protein as described above, which comprises, as cargo molecules, at least one antigen or antigenic epitope and a TLR peptide agonist, and in such a preferred complex according to the present invention the cell penetrating peptide, the at least one antigen or antigenic epitope and the TLR peptide agonist are positioned in N-terminus→C-terminus direction of the main chain of said complex in the order:
(a) cell penetrating peptide—at least one antigen or antigenic epitope—TLR peptide agonist; or
(b) TLR peptide agonist—cell penetrating peptide—at least one antigen or antigenic epitope,
wherein the cell penetrating peptide, the at least one antigen or antigenic epitope and the
TLR peptide agonist may be linked by a further component, in particular by a linker or a spacer.
Nucleic Acid Encoding the Cell Penetrating Peptides and the Complexes According to the Invention
In another aspect the present invention provides a nucleic acid encoding the cell penetrating peptide according to the present invention as described herein and/or the complex according to the present invention as described herein, wherein the complex is a polypeptide or a protein. In particular, the present invention provides polynucleotides encoding the cell penetrating peptide according to the present invention and/or the complex according to the present invention, as defined above.
In this context, nucleic acids preferably comprise single stranded, double stranded or partially double stranded nucleic acids, preferably selected from genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complimentary RNA/DNA sequences with or without expression elements, a mini-gene, gene fragments, regulatory elements, promoters, and combinations thereof.
Preferably, the invention relates to a nucleic acid encoding the cell penetrating peptide as described above and/or the complex as described above, which is in particular a polypeptide or protein, said complex comprising a cell penetrating peptide and a peptidic cargo molecule, e.g. at least one antigen or antigenic epitope, which is a polypeptide or protein, and/or at least one TLR peptide agonist, wherein the cell penetrating peptide, the at least one antigen or antigenic epitope, and the at least one TLR peptide agonist are covalently linked, optionally with peptidic spacer(s) or linker(s) as described herein. If more than one antigen or antigenic epitope, which is a polypeptide or protein, is comprised by said complex, the more than one antigens or antigenic epitopes are also covalently linked, optionally with peptidic spacer(s) or linker(s) as described herein.
Particularly preferably the nucleic acid according to the present invention encodes the cell penetrating peptide as described above and/or the complex as described above, which is a (recombinant) fusion protein comprising the cell penetrating peptide as described above and at least one cargo molecule, which is preferably TLR agonist as described above and at least one, preferably at least two, more preferably at least three, even more preferably at least four, particularly preferably at least five, most preferably at least six antigens or antigenic epitopes as described above, preferably arranged in a consecutive manner as described above.
Production and Purification of the Cell Penetrating Peptides and Complexes According to the Invention
According to a further aspect the present invention provides a vector, in particular a recombinant vector, comprising a nucleic acid according to the present invention as described above.
The term “vector”, as used in the context of the present invention, refers to a nucleic acid molecule, preferably to an artificial nucleic acid molecule, i.e. a nucleic acid molecule which does not occur in nature. A vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector is a vector which allows the convenient storage of a nucleic acid molecule. Thus, the vector may comprise a sequence corresponding, e.g., to a desired antibody or antibody fragment thereof according to the present invention. An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins. For example, an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a promoter sequence. A cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bacteriophage vector. A transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors. A vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector. Preferably, a vector is a DNA molecule. For example, a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication. Preferably, a vector in the context of the present application is a plasmid vector. Preferably, a vector in the context of the present application is an expression vector.
Cells transformed with a vector according to the present invention are also included within the scope of the invention. Examples of such cells include, but are not limited to, bacterial cells, e.g. E. coli, and eukaryotic cells, e.g., yeast cells, animal cells or plant cells. In one embodiment the cells are mammalian, e.g., human, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Accordingly, the present invention also relates to a cell expressing the antibody, or the antigen binding fragment thereof, according to the present invention; or comprising the vector according to the present invention.
In particular, a cell may be transfected with a vector according to the present invention, preferably with an expression vector. The term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably into eukaryotic cells. In the context of the present invention, the term “transfection” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, preferably into eukaryotic cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g. based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine etc. Preferably, the introduction is non-viral.
Numerous expression systems can be used, including without limitation chromosomes, episomes, and derived viruses. More particularly, the vector according to the present invention, in particular the recombinant vector used, can be derived from bacterial plasmids, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses such as baculovirus, papilloma viruses such as SV40, vaccinia viruses, adenoviruses, fox pox viruses, pseudorabies viruses, retroviruses.
For example, such vectors, in particular recombinant vectors, can equally be cosmid or phagemid derivatives. The nucleotide sequence, in particular the nucleic acid according to the present invention, may be inserted in the recombinant expression vector by methods well known to a person skilled in the art such as, for example, those described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.
The vector, in particular the recombinant vector, may also include nucleotide sequences that control the regulation of the expression, in particular of the nucleic acid according to the present invention, as well as nucleotide sequences permitting the expression, the transcription, and the translation, in particular of the nucleic acid according to the present invention. Typically, these sequences are selected according to the host cells used.
Thus, for example, an appropriate secretion signal can be integrated in the vector according to the present invention, in particular in a recombinant vector, so that the polypeptide or protein encoded by the nucleic acid according to the present invention, will be directed, for example towards the lumen of the endoplasmic reticulum, towards the periplasmic space, on the membrane or towards the extracellular environment. The choice of an appropriate secretion signal may facilitate subsequent protein purification.
In yet another aspect the present invention provides a host cell comprising the vector, in particular the recombinant vector, according to the present invention.
The introduction of the vector, in particular the recombinant vector, according to the present invention into a host cell can be carried out according to methods that are well known to a person skilled in the art, such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., 2nd ed., McGraw-Hill Professional Publishing, 1995, and MOLECULAR CLONING: A LABORATORY MANUAL, supra, including for example transfection as described above, e.g. by calcium phosphate, by DEAE dextran, or by cationic lipids; microinjection, electroporation, transduction or infection.
The host cell can be, for example, bacterial cells such as E. coli, cells of fungi such as yeast cells and cells of Aspergillus, Streptomyces, insect cells, and/or any cell line, e.g. Chinese Hamster Ovary cells (CHO), C127 mouse cell line, BHK cell line of Syrian hamster cells, Human Embryonic Kidney 293 (HEK 293) cells. Preferably, the host cell according to the present invention is mammalian, e.g., human, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Dendritic cells and dendritic cell lines are particularly preferred as a host cell. Typically, the choice of a culture medium depends in particular on the choice of the cell type and/or the cell line, whereby the skilled person is aware of suitable culture media, which are appropriate for a selected cell type and/or cell line.
The host cells can be used, for example, to express a polypeptide or protein, in particular the cell penetrating peptide according to the present invention and/or the complex according to the present invention, on the basis of the vector and/or the nucleic acid according to the present invention. After purification by standard methods, the expressed polypeptide or protein, in particular the cell penetrating peptide according to the present invention and/or the complex according to the present invention, can be used in a method as described hereinafter.
Accordingly, the present invention also provides a method for preparing the cell penetrating peptide according to the present invention and/or the complex according to the present invention, in particular wherein the complex is a polypeptide or protein. Said method comprises the steps of:
For protein extraction commercially available kits and/or reagents may be used, for example BugBuster™ from Novagen.
Preferably, the method for preparing the cell penetrating peptide according to the present invention and/or the complex according to the present invention as described above further comprises the following step:
wherein step (iii) follows step (ii) as described above.
Moreover, it is preferred that the method for preparing the cell penetrating peptide according to the present invention and/or the complex according to the present invention as described above further comprises the following step:
wherein step (iv) follows step (ii), or, if present, step (iii) as described above.
In addition, the cell penetrating peptide according to the present invention and/or the complex according to the present invention may also be prepared by synthetic chemistry methods, for example by solid-phase peptide synthesis.
Purification of those peptides or proteins may be carried out by means of any technique known in the art for protein/peptide purification. Exemplary techniques include ion-exchange chromatography, hydrophobic interaction chromatography, and immunoaffinity methods.
Thus, the present invention also provides a method for preparing the cell penetrating peptide according to the present invention and/or the complex according to the present invention comprising the steps of:
(i) chemically synthesizing said complex; and
(ii) purifying said complex.
Preferably, in the method for preparing the cell penetrating peptide according to the present invention and/or the complex according to the present invention, the cell penetrating peptide and/or the complex, which are chemically synthesized in step (i) and purified in step (ii), comprise (a) an amino acid sequences as described herein for the cell penetrating peptide as described herein and/or (b) an amino acid sequences as described herein for the cell penetrating peptide and an amino acid sequence as described herein for one or more cargo molecules such as a TLR peptide agonist, and, optionally if the at least one antigen and/or antigenic epitope is a peptide or a protein, an amino acid sequence as described herein for an antigen or antigenic epitope.
Alternatively, the present invention also provides a method for preparing the complex according to the present invention, wherein
Cells Loaded with the Cell Penetrating Peptide and/or with the Complex According to the Invention
In yet another aspect the present invention relates to a cell loaded with the cell penetrating peptide according to the present invention and/or the complex according to the invention.
For example, the cells loaded with the cell penetrating peptide according to the present invention and/or the complex according to the invention are cells from a subject to be treated, in particular isolated cells from a subject to be treated, i.e. cells isolated from a subject to be treated.
As used in the context of the present invention, the term “subject” refers in particular to mammals. For example, mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents and the like. More preferably, the term “subject” refers to a human subject.
As used in the context of the present invention, “treatment” and “treating” and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, a symptom or a condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, a condition, a symptom or an adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a mammal, in particular in a human, and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but the outbreak of the disease has not yet occurred and/or the disease has not yet been diagnosed in this subject, for example a preventive early asymptomatic intervention; (b) inhibiting the disease, i.e., arresting or slowing down its development; or (c) relieving the disease, i.e., causing an at least partial regression of the disease and/or of at least one of its symptoms or conditions such as improvement or remediation of damage. In particular, the methods, uses, formulations and compositions according to the invention are useful in the treatment of cancers or infectious diseases and/or in the prevention of evolution of cancers into an advanced or metastatic stage in subjects with early stage cancer, thereby improving the staging of the cancer. When applied to cancers, prevention of a disease or disorder includes the prevention of the appearance or development of a cancer in an individual identified as at risk of developing said cancer, for instance due to past occurrence of said cancer in the circle of the individual's relatives, and prevention of infection with tumor promoting pathogens such as, for example, Epstein-Barr virus (EBV), Human papillomavirus (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human Herpes virus 8 (HHV8), human T-cell leukemia virus type 1 (HTLV-1), Merkel cell polyomavirus (MCV) and Helicobacter pylori. Also covered by the terms “prevention/treatment” of a cancer is the stabilization or delay of an already diagnosed cancer in an individual. By “stabilization”, it is meant the prevention of evolution of cancer into advanced or metastatic stage in subjects with early stage cancer.
Preferably, the cell loaded with the complex according to the invention is an antigen-presenting cell (APC). Preferably, the antigen presenting cell is selected from the group consisting of a dendritic cell (DC), a macrophage and a B-cell. Dendritic cells, in particular dendritic cells (conventional and/or plasmacytoid) isolated from a subject to be treated, are more preferred.
Methods to isolate antigen-presenting cells, in particular dendritic cells, from a subject are known to the skilled person. They include harvesting monocytes or hematopoietic stem cells from bone marrow, cord blood, or peripheral blood. They also include the use of embryonic stem (ES) cells and induced pluripotent stem cells (iPS). Antigen presenting cells, in particular dendritic cells or their precursors, can be enriched by methods including elutriation and magnetic bead based separation, which may involve enrichment for CD14+0 precursor cells.
Methods to load the cell penetrating peptide according to the present invention and/or the complex according to the present invention into the cells, preferably into the above-mentioned antigen presenting cells, more preferably into dendritic cells, and further to prepare such cells before administration to a subject are known to one skilled in the art. For example, preparation of dendritic cells can include their culture or differentiation using cytokines that may include for example GM-CSF and IL-4. Dendritic cell lines may also be employed. Loading of the complex of the invention into the cells, preferably into APC, more preferably into the dendritic cells, can involve co-incubation of the complex of the invention with the cells in culture, making use of the intrinsic properties of the cell penetrating peptide comprised by the complex according to the invention (i.e. its internalization ability). Further culture of the cells, e.g. the dendritic cells, thus loaded to induce efficient maturation can include addition of cytokines including IL-1β, IL-6, TNFα, PGE2, IFNα, and adjuvants which may include poly-IC, poly-ICLC (i.e. a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), and further TLR agonists and NLR (nucleotide-binding oligomerization domain-like receptors) agonists.
According to a further aspect the present invention also concerns imaging cells used for cell therapy, such as stem cells, dendritic cells, T cells or natural killer cells, wherein the cells are loaded with the complex according to the invention, which may further comprise an imaging agent.
The present invention also provides a method for preparing cells, in particular antigen presenting cells, loaded with the cell penetrating peptide according to the present invention and/or the complex according to the present invention as described above, said method comprising the steps of:
Preferably, the cells are loaded with the complex according to the present invention, wherein the complex is a polypeptide or a protein.
Preferably, the cells loaded with a complex(es) according to the present invention comprising at least one antigen or antigenic epitope present the at least one antigen or antigenic epitope comprised by said complex at the cell surface in an MHC class I context and/or in an MHC class II context as described herein.
Compositions and kits According to the Present Invention
According to another aspect, the invention provides a composition comprising at least one component selected from:
(i) a CPP according to the present invention as described above,
(ii) a complex according to the present invention as described above,
(iii) a nucleic acid according to the present invention as described above,
(iv) a vector according to the present invention as described above,
(v) a host cell according to the present invention as described above, and
(vi) a cell loaded with a complex according to the present invention as described above.
Preferably, the composition according to the present invention comprises the complex according to the present invention as described above.
The composition according to the present invention may also comprises more than one of the above components (i) to (vi). For example, the composition according to the present invention may comprise at least two different complexes under (ii), at least two different nucleic acids under (iii), at least two different vectors under (iv), at least two different host cells under (v), and/or at least two different cells under (vi); e.g., the composition of the invention may comprise at least two different complexes (ii) and/or at least two different nucleic acids (iii).
For example, the different complexes (ii) comprised by the composition as described above may differ in the at least one cargo molecule, for example in the antigens or antigenic epitopes or in the subsets of more than one antigen or antigenic epitope, or in the TLR peptide agonist. Accordingly, the different nucleic acids (iii) comprised by the composition as described above may differ in that they encode such different complexes; the different vectors (iv) comprised by the composition as described above may differ in that they comprise such different nucleic acids; the different host cells (v) comprised by the composition as described above may differ in that they comprise such different vectors; and the different cells loaded with a complex (vi) comprised by the composition as described above may differ in that they are loaded with such different complexes.
The present invention also provides the cell penetrating peptide and/or the complex as described herein or cells loaded with said CPP and/or said complex, as described herein, for use as a medicament, in particular as a vaccine. In particular, the composition according to the present invention is preferably a vaccine.
Thus, the present invention also provides a vaccine comprising at least one component selected from:
Preferably, the vaccine according to the present invention comprises the complex according to the present invention as described above.
Thereby, the above details described for the composition according to the present invention regarding more than one of the components (i) to (vi), also apply for the vaccine according to the present invention.
As used in the context of the present invention, the term “vaccine” refers to a biological preparation that provides innate and/or adaptive immunity, typically to a particular disease, preferably cancer. Thus, a vaccine supports in particular an innate and/or an adaptive immune response of the immune system of a subject to be treated. For example, an antigen or antigenic epitope of the complex according to the present invention typically leads to or supports an adaptive immune response in the patient to be treated, and/or a TLR peptide agonist of the complex according to the present invention may lead to or support an innate immune response.
The inventive composition, in particular the inventive vaccine, may also comprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicle as defined below for the inventive pharmaceutical composition. In the specific context of the inventive composition, in particular of the inventive vaccine, the choice of a pharmaceutically acceptable carrier is determined in principle by the manner in which the inventive composition, in particular the inventive vaccine, is administered. The inventive composition, in particular the inventive vaccine, can be administered, for example, systemically or locally. Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes. Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, intranodal and sublingual injections. More preferably, inventive composition, in particular the vaccines, may be administered by an intradermal, subcutaneous, intranodal or intramuscular route. Even more preferably, the inventive composition, in particular the vaccine, may be administered by subcutaneous, intranodal or intramuscular route. Particularly preferably, the inventive composition, in particular the vaccines, may be administered by subcutaneous or intranodal route. Most preferably, the inventive composition, in particular the vaccines, may be administered by subcutaneous route. Inventive composition, in particular the inventive vaccines, are therefore preferably formulated in liquid (or sometimes in solid) form.
The suitable amount of the inventive composition, in particular the inventive vaccine, to be administered can be determined by routine experiments with animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like. If the inventive composition, in particular the inventive vaccine, is to be administered orally, tablets, capsules and the like are the preferred unit dose form. The pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art.
The inventive composition, in particular the inventive vaccine, can additionally contain one or more auxiliary substances in order to further increase its immunogenicity. A synergistic action of the inventive complex as defined above and of an auxiliary substance, which may be optionally contained in the inventive vaccine as described above, is preferably achieved thereby. Depending on the various types of auxiliary substances, various mechanisms can come into consideration in this respect. For example, compounds that permit the maturation of dendritic cells (DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class of suitable auxiliary substances. In general, it is possible to use as auxiliary substance any agent that influences the immune system in the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, such as GM-CSF, which allow an immune response produced by the immune-stimulating adjuvant according to the invention to be enhanced and/or influenced in a targeted manner. Particularly preferred auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, that further promote the innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.
Further additives which may be included in the inventive vaccine are emulsifiers, such as, for example, Tween®; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives.
The inventive composition, in particular the inventive vaccine, can also additionally contain any further compound, which is known to be immune-stimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.
Another class of compounds, which may be added to an inventive composition, in particular to an inventive vaccine, in this context, may be CpG nucleic acids, in particular CpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid preferably contains at least one or more (mitogenic) cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According to a first preferred alternative, at least one CpG motif contained in these sequences, in particular the C (cytosine) and the G (guanine) of the CpG motif, is unmethylated. All further cytosines or guanines optionally contained in these sequences can be either methylated or unmethylated. According to a further preferred alternative, however, the C (cytosine) and the G (guanine) of the CpG motif can also be present in methylated form.
Moreover, the present invention also provides the complex as described above or cells loaded with said complex or a composition or a vaccine as described above for use in the prevention and/or treatment of diseases or disorders including for example cancers, hematological disorders, infectious diseases, autoimmunity disorders and transplant rejections, whereby cancer is preferred.
In addition, the present invention also provides the complex as described above or cells loaded with said complex or a composition as described above for use as an imaging or diagnostic composition.
The present invention also provides a pharmaceutical composition, in particular a vaccine composition as described above, and a method for treating a subject, preferably a mammalian subject, and most preferably a human subject, who is suffering from a disease or disorder, in particular from a disorder that can be treated by immunotherapy, such as cancers, infectious diseases, autoimmunity disorders and transplant rejections.
In particular, the present invention provides a pharmaceutical composition comprising at least one complex according to the present invention or at least one cell loaded with a complex according to the present invention, and optionally a pharmaceutically acceptable carrier and/or vehicle, or any excipient, buffer, stabilizer or other materials well known to those skilled in the art. Preferably, the pharmaceutical composition comprises at least one complex according to the present invention or at least one cell loaded with a complex according to the present invention, and a pharmaceutically acceptable carrier.
As a further ingredient, the inventive pharmaceutical composition may in particular comprise a pharmaceutically acceptable carrier and/or vehicle. In the context of the present invention, a pharmaceutically acceptable carrier typically includes the liquid or non-liquid basis of the inventive pharmaceutical composition. If the inventive pharmaceutical composition is provided in liquid form, the carrier will typically be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrate etc. buffered solutions. Particularly for injection of the inventive pharmaceutical composition, water or preferably a buffer, more preferably an aqueous buffer, may be used, containing a sodium salt, preferably at least 30 mM of a sodium salt, a calcium salt, preferably at least 0.05 mM of a calcium salt, and optionally a potassium salt, preferably at least 1 mM of a potassium salt. According to a preferred embodiment, the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Without being limited thereto, examples of sodium salts include e.g. NaCl, Nal, NaBr, Na2CO3, NaHCO3, Na2SO4, examples of the optional potassium salts include e.g. KCl, Kl, KBr, K2CO3, KHCO3, K2SO4, and examples of calcium salts include e.g. CaCl2, Cal2, CaBr2, CaCO3, CaSO4, Ca(OH)2. Furthermore, organic anions of the aforementioned cations may be contained in the buffer. According to a more preferred embodiment, the buffer suitable for injection purposes as defined above, may contain salts selected from sodium chloride (NaCl), calcium chloride (CaCl2) and optionally potassium chloride (KCl), wherein further anions may be present additional to the chlorides. CaCl2 can also be replaced by another salt like KCl. Typically, the salts in the injection buffer are present in a concentration of at least 30 mM sodium chloride (NaCl), at least 1 mM potassium chloride (KCl) and at least 0,05 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects. Reference media are e.g. liquids occurring in “in vivd” methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in “in vitro” methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Saline (0.9% NaCl) and Ringer-Lactate solution are particularly preferred as a liquid basis.
However, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well for the inventive pharmaceutical composition, which are suitable for administration to a subject to be treated. The term “compatible” as used herein means that these constituents of the inventive pharmaceutical composition are capable of being mixed with the complex according to the present invention as defined above in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive pharmaceutical composition under typical use conditions. Pharmaceutically acceptable carriers, fillers and diluents must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject to be treated. Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
The inventive pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, intranodal and sublingual injection or infusion techniques. Preferably, the inventive pharmaceutical composition may be administered intradermally, intramuscularly, intranodally or subcutaneously. More preferably the inventive pharmaceutical composition may be administered intramuscularly, intranodally or subcutaneously. Even more preferably the inventive pharmaceutical composition may be administered intranodally or subcutaneously. Most preferably, the inventive pharmaceutical composition may be administered subcutaneously.
Preferably, the inventive pharmaceutical composition may be administered by parenteral injection, more preferably by subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, intranodal and sublingual injection or via infusion techniques. Sterile injectable forms of the inventive pharmaceutical compositions may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1.3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation of the inventive pharmaceutical composition.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will preferably be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required. Whether it is a polypeptide, peptide, or nucleic acid molecule, other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.
The inventive pharmaceutical composition as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient, i.e. the inventive transporter cargo conjugate molecule as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
The inventive pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the inventive pharmaceutical composition may be formulated in a suitable ointment, containing the inventive immunostimulatory composition, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the inventive pharmaceutical composition can be formulated in a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
In this context, prescription of treatment, e.g. decisions on dosage etc. when using the above pharmaceutical composition is typically within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 16th edition, Osol, A. (ed), 1980.
Accordingly, the inventive pharmaceutical composition typically comprises a “safe and effective amount” of the components of the inventive pharmaceutical composition, in particular of the complex according to the present invention as defined above and/or cells loaded with said complex. As used herein, a “safe and effective amount” means an amount of the complex according to the present invention that is sufficient to significantly induce a positive modification of a disease or disorder, i.e. an amount of the complex according to the present invention or cells loaded with said complex, that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought. An effective amount may be a “therapeutically effective amount” for the alleviation of the symptoms of the disease or condition being treated and/or a “prophylactically effective amount” for prophylaxis of the symptoms of the disease or condition being prevented. The term also includes the amount of active complex sufficient to reduce the progression of the disease, notably to reduce or inhibit the tumor growth or infection and thereby elicit the response being sought, in particular such response could be an immune response directed against the antigens or antigenic epitopes comprised in by the complex (i.e. an “inhibition effective amount”). At the same time, however, a “safe and effective amount” is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment. A “safe and effective amount” of the components of the inventive pharmaceutical composition, particularly of the complex according to the present invention as defined above, will furthermore vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the activity of the specific components a), b), and c) of the complex according to the present invention as defined above, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the accompanying doctor. The inventive pharmaceutical composition may be used for human and also for veterinary medical purposes, preferably for human medical purposes, as a pharmaceutical composition in general or as a vaccine.
Pharmaceutical compositions, in particular vaccine compositions, or formulations according to the invention may be administered as a pharmaceutical formulation which can contain a complex according to the present invention in any form described herein.
The terms “pharmaceutical formulation” and “pharmaceutical composition” as used in the context of the present invention refer in particular to preparations which are in such a form as to permit biological activity of the active ingredient(s) to be unequivocally effective and which contain no additional component which would be toxic to subjects to which the said formulation would be administered.
In the context of the present invention, an “efficacy” of a treatment can be measured based on changes in the course of a disease in response to a use or a method according to the present invention. For example, the efficacy of a treatment of cancer can be measured by a reduction of tumor volume, and/or an increase of progression free survival time, and/or a decreased risk of relapse post-resection for primary cancer. More specifically for cancer treated by immunotherapy, assessment of efficacy can be by the spectrum of clinical patterns of antitumor response for immunotherapeutic agents through novel immune-related response criteria (irRC), which are adapted from Response Evaluation Criteria in Solid Tumors (RECIST) and World Health Organization (WHO) criteria (J. Natl. Cancer Inst. 2010, 102(18): 1388-7397). The efficacy of prevention of infectious disease is ultimately assessed by epidemiological studies in human populations, which often correlates with titres of neutralizing antibodies in sera, and induction of multifunctional pathogen specific T cell responses. Preclinical assessment can include resistance to infection after challenge with infectious pathogen. Treatment of an infectious disease can be measured by inhibition of the pathogen's growth or elimination of the pathogen (and, thus, absence of detection of the pathogen), correlating with pathogen specific antibodies and/or T cell immune responses.
Pharmaceutical compositions, in particular vaccine compositions, or formulations according to the invention may also be administered as a pharmaceutical formulation which can contain antigen presenting cells loaded with a complex according to the invention in any form described herein.
The vaccine and/or the composition according to the present invention may also be formulated as pharmaceutical compositions and unit dosages thereof, in particular together with a conventionally employed adjuvant, immunomodulatory material, carrier, diluent or excipient as described above and below, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral (including subcutaneous and intradermal) use by injection or continuous infusion.
In the context of the present invention, in particular in the context of a pharmaceutical composition and vaccines according to the present invention, injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
Examples of suitable adjuvants and/or immunomodulatory materials in the context of the present invention include MPL® (Corixa), aluminum-based minerals including aluminum compounds (generically called Alum), ASO1-4, MF59, CalciumPhosphate, Liposomes, Iscom, polyinosinic:polycytidylic acid (polylC), including its stabilized form poly-ICLC (Hiltonol), CpG oligodeoxynucleotides, Granulocyte-macrophage colony-stimulating factor (GM-CSF), lipopolysaccharide (LPS), Montanide, polylactide co-glycolide (PLG), Flagellin, Soap Bark tree saponins (QS21), amino alkyl glucosamide compounds (e.g. RC529), two component antibacterial peptides with synthetic oligodeoxynucleotides (e.g. IC31), Imiquimod, Resiquimod, Immunostimulatory sequences (ISS), monophosphoryl lipid A (MPLA), Fibroblast-stimulating lipopeptide (FSL1), and anti-CD40 antibodies.
Compositions, in particular pharmaceutical compositions and vaccines, according to the present invention may be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents include but are not limited to poly(ethylene glycol), glycerol, bovine serum albumin, Tween®, Span®.
Compositions, in particular pharmaceutical compositions and vaccines, according to the present invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.
Compositions, in particular pharmaceutical compositions and vaccines, according to the present invention may also be solid compositions, which may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
Compositions, in particular pharmaceutical compositions and vaccines, according to the present invention may also be administered in sustained release forms or from sustained release drug delivery systems.
Moreover, the compositions, in particular pharmaceutical compositions and vaccines, according to the present invention may be adapted for delivery by repeated administration.
Further materials as well as formulation processing techniques and the like, which are useful in the context of compositions, in particular pharmaceutical compositions and vaccines, according to the present invention or in the context of their preparation are set out in “Part 5 of Remington's “The Science and Practice of Pharmacy”, 22″d Edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins”.
According to still another aspect, the present invention provides a method of preparing a pharmaceutical composition according to the present invention comprising the step of mixing complex according to the present invention or cells, in particular antigen-presenting cells, loaded with a complex according to the present invention, and a pharmaceutically acceptable carrier.
Accordingly, the complex according to the present invention and the cell loaded with a complex according to the present invention may be used (for the preparation of a pharmaceutical composition and/or the preparation of a vaccine) for the prevention, treatment and/or amelioration of any of the diseases and disorders as defined herein, in particular those that can be treated or prevented by immunotherapy such as cancers and infectious diseases.
In another aspect, the invention provides imaging or diagnosis compositions. A still other aspect concerns methods for delivering an imaging agent and methods for diagnosing a disease or disorder in a subject, preferably a mammalian subject, and most preferably a human subject who is suspected of suffering from a medical disorder, and in particular a cancer, infectious disease, autoimmunity disorder and transplant rejection.
The formulations and modes of administration described herewith for the pharmaceutical compositions can also be suitable to the imaging or diagnosis compositions according to the invention.
Preferably, the pharmaceutical composition as described herein comprises at least two different complexes according to the present invention.
In a further aspect, the present invention also relates to a kit-of-parts comprising at least one of:
In particular, the kit-of-parts of the invention may comprise more than one component (i) to (vi). For example, the kit-of-parts according to the present invention may comprise at least two different complexes under (ii), at least two different nucleic acids under (iii), at least two different vectors under (iv), at least two different host cells under (v), and/or at least two different cells under (vi); e.g., the kit-of-parts of the invention may comprise at least two different complexes (ii) and/or at least two different nucleic acids (iii).
The various components of the kit-of-parts may be packaged in one or more containers. The above components may be provided in a lyophilized or dry form or dissolved in a suitable buffer. The kit may also comprise additional reagents including, for instance, preservatives, growth media, and/or buffers for storage and/or reconstitution of the above-referenced components, washing solutions, and the like. In addition, the kit-of-parts according to the present invention may optionally contain instructions of use.
Moreover, the present invention also provides a vaccination kit for treating, preventing and/or stabilizing a cancer or an infectious disease, comprising the pharmaceutical composition according to the present invention or a vaccine according to the present invention and instructions for use of said pharmaceutical composition or of said vaccine.
In addition, the compositions and/or the kit-of-parts according to the present inventions may be used in imaging techniques and/or in diagnosis of disease or disorder as disclosed herein.
Use and Methods According to the Invention
In another aspect, the present invention provides the use of any one of: (i) the complex according to the present invention, (ii) the pharmaceutical composition according to the present invention and/or (ii) cells, such as antigen-presenting cells, loaded with a complex according to the present invention, as a medicament.
In particular, the present invention provides the use of any one of: (i) the complex according to the present invention, (ii) the pharmaceutical composition according to the present invention and/or (ii) cells, such as antigen-presenting cells, loaded with a complex according to the present invention (for the preparation of a medicament) for the prevention, treatment or stabilization of a disease or disorder, such as those which can be treated by immunotherapy, including cancers, infectious diseases, autoimmunity disorders, hematological diseases and transplant rejections. Preferably, the disease to be prevented and/or treated is cancer and/or a hematological disorder, preferably a malignant neoplasm of the brain or a malignant neoplasm of lymphoid, hematopoietic and related tissue, most preferably glioblastoma.
Accordingly, the present invention provides any one of: (i) the complex according to the present invention, (ii) the pharmaceutical composition according to the present invention and/or (ii) cells, such as antigen-presenting cells, loaded with a complex according to the present invention, for use in the prevention, treatment or stabilization of a disease or disorder, such as those which can be treated by immunotherapy, including cancers, infectious diseases, autoimmunity disorders, hematological diseases and transplant rejections.
The present invention also provides a complex according to the present invention, which allows the transport and presentation of the at least one antigen or antigenic epitope comprised by the complex at the cell surface of antigen presenting cells in an MHC class I and/or MHC class II context, for use in vaccination and/or immunotherapy.
According to another aspect, the present invention provides a method of preventing, treating and/or repressing a disease or disorder such as those which can be treated by immunotherapy, including cancers, infectious diseases, autoimmunity disorders, hematological diseases and transplant rejections in a subject, wherein said method comprises administering any one of: (i) a complex of the invention, (ii) cells, such as antigen-presenting cells, loaded with a complex of the invention, or (iii) a pharmaceutical composition of (i) or (ii), to said subject.
Moreover, the present invention provides a method for eliciting or improving, in a subject, an immune response against one or multiple epitopes that is dependent on CD4+ helper T cells and/or CD8+ cytotoxic T cells, wherein said method comprises administering any one of: (i) a complex according to the present invention, and/or (ii) cells, such as antigen-presenting cells, loaded with said complex, or (iii) a pharmaceutical composition of (i) or (ii), to said subject.
An immune response that is dependent on CD4+ and/or CD8+ response can be determined by evaluating an inflammatory response, a pro-inflammatory cytokine response, including an increase in the expression of one or more of IFN-γ, TNF-α and IL-2 mRNA or protein relative to the level before administration of the compounds of the invention. It can also be measured by an increase in the frequency or absolute number of antigen-specific T cells after administration of the compounds of the invention, measured by HLA-peptide multimer staining, ELISPOT assays, and delayed type hypersensitivity tests. It can also be indirectly measured by an increase in antigen-specific serum antibodies that are dependent on antigen-specific T helper cells.
The present invention also provides a method for eliciting or improving, in a subject, an immune response against one or multiple antigens or antigenic epitopes that is restricted by multiple MHC class I molecules and/or multiple MHC class II molecules, wherein said method comprises administering any one of: (i) a complex according to the present invention, and/or (ii) cells, such as antigen-presenting cells, loaded with said complex, or (iii) a pharmaceutical composition of (i) or (ii), to said subject.
A method for eliciting or improving, in a subject, an immune response against multiple epitopes that is restricted by multiple MHC class I molecules and/or multiple MHC class II molecules can be determined by evaluating a cytokine response, including an increase in the expression of one or more of IFN-γ, TNF-α and IL-2 mRNA or protein relative to the level before administration of the compounds of the invention, after in vitro stimulation of T cells with individual peptides binding to discrete MHC class I and class II molecules on antigen presenting cells. Restriction to different MHC molecules can also be validated by using antigen presenting cells expressing different MHC molecules, or by using MHC blocking antibodies. It can also be measured by an increase in the frequency or absolute number of antigen-specific T cells after administration of the compounds of the invention, measured by HLA-peptide multimer staining, which uses multimers assembled with discrete MHC molecules.
Preferably, in the methods for eliciting or improving an immune response against one or multiple antigens or antigenic epitopes according to the present invention, the immune response is directed against one or multiple epitopes of a tumor-associated antigen or a tumor-specific antigen as, for instance, a combination of glioma epitopes such as those described in Novellino et al. (2005, Cancer Immuno Immunother, 54(3):187-207) and Vigneron et al. (2013, Cancer Immun. 13:15).
Alternatively or additionally, the immune response may be directed against multiple epitopes of an antigenic protein from a pathogen.
The methods according to the present invention as described herein, may be for eliciting or improving, in a subject, an immune response against one or multiple epitopes that is restricted by MHC class I molecules and/or MHC class II molecules.
In another aspect, the present invention provides the use of any one of: (i) a complex according to the present invention, and/or (ii) cells, such as antigen-presenting cells, loaded with the complex according to the present invention, for the preparation of an imaging composition for imaging techniques or for the preparation of a diagnosis composition for diagnosing a disease or disorder, respectively. The diseases or disorders that can be diagnosed with the invention include those which can be treated by immunotherapy, for instance cancers, infectious diseases, autoimmunity disorders and transplant rejections.
In particular, the complex according to the present invention, the cell, such as antigen-presenting cell, loaded with the complex according to the present invention, the inventive composition, the inventive pharmaceutical composition or the inventive vaccine may be utilized in diagnosis as a diagnostic tool, e.g. in (in vivo or in vitro) assays, e.g. in immunoassays, to detect, prognose, diagnose, or monitor various conditions and disease states of disorders or diseases mentioned.
As an example, (in vitro) assays may be performed by delivering the complex according to the present invention, the cell, such as antigen-presenting cell, loaded with the complex according to the present invention, the inventive composition, the inventive pharmaceutical composition or the inventive vaccine to target cells typically selected from e.g. cultured animal cells, human cells or micro-organisms, and to monitor the cell response by biophysical methods typically known to a skilled person. The target cells typically used therein may be cultured cells (in vitro) or in vivo cells, i.e. cells composing the organs or tissues of living animals or humans, or microorganisms found in living animals or humans. Particularly preferable in this context are so called markers or labels, which may be contained in the complex according to the present invention.
According to a further aspect, the invention provides a method of diagnosing a disease or disorder in a subject, wherein said method comprises administering any one of: (i) a complex of the invention, (ii) cells, such as antigen-presenting cells, loaded with the complex of the invention, or (iii) a pharmaceutical formulation of (i) or (ii), to said subject or to said subject's sample ex vivo.
Accordingly, the present invention also provides a diagnostic composition comprising the cell penetrating peptide according to the present invention and/or the complex according to the present invention. Such a diagnostic composition may be used in a method of diagnosing a disease or disorder in a subject as described above.
According to another aspect, the present invention provides an imaging method wherein said method comprises using, in vitro, ex vivo or in vivo, any one of: (i) a complex of the invention, (ii) cells, such as antigen-presenting cells, loaded with the complex of the invention, or (iii) a pharmaceutical formulation of (i) or (ii).
Preferably, uses and methods according to the present invention comprise administration of a complex according to the invention.
Moreover, uses and methods according to the present invention comprise administration of more than one complex, cells, or pharmaceutical formulation according to the invention. For example, in the uses and methods according to the present invention, at least two different complexes are used or administered, wherein each complex comprises at least one antigen or antigenic epitope and said antigen or antigenic epitope or (if more than one antigen or antigenic epitope is comprised by said complex) said subset of antigens or antigenic epitopes are different between the two complexes.
Moreover, in the uses and methods according to the present invention, the cells according to the present invention may be antigen presenting cells, in particular dendritic cells, more preferably dendritic cells from the subject to be treated.
Diseases to be Treated or Prevented
The term “disease” as used in the context of the present invention is intended to be generally synonymous, and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
Diseases to be treated and/or prevented by use of the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine include cancer, hematological disorders, infectious diseases, autoimmunity disorders and transplant rejections. Thereby, treatment and/or prevention of cancer and infectious diseases is preferred and treatment and/or prevention of cancer is more preferred. For cancer, a malignant neoplasm of the brain or a malignant neoplasm of lymphoid, hematopoietic and related tissue are preferred and glioblastoma is more preferred.
Preferably, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine, may be used for (the preparation of a medicament for) the prophylaxis, treatment and/or amelioration of cancer or tumor diseases, including diseases caused by defective apoptosis, preferably selected from acusticus neurinoma, anal carcinoma, astrocytoma, basalioma, Behcet's syndrome, bladder cancer, blastomas, bone cancer, brain metastases, brain tumors, brain cancer (glioblastomas), breast cancer (mamma carcinoma), Burkitt's lymphoma, carcinoids, cervical cancer, colon carcinoma, colorectal cancer, corpus carcinoma, craniopharyngeomas, CUP syndrome, endometrial carcinoma, gall bladder cancer, genital tumors, including cancers of the genitourinary tract, glioblastoma, gliomas, head/neck tumors, hepatomas, histocytic lymphoma, Hodgkin's syndromes or lymphomas and non-Hodgkin's lymphomas, hypophysis tumor, intestinal cancer, including tumors of the small intestine, and gastrointestinal tumors, Kaposi's sarcoma, kidney cancer, kidney carcinomas, laryngeal cancer or larynx cancer, leukemia, including acute myeloid leukaemia (AML), erythroleukemia, acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), and chronic lymphocytic leukaemia (CLL), lid tumor, liver cancer, liver metastases, lung carcinomas (=lung cancer=bronchial carcinoma), small cell lung carcinomas and non-small cell lung carcinomas, and lung adenocarcinoma, lymphomas, lymphatic cancer, malignant melanomas, mammary carcinomas (=breast cancer), medulloblastomas, melanomas, meningiomas, Mycosis fungoides, neoplastic diseases neurinoma, oesophageal cancer, oesophageal carcinoma (=oesophageal cancer), oligodendroglioma, ovarian cancer (=ovarian carcinoma), ovarian carcinoma, pancreatic carcinoma (=pancreatic cancer), penile cancer, penis cancer, pharyngeal cancer, pituitary tumour, plasmocytoma, prostate cancer (=prostate tumors), rectal carcinoma, rectal tumors, renal cancer, renal carcinomas, retinoblastoma, sarcomas, Schneeberger's disease, skin cancer, e.g. melanoma or non-melanoma skin cancer, including basal cell and squamous cell carcinomas as well as psoriasis, pemphigus vulgaris, soft tissue tumours, spinalioma, stomach cancer, testicular cancer, throat cancer, thymoma, thyroid carcinoma, tongue cancer, urethral cancer, uterine cancer, vaginal cancer, various virus-induced tumors such as, for example, papilloma virus-induced carcinomas (e.g. cervical carcinoma =cervical cancer), adenocarcinomas, herpes virus-induced tumors (e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma, cervix carcinoma), heptatitis B-induced tumors (hepatocell carcinomas), HTLV-1- and HTLV-2-induced lymphomas, vulval cancer, wart conditions or involvement, etc. In the present context, the terms “therapy” and “therapeutic” preferably mean to have at least some minimal physiological effect upon being administered to a living body. For example, a physiological effect upon administering a “therapeutic” anti-tumor compound may be the inhibition of tumor growth, or decrease in tumor size, or prevention reoccurrence of the tumor. Preferably, in the treatment of cancer or neoplastic disease, a compound which inhibits the growth of a tumor or decreased the size of the tumor or prevents the reoccurrence of the tumor would be considered therapeutically effective. The term “anti-tumor drug” therefore preferably means any therapeutic agent having therapeutic effect against a tumor, neoplastic disease or cancer.
Examples of cancers include brain cancer, prostate cancer, breast cancer, ovarian cancer, esophageal cancer, lung cancer, liver cancer, kidney cancer, melanoma, gut carcinoma, lung carcinoma, head and neck squamous cell carcinoma, chronic myeloid leukemia, colorectal carcinoma, gastric carcinoma, endometrial carcinoma, myeloid leukemia, lung squamous cell carcinoma, acute lymphoblastic leukemia, acute myelogenous leukemia, bladder tumor, promyelocytic leukemia, non-small cell lung carcinoma, sarcoma.
The cancer may be a solid tumor, blood cancer, or lymphatic cancer. The cancer may be benign or metastatic.
Preferably, the cancer to be prevented and/or treated is a glioma, more preferably highly invasive glioblastoma multiforme (GBM). Gliomas are the most frequent form of primary brain tumors in adults, with glioblastoma multiforme (GBM) having the poorest prognosis. This tumor is notorious for its highly invasive and aggressive behavior. In particular, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be used in conjunction with existing modalities for glioma, more specifically highly invasive GBM. T lymphocytes can actively seek out neoplastic cells in the brain, and have the potential to safely eliminate specific tumor cells without damaging the surrounding healthy tissues.
Moreover, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be used for (the preparation of a medicament for) the prophylaxis, treatment and/or amelioration of infectious diseases, preferably viral, retroviral, bacterial or protozoological infectious diseases. Such infectious diseases are typically selected from AIDS, anthrax, Japanese encephalitis, bacterial infectious diseases such as miscarriage (prostate inflammation), anthrax, appendicitis, borreliosis, botulism, Camphylobacter, Chlamydia trachomatis (inflammation of the urethra, conjunctivitis), cholera, diphtheria, donavanosis, epiglottitis, typhus fever, gas gangrene, gonorrhoea, rabbit fever, Heliobacter pylori, whooping cough, climatic bubo, osteomyelitis, Legionnaire's disease, chicken-pox, condyloma acuminata, cytomegalic virus (CMV), dengue fever, early summer meningoencephalitis (ESME), Ebola virus, colds, fifth disease, foot-and-mouth disease, herpes simplex type I, herpes simplex type II, herpes zoster, HSV, infectious diseases caused by parasites, protozoa or fungi, such as amoebiasis, bilharziosis, Chagas disease, Echinococcus, fish tapeworm, fish poisoning (Ciguatera), fox tapeworm, athlete's foot, canine tapeworm, candidosis, yeast fungus spots, scabies, cutaneous Leishmaniosis, lambliasis (giardiasis), lice, onchocercosis (river blindness), fungal diseases, bovine tapeworm, schistosomiasis, porcine tapeworm, toxoplasmosis, trichomoniasis, trypanosomiasis (sleeping sickness), visceral Leishmaniosis, nappy/diaper dermatitis or miniature tapeworm, infectious erythema, influenza, Kaposi's sarcoma, Lassa fever, Leishmaniasis, leprosy, listeriosis, Lyme borreliosis, malaria, Marburg virus infection, measles, meningitis, including bacterial meningitis, molluscum contagiosum, mononucleosis, mumps, Mycoplasma hominis, neonatal sepsis (Chorioamnionitis), noma, Norwalk virus infection, otitis media, paratyphus, Pfeiffer's glandular fever, plague, pneumonia, polio (poliomyelitis, childhood lameness), pseudo-croup, rabies, Reiter's syndrome, Rocky Mountain spotted fever, Salmonella paratyphus, Salmonella typhus, SARS, scarlet fever, shingles, hepatitis, smallpox, soft chancre, syphilis, tetanus,three-day fever, tripper, tsutsugamushi disease, tuberculosis, typhus, vaginitis (colpitis), viral diseases caused by cytomegalovirus (CMV), orthopox variola virus, orthopox alastrim virus, parapox ovis virus, molluscum contagiosum virus, herpes simplex virus 1, herpes simplex virus 2, herpes B virus, varicella zoster virus, pseudorabies virus, human cytomegaly virus, human herpes virus 6, human herpes virus 7, Epstein-Barr virus, human herpes virus 8, hepatitis B virus, chikungunya virus, O'nyong'nyong virus, rubivirus, hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellow fever virus, louping ill virus, St. Louis encephalitis virus, Japan B encephalitis virus, Powassan virus, FSME virus, SARS, SARS-associated corona virus, human corona virus 229E, human corona virus Oc43, Torovirus, human T cell lymphotropic virus type I, human T cell lymphotropic virus type II, HIV (AIDS), i.e. human immunodeficiency virus type 1 or human immunodeficiency virus type 2, influenza virus, Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Junin virus, Machupo virus, Borna disease virus, Bunyamwera virus, California encephalitis virus, Rift Valley fever virus, sand fly fever virus, Toscana virus, Crimean-Congo haemorrhagic fever virus, Hazara virus, Khasan virus, Hantaan virus, Seoul virus, Prospect Hill virus, Puumala virus, Dobrava Belgrade virus, Tula virus, sin nombre virus, Lake Victoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, Ivory Coast Ebola virus, influenza virus A, influenza virus B, influenza viruses C, parainfluenza virus, measles virus, mumps virus, respiratory syncytial virus, human metapneumovirus, vesicular stomatitis Indiana virus, rabies virus, Mokola virus, Duvenhage virus, European bat lyssavirus 1+2, Australian bat lyssavirus, adenoviruses A-F, human papilloma viruses, condyloma virus 6, condyloma virus 11, polyoma viruses, adeno-associated virus 2, rotaviruses, or orbiviruses, Varicella including Varizella zoster, and malaria parasite (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi), viral infectious diseases such as AIDS, infectious diseases caused by Condyloma acuminata, hollow warts, Dengue fever, three-day fever, Ebola virus, cold, early summer meningoencephalitis (FSME), flu, shingles, hepatitis, herpes simplex type I, herpes simplex type II, Herpes zoster, influenza, Japanese encephalitis, Lassa fever, Marburg virus, warts, West Nile fever, yellow fever, etc.
Examples of infectious diseases include diseases caused by viruses, bacteria, fungi, protozoa and multicellular parasites. They include, for instance, Amoebiasis, Anthrax, Buruli Ulcer (Mycobacterium ulcerans), Caliciviruses associated diarrhoea, Campylobacter diarrhoea, Cervical Cancer (Human papillomavirus), Chlamydia trachomatis associated genital diseases, Cholera, Crimean-Congo haemorrhagic fever, Dengue Fever, Diptheria, Ebola haemorrhagic fever, Enterotoxigenic Escherichia coli (ETEC) diarrhoea, Gastric Cancer (Helicobacter pylori), Gonorrhea, Group A Streptococcus associated diseases, Group B Streptococcus associated diseases, Haemophilus influenzae B pneumonia and invasive disease, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E diarrhoea, Herpes simplex type 2 genital ulcers, HIV/AIDS, Hookworm Disease, Influenza, Japanese encephalitis, Lassa Fever, Leishmaniasis, Leptospirosi, Liver cancer (Hepatitis B), Liver Cancer (Hepatitis C), Lyme Disease, Malaria, Marburg haemorrhagic fever, Measles, Mumps, Nasopharyngeal cancer (Epstein-Barr virus), Neisseria meningitidis Meningitis, Parainfluenza associated pneumonia, Pertussis, Plague, Poliomyelitis, Rabies, Respiratory syncytial virus (RSV) pneumonia, Rift Valley fever, Rotavirus diarrhoea, Rubella, Schistosomiasis, Severe Acute Respiratory Syndrome (SARS), Shigellosis, Smallpox, Staphylococcus aureus associated diseases, Stomach Cancer (Helicobacter pylori), Streptococcus pneumoniae and invasive disease, Tetanus, Tick-borne encephalitis, Trachoma, Tuberculosis, Tularaemia, Typhoid fever, West-Nile virus associated disease, Yellow fever.
Moreover, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be used for (the preparation of a medicament for) the prophylaxis, treatment and/or amelioration of autoimmune disorders, for example autoimmune diseases of the CNS, auto-inflammatory diseases, Celiac disease; Sjogren's syndrome, systemic lupus erythematosus etc. Typically, autoimmune diseases arise from an abnormal immune response of the body against substances and tissues normally present in the body (autoimmunity). This may be restricted to certain organs (e.g. in autoimmune thyroiditis) or may involve a particular tissue in different places (e.g. Goodpasture's disease which may affect the basement membrane in both the lung and the kidney). Autoimmune diseases may be classified by corresponding type of hypersensitivity: type I (i.e. urticaria induced by autologous serum), type II, type III, or type IV.
Examples of autoimmune diseases include Blau syndrome, Bullous pemphigoid, Cancer, Castleman's disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Contact dermatitis, Cranial arteritis, CREST syndrome, Crohn's disease, Cushing's Syndrome, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler's syndrome, lupus, Discoid lupus erythematosus, Eczema, Acute disseminated encephalomyelitis (ADEM), Addison's disease, Agammaglobulinemia, Amyotrophic lateral sclerosis (Also Lou Gehrig's disease; Motor Neuron Disease), Ankylosing Spondylitis Antiphospholipid syndrome, Antisynthetase syndrome, Atopic dermatitis, Autoimmune aplastic anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune urticarial, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaff's encephalitis, Endometriosis, Enthesitis-related arthritis, Eosinophilic gastroenteritis, Epidermolysis bullosa acquisita, Erythroblastosis fetalis, Evan's syndrome, Fibrodysplasia ossificans, Fibrosing alveolitis (or Idiopathic pulmonary fibrosis), Gastritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephelopathy, Hashimoto's thyroiditis, Gestational Pemphigoid, Hidradenitis suppurativa, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (Autoimmune thrombocytopenic purpura), IgA nephropathy, Occular cicatricial pemphigoid, Inclusion body myositis, Rheumatoid arthritis, Chronic inflammatory Rheumatic fever, demyelinating polyneuropathy, Sarcoidosis, Palindromic rheumatism, Interstitial cystitis, Juvenile idiopathic Schizophrenia, PANDAS (pediatric arthritis aka Juvenile autoimmune rheumatoid arthritis), Schmidt syndrome, neuropsychiatric Kawasaki's disease another form of APS, Schnitzler syndrome, Paraneoplastic cerebellar myasthenic syndrome, Leukocytoclastic Serum Sickness, Lichen planus, Sjögren's syndrome, Lichen sclerosus, Parsonage-Tumer, Linear IgA disease, Still's disease, Pemphigus vulgaris, Lupoid hepatitis, Autoimmune hepatitis, Stiff person syndrome, Pernicious anaemia, Subacute bacterial endocarditis (SBE), POEMS syndrome, Lupus erythematosus, Sweet's syndrome, Sympathetic ophthalmia, Meniere's disease, Systemic lupus, Primary biliary cirrhosis, Miller-Fisher syndrome, Takayasu's arteritis, cholangitis, Progressive inflammatory neuropathy, Mucha-Habermann disease, Psoriasis, Psoriatic arthritis, Pyoderma gangrenosum, Multiple sclerosis, Pure red cell aplasia, Rasmussen's encephalitis, Myasthenia gravis, Transverse myelitis, Raynaud phenomenon, Microscopic colitis, Ulcerative colitis, Myositis, idiopathic inflammatory bowel disease (IBD), Neuromyelitis optica, Devic's disease, and Neuromyotonia.
Moreover, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be used for (the preparation of a medicament for) the prophylaxis, treatment and/or amelioration of hematological disorders, which are typically disorders which primarily affect the blood. Thereby, Hematological malignancies are preferred.
Examples of hematological diseases include myeloid disorders, including Hemoglobinopathies (congenital abnormality of the hemoglobin molecule or of the rate of hemoglobin synthesis), e.g. Sickle-cell disease, Thalassemia, Methemoglobinemia; Anemias (lack of red blood cells or hemoglobin), e.g. Iron deficiency anemia, Megaloblastic anemia including Vitamin B12 deficiency, Pernicious anemia, and Folate deficiency, Hemolytic anemias (destruction of red blood cells) including Genetic disorders of RBC membrane such as Hereditary spherocytosis, Hereditary elliptocytosis, and Congenital dyserythropoietic anemia, Genetic disorders of RBC metabolism such as Glucose-6-phosphate dehydrogenase deficiency (G6PD), and Pyruvate kinase deficiency, Immune mediated hemolytic anemia (direct Coombs test is positive) such as Autoimmune hemolytic anemia including Warm antibody autoimmune hemolytic anemia (such as Idiopathic, Systemic lupus erythematosus (SLE), and Evans' syndrome (antiplatelet antibodies and hemolytic antibodies)) and Cold antibody autoimmune hemolytic anemia (such as Idiopathic cold hemagglutinin syndrome, Infectious mononucleosis, and Paroxysmal cold hemoglobinuria), Alloimmune hemolytic anemia including Hemolytic disease of the newborn (HDN) (such as Rh disease (Rh D),
ABO hemolytic disease of the newborn, Anti-Kell hemolytic disease of the newborn, Rhesus c hemolytic disease of the newborn, Rhesus E hemolytic disease of the newborn, and other blood group incompatibility (RhC, Rhe, Kid, Duffy, MN, P and others)), Drug induced immune mediated hemolytic anemia including Penicillin (high dose) and Methyldopa, Hemoglobinopathies (i.e. where these is an unstable or crystalline hemoglobin), Paroxysmal nocturnal hemoglobinuria (rare acquired clonal disorder of red blood cell surface proteins), Direct physical damage to RBCs including Microangiopathic hemolytic anemia and Secondary to artificial heart valve(s), Aplastic anemia such as Fanconi anemia, Diamond-Blackfan anemia (inherited pure red cell aplasia), and Acquired pure red cell aplasia; Decreased numbers of cells, e.g. Myelodysplastic syndrome, Myelofibrosis, Neutropenia (decrease in the number of neutrophils), Agranulocytosis, Glanzmann's thrombasthenia, and Thrombocytopenia (decrease in the number of platelets) including Idiopathic thrombocytopenic purpura (ITP), Thrombotic thrombocytopenic purpura (TTP), and Heparin-induced thrombocytopenia (HIT); Myeloproliferative disorders (Increased numbers of cells), e.g. Polycythemia vera (increase in the number of cells in general), Erythrocytosis (increase in the number of red blood cells), Leukocytosis (increase in the number of white blood cells), Thrombocytosis (increase in the number of platelets), and Myeloproliferative disorder; Coagulopathies (disorders of bleeding and coagulation), e.g. Thrombocytosis, Recurrent thrombosis, Disseminated intravascular coagulation, Disorders of clotting proteins including Hemophilia such as Hemophilia A, Hemophilia B (also known as Christmas disease), and Hemophilia C, Von Willebrand disease, Disseminated intravascular coagulation, Protein S deficiency, and Antiphospholipid syndrome, and Disorders of platelets including Thrombocytopenia, Glanzmann's thrombasthenia, and Wiskott-Aldrich syndrome. Moreover, examples of hematological diseases also include Hematological malignancies including Hematological malignancies, e.g. Lymphomas including Hodgkin's disease and Non-Hodgkin's lymphoma such as Burkitt's lymphoma, Anaplastic large cell lymphoma, Splenic marginal zone lymphoma, Hepatosplenic T-cell lymphoma, and Angioimmunoblastic T-cell lymphoma (AILT), Myelomas such as Multiple myeloma, Waldenström macroglobulinemia, and Plasmacytoma, Leukemias such as Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenous leukemia (AML), Chronic Idiopathic Myleofibrosis (MF), Chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), Chronic neutrophilic leukemia (CNL), Hairy cell leukemia (HCL), T-cell large granular lymphocyte leukemia (T-LGL), and Aggressive NK-cell leukemia. Moreover, examples of hematological diseases also include miscellaneous haematological diseases including Hemochromatosis, Asplenia, Hypersplenism such as Gauchers disease, Monoclonal gammopathy of undetermined significance, Hemophagocytic lymphohistiocytosis, and Tempi syndrome. Moreover, examples of hematological diseases also include Hematological changes secondary to non-hematological disorders including Anemia of chronic disease, Infectious mononucleosis, AIDS, Malaria, and Leishmaniasis.
Moreover, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be used for (the preparation of a medicament for) the prophylaxis, treatment and/or amelioration of transplant rejection, including e.g. graft-versus-host reaction. Transplant rejection includes hyperacute rejection, acute rejection and chronic rejection of a transplant. Examples of transplant rejection include skin, kidney, heart, lung, pancreas, liver, blood cell, bone marrow, cornea, accidental severed limb, in particular fingers, hand, foot, face, nose, bone, cardiac valve, blood vessel or intestine transplant rejection reaction.
Mode of Administration
The complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be administered in any manner as described above, including orally, parenterally, intravenously, rectally, or combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intra-arterial, intra-peritoneal, subcutaneous, intradermal and intramuscular. The complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may also be preferably administered via topical, intratumoral, intradermal, subcutaneous, intramuscular, intranasal, or intranodal route. The complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may also be administered in the form of an implant, which allows slow release of the compositions as well as a slow controlled i.v. infusion. For example, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be administered subcutaneously.
The administration of complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may require multiple successive injections. Thus, the administration may be repeated at least two times, for example once as primary immunization injections and, later, as booster injections.
In particular, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be administered repeatedly or continuously. The complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be administered repeatedly or continuously for a period of at least 1, 2, 3, or 4 weeks; 2, 3, 4, 5, 6, 8, 10, or 12 months; or 2, 3, 4, or 5 years.
Moreover, the cell penetrating peptide and the at least one cargo molecule, e.g. at least one antigen or antigenic epitope and a TLR peptide agonist as described herein, composing the complex according to the present invention may be contained in separate compositions which are mixed just before administration or which are administered simultaneously to the subject in need thereof.
According to one approach, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be administered directly to a patient using the administration routes as described above, in particular for pharmaceutical compositions. Alternatively, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine may be administered to a patient using an ex vivo approach, e.g. by introducing the pharmaceutical composition, the vaccine or the inventive transporter cargo conjugate molecule as defined above into cells, preferably autologous cells, i.e. cells derived from the patient to be treated, and transplanting these cells into the site of the patient to be treated, optionally subsequent to storing and/or culturing these cells prior to treatment.
The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties, subject conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.
Typically, for cancer treatment, the therapeutically effective dose of a complex according to the present invention is from about 0.01 mg to 5 mg per injection, in particular from about 0.1 mg to 2 mg per injection, or from about 0.01 nmol to 1 mmol per injection, in particular from 1 nmol to 1 mmol per injection, preferably from 1 pmol to 1 mmol per injection.
Typically, for cancer treatment, the therapeutically effective dose of an antigen presenting cell loaded with a complex according to the present invention is from about 0.2 million cells to 2 million cells per injection.
Combination Therapy
The administration of the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine in the methods and uses according to the invention can be carried out alone or in combination with a co-agent useful for treating and/or stabilizing the disease or disorder to be treated or repressed.
For instance, in the case of treatment, prevention, or stabilization of a cancer, the administration of the pharmaceutical compositions in the methods and uses according to the invention can be carried out in combination with substances used in conventional chemotherapy directed against solid tumors and for control of establishment of metastases or any other molecule that act by triggering programmed cell death e.g. for example a co-agent selected from Tumor Necrosis Family Members including, but not limited, to Fas Ligand and tumor necrosis factor (TNF)-related apoptosis inducing (TRAIL) ligand. According to a further embodiment, the administration of the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine in the methods and uses according to the present invention can be carried out in parallel of radiotherapy.
The invention encompasses the administration of the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine, wherein it is administered to a subject prior to, simultaneously or sequentially with other therapeutic regimens or co-agents useful for treating, and/or stabilizing a cancer and/or preventing cancer relapsing (e.g. multiple drug regimens), in a therapeutically effective amount. Said complex, cell, composition, vaccine or pharmaceutical composition, that is administered simultaneously with said co-agents can be administered in the same or different composition(s) and by the same or different route(s) of administration.
Said other therapeutic regimens or co-agents may be selected from the group consisting of radiation therapy, chemotherapy, surgery, targeted therapy (including small molecules, peptides and monoclonal antibodies), and anti-angiogenic therapy. Anti-angiogenic therapy is defined herein as the administration of an agent that directly or indirectly targets tumor-associated vasculature.
Accordingly, the present invention provides a pharmaceutical composition comprising a complex of the invention or a cell of the invention, in particular an antigen-presenting cell of the invention, combined with at least one co-agent useful for treating and/or stabilizing a cancer and/or preventing a cancer relapsing, and at least one pharmaceutically acceptable carrier.
Moreover, the complex according to the present invention; the cell, such as antigen-presenting cell, loaded with the complex according to the present invention; the inventive composition; the inventive pharmaceutical composition or the inventive vaccine can be administered after surgery where solid tumors have been removed as a prophylaxis against relapsing and/or metastases.
Moreover, the administration of the imaging or diagnosis composition in the methods and uses according to the invention can be carried out alone or in combination with a co-agent useful for imaging and/or diagnosing the suspected disease or disorder.
Subjects
The present invention can be applied to any subject suffering from any disease or disorder, depending on the specificity in particular of the at least one antigen or antigenic epitope comprised by the complex according to the present invention. In particular, the therapeutic effect of said complex may be to elicit an immune response directed against said antigens or antigenic epitopes, in particular a response that is dependent on CD4+ helper T cells and/or CD8+cytotoxic T cells and/or that is restricted by MHC class I molecules and/or MHC class II molecules.
Preferably, subjects according to the invention are subjects suffering from a cancer, for instance from a cancer of the brain, colon, head or neck, or from a cervical cancer. More preferably, subjects according to the invention are subjects suffering from a brain cancer including glioma.
It is also preferred that subjects according to the invention have been subjected to a surgical removal of a tumor.
Alternatively, subjects according to the invention are subjects suffering from an infectious disease.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.
All references cited herein are herewith incorporated by reference.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.
In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.
Different ZEBRA CPP truncations were designed and synthesized as shown in
Sci. 101, 16460-16465). Purity and integrity of each peptide were routinely verified by HPLC and mass spectrometry. The amino acid sequences of the different ZEBRA CPP truncations are shown in the following:
The objective of this study was to select the best ZEBRA CPP truncations in vitro and in vivo from the different ZEBRA CPP truncations designed and synthesized in Example 1.
To this end, each Zebra CPP truncation (Z12 to Z20) was conjugated to the OVACD8 epitope. Accordingly, the following fusion peptides/fusion proteins were synthesized as described in Example 1:
For comparison, the following OVACD8 peptide (without any CPP) was also synthesized:
The functionality of the different ZEBRA CPP truncations was validated both in vitro and in vivo, based on the capacity to stimulate MHC class-I restricted CD8 T cells specific for a model antigen OVA (OVA257-264-specific CD8+ T cells from OT-1 T cell receptor (TCR) transgenic mice). This capacity should reflect transport of the OVA antigen cargo into APCs. The functional read-out of these experiments was proliferation of OVA-specific CD8 T cells in vitro (
To assess the proliferation of OVA-specific CD8 T cells in vitro, bone marrow derived dendritic cells (BMDCs) were prepared from C57BL/6 mice as previously described (Santiago-Raber (2003), J. Exp. Med. 197, 777-788), and used at day 9-10 of culture.
BMDCs were loaded with 0.3 μM of each Zebra CPP truncation (Z13 to Z20) conjugated to OVACD8 epitope as described above for 4 hours at 37° C., washed 3 times, then matured overnight at 37° C. with 100 ng/ml LPS (from Salmonella abortus, equi S-form, Enzo Life Sciences). Antigen-loaded mature BMDC were then mixed at a ratio of 1:10 with OT-1 T cell receptor (TCR) transgenic mice splenocytes that had been stained with 10 μM 5-(and 6) Carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies). After 4 days co-incubation of spleen cells and BMDCs, antigen-specific proliferation was assessed by flow cytometry, measuring CFSE dilution on CD8 T cells and live-gated cells. OT-1 mice express TCR specific for MHC class I restricted OVA257-264. Results are shown in
To assess the induction of OVA-specific CD8 T cells in vivo, C57/BL6 mice were assigned to nine different groups (Z12 to Z20, respectively). Each group of mice was vaccinated by subcutaneous injection of 10 nmol of one selected Zebra CPP truncation (one selected from Z12 to Z20) conjugated to OVACD8 epitope as described above with 100 μg of anti-CD40 at week0 and week2. Mice were also injected with 50 μg of Hiltonol i.m. (right hind leg). Mice were bled 1 wk after the 2nd vaccination in order to assess OVA257-264-specific CD8 T cells. Results are shown in
Taken together, the data show that truncated CPPs generally conserved their immunogenicity. In general, the in vivo vaccination experiment (
In order to more directly assess the transduction capacities of the selected ZEBRA CPP truncations Z13, Z14, Z15 and Z18 fluorescein-conjugated constructs (Z13OVACD8FAM, Z14OVACD8FAM, Z15OVACD8FAM and Z18OVACD8FAM) were synthesized. To this end, 5-(and-6)-carboxyfluorescein (mixed isomers; e.g. ThermoFisher Scientific catalogue no. C194) was added to each of Z13OVACD8 (SEQ ID NO: 29), Z14OVACD8 (SEQ ID NO: 30), Z15OVACD8 (SEQ ID NO: 31) and Z18OVACD8 (SEQ ID NO: 34).
These fluorescein-conjugated constructs (Z13OVACD8FAM, Z14OVACD8FAM, Z15OVACD8FAM and Z18OVACD8FAM) were tested for their ability to transduce different human and mouse cell types, in particular (i) cells with high phagocytosis capacity, namely dendritic cells (DCs) of human (BMDCs) and mice origin (mo-DCs), and (ii) cells with poor phagocytosis capacity, namely T cells from human, K562, or mouse, EL4, origin). The EL-4 thymoma cell line was maintained in complete RPMI 1640 medium. The K562 cell line was maintained in complete Iscove's modified Dulbecco's medium. BMDC, mo-DCs, EL-4 or K562 cells were incubated for 4 h at 37° C. with the fluorescein-conjugated constructs as described above (Z13OVACD8FAM, Z14OVACD8FAM, Z15OVACD8FAM or Z18OVACD8FAM). For removal of membrane bound peptide prior to cell transduction, a 30s wash with an acidic buffer (0.2 M Glycine, 0.15 M NaCl, pH 3) was performed. Cells were then analyzed by flow cytometry (FACS). Results are shown in
Transduction efficacy was different depending on the cell type. Z13 and Z14 were rapidly entering in the majority of the cells. In contrast, the kinetic of transduction for Z15 and Z18 was slower. Overall, transduction efficacy was higher in human DCs, which might be correlated to phagocytic activity. However, high transduction was also observed in EL4 cells. All the peptides showed a modest transduction in dendritic cells of murine origin. For all cell types, Z18 exhibited however a lower transduction efficacy compared to other ZEBRA CPP truncations.
In order to assess the capacity of the ZEBRA CPP truncations to stimulate an integrated immune response including CD4 T cells, antigenic cargoes were produced that included both CD8 and CD4 T cell epitopes from OVA, and conjugated to Z13, Z15 and Z18. Accordingly, the following constructs (Z13OVACD8CD4, Z15OVACD8CD4 and Z18OVACD8CD4) were synthesized:
Processing and presentation of the CD8 and CD4 T cell epitopes were monitored, as described in Example 2 for CD8 T cell epitopes, by measuring the in vitro proliferation of naïve OT-1 T cells (CD8 T cell epitopes;
The data indicate that proliferation of both CD8 and CD4 T cells occurred in vitro with all tested ZEBRA CPP truncations (Z13, Z15 and Z18), confirming an efficient transduction of the DCs and delivery of the cargo into antigen processing pathways for both MHC class I and MHC class II. However, Z18 was less efficient that Z13 or Z15.
In order to assess the capacity of the ZEBRA CPP truncations to promote epitope presentation by human DCs, the following constructs (Z13-MART1, Z14-MART1, Z15-MART1 and Z18-MART1, with each of the Z13, Z14, Z15 and Z18 CPP conjugated to the HLA-A2 epitope of the tumor associated antigen (TAA) MART1)) were synthesized:
These constructs (Z13, Z14, Z15, and Z18 CPPs conjugated to the HLA-A2 epitope of the tumor associated antigen (TAA) MART1) were assessed for their capacity to promote epitope presentation on MHC class I by human DCs. To this purpose, DCs from an HLA-A2+ donor were prepared from elutriated monocytes, cultured in CelIGro DC medium (CellGenix, Freiburg, Germany) with granulocyte-macrophage colony-stimulating factor (GM-CSF; 2500 U/ml) (Leucomax; Schering-Plough, Kenilworth, N.J.) and interleukin-4 (IL-4; 1000 U/ml) (CellGenix). The immature DCs were matured with cytokines: IL-1β (10 ng/ml), IL-6 (1000 U/ml), tumor necrosis factor α (TNFα; 10 ng/ml) (all from CellGenix) and prostaglandin E2 (1 μg/ml) (Sigma-Aldrich). DCs were rested in CellGro DC medium for 2 hours and loaded with 1 μM Scramble-MART1, Z13-MART1, Z14-MART1, Z15-MART-1 or Z18-MART1 for 4 hrs at 37° C. before being washed once and plated at 300 000 cells per well in round-bottomed 96-well plates. Non-peptide loaded DCs were used as a negative control. T cells were expanded from peripheral blood mononuclear cells using Dynabeads ClinExVivo CD3/CD28 (Life Technologies, Oslo, Norway). Expanded T cells (day 10 post activation) were transfected with the DMFS TCR specific for MART1 by mRNA electroporation. T cells were co-incubated with DCs at a ratio of 1:2 for 5 hours in the presence of GolgiPlug and GolgiStop (both BD Biosciences) before intracellular staining was performed using the following antibodies after FcR blocking; CD4 (RPAT4), CD8 (RPAT8), CD107a (H4A3) (BD Biosciences), IFN-γ (4S.B3) and TNF-α (MAb11) (BD Biosciences), all from eBioscience except where noted. Fixation and permabilization was performed using the PerFix kit from Beckman Coulter according to manufacturer's instructions. Cells were analysed using an LSR II flow cytometer (BD Biosciences) and results were processed with Kaluza (Beckman Coulter) software. All conditions were tested in duplicate, and the experiment was repeated up to three times.
These experiments showed an efficient stimulation of human MART1-specific CD8 T cells that was CPP dependent (
To extend the previous findings to in vivo vaccination, Z13, Z14, Z15 and Z18 conjugated to antigenic cargo containing both CD8 and CD4 T cell epitopes from OVA (as described in Example 4 for Z13, Z15 and Z18) was synthesized. For Z14 the construct was as follows:
The following construct without ZEBRA CPP was used as control:
Firstly, those constructs were tested with Hiltonol adjuvant (a TLR3 agonist). To this end, mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection of 10 nmol of OVACD8CD4 (the cargo without Zebra CPP truncation; as described above), Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4, Z18OVACD8CD4 (as described in Examples 4 and 6) and i.m. injection of 50 μg of Hiltonol. One week after the last vaccination, mice were bled for assessing OVA257-264-specific CD8 T cells by FACS MHC-peptide multimer staining and elispot analysis. The enzyme-linked immunospot (ELISPOT) assay for detection of peptide-specific gamma interferon (IFN-γ)-secreting T cells was performed essentially as described previously (Miyahira (1995), J. Immunol. Methods, 181, 45-54) For analysis of ex vivo cytokine secretion, splenocytes were incubated o.n. in ELISPOT plates in the presence or absence of 5 μM of OVA257-264 or OVA323-339. The number of peptide-specific IFN-γ-producing cells was calculated by subtracting the number of IFN-γ-secreting cells cultured without peptide to that obtained with cells cultured with peptide. Results are shown in
Next, those constructs were tested with Pam3CSK4 adjuvant (a TLR2 agonist). To this end, mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection of 10 nmol of OVACD8CD4 (the cargo without Zebra CPP truncation; as described above), Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4, Z18OVACD8CD4 (as described in Examples 4 and 6) and of 20 μg of Pam3CSK4. One week after the last vaccination, Elispot analysis was performed on spleen cells as described above. Results are shown in
Thereafter, the constructs were tested with MPLA adjuvant (a TLR4 agonist). To this end, mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection of 10 nmol of OVACD8CD4 (the cargo without Zebra CPP truncation; as described above), Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4, Z18OVACD8CD4 (as described in Examples 4 and 6) and of 20 μg of MPLA. One week after the last vaccination, Elispot analysis was performed on spleen cells as described above. Results are shown in
Taken together, the data showed that: i) Z18 induced the highest CD8 and CD4 immune response when combined with Pam3CSK4 whereas ii) Z13 is the most efficacious when combined with MPLA; iii) in contrast, for any adjuvant, the Z15 CPP elicited low CD8 T cell immune responses.
To extend the previous findings to self antigen-specific immune response, Z13, Z14 and Z18 conjugated to antigenic cargo containing a self antigen (CD8 epitope from gp100) and CD4 T cell epitope from OVA were synthesized.
Firstly, those constructs were tested with Hiltonol or MPLA adjuvant. To this end, mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection (right flank) of 10 nmol of Z13OVACD4gp100CD8, Z14OVACD4gp100CD8, Z18OVACD4gp100CD8 and i.m. injection of 50 μg of Hiltonol (right hind leg) or s.c. injection of 20 μg of MPLA. One week after the last vaccination, spleen cells were restimulated in vitro for 7 days with gp100CD8 peptide and stained as follow. Briefly, for surface staining, after FcR blocking, the following mAb were used: CD4 (RMA4-4), CD8 (53-6.7), CD11b (M1/70), CD19 (6D5), all from BD Biosciences. Dead cells were identified with LIVE/DEAD yellow fluorescent reactive dye (L34959) from Life Technologies and were excluded from analyses. MHC-peptide multimers were from Immudex (Copenhagen, Denmark). Multimer gating strategy used a dump gate (CD4, CD11b, CD19) and excluded dead cells. Results are shown in
The data indicated that: i) Z18 did not elicit self antigen-specific CD8 T cell responses; ii) Z13 and Z14 are able to promote high self antigen-specific CD8 immune response when combined with MPLA or Hiltonol.
To investigate the effects of different ZEBRA CPP truncation-based vaccines on tumor growth and survival, the effects of the constructs Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4 and Z18OVACD8CD4 (as described in Examples 4 and 6) were investigated in the EG.7-OVA s.c. model. On d0, C57BL/6 mice were implanted s.c. with 3×105 EG7-OVA tumor cells in the left flank and assigned to five different groups (control, Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4 and Z18OVACD8CD4). Mice were vaccinated three times (namely, at d5, d13 and d21 after tumor implantation) by s.c. injection of either 10 nmol of Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4 or Z18OVACD8CD4 and 20μg of MPLA in the right flank. Tumor size was measured with a caliper.
Results are shown in
Based on the results of Example 7, the best ZEBRA CPP truncations Z13 and Z14 were selected for further investigation, namely in a complex according to the present invention. To this end, two fusion proteins “Z13Mad5Anaxa” and “Z14Mad5Anaxa” were designed and synthesized. The two fusion proteins differed only in the cell penetrating peptides (Z13 or Z14). Both fusion proteins comprise—in addition to the CPP (Z13 or Z14)-(i) the protein “MAD5”, which contains various epitopes of different antigens, namely OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopes, and (ii) the TLR2 peptide agonist “Anaxa”.
In the following, the amino acid sequences of “Z13Mad5Anaxa” and “Z14Mad5Anaxa” are shown:
LLLKESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQD
FDAE
To investigate the CD8 T cell immune responses in more detail, C57BL/6 mice were assigned to three different groups (3-4 mice per group): naive, Z13Mad5Anaxa or Z14Mad5Anaxa.
C57BL/6 mice of the Z13Mad5Anaxa group and of the Z14Mad5Anaxa group were vaccinated five times (Week1, Week2, Week4, Week6 and Week8) s.c. with 2 nmol of either Z13Mad5Anaxa or Z14Mad5Anaxa. Nine days after the 5th vaccination, mice were euthanized, organs recovered and multimer staining was performed to identify the percentage of SIINFEKL-specific CD8 T cells in the spleen, bone marrow and draining lymph nodes (inguinal and axillary).
The results are shown in
To further investigate the CD8 T cell effector function after vaccination with complexes with different CPPs, in the same groups of mice as described above Elispot assay was performed on spleen cells stimulated with SIINFEKL OVACD8 peptide (SEQ ID NO: 37) nine days after the 5th vaccination in order to quantify IFN-γ producing cells.
The results are shown in
To investigate the CD4 T cell responses after vaccination with complexes with different CPPs, in the same groups of mice as described above Elispot assay was performed on spleen cells stimulated with OVACD4 peptide (SEQ ID NO: 52) nine days after the 5th vaccination in order to quantify IFN-γ producing cells.
The results are shown in
In addition, in the above described groups of mice, intracellular staining was performed on spleen cells stimulated with SIINFEKL OVACD8 peptide (SEQ ID NO: 37) to identify CD107a+IFN-γ+TNF-α+ cells. Results are shown in
To investigate the effects of complexes having different cell penetrating peptides on tumor growth and survival the EG.7-OVA s.c. model was used. On d0 C57BL/6 mice were implanted s.c. with 3×105 EG7-OVA tumor cells in the left flank and assigned to three different groups (naïve, Z13Mad5Anaxa and Z14Mad5Anaxa). Mice were vaccinated twice at d5 and d13 after tumor implantation by s.c. injection of either 0.5 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa in the right flank.
Results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/000580 | Mar 2015 | EP | regional |
| PCT/EP2015/002244 | Nov 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/002598 | 12/22/2015 | WO | 00 |
