WO2006054262A2 - Use of phage display technique for identifying peptides capable of binding progenitor/stem cells, peptides thereby obtained and uses thereof - Google Patents

Abstract

The present application relates to the utilization of phage display technique, based on phage libraries of random sequence type or with defined amino acid sequences for identifying peptide sequences capable of binding human progenitor/stem cells, as well as peptides and derivatives thereof identified and isolated by such methods. The present invention further relates to methods of recognizing, monitoring and modifying progenitor/stem cells by use of complexes comprising said peptides and derivatives .

Description

USE OF PHAGE DISPLAY TECHNIQUE FOR IDENTIFYING PEPTIDES CAPABLE OF BINDING PROGENITOR/STEM CELLS, PEPTIDES THEREBY OBTAINED AND USES THEREOF

DESCRIPTION TECHNICAL FIELD OF THE INVENTION

The present application relates to the utilization of phage display technique, based on phage libraries of random sequence type or with defined amino acid sequences for identifying peptide sequences capable of binding human stem/progenitor cells, as well as peptides and derivatives thereof identified and isolated by such methods. The present invention further relates to methods of recognizing, monitoring and modifying progenitor/stem cells by use of complexes comprising said peptides and derivatives.

BACKGROUND OF THE INVENTION Features of Phage Display Methodology. Phage display technology is based on phage libraries and has been known for two decades as a technique for identifying amino acid sequences involved in molecular interactions. The technique envisages the construction of libraries by cloning random-sequence oligonucleotides (random library) or defined sequences (e.g., coding for one or more genes specific or corresponding to a pool of genes contained in a cell, tissue, organ or entire organism) in genes coding for capsid proteins of filamentous bacteriophages (Devlin et al . , 1990; Smith and Petrenko, 1997) . The peptides may be of variable length and each of them, with a different sequence, is displayed on a different phage particle. Most of the phage libraries utilized for phage display are based on filamentous bacteriophages of the M13 type as vectors whose genome is completely known. To generate a random sequence phage library, random-sequence oligonucleotides are usually cloned either at the 5' end of the gene III coding for a 42 kDa protein (glllp) or at the 5' end of the gene VIII coding for a 5.6 kDa protein (gVIIIp) . Since George Smith first described phage display- methodology in 1985 (Smith and Petrenko, 1997) , the presentation of peptides and exogenous proteins upon the surface of filamentous phages has become an important technique in the definition of binding sites between two or more interacting molecules (Clackson and Lowman, 2004) . By utilizing a given molecule as target it is" possible to enrich and select (biopanning) from the extremely heterogeneous mixture constituting the library, those recombinant phage particles that display onto their surface peptide sequences capable of binding the target . The sequence of these peptides thus selected can rapidly and effectively be obtained by sequencing the DNA that is encapsized in the same phage particles (Clackson and Lowman, 2004) .

The selection strategy characterizing the phage display procedure has been exploited in a number of ligand-ligate systems and has led to the identification of a number of peptide sequences, some among them not necessarily identical to natural ones but anyhow capable of reproducing the interaction with the target ligand. Some examples of biological applications of the phage display technique in vitro are the identification and subsequent analysis of antigenic epitomes, enzyme inhibitors and substrates, antagonists and agonists for various types of molecules and ligands for various types of receptors and for structural cell and tissue components. The procedure can then be exploited also for researching peptide sequences directed against multivalent and complex structures such as the entire surface of a cell (Clackson and Lowman, 2004) .

In the late '90s, Erkki Ruoslahti's group disclosed the possibility of exploiting the methodology also in vivo (in murine models) for researching peptide sequences with particular specificity for individual organs (Pasqualini et al . , 2002; Clackson and Lowman, 2004) . From these studies and subsequent investigations of the same research group, there emerged peptide sequences with high affinity for vascular structures; some of these sequences were exploited for targeting tumor neovascularization in an abrogative way (Pasqualini et al . , 2002) . Concomitantly, a number of scientific works demonstrated the applicability of the phage display methodology for selecting peptide sequences directed against individual cell phenotypes (both healthy and pathological) in culture (Nickling et al. , 2000; Zhang et al. , 2001) .

Stem or progenitor cells

Nowadays the enormous potentiality offered by cell therapy based on the use of multipotent stem cells or alike progenitor cells in the treatment of numerous pathologies, to date hardly curable, is well-known to a person skilled in the art. Among the different types of progenitor/stem cells, the cells with mesenchymal features MPC (MPC cells) , previously known as stromal cells and residing in bone marrow, already find clinical application in the hematology field, in particular in the context of autologous (and mini-allogenic) transplantation in oncology patients and in patients suffering from hereditary degenerative diseases and autoimmune dysfunctions, as well as in the field of organ and tissue transplantation. In the former cases, MPC can be advantageous in fostering an improved grafting of the hematopoietic stem cells CD34⁺ routinely transplanted to compensate for immune deficits. In this context, as well as in the field of organ transplantation, MPC are deemed to reduce the immune rejection response, the so-called graft-versus-host-disease.

Another potential clinical application of these cells is provided by their innate capability of differentiating into bone tissue cells and, as recently demonstrated at an experimental level, to promote growth and differentiation of endogenous bone precursors (osteoblasts) following transplantation. Hence, the apparent MPC capability of fostering a potential regeneration of cartilage or bone tissue damaged due to trauma or to hereditary and non-hereditary degenerative pathologies, e.g., osteoporosis, rheumatoid arthritis, arthrosis and advanced multiple myeloma, is of particular relevance in the effort to face the repair of said tissues. Lastly, various scientific works documented the cells' capability of being recalled by tumor lesions, following systemic infusion or local inoculation, and to release cytokines or other factors with direct or indirect antitumoral action (by suppression of cytotoxic lymphocyte response) genetically transferred beforehand into ex vivo cells (Studeny et al . , 2002) . Unfortunately, to date the use of progenitor/stem cells is hindered by numerous unsolved problems such as the lack of selective markers allowing their irrefutable singling out and ensuring their traceability post-transplantation, as well as the lack of transformation methods effective and safe to the patient increasing their potential therapeutic applicability. Hence, scope of the present invention is to offer novel tool s facilitating the in vitro, in vivo and ex vivo identifying, monitoring, isolating and manipulating of multipotent progenitor/stem cells. SUMMARY OF THE INVENTION The invention is based on an adaptation of the phage display technique for identifying peptide sequences capable of highly selectively binding progenitor/stem cells, like, e.g., MPC residing in human bone marrow, as well as capable of being at least partially internalized by the cells themselves. Synthetic peptides reproduced on the basis of the sequences identified in accordance with the invention and derivatives thereof not only bind the surface of target cells, but are then included by the latter, by an endocytosis process apparently mediated by clathrin complexes. Moreover, a large fraction of molecules entering the plasma membrane are translocated into the nucleus. Furthermore, the invention is based on the discovery that the peptides' capability of crossing the plasma membrane and entering into the nucleus is in no way limited by the presence of molecules of various nature bound to the peptide itself, which molecules are therefore carried into the cell . These features make the peptides of the invention highly effective tools not only in allowing the identification and ensuring the traceability of stem cells, but also in allowing molecular, genetic or chemical modification processes of target cells to be carried out both in vitro {ex vivo) and, possibly, directly in vivo.

Therefore, object of the present invention is a selection method devised to exploit the phage display technique in order to identify amino acid sequences having affinity for a target cell, comprising steps of: incubating a phage library with target progenitor/stem cells of human or animal origin and isolating the phage fraction bound to said cells. Advantageously, the method comprises one or more preliminary steps, wherein the phage library is sequentially incubated with one or more reagents selected from: cell types different from the target cell, solid supports used in the procedure and agents used for cell cultivation. A second object of the invention is a method of producing peptides having affinity for a target cell, characterized in that it comprises steps wherein ligand peptides having amino acid sequence selected according to the method of the invention are reproduced by chemical synthesis or by expression in modified host cell.

A third object of the invention are peptides obtainable by the method described hereinafter, e.g. the peptide having amino acid sequence HHSRSTL (SEQ ID N0:l) in linear or circular form, and all of its derivatives capable of interacting with, and of being internalized by, progenitor/stem cells.

Part of the invention are also peptide complexes comprising a peptide capable of interacting with, and being internalized by, a progenitor/stem cell bound to a molecule selected from synthetic molecules, molecules of protein, hydrocarbon, lipid nature or nucleic material, such as a structural or reporter gene, or a vector comprising said gene capable of modifying the host cell.

Further objects of the invention are peptide complexes comprising a peptide capable of interacting and being internalized by an MPC cell when bound to other molecules. Other objects of the invention are progenitor/stem cells tagged by bonding or incorporation of the above described, progenitor/stem cells carrying a substance having therapeutic action immobilized or incorporated by a complex described above and stem cells modified with a heterologous gene, or by a vector containing it, introduced into the cell by means of the described complex. Lastly, objects of the invention are also methods of preparing tagged stem cells, or methods of preparing stem cells carrying therapeutic substances, or methods of genetically modifying stem cells and clinical applications thereof in various therapeutic treatments.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: the figure illustrates a possible scheme of subtractive biopanning according to the invention. The first biopanning cycle results in the elimination of phage clones non-specifically binding the plastics supports used for cell culture. The second cycle removes phage clones yielding non-specific bonds with FCS (Fetal Calf Serum) components capable of adhering to the plastics surface of the cell culture plates. A minimum of two subsequent biopanning cycles eliminate phage clones capable of binding fibroblastic cells, utilized in this case as "subtractive cells" . A minimum of further two cycles remove phage clones capable of binding uterine smooth muscle cells (UtSMC) constituting a second subtractive cell phenotype. Finally, the remaining phage clones are incubated in 3-4 biopanning cycles repeated with target MSC cells.

Figure 2 : the figure reports in a graph the results of a fluorimetric assay of competition between a target cell-specific phage clone and the corresponding synthetic peptide.

Figure 3 : Panels A and B show the internalization into the nucleus of target MSC cells of phage clones containing the sequence HHSRSTL (SEQ ID NO:1) . In panel A, the cell has also been treated with phalloidin-TRITC to highlight the actyn cytoskeleton. Panels C and D highlight the lack of internalization in dermal fibroblasts and in endothelial cells deriving from umbilical cord veins (HUVEC) . Panel E shows the nuclear internalization of a fluorescein-tagged synthetic peptide having the sequence HHSRSTL (SEQ ID N0:l) . Panel F shows the internalization and the cytoplasmic localization of the synthetic peptide HHSRSTL conjugated to fluorescent microspheres (FITC) of 0.04 μm in diameter. Panel G shows the target cell transfection mediated by the peptide

HHSRSTL complex associated to a PNA-GFP vector (Gene

Therapy Systems, Inc) at +48h from complex incorporation.

Panel H highlights the lack of internalization of the fluorescent peptide shown in panel E in quiescent MSC cells.

DETAILED DESCRIPTION OP THE INVENTION The method of selecting and preparing peptides having affinity for target progenitor/stem cells, according to the present invention, entails the following stages.

1. Preparation and/or acquisition of a phage library in which the modified phages express, at their surface, heterologous oligopeptides in the form of fusion molecules fused to a coat protein of the virus. 2. Preparation of a target cell and optionally of one or more subtractive cells;

3. Repeated incubations of the phage library with the target cells and the subtractive cells, selection (biopanning/subtractive biopanning) and recovery of the phages bound and/or internalized by target cells;

4. Amplification of the phages recovered from the target cell through successive cycles of infection in bacteria, incubation with target cells and biopanning;

5. Determination of the peptide sequences exposed onto the surface of the phages amplified and isolated by DNA sequencing. PHAGE LIBRARIES

On the basis of literature protocols, to date phage libraries with random peptide sequences of various length and nature, in which peptides of up to 20 residues, e.g. 5, 7, 12 or 15, are exposed onto the phage surface are easily generated in suitably equipped laboratories. These libraries may be based on phage or "phagemid" vectors and can generate displays of the peptides at issue of a polyvalent or monovalent type, respectively (Clackson and Lowman, 2004) . Alike libraries can be produced with peptide fragments derived from a single protein, e.g. in an immunoglobulin, or produced on the basis of mRNAs deriving from particular cells or tissues, e.g. from hepatic gene library. Libraries of the first random type can also be easily obtained from commercial sources and are particularly suitable in the context of the present invention. The random peptides of said libraries are expressed onto the phage surface in the form of fusion protein comprising the peptide of interest bound to a surface protein of the filamentous phage M13, such as the capsid protein (protein pill) , the protein pVIII or the protein pVI (Smith and Petrenko, 1997; Larocca et al . , 1999; Devlin et al. , 1990; Clackson and Lowman, 2004) .

Random peptide libraries can comprise up to 10⁸-10⁹ different combinations of oligopeptides normally composed of 5 to 20 amino acid residues. For instance, in a specific embodiment of the invention different random libraries of M13 type were utilized, capable of expressing random peptides composed of 7 to 20 amino acid residues; therefrom, two libraries were selected, bearing: the first one, 7-residue circular oligopeptides, the second one, 12-residue linear oligopeptides. Of course, while the invention envisages the use of phage libraries, other peptide libraries, such as combinatorial libraries, may likewise be utilized in the scope of the present invention, though less preferred owing to significantly higher costs for their generation and use and to more complicated utilization procedures.

Embodiments of the present invention are based on the comparative use of commercially available 7-residue (Ph.D-7™ Phage Display Peptide Library Kit) and 12- residue (Ph.D-12™ Phage Display Peptide Library Kit) linear random type phage libraries and a 7-residue circular random library (Ph.D-C7C™ Phage Display Peptide Library Kit) . The peptides presented by phages of these libraries are fused with the surface protein pill of bacteriophage M13 by means of a flexible Gly-Gly-Gly-Ser bridge (clinker) for the 7- and 12-residue linear libraries and by a cysteine loop in the case of the circular library that comprised two terminal cysteines flanking the 7-aa sequence. The libraries, purchased from New England-Biolab (Boston, MA, USA) contained 2.8xlO⁹ clones (7 linear residues), 1.9xlO⁹ clones (12 linear residues) and 3.7xlO⁹ clones (7 circular residues) . Since the phage libraries utilized derived from the common cloning vector M13pl9 containing the gene lacZα, the phage plaques obtained during the various amplification stages of the subtractive biopanning appeared blue when plated on Xgal- and IPTG-containing medium. The host strain of E. coli utilized, ER2738, corresponds to an F⁺ strain with rapid growth rate and particularly suitable for the propagation of the phage M13. Factor F contains a mini-transposon giving resistance to tetracycline. Target cells Target cells utilized for selecting putative ligand peptide sequences are somatic adult or fetal progenitor/stem cells at any stage of their differentiation. Exemplary cells utilized in accordance with the invention are neural progenitor/stem cells deriving from fetal or adult central nervous system, progenitor/stem cells with fibroblastic/mesenchymal features (MPC) residing in human or animal bone marrow, fetal and adult hematopoietic progenitor/stem cells CD34⁺ and CD34^", umbilical cord progenitor/stem cells, progenitor/stem cells residing in human or animal bone marrow, adipose tissue progenitor/stem cells, mammary stroma progenitor/stem cells, prostate stroma progenitor/stem cells, gingival progenitor/stem cells, progenitor/stem cells of epithelial tissues (gastrointestinal tract, skin, respiratory tract, lung) , progenitor/stem cells residing in fetal and adult skeletal muscle, progenitor/stem cells residing in fetal and adult heart, progenitor/stem cells deriving from the placenta, progenitor/stem cells deriving from the decidua, progenitor/stem cells circulating in the peripheral blood and lymphatic circle, progenitor/stem cells residing in the peripheral nervous tissue, progenitor/stem cells residing in the liver, progenitor/stem cells residing in the pancreas, progenitor/stem cells residing in the spleen. An embodiment of the invention envisages the use of progenitor/stem cells residing in human or animal bone marrow with fibroblastic/mesenchymal features (MPC) . These cells exhibit, both in vitro and in vivo, a multipotent differentiation capability with preference for transformation into cell phenotypes of mesodermal origin, such as osteoblasts, chondrocytes, adipocytes and smooth muscle. Multipotent mesenchymal progenitor cells can easily be isolated from bone marrow of adult donors, both healthy and diseased, and can easily be amplified ex vivo through various culture steps obtaining excellent yields. In particular, within the scope of the invention there may be utilized MPC cells starting from the 4-5^th passage of in vitro amplification. Target cells are normally utilized in a semiconfluent state, or more scattered, after immobilization directly onto the bottom of a traditional cell culture plate, or of other support fostering cell adhesion and growth, and require no specific materials or molecular substrates.

TARGET CELL INCUBATION WITH PHAGE LIBRARIES AND SELECTION OF SPECIFIC PHAGE CLONES BY SUBTRACTIVE BIOPANNING

Prior to incubation with the target cell, phage libraries are subjected to sequential steps of preliminary selection envisaging the repeated incubation of the phage library with one or more cell types different from the target cell, defined here as "subtractive cells" , solid supports used for cell culture, and/or soluble substances used for cell cultivation. Purpose of this procedure, called "subtractive biopanning" , is to gradually eliminate peptides associated to phage clones that lack specificity for the target cell but can generate nonspecific bonds with other targets. For instance, when the target cells are MPC, the "subtractive biopanning" may be based on the following steps (Figure 1) . (1) A first elimination of the phage clones to which there are associated peptide sequences non-specifically interacting with plastics surfaces such as those for cell culture containers and/or binding bovine fetal serum components commonly utilized as supplement for animal and human cell cultivation. (2) A second incubation of the remaining phage clones of the library with primary cells of uterine smooth muscle selected as representing a cell having target cell-like features; (3) a third step implying the incubation of the remaining phage clones with a second cell phenotype, fibroblastic in this case, it also having homologies with the target cell; and (4) 3-4 steps entailing the repeated incubation of the remaining phage clones of the library with target cells, i.e., MPC. Hence, the subtractive procedure implies that following each incubation of the phage library with the subtractive target, i.e., plastics, serum or non-stem cells, or following each subtraction, phage clones reacting with the subtractive element, i.e., components non-cellular or ubiquitous for various cell types, are eliminated from the library, so as to create an enrichment of the phage clones containing peptide sequences with potentiality to selectively interact with the target progenitor/stem cells.

PEPTIDE SEQUENCES WITH HIGH SPECIFICITY FOR THE TARGET CELL

In the search for peptide sequences capable of being internalized by a specific cell phenotype, after a number of biopanning cycles against the target cell that are arbitrarily or empirically definable with pilot tests and usually of a number not lower than 3, cell-internalized phage clones are eluted, their DNA extracted and the fragment coding for the peptide PCR- amplified and sequenced to deduce the corresponding amino acid sequence, as described above. The number of phage clones to be sequenced is it also arbitrary, yet usually not lower than 50. The criterion conventionally adopted to establish the occurred enrichment of a specific sequence is of at least 3 clones on the total number of sequenced clone phages having the same amino acid sequence. By utilizing MPC cells as target cell, 4 selection cycles against said cell and an at least 5-fold enrichment criterion were adopted. Such an enrichment level was not detected in the first 50 clones and therefore additional 20 clones were selected for a pool of 70 phage clones that led to a 5-fold frequency of the peptide sequence HHSRSTL (SEQ ID NO:1) . The presence of clones bearing other sequences enriched up to 3-fold, such as DKTTASR (SEQ ID N0:12), EPRLTKA (SEQ ID NO:13) and TASQGRQ (SEQ ID NO:14) was detected as well, but the enrichment of the latter sequences was not deemed significant in the pool of the 70 clones isolated (Table 1) . This does not rule out a possible significance thereof detected by testing a larger number of clones.

Initially, the phage clone containing the aboveindicated sequence HHSRSTL was assayed for its capability of binding MPC cells and being internalized thereby. The specificity of this interaction was then checked by competition assays envisaging the concomitant incubation of the MPC cells with the phage clone at issue and increasing doses of the synthetic peptide HHSRSTL. Subsequent experiments with alike peptides conjugated to fluorophores (fluorescein, rhodamine) or biotinylated have confirmed the MPC-selectivity of the sequence. Actually, an analysis on more than 40 cell phenotypes derived from adult human body enabled to further confirm the selectivity of the peptide sequence, exhibiting low internalization levels in sole 9 cell types different from MPC and the missed nuclear translocation observed in MPC cells (Table 2) . Peptides meeting the functional conditions of selectivity and internalization capability with effectiveness equal to or greater than the 1% of the effectiveness exhibited by the peptide of sequence SEQ ID N0:l are part of the invention. These peptides may be produced in linear shape, circular or branched, and may consist in, or comprise one or more copies of the ligand peptide selected with or without spacers; e.g., polypeptides comprising various copies of the sequence SEQ ID N0:l and having formula R₁- (HHSRSTL-R₂-)_n-R_3/ where R₁, R₂ and R₃ are oligopeptides independently comprising 0, 1, 3, 6, 10, 50, 100 or more amino acid residues, usually 7 or more, and where n is an integer equal to 1, 2, 3, 4 or more, e.g. : HHSRSTLHHSRSTL, HHSRSTL-R₂- HHSRSTL. Other mimitope-type derivatives of sequence SEQ ID NO:1 may be linear, circular or branched peptides consisting in or comprising: 1) at least 5 consecutive amino acid residues of sequence SEQ ID N0:l, e.g.: HHSRSTV, AHSRSTL, HHYRSTL;

2) sequence SEQ ID NO:1 with substitutions of 1 to 5 amino acid residues, e.g. HHVRSTL, HRSRSGL, AHVRSSL; 3) sequence SEQ ID NO:1 with addition or elimination of at least one amino acid residue, e.g. : HHSRST, HHSRSTLL;

4) sequence SEQ ID NO:1, wherein one or more amino acid residues are chemically modified by acetylation, e.g.: HAcHSRSTL, HHSRSAcTL, HHACSRSTACL;

5) sequence SEQ ID N0:l, wherein one or more amino acid residues are chemically modified by alkylation, e.g.: H(R)HSRSTL, HHSRS(R)TL, HH(R)SRST(R)L;

6) sequence SEQ ID N0:l, wherein one or more amino acid residues are chemically modified by sulfonylation, e.g. :H(sulfJHSRSTL, HHSRS (sulf)TL, HHfSUlJfJSRSTfSuIfJL;

7) sequence SEQ ID NO:I₇ wherein one or more amino acid residues are chemically modified by alkoxy- carbamylation, e.g.: H(carb)HSRSTL, HHSRS (carb)TL, HH(carb)SRST(carb)L;

8) sequence SEQ ID NO:1 with isomerization of one or more amino acids, e.g. dHHSRSTL, dHdHSRSTL, HHdSRdSTL, where "d" stands for isoform "D" of the amino acid residue; 9) sequence SEQ ID NO:1 to which there has been added, with or without spacer, a non-amino acid residue, e.g.: HHSRSTL-x-R, R-x-HHSRSTL, where "R" is a lipid, glycolipid, hydrocarbon or glycosaminoglycane residue.

10) sequence SEQ ID NO:1 to which there is added a cysteine residue to allow binding to protein or nucleic complexes, e.g. HHSRSTLC.

An important aspect of the invention is represented by peptide complexes formed by the peptide, or derivatives thereof, according to any one of the options reported above, conjugated or assembled with a molecule or molecular complex selected from the group comprising nucleic acids, a radioactive molecule, a nuclear tracer such as Technetium-99, Gallium-67 or Indium-Ill, a fluorescent molecule, a cromophore molecule, an enzyme, an immuno-partner such as an antigen, hapten, antibody or parts thereof, one of the two partners of an affinity pair such as, e.g., avidin/biotin, a synthetic compound of organic or inorganic nature, a paramagnetic, supermagnetic, ferromagnetic microsphere, a virus, a virus-derived particle, a single molecule belonging to or produced by a virus, or a molecule produced by viral infection of prokaryote or eukaryote cells, a protein or other molecule of protein nature.

In the context of ligand peptide/substance complexes, a crucial possibility is that of forming complexes between the peptide and substances of polynucleotide nature, i.e. DNA or RNA sequences, with or without the intervention of other elements, either or not of protein nature, working as adapters for the construction of the complex (e.g. a PNA, peptide nucleic acid, molecule analogue) . Suchlike complexes comprises individual genes coding for one or more polypeptides, utilized individually or in combination, e.g. a marker protein, a structural protein of the cell, an enzyme, a cell-secreted factor, a factor altering the pharmacological resistance of the cell, a factor modifying the host/target cell gene expression, protein synthesis or metabolism, or other factor acting on the replicative, differentiative and migratory capability of the cell at issue. Genes belonging to the first class of compounds are, e.g. reporter genes coding for an easily recognizable protein, such as the gene GFP (Green Fluorescent Protein) or the β-galactosidase gene. The other examples refer to genes coding for a pharmacologically active protein, such as molecules active in anti-inflammatory and antitumoral treatment such as cytochines, e.g. the interferons (a, β and γ) interleukins, the tumor necrotic factors (TNF a. and β) and the stromal derived factor-1 (SDF-I) , the tissue factor (TF) , various types of signaling hormones and molecules (e.g., semaphorins, ephrins, hedgehogs, members of the Wnt family) or growth factors. Other useful genes are genes coding for transcription factors and other elements involved in the regulation of gene expression, intracellular signaling receptors and molecules, cyclins and molecules with positive and negative action regulating the cell cycle, plasma membrane components responsible for the interaction of cells with their microenvironment (extracellular matrix and other cells) , molecules with chemotherapeutic action, molecules active in fostering tissue regeneration, or molecules capable of providing a specific localization and tissue integration of the systemically infused.cells. Thanks to their effective internalization in the target cell, suchlike complexes find ample application in genetic modification processes of the cell, offering the enormous advantage of avoiding the use of viral vectors and therefore solving the problem of the potential hazardousness of the latter to the cell and to the patient's health. Moreover, MPC modification processes with the complexes of the invention highlighted how the transfection of said cells with genetic material carried by peptide HHSRSTL runs with yields much higher than those observed when the usual viral vectors are used. Actually, adenoviral vectors exhibit transfection efficiency similar to that obtained with the peptide object of the invention, yet the duration of transgenic expression of the construct transferred by said vectors is limited to 2-3 weeks. Retroviral vectors (to date not approved for human use) provide a transgenic expression of the cDNA insert contained therein and transferred to the target cell not older than 2 months with a transfection efficiency seldom exceeding the 50%. On the contrary, by transfeeting a gene through peptide HHSRSTL it is achieved a transgenic expression keeping its efficiency for up to 4 months. FEATURES OP IDENTIFIED PEPTIDE SEQUENCE INTERACTION AND OF PUTATIVE INTERNALIZATION MECHANISMS OF CORRESPONDING SYNTHETIC PEPTIDES

As described above, experimental data demonstrate that the peptide sequences identified with the method of the invention not only exhibit high selectivity of interaction with the target cell MPC, but are also internalized with remarkable effectiveness by this cell and partially translocated into the nucleus. In the case of peptide HHSRSTL, it was observed that more than the 80%, at times more than the 90%, of the MPC cells selectively incorporate the peptide ligand or its derivatives, subsequently translocating a considerable fraction thereof into the nucleus. Other data suggest that this inclusion occurs thanks to an active and constitutive transport mechanism through the plasma membrane, probably endocytosis-mediated at the level of cholesterol-rich membrane domains (lipid rafts) and fostered by clathrin-coated vesicles. Moreover, it was observed that the inclusion of peptides into the cell and/or the nucleus does not interfere with normal cell reproduction processes. Other experimental data highlight how the peptide incorporation phenomenon and its nuclear translocation strictly depend on: the anchoring of the cell to a solid plastics support or to extracellular matrix components (yet not polylysine and similar artificial substrates); the presence of bivalent ions; specific intracellular signaling; and the cytoskeleton participation (both microfilament and microtubule dynamics) , yet is not associated to the various cell cycle phases. Moreover, incorporation tests carried out with the fluorescent compound HHSRSTL-fluorescein, with and without untagged peptide competition demonstrated that the integration of the peptide complex in the target cell is a process with dose-dependent kinetics, occurring within 5 minutes from the contact between the complex and the cell and achieving saturation within 90 minutes. Maximum inclusion is achieved with starting concentrations of the complex (input) equal to 1-3 μM, and, at saturation, the maximum ratio of fluorescence intensity that can respectively be determined in the cytoplasm and in the nucleus (cytoplasm/nucleus) by confocal laser microscopy is of about 1.35.

MODIFIED PROGENITOR/STEM CELLS AND APPLICATIONS THEREOF

Progenitor/stem cells susceptible of being modified with the method of the invention are the cell typologies listed above. A first embodiment of the invention envisages the use of progenitor/stem cells MPC residing in human bone marrow. The modified cells of the invention are obtained by inclusion into the plasma membrane of tagging substances or of substances having therapeutic activity, as well as by integration in cell synthesis mechanisms of the genetic material required for intracellular expression of marker proteins or of substances having therapeutic action. Both in the former and in the latter situation, it has been verified that the material introduced into such a cell phenotype does not damage the cell and does not interfere with metabolism and normal cell replication processes. Modified cells, like e.g. the MPC, maintain an unaltered expression of the phenotype markers characteristic of the cell type, and preliminary DNA microarray studies highlight no significant alterations of the gene expression. These properties make the modified cells central to the invention suitable for all those clinical applications in which progenitor/stem cells are already used today, yet enriching their use with remarkable additional advantages. Among the latter, the main one is that deriving from the tagging of the cells to be infused by nuclear tracer such as Technetium-99, Gallium-67 or Indium-Ill in order to allow the monitoring of their short-term tissue distribution. This application may be extended to a genetic-molecular modification of the cells aimed at favoring an assessment of the in vivo distribution of the cells, even in the long term.

Even though many positive effects are found associated to a systemic (or, more seldom, local) infusion of MPC in various therapeutic situations, the destiny of these cells in humans following infusion is not yet known, that is, the tissue distribution (i.e., homing) or the integration and the differentiation destiny of the cells in the various anatomic districts in which they settle are not yet known. Hence, the object of the invention would provide a valid tool for approaching this problem. Moreover, the complexes of the invention are valid tools for isolating, selecting and enriching MPC-type stem cells contained in aphereses of peripheral blood, lymphatic samplings, bone marrow aspirates, lymph node aspirates, synovial fluids, follicular fluids or other samplings containing body fluids, or biopsies, or tissues or organs removed from alive or deceased subjects, healthy or diseased at the time of sampling or removal .

The invention offers the additional advantage of providing novel cell tagging or modification tools that are not of viral origin and therefore are completely innocuous to cells and organism. Of course, the traceability of the progenitor/stem cells can be ensured both by the use of cells modified ex vivo and reintroduced by infusion in the patient, and by an optional direct tagging in vivo with the complexes of the invention infused at a systemic or local level and made capable of recognizing and tagging the cells, endogenous or transplanted, in untagged form. The invention is of particular value for all therapeutic approaches foreseeing a combined use of genetically modified cells to be transplanted (i.e. a combination of cellular and genetic therapy) . Exemplary applications of such a combinatorial approach, within which the peptide system of the invention plays a central role, are illustrated hereinafter.

Several pre-clinical and clinical studies demonstrate how MPC cells contribute to alleviate the rejection response (graft-versus-host-disease) in organ transplantation situations (specifically demonstrated for liver, kidney and skin) and mini-allogenic transplants, when inoculated concomitantly to such surgery (Matthew et al . , 2003) . Therefore, also in this case genetic- molecular modifications to be made to the cells by means of peptide complexes object of the invention are of remarkable value.

Other applications of MPC cells modified by transfer of peptide complexes containing specific active molecules aim at enhancing the cells' intrinsic capabilities of fostering cartilage and bone tissue regeneration following trauma or a debilitating pathology. Moreover, it is surmised that in patients suffering from multiple myeloma and the entailed serious erosions of bone tissue, exogenous MPC cells may act as an additional source of preosteoblasts and/or as powerful inducers of the endogenous intramembraneous osteogenesis. On the basis of laboratory studies, as well as of previous pilot clinical studies, it is envisaged that progenitor/stem cells may be employed as cellular means for gene therapy adapted to the purpose of mending hereditary and non-hereditary genetic deficiencies, especially those affecting mesoderm-deriving connective tissues (Chamberlain et al . , 2004; Prockop, 2004) . This by applying the transplantation of the cells at issue in the adult, as well as in the intrauterine embryonic phase. To this possibility it is added the optional potentiation of the intrinsic capabilities of the MPC to modulate the immune system (as already mentioned above) in order to create therapeutic means to be exploited in the treatment of autoimmune pathologies.

Lastly, given the capability of the MPC cells, as well as of other progenitor/stem cells, of being attracted in vivo by tumor lesions, MPC cells modified with peptide complexes according to the invention appear to be effective vehicles of antitumoral compounds. Actually, the feasibility of such an approach of combined cellular-genetic antineoplastic therapy has recently been demonstrated by a study in which local lesions from lung cancer or lung cancer metastases have been abrogated by infusion of MPC cells transduced to express high levels of IFNα (to which the tumors at issue exhibited particular susceptibility; Studeny et al . , 2002) . EXAMPLES

Example 1: MPC target cell production MPC cells are easily obtainable from bone marrow following standard procedures by Ficoll centrifugation and plate culture starting from bone marrow aspirates obtained from healthy donors or by patients affected by various pathologies (however, yields may differ in both cases, both at an individual level and comparing various pathological situations) . To date, MPC cells are also available by means of various commercial sources and stem cell banks. Conventionally, MPC cells obtained as indicated above are cultivated and amplified in undifferentiated form in classic culture media, like, e.g., DMEM, with variable concentrations of 1-20% FCS serum and with the optional addition of FGF2 (5-10 ng/ml) . MPC cells grow exclusively in culture support- anchored form and may, when needed, be cultivated on substrates containing single extracellular matrix molecules or complexes thereof. Therapeutic approaches on humans involving MPC cells necessarily imply that the cells should first be propagated in vitro so as to obtain a number of them sufficient for transplantation. Hence, MPC cells were utilized at the 4^th-5^th passage with the following consensus surface features (based on a cumulative survey of literature data) : CD4/CD8^", CD10^low/", CD14/CD15/CD68^", CD19/CD20^", CD31^", CD34^", CD38^low/", CD44⁺, CD45^", CD47^", CD59⁺, CD71^", CD72⁺, CD73⁺, CD81⁺, CD90⁺, CD105⁺, CD117⁺, CD133^", CD162^", CD166\ CD184⁺, Stro-1^+/", PDGF-Ra⁺, FGFR- HIIb and FGF-R3IIIb/IIIc⁺. In this context, it has to be noted that a certain inter-donor variability was detected in the relative expression of these markers and, in particular, it was found that the cells from certain donors can be totally negative for the markers CD38, CD44 and CD105. Prior to phage selection, also the differentiation potential of the cells in adipogenic, chondrogenic and osteogenic lines was ascertained according to standard procedures.

Example 2: Selection of MPC high-specificity peptides through subtractive biopanning protocol

In the initial stage of this work, different typologies of random M13 phage libraries containing peptide sequences of 7 to 20 amino acids, in linear as well as in circular form were comparatively assayed. By subtractive biopanning protocol, semiconfluent MPC cells that had undergone no more than 4 passages from initial isolation from bone marrow sampling were assayed. This first analysis led to the singling out of a 7-residue circular peptide library such as that optimal for this type of experiments. By exploiting this library, 70 (arbitrary number) phage clones were obtained to be subjected to DNA reamplification, extraction and sequencing following standardized protocols (Big Dye Terminator Cycle Sequencing Technology - PE Biosytems, Foster, CA, USA) and an 8-channel, capillary (electrophoresis based) Beckman-Coulter automatic sequencer. The results are reported in Table 2. To ensure optimal specificity during the in vitro selection procedure a subtractive biopanning protocol was designed, based on different removal cycles of sequences characterized by non-specific and/or cell-ubiquitous bonds.

It is known from literature that non-stimulated MPC cells exhibit a myofibroblastic phenotype. Therefore, in said sequential subtraction procedure both primary fibroblasts and smooth cells were utilized (Figure 1) . Moreover, as the main aim was the identification of peptide sequences capable of functioning as putative vehicles for transporting substances into the MPC cells, the testing was directed at identifying phage clones effectively internalized in the target MPC cell.

The specific subtractive biopanning procedure was conducted by utilizing 10¹⁰ pfu of the phage library in 2 ml PBS containing 0.1% BSA, 1OmM MgCl₂ and 1 mM CaCl₂ and incubating the library with the target at 37° C for 30 min under gentle stirring. Then, the target cell population was rinsed a minimum of 5-6 times with the same PBS solution to remove phages not associated to the cells, whereas phages binding the cell surface were eluted using 2 ml elution buffer composed of 0.1 N glycin-HCl, at pH 2,2 for 10 min at O⁰C. No enriched peptide sequences were detected in this fraction. Then, phage clones contained into target cells were recovered by elution following cell lysis with 30 mM Tris-HCl, pH

8,0 and 1 mM EDTA for 1 hour at O⁰C and harvesting of phages in solutions (having first removed all cell residues capable of contaminating the phage population) .

Phage amplification was performed by utilizing 1 ml of the eluate and of the cell lysate obtained in each selection round on the target MPC cells and incubating said volume with 200 μl of the bacterial culture ER2738 or ER2537 (according to the phage library utilized) at 37⁰C overnight under stirring. Then, the culture was centrifuged at 7,000 rpm for 20 min at 4⁰C to remove the bacteria. Phages were then precipitated with 1/4 of the volume of the original solution of a mixture of 30% PEG 8000 in 1,6 M NaCl and an incubation at 4°C of at least 30 min. The phages thus precipitated were recovered by centrifugation at 8,000 rpm at 4°C for 20 min and pellet resuspension in 1 ml PBS. Phage fractions, amplified by agar culture according to standardized methods, were routinely preserved in PBS-glycerol (1:1 ratio) at -20⁰C. For the various subtraction phases and selection rounds against the target cell, phage library titrations were performed as described hereinafter, in order to ensure a constant input of 10¹⁰ pfu per each phase/round. A single ER2738 colony was inoculated in 10 ml LB Ix liquid medium added with tetracyclin in concentrations equal to 20 mg/ml and incubated under gentle stirring until reaching an optical density value (OD at 600 nm) of about 0.5, established by spectrophotometer.

To determine amino acid sequences contained in the phage clones isolated in the final stage of the biopanning and deduced by DNA sequencing, the former were amplified by RT-PCR to allow said sequence analysis. The PCR protocol was of a conventional type, based on Taq Polymed (Qiagen) and the following pair of primers: M13 sense, 5' -TGCAAAGCAAGCTGATAAACCG-3' (SEQ ID NO: 15) and antisense, 5' -ACAGACAACCCTCATAGTTAGCG-B' (SEQ ID NO:16) . Amplificates were analyzed on agarose gel following standardized methods for this procedure.

Tests repeated with 4 biopanning cycles on MPC cells cultivated on plastic in semiconfluence with the 7-mer circular library identified a peptide pool as reported in Table 2, from which it is recognized a 5-fold enrichment of the sequence HHSRSTL (SEQ ID NO: 1; sequences with lower enrichment levels were not deemed significant) .

TABLEl

CLONE NUMBER PEPTIDE CLONENUMBER PEPTIDE

01 DKTTASR 36 MAGSNAF

02 — 37 SEGLNRG

03 FPNTRAT 38 HHSRSTL

04 PYSSQDN 39 HHSRSTL

05 KPTFHGA 40 KTHPNSQ

06 LNGFTHA 41 DSHHSPL

07 LPRPPHD 42 —

08 DSQPSNW 43 IRLRMTQ

09 QSPSARS 44 ~

10 NSMHPRQ 45 ZURSLSR

11 NLMSRTH 46 PVTKLLI

12 — 47 —

13 IPSKART 48 WKKAKH

14 GNTNSLQ 49 GPLPSRF

15 RTAALTQ 50 GSPDAMN

16 EPRLTKA 51 —

16 TPNAGNK 52 EPRLTKA

18 SMPFKHV 53 TQNPSSS

19 ~ 54 NRNTQLH

20 MGSANMS 55 RPHDKGA

21 — 56 TASQGRQ

22 LRTAAAT 57 SNLSMNT

23 MFAHPAV 58 —

24 HHSRSTL 59 HHSRSTL

25 HAAPTLQ 60 SKYGHAG

26 — 61 —

27 DLTALHV 62 —

28 — 63 HSRASEM

29 TTAKQTY 64 KSAHPAV

30 TPHFWAK 65 PRQSPQL

31 — 66 TASQGRQ

32 HKNLSAT 67 LRLWLGI

33 — 68 HHSRSTL

34 LGVKAPS 69 —

35 ~ 70 DKTTASR

The enriched and amplified phage clone bearing said sequence proved to be effectively incorporated by a high number of MPC cells, yet unable to enter the fibroblastic or uterine smooth muscle cells used in the various biopanning stages, as well as unable to enter a battery of 12 cell lines including immortalized fibroblast cells and connective cells of connective tissue polymorphous sarcoma exhibiting marked similarities with MPC cells (Table 2) . Data bank researches and comparisons with peptide sequences capable of binding cells previously identified by in vitro or in vivo phage display, or with peptide sequences from combinatorial libraries, or other selection methods, demonstrated that the sequence identified here matches none of the sequences previously singled out for cell targeting. To perform further cell assays, the sequence was reproduced in the form of a synthetic linear peptide that was biotinylated, conjugated to fluorescent substances

(fluoresceine or rodhamine through a suitable spacer) , or coupled to differently sized fluorescent microspheres (FITC) , or conjugated to a PNA-GFP reporter vector. Flow cytometry analysis of confocal laser microscopy, both of real-time type and of traditional scanning type, showed that under optimal conditions all three kinds of peptide conjugates are internalized by the 70-90% of adherent MPC cells (Figure 2) . Moreover, tagged and untagged selected peptides could effectively compete with the original phage clone expressing the sequence, whereas peptides with phage clone-exhibited sequences not sufficiently enriched in the biopanning procedure could not. In addition, evidently a predominant portion of the peptide internalized in the cells was transported in the corresponding nuclei . Lastly, absorption tests and competition tests between FITC-conjugated peptides and non-conjugated peptides demonstrated that the incorporation into the cell is dose-dependent and that the optimal concentration of the peptide is of 1-3 μM. MPC cells rapidly internalized the peptide-spheres complexes composed of fluorescent microspheres with a diameter of up to 0.04 μm. Under optimal conditions, the ratio cytoplasm fluorescence intensity versus nucleus fluorescence intensity was of 1.35.

Example 4. MPC cell-specificity of peptide sequence HHSRSTL

Freshly isolated cells and cells from previous passages (i.e., up to the 3^rd passage) did not significantly internalize the selected peptide. However, for the envisaged clinical applications this fact does not represent a limit, since for both allogenic and autologous transplantation MPC cells are subjected to ex vivo propagation prior to infusion. Accordingly, it was important to ascertain that the peptide sequence were effective on cells extensively amplified in vitro and that it had high specificity for MPC cells isolated both from healthy and diseased individuals. In spite of the detection of some variability in the efficiency of peptide uptake in cells derived from healthy donors with respect to those obtained from patients suffering from various malignant tumors of hematological nature

(possibly due to bone marrow alterations caused by diseases and/or therapeutic treatments) , in all cases more than the 80% of the MPC cells internalized significant amounts of the peptide, most of which entered the nucleus.

Since under optimal conditions the peptide is incorporated by the 90% of the MPC cells isolated from healthy donors, it may be ruled out that the uptake capacity be restricted to a subpopulation of MPC cells. Likewise, it is possible that the minority of the MPC cells that did not incorporate the peptide might represent a subpopulation of progenitor cells with divergent properties. The MPC cells effectively internalized the peptide up to the 44^th duplication, when cultivated in the continuous presence of 10% FCS, with and without addition of 10 ng/ml FGF2, or when amplified by using the RepliCell system (by Aastrom Inc.) . Instead, no incorporation was observed when the cells were forced in suspension by short-term cultivation on poly-HEMA substrates, or by exposition to centrifugal forces as is conventionally done to increase viral particle entering. It is important to observe how the latter results demonstrate that the peptide cannot be physically forced into the MPC cells. Moreover, its incorporation efficiency does not seem to be significantly affected by the anchoring of the MPC cells to specific ECM substrates

(collagen types I, IV, VI, vitronectin, laminin-10 and fibronectin were tested) . On the other hand, in the presence of the peptide no cell detachment from any of these substrates was observed, indicating that the peptide does not intervene in the interactions of the cells with their substrate. In fact, a cell adhesion analysis based on the CAFCA system and on a panel of 8 representative ECM molecules, like fibronectin, vitronectin, collagen types I, IV and VI, laminin-1, laminin-5 and laminin-10) highlighted no differences in the MPC cells capability of adhering to said substrates.

Next, to determine the specificity of the selected peptide sequence with respect to other human cell types, over 40 cell types deriving from main tissues and/or organs of the human adult body, including the main hematopoietic phenotypes, were tested for their capability of internalizing and translocating the peptide sequence into the nucleus. The results reported in Table 2 demonstrated that a vast majority of these phenotypes did not internalize the peptide or exhibited limited uptake capability and incapability of translocating the peptide into the nucleus. It is interesting to observe in this context that the progenitor stem/multipotent cells from other tissues like prostate, umbilical cord, decidua and brain were not capable of incorporating the peptide. Therefore, the identified peptide sequence demonstrates a high MPC cell-selectivity and a marked efficiency of transfer into the nucleus in this specific cell type. On the other hand, the peptide has no structural or sequence analogy with peptide sequences known or characterized by α-helix of amphipathic nature, such as those with a documented capability of crossing the cell membrane and accumulating in the nuclear compartment. TABLE 2 .

Phenotype specificity¹ of the MPC-selective peptide sequence HHSRSTL

Cell code Phenotype Tissue source % Level Intracellular

Positivity of localization³ cells¹ uptake²

MPC⁴ mesenchymal Bone marrow ++++ cyto + nucleus

MPC -NHL⁵ mesenchymal Bone marrow +++(+) cyto + nucleus

MPC - MM⁷ mesenchymal Bone marrow ++++ cyto + nucleus

MMP - UCB mesenchymal umbilical cord + cyto

HSC (CD34⁺) hematopoietic peripheral — 0 blood⁸

B cells lymphocyte peripheral — 0 blood⁹

T cells lymphocyte peripheral — 0 —

(CD4/CD8) blood¹⁰

Mononucleated leukocytes bone marrow¹¹ « 0 —

HSCAEC endothelial subclavian + artery

HITAEC endothelial thoracic artery + cyto

HCAEC endothelial coronary artery — 0 —

HDMEC endothelial dermal 0 „ microvascular

HIAEC endothelial iliac artery -- 0

HMVEC endothelial microvascular — 0 ~

HPAEC endothelial pulmonary + cyto artery

HUMED endothelial neonatal aorta — 0 —

HAl-HECM endothelial adult aorta — 0 —

HUVEC endothelial umbilical cord — 0 —

AoSMC smooth muscle adult aorta — 0 —

CASMC smooth muscle coronary artery + cyto

HISM smooth muscle intestine — 0 —

Decidua mesenchymal decidua + cyto NHAC-kn chondrocyte articular 0 cartilage NHOst osteoblast bone 0

HPAd preadipocyte fat 0 —

NHEK-Ad keratinocyte adult skin + cyto

NHMC mesangial kidney 0 —

HNPU-5 epithelial pancreas ~ 0 —

HNPU-6 epithelial pancreas — 0 —

RPTEC epithelial kidney ~ 0 —

HBEpC epithelial lungs ++ cyto + (nucleus)

HFDPC epithelial dermal papillae ++ cyto + nucleus

PrEC epithelial prostate -- 0 —

HSKMC myocyte skeletal muscle ~ 0 —

HHC-I cardiomyocyte- heart ~ 0 —

NHeps hepatocyte liver -- 0 —

HNH-I hepatocyte liver -- 0 ~

NHDF fibroblast dermis + cyto

PrSC stromal prostate — 0 —

HCF fibroblast heart — 0 —

HLFa fibroblast lung — 0 —

HTU-5 thyrocyte thyroid — 0 —

HTU-6 thyrocyte thyroid -- 0 —

HEM melanocyte Dermis — 0 --

NSC stem cell¹² fetal brain — 0 —

NHA astrocyte fetal brain — 0 —

Oligo oligodendrocyte¹³ fetal brain — 0 —

Neurons neurons¹⁴ fetal brain — 0 —

¹As determined by confocal microscopy and/or FACS: "--"no detectable uptake; "+", "++","+++", indicates <25%, <50%, and <75% of the cells showing detectable peptide uptake, and "++++" indicating an uptake in >80% of the cells;

² Defined as ratio incorporated peptide into the given cell type versus the optimal one of MPC;

³ Intracellular compartmentalization pattern of internalized tagged peptide, as determined by confocal laser microscopy, cyto = diffuse cytoplasmic distribution; nucleus = nuclear accumulation; includes MPC isolated from bone marrow aspirates from 12 healthy donors; and 5 batches of 4-7^Λ passage, i.e. 5-9 population doublings, MPC from Cambrex Corp;

Includes MPC collected from 4 non-Hodgkin's lymphoma patients undergoing bone marrow transplantation. includes MPC collected from 3 chronic lymphocytic B cell leukemia patients undergoing bone marrow transplantation.

Includes MPC collected from 5 multiple myeloma patients undergoing bone marrow transplantation; Separated by magnetic immunobead procedures using anti-CD34 antibodies from peripheral aphereses of myeloma and lymphoma patients undergoing autologous bone marrow transplantation;

'Separated by magnetic immunobead procedures using anti-CD20 antibodies from healthy donors; '"includes CD4⁺/CD8^", CD47CD8⁺, CD4⁺/CD8⁺ e CD47CD8^" T lymphocyte populations isolated by sequential magnetic immunobead separation using anti-CD4 and anti-CD8 antibodies from healthy and diseased donors; " Corresponds to the mononucleated cell populations depleted of surface adherent MPC in various bone marrow aspirates or peripheral blood smears from healthy donors; ¹²Neuropheres derived from fetal brain;

¹³ Refers to 04/CNPase-ρositive cells generated from neurospheres following appropriate induction

¹⁴ Refers to dopaminergic and GABAergic neurons generated from neurospheres following appropriate induction;

¹ HFDPC were derived from hair follicle dermal papillae.

¹⁶ The cells were isolated in the laboratory from healthy and diseased donors or obtained from Cambrex Corp., Cell Application Inc. (San Diego, CA) and kindly donated by Francesco Curcio (Department of Immunopathology, University of Udine, Italy). They were all utilized at the 3^-5* passage (except for cells that were induced to differentiate into specific phenotypes).

Example 5: HHSRSTL peptide derivatives (SEQ ID NO: D

Peptides with sequences alternative to that of the peptide HHSRSTL (SEQ ID NO: 1), concerning the amino acid composition as well as chemical modifications of the same amino acids, were produced by traditional peptide synthesis in accordance to methods well known to a person skilled in the art. Examples of these peptides are reported hereinafter. HHSRSTV (SEQ ID NO: 2) , AHSRSTL (SEQ ID NO: 3) ,

HHYRSTL (SEQ ID NO: 4) ; HHVRSTL (SEQ ID NO: 5) , HRSRSGL

(SEQ ID NO: 6) , AHVRSSL (SEQ ID NO: 7) , HHSRST (SEQ ID

NO: 8), HHSRSTLL (SEQ ID NO: 9) , HAcHSRSTL, HHSRSAcTL,

HHACSRSTACL, H (CH3)HS (CH3)RSTL, HHSRS (CH3)TL, HHCCJfSjSRSTrCHJjL; HHSRSTLHHSRSTL (SEQ ID NO: 10), H (solJfjHSRSTL, HHSRS (sulf)TL, HH (sulf)SRST (sulf)L; H (carb)HSRSTL, HHSRS (carb)TL, HH (carb)SRST (carb)L; dHHSRSTL, dHdHSRSTL, HHdSRdSTL, HHSRSTLC (SEQ ID NO: 10); CHHSRSTLC (SEQ ID NO:11) . Example 6 : Peptide complexes preparation

Complexes between the peptide HHSRSTL (SEQ ID NO: 1) and biotin, or between the peptide and the fluorescent taggers fluorescein and rodhamine, were produced by utilizing a spacer and in accordance with known methods. In another experiment the peptide was conjugated to fluorescent microspheres (FITC) of various sizes or alternatively bound to a reporter vector PNA-GFP (Gene Therapy Systems Inc.) . This comprises the gene coding for the GFP (Green Fluorescent Protein) and, once integrated within the target cell, causes GFP expression. These complexes were assayed for their internalization capability into MPC cells, dermal fibroblasts, endothelial HUVEC cells and quiescent MSC cells. The results are reported in Figure 3. Example 7: Modified MPC Cells

Modified MPC cells were produced by spontaneous incorporation in DMEM medium, without serum, of peptide complexes comprising fluoresceinated peptides, peptides associated to fluorescent microspheres, peptides tagged with nuclear tracers or peptides associated to the PNA- GFP vector described above. This vector was also utilized to introduce into the target cell cDNA sequences coding for the following molecules: follistatine, noggin, BMP-4, BMP-7, gremlin or interleukin-12. For this purpose, there were utilized MPC cells at the 3^rd - 12^th passage.

BIBLIOGRAPHY

Barry, M.A., Dower, W.J., and Johnston, S.A. (1996) , Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nature Med. 2, 299-304.

Chamberlain, J.R., Schwarze, U., Wang, P.R., Hirata, R.K., Hankenson, K.D., Pace, J.M. , Underwood, R.A. , Song, K.M., Sussman, M., Byers, P.H., and Russell, D.W. (2004) Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 303, 1198-201.

Clackson, T., and Lowman, H.B. (2004) Phage display. A practical approach. Oxford University Press.

Devlin, J.J., Panganiban, L.C, and Devlin, P.E. (1990) . Random peptide libraries: a source of specific protein binding molecules. Science 249, 404-406.

Krampera, M., Glennie, S., Dyson, J., Scott, D.,

Laylor, R., Simpson, E., and Dazzi, F. (2003) Bone marrow mesenchymal stem cells inhibit the response of naϊve and memory antigen-specific T cells to their cognate peptide. Blood 101, 3722-3729.

Larocca, D., Kassner, P.D., Witte, A., Ladner, R.C, Pierce, G.F., and Baird, A. (1999) Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage. FASEB J. 13, 727-734. Le Blanc, K. (2003) Immunomodulatory effects of fetal and adult mesenchymal stem cells. Cytotherapy 5, 485-489.

Matthew, J.M., Garcia-Morales, R.O., Carreno, M., Jin, Y., Fuller, L., Blomberg, B., Cirocco, B., Burke, G.W. , Ciancio, G., Ricordi, C, Esquenazi, V., Tzakis, A.G., and Miller, J. (2003) Immune responses and their regulations by donor bone marrow cells in clinical organ transplantation. Transpl. Immunol. 11, 307-321.

Nicklin, S.A., White, S.J., Watkins, S.J., Hawkins, R.E., and Baker, A.H. (2000) . Selective targeting of gene transfer to vascular endothelium cells by use of peptides isolated by phage display. Circulation 102, 231-238. Pasqualini R, and Ruoslahti E. 1996. Organ targeting in vivo using phage display peptide libraries. Nature 380:364-369.

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Claims

1. A method of selecting amino acid sequences having affinity for a target cell, by phage display technique, comprising steps of: incubating a phage library with target progenitor/stem cells of human or animal origin and isolating the phage fraction bound to said cells.

2. The method of selecting according to claim 1, wherein in one or more preliminary steps the phage library is sequentially incubated with one or more reagents selected from: cell types different from the target cell, solid supports used in the procedure and agents used for cell cultivation.

3. The method according to claim 1 or 2, wherein the target stem cells are cells selected from the group comprising: neural progenitor/stem cells deriving from fetal or adult central nervous system; progenitor/stem cells of mesenchymal/stromal type (MPC) residing in adult bone marrow; fetal and adult hematopoietic progenitor/stem cells CD34⁺ and CD34^"; umbilical cord progenitor/stem cells; adipose tissue progenitor/stem cells; mammary stroma progenitor/stem cells; prostate stroma progenitor/stem cells; gingival progenitor/stem cells; progenitor/stem cells of epithelial tissues of gastrointestinal tract, skin, respiratory tract or lung,- progenitor/stem cells residing in fetal and adult skeletal muscle; progenitor/stem cells residing in fetal and adult heart; progenitor/stem cells deriving from the placenta; progenitor/stem cells deriving from the decidua,- progenitor/stem cells circulating in the peripheral blood and lymphatic circle; progenitor/stem cells residing in the peripheral nervous tissue; progenitor/stem cells residing in the liver, pancreas, spleen, fetal and adult thymus.

4. The method according to any one of the claims 1 to 3, wherein the phage library is a random library of peptides having 5 to 20 amino acid residues.

5. A method of producing peptides having affinity for a target cell, comprising steps wherein peptides having amino acid sequence selected according to the method of any one of the claims 1 to 4 are reproduced by chemical synthesis or by expression in modified host cell.

6. A peptide obtainable by the method according to claim 5.

7. A peptide having binding affinity for MPC cells, selected from the group comprising the peptide of amino acid sequence HHSRSTL (SEQ ID NO:1) in linear or circular form and the mimitope derivatives thereof having capability of binding to and internalizing into the MPC cell equal to or greater than the 1% of the effectiveness shown by the peptide of sequence SEQ ID NO:1.

8. The peptide according to claim 7, selected from the group of linear, circular or branched peptides comprising: one or more copies of sequence SEQ ID N0:l; at least 5 consecutive amino acid residues of sequence SEQ ID N0:l; sequence SEQ ID NO:1 with substitutions of 1 to 5 amino acid residues; sequence SEQ ID N0:l with addition or elimination of at least one amino acid residue; sequence SEQ ID N0:l chemically modified on one or more amino acid residues; sequence SEQ ID NO:1 with isomerization of one or more amino acid residues; sequence SEQ ID NO:1 to which there has been added, with or without spacer, a non-amino acid residue.

9. A peptide complex, characterized in that it comprises the peptide according to any one of the claims 6 to 8 bound to a molecule selected from the group comprising synthetic molecules, protein molecules, hydrocarbons, lipids, or nucleic material.

10. The complex according to claim 9, wherein the peptide is bound to a reporter molecule selected from a radioactive molecule, a fluorescent molecule, a cromophore molecule, an enzyme, an immuno-partner, an affinity partner, a microsphere.

11. A diagnostic agent for selective recognition and traceability of stem cells, comprising the complex according to claim 10.

12. The complex according to claim 9, wherein the peptide is bound to a gene, a vector comprising said gene, a virus, a viral particle, a particle belonging to a virus, a molecule belonging to a virus, an element deriving from a virus, or a molecule produced by viral infection of prokaryote or eukaryote cells.

13. A progenitor/stem cell modified by bonding or incorporating the complex according to claim 9.

14. The progenitor/stem cell according to claim 13, characterized in that it is an MPC cell.

15. A progenitor/stem cell tagged by bonding or incorporating the complex according to claim 10.

16. A progenitor/stem cell carrying a substance having therapeutic action immobilized or incorporated by the complex according to claim 9.

17. A progenitor/stem cell modified by the genetic material incorporated by the complex according to claim

12.

18. A method of genetically modifying an MPC-type progenitor/stem cell, comprising a step wherein the ex vivo or in vivo stem cell is treated with a complex according to claim 12 for a time sufficient to allow the intracellular incorporation of the complex.

19. A method of isolating, selecting or enriching MPC-type stem cells comprising steps wherein aphereses of peripheral blood, lymphatic samplings, bone marrow aspirates, lymph node aspirates, synovial fluids, follicular fluids or other samplings containing body fluids, or biopsies, or tissues or organs excised from alive or deceased subjects, healthy or diseased at the time of sampling or excising, are treated with complexes according to claim 9.

20. MPC-type stem cell modified ex vivo or in vivo according to any one of the claims 13 to 17 for use in a therapeutic method of:

(I) autologous transplantation of MPC cells alone, MPC cells cotransplanted with other stem cells, non-stem cells deriving from healthy or diseased, alive' or deceased subjects, or animal cells;

(II) allogenic or mini-allogenic transplantation' as per subclaim;

(III) autologous or allogenic transplantation of tissues or organs deriving from healthy or diseased subjects or animals, wherein MPC cells are cotransplanted or separately infused.

21. An MPC-type stem cell modified ex vivo or in vivo according to any one of the claims 13 to 17 for use in a therapeutic treatment aimed at:

(I) fostering reconstruction and/or regeneration in case of accident traumas, wearing out, degenerative hereditary diseases including autoimmune ones, tumors of hematopoietic, bone, cartilaginous, nervous, vascular or cutaneous tissues, or organ reconstruction/regeneration.

(II) gene therapy aimed at correcting hereditary or acquired genetic deficiencies, curing autoimmune, cardiovascular, neurologic, degenerative or viral pathologies, and antitumoral gene therapy; (III) antitumoral therapy.