Antibodies, uses thereof, pharmaceutical compositions, including fungal infection diagnosis methods, fungal infection diagnosis kits, and fungal infection treatment methods

Abstract

The present invention provides a monoclonal antibody against chitin oligomers using hybridoma technology. The antibody can be used as a tool for diagnosis and treatment of fungal infection. A pharmaceutical composition and a kit for treating fungal infection comprising the antibody are also disclosed. A method for diagnosing fungal infection using the antibody and the use of the antibody in preparing a drug for treating fungal infection are also disclosed.

Description

Field of the Invention

[0001] The present invention relates to the fields of diagnostic medicine, biotechnology and biopharmaceuticals. In particular, the present invention relates to the use of monoclonal antibodies for the treatment of fungal infections, as well as their use in the diagnosis of fungal infections in any human or animal subject. Background of the Invention

[0003] Fungi are eukaryotic saprophytes with rigid cell walls, numbering approximately 5 million species on Earth (1). In contrast to plants, which are also eukaryotic cells (including cell walls), fungal cells contain chitin in their cell walls, rather than cellulose, as in plant cells. This distinction should be emphasized.

[0004] Fungi present a large morphological diversity, including two main morphologies: yeasts, which are unicellular with round, oval or spherical shapes, and filamentous fungi, which have a hyphal shape and are multicellular. Yeasts are cells that reproduce by single or multiple budding. Filamentous fungi have a basic hyphal morphology that can be seven-layered or non-seven-layered. The existence of dimorphic fungi should be highlighted, which can live in both yeast and hyphal shapes, depending on environmental changes that induce the transition between morphologies.

[0005] The fungal kingdom is divided into four phyla: Ascomycota, Basidiomycota, Zygomycota, and Chitridiomycota. Ascomycota and Basidiomycota are hosts to a variety of animal and plant pathogens, including the human pathogens Cryptococcus neoformans, Cryptococcus gattii, Candida sp., Aspergillus sp., Histoplasma capsulatum, and Coccidioides immitis (2).

[0006] Systemic fungal disease caused by invasive fungal infections (IFIs) is one of the leading causes of death in the world (3). According to data submitted by the Global Action Fund for Fungal Infections (GAFFI), more than 300 million people of all ages experience some kind of serious fungal infection each year. It is estimated that more than 1.6 million people will die in this group, which is comparable to the data observed for tuberculosis and three times that of malaria.

[0007] The increase in the incidence of IFIs is mainly the result of a significant increase in the use of immunosuppressive conditions (including HIV infection) and immunosuppression-inducing drugs over the past few decades (4). In addition, in addition to invasive medical interventions (5), the use of broad-spectrum antineoplastic drugs and antibiotics can also directly increase IFIs.

[0008] Systemic fungal diseases can affect any host organ. The classification of fungal diseases in this category is dynamic and highly variable, as illustrated by the review, with species previously considered nonpathogenic now recognized, including pathogens that cause invasive fungal diseases (6). Such infections are very common in immunocompromised patients and are often associated with high mortality (4). The most common genera involved in systemic fungal infections include Candida, Pneumocystis, Histoplasma, Aspergillus, Cryptococcus, Mucor, Rhizopus, and Coccidioidomyces (Table 1).

[0009] Table 1: Estimated annual global numbers of deaths associated with fungal infections in people with HIV

[0010]

[0011] Fungal pathogens of humans are present in several natural habitats, but IFIs are usually observed in tropical and subtropical climates (7,8). It has been hypothesized that increasing climate change could alter the distribution of diseases, thus causing the emergence of fungal pathogens in new regions (7). For example, 100 cases of illness were registered in the northeastern United States due to Cryptococcus gattii belonging to molecular type VGII, which is usually found in South America (9). However, this outbreak was registered in the temperate region of the EUA (10). It is noteworthy that fungi have various traits that enable adaptation and colonization of new ecological niches, thus possessing the ability to survive under the physiological conditions of the host and switch between morphologies (11). The most common adaptive traits of fungal pathogens include the ability to grow at 37°C (12), changes in shape and size, the ability to escape macrophages (13,14), melanization, and biofilm formation (15).

[0012] Notably, species belonging to the genera Aspergillus, Candida, Cryptococcus, and Pneumocystis are responsible for approximately 90% of human deaths (11). Recent global estimates document more than 11 million cases of fungal infections associated with HIV-positive patients, resulting in more than 500,000 deaths per year (Table 1).

[0013] According to the Centers for Disease Control and Prevention (CDC, USA), fungi are the most common pathogens associated with mortality from microbial meningitis. It is estimated that approximately 1 million cases of meningitis caused by the fungus, Cryptococcus neoformans, occur each year, resulting in more than 200,000 deaths within 3 months of infection (16).

[0014] Despite the high mortality rates associated with different fungal infection types, the recommended treatment strategies have remained virtually the same since the 1950s, compared with the treatment of bacterial infections (18). Currently, there are four major classes of antifungal drugs: azoles, polyenes, pyrimidines, and echinocandins. Several other classes of drugs, including morpholines and allylamines, are used only as topical agents due to low efficacy or severe side effects when administered systemically.

[0015] However, the intensive and indiscriminate use of drugs belonging to this class has allowed the selection of multidrug-resistant isolates of antifungal drugs (19), which, although rare, have come to the fore, especially in the case of Candida species. Currently, the emergence of multidrug-resistant isolates of antifungal drugs also represents a major threat to global public health. An example of this serious problem occurred in early 2009, when Candida auris resistant to fluconazole (FLC) was described in East Asia. Currently, this species is widespread in five continents and its description is recognized as multidrug-resistant (20).

[0016] The current gold standard for the treatment of meningoencephalitis caused by cryptococci is a combination of amphotericin polyene B (AmB) and 5-fluorocytosine. AmB exhibits high nephrotoxicity and requires intravenous administration, which limits its use in areas without adequate medical infrastructure. It is estimated that in Europe (21), the cost of a 15-day intravenous treatment with liposomal AmB ranges from €10,000 to €20,000, while in Brazil this value can reach a monthly cost of R$250,000.00 (Source: SUSAssisting Center to Assess Health Technologies and Excellence-CCATES).

[0017] Therefore, the use of monoclonal antibodies in the fungal infection paradigm is considered very promising when searching for alternatives to reduce the side effects of antifungal treatment, as they can bind highly specifically to antigens expressed by fungi in the patient's body fluids and are also key tools in the field of clinical diagnostics.

[0018] The genus Cryptococcus is characterized by oval or spherical yeast cells surrounded by a capsule. Members of the genus belong to the phylum Basidiomycetes (22). Cryptococcus neoformans and Cryptococcus grubii, the pathogenic members, have been subdivided for decades into three variants and five serotypes based on capsular polysaccharide antigenic determinants: Cryptococcus neoformans var grubii (serotype A), Cryptococcus neoformans var neoformans (serotype D), Cryptococcus neoformans (hybrid AD) and Cryptococcus grubii (serotypes B and C) (23). Currently, based on molecular evidence, there is a proposal to reclassify Cryptococcus neoformans and Cryptococcus grubii into seven species (24).

[0019] Cryptococcus neoformans is a saprophytic, cosmopolitan fungus that is globally distributed and found in soil and in bird droppings (usually pigeons) in woodlands. It causes cryptococcosis primarily in immunosuppressed individuals. Cryptococcus gattii is found in tree trunks in tropical and subtropical regions, causing infection particularly in immunocompetent individuals (25).

[0020] The balance between the host immune system and fungal virulence is directly related to whether disease develops or not. Notably, human exposure to environments contaminated with fungi provides a means for establishing infection, as similarities have been found between clinical and environmental isolates from individuals with cryptococcosis (26).

[0021] Cryptococcosis in humans occurs primarily through inhalation of dried yeast cells or possibly basidiospores, which become deposited in the alveolar spaces ( Figure 1 The virulence of the infectious strain, inoculum size, and individual immune status are major factors in disease progression (27).

[0022] Depending on the host immune system, infection can be asymptomatic or have a latent form. In contrast, in immunocompromised individuals, cryptococcal cells proliferate and spread to multiple organs, especially the brain. In these cases, a meningoencephalitis background is common (28).

[0023] Notably, interactions between C. neoformans and environmental predators are considered important factors in the fungus’s evolution into a successful facultative intracellular pathogen. C. neoformans can survive in amoebae and can use the same pathogenic strategies in human macrophages, which provide a similar environment in several aspects. Therefore, it has been proposed that this environmental niche predation selects for virulence traits in cryptococci that enable pathogenicity in human hosts (29).

[0024] Phagocytes are the first line of defense of the organism against pathogenic fungi. Depending on several stimuli, the interaction between Cryptococcus and phagocytes can lead to infection control. However, phagocytes can increase the risk of disseminated fungal infections because they can carry live fungi to different tissues. The immune status of the individual is directly related to the destination of this interaction. Immunocompetent individuals usually prevent fungal dissemination through local cellular mechanisms. Immunocompromised patients develop an inflammatory response that enables pathogen replication (30,31) and subsequent dissemination.

[0025] Disease progression is directly related to the Th1 and Th2 spectrum and the resulting polarization of macrophages into M1 / M2 (32). Th1 cells produce large amounts of TNF and INFγ, which induce the activation of M1 macrophages, thereby eliminating cryptococci (33). Th2 cells produce cytokines involved in inflammatory responses, thereby inducing the proliferation of M2 macrophages, which have no antifungal activity and can promote fungal proliferation (30).

[0026] Notably, several molecules produced by Cryptococcus stimulate M2 macrophage responses (34,35). These molecules include arginase, urease, and laccase (30). This type of macrophage appears to play a central role in the dissemination of cryptococcal disease, since cryptococci can use these cells as a replication niche and, among other mechanisms, exit macrophages by nonlytic exocytosis (14). Due to this property, it has been hypothesized that macrophages can act as Trojan Horses, allowing cells that internalize Cryptococcus neoformans to cross the blood-brain barrier and reach the central nervous system (CNS) (36).

[0027] The preference of C. neoformans for SNC is associated with several factors. SNC may present a safer host for the fungus because the brain is an immune-privileged environment. Furthermore, the diphenolic substrate L-3,4-dihydroxyphenylalanine (L-DOPA) used by the fungus for melanin synthesis may allow it to persist in SNC. Melanization of C. neoformans and C. gattii protects the fungus from oxidative stress, phagocytosis, reduces antifungal effects, and alters immune criteria (27).

[0028] Clinical diagnosis of IFI is difficult due to the lack of signs and specific symptoms at the onset of the disease. Therefore, laboratory testing is essential to reduce morbidity and mortality outcomes.

[0029] Optimal features for developing diagnostic platforms include early pathogen detection, good sensitivity, ability to achieve differentiation at the species level, broad pathogen range detection (multiple capabilities), reliability, quantitative accuracy (ability to differentiate between disease and colonization), and non-invasiveness. No standard diagnostic test meets such criteria, and indeed, at different levels, several criteria are non-existent.

[0030] Identification of fungal pathogens is still based on direct observation of the organism by light microscopy, histopathology of infected tissues, and fungal culture (17,37). Although classical fungal culture and traditional serological techniques are important and necessary, there is a need for molecular techniques (antibodies and antigens, PCR, and sequencing) for fungal detection and identification to enable rapid and effective diagnosis, complementing traditional culture-based methods.

[0031] One of the greatest limitations to the use of cultures for IFI diagnosis is related to the time to result, as several pathogens, particularly filamentous fungi, grow slowly. Depending on the characteristics of the inoculum and fungal growth, cultures require an incubation time of at least 2–3 days, and for some species, several days to weeks. Positive cultures from nonsterile sources, including bronchoalveolar lavage (BAL) specimens, also require careful interpretation to distinguish fungal colonization from actual invasive pathogen isolation. Finally, fungal blood cultures, while noninvasive and highly specific, require long incubation times and are similarly insensitive, yielding reliable results in only 50% of cases involving Candida species and 10% of cases involving Aspergillus species (38).

[0032] In the case of fungal meningitis, the problem is even more serious, as an inaccurate or delayed diagnosis can lead to the death of the patient. Of note, in some cases, a biopsy is required to establish an accurate diagnosis, as cerebrospinal fluid cultures are not usually performed for diagnosis, especially in the case of fungal brain abscesses (39). In cryptococcosis, this type of problem can be easily solved by using the serum cryptococcal antigen (CrAg) test, which uses fungal antigens in the patient's serum. This test consists of an effective and inexpensive method that has prevented deaths in HIV-infected persons with lymphocyte counts T CD4 ≤ 100 / μL in areas with socioeconomic restrictions (40).

[0033] Biopsy is generally not a viable option for critically ill patients and those with suspected IFIs, including aspergillosis, because they are more likely to bleed due to thrombocytopenia (39). This test has demonstrated limited sensitivity and specificity (41) and requires appropriately trained personnel to identify the organism. Nevertheless, microscopic observation remains the gold standard for diagnosis of several IFIs (42).

[0034] The identification of fungal antigens in patient samples has led to significant progress in the field of IFI diagnostics. These molecules mainly include cell wall components ( Figure 2 Detection of such structures may indicate the development of IFI and is often detectable before clinical signs or symptoms of disease occur (43). These biomarkers include (1,3)-β-D-glucan (BDG), Aspergillus galactomannan (GM), Cryptococcal glucuronoxylomannan (GXM), and histoplasmin antigen.

[0035] As mentioned above, antigen detection can identify IFI early, guide treatment strategies, and assess the prognosis of disease response to treatment (38,45,46). BDG is present in most fungi, with the exception of Zygomycetes, Blastomyces dermatitidis, Mucoromycotina, Cryptococcus spp., and certain Basidiomycetes (e.g., Malassezia spp.) (45).

[0036] Although the specificity of BDG for specific fungal pathogens is limited and varies depending on the organism, it still has clear advantages over other techniques. In the case of invasive candidiasis, the sensitivity is approximately 70% compared with blood culture (47). In the case of Pneumocystis pneumonia, higher sensitivity (96%) and specificity (84%) were observed in serum compared with no more than 80% in the same assays for candidiasis and aspergillosis (48). It is important to emphasize that such diagnostic kits are already commercially available and are used to evaluate cases of pneumonia with Pneumocystis (Era Biology – Goldstream Fungal (1-3)-β-D-Glucan Test (GCT-110T – MiraVista Diagnostics – β-D-Glucan Assay).

[0037] On the other hand, the high costs associated with false-positive results in patients with both positive and negative Gram-positive bacteremia (37%) and false-positive results in multiple β-lactam antibiotic batches (33%) suggest that this diagnostic test may be limited, but it is useful in combination with other complementary diagnostic methods (49).

[0038] Galactomannan (GM) is a characteristic polysaccharide released by Aspergillus species during growth and can be detected in the blood circulation, serum, urine, and BAL during fungal growth in tissues by commercial tests (50). Since it is constitutively released by the fungus, GM may be a marker of prognosis and disease progression and / or response to treatment. Several immunoenzymatic methods are used to detect GM, but the most promising one is the sandwich ELISA because it is the most sensitive one and detects low concentrations of GM in clinical samples (51).

[0039] Depending on the patient population, such as transplant patients or patients with hematological diseases, the GM test presents better results, presenting a specificity of about 90% whenever it is tested in bronchoalveolar fluid (43,52). In pediatric patients, the test can produce false positives in about 80% of cases, a fact associated with breastfeeding, bacteremia or antibiotic use (43). Moreover, GM presents cross-reactivity with several fungal antigens, since mannan polysaccharides are found in the wall of several fungi.

[0040] However, there are challenges associated with this approach, including the risk of false-positive results due to cross-contamination or cross-reactivity, and the risk of false-negative results due to imperfect sensitivity of the test. As a general rule, laboratory diagnostic tests should not be used as stand-alone tests for the diagnosis of IFIs. Current fungal diagnostic tests should be used in combination with host assessment and radiographic characterization in order to optimally manage patients at risk (43).

[0041] Over the past decade, PCR testing has emerged as a promising assay for detecting fungal pathogens. The use of PCR is common in clinical practice and has many applications in guiding preventive or targeted therapy to control pathogens (43,50,53).

[0042] In fungi, PCR gene amplification typically involves variable regions of DNA sequence encoding the 18S, 5.8S, and 28S regions of ribosomal RNA (rRNA), as well as the internal transcribed spacer intervening regions known as ITS1 and ITS2 (54).

[0043] In addition to PCR, a variety of molecular method variations, including DNA sequencing, microarrays, and mass spectrometry, mass desorption ionization laser and ionization (MALDI) have been used to develop a wide range of molecular detection tests (43). However, the most commonly used molecular detection method is PCR followed by Sanger sequencing because it can be particularly useful when fungal cultures are negative, and PCR is not necessary when a tissue biopsy has been performed (55).

[0044] Over the past 30 years, the relevance of fungi as agents of human disease has increased dramatically (18). On the other hand, available therapeutic options are either inaccessible, toxic, or ineffective, thus making the search for new antifungal agents unquestionable (56).

[0045] As mentioned above, there are currently four major classes of antifungal drugs. These classes include azoles, polyenes, pyrimidines, and echinocandins. Several other classes of drugs, including morpholines and allylamines, are used only as topical agents due to low efficacy or severe side effects when administered systemically.

[0046] Azoles are the most widely used antifungal agents in clinical practice. These drugs target the ergosterol biosynthesis pathway and act primarily by inhibiting the key enzyme, lanosterol 14α-demethylase, encoded by the ERG11 gene (57).

[0047] FLC and itraconazole, broad-spectrum azoles, have been used since the 1990s for the treatment of different systemic infections due to their higher absorption capacity and lower toxicity than the originally developed azoles (58). However, they exhibit drug interactions with drugs used in chemotherapy or AIDS treatment (58,59). In addition, itraconazole and FLC are ineffective against some emerging pathogens, including Scedosporium, Fusarium, and Mucorales (60). However, the view that the intensity of fungal sensing of azoles is increasing remains popular (58). Other drugs in this class are under development, including new-generation triazoles. Some have been approved by the Food and Drug Administration (FDA) (57).

[0048] Polyenes include more than 200 molecules, most of which are produced by Streptomyces, but only three of them are clinically used: AmB, nystatin, and natamycin (58). These compounds complex with ergosterol in the plasma membrane (57). Its amphipathic structure allows insertion into the lipid bilayer and the formation of pores. The formation of pores destabilizes the plasma membrane, with the ensuing leakage of intracellular components, leading to cell lysis (61). Polyenes bind with lower affinity to cholesterol, the human analog of ergosterol. Cholesterol binding explains its high toxicity and the attendant side effects (62). Therefore, AmB is widely used for systemic infections, unlike nystatin and natamycin, which are only used topically for vaginal and skin infections due to their extreme toxicity (63).

[0049] AmB is poorly absorbed from the gastrointestinal tract, necessitating administration via the intravenous route, which is associated with severe side effects, particularly in the kidneys and liver (64). Of note, side effects including nausea, vomiting, and fever are common, but the most serious side effect is nephrotoxicity (65). Newer AmB formulations, including AmB liposome complexes, minimize these side effects but at the expense of increased drug cost (66,67).

[0050] Fluoropyrimidines are synthetic cytokine structural analogs, therefore, they inhibit nucleic acid synthesis (68). However, this class of drugs cannot be used in an isolated manner due to the reported potent mechanisms (69). The current gold standard for the treatment of meningoencephalitis caused by cryptococci is the combination of AmB with 5-fluorocytosine (5-FC), which minimizes the nephrotoxicity of AmB and reduces the potency of 5-FC due to lower drug doses and a shorter period of administration (64). It should be emphasized that 5-FC is not available in Brazil, which limits Brazilian patients’ access to optimal treatment.

[0051] Echinocandins are noncompetitive inhibitors of β(1-3)-glucan synthase, which catalyzes the polymerization of uridine diphosphate-glucose in β(1-3)-glucan, one of the structural components that maintain the integrity of the fungal cell wall (70). β(1-3)-glucan synthase inhibition leads to cell wall instability and the escape of intracellular components, resulting in cell lysis (71,72). However, in addition to Scedosporium apiospermum, S. prolificans, and Cladophialophora bantiana, several fungal pathogens are also partially or completely resistant to the action of echinocandins, including Cryptococcus neoformans and Cryptococcus gattii or species belonging to the genera Trichophyton and Fusarium (58). Nevertheless, echinocandins are good alternatives for combating other fungal infections. Most treatments that have failed with classical therapy with azoles or polyenes have been successful with the use of echinocandins.

[0052] The mechanisms of antifungal potency may begin with reduced effective drug concentrations, altered or overexpressed drug targets, and metabolic deviations (57).

[0053] Antibodies or immunoglobulins (Ig) are glycoproteins secreted by B cells that exhibit the ability to identify and / or neutralize organisms or antigens that are foreign to the host immune system (73). Igs are formed by two heavy protein chains and two light protein chains that have variable regions involved in antigen recognition and regions that perform molecular effector functions (73).

[0054] Antibody diversity is manifested by the amino acid sequence variability of the light chain variable region and the heavy chain variable region. The complementarity determining regions (CDRs) are highly variable sequences that directly contact the antigen to be recognized (73).

[0055] Antibodies can be divided into different classes and subclasses, called isotypes (73). Ig classes include IgM, IgG, IgA, IgD, and IgE, and IgA and IgG isotypes are subdivided into IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4, each of which exerts different effector functions (73).

[0056] Monoclonal antibodies (AcM) were first discovered in 1975 by Georges and César Milstein (74) by producing hybrid cells called hybridomas, which are produced by the fusion of two different cells (74). Hybridomas are produced by the fusion of B lymphocytes (previously immunized with the antigen of interest) with myeloma cells, thereby generating immortal cells and AcM producers ( Figure 4 ).

[0057] Given the high sensitivity and specificity of AcM, AcM has diverse applications in the fields of diagnosis and treatment, not only for infectious diseases caused by bacteria, viruses, and protozoa, but also for autoimmune diseases and tumors (75). In the past 30 years, approximately 80 types of AcM have been approved by the FDA for the treatment of several diseases, including cancer, chronic inflammatory diseases, neurodegenerative diseases, and infectious diseases (76).

[0058] The advent of hybridoma technology has significantly improved infectious disease immunodiagnosis because AcM overcomes the shortcomings of polyclonal antibodies and can provide identification of a single antigen, thereby generating reproducible and consistent results (77).

[0059] AcMs directed against pathogenic fungi are being used more frequently. Antibodies with therapeutic potential have been developed using antigens such as histone 2B of Histoplasma capsulatum (78), β-glucan of Candida albicans (79), glucosylceramide of Cryptococcus neoformans (80), melanin from several pathogens (81), and heat shock protein of Histoplasma capsulatum (82). Antibodies with diagnostic use include antibodies reactive against Histoplasma capsulatum antigens M and H and against an antigen of Cryptococcus spp. (CrAg) (83,84).

[0060] AcM administration in IFI depends on several factors, which necessarily include the AcM isotype, its titer, presentation via the major histocompatibility complex (MHC), and especially immune cell activation (85).

[0061] AcM against fungi can mediate three different effects: protection (inhibition of growth, immunomodulation of immune system responses, and neutralization of fungal effects in host tissues), disease enhancement (because it promotes disease dissemination, e.g., promoting phagocytosis of Cryptococcus neoformans), and virulence neutralization (by inhibiting protein or fungal polysaccharide release) (77). These effects are Figure 5 shown.

[0062] AcM can also play an indirect role in the defense against IFI through phagocytosis, complement system activation, cytotoxicity regulation, and phagosome maturation (86). They can also directly affect, for example, biofilm formation (87), polysaccharide release (88), dimorphism (89), gene expression (90), and signal transduction (86).

[0063] Immunotherapy studies have characterized protective AcMs against different targets and different fungal species including: β-1,3 glucan-A. fumigatus (91), Als3-Candida albicans (92), Hsp60-Histoplasma capsulatum (93), GXM-Cryptococcus neoformans (94), gp43-P. brasiliensis (95), p55-Pneumocystis species (96). These studies demonstrated that some fungal antigens elicit antibody-mediated protection during fungal infections.

[0064] Some of these antigenic targets to be maintained are applied to different fungi. As chitin, a polymer of N-acetylglucosamine, is one of the major components of the fungal cell wall, it has become a prominent target for new therapeutic strategies.

[0065] Therefore, fungal infection has become one of the major causes of disease in immunocompromised individuals and is a serious and underestimated public health problem (16), and drug resistance may also lead to a significant increase in morbidity and mortality in immunocompromised individuals worldwide.

[0066] Therefore, in the search for alternatives to reduce the side effects of antifungal therapy, the use of monoclonal antibodies in the fungal infection paradigm is considered very promising, as it can bind with high specificity to antigens expressed by fungi in the patient's body fluids and is also a key tool in the field of clinical diagnostics.

[0067] In addition, effective diagnostic techniques can identify IFI patients earlier than by cell culture alone. However, there are challenges associated with this approach, including the risk of false-positive results due to cross-contamination or cross-reactivity, and the risk of false-negative results due to imperfect sensitivity of the test. Therefore, there is a need to combine different tests related to host assessment (including intrinsic characteristics of risk patients).

[0068] Considering this background and due to the great complexity of the fungal cell surface structure, chitin oligomers were chosen as targets for the development of AcMs.

[0069] Therefore, the present invention discloses AcM against chitin oligomers for therapeutic use and fungal diagnosis in biological samples. Moreover, the present invention also reveals that the synergistic effect of the developed AcM and AmB leads to the survival of infected individuals due to binding to chitin oligomers in a mouse model infected with Cryptococcus neoformans.

[0070] The advantages of the present invention should be apparent from the description of the invention provided in this document. SUMMARY OF THE INVENTION

[0072] The object of the present invention is to provide monoclonal antibodies (AcM) for the treatment and diagnosis of fungal infections.

[0073] In particular, AcMs were developed that showed activity against fungal chitin oligomers.

[0074] In a first aspect, the present invention provides a monoclonal antibody comprising:

[0075] (i) the VH CDR1 sequence of SEQ ID NO: 1, the VH CDR2 of SEQ ID NO: 2, and the VH CDR3 of SEQ ID NO: 3; and (i) the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 of SEQ ID NO: 5, and the VL CDR3 of SEQ ID NO: 6; or

[0076] (i) the VH CDR1 sequence described in SEQ ID NO:7, the VH CDR2 described in SEQ ID NO:8, and the VH CDR3 described in SEQ ID NO:9; and (i) the VL CDR1 sequence described in SEQ ID NO:10, the VL CDR2 described in SEQ ID NO:11, and the VL CDR3 described in SEQ ID NO:12.

[0077] As a second aspect, the present invention foresees a pharmaceutical composition comprising the above monoclonal antibody. The pharmaceutical composition may also include AmB and / or FLC, and a pharmaceutically acceptable vehicle / excipient. The pharmaceutical composition is used to treat fungal infections.

[0078] As a third aspect, the present invention foresees the use of monoclonal antibodies for the preparation of a medicament for the treatment of fungal infections. The above use can be carried out in combination with polyenes and / or azoles, preferably AmB and / or FLC.

[0079] As a fourth aspect, the present invention foresees a method for diagnosing fungal infection comprising the steps of:

[0080] (i) providing the above-mentioned monoclonal antibody or composition and a sample obtained from an individual;

[0081] (ii) contacting the monoclonal antibody or composition described above with the biological sample to be tested for a sufficient period of time and under conditions sufficient to form an antigen / antibody complex; and

[0082] (iii) detecting the antigen / antibody complex formed in the previous stage by a detection technique capable of generating a detectable signal in the presence of the above antigen / antibody complex. The biological sample is selected from the group consisting of saliva, urine, serum, blood, bronchoalveolar lavage fluid, peritoneal fluid or fluid or any other biological fluid of an individual.

[0083] As a fifth aspect, the present invention foresees a fungal infection diagnostic kit comprising the above monoclonal antibody or composition. The kit may also include instructions for use. Moreover, the kit may also include an antigen / antibody complex detection medium, which may include a signal generating agent capable of generating a detectable signal.

[0084] As a sixth aspect, the present invention foresees a kit for treating fungal infections, characterized in that it comprises:

[0085] (i) the above monoclonal antibody or composition; and

[0086] (ii) antifungal agents,

[0087] (iii) instructions for using the components in combination.

[0088] As a seventh aspect, the present invention contemplates a method for treating fungal infection, comprising administering a therapeutically effective amount of the above-mentioned antibody or composition to an individual.

[0089] As an eighth aspect, the present invention contemplates an antibody or pharmaceutical composition for use in the treatment of a fungal infection in an individual.

[0090] BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Infectious cycle involving species of the genus Cryptococcus. The fungus can survive in soil and trees and infect several animals, including pigeons, which serve as vectors for the dispersal of fungal cells. In humans, pulmonary infection is established by inhalation of spores or dried yeast present in the environment and may develop into central nervous system (SNC) disease. (Modified from

[28] ).

[0092] Figure 2 Schematic diagram involving the cell wall structure and fungal capsule (modified from

[99] ).

[0093] Figure 3 The action of the four major classes of antifungal drugs is implicated. This diagram demonstrates the action of the four major classes of antifungal drugs. Flucytosine demonstrates its action in nucleic acid synthesis, while the sites of action of the other classes are in the plasma membrane or cell wall, either in ergosterol synthesis or in complex with ergosterol (azoles and AmB, respectively) or in complex with the β-glucan synthase echinocandins (echinocandins). (Modified from

[100] ).

[0094] Figure 4 Involves the development of AcMs by hybridoma technology. B lymphocytes fused with mouse myeloma cells generate hybridomas, which are immortal cells that produce antibodies, which are then selected by immunoassay technology to select the best clones that produce the AcM under study. Figure adapted from Abbas, A. et al. 2015

[073] .

[0095] Figure 5 Concerning the proposed mechanism of action of the antibodies against fungi, the mechanisms of action can be broadly divided into three categories including: A) direct inhibition of growth, B) neutralization of harmful effects of fungal products in host tissues, and C) immunomodulation and enhancement of innate immune mechanisms, which in the case of C. neoformans have no protective effect.

[0096] Figure 6 Figure 1. Mouse serum antibody titration curves obtained by ELISA. Balb / C mice were immunized intraperitoneally (ip) with Cryptococcus gattii and intraperitoneally and intravenously (iv) with chitin trisaccharide. Antibody serum titration was performed on the immunized animals at the end of immunization. (A) Serum titration of immunoglobulin M (IgM); (B) Serum titration of IgG. The curves with squares, triangles and circles represent different animals. The dotted line represents the pre-immunization serum cut-off line.

[0097] Figure 7 Involves isotyping of selected AcMs. This assay was performed using an ELISA indirect kit, which defines the presence of different immunoglobulin (Ig) isotypes. The selected AcMs involved AF1 / CC5 and HC6 / DD11 antibody isotyping, demonstrating the universality of detecting IgMs.

[0098] Figure 8Purification analysis involving AcM AF1 / CC5 and HC6 / DD11. Lane 1: Precision DualColor (Bio-Rad) molecular weight standard; Lanes 2 and 3: AcM AF1 / CC5 and HC6 / DD11, respectively; Lane 4: Precision DualColor (Bio-Rad) molecular weight standard. The major bands in fractions 2 and 3 correspond to the heavy chain (about 70 kDa) and light chain (molecular weight range of about 23-24 kDa) of IgM.

[0099] Fig. 9 Binding saturation curves involving AcM and Candida albicans.

[0100] Fig.10 ELISA saturation curves for detection of Cryptococcus neoformans and Candida albicans by AcM HC6 / DD11 and AF1 / CC5. (A) for antibody AF1 / CC5 and (B) for antibody HC6 / DD11.

[0101] Fig.11 Dot Blot saturation curves for detection of Cryptococcus neoformans and Candida albicans by AcM HC6 / DD11 and AF1 / CC5. (A) for antibody AF1 / CC5 and (B) for antibody HC6 / DD11.

[0102] Fig.12 IF analysis of AcMs HC6 / DD11 and AF1 / CC5 reacting with Candida albicans. The left image shows the fungal cells by differential contrast, the other images show the cells in fluorescence mode. The arrows indicate characteristic polarity markers of this target fungal type, which were confirmed for both AcMs and also identified in the overlay image (Calcofluor / Alexa 548).

[0103] Fig.13 Melanization assay. (A) Visual analysis of pigmentation of fungi grown in liquid medium supplemented with L-DOPA and treated with AcM; B and C) Graphical representation of relative densitometry of pigmentation. AcM HC6 / DD11 showed partial inhibition of pigmentation up to a concentration of 6.2 μg / ml (p<0.05), while AcM AF1 / CC5 showed complete inhibition up to a concentration of 6.2 μg / ml (p<0.001) and partial inhibition at concentrations of 3.2 and 1.6 μg / ml (p<0.05).

[0104] Fig.14XTT assay - Biofilm development. Biofilm development was measured indirectly by the XTT reduction assay. Fungi treated with AcM HC6 / DD11 and AF1 / CC5 showed significantly reduced biofilm development compared to untreated fungi (p<0.05). Black bars: untreated fungi, open bars: treated fungi. Washed cells A and B; unwashed cells C and D.

[0105] Fig.15 XTT assay - biofilm formation. Biofilm formation was measured indirectly by the XTT reduction assay. Fungus H99 treated with AcM HC6 / DD11 and AF1 / CC5 significantly reduced biofilm formation compared to untreated fungi and showed a similar behavior to fungi treated with antibody 18B7 (p<0.05). Black bars: untreated fungi, white bars: treated fungi. Washed cells A and B; unwashed cells C and D.

[0106] Fig.16 ELISA involving whole cells against Cryptococcus neoformans, Giardia lamblia, A549 cell line, E. coli and Staphylococcus aureus. Dashed lines represent reaction blanks.

[0107] Fig.17 Representative sensorgrams demonstrating the interaction of two AcMs towards chitotriose: (A) AcM HC6 / DD11 and (B) AF1 / CC5. Chitotriose concentration of 0.06 M is highlighted in green and chitotriose concentration of 0.1 M is highlighted in red.

[0108] Fig.18 Graphical representation of the fungicide synergistic effect of AcM HC6 / DD11 and AF1 / CC5 when added to 0.1 μg / ml AmB compared to AmB in isolated form at a concentration of 1 μg / ml (p<0.001), both showing synergistic effects from 6.2 μg / ml added to AmB 0.1 μg / ml (A and B). A) AcM induced partial synergistic effects at 1.6 and 3.2 μg / ml concentrations compared to AmB isolated at a concentration of 0.1 μg / ml (p<0.01); B) AcM induced partial synergistic effects at a concentration of 3.2 μg / ml compared to AmB isolated at a concentration of 0.1 μg / ml (p<0.01).

[0109] Fig.19Graphs of the synergistic effect involving AcMs HC6 / DD11 and AF1 / CC5 when added to FLC (A and B). There was a partial induced fungicide synergy of HC6 / DD11 and AF1 / CC5 added to 4 μg / ml FLC starting at a concentration of 3.2 μg / ml compared to FLC in isolation at a concentration of 4 μg / ml (p<0.01). Both AcMs showed partial induced fungicide effects whenever added to FLC at a concentration of 2 μg / ml, behaving similarly to FLC in isolation at a concentration of 4 μg / ml.

[0110] Fig. 20 The administration of AcM, which involves addition to AmB, has a protective effect in the neoformans infection paradigm. The survival curves of animals treated with PBS, AcM, AmB (0.25 mg / kg) and the synergistic group were compared. Compared with the other groups, the isolated treatment group died between 28 and 37 days, while the synergistic group showed 100% survival and had statistical relevance (p≤0.01) (n=7).

[0111] Fig.21 scFv fragments involving murine and humanized antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0113] Unless otherwise specified, each technical term and scientific term used herein has the same meaning as understood by technical experts of the subject matter related to the present invention. The terms used for the purpose of describing the present invention only describe specific achievements and are not intended to limit the scope of ideas. Unless otherwise specified, each number representing quantity, percentage and ratio and other numerical values ​​used in the descriptive reports and claims should be understood as being modified by the word "about" in each case. Therefore, unless otherwise specified, the numerical parameters provided in the descriptive reports and claims are approximate values ​​that can vary depending on the characteristics to be achieved.

[0114] The practice of the present invention will employ, unless otherwise indicated, standard methods of chemistry, biochemistry, recombinant DNA techniques and immunology within the skill of the art. Such techniques are fully explained in the literature. For example, see Fundamental Virology, 2nd edition, Volumes I & II (BN Fields & D.M. Knipe, eds.); Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir & C. C. Blackwell, eds., Blackwell Scientific Publications); T. Creighton, Proteins: Structures & Molecular Properties (W. H. Freeman & Company, 1993); A. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook, & cols., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick & N. Kaplan, eds., Academic Press, Inc.).

[0115] As used in this application, the term "amino acid" means an α-amino acid group that can be encoded by a nucleic acid directly or in a precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides called codons. Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "genetic code degeneracy". As used in this application, the term "amino acid" means naturally occurring α-amino acids, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0116] The terms "peptide", "polypeptide" or "protein" are used interchangeably and refer to a polymer of amino acids linked by peptide bonds, regardless of the number of amino acids constituting the chain. As used herein, polypeptides include their "variants" or "derivatives", which refer to polypeptides that include variations or modifications (e.g., chemical substitutions, deletions, additions or modifications) in the amino acid sequence relative to a reference polypeptide. Examples of chemical modifications include glycosylation, PEGylation, PEG alkylation, alkylation, phosphorylation, acetylation, amidation, and the like. Polypeptides can be artificially produced by cloning nucleotide sequences using DNA technology of recombinant DNA, or prepared by known chemical synthesis reactions.

[0117] More specifically, the term polypeptide of the present invention may also be understood as antigenic, polyantigenic or polyepitopic antigenic, wherein the polyepitopic antigen consists of different epitopes connected, which may or may not be interconnected by flexible or rigid linkers, and the epitopes are specific for a single pathogen or for different pathogens.

[0118] In a first achievement, the present invention provides murine AcMs directed against chitin or chitin oligomers produced by hybridoma technology.

[0119] The term "antibody" is any immunoglobulin associated with a specific epitope, including antibodies and fragments. The term includes polyclonal antibodies, monoclonal antibodies and chimeric antibodies, the latter of which are described in more detail in U.S. Patent Nos. 4.816.397 and 4.816.567. The term "antibody" includes wild-type immunoglobulin (Ig) molecules, typically including four full-length polypeptide chains, i.e., two heavy chains (H) and two light chains (L), or equivalent homologous Ig (e.g., a camel nanobody that includes only one heavy chain); including full-length functional mutants, variants or derivatives that retain the basic epitope binding characteristics of an Ig molecule, and including specific, bispecific, multispecific and dual variable domain antibodies; immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Within the meaning of the term "antibody", any "antibody fragment" is included.

[0120] The term "monoclonal antibody" is widely recognized in the art and refers to an antibody that is produced in large quantities in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically prepared by fusing antibody-producing B cells with rapidly growing cells, including tumor cells (also called "immortal" cells). The resulting hybrid cell, or hybridoma, is a clone that is capable of unlimited production of antibodies under normal culture conditions.

[0121] The term "hybridoma" is also conventionally recognized in the art and is understood by any subject matter expert with general knowledge to refer to a cell produced by fusing an antibody-producing cell with an immortal cell (e.g., a myeloma cell). Such a hybrid cell is capable of continuously providing antibodies.

[0122] The term "antigen" refers to an entity or fragment that can induce an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions responsible for antigenicity or antigenic determinants.

[0123] The fungal antigen used in the present invention can be selected from fungal cell wall components and oligomers. More specifically, the antigen can be selected from chitodisaccharide, chitotrisaccharide, chitotetrasaccharide, chitopentasaccharide, chitohexasaccharide and chitoheptasaccharide, and any other fungal antigens that are derived from chitin and oligomers and can induce an immune response. Preferably, the fungal antigen is chitotrisaccharide.

[0124] The fungal antigens of the present invention can be obtained from any species of the kingdom Fungi, more specifically any species of the phylum Ascomycota, Basidiomycota, Zygomycota and Sordidiaceae, even more specifically, Aspergillus, Candida, Cryptococcus and Pneumocystis. Thus, the species can be selected from Cryptococcus neoformans, Cryptococcus gattii, species of Candida, species of Aspergillus, Histoplasma capsulatum and Coccidioides immitis.

[0125] Furthermore, the antigen may be combined with an adjuvant capable of inducing an immune response.

[0126] An adjuvant is a compound that increases the immune response to an antigen when administered in conjunction with the antigen, but does not produce an immune response to the antigen when administered alone. Adjuvants can increase the immune response through a variety of mechanisms, including lymphocyte recruitment, B cell and / or T cell stimulation, and macrophage stimulation.

[0127] Suitable adjuvants of the present invention may be selected from, but not limited to, the following classes of compounds: (i) Aluminum salts (alum), including aluminum hydroxide, aluminum phosphate, and aluminum sulfate. Such adjuvants may be used with or without other specific immunostimulants, including MPL or 3-DMP, QS21, polymeric or monomeric amino acids, including polyglutamic acid or polylysine; (ii) oil-in-water emulsion formulations, including (a) MF59 (WO 90 / 14837), containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing some amount of MTP-PE), formulated into submicron particles using a microfluidizer such as Microfluidizer Model 110Y (Microfluidics, Newton Mass.), (b) SAF, containing 10% squalene, 0.4% polymer L121 blocked with Tween 80.5% Pluronic, and thr-MDP, microfluidized into a submicron emulsion or vortexed to produce an emulsion with larger particle size, and (c) Ribi TM (RAS) adjuvant system (Ribi Immunochem, Hamilton, Mont.), containing 2% squalene, 0.2% Tween80 and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimicolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox TM ); (iii) saponin adjuvants, including Stimulons (QS21, Aquila, Worcester, Mass.) or particles generated therefrom, including ISCOMs (immunostimulatory complexes) and ISCOMATRIX; (iv) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (v) cytokines, including interleukins, such as IL-1, IL-2 and IL-12, macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF) and / or chemokines, including CXCL10 and CCL5. Preferably, the adjuvant used is aluminum hydroxide (Al(OH)3).

[0128] Originally by & Milstein developed

[075] , immunization is performed in hybridomas, mice or other suitable host animals to generate lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization.

[0129] After immunization, antibody-producing cells (splenic lymphocytes) fuse with myeloma cells (malignant cells of primary tumors of the bone marrow) to produce a single fused cell hybrid (called a hybridoma) that inherits certain characteristics of lymphocytes and myeloma cell lines. A suitable fusing agent may be polyethylene glycol.

[0130] The hybridoma cells prepared in this manner are cultured in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, due to the lack of hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium selected for hybridomas should generally include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents cells from growing insufficiently in HGPRT.

[0131] Hybridomas secrete a single specific immunoglobulin type against an antigen; moreover, as myeloma cells, hybrid cells have an indeterminate cell division potential. The combination of these two characteristics provides different advantages over standard antisera. While antisera from animals used for vaccination are variable mixtures of polyclonal antibodies that are not completely reproducible, monoclonal antibodies are a single type of highly specific immunoglobulin.

[0132] The only type of immunoglobulin secreted by the hybridoma (monoclonal antibody) is specific for only one antigenic determinant, or epitope, in the antigen, which is a complex molecule with multiple antigenic determinants.

[0133] The culture medium in which the hybridoma cells are cultured is tested to determine the production of monoclonal antibodies directed against the antigen. Preferably, the binding of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, including immunoassay (RIA) or with enzyme-linked immunosorbent assay (ELISA).

[0134] After hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (98). Suitable culture media for such purposes include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo, for example, by injecting the cells intraperitoneally into mice.

[0135] Monoclonal antibodies produced by the hybridomas are suitably separated from the culture medium, ascites fluid, or serum by standard procedures for antibody purification, including affinity chromatography (e.g., using protein A or protein G-Sepharose) or exchange chromatography, ion chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and the like.

[0136] In this context, several specialized myeloma cell lines have been developed to produce hybridomas, for example, the following cell lines are well known in the art: X63-Ag8, NSI-Ag4 / 1, MPC11-45.6TG1.7, C63-Ag8.653, Sp2 / 0-Ag14, FO and S194 / 5XXO, BU.1, 210.RCY3.Agl.2.3, U-226AR, GM1500GTGAL2, U-226AR and GM1500GTGAL2. More specifically, the cell line is SP2 / 0 (Sp2 / 0-Ag14) ( CRL-1581 TM ).

[0137] Typically, after cell fusion, the resulting product is cultured in a selective medium, for example, HAT medium containing hypoxanthine, aminopterin and thymidine.

[0138] Such medium enables the hybrid cells to proliferate and prevents the growth of unfused myeloma cells, which normally would continue to divide indefinitely.

[0139] The myeloma cells used were mutants that lacked the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), so the rescue pathway could not be used. In the surviving hybrids, the B lymphocytes provided the genetic information to produce this enzyme. Since B lymphocytes themselves have a limited lifespan in culture (approximately two weeks), the only cells that can proliferate in HAT medium are hybrids formed from myeloma and spleen cells.

[0140] Since antibodies can vary in different ways, the term "antibody" is understood to encompass any molecule or substance with a binding domain having the desired specificity. Thus, the term encompasses antibody fragments, derivatives, functional equivalents, and antibody homologs, including any polypeptide, including any immunoglobulin binding domain, whether natural or synthesized in whole or in part. Thus, chimeric molecules are included, which include an immunoglobulin binding domain or an equivalent structure fused to another polypeptide. The cloning and expression of chimeric antibodies is provided in EP-A-0120694 and EP-A-0125023 and U.S. Patent Nos. 4.816.397 and 4.816.567.

[0141] It has been demonstrated that intact antibody fragments can perform antigen binding functions. Examples of binding fragments include (i) Fab fragments consisting of the domains VL, VH, CL and CH1; (ii) Fd fragments consisting of the VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single VH domain

[101] ; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment consisting of two Fab fragments bound together; (vii) single-chain Fv molecules (scFv) , wherein the VH domain and the VL domain are linked by a peptide linker that enables the two domains to bind to form an antigen binding site [102, 103]; (viii) multivalent antibody fragments (dimers, trimers and / or tetramers of scFv

[104] , (ix) bispecific single-chain Fv dimers (PCT / US92 / 09965), and (x) diabody multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804;

[105] ).

[0142] The expression "antibody molecule" and its various grammatical forms as used herein includes both intact immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.

[0143] The antibody molecule can be, for example, a complete immunoglobulin molecule, a substantially complete immunoglobulin molecule, and a portion of an immunoglobulin molecule containing a paratope, including portions known in the art, including Fab, Fab', F(ab')2, and F(v), which portions are preferably used in the therapeutic methods described herein.

[0144] The Fab and F(ab')2 portions of antibody molecules are prepared separately in substantially intact antibody molecules by widely known methods by proteolytic reactions with papain and pepsin. For example, see U.S. Pat. No. 4,342,566 to Theofilopolous et al. The Fab' antibody molecule portion is also well known and is produced from the F(ab')2 portion, followed by linking the two heavy chain portions with a disulfide bond such as mercapto-ethanol, followed by alkylation of the thiol protein with reagents including iodo-acetamide. Antibodies containing intact antibody molecules are preferred herein.

[0145] In addition to traditional hybridoma technology, there are several other well-known techniques for making monoclonal antibodies. Methods for making fully human antibodies are particularly useful. One method is phage display technology using affinity enrichment methods, which can be used to select several human antibodies that specifically bind to an antigen. Phage display is well described in the literature, and the development and screening of phage display libraries are well known in the art, for example [106, 107, 108, 109]. Fully human antibodies can also be made by immunizing transgenic mice with an immunogen that provides most of the heavy and light chains of human immunoglobulins. Examples of such mice are well known in the art, for example, (Abgenix, Inc.) and HuMAb-Mouse (Medarex, Inc.), see also U.S. Patent Nos. 6.207.418, 6.150.584, 6.111.166, 6.075.181, 5.922.545, 5.545.806 and 5,569,825. Antibodies can be prepared by standard techniques such as standard hybridoma technology or phage display.

[0146] Monoclonal antibodies obtained from species other than human (including mice) by hybridoma technology can be humanized, which means that genetically non-human antibodies are modified to be more human-like with the goal of preventing HAMA when infused into humans. Methods for antibody humanization are well known in the art, with the most commonly used methods including complementary determining region (CDR) grafting and surface modification (also known as neosurfacing). These methods have been widely described in the literature and patents, see, for example

[110] ; U.S. Patent Nos. 5.225.539, 5.530.101, 5.585.089, 5.859.205 and 6.797.492, each of which is incorporated herein by reference.

[0147] More specifically, the antibodies of the present invention include the following light chain and heavy chain nucleotide sequences; or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or degenerate sequences thereof:

[0148] i)HC6 / DD11

[0149] Heavy chain nucleotide sequence (VH):

[0150]

[0151] Light chain nucleotide sequence (VL):

[0152]

[0153] Sequence translation and associated CDRs using the Kabat definition method:

[0154] VH

[0155]

[0156] V L

[0157]

[0158] More specifically, the CDRs comprise the following sequences: (i) the VH CDR1 sequence described in SEQ ID NO: 1, the VH CDR2 described in SEQ ID NO: 2, and the VH CDR3 described in SEQ ID NO: 3; and (ii) the VL CDR1 sequence described in SEQ ID NO: 4, the VL CDR2 described in SEQ ID NO: 5, and the VL CDR3 described in SEQ ID NO: 6, or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0159] i)AF1 / CC5

[0160] Heavy chain nucleotide sequence (VH):

[0161]

[0162] Light chain nucleotide sequence (VL):

[0163]

[0164] Sequence translation and associated CDRs using the Kabat definition method

[0165] VH

[0166]

[0167] V L

[0168]

[0169] More specifically, the CDRs comprise the following sequences: (i) the VH CDR1 sequence described in SEQ ID NO:7, the VH CDR2 described in SEQ ID NO:8, and the VH CDR3 described in SEQ ID NO:9; and (ii) the VL CDR1 sequence described in SEQ ID NO:10, the VL CDR2 described in SEQ ID NO:11, and the VL CDR3 described in SEQ ID NO:12, or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0170] Mutations can be made in the DNA sequences encoding antibodies or peptides provided herein (including in CDRs) so that specific codons are replaced by codons encoding different amino acids. Such mutations are usually carried out by making as few changes as possible. This type of substitution mutation can be carried out so that the amino acid in the protein is replaced in a non-conservative manner (i.e., the codons of the amino acid belonging to an amino acid group with a specific size or feature are changed to the amino acid belonging to another group) or in a conservative manner (i.e., the codons of the amino acid belonging to an amino acid group with a specific size or feature are replaced to an amino acid belonging to the same group). Conservative replacements usually result in smaller changes in the structure and function of the resulting protein. Non-conservative changes may change the structure, activity or function of the resulting protein more. It should be recognized that the present invention includes sequences containing conservative changes that do not significantly change the resulting protein binding activity or characteristics.

[0171] The polypeptides of the present invention exhibit reproducibility with respect to sensitivity and specificity. This indicates that the developed protein can remain stable for a long time, thereby maintaining its responsiveness. It is possible that the use of a stock buffer composition protease inhibitor, the presence of a buffer denaturant or even due to its amino acid sequence may have contributed to its stability. Whenever diagnostic applications are of interest, a stable protein is a protein of consideration.

[0172] As a second achievement, the present invention discloses a pharmaceutical composition comprising the above monoclonal antibody.

[0173] Pharmaceutical compositions may include the AcM combinations of the present invention.

[0174] The above compositions may additionally include other antifungal agents.

[0175] Suitable antifungal agents can be selected from the group consisting of (1) to (5): (1) polyene antifungal agents, including natamycin, cytomycin, filipin, nystatin, amphotericin B, and candidiasis; (2) imidazole antifungal agents, including miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole; (3) triazole antifungal agents, including FLC, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, and terconazole; (4) allylamine antifungal agents, including terbinafine, amorolfine, naftifine, and butenafine; (5) echinocandin antifungal agents, including anidulafungin, caspofungin, and micafungin.

[0176] Preferably, the other antifungal agent is AmB or FLC.

[0177] Furthermore, the pharmaceutical composition may be formulated with pharmaceutically acceptable carriers or excipients and any other adjuvants and diluents known to the subject matter expert having general knowledge in this field.

[0178] Pharmaceutically acceptable carriers include excipients and adjuvants that can process active compounds in pharmaceutically acceptable preparations. Pharmaceutically acceptable media are well known in the art and are described in, for example, Gennaro, Alfonso, ed., Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing Co., Easton, Pa., which is a reference text in the art. More specifically, pharmaceutically acceptable excipients, carriers or stabilizers are non-toxic to the recipient organism at the dosages and concentrations used, and include buffers such as phosphoric acid, citric acid and other organic acids; antioxidants such as ascorbic acid and methionine; conservants such as octadecyldimethylbenzyl ammonium, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzilic acid, alkyl parabens such as methyl and propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; proteins such as albumin, gelatin or immunoglobulins; amino acids, monosaccharides, disaccharides and other carbohydrates such as glucose, mannose, sucrose, mannitol or sorbitol; polymer excipients such as polyvinyl pyrrolidone, Dextrin and polyethylene glycol; flavoring agents, sweeteners, antistatic agents; chelating agents, such as EDTA or EGTA; ion-releasing salts, such as sodium; metal complexes; nonionic surfactants, such as polysorbate 20 and 80; lipids, such as phospholipids, fatty acids, and steroids, such as cholesterol. Methods for preparing several pharmaceutical compositions are well known to experts in the field of pharmaceutical technology or should be obvious in light of the present invention.

[0179] Furthermore, the composition may include additives for the purpose of increasing ease of administration, storage capacity, degradation strength, bioavailability, half-life, providing isotonic preparations, etc. Common additives for the formulation of pharmaceutical compositions are well known in the art.

[0180] The pharmaceutically acceptable medium can be routinely selected based on the mode of administration of the peptide as well as the solubility and stability.

[0181] For example, formulations for intravenous administration may include sterile aqueous solutions which may also include buffers, diluents and other suitable additives.

[0182] Suitable formulations for parenteral administration include aqueous solutions of the active compound in water-soluble form, such as soluble salts in water. In addition, suspensions of the active compound may be administered, including suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions that may contain materials that increase the viscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. Alternatively, the suspension may also contain a stabilizer.

[0183] Pharmaceutical compositions for oral administration (at appropriate doses) may preferably, but not necessarily, be in the form of tablets or capsules of sizes standard for the pharmaceutical industry.

[0184] The concentration of the antibody of the invention in the pharmaceutical composition can vary widely and is selected based on, among other things, fluid volume, viscosity, etc. The particular mode of administration should be determinative. The composition may also include another antibody type.

[0185] For any compound, a preliminary estimate of the therapeutically effective dose can be made, whether in a cell culture assay (e.g., tumor cells) or in an animal model (usually mice, rabbits, dogs, or pigs). Animal models can also be used to define appropriate concentration ranges and routes of administration. Such information can then be used to define useful doses and routes of administration for humans.

[0186] In general, the amount of active ingredient that can be combined with a pharmaceutically acceptable medium to produce a single dosage form should vary depending on the individual to be treated and the specific mode of administration. The amount of active ingredient that can be combined with a pharmaceutically acceptable medium to produce a single dosage form should generally be the amount of the composition that produces a therapeutic effect. Typically, in one hundred percent, when combined with a pharmaceutically acceptable medium, the amount varies from about 0.01% to 99% of the active ingredient, preferably from about 0.1% to 70%, and more preferably from 1% to 30%.

[0187] The pharmaceutical compositions of the present invention can be administered by several routes of administration, including oral, sublingual, nasal, intravenous, intramuscular, intraperitoneal, intraarticular, subcutaneous, cutaneous, transdermal, etc., but are not limited to these.

[0188] As a third achievement, the present invention discloses the use of the above monoclonal antibody to prepare a drug for treating fungal infection. The use can be combined with other active antifungal agents, including AmB and / or FLC.

[0189] "Combination" may be understood as the administration of two or more therapeutic agents to treat a disease, condition, and / or disorder. Such administration includes co-administration of two or more therapeutic agents in a substantially simultaneous manner, for example in a single capsule having a fixed ratio of active ingredients, or in multiple capsules with each active agent separate. In addition, administration may be sequential.

[0190] The antibodies of the present invention can be administered in combination with other standard drugs to treat fungal infections. AmB, which has a broad antibacterial spectrum against yeasts and filamentous fungi, and FLC, which has a fungicidal effect against Cryptococcus, are preferred.

[0191] Both drugs act in ergosterol, with AmB binding directly to ergosterol to form a pore, while FLC binds to lanosterol 14α-demethylase, a key enzyme in ergosterol synthesis.

[0192] As a fourth achievement, the present invention discloses a method for diagnosing fungal infection, comprising:

[0193] (i) providing the above-mentioned monoclonal antibody or composition and a sample obtained from an individual;

[0194] (ii) contacting the monoclonal antibody or composition with the sample to be tested for a sufficient time and under conditions sufficient to form an antigen / antibody complex; and

[0195] (iii) detecting the antigen / antibody complex formed in the previous stage by a detection technique capable of generating a detectable signal in the presence of the above antigen / antibody complex.

[0196] The sample may be selected from the group consisting of saliva, urine, serum, blood, bronchoalveolar lavage fluid, peritoneal fluid or fluid or any other biological fluid of the individual.

[0197] The antibody may include a detectable marker, including a fluorescent, radioisotope, chemiluminescent agent, or an enzyme, including wild radish peroxidase, alkaline phosphatase, or luciferase.

[0198] Several fluorescent materials are known and can be used as markers. For example, they include fluorescein, rhodamine, auramine, Texas red, AMCA blue and fluorescein yellow. The specific detection material is an anti-rabbit antibody prepared in goats and conjugated to fluorescein by isothiocyanate. The peptide SLC34A2 or its binding partner can also be labeled with a radioactive element or with an enzyme. The radioactive marker can be detected by any currently available counting procedure. Preferred types can be selected from 3 H. 14 C. 32 P. 35 S. 36 C1. 51 Cr, 57 Co. 58 Co. 59 Fe, 90 Y. 123 I. 125 I, 131 I. 186 Re, 99 Tc, 67 Ga, 201 Tl and 111 In.

[0199] Enzyme markers are equally useful and can be detected by any colorimetric method, spectrophotometry, fluorospectrophotometric, amperometric or gas quantification method currently used. The enzyme is conjugated in the selected particle by a bridge molecule reaction (including carbodiimide, diisocyanate, glutaraldehyde, etc.). Many enzymes that can be used in these programs are known and can be used. Preferred enzymes include peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. For alternative labeling methods and material disclosures, reference is made to, for example, U.S. Patent Nos. 3.654.090, 3.850.752, 4.016.043 and 4.016.043.

[0200] The detection medium may be known in the art.A non-limiting example of a detection medium may be a conjugated antibody coupled to a compound capable of generating a detectable signal (thereby capable of generating a detectable signal).

[0201] Alternatively, the antibody can be bound to a solid support that can be adapted for assay automation. Suitable solid supports include, but are not limited to, glass or plastic inserts, tissue culture plates, microtiter wells, tubes, chips, or particles such as beads selected from, but not limited to, latex beads, polystyrene beads, or glass beads.

[0202] The present invention can be carried out by any method known in the art that can be used to connect the antibody to the solid support, including covalent and non-covalent binding, passive adsorption or binding of the binding moiety of the antibody and the solid support. Antigen and antibody binding can be carried out in any receiving container sufficient to accommodate the reagents. Examples of such receiving containers include microtiter plates, test tubes and microcentrifuge tubes.

[0203] Antibodies may also be used, particularly to differentiate invasive fungal infections from bacterial infections in hospital settings and in high-risk and / or immunocompromised patients.

[0204] As a fifth achievement, the present invention discloses a fungal infection diagnostic kit comprising the above monoclonal antibody or composition and instructions for use.

[0205] The kit may also include an antigen / antibody complex detection medium, which may include a signal generating agent capable of generating a detectable signal. For example, the antibody may carry a label and a detection medium that can detect the antibody whenever the antibody binds to its associated antigen.

[0206] The detection medium can be a fluorescent marker, such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), etc., an enzyme such as wild radish peroxidase (HRP), glucose oxidase or the like, a radioactive element, such as a gamma-ray emitting 125 I or 51 Cr, or a radioactive element that releases positrons, which, when encountering electrons present in the test solution, 11 C. 15 O or 13 N produces gamma rays. Binding can also be detected by other methods, such as by avidin-biotin. Moreover, the labeling agent can be any enzyme included in the group of oxidases (such as radish peroxidase), luciferase, peptidase (such as caspase-3), glucosidase (such as β-galactosidase) and phosphatase (such as alkaline phosphatase).

[0207] Detection medium binding is well known to experts in the subject technical field. Thus, the produced AcM can be metabolically labeled by incorporating amino acids containing radioisotopes into the culture medium, or can be conjugated or coupled to the detection medium via an activated functional group.

[0208] The fungal infection diagnostic kit suitable for the present invention may preferably be based on the following technologies: ELISA, Lateral Flow Immunochromatographic Assay and Liquid Microarray.

[0209] ELISA tests are based on antigen-antibody reactions that can be detected by enzymatic reactions.

[0210] The purified antigen is immobilized on a suitable solid support (eg, a polystyrene plate). Next, the sample is added to the wells, and if the sample is a positive individual for the above condition, the specific antibodies should bind to the antigen immobilized on the solid support.

[0211] After this stage, an antibody bound to a marker (peroxidase) is added to the antibody bound to the antigen. Thus, in the wells where antigen-antibody binding occurs, coloring occurs as a substrate is added to the marker.

[0212] Lack of staining indicates a lack of antibodies against the substrate antigen in the sample.

[0213] The lateral flow immunochromatographic assay consists of different overlapping membranes assembled on a supporting adhesive card. The antibodies are immobilized on a nitrocellulose membrane, which overlaps with a membrane that receives the sample to be tested in the proximal area of ​​the card. The membrane impregnated with the compound that reveals the reaction (conjugated) is located at the end of the nitrocellulose membrane, while the absorbent membrane is located in the distal area. The resulting strip is encapsulated in a plastic device.

[0214] The test uses a certain volume of the sample to be evaluated (serum, plasma or blood) and then immediately applies a buffer. The buffer solution enables the migration of the conjugated protein included in the sample.

[0215] Sample migration occurs by capillary action at room temperature to the test line area and control. The test is visually read after sample / buffer migration is complete, which usually occurs within 15 minutes. Observation of both the test and control lines indicates a positive result for the disease. No observation of the test line but a control line mark indicates a negative result, and failure to observe the control line invalidates the test.

[0216] Liquid microarray is an experiment using a polystyrene microsphere matrix with a diameter of 5.6 or 6.5 microns in suspension as a solid support for antibodies coupled by covalent bonding. Antibody-antigen reaction detection is carried out with the assistance of detection molecules (fluorophores), especially phycoetritin. In this system, a mixture of different types of microspheres can be used, which have uniform sizes but different fluorescence intensities released by them.

[0217] The conjugation was performed via covalent bonding between the carboxylated surface of the microspheres and the primary amines present in the antibody.

[0218] The microspheres have internal dyes with individual codes that distinguish single emission spectra. Thus, analysis of multiple analytes can be performed simultaneously, since each microsphere covalently coupled to a capture reagent can be distinguished by its spectrum.

[0219] The reaction is read by aspirating microspheres in a solution that is delivered to a special chamber. The microspheres are then focused in a continuous flow and individualized manner so that two lasers intercept one particle at a time and accurately identify each code.

[0220] As a sixth aspect, the present invention provides a fungal infection treatment kit, comprising

[0221] (i) the monoclonal antibody provided in claim 1 or the composition provided in any one of claims 2 to 4; and

[0222] (ii) antifungal agents;

[0223] (iii) instructions for using the components in combination.

[0224] As a seventh achievement, the present invention provides a method for treating fungal infection, comprising administering a therapeutically effective amount of the above-mentioned antibody or composition to an individual in need thereof.

[0225] As an eighth achievement, the present invention provides an antibody or composition for treating a fungal infection in a subject in need thereof.

[0226] Therapeutic uses may be envisaged for both human and veterinary uses. Preferably, the individual treated is a human in need of treatment.

[0227] The effective amount for a human subject will depend on the severity of the disease state, the individual's overall health, age, weight, sex, diet, time and frequency of administration, one or more drug combinations, reaction sensitivity, and tolerance / response to treatment. Thus, the dosage to be provided depends on several factors that cannot be measured before conducting clinical trial studies. However, subject matter experts know how to obtain adequate dosages for different treatments.

[0228] The above examples are for illustration only. They are only used for a better understanding of the developments provided by the present invention, but should not be used to limit the purpose described. Example

[0229] Example 1 - Cell types and growth conditions

[0230] The fungal species used included Cryptococcus neoformans (serotype A, clinical isolate H99ATCC 208821), Cryptococcus gattii (serotype B, strain R265 ATCC MYA-4093), Candida albicans (ATCC 90028), Cryptococcus neoformans without capsule (mutant Cap67 ​​ATCC 52817), Giardia lamblia (ATCC 30957), human lung lineage cells A549 (ATCC CCL-185), Escherichia coli (ATCC 9637) and Staphylococcus aureus (ATCC 25923). For in vitro and in vivo experiments, cells were cultured in minimal medium (glucose 15mM, MgSO4 10mM, KH2PO4 29.4mM, glycine 13mM, thiamine-HCl 3μM, pH 5.5) and kept at 30°C for 2 days under stirring. Cells were harvested by centrifugation, washed in PBS, and counted in a Neubauer chamber.

[0231] For hybrid cell development, the SP2 / 0 myeloma cell line (Sp2 / 0-Ag14) ( CRL-1581 TM ), and B cells from the spleen of animals previously immunized with chitin oligomers and the fungus Cryptococcus gattii. Culture was performed in DMEM (LONZA) medium supplemented with 6.4 mM glutamine / SFB 10%. All contents were transferred to a T-shaped 25 cm 2 bottle and incubate at 37°C / 5% CO 2 Incubate until desired viability is reached.

[0232] Example 2 - Animal Immunization

[0233] Mice were immunized as follows: Balb / C mice were immunized intraperitoneally (ip; 200 μl) every 15 days. Thus, two different strategies were used: in the first strategy, C. gattii (1×10 6The animals were immunized ip with 200 μg of free chitin trisaccharide (a trimer molecule composed of β-1,4-N-acetylglucosamine-β-1,4-GlcNAc units) using aluminum hydroxide (Al(OH)3–1.5 mg) as an adjuvant at an interval of 15 days, with the adjuvant and free chitin trisaccharide matching a 1:1 ratio (v / v). Finally, the animals were immunized intravenously (iv) with 50 μg of free chitin trisaccharide using PBS as a vehicle. The second strategy was identical to the first, except that an additional immunization with free chitin trisaccharide and Al(OH)3 was performed before the final iv injection. In both strategies, bleeding was performed at the end of immunization to check the antibody titer in the serum by indirect ELISA. Pre-immune serum was collected from each animal to be used as a screening control and cut off.

[0234] Example 3 - Fusion

[0235] After the fourth / fifth immunization, the animals were splenectomized to process the spleen cells for cell fusion with murine myeloma cells SP2 / 0 (ATCC), adapted from &Milstein 1975.

[0236] Splenocytes and SP2 / 0 were fused with the aid of 50% PEG 3000-3700 solution preheated at 37° C. Subsequently, cells were homogenized at 1×10 8 The entire suspension volume was transferred to 96-well plates for cell culture (Corning) at a ratio of 10 cells / 100 mL. 6 x 10 4 SP2 / 0 cells were used as a selection medium control. The plate was then incubated at 37°C, 5% CO2 for 24 hours, and one day after the initial incubation, 100 μL of DMEM medium supplemented with 6.4 mM glutamine / ATB1x / SFB 20% / hypoxanthine, aminopterin, thymidine (HAT) 2x was added to the wells of the plate to start the surviving hybrid cell selection process, which was obtained from a 14-day fusion process. The supernatant of the well was used for a specific indirect ELISA test for chitin trisaccharide.

[0237] Example 4 - Direct Elisa

[0238] Indirect ELISA (97) was performed twice: 1) to determine the antibody titer of the animal serum at the end of the immunization process; 2) to determine the polyclonal and monoclonal antibodies specific for chitin oligomers produced by the hybridoma.

[0239] Example 4.1 - ELISA for determination of animal serum titers

[0240] 96-well plates were coated with BSA-conjugated chitin trisaccharide at a concentration of 0.5 μg / ml in PBS and incubated overnight at 4°C. After incubation, the plates were incubated with PBS / BSA 1% at 37°C for 1 hour, followed by washing and adding different dilutions of animal serum and incubating at 37°C for 2 hours. The plates were washed 3 times with PBS / Tween 0.05%, and peroxidase-conjugated mouse anti-IgG and anti-IgM were added and incubated at 37°C for 2 hours. After incubation, the plates were washed as before and incubated with tetramethylbenzidine (TMB) and incubated at 37°C for 30 minutes. The reaction was stopped with 1N HCl and a 450nm reading was obtained in a spectrophotometer.

[0241] Example 4.2 - ELISA for determination of polyclonal and monoclonal antibodies

[0242] The same procedure as above was performed, except that the reactive primary antibody was derived from the hybridoma culture supernatant.

[0243] Example 5 - Polyclonal hybridoma selection

[0244] The positive polyclonal hybridomas in the ELISA test provided above were cloned by counting cells in a Neubauer chamber so that this cell suspension dilution finally presented a concentration of 1 cell / well in a final volume of 200 μl. The culture was incubated for 14 days at 37° C., 5% CO2, and colonies (monoclonal or polyclonal) were observed from the 5th day. The cultures that remained viable and monoclonal were again subjected to the ELISA test to check the specificity for the determined antigen.

[0245] Example 6 - Isotyping of selected clones

[0246] The clones previously selected by ELISA were isotyped by a commercial kit - Rapid ELISA Mouse mAb Isotyping Kit - ThermoFisher. Therefore, the supernatant of the colony culture was diluted 1:10 and added to a specific test plate. The kit quickly and efficiently determines the presence of murine isotypes IgG1, IgG2a, IgG2b, IgG3, IgA and IgM in the sample.

[0247] Example 7 - RNA extraction of selected monoclonal antibodies

[0248] RNA extraction was performed for each monoclonal antibody derived from culture flask T-25, where cells were collected only by centrifugation (400 g, room temperature for 10 minutes). Each monoclonal antibody pellet was used for total RNA extraction using a commercial kit, RNeasy Mini kit (Qiagen), following the manufacturer's protocol.

[0249] Example 8 - Polymerase chain reaction by reverse transcriptase (RT-PCR)

[0250] Prior to the RT-PCR stage, monoclonal antibody cDNA synthesis was performed by a commercial kit, Super Script III First-Strand Synthesis System (INVITROGEN).

[0251] Subsequently, PCR was performed with primer oligonucleotides selected from the publication of Zhou et al. in 1994, as they are described as universal primers for murine variable heavy chain (VH) and variable light chain (VL), as provided in Table 2. The sequences obtained were evaluated in the Kabat database.

[0252] RT-PCR was performed under the following conditions: for the VH chain, initial denaturation at 94°C / 5 min, denaturation at 94°C / 2 min, annealing at 48°C / 1 min, extension at 72°C / 1 min 30 sec, repeated 30 times, and a final extension at 72°C / 1 min. The conditions for the VL chain were the same as those for the VH chain, but the annealing temperature was 55°C / 1 min.

[0253] Band observation was performed on 1.5% agarose gel, and the size of VH was about 570 pb, and the size of VL was about 370 pb.

[0254] Table 2c - Synthetic oligonucleotides (starting from the 5' end) used in RT-PCR reactions to amplify cDNA encoding urine VH and VL.

[0255]

[0256]

[0257] Example 9 - DNA Sequencing of Selected Monoclonal Antibodies

[0258] DNA sequencing of the selected monoclonal antibodies was performed according to the protocol described in the commercial kit BigDye Terminator v3.1 (Life Technologies), and the same primers described in PCR were used (Example 8). Moreover, the identity of each sequencing was evaluated using the BLAST-Basic Local 54 alignment search tool (www.ncbi.nlm.nih.gov / blast) in the VH and VL sequences of the selected hybridomas. The sequences were evaluated using the SeqMan program (DNAStar), and CDR1, 2, and 3 were identified, and the gene sequences were submitted to the IgBlast tool (IgBlast Tool-NCBI-NIH; https: / / www.ncbi.nlm.nih.gov / igblast / ) for analysis.

[0259] Example 10 - Monoclonal Antibody (AcM) Purification

[0260] AcM purification was performed in three stages: PEG precipitation, size exclusion chromatography, and ion exchange chromatography in high performance liquid chromatography (AKTA Purifier 10; GE Healthcare).

[0261] For PEG precipitation, the culture supernatant was precipitated with 4% (p / v) polyethylene glycol (PEG 6000). The suspension was maintained by stirring for 3 hours at room temperature (TA), and the material was then centrifuged (1600 x g; 30 minutes; 4°C).

[0262] The supernatant obtained after centrifugation was subjected to a second precipitation with 6% (p / v) PEG 6000, and then centrifuged under the same conditions as above. The obtained precipitate was dissolved in a 15 mL volume of a buffer solution Tris-HCl 50 mM pH 8.0.

[0263] The material obtained after the second precipitation was fractionated by size exclusion chromatography (SEC) using a Superdex 200 high load 26 x 60 (320 mL) column at a flow rate of 3.0 mL / min, buffer solution Tris-HCl 50 mM pH=8.0 was used as eluent, and the collection volume was equal to 10 mL.

[0264] After selecting and pooling samples from SEC, anion exchange chromatography was performed in Pores HQ 10 x 100. Fraction elution was performed with two saline gradients (20% and 50%) at a flow rate of 5.0 mL / min in a buffer solution Tris-HCl 50 mM pH 8.0. Fractions were collected in 4.0 mL volumes.

[0265] The homogeneity of the samples obtained at each stage of the purification process was assessed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). To estimate the molecular weight (pM), Precision Plus Protein TM Dual Color (Bio-Rad) commercial standards. Proteins were visualized using Coomassie Blue R350 staining solution and were visualized using Gel Doc TM After image processing in the XR+ (BIO RAD) system, the TM Software evaluation results.

[0266] Example 11 - Affinity Constant Determination and Dissociation by Surface Plasmon Resonance (SPR)

[0267] SPR experiments were performed using a BIACORE X (GE Healthcare) system equipped with a chip sensor CM5. The joints tested included the use of amine coupling to immobilize AcM AF1 / CC5 and HC6 / DD11 (described later in the results). The two flow cell surfaces were activated for 7 minutes with a 1:1 mixture of 0.1M NHS (N-hydroxysuccinimide) and 0.1M EDC (3-(N,N-dimethylamine)propyl-N-ethylcarbodiimide) at a flow rate of 10μl / min. The joint was fixed in 10mM, pH 5.0 sodium acetate at a concentration of 100μg / ml. Ester waste was inactivated by injecting 1M, pH 8.0 ethanolamine for 7 minutes.

[0268] To collect kinetic binding data, the analyte BSA-(GlcNAc)3 was injected into two flow cells at 0.1 and 0.6 nM concentrations using buffer HBS-EP (10 mM HEPES, 150 mM NaCl, 3 MmEDTA and 0.005% P20) pH 7.4 at a flow rate of 5 μl / min and a temperature of 25°C.

[0269] The data were adjusted by a single interaction template (1:1) concentration of linker and analyte using the global data analysis option that allows setting all acquired plots simultaneously. All data were evaluated in BiaEvaluation 4.1 software.

[0270] Example 12 - Binding site determination assay

[0271] 96-well plates were coated with chitin trisaccharide-BSA at a concentration of 0.5 μg / ml in PBS and incubated overnight at 4°C, then incubated with 1% PBS / BSA at 37° for 1 hour. Subsequently, washing (PBS / Tween 0.05%) was performed, and a wheat germ agglutinin solution (WGA) was added, with a conjugated peroxidase at a concentration of 25 μg / ml used as a reaction control. Cold WGA was used to test the system in order to block the binding site of chitin trisaccharide-BSA with AcMm to check whether there was binding at chitin trisaccharide-BSA. Cold WGA was incubated at 37°C for 1 hour, then washed 3 times with PBS / Tween 0.05%. AcM was incubated at a concentration of 25 μg / ml, and then incubated at 37°C for 2 hours. The plates were washed three times with PBS / Tween 0.05% and incubated with peroxidase-conjugated mouse anti-IgM (1:10000) for another 2 hours at 37° C. The system was washed as before and incubated with TMB for serological development as described above.

[0272] Example 13 - Functional Test

[0273] Example 13.1 - Indirect ELISA for sound cells adapted from Stearns et al. 1999

[0274] Cryptococcus neoformans (H99), Candida albicans, Giardia lamblia, human lung cell line A549 (ATCC), Escherichia coli and Staphylococcus aureus were used for the test. The cells were washed three times in PBS and incubated at 4°C for 10 min. 7 The cells were suspended in poly-L-lysine (5 μg / ml in PBS) at a density of 10 cells / ml overnight to allow adhesion. The next day, the plates were blocked with PBS / BSA 5% and incubated at 37°C for 1 hour, then they were incubated with anti-chitin oligomer AcM at a concentration of 50 μg / ml at 37°C for 1 hour, and the anti-chitin oligomer AcM was diluted to 5 μg / ml. Then, they were washed 3 times with PBS / Tween 0.05%, anti-IgM mouse peroxidase diluted 1:5000 was added and incubated at 37°C for 2 hours. The plates were washed again, TMB was added, and incubated at 37°C for 30 minutes.

[0275] The reaction was stopped with 1N HCl and read with a spectrophotometer at 450 nm. 7 Serial dilutions were performed until the cell density reached 10 cells / ml for fungi and 10 cells / ml for other cell types. 7 The cell density was serially diluted to 10 4cells / ml, and tested the anti-chitin oligomer AcM at a concentration of 25 μg / ml, and then followed the same procedure as above.

[0276] In addition, similar experiments were performed with different concentrations of another chitin derivative, chitosan (deacetylated form), following the same procedure as described.

[0277] Example 13.2 - Dot blot for resistant cells adapted from Nimrichter et al. 2007

[0278] The density is 10 7 Cryptococcus neoformans (H99) and Candida albicans of 10 cells / ml were suspended in poly-L-lysine solution (5 μg / ml in PBS) until 10 cells / ml, and 10 μl were loaded into nitrocellulose membrane. Then, the same steps as ELISA were followed, but the concentration of anti-chitin oligomer AcM was 25 μg / ml. The membrane was cut and deposited in a 96-well plate, 50 μl TMB was added thereto and incubated at 37°C for 30 minutes. The volume was removed and transferred to a new plate, 1N HCl was added thereto to terminate the reaction, and read at 450 nm with a spectrophotometer.

[0279] Example 13.3 - Evaluation of the activity of anti-chitin oligomer AcM on healthy cells by immunofluorescence method adapted from Rodrigues et al. 2008

[0280] The fungal cells (10 6 Cells were fixed (cacodylate-paraformaldehyde buffer 4%; 30 min) and later blocked (PBS / BSA 1%; 1 h). They were then incubated with anti-chitin oligomer AcM (25 μg / mL; 1 h at 37°C). After washing with PBS, the cells were incubated with mouse anti-IgM antibody conjugated to Alexa 568 (SIGMA; 1:1000). After washing with PBS, the cells were incubated with 25 μM white calcofluor (calcofluor, Invitrogen) and washed again. The cell suspension was aggregated into a glass blade and evaluated under an Olympus AX70 microscope coupled to a camera system (QImaging Retiga 1300) and evaluated in the QCapture suite V2.46 software.

[0281] Example 13.4 - Minimum inhibitory concentration (MIC), adapted from Joffe et al. 2017

[0282] Cryptococcus neoformans cells were cultured at 10 5The density of 10 cells / well was cultured in RPMI 1640 buffered with MOPS at pH 7 in a 96-well plate in a final volume of 200 μl. The system was supplemented with AcM, AmB (1-0.1 μg / ml) or FLC (8-2 μg / ml) at a concentration of 25-0.05 μg / ml alone or in combination. After incubation at 37° C. for 48 hours under stirring, the cells were suspended by pipetting for spectrophotometer reading at a wavelength of 592 nm.

[0283] Example 13.5 - Biofilm formation, adapted from Joffe et al. 2017

[0284] Cryptococcus neoformans (H99 and Cap67) and Candida albicans were cultured in Sabouraud medium at 30°C for 24 hours. The cell suspension was centrifuged at 3000g for 5 minutes, washed 3 times in PBC, and suspended in minimal medium (20 mg / ml thiamine, 30 mm glucose, 26 mM glycine, 20 mM MgSO4, and 58.8 mM KH2PO4). They were then added to a 96-well plate (100 μl / well - 1x10 6 Cells / ml) and in the presence of AcM (HC6 / DD11 and AF1 / CC5) at a concentration of 25 μg / ml and anti-GXM AcM 18B7 (only for H99), cultured at 37°C for 48 hours, while being kept under stirring for 30 minutes so that AcM and fungi are completely and evenly mixed. As a control, the drug AmB was used at a concentration of 1 μg / ml.

[0285] Two analytical systems were run: in the first system, the cells were washed to remove non-adherent cells and retain only cells associated with the developing biofilm; in the second system, the cells were not washed and all cells, attached or not attached to the substrate, were quantified.

[0286] The metabolic activity of living cells was assessed in both assays by a method based on the reduction of XTT (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolyl hydroxide) at a wavelength of 492 nm in a spectrophotometer.

[0287] Example 13.6 - Melanization assay adapted from Walker et al. 2010

[0288] C. neoformans was cultured according to paragraph

[0221] , and the cell suspension (1x10 6Cells / ml) were cultured in minimal medium supplemented with 1 mM L-DOPA for 72 hours. Antibodies HC6 / DD11 and AF1 / CC5 were added to minimal medium at concentrations ranging from 25 to 0.05 μg / ml. The plates were centrifuged and the amount of pigment formation was defined by optical density after image scanning by an iBright FL1000 Invitrogen instrument.

[0289] Example 14 - Survival Test

[0290] Use PBS for 10 5 Balb / C mice (n=7) were lethally challenged ip with 100 μl of Cryptococcus neoformans (H199) cells and 2 hours later treated with 100 μl of AmB (2.5 mg / kg and 0.25 mg / kg), 100 μl of AcM (85 μg / ml) and 100 μl of a combined solution of AcM and AmB (0.25 mg / kg). Control animals were challenged with Cryptococcus neoformans (H99) alone or with 100 μl of a solution of fungus-free PBS, AmB (2.5 mg / kg) or AcM (85 μg / ml) alone (adapted from Liedke et al.). Survival was obtained and the analysis extended to up to 90 days post-infection, presenting a statistical value of p<0.005.

[0291] Example 15 - Molecular Modeling

[0292] The variable region sequences of HC6 / DD11 and AF1 / CC5 antibodies were obtained by sequencing and translated using the biocomputing tool ExPASy translation tool (https: / / web.expasy.org / translate / ) to obtain the corresponding amino acid sequences.

[0293] Sequence humanization was performed using the IMGT (http: / / imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) database to obtain a human germinal repertoire that was more identical (percent identity) to the VH and VL chains.

[0294] The humanized sequences obtained from the more homologous human library were aligned against the PDB (Protein Data Bank - https: / / www.rcsb.org) database (select mycoproteins) using the blastp (https: / / blast.ncbi.nlm.nih.gov) tool. The three-dimensional template development was performed using the Modeller 9.19 program.

[0295] Example 16 - Development of murine monoclonal antibodies against chitin oligomers by hybridoma technology

[0296] Example 16.1 - Titration of chitin trisaccharide in immune animals against Cryptococcus gattii

[0297] Two immunization strategies were developed, in both of which blood was drawn at the end of the last immunization to check antibody serum titers ( Figure 6 Regardless of the strategy used, a titer of 1:3200 was achieved for IgG and IgM against chitin trisaccharide-BSA in the sera used for screening. To illustrate the cut-off line, the absorbance values ​​obtained in the reaction with pre-immune sera were used. Three animals were used to proceed with the fusion.

[0298] Example 16.2 - Selection of AcM-producing hybridomas by ELISA

[0299] Three fusions were performed from splenectomies of three animals. Splenocytes were fused with Sp2 / 0 and 172 hybridomas were obtained, of which four produced antibodies reactive against chitotrisaccharide.

[0300] Four polyclonal antibody-producing hybridomas were cloned, generating 541 hybridomas producing AcM. From this group, 58 hybridomas reactive to chitotriose were selected, and 10 hybridomas were finally selected for subsequent studies, using chitotriose-BSA as the primary antigen, and these 10 hybridomas showed high reactivity in the ELISA test (optical density-DO ≥ 3x cutoff value).

[0301] Example 16.3-AcM purification

[0302] The two AcMs were purified by ion exchange chromatography and evaluated by polyacrylamide gel electrophoresis ( Figure 8 ).

[0303] Each IgM aliquot exhibited two major IgM bands corresponding to the heavy chain (about 70 kDa) and light chain (molecular weight range of about 23-24 kDa) of immunoglobulin M. Other bands observed indicated a reduced IgM fraction as well as micro protein and glycidic heterogeneity.

[0304] Example 16.4 - CDR Identification

[0305] RNA from hybridomas producing AcM AF1 / CC5 and HC6 / DD11 was extracted to generate cDNA and the VH and VL of the two AcMs were amplified by PCR. After amplification, the chains were sequenced and their CDRs were identified by the Kabat numbering system, which is a scheme for numbering amino acid residues in antibodies based on variable regions. After the CDRs were identified, they were compared by the immunoglobulin database IgBlast to check the identity of the AcMs with the immunoglobulins stored in GeneBank.

[0306] Thus, very high identity was achieved for both AcMs, but there were punctual differences in the HC6 / DD11 VH CDR (where there were amino acid changes relative to those deposited in GeneBank) and there were changes in the AF1 / CC5 VH and VL CDR3s, with the heavy chain CDR3 having a higher percentage of change, reaching 95% identity, relative to the 99% noted for the other CDR3s, as provided in Tables 3 and 4, respectively.

[0307] Table 3: AcM HC6 / DD11 CDR identification

[0308]

[0309]

[0310] Table 4: AcM AF1 / CC5 CDR identification

[0311]

[0312] Example 16.5 - ELISA using PL:

[0313] PL is a lysine polymer that can provide a positive charge to the surface of the bottle, site or blade that serves as the cell substrate. Fungi can have a cell wall formed by chitin, which is composed of long chains of N-acetylglucosamine and is a polymer with a negative charge. In addition to chitin, Cryptococcus neoformans also has a polysaccharide capsule composed of glucuronoxylomannan and galactoxylomannan, and the capsule structure also has a negative charge. Given this background, PL is used to change the plate charge (positive charge) and thus try to make cells adhere to such surfaces.

[0314] In this experiment, 10 7 The absorbance curve of Candida albicans was drawn with cells / ml. It was observed that both AcMs had reliable signals at a concentration of 25 μg / ml, because above this value, the curve began to enter a plateau phase, such as Fig. 9To examine the sensitivity of AcM detection, a cell saturation curve was plotted. The maximum sensitivity level of the two AcMs was determined to be 10 3 This conclusion was drawn because the reaction signal detection was considered to be positive 3 times the cut-off value (reaction blank). However, the signal obtained with antibody HC6 / DD11 was 4 and 10 5 cells / ml, while for AF1 / CC5, both concentrations of cells reached a plateau.

[0315] Example 16.6 - Dot Blot Using PL

[0316] This test uses the same principle as ELISA and is based on the charge presented by the fungi. Since the nitrocellulose membrane has a negative charge, PL is used to examine the binding of positive charges to the fungi and thus to the membrane.

[0317] like Fig.11 Supplied, initially diluted to 10 in 5 μg / ml PL 7 In this experiment, AcM HC6 / DD11 was demonstrated to be able to bind up to 10 cells / ml. 4 albicans binding at the level of 10 cells / ml, which is in contrast to Cryptococcus neoformans, which was determined to be 10 6 Each experiment was performed three independent experiments.

[0318] Example 16.7 - Immunofluorescence

[0319] Bioactivity validation tests were performed by reliable ELISA and dot blot assays. Therefore, AcM was used by immunofluorescence (IF) using the Candida albicans pathogen as an example method. AcM is very efficient in fungal cell wall labeling and showed the same profile whenever a standard marker (lectin) was used, data not shown. This experiment was performed 3 times and showed the same reaction pattern, but Fig.12 Separate experimental data are presented.

[0320] Example 16.8 - Blackening Test

[0321] The ability of C. neoformans to produce melanin represents the second most relevant virulence factor for it after the presence of a polysaccharide capsule. Therefore, it was proposed to evaluate the activity of AcM in terms of fungal cell wall melanin deposition.

[0322] The concentrations of AcM HC6 / DD11 and AF1 / CC5 were determined to be 0.2-25 μg / ml. L-DOPA was used as a substrate for melanization.

[0323] The pigmentation of Cryptococcus neoformans was assessed by observing brown to black sediment at the bottom of the 96-well plate. To record pigmentation or inhibition, the plate was photographed on a white surface (transparent bottom) to be able to distinguish pigmented from non-pigmented groups. It should be noted that each AcM dose will show fungal growth, but will have negative pigmentation or partial inhibition.

[0324] There was evidence that AcM HC6 / DD11 treatment partially inhibited melanization up to a concentration of 6.2 μg / ml (p<0.05), but there was no significant inhibition at lower concentrations (p>0.05). AcM AF1 / CC5 inhibited melanization at concentrations up to 6.2 μg / ml (p<0.001) and had a partial inhibitory effect at concentrations of 3.2 and 1.6 μg / ml (p<0.05), but there was no significant inhibition at lower concentrations (p>0.05). Fig.13 )

[0325] Example 16.9 - Biofilm Formation

[0326] Biofilm formation is of great importance in clinical medicine as it can make several diseases, including cryptococcosis, difficult to treat. Therefore, it was proposed to evaluate the role of AcM in biofilm formation by the XTT test.

[0327] AcM (25 μg / ml) was tested against three fungal species (Candida albicans, Cryptococcus neoformans - H99 and a non-capsulated mutant of Cryptococcus neoformans - Cap67) and the control was the anti-GXM AcM18B7 (25 μg / ml) against H99 only. Fig.14 A and 14B), the cell suspension was washed to remove cells that did not adhere to the substrate. AcM significantly inhibited biofilm formation compared to untreated cells that adhered directly to the substrate (p < 0.05). In the second assay ( Fig.14 C and 14D), unwashed cell suspensions, AcM again inhibited biofilm formation compared to cells not treated with antibody (p<0.05). In both assays, cell growth was observed.

[0328] AcM was compared with the anti-GXM AcM18B7 in terms of biofilm formation inhibition. For both washed and unwashed cell suspensions, AcM was able to significantly inhibit H99 biofilm formation compared to untreated cells and exhibited a similar behavior to the anti-GXM AcM 18B7 (p<0.05) ( Fig.15 ).

[0329] It was demonstrated that treatment with AcM affected biofilm formation compared to untreated fungi (p<0.05) and had a similar behavior to AmB. Each treatment tested affected only biofilm formation due to the presence of cell growth.

[0330] Example 16.10 - ELISA using PL against several cells

[0331] In this assay, the same principle was used to label fungi by ELISA using PL, but it was performed on different cell types to demonstrate that AcM is specific for chitin and therefore for fungi. For this purpose, the human lung cell line A549, Giardia lamblia, and Gram-negative and Gram-positive bacteria were used. The graph shows that at each cell density, AcM was not able to discriminate a specific target, while on the other hand, it showed the same results in discriminating the Cryptococcus neoformans target, indicating that AcM is target specific and detects the target at 10 6 cells / ml to maintain its sensitivity, such as Fig.16 The experiment was performed three times to maintain its reproducibility.

[0332] Example 16.11 - Minimum Inhibitory Concentration Determination

[0333] AcM fungicidal activity was tested by CIM test. Compared with the control of 1 μg / ml AmB and 8 μg / ml FLC ( Fig.18 and 19 ), no AcM showed fungicidal effect. It was evaluated whether the combination of AcM with AmB or FLC would trigger antifungal effects at sub-inhibitory concentrations, so AmB at a concentration of 1 μg / ml and FLC at 4 μg / ml and 2 μg / ml were combined with different concentrations of AcM. In order to evaluate whether there was a combined effect of AcM with the drug, it was used in isolated form as the basis of drug action.

[0334] When AcM was evaluated in combination with AmB for fungicidal effect, it was observed that both antibodies at a concentration of 6.2 μg / ml triggered the fungicidal effect of AmB at sub-inhibitory concentrations (p<0.001) compared to the isolated effect of the drug at its optimal concentration. However, the combination of AmB 0.1 μg / ml with antibodies HC6 / DD11 enhanced the fungicidal effect starting from a concentration of 1.6 μg / ml (p<0.01), while the combination effect with antibodies AF1 / CC5 showed fungicidal effect starting from a concentration of 3.2 μg / ml (p<0.01) compared to the isolated sub-inhibitory concentration of AmB ( Fig.18 ).

[0335] Regarding the combined effect of AcM and FLC, a partial fungicidal effect of the antibody was observed when combined with FLC (4 μg / ml) (3.2 μg / ml for both AcM) (p<0.01). Thus, the drug effect was enhanced compared to the isolated form at a concentration of 4 μg / ml. Moreover, because the drug effect was triggered, a combined fungicide effect of AcM and FLC at a concentration of 2 μg / ml was observed, reaching the level of the isolated form at a concentration of 4 μg / ml.

[0336] Example 16.12 - Survival Test

[0337] Use 1×10 5 Mice were lethally challenged by ip inoculation with 100% yeast Cryptococcus neoformans strain H99 cells. 2 hours later, they were treated with PBS (negative control), 85 μg / animal of isolated form of AcM (HC6 / DD11), 0.25 or 2.5 mg / kg of isolated form of AmB and synergistic form, AcM maintaining the tested concentration. The treatment was repeated twice more with an interval of 10 days.

[0338] Animals infected with Cryptococcus neoformans and treated with PBS died on day 28 after infection, while animals treated with AcM did not die until day 29, and the group of animals treated with only 0.25 mg / kg AmB died on day 37, and there was no death in the synergistic group (AcM-85 μg / animal-with AmB-0.25 mg / kg-). As expected, there was no death in the control group treated with the standard dose of AmB (2.5 mg / kg) (data not provided). Therefore, animals infected with Cryptococcus neoformans and treated with AcM HC6 / DD11 and sub-inhibitory concentrations of AmB achieved a 100% survival rate.

[0339] The synergistic group was statistically significant relative to the PBS and AcM treated groups (p≤0.001). The same was observed for the group treated with AmB (0.25 mg / kg) (p=0.01). The surviving animals in the groups treated with mAb and AmB were symptom-free at the end of the experiment (90 days) ( Fig. 20 ).

[0340] Example 16.13 - Comparative Modeling

[0341] To humanize mouse AcM, the light and heavy chains of AcM HC6 / DD11 and AF1 / CC5 were aligned with the human antibody database of IMGT (http: / / imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi). The sequences with higher homology to the heavy and light chains of HC6 / DD11 were IGHV3-11*01 and IGKV1-16*01, respectively, and to AF1 / CC5, respectively, IGHV3-73*01 and IGKV1-17*03. The amino acids that differed between the mouse and human sequences were replaced with amino acids present in the human antibody framework.

[0342] To obtain the three-dimensional structures of the murine and humanized HC6 / DD11 and AF1 / CC5 antibodies, molecular modeling methods were used. Mycoproteins were selected and chosen by local alignment of the PDB database by BLASTp (Basic Local Alignment Search Tool). Sequence 4UOR was used as template for murine AcM HC6 / DD11 and murine AF1 / CC5, with the following heavy and light chain sequences, respectively: 5IJK, 3NFT. For humanized HC6 / DD11, sequence 5F72 was used, and for AF1 / CC5 humanized heavy and light chains, 6MAM and 3NFP were used, respectively.

[0343] The three-dimensional template development was performed using the Modeller 9.19 program. The mouse and humanized templates were subjected to energy minimization using the Wincoot program and were aligned between them to check the predicted structures ( Fig.21 ).

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Sequence Listing <110> Oswald Cruz Foundation <120> Antibodies, uses thereof, pharmaceutical compositions, including fungal infection diagnosis methods, fungal infection diagnosis kits, and fungal infection treatment methods <130> R001010 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 1 Gly Phe Thr Phe Ser Asp Tyr Gly Met Ala 1 5 10 <210> 2 <211> 9 <212> PRT <213> Mouse <400> 2 Ile Ser Asn Leu Ala Tyr Ser Ile Tyr 1 5 <210> 3 <211> 12 <212> PRT <213> Mouse <400> 3 Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 4 <211> 10 <212> PRT <213> Mouse <400> 4 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mouse <400> 5 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Mouse <400> 6 Gln Gln Arg Ser Ser Tyr Pro Leu Thr 1 5 <210> 7 <211> 10 <212> PRT <213> Mouse <400> 7 Gly Phe Thr Phe Ser Asp Ala Trp Met Asp 1 5 10 <210> 8 <211> 9 <212> PRT <213> Mouse <400> 8 Ile Arg Ser Lys Ala Asn Asn His Ala 1 5 <210> 9 <211> 8 <212> PRT <213> Mouse <400> 9 His Arg Tyr Asp Gly Phe Asp Tyr 1 5 <210> 10 <211> 10 <212> PRT <213> Mouse <400> 10 Ser Ala Ser Ser Ser Ile Ser Tyr Met His 1 5 10 <210> 11 <211> 7 <212> PRT <213> Mouse <400> 11 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 12 <211> 8 <212> PRT <213> Mouse <400> 12 Gln Arg Ser Ser Tyr Pro Cys Thr 1 5 <210> 13 <211> 373 <212> DNA <213> Mouse <400> 13 ggcagggagc ggtgaccgtg gtccctgcgc cccagacatc gaagtaccag tagctactac 60 cgtagtaatc ccttgcacag tagtacatgg ctgtgtcctc agacctcaga ctgctcattt 120 ccaggtacag ggtgttcttg gcattctctc tagagatggt gaatcggccc gtcacagtgt 180 ctgcatagta gatactatat gccaaattac taatgaatgc tacccactca ggccccttcc 240 ctggagcctg tcgaacccac gccattccgt agtcactgaa agtgaatcca gaggctgcac 300 aggatagttt ccgggaccct ccaggctgca ctaagcctcc ccctgactcc tccagcttaa 360 cttgaccggt cga 373 <210> 14 <211> 331 <212> DNA <213> Mouse <400> 14 gcaaccaatt cctgcatctc caggggagag gtcaccataa cctgcagtgc cagctcaagt 60 gtaagttaca tgcactggtt ccagcagaag ccaggcactt ctcccaaact ctggatttat 120 agcacatcca acctggcttc tggagtccct gctcgcttca gtggcagtgg atctgggacc 180 tcttactctc tcacaatcag ccgaatggag gctgaagatg ctgccactta ttactgccag 240 caaaggagta gttacccgct cacgttcggt gctgggacca agctggagct gaaacgggct 300 gatgctgcac caactgtatc cctcgagacc a 331 <210> 15 <211> 124 <212> PRT <213> Mouse <400> 15 Ser Thr Gly Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser Asp Tyr Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Pro 35 40 45 Glu Trp Val Ala Phe Ile Ser Asn Leu Ala Tyr Ser Ile Tyr Tyr Ala 50 55 60 Asp Thr Val Thr Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn 65 70 75 80 Thr Leu Tyr Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met 85 90 95 Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp 100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Ala Pro Cys 115 120 <210> 16 <211> 110 <212> PRT <213> Mouse <400> 16 Ala Thr Asn Ser Cys Ile Ser Arg Gly Glu Val Thr Ile Thr Cys Ser 1 5 10 15 Ala Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly 20 25 30 Thr Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly 35 40 45 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 50 55 60 Thr Ile Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 65 70 75 80 Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu 85 90 95 Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Leu Glu Thr 100 105 110 <210> 17 <211> 364 <212> DNA <213> Mouse <400> 17 cggggaatgt gagagtggtg ccttggcccc agtagtcaaa gccgtcgtac ctatgcctcg 60 tacagtaata aatgccagtg tcttcagctc ttaagctgtt catttgcagg tagacactac 120 ttttggaatc atctcttgag atggtgaacc tccctttcac agactcagca tagtatgttg 180 catgattatt agctttgctt ctaatttcag caacccactc aagccccttc tctggagact 240 ggcggaccca gtccatccag gcgtcactaa aagtgaatcc agaggcagca caagagagtt 300 tcatggatcc tccaggttgc accaagcctc ctcctgactc ctccagctta acttgaccgg 360 tcga 364 <210> 18 <211> 336 <212> DNA <213> Mouse <400> 18 gattattttc ttgcatctca gggagaggtc accatgacct gcagtgccag ctcaagtata 60 agttacatgc actggtacca gcagaagcca ggcacctccc ccaaaagatg gatttatgac 120 acatccaaac tggcttctgg agtccctgct cgcttcagtg gcagtgggtc tgggacctct 180 tattctctca caatcagcag catggaggct gaagatgctg ccacttatta ctgccatcag 240 cggagtagtt acccatgcac gttcggtgct gggaccaagc tggagctgaa acgggctgat 300 gctgcaccaa ctgtatccct cgagaccaag accagc 336 <210> 19 <211> 121 <212> PRT <213> Mouse <400> 19 Ser Thr Gly Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30 Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 35 40 45 Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr 50 55 60 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 65 70 75 80 Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 85 90 95 Gly Ile Tyr Tyr Cys Thr Arg His Arg Tyr Asp Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Phe Pro 115 120 <210> 20 <211> 112 <212> PRT <213> Mouse <400> 20 Asp Tyr Phe Leu Ala Ser Gln Gly Glu Val Thr Met Thr Cys Ser Ala 1 5 10 15 Ser Ser Ser Ile Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Thr 20 25 30 Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 35 40 45 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr 50 55 60 Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln 65 70 75 80 Arg Ser Ser Tyr Pro Cys Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 85 90 95 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Leu Glu Thr Lys Thr Ser 100 105 110

Claims

1. An antibody that binds to chitin or chitin oligomers, characterized in that it comprises (i) the VH CDR1 sequence described in SEQ ID NO: 1, the VH CDR2 described in SEQ ID NO: 2, and the VH CDR3 described in SEQ ID NO: 3; and (ii) the VL CDR1 sequence described in SEQ ID NO: 4, the VL CDR2 described in SEQ ID NO: 5, and the VL CDR3 described in SEQ ID NO: 6; or (i) the VH CDR1 sequence described in SEQ ID NO:7, the VH CDR2 described in SEQ ID NO:8, and the VH CDR3 described in SEQ ID NO:9; and (ii) the VL CDR1 sequence described in SEQ ID NO:10, the VL CDR2 described in SEQ ID NO:11, and the VL CDR3 described in SEQ ID NO:

12. The antibody according to claim 1 , characterized in that it is a monoclonal antibody.

3. The antibody according to claim 2, characterized in that it is a murine antibody, a humanized antibody, a human antibody or a chimeric antibody.

4. A pharmaceutical composition, characterized in that it comprises the antibody according to claim 1, 2 or 3.

5. The pharmaceutical composition according to claim 4, characterized in that it comprises polyenes and / or azoles.

6. The pharmaceutical composition according to claim 5, characterized in that it comprises AmB and / or FLC.

7. The pharmaceutical composition according to claim 6, characterized in that it also comprises a pharmaceutically acceptable medium.

8. The pharmaceutical composition according to claim 6, characterized in that it also comprises a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 4, characterized in that it is designed for the treatment of fungal infections.

10. Use of the antibody according to any one of claims 1 to 3 in the preparation of a medicament for treating fungal infection.

11. The use according to claim 10, characterized in that the antibody is combined with polyenes and / or azoles.

12. Use according to claim 11, characterized in that the antibody is combined with AmB and / or FLC.

13. Use of the antibody according to any one of claims 1 to 3 in the preparation of a kit for use in a method for diagnosing fungal infection, wherein the method comprises the following steps: (i) providing the antibody and a sample obtained from a patient, (ii) contacting the antibody with the biological sample to be tested for a sufficient period of time and under conditions sufficient to form an antigen / antibody complex; and (iii) detecting the antigen / antibody complex formed in the previous stage by a detection technique capable of generating a detectable signal in the presence of the above antigen / antibody complex.

14. Use according to claim 13, characterized in that the biological sample is selected from the group comprising: saliva, urine, blood, bronchoalveolar lavage fluid, peritoneal fluid or liquid or any other biological fluid of a patient.

15. The use according to claim 13, characterized in that the biological sample is serum.

16. A fungal infection diagnostic kit, characterized in that it comprises the antibody according to claims 1-3.

17. The diagnostic kit according to claim 16, characterized in that it also includes instructions for use.

18. The diagnostic kit according to claim 16 or 17, characterized in that it further comprises an antigen / antibody complex detection medium, wherein the detection medium comprises a signal generating agent capable of generating a detectable signal.

19. A kit for treating fungal infection, characterized in that it comprises: (i) the antibody according to any one of claims 1 to 3; and (ii) antifungal agents, (iii) instructions for using the components in combination.

20. Use of the composition according to any one of claims 4 to 9 in the preparation of a medicament for treating fungal infection.

21. The antibody according to any one of claims 1 to 3, characterized in that The antibodies are designed for use in treating fungal infections in individuals.

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