Patent Application
20230406912
Fungal infections cause life-threatening diseases such as fungaemia, pneumonia, chronic pulmonary aspergillosis, bronchopulmonary aspergillosis and cryptococcosis (David S P et al., Lancet Infect Dis 2017; 17: e383-92; Darius A J et al., Lancet Infect Dis 2017; 17: e393-402). These pathologies affect mainly patients undergoing organ transplantation, surgery and neoplastic disease, immunocompromised subjects and premature infants. About 3,000,000 cases of chronic pulmonary aspergillosis, 223,100 cases of cryptococcal meningitis complicating HIV/AIDs, 700,000 cases of invasive candidiasis, 500,000 cases of Pneumocystis jirovecii pneumonia, 250,000 cases of invasive aspergillosis, 100,000 cases of disseminated histoplasmosis, over 10,000,000 cases of fungal asthma are reported annually (Bongomin F et al., J. Fungi 2017; 3:57). Of the five million fungal species (spp.) existent, only 300 are considered dangerous for humans, and ˜10% of them are recurrent. The most common fungi isolated in invasive diseases are Candida spp., Cryptococcus spp., and Aspergillus spp. The mortality rate for invasive candidiasis is about 40%, from 20 to 30% for cryptococcosis and 20% for aspergillosis. These data are referred to wealthy countries with a fully functional health-care system, while where resources are limited the death rate surpasses 50% (Nyazika TK et al., J. Med. Microbiol. 2016; 65:1281-1288). Candidiasis is the second most frequent fungal infection. Despite C. albicans is the most prevalent specie, there have been an increasing number of infections caused by non-albicans Candida spp. (NACs). This shift towards NACs appears to be due to the massive use of antifungal drugs leading to the selection of species with an innate resistance or higher tolerance. Together, C. albicans, C. krusei, C. parapsilosis and C. tropicalis represent the 80% of the total cases of infections and 49.5% of them are caused by NACs.
The discovery of new frightening fungal species makes the run for drugs more urgent. This is the case of C. auris which appeared for the first time in 2009 (Satoh K et al. Microbiol Immunol. 2009; 53:41-4) and spread rapidly all over the world. It commonly presents multidrug resistance to every class of antifungal drugs. The MIC of different strains for fluconazole range from 32 to ≥64 mg/L while for voriconazole the MIC is about 16 mg/L. 30% of the strains have a low susceptibility to Amphotericin B (MIC≥2 mg/L) and recent studies have confirmed an increasing resistance to echinocandins (MIC≥8 mg/L). C. auris is tolerant to high salt concentrations, where it tends to assume a rudimental pseudohyphal form, and to high temperatures (42° C.). C. auris can adhere to biotic and abiotic surfaces and colonize them for weeks and months becoming a serious problem for the invasive devices used in the hospitals. Until now, the leading classes of antifungals are: polyenes, pyrimidine analogues, azoles and echinocandins.
Toxicity represents the main issue of current and new antifungal agents. The action spectrum of antifungal agents should be balanced, not too limited but also not too broad: effective against several species but not subject to early resistance. They should be stable and have limited off-target interactions and a known pharmacokinetic. The way of administration may be chosen preferring the patient compliance and considering hypothetical comorbidities.
Despite several antifungal entities and new targets under investigation (Gintjee T J et al. J. Fungi 2020; 6:28; Perfect J R Nat Rev Drug Discov. 2017; 16:603-616), none of them has entered the market yet.
Among the novel therapeutic strategies to treat fungal diseases, the employment of monoclonal antibodies seems to be a great step forward. The antifungal sector attended the birth of several monoclonal antibodies (Di Mambro T et al. Front Pharmacol. 2019; 10:80). Matthews R C et al. in Antimicrob Agents Chemother. 2003; 47: 2208-2216 describe Mycograb, a recombinant antibody directed against fungal Hsp90, a member of the Heat Shock Proteins family. Mycograb consists of the antigen-binding variable domains of antibody heavy and light chains linked together to create a recombinant protein which is expressed in Escherichia coli. It does not have a Fc component. Benefits were not considered to outweigh the risks (EMEA/129723/2007) and the product did not entered the clinic.
mAb 2G8 has been described as a successful murine mAb able to specifically recognize β-1,3 glucans of pathogenic fungi. mAb 2G8 showed in vitro activity against Candida spp. and Aspergillus spp. It showed a strongly efficacy in vivo in a systemic mouse model of Candida infection and in a rat model of vaginal candidiasis (Torosantucci A et al. J Exp Med. 2005; 202: 597-606). The activity was verified also on Cryptococcus neoformans, confirming the capability to bind and inhibit the growth and capsule formation of this fungal species in vitro and in vivo (Rachini A et al. Infection and Immunity 2007; 75:5085-94). WO2006030318 describes 2G8 VH sequence (SEQ ID NO: 2) and VL sequence (SEQ ID NO: 1).
However, the murine source of this antibody precludes the possibility to use it in humans. At present, several attempts to obtain a humanised antibody from mAb 2G8, by using the state of the art technologies, failed.
Therefore, there is a strong need for novel, safe and efficient monoclonal antibodies based antifungal treatment.
It forms a first object of the present invention humanised monoclonal antibodies, or fragments or fusions proteins thereof, that recognize and bind β-1,3 glucans, favouring the human innate immunity against pathogenic fungi. In a second aspect, it is here described and claimed a combination of a) at least one humanised antibody that recognize and bind β-1,3 glucans and b) at least one antifungal agent, preferably an echinocandin and/or polyene macrolide.
Sequence Listings: SEQ ID NO: 1-28 are provided.
The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat, see Johnson G and Te Wua T, Nucleic Acids Res. 2000; 28: 214-218 for a review. This numbering system is used in the present specification except where otherwise indicated.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved Framework regions (FRs) and three Complementarity-determining regions (CDRs).
As used herein, the term ‘humanised antibody’ refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody). For a review, see Vaughan et al, Nature Biotech. 1998; 16: 535-539. In one embodiment, rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein are transferred to the human antibody framework. In one embodiment, only the specificity determining residues from one or more of the CDRs described herein are transferred to the human antibody framework. In another embodiment, only the specificity determining residues from each of the CDRs described herein are transferred to the human antibody framework.
When the CDRs or specificity determining residues are grafted, any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.
Thus, provided in one embodiment is a humanised antibody which binds β-1,3 glucans, wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs.
A murine anti β-1,3 glucans antibody, referred to herein as mAb 2G8, was described in WO2006030318 and the amino acidic sequences of the variable regions of this antibody are provided in
The CDRs sequences characterising the humanised antibodies according to the present invention have been surprisingly identified by the Applicant.
In an attempt to obtain a humanised version of the mAb 2G8, the authors of the present invention generated and then randomly combined several heavy and light chains and screened the resulting recombinant mAbs. Unexpectedly, only constructs comprising the CDRs listed below were biologically active and with an affinity for β-1,3 glucans higher than the affinity observed for murine 2G8.
Several hmAb 2G8 characterized by comprising the listed CDRs were produced and characterized and demonstrated capable to recognize a number of fungi.
In a first embodiment, here it is described and claimed a humanised antibody, or fragments or fusion proteins thereof, comprising an antigen-binding region that is specific for recognizing β-1,3 glucans, comprising:
In an embodiment, said humanised antibody, or fragments or fusion proteins thereof, comprises variable regions having CDR sequences of:
Antibodies or fragments thereof may be polyclonal or monoclonal antibodies. Monoclonal antibodies are preferred.
The antibodies include antibody-conjugates and molecules comprising the antibodies, such as chimeric molecules; chimeric receptors comprising one or more stimulatory, signalling, and/or costimulatory domains; and chimeric antigen receptors (CARs). Thus, an antibody includes, but is not limited to, full-length and native antibodies, as well as fragments and portions thereof retaining the binding specificities thereof, such as any specific binding portion thereof, including those having any number of, immunoglobulin classes and/or isotypes (e .g ., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab′)2, Fv, and scFv (single chain or related entity).
In some embodiments, the antibody is a full-length IgG class antibody.
In some embodiments, said antibody fragments are Antigen-binding fragment (Fab). In some embodiments, said fusion proteins are single-chain variable fragment (scFv), or multimeric formats such as multivalent scFvs (diabody, triabody, and tetrabody).
In an embodiment, an antibody of the present disclosure, or fragments or fusion proteins thereof, comprises a heavy chain, wherein the variable domain of the heavy chain comprises a sequence having at least 60% identity or similarity to the sequence H5 given in SEQ ID NO:22.
In one embodiment, an antibody of the present disclosure comprises a heavy chain, wherein the variable domain of the heavy chain comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence to the sequence H5 given in SEQ ID NO: 22.
“Identity”, as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. “Similarity”, as used herein, indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids which can often be substituted for one another include but are not limited to:
Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk A M, ed., Oxford University Press, New York, 1988).
In another embodiment, an antibody of the present disclosure comprises a light chain, wherein the variable domain of the light chain comprises a sequence having at least 60% identity or similarity to the sequence K1 given in SEQ ID NO: 15.
In one embodiment the antibody of the present disclosure comprises a light chain, wherein the variable domain of the light chain comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence K1 given in SEQ ID NO: 15.
In another embodiment of the disclosure, the antibody comprises a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO: 22 and the variable domain of the light chain comprises a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO: 15.
Suitably, the antibody comprises a heavy chain, wherein the variable domain of the heavy chain comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO: 22 and a light chain, wherein the variable domain of the light chain comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO: 15.
In an embodiment, the antibody comprises variable region sequences consisting of H5, SEQ ID NO: 22 for the heavy chain variable regions in combination with sequences consisting of K1, SEQ ID NO: 15 for the light chain variable regions.
In an embodiment, the humanised antibody is hmAb H5/K1.
The present invention also provides an isolated DNA sequence encoding the heavy and/or light chain(s) of an antibody molecule of the present invention. Suitably, the DNA sequence encodes the heavy or the light chain of an antibody molecule of the present invention. The DNA sequence of the present invention may comprise synthetic DNA, for instance produced by chemical processing, cDNA, genomic DNA or any combination thereof. DNA sequences which encode an antibody molecule of the present invention can be obtained by methods well known to those skilled in the art.
The present invention also relates to a cloning or expression vector comprising one or more DNA sequences of the present invention. Accordingly, provided is a cloning or expression vector comprising one or more DNA sequences encoding an antibody of the present invention. Suitably, the cloning or expression vector comprises two DNA sequences, encoding the light chain and the heavy chain of the antibody molecule of the present invention, respectively and suitable signal sequences.
The present invention also relates to a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding an antibody of the present invention. Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecule of the present invention.
The present invention also provides a process for the production of an antibody molecule according to the present invention comprising culturing a host cell containing a vector of the present invention under conditions suitable for leading to expression of protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.
As the antibodies of the present invention are useful in the treatment of fungal infections, the present invention also provides a pharmaceutical composition comprising an antibody molecule of the present invention in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier.
In a further aspect, it is here claimed a humanised antibody according to the present invention or a pharmaceutical composition comprising the same for use in the treatment or prophylaxis of infections of human cells.
In a preferred embodiment, said infections are mediated by pathogenic fungi, preferably selected in the group comprising the genus Candida, Aspergillus, Cryptococcus.
In a further embodiment, the Applicant has surprisingly found that antibodies with a specificity for β-1,3 glucans can be successfully used in association with antifungal agents.
In particular, the Applicant has now surprisingly found that the combination of antibodies with a specificity for β-1,3 glucans with at least one antifungal agents, preferably selected from echinocandins or polyene macrolide, has a synergistic effect in inhibiting the growth of fungi.
In the present description, the expression “synergistic effect” means that the combination of humanised antibodies with a specificity for β-1,3 glucans with at least one antifungal agents, preferably selected from echinocandins or polyene macrolide, inhibits the growth of fungi at a concentration lower than the sum of the concentrations of humanised antibodies with a specificity for β-1,3 glucans and of the antifungal agent used alone for the same strain to obtain the same inhibition. In other words, a synergistic effect means that the combination of humanised antibodies with a specificity for β-1,3 glucans and of the antifungal agent is effective in producing more than the additive effect of each component in the same strain.
Therefore, it is here claimed a combination of a) at least one humanised antibody which has specificity for β-1,3 glucans and b) one or more antifungal agents for use in the treatment or prophylaxis of infections of human cells.
Said combination is for simultaneous, separate or sequential use as a medicament.
In an embodiment, this combination can be a simple combination of pharmaceutical forms of the individual active ingredients, the dosage of which will be established according to modalities deriving from the application of the teachings of the present invention, that is to say, reduced doses such as to ensure the reciprocal synergism and, if so desired, the reduction or disappearance of side effects. In this case, the combination according to the present invention can also be in the form of a kit, i.e. a pack containing the individual dosage forms of the active ingredients and the instructions for their simultaneous, separate or sequential administration. Alternatively, the present invention provides for a new pharmaceutical composition containing the at least two active ingredients in a single dosage form.
In a preferred aspect, said antifungal agent is selected from echinocandins and/or polyene macrolide.
Preferably, said echinocandin is caspofungin, micafungin, or anidulafungin.
Preferably, said polyene macrolide is amphotericin B.
In an embodiment, said composition comprises a hmAb according to the present invention in combination with a further antifungal agent.
In an embodiment, said humanised antibody is hmAb 2G8 H5/K1.
In a further embodiment, it is here claimed a method of treatment or prophylaxis of fungal infections in a human comprising administering to a human in need thereof a therapeutically effective amount of a) at least one humanised antibody which has specificity for β-1,3 glucans and b) one or more antifungal agents, preferably selected from echinocandins or polyene macrolide.
In a preferred embodiment, said fungal infections are selected from candidiasis, aspergillosis, cryptococcosis, dermatomycoses, sporotrichosis and other subcutaneous mycoses.
Humanised antibodies according to the present invention are capable of inhibiting the binding of a microbial pathogen that contains β-1,3 glucans in the cell wall to a human cell, therefore blocking or limiting infection of human cells. Antibodies of the invention preferably bind a microbial pathogen that contains β-1,3 glucans in the cell wall and promote the recognition and phagocytosis of said complex by human macrophages favouring the human innate immunity against pathogenic fungi.
The present invention is further described by way of illustration only in the following examples.
The previously described mAb 2G8 VH amino acidic sequence (SEQ ID NO: 2) and 2G8 VL amino acidic sequence (SEQ ID NO: 1) have been analysed using IgBlastTool (https://www.ncbi.nlm.nih.gov/igblast/) murine databases IMGT mouse V genes, IMGT mouse D genes, IMGT mouse J genes.
The mAb 2G8 VH amino acidic sequence, SEQ ID NO: 2, has been analysed against the first 10 homologous germline sequences, i.e.
The results of the comparison, reported in
The nucleotide sequences of the first 10 homologous germline sequences identified above have been then compared with the nucleotide sequence of the prior art mAb 2G8 VH nucleotide sequence (SEQ ID NO: 6).
In the prior art mAb 2G8 VH sequence (SEQ ID NO: 6), the nucleotides codifying the 3 amino acids at the N terminus have been identified as lacking. The comparisons are reported in
Moreover, the comparison showed that the first domain of the heavy chain constant region, highlighted in the sequence reported in
From the above reported observations, the authors defined the 2G8 VHc sequence (SEQ ID NO: 8), containing 3 additional amino acids at the N terminus and stopping at amino acid Serin 116.
A similar approach was used to identify the sequence 2G8 VLc. The germline sequences with higher homology show a percentage that was over 78.9%. In
The authors defined a 2G8 VLc sequence (SEQ ID NO: 3), stopping at amino acid Lysin 112.
The newly defined amino-acidic and nucleotidic sequences, 2G8 VLc (SEQ ID NO: 7, SEQ ID NO: 3, respectively) and 2G8 VHc (SEQ ID NO: 4, SEQ ID NO: 8, respectively) are reported in
Candidate humanised sequences were selected starting from the murine one according to a classic CDR-grafting, consisting in selecting the CDR regions (defined according to the IMGT numbering scheme) of the murine sequence, and inserting them in the human germline with highest sequence similarity with the murine one in the Framework Regions (FR), as detailed in
H1 and K1 were transfected in a high-throughput microscale system. 3 ml of CHO-S cells culture were transfected using the EXPICHO Expression system (Thermo Scientific™) according to the information supplied by the manufacturer. After one week, supernatants were collected for further analysis. The antibody production on a medium scale was carried out with the same expression system.
Supernatants containing antibodies were centrifuged, filtered 0.22 μm to remove cells and debris and batch purified by affinity chromatography using TOYOPEARL AF-rProtein A HC-650F resin (Tosoh Bioscience LLC). Antibodies were eluted with Buffer Citrate 0.1 M pH 3, neutralized with an alkaline solution and dialyzed in PBS1X with slide-A-Lyzer (Thermo Scientific™). Purity of the antibody was checked by SDS-PAGE analysis that was performed under reducing/non-reducing conditions according to the standard method. Endotoxin levels were evaluated by Pierce™ LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific™) and the presence of aggregates analysed through HPLC-SEC analysis.
The activity of the purified antibody was then tested by ELISA assay. 96-well plates were coated with 50 μg/ml Laminarin (Sigma-Aldrich, L9634) in 0.05 M carbonate buffer pH 9.6 overnight at 4° C. Nonspecific interactions were blocked with 100 μL/well blocking solution, 3% (w/v) BSA in PBS-Tween 20 (8 g/L NaCl, 0.2 g/L KH2PO4, 2.9 g/L Na2HPO4·12 H2O, 0.2 g/L KCI, 0.05% (v/v) Tween 20, pH 7.4) at 37° C. for 1 h. The plates were then incubated with decreasing concentrations of hmAb 2G8_first approach (comparative purpose) (from 3.12 μg/ml to 0.003 μg/ml, each concentration was tested in triplicate) in blocking solution for 2 h at 37° C. At the same temperature and for the same time, 100 μL Goat anti-human-HRP diluted 1:1000 in blocking solution was poured in each well. After every single passage the plates were washed 5 times with PBS-Tween 20. To reveal the binding, 100 μl of 5 mg-ABTS tablet (Roche Diagnostics) dissolved in 12 ml of sodium citrate (0.05 M, pH 3) and supplemented with 1:1000 dilution hydrogen peroxide (Carlo Erba) was added and after 15, 30, 45 and 60 min the absorbance at 405 nm was measured. For the whole measuring time the plates were left in the dark. IC50 analysis of ELISA test was performed with Prism. The TEST was performed in triplicate.
The hmAb 2G8 H1/K1 was not active in an ELISA test.
Sequences obtained in Example 1, VHc and VLc sequences, SEQ ID NO: 4 and SEQ ID NO: 3, respectively, underwent CDR-grafting. Only the Framework Regions (FRW) present in the human germline gene database (www.ncbi.nlm.nih.gov/igblast) were analysed, according to Table 1.
The obtained sequences of humanised VH, named H2 (SEQ ID NO: 18) and VL, named K2 (SEQ ID ID NO: 17), are as follows:
The obtained sequences were launched in Protein Data Bank (PDB) and compared with the sequences found to be the most similar and with an available x-ray crystallographic resolution≤2 Å (www.rcsb.org). Results are reported in
H3 and K3 have been expressed as a recombinant Ab. Also in this case, the antibodies were not active in an ELISA test.
The humanization approach described in Clavero-Álvarez A et al. Scientific Reports 2018; 8:14820 has been followed, resorting to statistical modelling of the variable regions of human antibody sequences. VHc and VLc regions, SEQ ID NO: 8 and SEQ ID NO: 7, were aligned according to the AHo scheme and juxtaposed, to yield a unique VH-VL sequence with gaps of length 298, using IMGT numbering scheme and Kabat numbering scheme. The two protocols differ in the choice of the CDR regions to be kept fixed: in the first case, they coincided with residues chosen for the CDR-grafting protocol above, while in the second case, CDR residues were those corresponding to the Kabat numbering scheme.
This approach, that includes residue-residue correlations both within and between heavy and light chains, strongly depends on which residues are kept fixed, as belonging to CDR regions: starting from the murine sequence, the method implies mutating one residue at a time, choosing the mutation, at any site and to any amino-acid, that yields the greatest increase in the MG-score. This method does not guarantee to reach the highest scoring (i.e. the most humanlike sequence with the given CDRs), but it finds the local maximum closest to the initial murine sequence, with the smallest number of mutations (i.e. the shortest path to it).
The trajectories in sequence space thus produced were further analyzed with CamSol Intrinsic Server (http://www.mvsoftware.ch.cam.ac.uk/index.php/camsolintrinsic), to check explicitly their tendency towards aggregation. Since CamSol uses local sequence information for prediction, the latter could be affected by the presence of the artificial contiguity between the C-term of the VH chain and the N-term of the VL one in the alignment used in the design step above: for this reason, we analyse separately the VH and VL chains with CamSol, and also analyse scFV constructs, with a common linker between the two chains. This allows to separately pick, along the Kabat trajectory, the VH and VL sequences having the least tendency towards aggregation, among those that score above the murine/human threshold, and also the scFV that best copes with the requirements of having a good MG-score and a good solubility; the same is done for the other protocol, with the IMGT trajectory.
The humanization process based on the MG-score described above generated a trajectory that stopped at 42 mutations from the initial sequence in the IMGT definition of the CDRs, and at 35 mutations in the Kabat definition. The threshold between murine and human sequences is 6383 (Clavero-Álvarez A et al. 2018, cited), and we note that sequence can be considered as human starting from step 7 on both trajectories; on the other hand, the documentation of the CamSol Intrinsic tool states that regions with scores larger than 1 denote highly soluble regions, while scores smaller than −1 are indicative of poorly soluble ones. As a trade-off between the above consideration, we chose the VH sequence corresponding to sequence 19 and the VL of sequence 36 along the IMGT trajectory, and the VH of sequence 15 and VL of sequence 24 in the “Kabat” trajectory. Notice that in these latter sequences, the VHs are the same as those individually chosen before, but the VL is different, so that we end up with one choice of VH sequence and two choices of VL sequences for each trajectory. These sequences are listed below:
Among the identified and above listed sequences, only the sequence named H5/K5, when expressed as recombinant protein, showed some activity in an ELISA assay. However, the reactivity of this humanised mAb was lower than the activity observed for the murine 2G8.
Given the totally unsatisfying results obtained with the first, second and third state of the art approach, the authors random combined each one of the humanised VH sequences obtained with the three above reported approaches with each one of the humanised VL sequences.
Four different heavy chains (H1, H3, H5, H6) were combined with six different light chains (K1, K3, K5, K6, K7, K8), resulting in a series of humanised mAb randomly comprising the selected heavy and light chains.
The surnatants collected from the obtained humanised mAb were tested in ELISA test. Results are reported in Table 2.
The observed data are indicative of the fact that only antibodies comprising the 6 CDRs according to the present invention are effective in the ELISA assay, wherein antibodies that do not comprise at least one of the defined CDR do not sow the desired activity.
The activity observed with the humanised mAb comprising H5 (SEQ ID NO: 22) and K1 sequences (SEQ ID NO: 15) has been surprisingly high.
Four supernatants from hmAb according to the invention (H5/K3; H5/K1; H5/K7; H5/K5) were purified to confirm the data. 8 scalar dilutions, starting from 5 μg/ml, were tested. The results are shown in
A medium scale production was executed using the EXPICHO Expression and purified by affinity chromatography as above. The structural integrity and the absence of aggregates were confirmed by SDS-PAGE (
The purified humanised mAb 2G8 H5/K1 was then tested in an ELISA assay using laminarin as a target.
From an overnight inoculum, C. auris cells were washed with RPMI+MOPS (0.165 M, pH 7) and 3.0×106 cells were pelleted, suspended in Phosphate buffered saline (PBS) containing 3% (w/v) BSA (Bovine Serum Albumin—Sigma Aldrich) and put in contact with 12 μg/ml of the humanised mAb 2G8 for 1 hour at room temperature. The cells were then washed with PBS and marked with anti-human IgG FITC antibody 1:150 in PBS+3% BSA for 1 hour. The cells were then washed and fixed with paraformaldehyde 4% in PBS 1 hour at 4° C. After fixing and washing with PBS, the pellet was suspended in 400 μl of PBS and splitted in two tubes. The samples were analysed using flow cytometry and confocal microscopy, respectively.
A Flow cytometer (FACScanto II, BDBioscences, Erembodegem, Belgium), equipped with three lasers (488 nm, 633 nm, 405 nm) was employed to collect and quantitate FITC fluorescence from different samples. Both autofluorescence and fluorescence derived from a specific binding of FITC-conjugated secondary Ab were quantitated by Flow Cytometry.
The exemplificative confocal images, reported in
The growth inhibitory activity of humanised mAb 2G8 H5/K1 was tested as reported in W. Magliani et al, Nature Biotech. 1997; 15:155-158 with some modifications. In brief, 150-250 cells of C. auris in 10 μl of PBS were incubated with 100 μl of humanised mAb 2G8 at 250, 100 and 50 μg/ml (each concentration was tested in triplicate) and incubated for 18 h at 37° C. The inhibition was evaluated by seeding the yeast on PDA. The plates were incubated at 37° C. for 48 h and the inhibition was calculated by count of the CFU. The assay was performed in triplicate.
From the CFU counts, hmAb H5/K1 provided a statistically significant effect on the inhibition of the yeast cell growth at all the doses tested (
In order to investigate the protective effect of hmAb 2G8 H5/K1 in preventing fungal adhesion to human cells commonly known to be preferred targets, C. auris cells were left adhering to a monolayer of HeLa cells together with the humanised mAb H5/K1, to reproduce an infection-medication scenario. 1.0×104 cervical cancer cells HeLa were suspended in RPMI+*10% FBS (pH 7) and plated in a 96-well plate for 2 hours at 37° C.+5% CO2. After incubation, the cells not attached at the bottom of the wells were washed away and 1.0×104 cells of C. auris suspended in RPMI+MOPS (0.165 M, pH 7) were added to reach a 1:1 ratio HeLa:yeast cells. Together with the yeast, 12 μg/ml of the humanised mAb 2G8 were added. PBS was used in the control sample. The plate was left at 37° C. for 1 hour and after washing 5 times, HeLa cells were lysed with PBS 0.1% Triton X100 (15 min. RT). The suspension was plated in PDA plate and incubated at 37° C. for 48 hours. This experiment has been repeated two times in triplicate. Even when tested at a low dose, i.e. 12 μg/ml, hmAb 2G8 H5/K1 can afford a statistically significant reduction in fungal adhesion to mammalian cells of 51.5% (
To evaluate the susceptibility of C. auris to caspofungin, amphotericin B and their combination with hmAb 2G8, we followed the EUCAST antifungal MIC method (EUCAST E.DEF. 7.3). Antifungal drugs were purchased from Sigma-Aldrich.
The caspofungin and amphotericin B range concentrations analysed were reported in EUCAST document. Those concentrations were tested alone and in combination with 0.25, 2.5, 25 and 250 μg/ml of humanised antibody.
The plates microdilution and the yeast inoculum were prepared as reported in EUCAST document. The wells with 100 μl of 2X final antifungal drugs concentration in RPMI 2% G medium were inoculated with 100 μl (1-5×105 CFU/ml) of yeast suspension of C. auris. The monoclonal antibody was added to the samples of yeast suspension to test the combination drug-antibody. The plates were incubated at 37° C. for 24 and 48 hours and read after incubation at 405 nm. The assays were performed in triplicate.
The results are reported in
As reported in EUCAST, echinocandins' MIC50 is the lowest drug concentration giving inhibition of≥50% of that of the drug-free control (dotted line). For amphotericin B, MIC50 is the same as for echinocandins while MIC90 is considered as the lowest concentration giving a growth inhibition of≥90% of that of the drug-free control (dotted bold line). After 24 hours (A) the MIC breakpoints of caspofungin alone and in combination with hmAb 2G8 are fixed at 0.0625 μg/ml. At 48 hours (B), MIC50 is set at 0.25 μg/ml of caspofungin alone while at 0.125 μg/ml if in combination with 25 and 250 μg/ml of hmAb 2G8. This shows a reduction of 1 dilution of caspofungin concentration (from to 0.125 μg/ml). For what concerns amphotericin B, at 24 hours (C), the drug alone has a MIC50 and MIC90 at 0.5 μg/ml whereas, with 2.5 μg/ml of hmAb 2G8, MIC50 and 90 are respectively at 0.125 μg/ml and 0.25 μg/ml. With 25 and 250 μg/ml of hmAb 2G8, MIC50 shifts at 0.0625 and MIC90 at 0.125 μg/ml. At 48 hours (D), amphotericin B alone has its MIC50 and 90 at 1 μg/ml. With 2.5 μg/ml of hmAb, MIC90 is the same as for the AMB alone but MIC50 is at 0.5 μg/ml. The combination with 25 μg/ml of hmAb fixes MIC50 and 90 together at 0.25 μg/ml while with 250 μg/ml of the humanised mAb. MIC90 is at 0.25 μg/ml and MIC50 at 0.125 μg/ml.
To support and deepen the data coming from MIC assays, a time-kill curve was elaborated to evaluate how the hmAb 2G8 combined with caspofungin or amphotericin B could improve their basal activity on C. auris.
Results are reported in
Table 3 shows the improvement of the fungistatic effect thanks to the presence of hmAb 2G8 H5K1: the combination of CAS 0.25 μg/ml and hmAb 2G8 H5K1 ensures more than 1 log difference both at 24 and 48 hours and the combination with 0.125 μg/ml shows more than 1 log difference at 24 hours and more than half log at 48 hours.
Amphotericin B efficiency starts decreasing at 0.25 μg/ml. The fungicidal activity of Amphotericin B alone is obtained after 24 hours but, when administered in combination with hmAb 2G8 H5K1 the effect arises at 6 hours, as reported in table 4 and in
According to the standardized method (Pfaller et al, Clin Microbiol Rev. 2004), the synergic action between two agents is revealed with a difference≥2 log in their respective growth decrease after 24 hours. As showed in table 5, it is evident the strong synergy between amphotericin b and the humanised mAb 2G8 H5K1.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102020000026398 | Nov 2020 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/080372 | 11/2/2021 | WO |
