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Antibodies as a source of anti-infective peptides: an update Walter Magliani1, Laura Giovati1, Tecla Ciociola1, Martina Sperindè1, Claudia Santinoli1, Giorgio Conti1, Stefania Conti*,1 & Luciano Polonelli1

ABSTRACT This review focuses on antibodies (Abs) and their function in immune protection, with particular emphasis on microbicidal Abs. Some aspects of Abs and Ab–drug conjugates as targeting therapeutic agents are also discussed. The main aim, however, is devoted to Ab-derived peptides modulating functions of the immune system and to the latest experimental evidence of Abs as a source of anti-infective and antitumor peptides derived from their complementarity determining regions and constant regions. Antibodies & anti-infective immune protection All living multicellular organisms ensure their protection toward pathogens using sophisticated mechanisms of immune defense. During evolution, many different cells and molecules have been selected as components of two branches, innate and adaptive, of the immune system. While the former is considered the only active in invertebrates, both coexist in vertebrates. Previously thought to be functionally independent, they are currently considered intimately linked. Following engagement of innate immune defenses, the adaptive system is called into action by triggering humoral and/or cell (T lymphocyte)-mediated immunity in response to immunogenic components of the pathogen (antigens, Ags). Humoral immunity is mediated by antibodies (Abs), or immunoglobulins (Igs), produced and secreted by fully differentiated, mature B lymphocytes (plasma cells). Each Ab, elicited by and specific to an individual antigenic determinant (epitope), recognizes and binds to the Ag through a ‘lock and key’ interaction. Following immune complex formation, other cells and molecules of the immune system, such as macrophages and complement factors, can be recruited for the elimination of the Ag itself. Together, humoral and cellular immune responses can control pathogen’s replication and eliminate infected and altered cells. Importantly, adaptive immunity is characterized by immunological memory [1] . Following a first contact with the Ag, the immune system mounts a primary response, during which IgM first, then IgG are produced. A second stimulation by the same Ag may lead to a secondary response characterized by the production of IgG, IgA and/or IgE. Abs, constituting an extremely large repertoire of diversified molecules, represent a sophisticated product of evolution in vertebrates, which, together with the innate and cellular immunity, are essential to the integrity of the individual. Due to their features, Abs also represent an invaluable tool in biotechnology and biomedicine for research, diagnostics and therapy purposes. A detailed description of the structure and functions of Abs (summarized in Box 1) [2] is beyond the scope of this review. In the field of infectious disease, Abs play a major role in protection. Through noncovalent interactions between the epitope and their Ag binding site (paratope), Abs form immune complexes with

KEYWORDS

• antibodies • antibody-

derived immunomodulatory peptides • antibodyderived anti-infective peptides • antibodyderived antitumor peptides • cryptides • microbicidal antibodies • therapeutic antibodies

Department of Biomedical, Biotechnological & Translational Sciences, Microbiology & Virology Unit, University of Parma, 43125 Parma, Italy *Author for correspondence: Tel.: +39 052 103 3492; Fax: +39 052 190 3802; [email protected] 1

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Review  Magliani, Giovati, Ciociola et al. specific Ags, thus promoting their neutralization and elimination, via the engagement of other components of the immune system. By their ‘fragment crystallizable’ (Fc) region, in fact, Abs can interact with a number of specialized molecules, such as complement factors, cellular Fc receptors and other cell-signaling molecules. Fc region is responsible for the effector functions of the Ab molecule, but can also affect binding affinity and specificity of the Ab, contributing to Ag recognition [3,4] . Classical protective Ab functions include prevention of the entry of toxins and viruses into cells (neutralization), complement activation and complement-dependent cytotoxicity, opsonization and Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity (ADCC). The significance of ADCC-mediating Abs has recently been highlighted in influenza virus and HIV infections, and represents an important issue in vaccine development [5,6] . Abs functions account for passive transplacental immunoprotection and effectiveness of strategies for active (vaccination) and passive (immunoprophylaxis/immunotherapy) immunization. More recently, other nonclassical Ab functions have been discovered, that include antimicrobial activity, alteration of microbial signal transduction, immunomodulation through pro- or anti-inflammatory properties and modulation of microbial physiology [7] . Abs whose paratopes present catalytic activity constitute a class of enzyme-like catalysts with tailored specificities. These catalytic Abs can occur naturally and ubiquitously in healthy individuals, but are preferentially found in the

Ig repertoire of individuals with autoimmune disease. Their pathogenic or beneficial role still remains unclear [8,9] . Microbicidal Abs, characterized by diversified and incompletely clarified mechanisms of action, have aroused particular interest. ●●Microbicidal antibodies

Microbicidal Abs (‘antibiobodies’, killer Abs [KAbs]) are Abs that can function directly as effector molecules against microbial agents without the assistance of phagocytic cells or complement. First described as yeast killer toxin (KT)-like polyclonal anti-idiotypic Abs elicited by immunization of rabbits with a monoclonal Ab (mAb KT4) capable of neutralizing the fungicidal activity of a KT [10] , KAbs were subsequently detected in other experimental and natural conditions. Systemic and mucosal anticandidal protective KAbs could be elicited by parenteral or intravaginal immunization of mice or rats with mAb KT4. Similar natural anti­ receptor KAbs were consistently detected in the vaginal fluid of animals experimentally infected by Candida albicans cells bearing the receptor for KT (KTR), in the vaginal fluid of women afflicted with recurrent vaginal candidiasis and in the serum, saliva and/or bronchial washing of HIV-positive patients frequently infected by KTR-bearing fungi. Unlimited amounts of homogeneous and well-characterized KAbs were obtained through the generation of a monoclonal KAb and a recombinant KAb. Regardless of the mode of production, these KAbs proved to be microbicidal in vitro against many tested pathogenic agents, including fungi (yeasts and moulds),

Box 1. Antibodies: structure and function. ●● Antibodies (Abs) are large Y-shaped glycoproteins elicited by and specific to an individual antigenic determinant (epitope) expressed on a

‘non-self’ macromolecule, known as antigen (Ag) ●● Microorganisms and viruses are a mosaic of epitopes/Ags against which the Ab response is usually polyclonal, in other words, constituted by Abs with different specificities generated by different plasma cells ●● Abs consist of four polypeptide chains, two identical heavy (H) chains and two identical light (L) chains, linked by disulfide bonds, each composed by variable (V) and constant (C) domains ●● Within the ‘fragments Ag-binding’ VH and VL domains interact to form the Ag-combining site (paratope) that recognizes and binds the Ag with a ‘lock and key’ interaction ●● Within each V domain lie six hypervariable loops, known as ‘complementarity determining regions’ (L1, L2, L3, H1, H2 and H3), supported by four conserved ‘framework regions’ that define the positioning of the complementarity determining regions ●● ‘Fragment crystallizable’, composed by CH2 and CH3 domains, mediates the effector functions of the Ab molecule, once the Ab–Ag complex is formed, through the recruitment of other immune system cells and molecules, for the elimination of the Ag ●● Fragment crystallizable can also affect the affinity or kinetics of Ag binding ●● Abs can directly neutralize their target Ags, as for viruses and toxins (neutralizing Abs) ●● Abs can be responsible for complement-dependent cytotoxicity, opsonization and Ab-dependent cellular phagocytosis, and Abdependent cellular cytotoxicity

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Antibodies as a source of anti-infective peptides: an update  bacteria (Gram-positive cocci and Mycobacterium tuberculosis) and protozoa (Leishmania major and Leishmania infantum, Acanthamoeba castellanii). Their antibiotic-like activity was related to KAbs binding to KTRs on the microbial cell surface, mainly constituted by β-glucans, at least in fungi. When passively transferred, KAbs conferred a significant protection against infections by KTRbearing microorganisms. Even though the clinical relevance of these observations still needs to be assessed, KAbs may be part of the Ab repertoire and may play a relevant role in immune protection, reviewed in [11,12] . Candidacidal KAbs have also recently been elicited through the manipulation of the idiotypic cascade [13] . Based on the relevance of cell wall β-glucans as constituent of fungal KTRs and the protective role of anti-β-glucan Abs, a novel glycoconjugate vaccine (laminarin, a soluble β-glucan, conjugated with the diphtheria toxoid CRM197) has been proposed, able to efficiently protect against the major fungal p ­ athogens C. albicans and Aspergillus fumigatus [14] . Other KAbs were subsequently described. A mAb designated C7, produced against a cell wall stress mannoprotein of C. albicans, was able to react with the Als3 protein and enolase present in the fungal cell wall and to exert a direct candidacidal activity, other than inhibition of yeast filamentation and tissue adhesion. The killing activity was also directed against other clinically relevant fungi, including drugresistant species. More importantly, mAb C7 was protective in a murine model of systemic candidiasis. A genome-wide expression profiling to identify genes differentially expressed in response to mAb C7 allowed to clearly relate the candidacidal activity to the blockage of the reductive iron uptake pathway of the yeast [15–17] . Abs against spirochetes causing Lyme disease and relapsing fever, directly bactericidal in the absence of complement, have been described for some time [18,19] . More recently, the bactericidal activity against Borrelia burgdorferi has been related to the binding of the Ab V region to the outer surface protein B on the bacterial cell wall outer membrane (OM). Binding was followed by the formation of membrane blebs and openings with subsequent increased permeability. This resulted in bacterial osmotic lysis with a mechanism different from the one of the membrane attack complex formed after complement recruitment and reminiscent of the ‘carpet’ mechanism of action of cationic antimicrobial

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peptides. This bactericidal effect was not transferable to Escherichia coli expressing rOspB on its OM, suggesting the involvement in the phenomenon of peculiar features, such as the absence of LPS and the presence of cholesterol-containing glycolipids, in Borrelia OM [20,21] . Overall, these observations strengthen the hypothesis of the existence of a variety of KAbs directed against crucial microbial targets. Whether KAbs may have some clinical relevance, or the eliciting Ags could be immunodominant in natural infections, is not currently known. Even the interrelationship between KAbs and immune protection, their effective concentration and the possible interference of concomitant blocking Abs directed against the KAbs’ epitope or idiotype need to be established. Antibodies as targeting therapeutic agents Due to their specificity and effectiveness, since the end of 19th century, animal and human Abs have been used in serum prophylaxis and serum therapy against a variety of infectious diseases, not without significant adverse side effects, such as serum sickness and immediate hypersensitivity. With the advent of antibiotics, these medical practices have largely fallen into disuse. Many years later, mAb and recombinant Ab technologies provided the means to safely produce huge amounts of Abs and led to the renaissance of Ab-based therapies, mostly outside the field of infectious diseases. In June 1986, the US FDA approved the first unmodified murine mAb, muromonab-CD3, as immunosuppressive agent for the prophylaxis of acute organ transplant rejection [22] . The approval in 1997 of the first chimeric mAb, the anti-CD20 molecule rituximab, for use in cancer patients, initiated the era of the ‘targeted’ cancer therapy, in other words, the use of mAbs developed to inhibit oncogenic proteins or survival factors selectively expressed by tumors. Since then, more than 30 mAbs were approved in the USA and EU and a similar number is currently in Phase III clinical trials. Human and humanized mAbs are increasingly prevailing over chimeric mAbs. While cancer and autoimmune diseases remain the dominant therapeutic indications, mAbs for the treatment or prevention of other human pathological conditions, such as infectious, cardiovascular and neurodegenerative diseases, and organ transplantation have also been marketed or are in the late

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Review  Magliani, Giovati, Ciociola et al. stages of the drug pipeline [23,24] . Inhibition of proinflammatory cytokines, such as TNF-α, and cytokine receptors, such IL-6R and others, is a common mechanism of action of mAbs in the therapy of relevant chronic inflammatory diseases. The therapeutic armamentarium approved for the treatment of hematopoietic and solid neoplasms includes mAbs targeting tumor-associated Ags, mAbs neutralizing trophic signals provided by the tumor stroma and immunostimulatory mAbs, that exert their antineoplastic activity via distinct mechanisms. MAbs in IgG format are characterized by a prolonged circulatory half-life, due to their recycling, and can mediate effector functions, such as complement-dependent cytotoxicity, ADCC and Ab-dependent cellular phagocytosis, by interacting with Fcγ receptors expressed on natural killer cells, macrophages and other leukocytes [23] . The recent approval of two effective Ab– drug conjugates has enabled a new form of targeted cancer chemotherapy. This has spurred tremendous research in this field, leading to a plethora of Ab–drug conjugates in clinical development [25–27] . In the field of infectious diseases, Abs have not had the same success, despite numerous current ongoing studies and the actual requirement for new therapeutic tools to address unmet medical needs associated with microbial and viral infections. The emergence and spread of new pathogenic agents for which no therapy exists and of microorganisms highly resistant to currently available drugs, and the shortage in the drug pipeline of new anti-infective agents, particularly those with novel mechanisms of action, are some of the reasons that underline the critical relevance of having novel anti-infective therapeutic options [28–30] . Abs could have great potential in this field, but their use in the treatment of infectious disease is still at its infancy. Despite many candidates are in clinical development, to date only two mAbs are approved for clinical use. Palivizumab (Synagis ®), a humanized neutralizing mAb against the viral F glycoprotein, is indicated for prevention of respiratory syncytial virus infection, the most common lower respiratory tract infection in infants. Given monthly intramuscularly over the season at risk (from October–November to March–April in temperate climates), palivizumab significantly reduces severe infections, as attested by the decrease in hospitalization and

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intensive care unit admission and mechanical ventilation rates. However, due to the high costs associated with palivizumab prophylaxis, several limitations to its use have been introduced by national boards [31,32] . More recently, the FDA approved raxibacumab, a recombinant human IgG1 mAb directed against Bacillus anthracis protective antigen, to treat and prevent inhalational anthrax [33] . Many other mAbs against relevant infectious agents have not performed as well as anticipated in clinical trials. As mAbs magnitude could pose significant limitations to their possible therapeutic applications, Ag-binding fragments with significant reduction in size, such as single-chain Fragment variable, diabodies, minibodies, nanobodies, small Ab mimetics and others, have been produced. These products are more straightforward to manufacture also in nonmammalian systems, can bind epitopes inaccessible to conventional large mAbs and more readily penetrate through tissue and solid tumors. Of course, Ab fragments suffer from some potential drawbacks, including lack of Fc effector functions and recycling, and short in vivo half-life. Plenty of different Ab-derived constructs and methods for their production have been proposed and widely reviewed elsewhere and will not be considered here. Antibodies as a source of antimicrobial peptides In 1970, tuftsin was firstly described as an Ab-derived phagocytosis-stimulating tetrapeptide  [34] . Its sequence (TKPR) is present in the CH2 domain of the Fc segment of all four classes of IgGs and is probably split from the parent carrier molecule under the action of two specific enzymes: a splenic endocarboxypeptidase and a leucokininase located in the outer membrane of neutrophils. Several studies have shown that this natural peptide is characterized by a broad spectrum of biological activities, including immunostimulatory, neurotrophic, antimicrobial and antitumor activities in vitro and in vivo (reviewed in [35]). Similar studies led to the discovery of other in vitro IgG-derived peptides, from the Fc-region, such as rigin, p24 peptide and immunorphin, or from the V region, such as immunocortin. These peptides can act as signaling molecules mostly involved in the regulation of the immune response. Even though at present there are no data on generation of similar peptides in vivo, it is conceivable that they can

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Antibodies as a source of anti-infective peptides: an update  actually be produced under physiological conditions in the body [36] . Proceeding in studies previously carried out on KAbs, since 2003 our group has been involved in selecting peptides derived first from the V region, then from the Fc region of Abs, and in characterizing their antimicrobial, antiviral, ­immunomodulatory and antitumor activities. ●●Complementarity determining regions-

related anti-infective & antitumor peptides

Given the availability of a well-characterized recombinant K Ab, endowed with a widespectrum KT-like microbicidal activity [37] , in an attempt to establish a possible correlation between amino acid sequence and candidacidal activity, a number of peptides including the six complementarity determining regions (CDRs) and many related decapeptides were synthesized and tested in vitro against the model microorganism C. albicans. This allowed the selection of the most active peptide, called P6 (EKVTMTCSAS), comprising the first three amino acids of CDR L1 (SAS) in C-terminal position (Figure 1) . The substitution of the first residue with alanine generated a significantly more active peptide, called ‘killer peptide’ (KP, AKVTMTCSAS) [38] . As extensively and recently reviewed, subsequent studies demonstrated that KP is characterized by a broad spectrum of antimicrobial activity and other additional biological properties, exerted through different mechanisms of action, and can retain its in vitro fungicidal activity also as dextrorotatory isomer. Briefly, KP showed a significant antifungal activity at micromolar concentration against many relevant pathogenic yeasts and molds in vitro and in vivo, irrespective of their possible resistance to conventional antifungal agents. No KP-resistant strain was found within a Saccharomyces cerevisiae nonessential gene deletion collection, suggesting that a resistant phenotype is not compatible with viability [39] . The peptide seems to exert its fungicidal activity by interacting with superficial molecules, such as β-1,3-D-glucans. KP treatment of C. albicans cells resulted in gross alterations, in other words, cell wall swelling, membrane collapse, chromatin condensation and nucleus fragmentation, similar to what is observed in cells treated with classical apoptotic agents [12] . KP also showed antimicrobial activity against relevant pathogenic bacteria and protozoa as well as antiviral activity against HIV-1 and influenza

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A virus [40–43] . KP treatment of Leishmania spp. resulted in cell death without any apparent membrane involvement, with a cell degeneration similar to an autophagic process [44] . Different and specific mechanisms of action are involved in KP antiviral activity, based on downregulation of CCR5 coreceptor expression on the cell surface of peripheral blood mononuclear cells and/or steric block of gp120 in HIV-1 [45] , and inhibition of the late phase of viral multiplication in different influenza A virus strains, with reduction of membrane protein M1 and hemagglutinin, and prevention of mature viral particles production [46] . In addition, KP showed immunomodulatory properties that possibly contributed to the therapeutic effect in experimental models of infection, being able to bind, through different specific receptors, to dendritic cells and macrophages, modulating the expression of costimulatory and major histocompatibility complex molecules [51] . So many different activities in vitro, ex vivo and particularly in vivo are attributable to peculiar KP chemical, physical and structural properties. The dimeric functional unit of the peptide can spontaneously and reversibly selfaggregate in a β-sheet structure with the formation of hydrogel-like aggregates, favored by the distribution along the sequence of hydroxylated amino acids intercalated with hydrophobic residues. KP self-assembly is significantly catalyzed by β-1,3-D-glucans, either soluble or exposed on the surface of C. albicans cells. This peculiar behavior probably accounts for the significant KP activity in vivo, providing protection against proteases and ensuring a slow release of the active peptide over time [52] . Several other microbicidal CDR peptides have been identified over the last few years from wellcharacterized murine and human mAbs, regardless of their specificity for a given Ag (Figure 1) . CDR peptides of different origin and sequence, primarily germline encoded CDR1 and CDR2 of L and H chains, showed, at micromolar concentrations, differential antimicrobial and antiviral activities in vitro, ex vivo and/or in vivo against selected pathogens, such as C. albicans and HIV-1, conceivably involving different mechanisms of action [48] . Besides, a tyrosinesulfated peptide derived from the CDR H3 of an HIV-1-neutralizing Ab, E51, that recognizes the CCR5-binding region of gp120, has been demonstrated to bind gp120 and inhibit HIV-1 infection; additional Ab-derived CCR5-mimetic

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CDR-related peptides

Alanine substituted derivatives

E10S (P6)

E1A P6 (KP, A10S)

CDR-peptides K17T (C7L1) N9R (C7L3)

G5H (C7/pc42 H1)

Y3A G5H N6A Y16K

Y16K (C7/pc42 H2) F15A Y16K Y16H (pc42 L1)

L7S (pc42 L2)

Q9S (HuA L3) G10Y (moA H3) Fc-peptides H4L N1A N10K N10K L8A N10K T11F

D5A T11F

Figure 1. Peptides derived from variable and constant regions of antibodies endowed with anti-infective and/or antitumor activity. Stick representation, extended conformation; color of amino acids (Gly, Ala, Ser, Thr: orange; Cys, Val, Ile, Leu, Pro, Phe, Tyr, Met, Trp: green; Asn, Gln: magenta; Asp, Glu: red; Lys, Arg: blue) is according to Lesk [47]. C7, pc42, HuA, and MoA: selected monoclonal antibodies with different specificities (for details see [38,48–50]).

peptide variants showed enhanced recognition and neutralization of HIV-1 [53,54] . Even a synthetic peptide with sequence identical to CDR H3 of a murine mAb specific for difucosyl human blood group A, without possessing direct candidacidal properties, proved to have immunomodulatory activity and to be therapeutic against experimental systemic candidiasis [49] .

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In recent years, different CDR peptides characterized by in vitro and/or in vivo antitumor activity have also been identified. Among them, a 16-residue peptide, YISCYNGATSYNQKFK, derived from CDR H2 of the previously described KAb mAbC7 (C7 H2), showed significant antitumor activity in vitro and in vivo, being able to inhibit the germination of human umbilical vein endothelial

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Antibodies as a source of anti-infective peptides: an update  cells and lung colonization by melanoma cells in mice. C7 H2 proved to induce apoptosis in melanoma and leukemia cells, by interacting with β-actin and altering the cytoskeleton dynamics, and inducing the abundant production of reactive oxygen species. The peptide proved to be nontoxic in vivo in healthy mice and in vitro against noncancer cell lines. The different susceptibility to the peptide of cancer cells is presumably due to the greater exposure of β-actin at the cell surface and higher F-actin dynamics at the tumor cell extensions, faster internalization and response to the peptideinduced apoptosis [55] . Synthetic peptides representing CDRs of two antimelanoma murine mAbs (A4 and A4M), in particular A4 CDR H3 and A4M CDRs L1 and L2, showed significant cytotoxic activity in vitro, leading tumor cells to apoptosis. A4M L1 and L2 also inhibited angiogenesis of human umbilical vein endothelial cells in vitro  [56] . More recently, Figuereido  et al. described the differential antitumor activity of CDR peptides from different mAbs, reported to specifically recognize antigenic epitopes on eukaryotic and prokaryotic cells, and viruses. By searching databases, they selected CDR sequences with more than 80% homology within a family of Igs, giving priority to CDRs L1, L3, H2 and H3. These CDRs, on the basis of previous observations, are apparently more often involved in bioactivity. Four CDRs from different Abs showed in vitro cytotoxicity against murine melanoma and a panel of human tumor cell lineages by inducing programmed cell death. Importantly, these peptides also exerted antimetastatic activity in a mouse syngeneic melanoma model. Other CDR peptides were also protective in the in vivo model, without showing significant cytotoxicity against tumor cells in vitro. These peptides also induced significant production of nitric oxide in macrophages. Thus, the antitumor protection by CDR peptides could be the result of a direct antitumor activity as well as of activity as immune adjuvants able to modulate innate immune cells [57] . CDR-derived peptides with other biological activities have been described. From the sequence of the hypervariable region of the L chain of an mAb (B6B21), shown to be a glycine-site partial agonist of the N-methyld-aspartate receptor, were generated glyxins, a family of glycine-site specific N-methyl-daspartate receptor modulators. Some of them,

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such as the amidated tetrapeptide GLYX-13, being able to cross the blood–brain barrier, may have therapeutic potential as cognitive enhancers and have been proposed as a novel class of antidepressants [58] . Although of great interest, the variety of biological activities exhibited by CDR and CDRrelated peptides may be considered of relative importance from an immunological point of view, since it is unlikely that such peptides could be physiologically released in vivo and, in any case, in adequate amounts. From this point of view, peptides derived from more conserved Ab regions, such as Fc, may have a greater relevance. ●●Fc-derived anti-infective peptides

Peptides encompassing sequences of the Fc region (Fc-peptides) from the major classes of Igs (IgG, IgM, IgA) were selected, synthesized and evaluated for their biological activities (Figure 1) . The selection was carried out by using available bioinformatics tools and according to different criteria, including peptide length (maximum 12 amino acids), potential cleavage sites and probability of cleavage by physiological proteases, conserved sequence in human and animal Igs, isoelectric point, net charge and alternation of hydrophobic/hydrophilic amino acids, with regard to features common to peptides with known biological activity. Among the three selected peptides, H4L (HEAL, present in all mammalian IgGs, IgMs and IgAs), N10K (NQVSLTCLVK, conserved in IgG1s of different organisms) and T11F (TCRVDHRGLTF, present in human IgMs), N10K and T11F showed a significant fungicidal activity at micromolar concentrations against the tested pathogenic yeasts, including strains resistant to conventional antifungal drugs. Furthermore, N10K, selected as a proof-of-concept of the therapeutic potential of fungicidal Fc-peptides, showed a significant therapeutic activity in consolidated models of murine systemic and vaginal candidiasis. N10K aggregated and bound to C. albicans cells causing alterations that suggest the activation of an autophagic process [50] . N10K proved also to display an immunomodulatory activity toward human monocytes in vitro, resulting in an increased production of proinflammatory cytokines IL-6, IL-12p40, IL-1β and TNF-α. This process enhanced phagocytosis of nonopsonized C. albicans cells by monocytes [59] .

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Review  Magliani, Giovati, Ciociola et al. Further ongoing studies will better characterize N10K and other Fc-peptides, their mechanism of action and other possible biological activities. ●●Ab-derived peptides: structure–function

relationship

In an attempt to establish a possible correlation between structure and function, most of the previously described Ab-derived peptides were further investigated after alanine scanning. Alanine substitution, which might be considered as a surrogate of natural point mutations, was used to establish the functional relevance of each amino acid on structure and biological activity of Ab-derived peptides. Alanine-substituted derivatives (asds) proved to display differential antimicrobial, antiviral and antitumor activities; indeed, while the substitution of some amino acids did not have significant effects, the replacement of other residues could enhance or even abolish the biological activity of the parental peptide. KP is the most obvious example of asd with significantly increased activity in comparison with the original Ab-derived P6 peptide [38] , but similar behaviors were observed with asds from CDR-derived [48] and Fc-peptides [50] . Extensive studies using circular dichroism (CD) spectroscopy, transmission and scanning electron microscopy and confocal microscopy shed some light on aspects of peptide structure– activity relationship. The best-characterized Ab-derived peptide, KP, can easily dimerize in nonreducing conditions, due to the formation of intermolecular disulfide bonds between cysteine residues in position 7. As previously mentioned, the dimer represents the functional unit of the peptide and can spontaneously and reversibly assemble in antiparallel β-sheet structures producing an organized network of fibril-like structures easily observed by electron microscopy. KP active form could be released over time, depending on concentration and temperature, accounting for its in vivo therapeutic activity [52] . CD studies demonstrated an analogous ability of N10K Fc peptide to spontaneously aggregate over time, progressing from a typical random coil conformation to a rich β-sheet structure. Also in this case, the alternation of hydrophobic/hydrophilic residues favored the acquisition of a β-sheet structure and the formation of large fibril-like aggregates. In comparison to KP, the presence of more

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hydrophobic residues could explain the faster aggregation process of N10K (hours compared with days) [50] . N10K asds peptides showed different behaviors in terms of self-aggregation and antifungal activity. The asd N1A, in particular, proved to be the most active peptide and showed the ability to aggregate into fibril-like structures either spontaneously in a few days or instantly in the presence of C. albicans cells. The second most active asd peptide, L8A, did not present self-aggregating properties even after months following solubilization, but it was able to instantly aggregate in the presence of C. albicans cells. Both peptides induced apoptosis in C. albicans cells [60] . Studies on asds of the Fc-peptide T11F demonstrated a decrease in candidacidal activity after substitution of positively charged residues, as expected according to the recognized antimicrobial activity of cationic peptides characterized by a net positive charge. A dramatic reduction in activity was also caused by the replacement of the cysteine residue, responsible for peptide dimerization through the formation of a disulphide bridge. CD spectrum of T11F was indicative of the reversible formation of a polyproline II helix with a partial transition to a β-structure organization, as confirmed by nuclear magnetic resonance [61] . These observations indicated the critical role of some residues in self-aggregation processes, other than in the biological activity in vitro, emphasizing the potential of manipulating the amino acid sequence. Overall, the ability of Ab-derived peptides to reversibly self assemble and generate hydrogellike structures might represent a key feature for a new class of therapeutic auto-delivering peptides, as the aggregation property ensures protection against proteases and sustained release of the peptide active form, thus extending over time its biological effects in vivo. Conclusion & future perspective In recent decades, infectious diseases have returned to the fore, due to various events. The urgent medical need of new effective therapeutic approaches is emphasized by rising incidence of multidrug-resistant microorganisms and viruses, emergence and re-emergence of new and old etiologic agents, fears for the globalization of highly dangerous viruses, such as influenza and Ebola, and continuous increase of immunocompromised or otherwise debilitated individuals highly susceptible to opportunistic

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Antibodies as a source of anti-infective peptides: an update  pathogens. The increasing worldwide incidence of cancer is another challenging public health problem. Therapeutic mAbs are representing a successful response, at least in some clinical conditions, and already benefitted millions of patients worldwide. Ab-based treatments could also provide new therapeutic options toward emerging pathogens for which no therapy is currently available. Although advances in recombinant DNA technology enabled the generation of engineered Abs with significant improvements in terms of immunogenicity and safety [62] , serious adverse reactions associated with mAb treatment are reported and the absolute safety of every fresh stock of mAbs is hard to be ensured [63] . Fragility of Abs, that are perishable and require refrigeration as well as systemic administration, contributes to their high cost that remains a major obstacle to their widespread use. In the field of infectious disease, there may be some additional concerns. Many pathogens are antigenically variable, so widespread use of Ab-based therapies could produce selective pressure on microbial populations for the emergence of escape, Ab-resistant mutants. The use of Ab cocktails and Ab therapy combined with chemotherapy could reduce this possibility. Close attention should also be paid to the choice of anti-infective therapeutic Abs, as disease-enhancing mAbs have been reported pertaining to toxins [64] , bacteria [65] , fungi [66] and viruses [67] . More recently, innovative approaches have been proposed, such as radioimmunotherapy, exploiting the ability of Abs, after binding to microbial Ags, to deliver radionuclides that emit cytocidal radiation [68,69] . In this scenario, Ab-derived peptides could represent a promising therapeutic improvement in terms of immunogenicity, toxicity and safety, compared with Abs or their recombinant derivatives. Ab-derived peptides could be considered cryptides, in other words, biologically active peptides hidden in larger proteins [70] . The reported high frequency of bioactive CDR- and Fc-related peptides from the inside of Abs unfolds interesting perspectives from at least two different points of view. First, these observations suggest that Abs could represent an inexhaustible source of anti-infective, antitumor and immunomodulatory peptides by providing leading molecules to devise new target-directed therapeutic tools to different human diseases. Second, the possible production of Ab-derived peptides in vivo

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Review

would open a new scenario on the role of Abs in ­immunity and homeostasis. As already widely demonstrated, many biologically active peptides have been synthesized from the sequence of Abs, independent of their specificity or isotype. Sequences of Abs deposited in databases are widely available to select any peptide of interest to be tested for potential biological activities. Small sized synthetic peptides can be manufactured easily and at low cost. Moreover, peptide variants or peptidomimetics (asds, D-enantiomers, peptides containing non-natural amino acids resistant to proteases or new functional groups, or conjugated with conventional drugs, radionuclides or cytotoxic compounds) could be synthesized to positively affect bioactivity, stability against proteolysis, pharmacokinetics and/or pharmacodynamics, thus enhancing the therapeutic potential of these molecules. Even though peptides are generally considered to be poor drug candidates because of their low oral bioavailability and propensity to be rapidly metabolized, new recent synthetic strategies and alternative routes of administration have enabled the development of peptide-based drugs, such as hormones, agonists or antagonists for receptors implicated in oncology and inflammation, and antimicrobial peptides. A large number of them are now marketed or represent great promises for the coming years [71] . Pros, cons and open questions about the use of Ab-derived anti-infective peptides are summarized in Table 1. An additional opportunity could be the design and synthesis of consensus peptides identified on the basis of structure–activity relationships of the large number of bioactive Ab-peptides described so far, in the attempt to optimize and enhance their ­biological activity and stability. A further, more intriguing implication related to Ab-derived peptides is the hypothesis that at least some of them may be released in vivo by physiological proteolysis, thus allowing a biological function of Abs over their half-life. If this were true, bioactive Ab-derived peptides should be found in biological fluids/tissues of individuals, opening a new scenario on the possibility that Ab fragments can act as effectors of anti-infective and antitumor immune responses in a manner reminiscent of molecules of innate immunity. Even though complete analysis of the human proteome/peptidome is a daunting task, very sensitive analytical methods, such as MS, are allowing the detection of hundreds to

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Review  Magliani, Giovati, Ciociola et al. Table 1. Pros, cons and questions about the use of antibody-derived antimicrobial and antiviral peptides. Pros

Cons

Questions

Unlimited sourcing Easiness of synthesis Low cost of production Stability Water solubility Exclusive mode of action Lack of toxicity Lack of immunogenicity Opportunity of engineering†

Instability Proteases susceptibility Water insolubility            

Oral bioavailability Emergence of resistance Pharmacokinetics Pharmacodynamic Molecular mechanisms of action Impact on microbiota      

Potential overcoming of cons.

†

thousands of endogenous peptides in the blood. Although origin, physiological role and significance of most of these peptides in different clinical conditions have still largely to be clarified  [72] , the detection of Ab-derived cryptides could be of relevance. Ongoing studies using a liquid chromatography-electrospray ionization-high resolution multistage MS method until now failed in detecting previously described Fc peptides in the serum of individuals. However, other Ab-derived peptides have been detected. One of them, a 40-amino acid phosphopeptide from the C region of IgMs, is characterized by fungicidal activity in vitro and in vivo without showing toxic or genotoxic effects against mammalian cells (manuscript in preparation). These observations, along with the previous on tuftsin, may shade the traditional distinction between adaptive and innate immunity and support the hypothesis that the immune system, during evolution, may have adopted unconventional mechanisms for host defense against microbial infections. In this perspective, could Abs be the result of the association and combination of peptides that ancestrally possessed an intrinsic, different biological function? Studies are underway to test the potential microbicidal activity of Ab-peptides synthesized on the basis of the sequences, present in available databases, of proteins encoded by genes belonging to the family of Igs (genes V, J and C for the L chain and V, D, J and C for the H chain). Some of them are proving to exert differential in vitro and in vivo antimicrobial activities without showing toxic or genotoxic effects [73] . Thus, Abs could be the product of an evolutionary process by which the association of ancestral genes, encoding for

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peptides/proteins characterized by nonspecific multiple biological activities, could have given rise to a complex cluster of genes that currently determine the production of Igs with highly ­specific activity. Much still needs to be assessed to confirm these observations and hypothetical correlations, but current data suggest assigning Abs new relevant functions. Overall, Abs can represent an unlimited source of leading structures for the rational design of a new class of antitumor, antimicrobial, antiviral and immunomodulatory therapeutic compounds in their own right or in synergy with existing drugs. In an era of resurgence of infectious diseases, increasingly due to multiresistant, difficult-totreat or untreatable pathogenic microorganisms and viruses, and of widespread incidence of cancer, the need for obtaining new effective therapeutic compounds is more and more pressing. Ab-derived peptides and other natural antimicrobial peptides could represent a promising exploitable alternative [74,75] . Easy and low-cost production and handling, availability of new expression systems, and delivery mechanisms are expected to favor the obtainment of a new generation of therapeutic agents under good manufacturing practice conditions. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or ­pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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Antibodies as a source of anti-infective peptides: an update 

Review

EXECUTIVE SUMMARY Antibodies & anti-infective immune protection ●●

Antibodies (Abs) are relevant effectors of the anti-infective humoral adaptive immunity.

●●

Microbicidal Abs can function directly as effector molecules against microbial agents without the assistance of phagocytic cells or complement.

Antibodies as targeting therapeutic agents ●●

Due to their specificity and effectiveness, Abs have been used widely in the past in serum prophylaxis and serum therapy against a variety of infectious diseases.

●●

In their monoclonal and recombinant formats, Abs, mostly human or humanized, are now widely used as therapeutic agents in the ‘targeted’ cancer therapy.

●●

In the last decades therapeutic Abs have represented one of the major clinical successes, with also a major economic impact in the field of cancer and autoimmune diseases.

●●

In the field of infectious diseases, despite their recognized potential, Abs still have very limited applications.

Antibodies as a source of antimicrobial peptides ●●

A killer peptide derived from the variable region of a recombinant microbicidal Ab, displayed significant in vitro, ex vivo and/or in vivo antifungal, antibacterial, antiprotozoan, antiviral and immunomodulatory properties mediated by different mechanisms of action.

●●

Complementarity determining region- and fragment crystallizable-related peptides can display differential

antimicrobial, antiviral and/or antitumor activities, irrespective of the specificity of the native Ab for a given antigen. ●●

Some Ab-derived peptides showed the ability to spontaneously and reversibly self assemble, a property that might explain their significant in vivo antimicrobial and/or antitumor activity.

Ab-derived anti-infective & antitumor peptides: main implications ●●

Abs could represent an unlimited source of peptides as leading structures for the rational design of a new class of therapeutic compounds to be used alone or in synergy with existing drugs.

●●

Along with other peptides, such as natural antimicrobial peptides, Ab-derived peptides could represent new therapeutic anti-infective and anticancer options.

●●

Ab-derived, small-sized synthetic peptides can be manufactured easily and at a low cost, and peptidomimetics could be obtained with improved pharmacokinetic and/or pharmacodynamic properties.

●●

The demonstrated or highly probable production in vivo of bioactive Ab-derived peptides, that may be released by physiological proteolysis of Abs, suggests the possibility that Ab fragments can act as effectors of anti-infective and antitumor immune responses in a manner reminiscent of molecules of innate immunity.

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