HUMANISED ANTIBODIES AGAINST PATHOGENIC FUNGI

Abstract

The present invention relates to humanised antibody molecules having specificity for -1,3 glucans and compositions comprising the same. The present invention also relates to the therapeutic uses of the antibody molecules, wherein said use is in the treatment or prophylaxis of fungal infections. It is further claimed a synergistic combination of a) at least one humanised antibody which has specificity for -1,3 glucans and b) one or more antifungal agents for use in the treatment or prophylaxis of infections of human cells.

Claims

1. A humanised antibody which has specificity for (-1,3 glucans of pathogenic fungi, or fragments or fusion proteins thereof, comprising: (i) a CDRH1 having at least 90% of identity with the sequence SYWLE (SEQ ID NO: 10. (ii) a CDRH2 having at least 90% of identity with the sequence TABLE-US-00011 (SEQIDNO:12) EILPGSGSTNYNEKFKG (iii) a CDRH3 having at least 90% of identity with the sequence EGWYFDV (SEQ ID NO: 14) (iv) a CDRL1 having at least 90% of identity with the sequence QSLLYSNGNTH (SEQ ID NO: 9) (v) a CDRL2 having at least 90% of identity with the sequence LVS (SEQ ID NO: 11), and (vi) a CDRL3 having at least 90% of identity with the sequence VQGTHFPYT (SEQ ID NO: 13).

2. A humanised antibody or fragments or fusion proteins thereof according to claim 1, comprising variable regions having CDR sequences of: (i) CDRH1 comprising sequence SYWLE (SEQ ID NO: 10) (ii) CDRH2 comprising sequence EILPGSGSTNYNEKFKG (SEQ ID NO: 12) (iii) CDRH3 comprising sequence EGWYFDV (SEQ ID NO: 14) (iv) CDRL1 comprising sequence QSLLYSNGNTH (SEQ ID NO: 9) (v) CDRL2 comprising sequence LVS (SEQ ID NO: 11), and (vi) CDRL3 comprising sequence VQGTHFPYT (SEQ ID NO: 13).

3. A humanised antibody or fragments or fusion proteins thereof according to claim 1, comprising 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.

4. A humanised antibody or fragments or fusion proteins thereof according to claim 1, comprising 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.

5. A humanised antibody or fragments or fusion proteins thereof according to claim 1, comprising 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.

6. A humanised antibody or fragments or fusion proteins thereof according to claim 1, comprising a variable region consisting of H5, SEQ ID NO: 22 for the heavy chain variable regions in combination with a variable region consisting of K1, SEQ ID NO: 15 for the light chain variable regions.

7. An isolated DNA sequence encoding the heavy and light chain of an antibody according to claim 1.

8. A cloning or expression vector comprising one or more DNA sequences according to claim 7.

9. A host cell comprising one or more cloning or expression vector selected from a cloning vector comprising a DNA sequence encoding a heavy and light chain according to claim 7 or a cloning or expression vector comprising a DNA sequence encoding the light chain of an antibody and a cloning or expression vector comprising a DNA sequence encoding the heavy chain of an antibody.

10. A pharmaceutical composition comprising an antibody according to claim 1, in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier and optionally additionally comprising other active ingredients.

11. A combination of a) at least one humanised antibody which has specificity for -1,3 glucans and b) one or more antifungal agents for use in the treatment or prophylaxis of infections of human cells.

12. A combination for use according to claim 11, wherein said at least one humanised antibody is as defined in claim 1.

13. A combination for use according to claim 11, wherein said one ore more antifungal agent is selected from echinocandins and/or polyene macrolide.

14. A pharmaceutical composition comprising a) at least one humanised antibody which has specificity for -1,3 glucans and b) one or more antifungal agents with one or more of a pharmaceutically acceptable excipient, diluent or carrier.

15. A humanised antibody according to claim 1 or a pharmaceutical composition, for use in the treatment or prophylaxis of infections of human cells.

16. A combination for use according to claim 11 or a humanised antibody or a pharmaceutical composition for use wherein said infections are mediated by pathogenic fungi.

17. A combination for use or a humanised antibody or a pharmaceutical composition for use according to claim 16, wherein said infections is selected from candidiasis, aspergillosis, cryptococcosis, dermatomycoses, sporotrichosis and other subcutaneous mycoses.

Description

DESCRIPTION OF THE FIGURES

[0010] FIG. 1: Comparison of the 10 germlines amino acidic sequences and SEQ ID NO: 2, (2G8 Heavy chain variable region according to WO2006030318).

[0011] FIG. 2: Comparison of the 10 germline nucleotide sequences and SEQ ID NO: 6, (VH2G8patent: Heavy chain variable region according to WO2006030318).

[0012] FIG. 3: Comparison between the C terminal portion of SEQ ID NO: 6 (grey) and the constant regions IGHV accession number IMGT EF392837.

[0013] FIG. 4: Comparison of the 10 germline nucleotide sequences and SEQ ID NO: 1, (2G8 Light chain variable region according to WO2006030318).

[0014] FIG. 5: Comparison between the described murine 2G8 VL sequence and the here identified 2G8 VL.sub.c sequence: (A) nucleotide SEQ ID NO: 5 (VL2G8patent) versus SEQ ID NO: 7 (VL2G8correct); (B) amino acids, SEQ ID NO: 1 (VL2G8PATENT); versus SEQ ID NO: 3 (VL2G8CORRECT); .

[0015] FIG. 6: Comparison between the described murine 2G8 VH sequence and the here identified 2G8 VH.sub.c sequence, (A) nucleotide SEQ ID NO: 6 (VH2G8patent) versus SEQ ID NO: 8 (VH2G8correct); (B) amino acids, SEQ ID NO: 2 (VH2G8PATENT) versus SEQ ID NO: 4 (VH2G8CORRECT).

[0016] FIG. 7: Humanisation of the 2G8 antibody, first approach. (A) Comparison between the 2G8 VH.sub.c sequence (SEQ ID NO: 4) with the most homologous human germline IGHV1-46*01; (B) amino-acids comparison between the 2G8 VH.sub.c sequence (SEQ ID NO: 4, VH2G8MURINE) and H1 2G8 (SEQ ID NO: 16, VH2G8HUMANISED); (C) Comparison of the 2G8 VL.sub.c sequence (SEQ ID NO: 3) with the most homologous human germ line IGKV2-30*01.

[0017] FIG. 8: Humanisation of the 2G8 antibody, second approach. Comparison of 2G8 H2 sequence (SEQ ID NO: 18) and 2G8 K2 sequence (SEQ ID NO: 17) with sequences identified as most similar in PDB.

[0018] FIG. 9: ELISA assays performed by using the indicated antibodies.

[0019] FIG. 10: Characterization of the hmAb 2G8 H5/K1 by (A) SDS-PAGE and (B) HPLC-SEC analysis.

[0020] FIG. 11: ELISA assays evaluating laminarin binding.

[0021] FIG. 12: (A) FITC-fluorescence signal from control and hmAb 2G8 H5/K1 treated samples. (B) fold of increase in treated versus control samples.

[0022] FIG. 13: exemplificative confocal images (A, B) control; (C, D) hmAb 2G8 H5/K1 treated samples.

[0023] FIG. 14: Yeast cells growth inhibition in the absence (CTR) or in the presence of the indicated amount of hmAb 2G8 5H/K1.

[0024] FIG. 15: Fungal adhesion to mammalian cells in the presence or in the absence (CTR) of hmAb 2G8 5H/K1.

[0025] FIG. 16: MIC assays of caspofungin alone (CAS) and in combination with different concentrations of humanised mAb 2G8 at 24 h (A) and 48 h (B). MIC assays of amphotericin B alone (AMB) and in combination with different concentrations of humanised mAb 2G8 at 24 (C) and 48 (D) hours.

[0026] FIG. 17: time-kill curve of caspofungin alone (A) or in combination with hmAb 2G8 (B).

[0027] FIG. 18: time-kill curve of amphotericin B alone (A) or in combination with hmAb 2G8 (B).

[0028] Sequence Listings: SEQ ID NO: 1-28 are provided.

DETAILED DESCRIPTION OF THE INVENTION

[0029] 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.

[0030] 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).

[0031] 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.

[0032] 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.

[0033] Thus, provided in one embodiment is a humanised antibody which binds -1,3 glucans, wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs.

[0034] A murine anti -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 FIG. 1 and in FIG. 4 (SEQ ID NO: 2, SEQ ID NO: 1), respectively.

[0035] The CDRs sequences characterising the humanised antibodies according to the present invention have been surprisingly identified by the Applicant.

[0036] 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 -1,3 glucans higher than the affinity observed for murine 2G8.

[0037] Several hmAb 2G8 characterized by comprising the listed CDRs were produced and characterized and demonstrated capable to recognize a number of fungi.

[0038] 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 -1,3 glucans, comprising: [0039] (i) a CDRH1 having at least 90% of identity with the sequence SYWLE (SEQ ID NO: 10) [0040] (ii) a CDRH2 having at least 90% of identity with the sequence EILPGSGSTNYNEKFKG (SEQ ID NO: 12) [0041] (iii) a CDRH3 having at least 90% of identity with the sequence EGWYFDV (SEQ ID NO: 14) [0042] (iv) a CDRL1 having at least 90% of identity with the sequence QSLLYSNGNTH (SEQ ID NO: 9) [0043] (v) a CDRL2 having at least 90% of identity with the sequence LVS (SEQ ID NO: 11), and [0044] (vi) a CDRL3 having at least 90% of identity with the sequence VQGTHFPYT (SEQ ID NO: 13).

[0045] In an embodiment, said humanised antibody, or fragments or fusion proteins thereof, comprises variable regions having CDR sequences of: [0046] (i) CDRH1 comprising sequence SYWLE (SEQ ID NO: 10) [0047] (ii) CDRH2 comprising sequence EILPGSGSTNYNEKFKG (SEQ ID NO: 12) [0048] (iii) CDRH3 comprising sequence EGWYFDV (SEQ ID NO: 14) [0049] (iv) CDRL1 comprising sequence QSLLYSNGNTH (SEQ ID NO: 9) [0050] (v) CDRL2 comprising sequence LVS (SEQ ID NO: 11), and [0051] (vi) CDRL3 comprising sequence VQGTHFPYT (SEQ ID NO: 13).

[0052] Antibodies or fragments thereof may be polyclonal or monoclonal antibodies. Monoclonal antibodies are preferred.

[0053] 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).

[0054] In some embodiments, the antibody is a full-length IgG class antibody.

[0055] 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).

[0056] 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.

[0057] 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.

[0058] 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: [0059] phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); [0060] lysine, arginine and histidine (amino acids having basic side chains); [0061] aspartate and glutamate (amino acids having acidic side chains); [0062] asparagine and glutamine (amino acids having amide side chains); and [0063] cysteine and methionine (amino acids having sulphur-containing side chains).

[0064] Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk A M, ed., Oxford University Press, New York, 1988).

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In an embodiment, the humanised antibody is hmAb H5/K1.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] In a preferred embodiment, said infections are mediated by pathogenic fungi, preferably selected in the group comprising the genus Candida, Aspergillus, Cryptococcus.

[0078] In a further embodiment, the Applicant has surprisingly found that antibodies with a specificity for -1,3 glucans can be successfully used in association with antifungal agents.

[0079] In particular, the Applicant has now surprisingly found that the combination of antibodies with a specificity for -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.

[0080] In the present description, the expression synergistic effect means that the combination of humanised antibodies with a specificity for -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 -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 -1,3 glucans and of the antifungal agent is effective in producing more than the additive effect of each component in the same strain.

[0081] Therefore, it is here claimed a combination of a) at least one humanised antibody which has specificity for -1,3 glucans and b) one or more antifungal agents for use in the treatment or prophylaxis of infections of human cells.

[0082] Said combination is for simultaneous, separate or sequential use as a medicament.

[0083] 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.

[0084] In a preferred aspect, said antifungal agent is selected from echinocandins and/or polyene macrolide.

[0085] Preferably, said echinocandin is caspofungin, micafungin, or anidulafungin.

[0086] Preferably, said polyene macrolide is amphotericin B.

[0087] In an embodiment, said composition comprises a hmAb according to the present invention in combination with a further antifungal agent.

[0088] In an embodiment, said humanised antibody is hmAb 2G8 H5/K1.

[0089] 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 -1,3 glucans and b) one or more antifungal agents, preferably selected from echinocandins or polyene macrolide.

[0090] In a preferred embodiment, said fungal infections are selected from candidiasis, aspergillosis, cryptococcosis, dermatomycoses, sporotrichosis and other subcutaneous mycoses.

[0091] Humanised antibodies according to the present invention are capable of inhibiting the binding of a microbial pathogen that contains -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 -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.

[0092] The present invention is further described by way of illustration only in the following examples.

EXAMPLE

Example 1: Definition of the Murine 2G8 VH.SUB.c .and VL.SUB.c .Sequences

[0093] 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.

[0094] The mAb 2G8 VH amino acidic sequence, SEQ ID NO: 2, has been analysed against the first 10 homologous germline sequences, i.e. [0095] IGHV1-9*01 (accession number AC090843), [0096] IGHV1-63*02 (accession number D14633), [0097] IGHV1-55*01 (accession number AC074329), [0098] IGHV1-56*01 (accession number AC087166), [0099] IGHV1-77*01 (accession number AC160473), [0100] IGHV1-54*02 (accession number X02064), [0101] IGHV1-56*02 (accession number AF304551), [0102] IGHV1S61*01 (accession number D14634), [0103] IGHV1-66*01 (accession number AC073939).

[0104] The results of the comparison, reported in FIG. 1, highlighted an homology higher than 88.4%.

[0105] 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).

[0106] 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 FIG. 2, highlighted in grey the lacking nucleotides.

[0107] Moreover, the comparison showed that the first domain of the heavy chain constant region, highlighted in the sequence reported in FIG. 3, is not to be considered part of FR4 as can be seen from a comparison of this sequence with the constant region of another murine antibody i.e. IGHV accession number IMGT EF392837, detailed in the same FIG. 3.

[0108] From the above reported observations, the authors defined the 2G8 VH.sub.c sequence (SEQ ID NO: 8), containing 3 additional amino acids at the N terminus and stopping at amino acid Serin 116.

[0109] A similar approach was used to identify the sequence 2G8 VL.sub.c. The germline sequences with higher homology show a percentage that was over 78.9%. In FIG. 4 is reported the comparison versus the first 10 homologous germline sequences listed below: [0110] IGKV1-133*01 (accession number Z72382), [0111] IGKV1-135*01 (accession number Z72384), [0112] IGKV1-131*01 (accession number AJ231197), [0113] IGKV1-132*01 (accession number AJ231198), [0114] IGKV1-35*01 (accession number AJ231200), [0115] IGKV1-110*02 (accession number M28132), [0116] IGKV1-110*01 (accession number D00080), [0117] IGKV1-122*01 (accession number D00082), [0118] IGKV1-117*02 (accession number M28134), [0119] IGKV1-117*01 (accession number D00081).

[0120] The authors defined a 2G8 VL.sub.c sequence (SEQ ID NO: 3), stopping at amino acid Lysin 112.

[0121] The newly defined amino-acidic and nucleotidic sequences, 2G8 VL.sub.c (SEQ ID NO: 7, SEQ ID NO: 3, respectively) and 2G8 VH.sub.c (SEQ ID NO: 4, SEQ ID NO: 8, respectively) are reported in FIG. 5A, FIG. 5B and FIG. 6A, FIG. 6B, respectively, in comparison to the sequences described in WO2006030318.

Example 2: Obtainment of Humanised Antibodies

Example 2A, First Approach

[0122] 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 FIG. 7. FIG. 7A shows the humanisation of the 2G8 VH.sub.c sequence (SEQ ID NO: 4) obtained in Example 1 with the most homologous human germline IGHV1-46*01, accession number X92343. FIG. 7B shows the amino-acids comparison from murine VH.sub.c 2G8 (VH2G8MURINE, SEQ ID NO: 4) and the here obtained humanised VH 2G8, named H1 (VH2G8HUMANISED, SEQ ID NO: 16). FIG. 7C shows the humanisation of the 2G8 VL.sub.c sequence (SEQ ID NO: 3) with the cited most homologous human germline IGKV2-30*01. The here obtained sequence of humanised VH, named H1 (SEQ ID NO: 16) and VL, named K1 (SEQ ID ID NO: 15), are as follows:

TABLE-US-00002 H1, SEQIDNO:16 QVQLVQSGAEVKKPGASVKVSCKASGYTLSSYWMHWRQAP GQGLEWMGIILPGSGSTSYAQKFQGRVTMTRDTSTSTVYM ELSSLRSEDTAVYYCAREGWYFDVWGAGTTVTVSS K1, SEQIDNO:15 DVVMTQSPLSLPVTLGQPASISCRSSQSLLYSNGNTHLNW FQQRPGQSPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCVQGTHFPYTFGGGTKLEIK
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.

[0123] Supernatants containing antibodies were centrifuged, filtered 0.22 m 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.

[0124] The activity of the purified antibody was then tested by ELISA assay. 96-well plates were coated with 50 g/ml Laminarin (Sigma-Aldrich, L9634) in 0.05 M carbonate buffer pH 9.6 overnight at 4 C. Nonspecific interactions were blocked with 100 L/well blocking solution, 3% (w/v) BSA in PBS-Tween 20 (8 g/L NaCl, 0.2 g/L KH.sub.2PO.sub.4, 2.9 g/L Na.sub.2HPO.sub.4.Math.12 H.sub.2O, 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 g/ml to 0.003 g/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 L 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 l 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.

[0125] The hmAb 2G8 H1/K1 was not active in an ELISA test.

Example 2B, Second Approach

[0126] Sequences obtained in Example 1, VH.sub.c and VL.sub.c 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.

TABLE-US-00003 TABLE 1 Germiline genes databases Selection Organism for query sequence Human Germline V gene Database IMGT Human V genes (F + ORF + in-frame P) Germline D gene Database IMGT Human D genes (F + ORF) Germline J gene Database IMGT Human V genes (F + ORF)

[0127] The obtained sequences of humanised VH, named H2 (SEQ ID NO: 18) and VL, named K2 (SEQ ID ID NO: 17), are as follows:

TABLE-US-00004 H2, SEQIDNO:18 QVQLVQSGAEVKKPGASVKVSCKASGYTLSSYWLHWVQQS PGQGLEWMGEILPGSGSTNYAQKFQGRVTITADKSTSTAY MELSSLRSEDTAVYYCAREGWYFDVWGAGTTVTVSS K2, SEQIDNO:17 DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGNTHLNW FQQRPGQSPRRLIYLVSNRASGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCVQGTHEPYTFGGGTKLEIK

[0128] 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 resolution2 (www.rcsb.org). Results are reported in FIG. 8. The alignment of the three most homologous sequences emerging from PDB database analyses showed that only one amino acids was different (E50W), and for this reason it has been mutagenized inserting W in the VH sequence. Furthermore, we back mutated additional three amino acids in the VH sequence to reduce the risk of protein aggregation (M48I, R67K, V68A) and three amino acids in the VL sequence (R24K, K44R, S68T). This approach generated the following sequences of humanised VH, named H3 (SEQ ID NO: 20) and VL, named K3 (SEQ ID NO: 19):

TABLE-US-00005 H3, SEQIDNO:20 QVQLVQSGAEVKKPGASVKVSCKASGYTLSSYWIHWVRQA PGQGLEWIGWILPGSGSTNYAQKFQGKATITADKSTSTAY MELSSLRSEDTAVYYCAREGWYFDVWGQGTLVTVSS K3, SEQIDNO:19 DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGNTHLNW YLQRPGQSPQLLIYLVSNRASGVPDRFTGSGSGTDFTLKI SRVEAEDVGVYYCVQGTHFPYTFGGGTKVEIK
H3 and K3 have been expressed as a recombinant Ab. Also in this case, the antibodies were not active in an ELISA test.

Example 2C, Third Approach

[0129] The humanization approach described in Clavero-lvarez A et al. Scientific Reports 2018; 8:14820 has been followed, resorting to statistical modelling of the variable regions of human antibody sequences. VH.sub.c and VL.sub.c 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.

[0130] 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).

[0131] 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.

[0132] 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-lvarez 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:

TABLE-US-00006 KABAT K5, SEQIDNO:21 DIVMTQSPLSLPVTLGQPASISCKSSQSLLYSNGNTHLNW YQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCVQGTHEPYTFGGGTKLEIK H5, SEQIDNO:22 QVQLQQSGAELKKPGASVKISCKASGYTLSSYWLEWVRQR PGQGLEWIGEILPGSGSTNYNEKFKGRATFTADTSTNTAY MELSSLTSEDSAVYYCAREGWYFDVWGQGTTVTVSS IMGT K6, SEQIDNO:23 DIVMTQSPLSLPVTLGQPASISCRSSQSLLYSNGNTHLNW YQQRPGQSPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCMQGTHFPYTFGGGTKLEIK H6, SEQIDNO:24 QVQLQQSGAELKKPGASVKISCKASGYTLSSYWIHWVRQR PGQGLEWIGEILPGSGSTNYNEKFKGRATFTADTSTNTAY MELSSLTSEDSAVYYCAREGWYFDVWGQGTTVTVSS KABAT K7, SEQIDNO:25 DIVMTQSPLSLPVTLGQPASISCKSSQSLLYSNGNTHLNW YLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCVQGTHFPYTFGGGTKLEIK H7, SEQIDNO:26 QVQLQQSGAELKKPGASVKISCKASGYTLSSYWLEWVRQR PGQGLEWIGEILPGSGSTNYNEKFKGRATFTADTSTNTAY MELSSLTSEDSAVYYCAREGWYFDVWGQGTTVTVSS IMGT K8, SEQIDNO:27 DIVMTQSPLSLPVTLGQPASISCKSSQSLLYSNGNTHLNW YLQRPGQSPKRLIYLVSKRDSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCMQGTHFPYTFGGGTKLEIK H8, SEQIDNO:28 QVQLQQSGAELKKPGASVKISCKASGYTLSSYWIHWVRQR PGQGLEWIGEILPGSGSTNYNEKFKGRATFTADTSTNTAY MELSSLTSEDSAVYYCAREGWYFDVWGQGTTVTVSS
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.

Example 2D, Fourth Approach

[0133] 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.

[0134] 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.

[0135] The surnatants collected from the obtained humanised mAb were tested in ELISA test. Results are reported in Table 2.

TABLE-US-00007 TABLE 2 Humanized mAbs Abs 405 nm H1 K1 0.35 H3 K1 0.4 H5 K1 3.48 H5 K3 2.47 H5 K5 1.61 H5 K7 0.82 H6 K1 0.27

[0136] 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.

[0137] The activity observed with the humanised mAb comprising H5 (SEQ ID NO: 22) and K1 sequences (SEQ ID NO: 15) has been surprisingly high.

Example 3: Characterization of H5/K1 Recombinant, Humanised Anti -1,3 Glucan Antibody

[0138] 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 g/ml, were tested. The results are shown in FIG. 9 and confirm the combination heavy:light H5/K1 being the best performer in ELISA assay.

[0139] 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 (FIG. 10A) and HPLC-SEC analysis (FIG. 10B).

[0140] The purified humanised mAb 2G8 H5/K1 was then tested in an ELISA assay using laminarin as a target. FIG. 11 shows the activity of hmAb 2G8 H5/K1 against Laminarin compared to the murine one, the mAb 2G8 according to WO2006030318. The IC50 values of the humanised and murine mAbs estimated from these data were respectively 0.06 g/ml and 0.120 g/ml. Thus, the hmAb 2G8 H5/K1 performs better than the original murine one.

Example 4: Recognition of Pathogenic Fungi Expressing -1,3-glucan and Effect of hmAb 2G8 H5/K1 on the Same

[0141] From an overnight inoculum, C. auris cells were washed with RPMI+MOPS (0.165 M, pH 7) and 3.010.sup.6 cells were pelleted, suspended in Phosphate buffered saline (PBS) containing 3% (w/v) BSA (Bovine Serum AlbuminSigma Aldrich) and put in contact with 12 g/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 l of PBS and splitted in two tubes. The samples were analysed using flow cytometry and confocal microscopy, respectively.

[0142] 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. FIG. 12A shows fluorescence signal from control samples (on the left) and from treated samples (on the right). The histogram CTRL evaluates the background from aspecific binding of FITC-conjugated secondary Ab, enabling to fix the threshold defining the positivity for the humanised mAb H5/K1 (treated). 97% of events express the antigen (treated panel). The expression was precisely quantitated by MFI values: the fold of increase is 164, when compared to control (FIG. 12B). Light grey events in FIG. 12A histograms represent cells outside the scatter area mainly occupied by the cells: i.e. aggregates or increase in cell size (as during senescence).

[0143] The exemplificative confocal images, reported in FIG. 13, show the ability of the hmAb H5/K1 to recognize and bind -1,3-glucans on the surface of C. auris (white dots corresponding to the FITC signal).

Example 5: Growth Inhibition Assay

[0144] 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 l of PBS were incubated with 100 l of humanised mAb 2G8 at 250, 100 and 50 g/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.

[0145] 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 (FIG. 14) ranging over 70% for 250 and 100 g/ml and over 60% for 50 g/ml.

Example 6: Adhesion Assay

[0146] 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. 1.010.sup.4 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% CO.sub.2. After incubation, the cells not attached at the bottom of the wells were washed away and 1.010.sup.4 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 g/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 g/ml, hmAb 2G8 H5/K1 can afford a statistically significant reduction in fungal adhesion to mammalian cells of 51.5% (FIG. 15).

Example 7: Synergistic Effect, MIC Assay

[0147] 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.

[0148] 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 g/ml of humanised antibody.

[0149] The plates microdilution and the yeast inoculum were prepared as reported in EUCAST document. The wells with 100 l of 2X final antifungal drugs concentration in RPMI 2% G medium were inoculated with 100 l (1-510.sup.5 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.

[0150] The results are reported in FIG. 16.

[0151] As reported in EUCAST, echinocandins' MIC50 is the lowest drug concentration giving inhibition of50% 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 of90% 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 g/ml. At 48 hours (B), MIC50 is set at 0.25 g/ml of caspofungin alone while at 0.125 g/ml if in combination with 25 and 250 g/ml of hmAb 2G8. This shows a reduction of 1 dilution of caspofungin concentration (from to 0.125 g/ml). For what concerns amphotericin B, at 24 hours (C), the drug alone has a MIC50 and MIC90 at 0.5 g/ml whereas, with 2.5 g/ml of hmAb 2G8, MIC50 and 90 are respectively at 0.125 g/ml and 0.25 g/ml. With 25 and 250 g/ml of hmAb 2G8, MIC50 shifts at 0.0625 and MIC90 at 0.125 g/ml. At 48 hours (D), amphotericin B alone has its MIC50 and 90 at 1 g/ml. With 2.5 g/ml of hmAb, MIC90 is the same as for the AMB alone but MIC50 is at 0.5 g/ml. The combination with 25 g/ml of hmAb fixes MIC50 and 90 together at 0.25 g/ml while with 250 g/ml of the humanised mAb. MIC90 is at 0.25 g/ml and MIC50 at 0.125 g/ml.

Example 8: Synergistic Effect, Time-kill Curve

[0152] 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.

[0153] Results are reported in FIG. 17. At 24 h and 48 h, when caspofungin efficiency at 0.25 and 0.125 g/ml starts decreasing, the presence of hmAb ameliorated the fungistatic activity. In particular with 0.125 g/ml caspofungin, it resulted in a recovery slower than the one with CAS alone while the combination of 0.25 g/ml+250 g/ml of hmAb 2G8 H5K1 maintained a constant trend all over time.

[0154] Table 3 shows the improvement of the fungistatic effect thanks to the presence of hmAb 2G8 H5K1: the combination of CAS 0.25 g/ml and hmAb 2G8 H5K1 ensures more than 1 log difference both at 24 and 48 hours and the combination with 0.125 g/ml shows more than 1 log difference at 24 hours and more than half log at 48 hours.

TABLE-US-00008 TABLE 3 24 h 48 h CAS 0.25 g/ml 1.11 1.71 CAS 0.25 g/ml + hmAb 2G8 0.02 0.46 CAS 0.125 g/ml 1.88 2.10 CAS 0.125 g/ml + hmAb 2G8 0.87 1.51

[0155] Amphotericin B efficiency starts decreasing at 0.25 g/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 FIG. 18. Moreover, the combination shows a better effect at almost each concentration of Amphotericin B, the synergistic effect is still3 log at 24 h with 0.25 g/ml Amphotericin B (table 4).

TABLE-US-00009 TABLE 4 0 h 2 h 4 h 6 h 8 h 24 h 48 h AMB 4 g/ml 0.06 3 Log 3 Log 3 Log 3 Log 3 Log 3 Log AMB 4 + hmAb 2G8 0.08 3 Log 3 Log 3 Log 3 Log 3 Log 3 Log AMB 2 g/ml 0.05 3 Log 3 Log 3 Log 3 Log 3 Log 3 Log AMB 2 + hmAb 2G8 0.10 3 Log 3 Log 3 Log 3 Log 3 Log 3 Log AMB 1 g/ml 0.05 1.77 3 Log 3 Log 3 Log 3 Log 3 Log AMB 1 + hmAb 2G8 0.05 2.16 3 Log 3 Log 3 Log 3 Log 3 Log AMB 0.5 g/ml 0.05 0.21 0.41 0.86 1.95 3 Log 3 Log AMB 0.5 + hmAb 2G8 0.09 0.22 0.78 3 Log 3 Log 3 Log 3 Log AMB 0.25 g/ml 0.07 0.10 0.02 0.02 0.02 0.19 2.13 AMB 0.25 + hmAb 2G8 0.11 0.13 0.25 1.88 1.95 3 Log 2.66 AMB 0.125 g/ml 0.01 0.11 0.07 0.42 0.80 2.28 2.44 AMB 0.125 + hmAb 2G8 0.09 0.20 0.21 1.31 1.42 2.88 0.91 AMB 0.0625 g/ml 0.00 0.07 0.14 0.59 0.97 2.59 2.57 AMB 0.0625 + hmAb 2G8 0.09 0.15 0.09 0.46 0.84 1.45 2.17 AMB 0.03125 g/ml 0.01 0.05 0.19 0.62 1.04 2.65 2.85 AMB 0.03125 + hmAb 2G8 0.07 0.13 0.01 0.03 0.18 1.14 2.26

[0156] According to the standardized method (Pfaller et al, Clin Microbiol Rev. 2004), the synergic action between two agents is revealed with a difference2 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.

TABLE-US-00010 TABLE 5 24 h 48 h log AMB 0.25AMB 0.25 + hmAb 2G8 4.95 4.79 log AMB 0.125AMB 0.125 + hmAb 2G8 5.16 3.35 log AMB 0.0625AMB 0.0625 + hmAb 2G8 4.04 0.4