WO2011060393A1 - Antimicrobial, antiviral, anticancer and immunomodulatory peptides and uses therefore - Google Patents

Abstract

Polypeptides derived from constant domains of antibody light (L) and/or heavy (H) chains as well as from complementary determining regions (CDRs) of immunoglobulin variable regions are disclosed possessing broad spectrum biological activities including, among others, antifungal, antibacterial, antiviral, anticancer and/or immunomodulatory activity in vitro, ex vivo and/or in vivo.

Description

ANTIMICROBIAL, ANTIVIRAL, ANTICANCER AND IMMUNOMODULATORY

PEPTIDES AND USES THEREFORE

SEQUENCE LISTING

[001] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 15, 2010, is named RAS1001P.txt and is 3,049 bytes in size.

FIELD OF THE INVENTION

[002] This invention relates to the field of immunology, particularly to peptides derived from antibody light (L) and heavy (H) chain amino acid sequences, and derivatives thereof, that are active in immunologically related processes. More specifically, this invention relates to the use of such peptides possessing antibacterial, antifungal, antiviral, antitumor and/or immunomodulatory activities in hosts to which said peptides are introduced, for treating fungal, viral, and bacterial infections and immunologic and cancerous disorders in mammals including humans, farm and other domesticated and/or zoo-kept animals.

BACKGROUND OF THE INVENTION

[003] The following information includes subject matter that may be useful in understanding the present invention. It is not an admission that any such information is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[004] Immunoglobulins (Igs) otherwise commonly referred to as antibodies (Abs) are composed of two identical sized L chains (23kD) and two identical H chains which range in size, (i.e., between 50-70kD). The chains are connected by inter-chain disulfide bonds; the H and L chains and the two H chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds varies among different Ig molecules. There are also intra-chain disulfide bonds within each of the polypeptide chains. Figure 1 depicts, schematically, generally accepted aspects of Ab L and H chain construction. As can be understood by one of skill in the art, each of the H and L chains comprise a "constant" (C) region and a "variable" (V) region. The C region of the H chains makes up the greater portion of the amino acid sequence making up the Ab. The H chain C region is broken up into recognized domains labeled CHI -4. L chains only have one C region domain labeled CL. Igs also have a "hinge" region. This is the region at which the arms of the Ab molecule form a Y. It is called the hinge region because there is some flexibility in the molecule at this region.

[005] When the amino acid sequences of many different H chains and L chains were compared, it became clear that both the H and L chain could be divided into two regions based on variability in the amino acid sequences and each L and H chain comprised a V and a C region, as stated above, i.e., 1. L chain - V_L (110 amino acids) and C_L (110 amino acids), 2. H chain - V_H (110 amino acids) and C_H (330-440 amino acids). Igs are further structured in that they are folded into globular regions each of which contains an intra- chain disulfide bond (Figure 1). These regions are the earlier referenced domains (Fig. 1). Specifically, L chain domain, i.e., V_L and C_L, and the H chain domains - V_H, C_HI - C_H3 (or C_H4). Additionally, carbohydrates in the form of oligosaccharides are attached to the C_H2 domain in most Igs. However, in some cases carbohydrates may also be attached at other locations.

[006] Comparisons of the amino acid sequences of the V regions of Igs show that most of the variability resides in three regions called the hypervariable regions (HVRs) or the complementarity determining regions (CDRs). CDRs are found in both V_H and V_L. Finally, the regions between the CDRs in the V region are called the framework regions.

[007] Various amino acid sequence segments of Ab V sequences of L and H chains, specifically CDRs or CDR-related peptides, can possess antimicrobial (i.e., antibacterial and/or antifungal, whereby by antifungal is meant antiyeast and/or antimold), antiviral, anticancer and/or immunomodulatory activities that may be beneficial against certain fungal, bacterial and viral infections and immunological or cancer disorders (Polonelli et al., 2003, Cenci et al., 2006, Polonelli et al., 2008). The fact that amino acid sequences comprising less than the whole Ab, normally consisting of a complete H and L chains, can possess antimicrobial, antiviral anticancer and/or immunomodulatory activities is surprising and suggests that beyond the half life of a typical Ig, fragments of the whole Ig molecule may have biological actions such as effectively influence the antiinfective and anticancer cellular immune response in a way reminiscent of regulatory peptides of innate immunity.

[008] The finding that subsets of amino acid sequences within the V regions of the L and H chains have anti-infective activity, though nonetheless surprising, is comprehendible when considering the multiplicity of variation in amino acid sequence capable within the V region. A partly digested Ab could comprise just such a region that otherwise was specific for a family of microorganisms. However, it is not intuitive that the C region amino acid segments could harbor such activities as it is the conventional wisdom that the C regions merely play a structural role in presenting the active site (i.e., V region) to the antigen as well as provide attachment to cell membranes and effectors such as complement and receptors on immune effector cells termed Fc receptors. Given the ongoing need to advance the medical sciences, we disclose herein a new class of polypeptide within the CDR-derived peptide family that exhibits immune modulating activities. We further disclose the surprising and novel finding of a class of polypeptide derived from, exclusively, C region sections of Abs , whether L or H chain exhibiting any of antibacterial, antifungal, antiyeast, antimold, antiviral, anticancer and immune modulatory activities against a wide spectrum of microbial agents and tumor and immunological disorders.

[009] Additionally, it is well established that many invasive bacterial, and fungal and viral infections are difficult to treat. To date there have been some advancement in the development of antibacterial, antifungal, and antiviral agents for use in treatment regimens.

[010] Yet, over the last 3 decades, there has been a rise in the prevalence of opportunistic fungal infections concomitantly with an increase in the frequency of solid- organ and hematopoietic stem cell transplants (HSCTs), more aggressive chemotherapy, the AIDS epidemic, and advances in critical care. While Candida spp. and Aspergillus spp. remain the most common causes of invasive fungal infections (IFIs) in immune- compromised hosts, infections due to other fungi are seen with increased frequency (Arendrup, 2009; Erjavec et al, 2009; Zilberberg and Shorr, 2009). IFIs have historically been associated with high morbidity and mortality, partly because of the limitations of available antifungal therapies and difficulties in making a rapid and accurate diagnosis (Sable et al, 2008). In addition to being a growing clinical challenge, drug resistant and multidrug-resistant human pathogenic fungi are also neglected potential bioterrorism agents (Casadevall and Pirofski, 2006). In particular, human pathogenic fungi are easily obtainable from the environment, highly dispersible and can cause significant disease after inhalation with relatively low inocula (Casadevall and Pirofski, 2006).

[011] Since the late 1950s, the standard of care for treatment of serious fungal infections had been amphotericin B, an intravenous (IV)-only agent with significant toxicity (Kauffman and Carver, 2008; Comely et al, 2009; Moen et al, 2009; Rogers and Frost, 2009). The 1990s saw the introduction of lipid formulations of amphotericin B, as well as the triazoles fluconazole and itraconazole (Fera et al., 2009; Moen et al., 2009; Rogers and Frost, 2009). Although these agents offered clear advantages over amphotericin B, they were limited by formulation, spectrum of activity, and/or the development of resistance. In the past decade, there have been major advances in therapy. Broader- spectrum triazoles (voriconazole and posaconazole) and the new echinocandin class of antifungals (caspofungin, micafungin, and anidulafungin) have been introduced in the current decade, and noninvasive diagnostic methods have improved (Fera et al, 2009; Gergis et al, 2009; Rogers and Frost, 2009).

[012] Triazole antifungal drugs, such as fluconazole, voriconazole, itraconazole, and posaconazole, owe their success to better clinical safety profiles than the single effective fungicidal agent, amphotericin B, that was available before their introduction. However, amphotericin B formulations are still employed for clinically challenging infections like deep candidal infections. Resistance to azoles is emerging in species that were previously susceptible, in particular Candida albicans. Echinocandins competitively inhibit the synthesis of an essential cell wall component, the 1,3-P-glucan, of Candida spp. and Aspergillus spp., but are generally inactive against other fungi, such as Cryptococcus neoformans (Rogers and Frost, 2009). All agents of this class are of parenteral formulations, with no oral preparations available. Reduced sensitivity to echinocandins, although uncommon, can result from mutations or overexpression of their target, 1,3-β- glucan synthases (Cappelletty and Eiselstein-McKitrick, 2007; Kauffman and Carver, 2008; Walker et al, 2008). [013] Although several antifungals have been licensed in the last 5 years, some patients remain difficult to treat. In particular IFI in immunocompromised patients, such as HSCT recipients and patients with acute leukemia during periods of profound neutropenia, are an increasingly common cause of mortality (Arendrup, 2009; Erjavec et al., 2009; Zilberberg and Shorr, 2009).

[014] Thus, there is a need for new antifungal agents with a broad spectrum of activity, limited resistance potential, favorable safety profile and limited drug interactions. The main reasons for this need include intrinsic or acquired antifungal resistance, toxicity of existing agents with broad spectrum of activity, limited spectrum of activity of some of the safest available compounds, organ dysfunction preventing the use of some existing agents, and drug interactions of existing agents. To address this unmet clinical need, the present invention provides a heretofore untold novel class of polypeptides derived from both CDR and C regions of Igs, said peptides possessing a variety of activities including antifungal, antiyeast, antimold, antibacterial, antiviral, anticancer as well as immunomodulatory by direct action of the peptides, all the above of which activities are directly amenable to their respective effective use in treatment regimens.

SUMMARY OF THE INVENTION

[015] In a first embodiment, the invention comprises peptides derived from either the L or H chains of Igs. i.e., Abs. In a particularly preferred embodiment, invention peptides are derived from the C regions of the L and H chains of any class of Igs. In this aspect, C regions are meant to include C_H regions 1, 2, 3, and 4, the C_L region and the hinges, as such regions are well understood to those of ordinary skill in the arts. The discovery by the present inventors that peptide segments of the C regions possess antibacterial, antifungal, antiyeast, antimold, antiviral, anticancer and or immunomodulatory activities is novel as prior understanding of the skilled artisan is that only the V regions of L and H chains, and in particular the CDR-related segments thereof, possessed such activities.

[016] In a second embodiment, peptides of the present invention possess a broad spectrum of activity, whether CDR-related or strictly non-CDR C region-derived amino acid sequence, comprising any or all of antimicrobial, antibacterial, antifungal, antiyeast, antimold, antiviral, anticancer and immunomodulatory activity, regardless of their origin in either C_L or C_H or, with respect to CDR-related peptides, V_L or V_H regions, exclusive of specific known amino acid sequences derived from any of said origins with known antimicrobial, antiviral or anticancer activity.

[017] In a third embodiment, individual peptides of the invention exhibit alone or in combination any of antimicrobial, antibacterial, antifungal, antiyeast, antimold, antiviral, anticancer and immunomodulatory activity in vivo in a host mammal. In a related embodiment, the antimicrobial, antibacterial, antifungal, antiyeast, antimold, antiviral, anticancer and immunomodulatory activity of the peptides is unrelated to any activity of the complete Ig from which they are derived.

[018] In another embodiment, the peptides can be naturally occurring peptides derived from L or H chains of Igs or synthetic derivatives thereof wherein one or more amino acids of the peptide sequences are substituted with other amino acids such as alanine, for example. In a related embodiment, the peptides of the invention are generally between 4 and 20 amino acids in length, more usually between 4 and 16 amino acids in length, and even more typically between 4 and 12 amino acids in length. Most commonly the peptides are between 4 and 10 or 4 and 11 amino acids in length. Further, the amino acid sequences may possess a beta sheet secondary structure. In additional embodiments, the polypeptide sequences of the invention of identifying, isolating, and using polypeptide sequences derived exclusively from C domains of Igs can include sequences not only of 4 to 20 amino acids in length, but also of such lengths wherein there is at least 90% sequence identity with the native isolated sequence. Thus, for every 10 amino acids in such a C region derived polypeptide, one amino acid can be substituted for an amino acid not naturally found in that sequence location without negatively impacting the efficacy of said peptide's antimicrobial, antiviral immunomodulatory or anticancer activities. In a related embodiment, peptides of the invention derived from the CDR domaine, identified by Seq. Id. Nos. 5-13 possess 90% sequence identity with one another and with Seq. Id. No 4, and further, all of these (i.e., Seq. Id. Nos. 4-13) possess a similar activity against a target, here immune cells.

[019] In another embodiment, the peptide having antimicrobial, antibacterial, antifungal, antiyeast, antimold, antiviral, anticancer, and/or immunomodulatory activity can be used in therapeutic regimens by topical and/or systemic administration. In such administration, topical application can comprise cream and ointment bases including solvents, salts and absorbents as are well known in the arts. Other active and inert reagents can be included as appropriate for the healing arts.

[020] Further still, in yet another embodiment, certain peptides of the disclosure are CDR-derived peptides that induce a protective anticandidal cellular immune response exclusively through an immunomodulatory activity despite of their not possessing any direct candidacidal activity themselves. In a particularly preferred embodiment, the immunomodulatory activity was demonstrated by activities of synthetic peptides derived from the CDRs of a mouse monoclonal Ab (MoA) specific for the difucosyl human blood group A (Gabrielli et al, 2009).

BRIEF DESCRIPTION OF THE DRAWINGS

[021] The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings are provided to the Patent and Trademark Office with payment of the necessary fee.

[022] Figure 1 shows the Ab domain structure as is well known in the arts, namely, the two identical H chains comprising 3 to 4 C region domains (CHI -4), a V region (VH), itself comprising CDRs, and a hinge region as appropriately labeled. Also, the Ab has L chains comprising a C region (CL) and a V region (VL).

[023] Figures 2A, B, and C show three basic classes of Ig molecules, namely IgG (Fig. 2A), IgM (Fig. 2B) and IgA (Fig. 2C), each represented in the form of a linear amino acid numbered bar chart, the V end region of the Ig amino acid sequence to the left and C region to the right. Each chart further indicates sections of C region super families making up parts of the C region domains. Each class of Ig, IgG, IgM, or IgA, is disclosed to have peptides, with antimicrobial, antiviral, anticancer and immunomodulatory activities, of the invention, each such peptide, N10K, T11F, and H4L , for example, as further described below, located in one or another portion of the Ig's C region.

[024] Figure 3 shows the in vitro activity of N10K against C. albicans SC5314 strain. Amount of N10K is in micrograms/ml. [025] Figures 4A and B show the in vivo activity of N10K against systemic candidiasis. In Fig. 4A the survival rate of subject mice is depicted. In Fig. 4B the yeast recovery rate per kidney is graphed.

[026] Figure 5 shows the in vitro activity of N10K against caspofungin resistant C. albicans strain UM4. Amount of N10K is in micrograms/ml.

[027] Figure 6 shows the in vitro activity of N10K against a caspofungin-resistant Saccharomyces cerevisiae strain YGR032W. Amount of N10K is in micrograms/ml.

[028] Figure 7 shows the in vitro activity of N10K against C. neoformans 6995 strain. Amount of N10K is in micrograms/ml.

[029] Figure 8 shows the in vitro activity of N10K against Malassezia furfur 101 strain. Amount of N10K is in micrograms/ml.

[030] Figure 9 shows the in vitro activity of N10K against Aspergillus fumigatus 1163 strain. Amount of N10K is in micrograms/ml.

[031] Figure 10 shows the in vitro activity of N10K against Staphylococcus aureus 29213 strain. Amount of N10K is in micrograms/ml.

[032] Figure 11 shows the in vitro activity of N10K against Escherichia coli ATCC 25922 strain. Amount of N10K is in micrograms/ml.

[033] Figure 12 shows the in vitro activity of N10K against Klebsiella pneumoniae ATCC 700603 strain. Amount of N10K is in micrograms/ml.

[034] Figure 13 shows the in vitro activity of N10K against Pseudomonas aeruginosa ATCC 25853 strain. Amount of N10K is in micrograms/ml.

[035] Figures 14A and B show the in vitro activity of N10K against HIV IIIB (X4). In Fig. 14A the antiviral activity is depicted wherein the peptide concentration was 10 micrograms/ml administered every 4 days post viral infection. Fig. 14B is a graph showing that where cells were preincubated with N10K at the concentration of 10 micrograms/ml, a lesser antiviral activity is still present. Y axis shows concentration of HIV p24 (pg/ml). [036] Figure 15 shows the in vitro activity of NIOK against HIV BaL (R5). The antiviral activity is depicted wherein the peptide concentration was 10 micrograms/ml administered every 4 days post viral infection. Y axis shows concentration of HIV p24 (pg/ml).

[037] Figure 16 is a graph showing the in vitro activity of N10K against B16F10-Nex2 melanoma cells. Specifically, as the concentration of N10K increases (mM), the viability of the cancer cells decreases.

[038] Figure 17 shows the expression of Dectin-1 by monocytes stimulated by N10K. NC is negative control, NS is absence of heat inactivated (h.i.) CA-6. N10K at 10 microgram/ml.

[039] Figures 18 A, B, and C show the phagocytosis of non opsonised C. albicans CA-6. Fig. 18A is a graph of % of phagocytic cells. Fig. 18B shows FACS indicating the medium number of yeast particles adhered or ingested by each monocyte. Fig. 18C is a Table showing the mean number of attached and ingested C. albicans.

[040] Figures 19A and B show the production of cytokines IL-12p40 (Fig. 19A) and IL-6 (Fig. 19B) in human PBMCs stimulated by N10K (10 micrograms/ml).

[041] Figure 20 shows the in vitro activity of T11F against C. albicans SC5314 strain. Amount of Tl IF is in micrograms/ml.

[042] Figure 21 shows the in vitro activity of T11F against caspofungin resistant C. albicans strain UM4. Amount of Tl IF is in micrograms/ml.

[043] Figure 22 shows the in vitro activity of T11F against a caspofungin-resistant S. cerevisiae strain YGR032W. Amount of Tl IF is in micrograms/ml.

[044] Figure 23 shows the in vitro activity of T11F against C. neoformans 6995 strain. Amount of Tl IF is in micrograms/ml.

[045] Figure 24 shows the in vitro activity of Tl IF against M.furfur 101 strain. Amount of Tl IF is in micrograms/ml.

[046] Figure 25 shows the in vitro activity of T11F against A. fumigatus 1163 strain. Amount of Tl IF is in micrograms/ml. [047] Figure 26 shows the in vitro activity of Tl lF against S. aureus 29213 strain. Amount of Tl IF is in micrograms/ml.

[048] Figure 27 shows the in vitro activity of Tl lF against E. coli ATCC 25922 strain. Amount of Tl IF is in micrograms/ml.

[049] Figure 28 shows the in vitro activity of Tl lF against K. pneumoniae ATCC 700603 strain. Amount of Tl IF is in micrograms/ml.

[050] Figure 29 shows the in vitro activity of Tl IF against P. aeruginosa ATCC 25853 strain. Amount of Tl IF is in micrograms/ml.

[051] Figure 30 shows the in vitro activity of Tl IF against HIV BaL (R5). The antiviral activity is depicted wherein the peptide concentration was 10 micrograms/ml administered every 4 days post viral infection. Y axis shows concentration of HIV p24 (pg/ml).

[052] Figure 31 shows the in vitro activity of Tl lF against B16F10-Nex2 melanoma cells. (mM concentration directly associated with decrease in cancer cell viability).

[053] Figure 32 shows the in vitro activity of H4L against C. albicans SC5314 strain. Amount of H4L is in micrograms/ml.

[054] Figure 33 shows the in vitro activity of H4L against C. neoformans 6995 strain. Amount of H4L is in micrograms/ml.

[055] Figure 34 shows the in vitro activity of H4L against M. furfur 101 strain. Amount of H4L is in micrograms/ml.

[056] Figure 35 shows the in vitro activity of H4L against A. fumigatus 1163 strain. Amount of H4L is in micrograms/ml.

[057] Figure 36 shows the in vitro activity of H4L against S. aureus 29213 strain. Amount of H4L is in micrograms/ml.

[058] Figure 37 shows the in vitro activity of H4L against E. coli ATCC 25922 strain. Amount of H4L is in micrograms/ml.

[059] Figure 38 shows the in vitro activity of H4L against K. pneumoniae ATCC 700603 strain. Amount of H4L is in micrograms/ml. [060] Figure 39 shows the in vitro activity of H4L against P. aeruginosa ATCC 25853 strain. Amount of H4L is in micrograms/ml.

[061] Figure 40 shows the in vitro activity of H4L against HIV BaL (R5). The antiviral activity is depicted wherein the peptide concentration was 10 micrograms/ml administered every 4 days post viral infection. Yaxis shows concentration of HIV p24 (pg/ml).

[062] Figure 41 shows the in vitro activity of MoA V_HCDRH₃ against M. furfur 101 strain. Amount of MoA V_HCDR₃ is in micrograms/ml.

[063] Figure 42 shows the in vitro activity of MoA V_HCDRH₃ against A. fumigatus 1163 strain. Amount of MoA V_HCDR₃ is in micrograms/ml.

[064] Figure 43 shows the in vitro activity of MoA V_HCDRH₃ against S. aureus 29213 strain. Amount of MoA V_HCDR₃ is in micrograms/ml.

[065] Figure 44 shows the in vitro activity of MoA V_HCDRH₃ against E. coli ATCC 25922 strain. Amount of MoA V_HCDR₃ is in micrograms/ml.

[066] Figure 45 shows the in vitro activity of MoA V_HCDRH₃ against K. pneumoniae ATCC 700603 strain. Amount of MoA V_HCDR₃ is in micrograms/ml.

[066] Figure 46 shows the in vitro activity of MoA V_HCDRH₃ against P. aeruginosa ATCC 25853 strain. Amount of MoA V_RCDR₃ is in micrograms/ml.

[068] Figure 47 shows TNF-a production by peritoneal murine macrophages (PM) stimulated with MoA CDRs.

[069] Figure 48 shows IL-6 production by PM stimulated with MoA CDRs.

[070] Figure 49 shows TNF-a production by peritoneal murine neutrophils (PMN) stimulated with MoA CDRs.

[071 ] Figure 50 shows IL-6 production by PMN stimulated with MoA CDRs.

[072] Figure 51 shows MoA V_HCDR₃ uptake by different cell populations, namely dendritic, PM, PMN, and T cells.

[073] Figures 52A and B shows the kinetics of biotin-labelled MoA V_HCDR uptake by PM. In Fig. 52A data are reported as the mean fluorescence intensity (MFI) (upper panel) and percentage of positive cells (lower panel). *, P<0.05 (b-VHCDR3 -treated vs untreated cells, n = 7). In Fig. 52B shows the uptake of b-VHCDR3 by PM by fluorescent microscopy.

[074] Figure 53 shows Phospho-Akt activation in murine macrophages stimulated with MoA V_HCDR₃. After incubation of murine macrophages for 1 hr in the presence or absence (NS) of MoA V_HCDR₃, LPS, negative control (NC) or irrelevant peptide (SP) (all at 10 mg/ml), cell lysates were subjected to Western blotting. Membranes were incubated with Abs to pAkt and Akt; pAkt was normalized against Akt (A) *, P<0.05 (treated vs untreated cells, n =5).

[075] Figure 54 shows the production of TNF-a in murine macrophages stimulated with MoA V_RCDR₃. After incubation of murine macrophages for 1 hr in the presence or absence (NS) of MoA V_HCDR₃, LPS, negative control (NC) or irrelevant peptide (SP) (all at 10 mg/ml), TNF-alpha level was evaluated in culture supernatants by specific ELISA assays; *, P<0.05 (treated vs untreated cells, n = 5).

[076] Figure 55 shows phospho-IkBa activation in PM stimulated with MoA V_HCDR₃. Murine macrophages were incubated for 1 hr in the presence or absence (NS) of MoA V_HCDR₃, LPS, negative control (NC) or irrelevant peptide (SP) (all at 10 mg/ml) with or without wortmannin. After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to plkBa and IkBa; plkBa was normalized against IkBa. *, P<0.05 (treated vs untreated cells, n = 5);+, P<0.05 (wortmannin-treated vs wortmannin-untreated cells, n = 5).

[077] Figure 56 shows TNF-a gene expression in PM stimulated with MoA V_HCDR₃. Murine macrophages were cultured for 1, 6 and 18 hr as above described. After incubation, total RNA was isolated and analyzed for mRNA expression with RT-PCR. Transcript copy numbers were determined by qPCR. Copy numbers were normalized against the copy number of the GADPH gene (B). *, P<0.05 (treated vs untreated cells, n = 5).

[078] Figures 57A, B, and C show the expression of TLR-4 in PM stimulated with MoA V_HCDR₃. Specifically, Fig. 57A shows a graph depicting expression of TLR-4 in murine macrophages stimulated with MoA V_HCDR₃ for 1 and 6 hr in the presence or absence (NS) of MoA V_HCDR₃, LPS or negative control peptide (NC) (all 10 mg/ml). After incubation of murine macrophages, total RNA was isolated and analyzed for mRNA expression with RT-PCR. Transcript copy numbers normalized against the copy number of the GADPH. *, P<0.05 (VHCDR3 treated vs untreated cells, n = 5). Figure 57B shows induction of TLR4 by MoA V_HCDR₃. After incubation of murine macrophages, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to TLR- 4 and actin. TLR-4 production was normalized against actin. *, P>0.05 (MoA V_HCDR₃- treated vs untreated cells, n = 5). Error bars, s.e.m. Figure 57C shown expression of TLR- 4 in murine macrophages stimulated with MoA V_RCDR₃ by FACScan flow cytometry. After incubation, permeabilized cells were reacted with RPE-labelled mAb to TLR-4 and analyzed by FACScan flow cytometry. Values represent the percentage of positive cells.

[079] Figures 58A, B, C and D show the TNF-a induced TLR-4 expression in PM stimulated with MoA V_HCDR₃. Specifically, Figure 58A shows TNF-a induction in murine macrophages in the presence or absence (NS) of MoA V_HCDR₃ , VHCDR3, LPS or NC (all 10 mg/ml) for 1 hr. TNF- a level was evaluated in culture supernatants by specific ELISA assay. *, P<0.05 (treated vs untreated cells, n = 7). Figure 58B shows TNF-a induced TLR-4 expression in PM stimulated with MoA V_HCDR₃ by FACScan flow cytometry. Murine macrophages were cultured as in Figure 61 A and were then permeabilized cells and reacted with RPE-labeled mAb to TLR-4 and analyzed by FACScan flow cytometry. P<0.05 (VHCDR3 plus mAb to TNF-a treated vs VHCDR3 treated cells, n = 7). Figures 58C shows TNF-a induced TLR-4 expression by western blot in PM stimulated with MoA V_HCDR₃ as in Figure 58A. After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to TLR-4 and actin. TLR-4 production was normalized against actin (C) *, P<0.05 (VHCDR3 plus mAb to TNF-a treated vs VHCDR3 treated cells, n = 5). Figure 58D shows the expression level of TLR-4 gene by RT-PCR in murine macrophages in PM stimulated with MoA V_HCDR₃ as in Figure 58A. Copy numbers were normalized against the copy number of the GADPH gene (D). *, P<0.05 (VHCDR3 plus mAb to TNF-a treated vs VHCDR3 treated cells, n = 5). Error bars,s.e.m.

[080] Figures 59A and B are graphs showing the in vivo activity of MoA V_HCDR₃ against systemic candidiasis. In Fig. 59A, the survival rate of subject mice is depicted. In Fig. 59B, the yeast recovery rate per kidney is graphed. [081] Figure 60 is a pictoral graph depicting the mechanism of TLR-4 upregulation induced in PM by MoA V_HCDR₃.

DETAILED DESCRIPTION OF THE INVENTION

[082] In a first embodiment, polypeptides are disclosed that exhibit a variety of activities comprising 1.) antimicrobial, which includes antibacterial, antifungal, (antifungal includes antimold and antiyeast); 2.) antiviral; 3.) anticancer; and 4.) immunomodulatory activities. In a related and preferred embodiment, the peptides are derived from C regions of Ig molecules. Of particularly preferred embodiment, the subject peptides can be found and identified in each of at least three different classes of Igs, namely, IgG, IgM, and IgA as shown in Table I below. Specifically, as disclosed in Table I and in Figure 2, peptide N10K is derived from a C region of the IgG class Ig , peptide Tl IF is derived from a C region of the IgM class Ig, peptide H4L is derived from a C region of each of the IgG, IgA, and IgM class Igs, and the MoA VHCDR3, is derived from a CDR region of a murine mAb directed to difucosyl human blood group A substance.

TABLE I

[083] In a further preferred embodiment, the disclosed polypeptide sequences, possessing a broad spectrum of antimicrobial (including antibacterial and antifungal, which includes antiyeast and antimold activities), antiviral, anticancer, and/or immunomodulatory activities, manifest their activities either by direct "cidal" action against the bacterium or fungus (mold or yeast) or through inhibition of virus replication or cancer cell growth or indirect action by immunomodulatory therapeutic action in the host mammal.

[084] In a preferred embodiment, we disclose peptides derived from the C region of three Ig families, specifically, IgM, IgA, and IgG, each with at least one of antimicrobial, antiviral, anticancer and/or immunomodulatory activities, and a new class of peptides from a CDR region manifesting immunomodulatory activity.

[085] In a further embodiment, the selection of peptides of the C region has been made according to Databank PIR "Protein Information Resource": http://pir.georgetown.edu/. For the research of the sequence the following analyses has been performed by using the relative software: BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi); multiple alignment with ClustalW (http://www.ebi.ac.uk/Tools/clustalw/); peptide cutter (http://expasy.org/tools/peptidecutter/) with cathepsins, trypsins and/or chymotrypsin- high specificity; calculation of Isoelectric Point (pi) with The Sequence Manipulation Suite 2 (http://www.bioinformatics.org/sms2/index.html). Based on the previous experience acquired in the study of biologically active CDR-related peptides, the definition of peptides of interest in the C region within each Ig class (Figure 2) has been made according to: peptide length, proteolytic enzymes involved, percentage of cut, conserved amino acids (human Ig/different organisms), pi, and alternation of hydrophobic/hydrophilic residues in the sequence. The sequences, same or similar to those fragments which would be obtained upon digestion, might support the hypothesis that, beyond the half life of a typical Ig, fragments of the whole molecule may effectively influence the antiinfective and anticancer cellular immune response in a way reminiscent of regulatory peptides of innate immunity.

[086] In a preferred embodiment, the CDR selections were made from the V region of a mouse mAb (IgM) specific for the difucosyl human blood group A substance (MoA) on the basis of the previously described sequences of VH and VL chain (Nickerson, 1995). Additionally, peptides of the invention include alanine-scanning variants peptides of the parent mouse MoA V_HCDR₃ peptide. [087] Table II shows the sequences of the peptides of the present invention.

Table II

[088] In still a further embodiment, selected peptides of the C and V regions have been chemically synthesized and verified to possess antimicrobial, antiviral, anticancer and/or immunomodulatory activity. Moreover, recent data from CDR derived peptides, not here presented, implies a beta (β) sheet secondary structure of at least some of the C region peptides of interest. Such a structure may be common to immune competent C domain derived peptides. Moreover, we find that as exampled by the alanine substituted peptides Seq Id. Nos. 5-13, these peptides can still manifest activity with at least a 10% change in their respective amino acid sequences. Thus, we conceive and comprehend the claimed invention to include amino acid sequences possessing at least 90% sequence identity with polypeptides derived from C and CDR regions of Igs possessing any of antibacterial, antifungal, antiyeast, antimold, anticancer, antiviral, and immunomodulatory activities. As shown in Table III below, the broad spectrum of activity is disclosed by the variety of pathogenic bacteria, fungi, virus, and cancer cells.

Table III

Inventio Assay type Target Genus and Reference Activity n Peptide species strain

In vitro Fungus S. cerevisiae YGR032W cidal

(caspofungin

resistant)

In vitro Fungus C. neoformans 6995 cidal

In vitro Fungus M. furfur 101 cidal

In vitro Fungus A. fumigatus 1163 cidal

In vitro Bacterium S. aureus 29213 cidal

In vitro Bacterium E. coli ATCC25922 cidal

In vitro Bacterium K. pneumoniae ATCC700603 cidal

In vitro Bacterium P. aeruginosa ATCC25853 cidal

In vitro Virus HIV-1 inhibitory

In vitro Cancer B16F10-Nex2 inhibitory cells melanoma cell

Cancer SKmel28 inhibitory cells melanoma cell

Cancer SKmel25 inhibitory cells melanoma cell

H4L In vitro Fungus C. albicans SC5314 cidal

In vitro Fungus C. neoformans 6995 cidal

In vitro Fungus M. furfur 101 cidal

In vitro Fungus A. fumigatus 1163 cidal

In vitro Bacterium S. aureus 29213 cidal

In vitro Bacterium E. coli ATCC25922 cidal

In vitro Bacterium K. pneumonia ATCC700603 cidal

In vitro Bacterium P. aeruginosa ATCC25853 cidal

In vitro Virus HIV-1 inhibitory

MOAV_RC In vitro Fungus M. furfur 101 cidal DR₃

In vitro Fungus A. fumigates 1163 cidal

In vitro Bacterium S. aureus 29213 cidal

In vitro Bacterium E. coli ATCC25922 cidal

In vitro Bacterium K. pneumonia ATCC700603 cidal

In vitro Bacterium P. aeruginosa ATCC25853 cidal

In vitro Immune Murine Stimulation cells macrophages of TNF-a,

IL-6

In vivo Fungus C. albicans CA-6 Indirectly cidal up regulation of TNF-a, TLR-4 [089] Activity Data for Invention peptides is presented below in the following

Experimental sections

I. Data for NIOK peptide

[090] In vitro activity of N10K against C. albicans SC5314 strain. The candidacidal activity of N10K peptide against C. albicans was assessed by a conventional colony forming unit (CFU) assay as previously described (Polonelli et al, 2003; Manfredi et al, 2005). Briefly, cells of C. albicans SC5314 were incubated at 37°C for 6 hours (hr) in the presence of N10K at the concentration indicated of 20, 12.5 or 6.25 μg/ml, or in distilled water as control (C). After incubation, cell suspensions were plated on Sabouraud dextrose agar and incubated at 30°C for 48 hours when CFU were counted (** P<0.01 for N10K treated vs. untreated cells, by t test). As disclosed in Figure 3, peptide N10K showed candidacidal activity in vitro against cells of C. albicans SC5314 strain. Based on several independent replications, an EC₅₀ of 10.04 x 10^"6 mol/liter (95% confidence intervals 9.209-10.956 x 10^"6) was determined.

[091] In vivo activity of N10K against systemic candidiasis caused in immunocompetent mice by the highly virulent C. albicans CA-6 strain. The anticandidal therapeutic activity of N10K was evaluated in a murine model of systemic candidiasis. Groups of 8 Balb/c mice were infected intravenously with 2>< 10⁶ cells of the highly virulent strain C. albicans CA-6 and given 50μg of peptide N10K intraperitoneally 1, 24 and 48 hr after infection. Animals treated with an irrelevant peptide (SP) served as a negative control. Survival curves of infected mice were evaluated according to Mantel- Cox Logrank test and the difference between experimental and control groups resulted significant (* P<0.05 N10K vs SP treated mice) (Figure 4A). CFU recovery from the kidneys of mice was determined 7 and 12 days after fungal infection. (* P<0.05 (N10K treated vs untreated mice) (Figure 4B).

[092] In vitro activity of N10K against caspofungin resistant C. albicans strain UM4. The candidacidal activity of N10K peptide against caspofungin resistant C. albicans strain has been evaluated by a conventional CFU assay (Polonelli et al., 2003). Briefly, Cells of C. albicans UM4, a clinical isolate from University of Milan, have been incubated at 37°C for 6 hours in the presence of N-10-K at the concentration of 20 or 10 μ§/πι1, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 5, N10K showed fungicidal activity against a caspofungin resistant C. albicans strain (*** P<0.001, N-10- K treated vs untreated cells, t test).

[093] In vitro activity of N10K against a caspofungin-resistant S. cerevisiae strain YGR032W. The fungicidal activity of N10K peptide against a caspofungin-resistant S. cerevisiae strain has been evaluated by a conventional CFU assay (Conti et al, 2008). Briefly, Cells of S. cerevisiae YGR032W, a FSK2 deleted strain, have been incubated at 37°C for 6 hours in the presence of N10K at the concentration of 20 or 10 μg/ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 6, N10K showed fungicidal activity against a caspofungin resistant strain of S. cerevisiae {*** P<0.001, N10K treated vs untreated cells, t test).

[094] In vitro activity of N10K against C. neoformans 6995 strain. The fungicidal activity of N10K peptide against C. neoformans has been evaluated by a conventional CFU assay (Cenci et al, 2004). Cells of C. neoformans 6995 have been incubated at 37°C for 6 hours in the presence of N10K at the concentration of 20, 10 or 5 μg/ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 7, N10K showed fungicidal activity against C. neoformans {*** PO.001, ** PO.01 N-10-K treated vs untreated cells, t test). The results of multiple experiments allowed the determination for the EC50 value as 5.155 10^"6 mol/liter (95% confidence intervals 5.108-5.203 10^"6).

[095] In vitro activity of N10K against M. furfur 101 strain. The microbicidal activity of N10K peptide against Malassezia furfur has been evaluated by a conventional CFU assay. Cells of M. furfur 101 have been incubated at 30°C for 6 hours in the presence of N10K at the concentration of 10, 8 or 3 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar added with Tween 20 (1%), then incubated at 30°C and observed for CFU enumeration after 72 hours. As disclosed in Figure 8, N10K showed fungicidal activity against M. furfur (** PO.01, * P<0.05, N10K treated vs untreated cells, t test).

[096] In vitro activity of N10K against A. fumigatus 1163 strain. The microbicidal activity of NIOK peptide against A. fumigatus has been evaluated by a conventional CFU assay. Conidia of A. fumigatus 1163 have been incubated at 30°C for 18 hours in the presence of NIOK at the concentration of 100 μg/ml, or in distilled water as control growth. After the incubation period, the conidial suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 9, N10K showed fungicidal activity against A. fumigatus (** P<0.01, * P<0.05, treated vs untreated cells, t test).

[097] In vitro activity of N10K against S. aureus 29213 strain. The microbicidal activity of N10K peptide against S. aureus has been evaluated by a conventional CFU assay. Cells of S. aureus 29213 have been incubated at 37°C for 5 hours in the presence of N10K at the concentration of 50, 45 or 40 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 10, N10 K showed fungicidal activity against S. aureus (*** P<0.001, N10K treated vs untreated cells, t test).

[098] In vitro activity of N10K against E. coli ATCC 25922 strain. The bactericidal activity of N10K peptide against E. coli has been evaluated by a conventional CFU assay. Cells of E. coli ATCC 25922 have been incubated at 37°C for 5 hours in the presence of N10K at the concentration of 50, 20 or 10 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 11, N10K showed bactericidal activity against E. coli {*** P<0.001, ** P<0.01, N10K treated vs untreated cells, t test).

[099] In vitro activity of N10K against K. pneumoniae ATCC 700603 strain. The bactericidal activity of N10K peptide against K. pneumoniae has been evaluated by a conventional CFU assay. Cells of K. pneumoniae ATCC 700603 have been incubated at 37°C for 5 hours in the presence of N10K at the concentration of 100, 70 or 50 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 12, N10K showed bactericidal activity against K. pneumoniae {*** P<0.001, ** P<0.01, N10K treated vs untreated cells, t test).

[100] In vitro activity of N10K against P. aeruginosa ATCC 25853 strain. The bactericidal activity of N10K peptide against P. aeruginosa has been evaluated by a conventional CFU assay. Cells of P. aeruginosa ATCC 25853 have been incubated at 37°C for 5 hours in the presence of N10K at the concentration of 50, 40 or 30 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 13, N10K showed bactericidal activity against P. aeruginosa {*** P<0.001, N10K treated vs untreated cells, t test).

[101] In vitro activity of N10K against HIV-1. PBMC from healthy donors were cultured at the concentration of 2>< 10⁶ cells/ml in 96 wells plates with RPMI 1640 supplemented with 10% FBS, 1% glutamine and 20UI/ml rIL-2 for 24hr before treatment with N10K, then the peptide was added at increasing concentrations (1, 10 and 20 μg/ml) for 24 hours. AlamarBlue was added (10%v/v) and incubated for 4 hours at 37°C. Cells viability was determined by the AlamarBlue Assay (Biosource International, Inc.). The absorbance was measured with an ELISA plate reader (Tecan Sunrise Absorbance Reader) at the double wavelength of 570/595nm. AlamarBlue added to the complete RPMI 1640 medium was used as blank. N10K demonstrated not to be cytotoxic for PBMC when it was employed in the 1-20 μg/ml concentration range, so the lowest and the intermediate doses of 1 and 10 μg/ml were elected for all the experiments. Moreover we could exclude the induction of apoptotic/necrotic effects on the U937 cell line performing the flow cytometry analysis after annexine-V/ propidium iodide staining. To verify the anti-HIV-1 activity, an in vitro infection applying two different experimental approaches was performed. PBMC from 3 healthy donors were purified by Ficoll gradient centrifugation, mixed in pool and cultured in RPMI 1640 medium (10% FBS, 1% glutamine and 1% penicillin streptomycin). Before infection cells were stimulated for 24 hours with PHA (5 μg/ml) and after with rIL-2 (20UI/ml). The first protocol consists in infecting PBMCs with HIV IIIB (X4) or BaL (R5) (0.5 m.o.i.) for two hours, washing twice and culturing in 96 wells plates for 12 days with 10 μg/ml of the compounds added together with rIL-2 every 4 days. The second protocol consists instead in pre-incubating PBMCs for two hours at 37°C with the peptides (10 μg/ml), infecting with HIV IIIB or BaL (0,5 m.o.i.) for two hours, washing twice and culturing for 12 days. rIL-2 must be added to cells every 4 days (20UI/ml). In both protocols PBMCs are collected at the days 8 and 12 of infection. Viral replication was evaluated measuring the concentration of the p24 antigen in the culture supematants by the HIV p24 ELISA Ultrasensitive detection kit (PerkinElmer, Inc.). In the repeatedly performed in vitro infection assays the N10K peptide showed a significant antiviral activity against both the viral strains when employed in the concentration range of 1-10 μg/ml. No marked difference was noticed in the two concentrations induced effects. However, this viral inhibition was exclusively observed in PBMCs treated every 4 days after infection with the peptide (See Figs. 14A and 15), whereas in the pre-incubation protocol the viral replication seem to be normal (R5 strain) or slightly, but not significantly, reduced (X4 strain) (See Fig. 14B). In comparison to the untreated controls, a relevant HIV inhibition was yet observed after 8 days of infection and this reduction continued to persist also at the 12^th day of culture. Of interest, when observed to the microscope, infected PBMCs appeared to cluster together after N10K treatment. To verify whether N10K induce a surface adhesion effect only in infected cells or also in the normal one, PBMC from tree healthy donors were purified by Ficoll gradient centrifugation, mixed in pool and cultured in RPMI 1640 medium (10%FBS, 1% glutamine and 1% penicillin streptomycin). Cells were seeded at the concentration of 500.000, 300.000 and 100.000 cells/well in a 96 wells plate for 7 days in presence of 20UI/ml rIL-2 and 10 μg/ml peptides. The compounds and rIL-2 were added after 4 days of culture. At the 7th day of culture cells were observed at the microscope to analyze the aggregation effect. Interestingly, this attitude to aggregate in cluster wasn't parallelly observed in the infection controls and in the N10K treated uninfected PBMCs and it was exclusive for N10K but not for the other peptides. Concerning the possible mechanism of action by which the N10K peptide explains its antiviral activity, we firstly evaluated the syncytium formation attitude of N10K treated CD4+ cells in comparison to the untreated controls. CH033T, HeLa ADA and HeLa LAI cell lines, which express constitutively the gpl20 on their surface, were seeded at the concentration of 250.000 cells/well in 6 wells plate and cultured for 24hr with D-MEM high glucose medium (10%FBS, 1% glutamine, 1% penicillin streptomycin and 1% Sodium Pyruvate; G418 was added to the CH033T cells culture). The following day, CD4+T cells from 3 healthy donors were isolated by positive selection (Miltenyi Biotec Inc.), pooled together and co-cultured in complete RPMI1640 medium at the concentration of 1X10⁶ cells/well with the CH033T and HeLa cell lines in presence of peptides (10 μ§/ηι1). Untreated cells were used as controls. Syncytiums formation was observed after 18 hours incubation at 37°C. The syncytiums formation was evident both in controls and treated samples. This observation evidences that the N10K doesn't work on the interaction between the CD4 receptor and the viral gpl20 (Figures 14 and 15).

[102] In vitro activity of N10K against B16F10-Nex2, SKmel28 and SKmel25 melanoma cells. Peptide N10K and the relative scramble peptide (SP) used as negative control were diluted from 1 mM to 0.05 mM in RPMI with 10% FCS and incubated with B16F10-Nex2, SKmel28 and SKmel25 cells (5 l0³ cells/well) in 100 mL per well for 12 hr at 37uC. Each peptide was tested in triplicate. After 12 hr, the cytotoxic activities of the peptides were determined by measuring cell viability by Trypan Blue exclusion. A 50% inhibition of cell growth was taken as a comparative index of cytotoxicity (EC₅₀). As disclosed in Figure 16, N10K showed activity against B16F10-Nex2 melanoma cells. Similar results were obtained for SKmel28 and SKmel25 cells, data not here provided.

[103] In vitro immunomodulatory activity of N10K on human immune cells. Human monocytes or PMN (both 10 l0⁶/ml) were incubated in RPMI 1640 plus 10% FCS for 18 hr or 6 hr respectively in the presence or in the absence (NS) of LPS, negative control (NC) and peptides (all 10 μg/ml). After incubation culture supernatants were collected and tested for cytokines production by specific ELISA. As disclosed in Figure 17, N10K stimulated the expression of Dectin-1 in human monocytes, incubated 30 minutes in presence or absence (NS) of h . CA-6 (E/T 1 : 10), NC or N10K (both 10 μg/ml). The phagocytosis of non opsonized h . CA-6 is shown in Figure 18. Monocytes were incubated for 30 minutes in presence or absence (NS) of NC or N10K(both 10 μg/ml) or Cytochalasin D (30 μΜ). After incubation cells have been stimulated for 30 minutes with non opsonized hi. CA-6 (E/T 1 : 10). The percentage of phagocytosis (Fig. 18A) and the medium number of yeast particles adhered or ingested by each monocyte (phagocytic index) (Fig. 18B) have been determined by cytofluorimetric analysis. In Table I (Fig. 18C) is shown the mean number of attached and ingested C. Albicans for phagocytosing PBM. Cytokines production induced by N10K is disclosed in Figure 19. PBMC have been incubated 30 minutes in presence or absence (NS) of NC or N10K (both 10 μg/ml). After incubation PBMC have been stimulated for 18 h with LPS (10 μg/ml) or hi. CA-6 (E/T 1 : 10). The levels of IL-12p40 and IL-6 have been evaluated in the supernatants by ELISA Kit.

2. Data for Tl IF peptide

[104] In vitro activity of T11F against C. albicans SC5314 strain. The candidacidal activity of T11F peptide against C. albicans was assessed by a conventional CFU assay. Cells of C. albicans SC5314 were incubated at 37°C for 6 hours in the presence of T11F at the concentrations of 5, 3 or 2 μg/ml, or in distilled water as control (C). After incubation, cell suspensions were plated on Sabouraud dextrose agar and incubated at 30°C for 48 hours when CFU were counted (** P<0.01, * P<0.05, T11F treated vs. untreated cells, t test). As disclosed in Figure 20, peptide T11F showed candidacidal activity in vitro against cells of C. albicans SC5314 strain. Based on several independent replications, an EC₅₀ of 1.599 x 10^"6 mol/liter (95% confidence intervals 1.017-2.514 x 10^"6) was determined.

[105] In vitro activity of T11F against caspofungin resistant C. albicans strain UM4.

The candidacidal activity of Tl IF peptide against caspofungin resistant C. albicans strain has been evaluated by a conventional CFU assay. Cells of C. albicans UM4, a clinical isolate from University of Milan, have been incubated at 37°C for 6 hours in the presence of Tl IF at the concentration of 5 or 2 μ^ιηΐ, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 21, T11F showed candidacidal activity against a caspofungin resistant C. albicans strain (*** P<0.001, T-l 1-F treated vs untreated cells, t test).

[106] In vitro activity of T11F against a caspofungin-resistant S. cerevisiae strain YGR032W. The fungicidal activity of T11F peptide against a caspofungin-resistant S. cerevisiae strain has been evaluated by a conventional CFU assay. Cells of S. cerevisiae YGR032W, a FSK2 deleted strain, have been incubated at 37°C for 6 hours in the presence of Tl IF at the concentration of 20, 10 or 5 μ^ιηΐ, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 22, T11F showed fungicidal activity against a caspofungin- resistant S. cerevisiae strain (*** PO.001, T-l 1-F treated vs untreated cells, t test). [107] In vitro activity of TllF against C. neoformans 6995 strain. The fungicidal activity of Tl lF peptide against C. neoformans has been evaluated by a conventional CFU assay. Cells of C. neoformans 6995 have been incubated at 37°C for 6 hours in the presence of Tl IF at the concentration of 10, 5 or 4 μg/ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 23, Tl lF showed fungicidal activity against C. neoformans {*** P<0.0001, T-l l-F treated vs untreated cells, t test). The results of multiple experiments allowed the determination for the EC₅₀ value as 2.693 x 10^"6 mol/liter (95% confidence intervals 2.692-2.694 x 10^"6).

[108] In vitro activity of TllF against M. furfur 101 strain. The fungicidal activity of Tl lF peptide against M. furfur has been evaluated by a CFU assay. Cells of M. furfur 101 have been incubated at 30°C for 6 hours in the presence of Tl IF at the concentration of 2, 1 or 0.5 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar added with Tween 20 (1%), then incubated at 30°C and observed for CFU enumeration after 72 hours. As disclosed in Figure 24, Tl lF showed fungicidal activity against M. furfur {*** PO.001,** P<0.01, * P<0.05, Tl lF treated vs untreated cells, t test).

[109] In vitro activity of TllF against A. fumigatus 1163 strain. The fungicidal activity of Tl IF peptide against A. fumigatus has been evaluated by a conventional CFU assay. Conidia of A. fumigatus 1163 have been incubated at 30°C for 18 hours in the presence of Tl lF at the concentration of 100 μ^ιηΐ, or in distilled water as control growth. After the incubation period, the conidial suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 25, Tl IF showed fungicidal activity against A. fumigatus (* P<0.05, treated vs untreated cells, t test).

[110] In vitro activity of TllF against S. aureus 29213 strain. The bactericidal activity of Tl lF peptide against S. aureus has been evaluated by a conventional CFU assay. Cells of S. aureus 29213 have been incubated at 37°C for 5 hours in the presence of Tl lF at the concentration of 50, 40 or 30 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 26, T11F showed bactericidal activity against S. aureus {*** P<0.001, Tl IF treated vs untreated cells, t test).

[I l l] In vitro activity of T11F against E. coli ATCC 25922 strain. The bactericidal activity of Tl IF peptide against E. coli has been evaluated by a conventional CFU assay. Cells of E. coli ATCC 25922 have been incubated at 37°C for 5 hours in the presence of Tl IF at the concentration of 5, 3 or 2 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 27, T1 1F showed bactericidal activity against E. coli (*** P<0.001, T11F treated vs untreated cells, t test).

[112] In vitro activity of T11F against K. pneumoniae ATCC 700603 strain. The bactericidal activity of Tl IF peptide against K. pneumoniae has been evaluated by a CFU assay. Cells of K. pneumoniae ATCC 700603 have been incubated at 37°C for 5 hours in the presence of Tl IF at the concentration of 100, 80 or 60 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 28, T11F showed bactericidal activity against K. pneumoniae (** PO.01, * P<0.05 Tl IF treated vs untreated cells, t test).

[113] In vitro activity of T11F against P. aeruginosa ATCC 25853 strain. The bactericidal activity of T11F peptide against P. aeruginosa has been evaluated by a conventional CFU assay. Cells of P. aeruginosa ATCC 25853 have been incubated at 37°C for 5 hours in the presence of Tl IF at the concentration of 10, 5 or 2 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 29, Tl IF showed bactericidal activity against P. aeruginosa (** PO.01, Tl IF treated vs untreated cells, t test).

[114] In vitro activity of T11F against HIV-1. PBMC from healthy donors were cultured at the concentration of 2>< 10⁶ cells/ml in 96 wells plates with RPMI 1640 supplemented with 10% FBS, 1% glutamine and 20UI/ml rIL-2 for 24h before treatment with T11F, then the peptide was added at increasing concentrations (1, 10 and 20 μg/ml) for 24 hours. AlamarBlue was added (10%v/v) and incubated for 4 hours at 37°C. Cells viability was determined by the AlamarBlue Assay (Biosource International, Inc.). The absorbance was measured with an ELISA plate reader (Tecan Sunrise Absorbance Reader) at the double wavelength of 570/595nm. AlamarBlue added to the complete RPMI1640 medium was used as blank. Tl IF demonstrated not to be cytotoxic for PBMC when it was employed in the 1-20 μg/ml concentration range, so the lowest and the intermediate doses of 1 and 10 μg/ml were elected for all the experiments. Moreover we could exclude the induction of apoptotic/necrotic effects on the U937 cell line performing the flow cytometry analysis after annexine-V/ propidium iodide staining. To verify the anti-HIV-1 activity, an in vitro infection applying two different experimental approaches was performed. PBMC from 3 healthy donors were purified by Ficoll gradient centrifugation, mixed in pool and cultured in RPMI 1640 medium (10% FBS, 1% glutamine and 1% penicillin streptomycin). Before infection cells were stimulated for 24 hours with PHA (5 μg/ml) and after with rIL-2 (20UI/ml). The first protocol consists in infecting PBMCs with HIV IIIB (X4) or BaL (R5) (0.5 m.o.i.) for two hours, washing twice and culturing in 96 wells plates for 12 days with 10 μg/ml of the compounds added together with rIL-2 every 4 days. The second protocol consists instead in pre-incubating PBMCs for two hours at 37°C with the peptides (10 μg/ml), infecting with HIV IIIB or BaL (0,5 m.o.i.) for two hours, washing twice and culturing for 12 days. rIL-2 must be added to cells every 4 days (20UI/ml). In both protocols PBMCs are collected at the days 8 and 12 of infection. Viral replication was evaluated measuring the concentration of the p24 antigen in the culture supematants by the HIV p24 ELISA Ultrasensitive detection kit (PerkinElmer, Inc.). T11F keep low the BaL replication only if it was supplied every 4 days after infection (Figure 30).

[115] In vitro activity of T11F against B16F10-Nex2, SKmel28 and SKmel25 melanoma cells. Peptide T11F and the relative scramble peptide (SP) used as negative control were diluted from 1 mM to 0.05 mM in RPMI with 10% FCS and incubated with B16F10-Nex2, SKmel28 and SKmel25 cells (5 l0³ cells/well) in 100 mL per well for 12 hr at 37uC. Each peptide was tested in triplicate. After 12 hr, the cytotoxic activities of the peptides were determined by measuring cell viability by Trypan Blue exclusion. A 50% inhibition of cell growth was taken as a comparative index of cytotoxicity (EC₅₀). As disclosed in Figure 31, T11F showed activity against B16F10-Nex2 melanoma cells. Similar results, not here provided, were found with both SKmel28 and SKmel25 cells. 3. Data for H4L peptide

[116] In vitro activity of H4L against C. albicans SC5314 strain. The candidacidal activity of H4L peptide against C. albicans has been evaluated by a conventional CFU assay. Cells of C. albicans SC5314 have been incubated at 37°C for 6 hours in the presence of H4L at the concentration of 100 μg/ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 32, H4L showed candidacidal activity against C. albicans {*** P<0.001, treated vs untreated cells, t test).

[117] In vitro activity of H4L against C. neoformans 6995 strain. The fungicidal activity of H4L peptide against C. neoformans has been evaluated by a conventional CFU assay. Cells of C. neoformans 6995 have been incubated at 37°C for 6 hours in the presence of H4L at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 33, H4L showed fungicidal activity against C. neoformans {*** P<0.001, treated vs untreated cells, t test).

[118] In vitro activity of H4L against M. furfur 101 strain. The fungicidal activity of H4L peptide against M. furfur has been evaluated by a conventional CFU assay. Cells of M. furfur 101 have been incubated at 30°C for 6 hours in the presence of peptides at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar added with Tween 20 (1%), then incubated at 30°C and observed for CFU enumeration after 72 hours. As disclosed in Figure 34, H4L showed fungicidal activity against M. furfur {*** P<0.001, treated vs untreated cells, t test).

[119] In vitro activity of H4L against A. fumigatus 1163 strain. The fungicidal activity ofH4L peptide against A. fumigatus has been evaluated by a CFU assay. Conidia of A. fumigatus 1163 have been incubated at 30°C for 18 hours in the presence of H4L at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 35, H4L showed fungicidal activity against A. fumigatus (* P<0.05, treated vs untreated cells, t test).

[120] In vitro activity of H4L against S. aureus 29213 strain. The bactericidal activity of H4L peptide against S. aureus has been evaluated by a conventional CFU) assay. Cells of S. aureus 29213 have been incubated at 37°C for 5 hours in the presence of peptides at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 36, H4L showed bactericidal activity against S. aureus {*** P<0.001, treated vs untreated cells, t test).

[121] In vitro activity of H4L against E. coli ATCC 25922 strain. The bactericidal activity of H4L peptide against E. coli has been evaluated by a conventional CFU assay. Cells of E. coli ATCC 25922 have been incubated at 37°C for 5 hours in the presence of H4L at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 37, H4L showed bactericidal activity against E. coli {*** P<0.001, treated vs untreated cells, t test).

[122] In vitro activity of H4L against K. pneumoniae ATCC 700603 strain. The bactericidal activity of H4L peptide against K. pneumoniae has been evaluated by a conventional CFU assay. Cells of K. pneumoniae ATCC 700603 have been incubated at 37°C for 5 hours in the presence of H4L at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 38, H4L showed bactericidal activity against K. pneumoniae {*** P<0.001, treated vs untreated cells, t test).

[123] In vitro activity of H4L against P. aeruginosa ATCC 25853 strain. The microbicidal activity of H4L peptide against P. aeruginosa has been evaluated by a conventional CFU assay. Cells of P. aeruginosa ATCC 25853 have been incubated at 37°C for 5 hours in the presence of H4L at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 39, H4L showed bactericidal activity against P. aeruginosa.

[124] In vitro activity of H4L against HIV-1. PBMC from healthy donors were cultured at the concentration of 2>< 10⁶ cells/ml in 96 wells plates with RPMI 1640 supplemented with 10% FBS, 1% glutamine and 20UI/ml rIL-2 for 24hr before treatment with H41, then the peptide was added at increasing concentrations (1, 10 and 20 μg/ml) for 24 hours. AlamarBlue was added (10%v/v) and incubated for 4 hours at 37°C. Cells viability was determined by the AlamarBlue Assay (Biosource International, Inc.). The absorbance was measured with an ELISA plate reader (Tecan Sunrise Absorbance Reader) at the double wavelength of 570/595nm. AlamarBlue added to the complete RPMI 1640 medium was used as blank. H4L demonstrated not to be cytotoxic for PBMC when it was employed in the 1-20 μg/ml concentration range, so the lowest and the intermediate doses of 1 and 10 μg/ml were elected for all the experiments. Moreover we could exclude the induction of apoptotic/necrotic effects on the U937 cell line performing the flow cytometry analysis after annexine-V/ propidium iodide staining. To verify the anti-HIV-1 activity, an in vitro infection applying two different experimental approaches was performed. PBMC from 3 healthy donors were purified by Ficoll gradient centrifugation, mixed in pool and cultured in RPMI 1640 medium (10% FBS, 1% glutamine and 1% penicillin streptomycin). Before infection cells were stimulated for 24 hours with PHA (5 μg/ml) and after with rIL-2 (20UI/ml). The first protocol consists in infecting PBMCs with HIV IIIB (X4) or BaL (R5) (0.5 m.o.i.) for two hours, washing twice and culturing in 96 wells plates for 12 days with 10 μg/ml of the compounds added together with rIL-2 every 4 days. The second protocol consists instead in pre-incubating PBMCs for two hours at 37°C with the peptide (10 μg/ml), infecting with HIV IIIB or BaL (0,5 m.o.i.) for two hours, washing twice and culturing for 12 days. rIL-2 must be added to cells every 4 days (20UI/ml). In both protocols PBMCs are collected at the days 8 and 12 of infection. Viral replication was evaluated measuring the concentration of the p24 antigen in the culture supematants by the HIV p24 ELISA Ultrasensitive detection kit (PerkinElmer, Inc.). In fact, the H4L peptide administered to cell prior to the viral infection seemed to control HIV IIIB replication only during the first 8 days of culture, and kept low the BaL replication till the day 8 of infection only if it was supplied every 4 days after infection (Figure 40).

4. Data for MoA VhCDR3 peptide

[125] In vitro activity of MoA V_HCDR₃ against M. furfur 101 strain. The fungicidal activity of MoA V_HCDR₃ peptide against M. furfur has been evaluated by a conventional CFU assay. Cells of M. furfur 101 have been incubated at 30°C for 6 hours in the presence of peptides at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar added with Tween 20 (1%), then incubated at 30°C and observed for CFU enumeration after 72 hours. As disclosed in Figure 41, MoA V_HCDR₃ showed fungicidal activity against M. furfur.

[126] In vitro activity of MoA V_HCDR₃ against A. fumigatus 1163 strain. The fungicidal activity of H4L peptide against A. fumigatus has been evaluated by a CFU assay. Conidia of A. fumigatus 1163 have been incubated at 30°C for 18 hours in the presence of MoA V_HCDR₃ at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Sabouraud dextrose agar, then incubated at 30°C and observed for CFU enumeration after 48 hours. As disclosed in Figure 42, MoA V_HCDR₃ showed fungicidal activity against A. fumigatus (* P<0.05, treated vs untreated cells, t test).

[127] In vitro activity of MoA V_HCDR₃ against S. aureus 29213 strain. The microbicidal activity of MoA V_HCDR₃ peptide against S. aureus has been evaluated by a conventional CFU assay. Cells of S. aureus 29213 have been incubated at 37°C for 5 hours in the presence of MoA V_HCDR₃ at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 43, MoA V_HCDR₃ has showed bactericidal activity against S. aureus (* P<0.05, treated vs untreated cells, t test).

[128] In vitro activity of MoA V_HCDR₃ against E. coli ATCC 25922 strain. The bactericidal activity of MoA V_HCDR₃ peptide against E. coli has been evaluated by a conventional CFU assay. Cells of E. coli ATCC 25922 have been incubated at 37°C for 5 hours in the presence of MoA V_HCDR₃ at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 44, MoA V_HCDR₃ showed bactericidal activity against E. coli (*** P<0.001 , treated vs untreated cells, t test).

[ 129] In vitro activity of MoA V_HCDR₃ against K. pneumoniae ATCC 700603 strain.

The bactericidal activity of MoA V_HCDR₃ peptide against K. pneumoniae has been evaluated by a conventional CFU assay. Cells of K. pneumoniae ATCC 700603 have been incubated at 37°C for 5 hours in the presence of MoA V_HCDR₃ at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 45, MoA V_HCDR₃ showed bactericidal activity against K. pneumoniae.

[ 130] In vitro activity of MoA V_HCDR₃ against P. aeruginosa ATCC 25853 strain.

The bactericidal activity of MoA V_HCDR₃ peptide against P. aeruginosa has been evaluated by a CFU assay. Cells of P. aeruginosa ATCC 25853 have been incubated at 37°C for 5 hours in the presence of MoA V_HCDR₃ at the concentration of 100 μg /ml, or in distilled water as control growth. After the incubation period, the cell suspensions have been plated on Mueller Hinton agar, then incubated at 37°C and observed for CFU enumeration after 24 hours. As disclosed in Figure 46, MoA V_HCDR showed bactericidal activity against P. aeruginosa (*** P<0.001 , treated vs untreated cells, t test).

[ 131 ] In vitro immunomodulatory activity of MoA V_HCDR₃ on murine immune cells. Figures 47-51 are graphs showing TNF-a and IL-6 production by PM and PMN stimulated with MoA CDRs and mouse MoA V_HCDR₃ uptake by different cell populations. Mab MoA CDR sequences experiments with PM are shown in Figures 47 and 48. Mab MoA CDR sequences experiments with PMN are shown in Figures 49 and 50. Cells were cultured in the presence or absence (NS) of human and/or mouse CDRs, lipopolisaccharide (LPS), or negative control peptide (NC) (all 10 μg/ml) for 18 hr. Both PM and PMN cell populations were 5 ^X 10⁶/ml. After incubation, TNF-a and IL-6 levels were evaluated in culture supernatants by specific ELISA assays (* P<0.05 treated vs untreated cells, n = 7). In Figure 51, dendritic cells (DC), PM, PMN, and T cells (all l x l0⁶/ml) were incubated for 1 hr in the presence or absence (NS) of biotinylated (b)- MoA V_HCDP ₃ or b-NC (both 10 μg/ml). After incubation, permeabilized cells were reacted with FITC-labelled mAb to biotin (b) and analyzed by FACScan flow cytometry. Data are reported as the percentage of positive cells *, <0.05 (b-V_HCDR₃ treated vs untreated cells, n = 5). Error bars, s.e.m. Figures 52A and B show graphs and colored cell staining micrograph of kinetic of V_HCDR₃ uptake by PM. PM (l x l0⁶/ml) were incubated for different times with b-VnCDR₃ or b-NC (all 10 μg/ml). After incubation, permeabilized cells were reacted with FITC-labelled mAb to biotin and analyzed by FACScan flow cytometry. Data are reported as the mean fluorescence intensity (MFI) and percentage of positive cells (Figure 52A) (* P<0.05 b-VnCDR₃-treated vs untreated cells, n = 7). Error bars, s.e.m. In selected experiments, cells were incubated for 1 hr as above described, reacted with FITC-labelled mAb to biotin in the presence of Evans' Blue as a counter stain, and subsequently examined under fluorescent light microscopy (Figure 52B). Note the green fluorescence of b-VnCDR₃ treated cells. Original magnification 20 x. In Figures 53 and 54 is shown the phospho-Akt activation and TNF-a production in PM stimulated with Mo A V_HCDR₃. PM (3x l0⁶/ml) were stimulated for 1 hr in the presence or absence (NS) of MoA V_HCDR₃, LPS, an irrelevant control peptide (NC) or a scrambled pepetide (SP) (all 10 μg/ml). After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to pAkt and Akt; pAkt was normalized against Akt (Figure 53) *, <0.05 (treated vs untreated cells, n = 5). Figure 54 shows the production of TNF-alpha in murine macrophages stimulated with MoA V_HCDR₃. PM (5x l0⁶/ml) were stimulated for 18 hr as above described. After incubation, TNF-a level was evaluated in culture supernatants by specific ELISA assays. (Figure 54) *, <0.05 (treated vs untreated cells, n = 5). In Figures 55 and 56 is shown the phospho- IkBa activation and TNF-a gene expression in PM stimulated with MoA V_HCDR₃. PM (3x l0⁶/ml) were stimulated for 1 hr in the presence or absence (NS) of wortmannin (4 nM), MoA V_HCDR₃, LPS or NC (all 10 μg/ml). After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to plkBa and IkBa; plkBa was normalized against IkBa. (Figure 55) *, <0.05 (treated vs untreated cells, n = 5);†, <0.05 (wortmannin-treated vs wortmannin-untreated cells, n = 5). For testing the expression level of TNF-a gene, PM (l x l0⁶/ml) were cultured for 1, 6 and 18 hr as above described. After incubation, total RNA was isolated and analyzed for mRNA expression with RT-PCR (Figure 56). Transcript copy numbers were determined by qPCR using cDNA as a template. Copy numbers were normalized against the copy number of the GADPH gene (B). (* P<0.05 (treated vs untreated cells, n = 5). Error bars, s.e.m. In Figures 57 is shown the expression of TLR-4 in PM stimulated with MoA V_HCDR₃. PM (l l0⁶/ml) were cultured for 1 and 6 hr in the presence or absence (NS) of MoA V_HCDR₃, LPS or NC (all 10 μg/ml). After incubation, total RNA was isolated and analyzed for mRNA expression with RT-PCR. Transcript copy numbers were determined by qPCR using cDNA as a template. Copy numbers were normalized against the copy number of the GADPH gene (Figure 57 A) (* P<0.05 (MoA V_HCDR₃ treated vs untreated cells, n = 5). PM (3 l0⁶/ml) were cultured for 1 and 3 hr as above described. After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to TLR-4 and actin. TLR-4 production was normalized against actin (Figure 57 B) (*, P<0.05 MoA V_HCDR₃-treated vs untreated cells, n = 5). Error bars, s.e.m. PM (l x l0⁶/ml) were incubated for 1 hr as above described. After incubation, permeabilized cells were reacted with RPE-labelled mAb to TLR-4 and analyzed by FACScan flow cytometry. Values represent the percentage of positive cells (Figure 57 C). TNF-a induced TLR-4 expression in PM stimulated with MoA V_HCDR₃ (Figure 58). PM (5x l0⁶/ml) were cultured for 1 hr in the presence or absence (NS) of MoA V_HCDR3, LPS or NC (all 10 μg/ml). After incubation, TNF-a level was evaluated in culture supernatants by specific ELISA assay (Figure 58A) (* P<0.05 treated vs untreated cells, n = 7). PM (l x l0⁶/ml) were cultured for 1 hr with V_HCDR₃, LPS or NC (all 10 μ^ιηΐ), in the presence or absence (NS) of mAb to TNF-a (0.5 μg/ml). After incubation, permeabilized cells were reacted with RPE-labelled mAb to TLR-4 and analyzed by FACScan flow cytometry. Values represent the percentage of positive cells (Figure 58B) (* P<0.05 V_HCDR₃ plus mAb to TNF-a treated vs MoA V_HCDR₃ treated cells, n = 7). PM (3x l0⁶/ml) were cultured for 1 hr as above described. After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to TLR-4 and actin. TLR-4 production was normalized against actin (Figure 58C) (* P<0.05 V_HCDR₃ plus mAb to TNF-a treated vs MoA V_HCDR₃ treated cells, n = 5). For testing the expression level of TLR-4 gene, PM (l x l0⁶/ml) were cultured for 1 hr as above described. After incubation, total RNA was isolated and analyzed for mRNA expression with RT-PCR. Transcript copy numbers were determined by qPCR using cDNA as a template. Copy numbers were normalized against the copy number of the GADPH gene (Figure 58D) (* P<0.05 MoA V_HCDR₃ plus mAb to TNF-a treated vs V_HCDR₃ treated cells, n = 5). Error bars, s.e.m.

[ 132] Given that V_HCDR₃ is able to induce a state of activation in PM, we tested whether this condition could influence the course of infection in a mouse experimental model of systemic candidiasis, despite the proven non-candidacidal properties of the peptide. Mice were infected intravenously with the opportunistic fungus C. albicans and treated with mouse V_HCDR₃ or V_LCDR₃ (used as a negative control) intraperitoneally 4 hr before, and 1 and 2 days after infection. Animal survival and fungal burden in kidneys were evaluated in different groups of mice. Percent survival and determination of fungal clearance from kidneys of Balb/c mice challenged with C. albicans (CA-6) and treated with MoA V_HCDR₃ or MoA V_LCDR₃ are disclosed in Figures 59. Percent survival of infected mice was evaluated according to Mantel-Cox Log rank test and the difference among experimental groups resulted significant (* P<0.05, n = 7) (Figure 59A). CFU recovery from the kidneys of mice was determined 5, 7 and 12 days after fungal infection (Figure 59B) (* P<0.05 MoA V_HCDR₃ treated vs .untreated mice, n = 7). Error bars, s.e.m. Results, reported in Figure 59A, showed a significant increase in survival for infected mice treated with MoA V_HCDR₃ as compared to infected mice untreated or treated with the non immunomodulatory MoA V_LCDR₃. In the same experimental conditions, CFU recovery from kidneys showed an impressive decrease when mice infected with C. albicans were treated with MoA V_HCDR₃ as compared to negative control mice (Figure 59B). Figure 60 is a pictoral diagram delineating the mechanism of TLR-4 upregulation induced in PM by MoA V_HCDR₃. AS a proof of concept, here we demonstrate that a synthetic peptide with sequences identical to MoA V_RCDR₃ of a mouse IgM mAb specific for difucosyl human blood group A substance (MoA) could display a potent immunomodulatory activity thus exerting a therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. Significantly, the aminoacidic residue at position 5 (N) proved to be functionally critical for the immunostimulatory properties of MoA V_HCDR₃, as its substitution with alanine resulted in a loss of TNF-a production capacity. One possible scenario suggested by these data is that selected short sequences representative of the CDRs of Abs could be strongly involved in inflammatory responses and, as a consequence, in chronic inflammatory processes. MoA V_HCDR₃ peptide is able, indeed, to stimulate PM to produce TNF-a, and this could be instrumental in inducing inflammation. As a matter of fact, TNF-a is considered a classical cytokine of chronic inflammatory disease.

[133] PM perform a central task in both the innate and adaptive immune systems. The life and function of these cells are characterized by significant functional versatility. PM ingest foreign materials, present Ags to T lymphocytes in association with the MHC, and can kill microbes and tumor cells upon activation by cytokines and/or T cells. In addition, they eliminate damaged or apoptotic cells. Conversely, PM can also release copious amounts of toxic metabolites that can promote tissue damage during antimicrobial defence responses.

[134] Our evidence reports that PM very rapidly take up the MoA V_HCDR₃ peptide, and 18 hr post treatment this peptide is still associated to the cells. It is possible that MoA V_RCDR₃ could be continuously internalized and degraded within 18 hr; alternatively, the peptide could be retained by cells for 18 hr and subsequently degraded or expelled.

[135] PI3K has been linked to an extraordinarily diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. Many of these functions relate to the ability of PI3K to activate Akt. The interaction of MoA V_HCDR₃ with PM induces Akt activation that finally leads to phosphorylation of IkBalpha with consequent translocation of NFkB into the nucleus. These molecular events are responsible for cellular activation and subsequent transcription of proinflammatory cytokine genes such as TNF-a. Indeed, this pathway of activation is also confirmed by the inhibition of TNF-a production after blocking the specific Akt signalling pathway. Similarly, involvement of p38 MAPK activation was detected using a specific inhibitor of this pathway. As a matter of fact, TNF-a mRNA was detected 1 hr post stimulation with MoA V_HCDR₃, suggesting that the signal transduction pathway from Akt leads to cytokine gene expression, as depicted in Figure 60.

[136] Given that TNF-a is believed to be a positive regulator of TLR-4 expression, and that the ability of cells to respond to several microbial motifs depends on TLR-4 expression, we found that, in our experimental system, MoA V_HCDR₃ up-regulates TLR- 4. The stimulation of TLR-4 leads to cellular activation, and this effect could reinforce the capacity of the peptide to induce inflammatory responses. Moreover, when considering that TLR-4 up-regulation is completely blocked by neutralizing TNF-a, one could posit that the over expression of TLR-4 is secondary and dependent on TNF-a production.

[137] Convincing arguments point to the key role of TLR-4 in microbial antigen recognition in relation to its protective response. In particular, the antigenic structures of the opportunistic fungus C. albicans are recognized by TLR-4. In our experimental system, a significant increase in survival and a drastic decrease in fungal growth in the kidney, the target organ for C. albicans, was surprising, given that MoA V_HCDR₃ is ineffective against C. albicans cells in vitro. A possible explanation for this could be that natural immune cells are activated by MoA V_HCDR₃ treatment and more prone to ingest and kill C. albicans. Additionally, increased TLR-4 expression on PM could facilitate C. albicans recognition with consequent more prompt and efficient immune response. The rapid clearance of C. albicans observed in vivo is particularly relevant, given that MoA V_HCDR₃ does not show any direct candidacidal activity. A simple peptide derived from a mAb specific for difucosyl human blood group A substance, is endowed with potent immunoregulatory effects that are intrinsically able to control the course of a microbial infection.

[138] Whether a proteolytic release of modulatory fragments may physiologically occur beyond the half life of Igs is an intriguing hypothesis that would account for the apparent redundancy in their production. Nature may have provided extrinsic activities to peptides integrated in evolutionary molecules such as Abs in a way reminiscent of human cationic peptides that play an innate immune regulatory role in host defence. Overall these findings suggest that Ab-derived peptides can act likewise effectors of the innate immune response opening a new scenario about their interplay with the cellular immune response.

[139] In further embodiments, the invention peptides can be used in various methodologies for treating invasive bacterial, fungal, yeast, mold, and viral infections as well as be useful for treating cancer disorders or other therapies that benefit from immune modulation such as modulations that otherwise affect such factors as cytokines. In preferred embodiments, it is contemplated that such treatments can include both topical and systemic applications as in our data the peptides prove to have exceptionally low toxicity. The peptides of the invention can be administered to a mammal infected with a fungus, topically or systemically, such as in the case of an infection with Aspergillus sp. a yeast such as in the case of vulvovaginal candidiasis caused by C. albicans, or a mammal infected with a bacterium such as E. coli. Other invasive organisms contemplated for treatment in mammals by administration of the peptide of the invention topically or systemically include Mycobacterium tuberculosis, Cryptococcus spp., Fusarium spp., Scedosporium spp., Histoplasma capsulatum, Blastomyces dermatitidis, Zygomycetes and dematiaceous fungi.

Treatment Applications

[140] Topical administration can be cream or ointment based, the respective formulation comprising active and inert materials as are commonly known for such topical treatments and at concentrations as proven useful in mammalian models. Systemic administration can be by injection wherein the formulary of the injectate comprises salts and solutions well known for administering peptides in such fashion. For example, concentration of the invention peptides identified by Seq. Id Nos. 1-13 in either topical or systemic formulations can comprise concentration ranges of between 2 and 100 micrograms/ml, more typically any of 2-5 micrograms/ml, 3-5 micrograms/ml, 5-10 micrograms/ml, 5-20 micrograms/ml, 30-50 micrograms/ml, 40-50 micrograms/ml, 50-100 micrograms/ml. In some applications, as little as 1-2 micrograms/ml is effective. Other concentrations include, 3, 5, 10, 20, 50, and 100 micrograms/ml.

[141] Additional, treatment regimens include length of time periods for which treatment by topical or systemic application should be made. In preferred embodiments treatment regimens contemplate delivery over at least 4 days. In a particularly preferred embodiment, application or otherwise delivery of the antimicrobial, antibacterial, antifungal, antiyeast, antimold, antiviral, or immunomodulatory polypeptide is made between one and four days and alternatively every four days or (24hr period), or alternatively, continuously over a period of at least 4 days with application ranging from once, twice, three or even four times per day and depending upon the ultimate dosage concentration used. Dosing can be carried on for periods of up to one month or more. [142] Various publications are cited herein which are hereby incorporated by reference in their entirety.

[143] As will be apparent to those skilled in the art in which the invention is addressed, the present invention may be embodied in forms other than those specifically disclosed above without departing from the spirit or potential characteristics of the invention. Particular embodiments of the present invention described above are therefore to be considered in all respects as illustrative and not restrictive. The scope of the present invention is as set forth in the appended claims and equivalents thereof rather than being limited to the examples contained in the foregoing description.

REFERENCES

Arendrup MC (2009) Invasive fungal infections: past achievements and challenges ahead.

Clin Microbiol Infect 15:599-601.

Cappelletty D, Eiselstein-McKitrick K (2007) The echinocandins. Pharmacotherapy

27:369-388.

Casadevall A, Pirofski LA (2006) The weapon potential of human pathogenic fungi. Med

Mycol 44:689-96. Cenci E., Bistoni F., Mencacci A., Perito S., Magliani W., Conti

S., Polonelli L., Vecchiarelli A. (2004): A synthetic peptide as a novel anticryptococcal agent. Cellular Microbiology, 6:953-961.

Cenci E., Bistoni F., Mencacci A., Perito S., Magliani W., Conti S., Polonelli L.,

Vecchiarelli A. (2004): A synthetic peptide as a novel anticryptococcal agent.

Cellular Microbiology, 6:953-961.

Cenci E, Pericolini E, Mencacci A, Conti S, Magliani W, Bistoni F, Polonelli L,

Vecchiarelli A (2006) Modulation of phenotype and function of dendritic cells by a therapeutic synthetic killer peptide. J Leukoc Biol 79:40-45.

Conti S, Magliani W, Giovati L, Libri I, Maffei DL, Salati A, Polonelli L (2008)

Screening of a Saccharomyces cerevisiae nonessential gene deletion collection for altered susceptibility to a killer peptide. New Microbiol 31 : 143-145.

Comely OA, Maertens J, Bresnik M, Ullmann AJ, Ebrahimi R, Herbrecht R (2009)

Treatment outcome of invasive mould disease after sequential exposure to azoles and liposomal amphotericin B. J Antimicrob Chemother.

Erjavec Z, Kluin-Nelemans H, Verweij PE (2009) Trends in invasive fungal infections, with emphasis on invasive aspergillosis. Clin Microbiol Infect 15:625-633.

Fera MT, La Camera E, De Sarro A (2009) New triazoles and echinocandins: mode of action, in vitro activity and mechanisms of resistance. Expert Rev Anti Infect Ther

7:981-998.

Gabrielli E, Pericolini E, Cenci E, Ortelli F, Magliani W, Ciociola T, Bistoni F, Conti S, Vecchiarelli A, Polonelli L. Antibody complementarity-determining regions (CDRs): a bridge between adaptive and innate immunity. PLoS One. 2009 Dec 4;4(12):e8187.

Gergis U, Markey K, Greene J, Kharfan-Dabaja M, Field T, Wetzstein G, Schell MJ, Huang Y, Anasetti C, Perkins J (2009) Voriconazole provides effective prophylaxis for invasive fungal infection in patients receiving glucocorticoid therapy for GVHD. Bone Marrow Transplant.

Kauffman CA, Carver PL (2008) Update on echinocandin antifungals. Semin Respir Crit Care Med 29:211-219.

Kawai T, Akira S (2005) Pathogen recognition with Toll-like receptors. Curr Opin Immunol 17:338-344.

Manfredi M, McCullough MJ, Conti S, Polonelli L, Vescovi P, Al-Karaawi ZM, Porter SR (2005) In vitro activity of a monoclonal killer anti-idiotypic antibody and a synthetic killer peptide against oral isolates of Candida spp. differently susceptible to conventional antifungals. Oral Microbiol Immunol 20:226-232.

Moen MD, Lyseng-Williamson KA, Scott LJ (2009) Liposomal amphotericin B: a review of its use as empirical therapy in febrile neutropenia and in the treatment of invasive fungal infections. Drugs 69:361-392. Nickerson KG, Tao MH, Chen HT, Larrick J, Kabat EA (1995) Human and mouse monoclonal antibodies to blood group A substance, which are nearly identical immunochemically, use radically different primary sequences. J Biol Chem 270: 12457-12465.

Polonelli L, Magliani W, Conti S, Bracci L, Lozzi L, Neri P, Adriani D, De Bernardis F, Cassone A (2003) Therapeutic activity of an engineered synthetic killer antiidiotypic antibody fragment against experimental mucosal and systemic candidiasis. Infect Immun 71 :6205-6212.

Polonelli L., Ponton J., Elguezabal N., Moragues M.D., Casoli C, Pilotti E., Ronzi P., Dobroff A.S., Rodrigues E.G., Juliano M.A., Maffei D.L., Magliani W., Conti S., Travassos L.R. (2008) Antibody complementarity-determining regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities. PLoS ONE, 3: e2371. doi: 10.1371/journal.pone.0002371

Rogers TR, Frost S (2009) Newer antifungal agents for invasive fungal infections in patients with haematological malignancy. Br J Haematol 144:629-641.

Romani L, Mencacci A, Cenci E, Spaccapelo R, Mosci P, Puccetti P, Bistoni F (1993) CD4+ subset expression in murine candidiasis. Th responses correlate directly with genetically determined susceptibility or vaccine -induced resistance. J Immunol 150:925-931.

Sable CA, Strohmaier KM, Chodakewitz JA (2008) Advances in antifungal therapy.

Annu Rev Med 59:361-379.

Sevilla MJ, Robledo B, Rementeria A, Moragues MD, Ponton J (2006) A fungicidal monoclonal antibody protects against murine invasive candidiasis. Infect Immun 74:3042-3045.

Walker LA, Munro CA, de Bruijn I, Lenardon MD, McKinnon A, Gow NA (2008) Stimulation of chitin synthesis rescues Candida albicans from echinocandins. PLoS Pathog 4:el000040.

Zilberberg MD, Shorr AF (2009) Fungal infections in the ICU. Infect Dis Clin North Am 23:625-642.

Claims

WHAT IS CLAIMED IS:

1. An antimicrobial polypeptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos. 1, 2, 3, and 4.

2. The antimicrobial peptide of claim 1 wherein the peptide is an anti-bacterial peptide.

3. The antimicrobial peptide of claim 1 wherein the peptide is an anti-fungus peptide.

4. The antimicrobial peptide of claim 1 wherein the peptide is an anti-yeast peptide.

5. The antimicrobial peptide of claim 1 wherein the peptide is an anti-mold peptide.

6. An antibacterial peptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos. 1, 2, 3, and 4.

7. An anti-fungal peptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos.l, 2, 3, and 4.

8. An anti-mold peptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos.l, 2, 3, and 4.

9. An anti-yeast peptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos. 1, 2, 3, and 4.

10. A method of using an antimicrobial peptide in a therapeutic regimen for treating any of a bacterial, fungal, yeast, or mold infection in a mammal comprising administering, either topically or systemically, to said mammal a therapeutically beneficial dosage of said antimicrobial peptide and wherein said peptide is selected from the group consisting of Seq Id Nos. 1, 2, 3, and 4.

11. The method of claim 10 wherein the method comprises administration of said peptide topically.

12. The method of claim 10 wherein the method comprises administration of said peptide systemically.

13. The method of claim 10 wherein the peptide is used to treat a bacterial infection.

14. The method of claim 10 wherein the peptide is used to treat a fungal infection.

15. The method of claim 10 wherein the peptide is used to treat a yeast infection.

16. The method of claim 10 wherein the peptide is used to treat a mold infection.

17. A kit comprising a container containing a therapeutic amount of an antimicrobial peptide of claim 1 and a specification and use instructions document.

18. An antiviral peptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos. 1, 2, and 3.

19. An antitumoral peptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos. 1, and 2.

20. An immunomodulatory peptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

21. An anticancer peptide selected from the group consisting of sequences of amino acids as identified by Seq Id Nos. 1, and 2.

22. A method of using an antiviral peptide in a therapeutic regimen for treating a viral infection or disorder in a mammal comprising administering, either topically or systemically, to said mammal a therapeutically beneficial dosage of said antiviral peptide and wherein said peptide is selected from the group consisting of Seq Id Nos. 1, 2, and 3.

23. A method of using an antitumoral peptide in a therapeutic regimen for treating a cancerous disorder in a mammal comprising administering, either topically or systemically, to said mammal a therapeutically beneficial dosage of said antitumoral peptide and wherein said peptide is selected from the group consisting of Seq Id Nos. 1 , and 2.

24. A method of using an anticancer peptide in a therapeutic regimen for treating a cancerous disorder in a mammal comprising administering, either topically or systemically, to said mammal a therapeutically beneficial dosage of said anticancer peptide and wherein said peptide is selected from the group consisting of Seq Id Nos. 1 , and 2.

25. A method of using an immunomodulatory peptide in a therapeutic regimen for treating any of a microbial, bacterial, fungal, or viral infection , or cancerous or immune disorder in a mammal comprising administering, either topically or systemically, to said mammal a therapeutically beneficial dosage of said immunomodulatory peptide and wherein said peptide is selected from the group consisting of Seq Id Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

26. A polypeptide derived from or having sequence identity with an amino acid sequence of a mammalian immunoglobulin Light (L) or Heavy (H) chain constant domain, said polypeptide of a length of between 3 and 20 amino acids and that exhibits any of antibacterial, antifungal, antiviral, anticancer and immunomodulatory activities.

27. A polypeptide derived from or having sequence identity with an amino acid sequence of a mammalian immunoglobulin Light (L) or Heavy (H) chain constant domain, said polypeptide of a length of between 4 and 11 amino acids and that exhibits any of antibacterial, antifungal, antiviral, anticancer and immunomodulatory activities.

28. A polypeptide derived from or having at least 90% sequence identity with an amino acid sequence of a mammalian immunoglobulin Light (L) or Heavy (H) chain constant domain, said polypeptide of a length of between 4 and 11 amino acids and that exhibits any of antibacterial, antifungal, antiviral, anticancer and immunomodulatory activities.

29. The peptide of any of claims 26, 27 and 28 wherein said peptide is selected from the group consisting of Seq. Id. Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

30. The peptide of claim 29 wherein said peptide forms a beta sheet secondary structure.

31. A polypeptide having antimicrobial activities comprising:

between 4 and 11 amino acids said polypeptide having at least 90% sequence identity with an Ig L or H chain C region amino acid sequence.

32. The polypeptide of claim 31 wherein said sequence is selected from the group consisting of Seq ID Nos. 1, 2, 3, and 4.

33. A polypeptide having antiviral activities comprising:

between 4 and 11 amino acids said polypeptide having at least 90%> sequence identity with an Ig L or H chain C region amino acid sequence.

34. The polypeptide of claim 33 wherein said sequence is selected from the group consisting of Seq ID Nos. 1, 2, and 3.

35. A polypeptide having anticancer activities comprising:

between 4 and 11 amino acids said polypeptide having at least 90%> sequence identity with an Ig L or H chain C region amino acid sequence.

36. The polypeptide of claim 35 wherein said sequence is selected from the group consisting of Seq ID Nos. 1 and 2.

37. A polypeptide having immunomodulatory activities comprising:

between 4 and 11 amino acids said polypeptide having at least 90%> sequence identity with an Ig L or H chain C region amino acid sequence.

38. The polypeptide of claim 37 wherein said sequence is selected from the group consisting of Seq ID Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

39. A bacteriacidal polypeptide comprising:

a sequence of between 4 and 11 amino acids that in-part exhibits a beta sheet secondary structure, said sequence comprising sequence identity with an IgG or IgM H or L chain C region amino acid sequence.

40. The polypeptide of claim 39 wherein said sequence is selected from Seq Id Nos. 1, 2, 3, and 4.

41. A bactericidal polypeptide comprising:

a sequence of between 4 and 11 amino acids that in-part exhibits a beta sheet secondary structure, said sequence comprising at least 90% sequence identity with an IgG or IgM H or L chain C region amino acid sequence.

42. The polypeptide of claim 41 wherein said sequence is selected from Seq Id Nos.

1, 2, 3, and 4.

43. A method of treating any of a bacterial, fungal, or viral infection of a mammal comprising delivering to said mammal a therapeutically effective amount of a polypeptide selected from the group consisting of polypeptides identified by Seq Id Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.

44. The method of claim 43 wherein said polypeptide is delivered in an appropriate dosing to achieve an effective concentration selected from the group consisting of a range between 1-2 ug/ml, 2-5 ug/ml, 3-5ug/ml, 5-10 ug/ml, 5-20 ug/ml, 30-50 ug/ml, 40-50 ug/ml, 50-100 ug/ml.

45. The method of claim 44 wherein said concentration is selected from the group consisting of 2ug/ml, 5ug/ml, lOug/ml, 30 ug/ml, 50 ug/ml, and 100 ug/ml.

46. The method of claim 45 wherein said delivery is either by topical or systemic presentation to said mammal and administered either continuously or intermittently to said mammal at dosing frequencies of any of once, twice, three times, or four times in one day, ( 24hr period) from one day up to one month.