Patent Application
20120121574
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 May 4, 2011, is named RAS1001C.txt and is 10,421 bytes in size.
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.
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.
Immunoglobulins (Igs) otherwise commonly referred to as antibodies (Abs) are composed of two identical sized L chains (23 kD) and two identical H chains which range in size, (i.e., between 50-70 kD). 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.
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—VL (110 amino acids) and CL (110 amino acids), 2) H chain—VH (110 amino acids) and CH (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 (
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 VH and VL. Finally, the regions between the CDRs in the V region are called the framework regions.
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, Gabrielli et al., 2009). 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.
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 and antimold), antiviral, anticancer and immunomodulatory activities against a wide spectrum of microbial agents and tumor and immunological disorders.
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.
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).
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; Cornely 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).
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-β-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).
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).
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 and 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.
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 CH regions 1, 2, 3, and 4, the CL 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 and 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.
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 and antifungal) antiviral, anticancer and immunomodulatory activity, regardless of their origin in either CL or CH or, with respect to CDR-related peptides, VL or VH regions, exclusive of specific known amino acid sequences derived from any of said origins with known antimicrobial, antiviral or anticancer activity.
In a third embodiment, individual peptides of the invention exhibit alone or in combination any of antimicrobial (antibacterial and antifungal), antiviral, anticancer and immunomodulatory activity in vivo in a host mammal. In a related embodiment, the antimicrobial (antibacterial and antifungal), antiviral, anticancer and immunomodulatory activity of the peptides is unrelated to any activity of the complete Ig from which they are derived.
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 of the bioactive antibody peptides possess a primary family structural motif that as one of ordinary skill in the art will recognize comprises amino acids of one type (e.g., polar, nonpolar, charged, noncharged, bulky versus small R group, etc.) interchanged with amino acids of another type. Specifically, with respect to the invention polypeptide family comprising broad spectrum antimicrobial, anticancer, immunomodulatory, and other activities, the structural unity is found in the general formula XZ*1X1Z*11XZ (consensus sequence 1) as described in detail below. Additionally, the peptides can be described as possessing further unity through their respective relationships found in alternative structural formulas as also disclosed herein (consensus sequences 2, 3, 4, 5 and 6). With respect to said formulas, amino acid substitutions are allowable as one of skill will recognize the evolutionarily acceptable amino acid substitutions commonly found in peptide families possessing similar activities. Further still, the peptides may also possess a beta sheet secondary structure. In additional embodiments, the naturally occurring Ig C region and CDR region peptides, as well as their alanine, serine, or otherwise 80% and 90% sequence identity with the native isolated sequence possess a similar primary structure given the evolutionarily accepted equivalent amino acids substitutions as depicted in Table IV. Thus, for every 10 amino acids in such a C region derived polypeptide, one or two amino acids can likely be substituted for an amino acid not naturally found in that sequence location while preserving considerable activity or even improving the activity of said peptide's antimicrobial, antiviral immunomodulatory or anticancer activities and remaining within a base level structural unity based on similar charge configurations through the consensus formulas. In a related embodiment, peptides of the invention derived from the C domain, identified by Seq. Id. Nos. 14-24 possess 90% sequence identity with one another and with Seq. Id. No. 1, while Seq. Id. No. 25 possesses an 80% sequence identity with Seq. Id. No. 1. Peptides of the invention derived from the C domain, identified by Seq Id. Nos. 27-37 possess 90% sequence identity with one another and with Seq. Id. No. 2. Additionally, peptides of the invention derived from the CDR domain 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.
In another embodiment, the peptide having antimicrobial (antibacterial and antifungal), 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.
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 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).
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.
In a first embodiment, polypeptides are disclosed that exhibit a variety of activities comprising 1) antimicrobial, which includes antibacterial (antibacterial includes anti-Gram positive and anti-Gram negative bacteria), 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
In a further preferred embodiment, the disclosed polypeptide sequences, possessing a broad spectrum of antimicrobial (including antibacterial which includes anti-Gram positive and anti-Gram negative bacteria, 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 (Gram positive or Gram negative, mold or yeast) or through inhibition of virus replication or cancer cell growth or indirect action by immunomodulatory therapeutic action in the host mammal.
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.
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 (blast.ncbi.nlm.nih.gov/Blast.cgi); multiple alignment with ClustalW (www.ebi.ac.uk/Tools/clustalw/); peptide cutter (expasy.org/tools/peptidecutter/) with cathepsins, trypsins and/or chymotrypsin-high specificity; calculation of Isoelectric Point (pI) with The Sequence Manipulation Suite 2 (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 (
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 VHCDR3 peptide.
Table II shows the sequences of the peptides of the present invention.
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 for 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. 5-23, and Seq. Id. Nos. 27-37, these peptides can manifest activity with at least a 10% change in their respective amino acid sequences from the naturally occurring peptide at almost any position. Further, as exampled by Seq. Id. No. 25, peptides can manifest activity with at least 20% change in their amino acid sequences from the naturally occurring peptide. Thus, we conceive and comprehend the claimed invention to include amino acid sequences possessing at least between 80% and 90% sequence identity with polypeptides derived from C and CDR regions of Igs possessing any of antibacterial, antifungal (antiyeast and 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.
Candida albicans
C. albicans
C. albicans
Saccharomyces
cerevisiae
Cryptococcus
neoformans
Malassezia furfur
Aspergillus
fumigatus
Staphylococcus
aureus
Escherichia coli
Klebsiella
pneumoniae
Pseudomonas
aeruginosa
C. albicans
C. albicans
C. albicans
C. albicans
S. cerevisiae
C. neoformans
M. furfur
S. aureus
E. coli
K. pneumoniae
P. aeruginosa
C. albicans
C. albicans
C. neoformans
M. furfur
A. fumigatus
S. aureus
E. coli
K. pneumoniae
P. aeruginosa
M. furfur
A. fumigatus
S. aureus
E. coli
K. pneumoniae
P. aeruginosa
C. albicans
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 microgram/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 versus untreated cells, by t test). As disclosed in
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 50 micrograms 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 versus SP treated mice) (
In vivo activity of N10K against vaginal candidiasis caused in mice by the fluconazole-susceptible C. albicans SA40 strain. The anticandidal therapeutic activity of N10K was evaluated in a murine model of vaginal candidiasis. Groups of 5 mice were injected subcutaneously with 0.02 mg of estradiol benzoate in 100 μl of saline solution, 48 hr yeast challenge and weekly thereafter. Then, mice given intravaginally 106 cells of the fluconazole-susceptible C. albicans strain SA40 in 20 μl of saline solution on day 0 and were sampled for intravaginal CFU. N10K, 25 micrograms, was intravaginally administered in comparison to an irrelevant peptide (SP) as control, 1, 24 and 48 hours after the infectious challenge and sampled for intravaginal CFU at days 1, 2, 5, 7, 14, 21, and 28. The statistical significance was assessed by two-tailed Student's t test. On days 1, 2, 5, 7, 14, and 21, the differences in the CFU vaginal counts between N10K treated, untreated and SP treated animals were statistically significant (P<0.05) (Table V).
In vivo activity of N10K against vaginal candidiasis caused in mice by the fluconazole-resistant C. albicans AIDS68 strain. The anticandidal therapeutic activity of N10K was evaluated in a murine model of vaginal candidiasis. Groups of 5 mice were injected subcutaneously with 0.02 mg of estradiol benzoate in 100 μl of saline solution, 48 hr yeast challenge and weekly thereafter. Then, mice given intravaginally 106 cells of the fluconazole-resistant C. albicans strain AIDS68 in 20 μl of saline solution on day 0 and were sampled for intravaginal CFU. N10K, 25 micrograms, was intravaginally administered in comparison to an irrelevant peptide (SP) as control, 1, 24 and 48 hours after the infectious challenge and sampled for intravaginal CFU at days 1, 2, 5, 7, 14, 21, and 28. The statistical significance was assessed by two-tailed Student's t test. On days 1, 2, 5, 7, 14, and 21, the differences in the CFU vaginal counts between N10K treated, untreated, SP treated and fluconazole treated animals were statistically significant (P<0.05) (Table VI).
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 micrograms/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
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 micrograms/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
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 micrograms/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
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 micrograms/ml, or in distilled water as control growth. After the incubation period, the cell suspensions were 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
In vitro activity of N10K against A. fumigatus 1163 strain. The microbicidal activity of N10K 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 N10K at the concentration of 100 micrograms/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
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 micrograms/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
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 micrograms/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
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 micrograms/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
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 micrograms/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
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 20 UI/ml rIL-2 for 24 hr before treatment with N10K, then the peptide was added at increasing concentrations (1, 10 and 20 micrograms/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 570/595 nm. AlamarBlue added to the complete RPMI1640 medium was used as blank. N10K demonstrated not to be cytotoxic for PBMC when it was employed in the 1-20 micrograms/ml concentration range, so the lowest and the intermediate doses of 1 and 10 micrograms/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 micrograms/ml) and after with rIL-2 (20 UI/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 micrograms/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 micrograms/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 (20 UI/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 supernatants 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 micrograms/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
In vitro activity of N10K against B16F10-Nex2, SKme128 and SKme125 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, SKme128 and SKme125 cells (5×103 cells/well) in 100 microliters per well for 12 hr at 37° C. 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
In vitro immunomodulatory activity of N10K on human immune cells. Human monocytes or PMN (both 10×106/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 micrograms/ml). After incubation culture supernatants were collected and tested for cytokines production by specific ELISA.
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 micrograms/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 versus untreated cells, t test). As disclosed in
In vivo activity of T11F against vaginal candidiasis caused in mice by the fluconazole-susceptible C. albicans SA40 strain. The anticandidal therapeutic activity of T11F was evaluated in a murine model of vaginal candidiasis. Groups of 5 mice were injected subcutaneously with 0.02 mg of estradiol benzoate in 100 μl of saline solution, 48 hr yeast challenge and weekly thereafter. Then, mice given intravaginally 106 cells of the fluconazole-susceptible C. albicans strain SA40 in 20 μl of saline solution on day 0 and were sampled for intravaginal CFU. T11F, 25 micrograms, was intravaginally administered in comparison to an irrelevant peptide (SP) as control, 1, 24 and 48 hours after the infectious challenge and sampled for intravaginal CFU at days 1, 2, 5, 7, 14, 21, and 28. The statistical significance was assessed by two-tailed Student's t test. On days 1, 2, 5, 7, 14, and 21, the differences in the CFU vaginal counts between T11F treated, untreated and SP treated animals were statistically significant (P<0.05) (Table VII).
In vivo activity of T11F against vaginal candidiasis caused in mice by the fluconazole-resistant C. albicans AIDS68 strain. The anticandidal therapeutic activity of T11F was evaluated in a murine model of vaginal candidiasis. Groups of 5 mice were injected subcutaneously with 0.02 mg of estradiol benzoate in 100 μl of saline solution, 48 hr yeast challenge and weekly thereafter. Then, mice given intravaginally 106 cells of the fluconazole-resistant C. albicans strain AIDS68 in 20 μl of saline solution on day 0 and were sampled for intravaginal CFU. T11F, 25 micrograms, was intravaginally administered in comparison to an irrelevant peptide (SP) as control, 1, 24 and 48 hours after the infectious challenge and sampled for intravaginal CFU at days 1, 2, 5, 7, 14, 21, and 28. The statistical significance was assessed by two-tailed Student's t test. On days 1, 2, 5, 7, 14, and 21, the differences in the CFU vaginal counts between T11F treated, untreated, SP treated and fluconazole treated animals were statistically significant (P<0.05) (Table VIII).
In vitro activity of T11F against caspofungin resistant C. albicans strain UM4. The candidacidal activity of T11F 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 T11F at the concentration of 5 or 2 micrograms/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
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 T11F at the concentration of 20, 10 or 5 micrograms/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
In vitro activity of T11F against C. neoformans 6995 strain. The fungicidal activity of T11F 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 T11F at the concentration of 10, 5 or 4 micrograms/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
In vitro activity of T11F against M. furfur 101 strain. The fungicidal activity of T11F 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 T11F at the concentration of 2, 1 or 0.5 micrograms/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
In vitro activity of T11F against A. fumigatus 1163 strain. The fungicidal activity of T11F 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 T11F at the concentration of 100 micrograms/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
In vitro activity of T11F against S. aureus 29213 strain. The bactericidal activity of T11F 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 T11F at the concentration of 50, 40 or 30 micrograms/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
In vitro activity of T11F against E. coli ATCC 25922 strain. The bactericidal activity of T11F 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 T11F at the concentration of 5, 3 or 2 micrograms/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
In vitro activity of T11F against K. pneumoniae ATCC 700603 strain. The bactericidal activity of T11F 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 T11F at the concentration of 100, 80 or 60 micrograms/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
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 T11F at the concentration of 10, 5 or 2 micrograms/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
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 20 UI/ml rIL-2 for 24 hr before treatment with T11F, then the peptide was added at increasing concentrations (1, 10 and 20 micrograms/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 570/595 nm. AlamarBlue added to the complete RPMI1640 medium was used as blank. T11F demonstrated not to be cytotoxic for PBMC when it was employed in the 1-20 micrograms/ml concentration range, so the lowest and the intermediate doses of 1 and 10 micrograms/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 micrograms/ml) and after with rIL-2 (20 UI/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 micrograms/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 micrograms/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 (20 UI/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 supernatants 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 (
In vitro activity of T11F against B16F10-Nex2, SKme128 and SKme125 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, SKme128 and SKme125 cells (5×103 cells/well) in 100 microL per well for 12 hr at 37 u° C. 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
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 micrograms/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
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 micrograms/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
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 micrograms/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
In vitro activity of H4L 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 H4L at the concentration of 100 micrograms/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
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 micrograms/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
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 micrograms/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
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 micrograms/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
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 micrograms/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
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 20 UI/ml rIL-2 for 24 hr before treatment with H41, then the peptide was added at increasing concentrations (1, 10 and 20 micrograms/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 570/595 nm. AlamarBlue added to the complete RPMI1640 medium was used as blank. H4L demonstrated not to be cytotoxic for PBMC when it was employed in the 1-20 micrograms/ml concentration range, so the lowest and the intermediate doses of 1 and 10 micrograms/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 micrograms/ml) and after with rIL-2 (20 UI/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 micrograms/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 micrograms/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 (20 UI/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 supernatants 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 (
In vitro activity of MoA VHCDR3 against M. furfur 101 strain. The fungicidal activity of MoA VHCDR3 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 micrograms/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
In vitro activity of MoA VHCDR3 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 VHCDR3 at the concentration of 100 micrograms/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
In vitro activity of MoA VHCDR3 against S. aureus 29213 strain. The microbicidal activity of MoA VHCDR3 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 VHCDR3 at the concentration of 100 micrograms/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
In vitro activity of MoA VHCDR3 against E. coli ATCC 25922 strain. The bactericidal activity of MoA VHCDR3 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 VHCDR3 at the concentration of 100 micrograms/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
In vitro activity of MoA VHCDR3 against K. pneumoniae ATCC 700603 strain. The bactericidal activity of MoA VHCDR3 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 VHCDR3 at the concentration of 100 micrograms/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
In vitro activity of MoA VHCDR3 against P. aeruginosa ATCC 25853 strain. The bactericidal activity of MoA VHCDR3 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 VHCDR3 at the concentration of 100 micrograms/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
In vitro immunomodulatory activity of MoA VHCDR3 on murine immune cells.
Given that VHCDR3 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 VHCDR3 or VLCDR3 (used as a negative control) intraperitoneally 4 hr before (200 micrograms), and 1 (100 micrograms) and 2 (100 micrograms) 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 VHCDR3 or MoA VLCDR3 are disclosed in
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 defense responses.
Our evidence reports that PM very rapidly take up the MoA VHCDR3 peptide, and 18 hr post treatment this peptide is still associated to the cells. It is possible that MoA VHCDR3 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.
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 VHCDR3 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-α. Indeed, this pathway of activation is also confirmed by the inhibition of TNF-α 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-α mRNA was detected 1 hr post stimulation with MoA VHCDR3, suggesting that the signal transduction pathway from Akt leads to cytokine gene expression, as depicted in
Given that TNF-α 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 VHCDR3 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-α, one could posit that the over expression of TLR-4 is secondary and dependent on TNF-α production.
In relation to its protective response, there are convincing arguments pointing to the key role of TLR-4 in microbial antigen recognition. 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 VHCDR3 is ineffective against C. albicans cells in vitro. A possible explanation for this could be that natural immune cells are activated by MoA VHCDR3 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 VHCDR3 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.
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 defense. 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.
In Vitro Comparative Activity of N10K and N10K Alanine-Substituted Derivatives (asd) Against Candida albicans SC5314 Strain
Derivatives of the decapeptide N10K were obtained by alanine scanning in order to improve its candidacidal activity and identify the functional contribution of each residue. The alanine-substituted derivatives (asd), defined according to the position held by the alanine-substituted aminoacid, were tested against C. albicans by a conventional colony forming unit (CFU) as previously described (Polonelli et al., 2003). Briefly, cells of C. albicans SC5314 were incubated at 37° C. for 6 hours in the presence of N10K or N10K asd at different scalar concentrations or distilled water as control. After incubation, cell suspensions were plated on Sabouraud dextrose agar and incubated at 30° C. for 48 hours when CFU were counted. Based on several independent replications, the EC50 value of each peptide was calculated by nonlinear regression analysis using Graph Pad Prism 4.01 software, San Diego, Calif., USA. As disclosed in Table IX, N10K asd showed differential candidacidal activity in vitro against cells of C. albicans SC5314 strain in comparison to N10K. We note that with respect to the present experiments in this example NK10 and its derivatives show, generally, a higher activity level than the activity levels observed in NK10 peptide experiments presented in Example I. This is because the peptides, purified for use in the present example, were processed to a greater extent than for the NK10 in the experiments discussed in Example I suggesting that per unit used, the concentration of active peptide in the experiments of this example is greater per unit volume.
Thus, as indicated by the data presented in Table IX, alanine substitutions of NK10 peptide sequence show activity against yeast type fungus.
In Vitro Immunomodulatory Activity of N10K and N10K asd on Human Immune Cells.
The same battery of N10K asd was tested for their immunomodulatory activity on human monocytes or polymorphonucleated leukocyte PMN as previously described (Gabrielli et al., 2009). In particular, monocytes or PMN (both 10×106/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 as a positive control, an inactive peptide (MSTAVSKCAT, Seq. ID. No. 26) as a negative control (NC) and N10K and N10k asd peptides (all 10 μg/ml). After incubation culture supernatants were collected and tested for cytokines production by specific ELISA. As disclosed in
Specifically,
The microbicidal activity of T-11-F peptide alanine scanning derivatives (Seq. Id. Nos. 27 to 37) were evaluated by a conventional colony forming unit (CFU) assay. Cells of C. albicans SC5314 were incubated at 37° C. for 6 hours in the presence of peptides at the concentration of 5, 3 or 2 micrograms/ml, or in distilled water as control growth. After the incubation period, the cell suspensions were plated on Sabouraud dextrose agar, then incubated at 30° C. and observed for CFU enumeration after 48 hours.
As shown in
In vitro microbicidal activity of N10K derivative with 2 amino acid substitutions, or 80% sequence identity with native N10K, namely peptide Seq Id. No. 25 was tested against Candida albicans SC5314 as determined by Colony Forming Unit assay. As shown in Table X, candidacidal activity is present in a peptide derived from N10K with two amino acids substitutions.
In further embodiments, the invention peptides can be used in various methodologies for treating invasive bacterial, fungal (yeast and 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.
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 2, 3, 5, 10, 20, 30, 40, 50, 60, 80, and 100 micrograms/ml.
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 and antifungal), antiviral, or immunomodulatory polypeptide is made between one and four days and alternatively every four days or (24 hr 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.
In yet additional embodiments of the current invention, the naturally occurring Ig C region and CDR region peptide, as well as their alanine, serine, or otherwise 80 to 90% identity substituted derivatives, possess a similar primary structure given the evolutionarily accepted equivalent amino acid substitutions as one of ordinary skill in the art will understand as depicted in Table IV. Thus, at the base level of characterization, the amino acid sequences of the invention possess unity of invention based on the similar charge configurations of the various possible R groups positioned in sequence to one another as illuminated through the consensus formula XZ*1X1Z*11XZ as shown in Table IV. The varied amino acid sequence type motif of the present formula provides support for the conception that the active motif comprises between one and three large aromatic, or neucleophilic, or otherwise charged (basic or acid) amino acid groups (can be paired amino acids) that are spaced from one another (pairs, if present, are spaced from other pairs) by between two to three amino acids that are themselves either small, aliphatic, or hydrophopic (non polar or small polar R groups). Each of X, *, Z, and 1 represent amino acids of varying types having structural and/or evolutionary similarity or interchangeability. For example, amino acid “Z” is a structural amino acid. Amino acid Z, and the combination Z*, (where “*” represents a second structural amino acid, if present, making a double amino acid structural motif “Z*”), are generally large charged (basic or acidic), aromatic or bulky amino acids, or that possess cross-linking capability, such a Cysteine, in the polypeptide number 2-3, 6-7-8, and 10-11th positions for 10 or 11 amino acid polypeptides. Thus depending upon the total length of the active polypeptide of the invention the position number of the type of amino acid can vary within discernable limits and still remain within the overriding formula disclosed above. Specifically, Z and * amino acids are also defined as any of Histidine (H), Cysteine (C), Glutamine (Q), Phenylalanine (F), Tryptophan (W), Tyrosine (Y), Lysine (K), Glutamic acid (E), and Arginine (R). With respect to amino acid “1” in polypeptide positions number 3-4, 5-6, 8-9, the amino acids in these positions are hydrophobic nonpolar and generally minimal sized R groups. Specifically, amino acids in “1” position can be Glycine (G), Valine (V), Leucine (L), Isoleucine (I), Methionine (M), and Alanine (A). Amino acids of the “X” type are generally small polar oxygen containing R group in the 4-5 and 9-10 amino acid positions. Amino acids of the X type can be Threonine (T), Serine (S), Asparagine (N), and Aspartic acid (D). We further note that the 1-X amino acid combinations can be mixed in sequence position. The consensus 1 formula may also be expressed in the alternative as consensus sequences 2 or 3 wherein the amino acid choice “*” is represented as “B” and can be cystine (C), Glutamine (Q), Glutamic acid (E), and Arginine (R), Tyrosine (Y), and Phenylalanine (F), the consensus 2 providing a listing of those amino acids interchangeable for each position, and consensus formula 3 providing a further derivation wherein generally hydrophobic aliphatic and small polar uncharged R group amino acids, labeled “Hyd” which can be any of amino acids Glycine (G), Valine (V) Leucine (L), Isoleucine (I), Methionine (M), and Alanine (A), Serine (S), Threonine (T), Asparagine (N), and Aspartic acid (D). A fourth consensus alignment motif is provided in the formula “Z-B-Hyd-Hyd” which can be active in singular and multiple repeat motifs as in T11F sequence motifs CRVD (Seq. Id. No. 38) and HRGL (Seq. Id. No. 39), or sequence H4L and its “HEAL” (Seq. Id. No. 3) amino acid motif, or N10K sequence motif “TCLV” (Seq. Id. No. 40), or MoA VHCDR3 sequence motif “QYGN” (Seq. Id. No. 41). Still further, another consensus motif is discernable in consensus 5 recognizing a motif of six amino acids wherein the terminal 5′ and 3′ amino acids are of the “Hyd” type while the inner amino acids possess the motif “Hyd-X-Z-B-Hyd-Hyd” as found in T11F (VDHRGL) (Seq. Id. No. 42), N10K (VSLTCL) (Seq. Id. No. 43), and MoA VHCDR3 “GNLWFA” (Seq. Id. No. 44). Further still a sixth consensus motive (consensus 6) is discernable in that a combination of consensus 4 and 5 but further defined where spacing allows for a “1” amino acid type present with a Histidine in “Z” position. Although each of the six formula motifs are discernable alone, they further in combination with one another provide for unity of invention with one another.
Various publications are cited herein which are hereby incorporated by reference in their entirety.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/US2010/056763 | Nov 2010 | US | national |
This invention is filed as a Continuation-in-Part and claims the benefit of PCT/US10/56763 filed Nov. 15, 2010, and Provisional application Ser. No. 61/261,738 filed Nov. 16, 2009.
