WO2017004563A1 - Multi-specific binding compounds - Google Patents

Abstract

This invention relates to multi-specific binding compounds, including antibodies, for the treatment of microbial, such as fungal infections. In particular, the invention concerns binding compounds having binding affinity to a microbial, such as fungal polysaccharide and an immune cell, their preparation and use in the treatment of microbial, e.g., fungal, diseases and conditions.

Description

MULTI-SPECIFIC BINDING COMPOUNDS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of the filing date of U.S. Provisional Patent Application Serial No. 62/188,459, filed on July 2, 2015, the disclosure of which application is herein incorporated by reference in its entirety, and U.S. Provisional Patent Application Serial No. 62/269,831, filed on December 18, 2015, the disclosure of which application is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to multi-specific binding compounds and their preparation and use.

In particular, the invention concerns binding compounds having binding affinity to a microbial polysaccharide, such as fungal polysaccharide, and an immune cell, their preparation and use in the treatment of microbial, e.g., fungal, diseases and conditions.

BACKGROUND OF THE INVENTION

Fungal infections are prevalent in several clinical settings, particularly in immunocompromised patients. Immunocompromised patients provide perhaps the greatest challenge to modern health care delivery. During the last three decades there has been a dramatic increase in the frequency of fungal infections in these patients (Herbrecht, Eur. J. Haematol., 56: 12, 1996; Cox et al, Curr. Opin. Infect. Dis., 6:422, 1993). Deep-seated mycoses are increasingly observed in patients undergoing organ transplants and in patients receiving aggressive cancer chemotherapy (Alexander et al, Drugs, 54:657, 1997). The most common pathogens associated with invasive fungal infections are the opportunistic yeast, Candida albicans, and the filamentous fungus, Aspergillus fumigatus (Bow, Br. J. Haematol, 101 : 1, 1998; Wamock, J. Antimicrob. Chemother., 41 :95, 1998). Adding to the increase in the numbers of fungal infections is the emergence of Acquired Immunodeficiency Syndrome (AIDS) where virtually all patients become affected with some form of mycoses during the course of the disease (Alexander et al, Drugs, 54:657, 1997; Hood et al, J. Antimicrob. Chemother., 37:71,

1996) . The most common organisms encountered in these patients are Cryptococcus neoformans, Pneumocystis carinii, and C. albicans (HIV/ AIDS Surveillance Report, 1996, 7(2), Year-End Edition; Polis, M. A. et al, AIDS: Biology, Diagnosis, Treatment and Prevention, fourth edition,

1997) . New opportunistic fungal pathogens such as Penicillium marneffei, C. krusei, C. glabrata, Histoplasma capsulatum, and Coccidioides immitis are being reported with regularity in immunocompromised patients throughout the world.

The development of antifungal treatment regimens has been a continuing challenge. Currently available drugs for the treatment of fungal infections include amphotericin B, a macrolide polyene that interacts with fungal membrane sterols, flucytosine, a fluoropyrimidine that interferes with fungal protein and DNA biosynthesis, and a variety of azoles {e.g., ketoconazole, itraconazole, and fluconazole) that inhibit fungal membrane-sterol biosynthesis (Alexander et al, Drugs, 54:657, 1997). Even though amphotericin B has a broad range of activity and is viewed as the "gold standard " of antifungal therapy, its use is limited due to infusion-related reactions and nephrotoxicity (Wamock, J Antimicrob. Chemother., 41 :95, 1998). Flucytosine usage is also limited due to the development of resistant microbes and its narrow spectrum of activity. The widespread use of azoles is causing the emergence of clinically-resistant strains of Candida spp. Due to the problems associated with the current treatments, there is an ongoing search for new treatments.

The major components of fungal cell walls are polysaccharides, including glucans, glycogen-like compounds, mannans (mannose polymers), chitosan (glucosamine polymers), and galactans (galactose polymers). Fucose, rhamnose, xylose, and uronic acids may also be present in small amounts. Glucans are glucose-containing polysaccharides found inter alia in fungal cell walls, a-glucans include one or more a-linkages between glucose subunits and β-glucans include one or more β-linkages between glucose subunits. Within a typical fungal cell wall, P-l,3-glucan microfibrils are interwoven and crosslinked with chitin microfibrils to form the inner skeletal layer, whereas the outer layer consists of beta-l,6-glucan and mannoproteins, linked to the inner layer via chitin and P-l,3-glucan. Of the glucans, the most common in the fungal cell wall are in the β-configuration. Polymers with (β-1,3)- and (P-l,6)-linked glucosyl units with various proportions of 1,3- and 1,6- linkages are common cell wall components. Many fungi, and yeasts in particular, have soluble peptidomannans within a matrix of a- and β-glucans, as part of the outer portion of their cell wall.

Cellular immunity is critical for successful host defence against microginal, e.g., fungal infections. Monoclonal antibody (MAb) 2H1, which binds to the capsular glucuronoxylomannan (GXM) of the fungus Cryptococcus neoformans, prolonged survival and decreased fungal burden in an experimental murine model of infection, and was suggested for combined use with Fluconazole (FLU) or amphotericin B (AmB) (Mukherjee et al , Antimicrob. Agent Chemother. , 39(7): 1398-1405, 1995; Mukherjee et al, Antimicrob. Agent Chemother., 38(3):580-587, 1994). Cell-surface mannoproteins are the dominant antigenic components of C. albicans and antibodies to mannan, proteases and heat shock proteins have been associated with protection against infection (Polonelli et al, Med. Mycol, 38, Suppl. 1 : 281-92, 2000). Other antigens identified as stimulating protective cellular immune responses include members of the aspartyl proteinase (Sap2) family; the 65 kDa mannoprotein (MP65); adhesion molecules isolated from phosphomannan cell wall complexes; peptides which mimic epitopes from the mannan portion of the phosphomannan complex of Candida; and hemolysin-like proteins (Polonelli et al , supra).

In C. albicans, 50-70% of the cell wall is composed of β-1,3- and β-Ι,ό-glucans.

Protective antibodies against C. albicans β-Ι,ό-glucan have been generated in mice (US Patent No. 8,414,889). Mice in which anti β-Ι,ό-glucan antibodies were raised by idiotypic vaccination with mannoprotein-depleted C. albicans cells were shown to have some protection against systemic challenge by C. albicans. Furthermore, mice passively immunised with these anti-β- 1,6-glucan antibodies demonstrated a raised level of protection against C. albicans. An IgGl monoclonal antibody directed against A. fumigatus is disclosed in Chaturvedi et al , Clinical and Diagnostic Leboratory Immunology 2005, 12(9): 1063-1068. The antibody was reported to protect against experimental murine aspergillosis.

In view of the wide-spread occurrence of systemic invasive fungal infections in hospital setting, and the associated high mortality rates, there is a great need for new, effective, and less toxic treatment options.

SUMMARY OF THE INVENTION

Disclosed herein are multi-specific binding compounds and their uses in the treatment of microbial, e.g., fungal infections.

In one aspect, the invention concerns a multi-specific binding compound comprising a first polypeptide having binding affinity to a fungal polysaccharide linked to a second polypeptide having binding affinity to an immune cell.

In one embodiment, in the multi-specific binding compound: (a) the first polypeptide is an antibody, an antigen-binding fragment thereof, an antibody-like molecule, or an immunoadhesin; or (b) the second polypeptide is an antibody, an antigen-binding fragment thereof, or an antibody-like molecule; or (c) the first polypeptide is an antibody, an antigen- binding fragment thereof, an antibody-like molecule, or an immunoadhesin and the second polypeptide is an antibody, an antigen-binding fragment thereof, or an antibody-like molecule.

In another embodiment, the first polypeptide in the multi-specific binding compounds herein is an antibody, or an antigen-binding fragment thereof, having binding affinity to a fungal polysaccharide. In all various embodiments of the invention, the fungal polysaccharide can be, without limitation, a fungal cell wall polysaccharide, such as a glucan, a chitin, or a mannan (polymers consisting of repeating units of, respectively, glucose, N-acetylglucosamine, and mannose), preferably a glucan, such as a P-l,3-glucan or a β-Ι,ό-glucan.

In a further embodiment, the first polypeptide in the multi-specific binding compounds herein is an immunoadhesin. In a preferred embodiment, the first polypeptide is an immunoadhesin comprising a binding sequence of a fungal polysaccharide binding protein, such as a Dectin (including Dectins-1 and -2), fused to an immunoglobulin (Ig) heavy chain constant region sequence, such as the Fc region of an immunoglobulin. In a preferred embodiment, the first polypeptide is an immunoadhesin comprising an extracellular domain sequence of a Dectin receptor, such as a Dectin-1 or Dectin-2 receptor, preferably a Dectin-1 receptor, fused to the Fc region of an immunoglobulin.

In all various embodiments of the invention, the immune cell targeted by the second polypeptide present in the multi-specific binding compounds herein preferably is a T-cell, and the second polypeptide preferably binds to a T-cell antigen, such as a component of the CD3 complex, preferably CD3s.

In all various embodiments of the invention, at least one of the first and second polypeptides may be linked to one or more water-soluble polymers. Preferably, at least one of the first and second polypeptides may be linked, directly or indirectly through a linking moiety, to one or more water-soluble polymers.

In all various embodiments of the invention, at least one of the first and second polypeptides may be linked to one of more polyethylene glycol molecules. Preferably, at least one of the first and second polypeptides may be linked, directly or indirectly through a linking moiety, to one or more polyethylene glycol polymers.

In all various embodiments, at least one of the first and second polypeptides may be linked to at least one half-life extending moiety, such as, for example, a half-life extending moiety selected from the group consisting of biocompatible fatty acids and derivatives thereof, hydroxy alkyl starch (HAS), hydroxy ethyl starch (HES), polyethelene glycol (PEG), hyaluronic acid (HA), fleximers, dextran, poly-sialic acids (PSA), Fc domains, transferrin, albumin, elastin- like (ELP) peptides, XTEN polymers, albumin binding peptides and combinations thereof. Preferably, at least one of the first and second polypeptides may be linked, directly or indirectly through a linking moiety, to at least one half-life extending moiety.

In another aspect, the invention concerns a multi-specific binding compound, comprising a first antibody, or an antigen-binding fragment thereof, comprising a first binding domain to a fungal cell wall polysaccharide and a second antibody, or an antigen-binding fragment thereof, comprising a second binding domain to a T-cell antigen.

In one embodiment, the fungal cell wall polysacchaccharide is a glucan, a chitin, or a mannan.

In another embodiment, the fungal cell wall polysaccharide is a glucan.

In yet another embodiment, the fungal cell wall polysaccharide is a β-glucan.

In a further embodiment, the β-glucan is a P-l,3-glucan or a β-Ι,ό-glucan, preferably a β- 1,3-glucan.

In a different embodiment, the T-cell antigen is a component of the CD3 complex, preferably CD3s.

In all various embodiments of the invention, the the first and/or second antibody, or antigen-binding fragment, may be human, humanized, or chimeric.

In all various embodiments of the invention, the first and/or second antibody, or antigen- binding fragment, may be cross-species reactive.

In all various embodiments of the invention, the first and/or second antibody, or antigen- binding fragment, may have cidal activity against a microbial pathogen, such as a fungal pathogen.

In all embodiments, the antigen-binding fragment may be, for example, a single-domain antibody, a Fab, Fab', F(ab')₂, scFv, or (scFv)₂ fragment.

In all embodiments, the first and second antibodies, or antigen-binding fragments, may be linked to each other. In various embodiments, the linkage is through direct fusion or through a linker.

In various embodiments, the linker is a peptide or polypeptide linker, such as a substituted or unsubstituted hydrocarbyl, including, without limitation, a (G4S)n linker, where n = 1-9, preferably (G4S)3 (SEQ ID NO: 15), a BGLl linker (SEQ ID NO: 16) or a BGL2 linker (SEQ ID NO: 13).

In other embodiments, the linker is acylated.

In still other embodiments, the linker comprises one or more ethylene glycol subunits.

In a further aspect, the invention concerns a multi-specific single chain antibody, comprising a first binding domain for beta-l,3-glucan (B13G) linked through a linker (L) to a second binding domain for human CD3s (CD3s), each of the first and second binding domains comprising a heavy chain variable region (VH) and a light chain variable region (VL), the corresponding VH and VL regions being arranged, from N-terminus to C- terminus, in an order selected from

VH(B 13 G)-VL(B 13G)-L-VH(CD3)-VL(CD3),

VH(B 13 G)-VL(B 13 G)-L- VL(CD3)- VH(CD3),

VL(B13G)-VH(B13G)-L-VH(CD3)-VL(CD3),

VL(B13G)-VH(B13G)-L-VL(CD3)-VH(CD3),

VH(CD3)-VL(CD3)-L-VH(B 13G)-VL(B 13 G),

VH(CD3)-VL(CD3)-L-VL(B 13G)-VH(B 13 G),

VL(CD3)-VH(CD3)-L-VH(B 13G)-VL(B 13 G), and

VL(CD3)-VH(CD3)-L-VL(B 13G)-VH(B 13 G)

In a still further aspect, the invention concerns a multi-specific antibody comprising:

i) a first light chain Complementary Determining Region (Li-CDR) comprising a Li-CDRl, a Li-CDR2, or a Li-CDR3, wherein Li-CDRl, L CDR2 and Li- CDR3 are at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or at least about 99% homologous, or are substantially identical, to parental OKT3 antibody or parental UCHT1 antibody L-CDR1, L- CDR2 and L-CDR3, respectively;

ii) a first heavy chain Complementary Determining Region (Hi-CDR) comprising a Hi-CDRl, a H CDR2, or a H CDR3, wherein H CDRl, H CDR2 and ¾- CDR3 are at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or at least about 99% homologous, or are substantially identical, to parental OKT3 antibody or parental UCHT1 antibody H-CDR2 and H-CDR3, respectively, iii) a second light chain Complementary Determining Region (L₂-CDR) comprising a L₂-CDR1, a L₂-CDR2, or a L₂-CDR3, wherein L₂-CDR1, L₂- CDR2 and L₂-CDR3 are at least about 90%, or at least about 92%, or at least about 95%), or at least about 98%>, or at least about 99% homologous, or are substantially identical, to parental 2G8 antibody L₂-CDR1, L₂-CDR2 and L₂- CDR3, respectively; and

iv) a second heavy chain Complementary Determining Region (H₂-CDR) comprising a H₂-CDR1, a H₂-CDR2, or a H₂-CDR3, wherein H₂-CDR1, H₂- CDR2 and H₂-CDR3 are at least about 90%, or at least about 92%, or at least about 95%), or at least about 98%>, or at least about 99% homologous, or are substantially identical to parental 2G8 antibody H-CDR2 and H-CDR3, respectively, or an antigen-binding fragment thereof.

In yet another aspect, the invention concerns a tetravalent multi-specific antibody comprising two heavy and light chain pairs having binding affinity to a fungal cell wall polysaccharide, and a binding domain having binding affinity to a T-cell antigen, such as the CD3 complex, e.g., CD3s, linked to the C-terminus of each light chain of the anti -fungal cell wall polysaccharide antibody through a linker, or an antigen-binding fragment thereof. The T- cell binding domain preferably is a single-chain antibody (scFv) fragment binding to the CD3 complex, e.g., CD3s.

In one embodiment, the fungal cell wall polysacchaccharide is a glucan, a chitin, or a mannan.

In another embodiment, the fungal cell wall polysaccharide is a glucan.

In yet another embodiment, the fungal cell wall polysaccharide is a β-glucan.

In a further embodiment, the β-glucan is a β- 1,3 -glucan or a β-Ι,ό-glucan, preferably a β- 1,3-glucan.

In one embodiment, the antigen-binding fragment of the multi-specific antibody is selected from the group consisting of a single-domain antibody, Fab, Fab', F(ab')₂, scFv, and (scFv)₂ fragments.

In another embodiment, the multi-specific antibody (including tetravalent multi-specific antibodies), or antigen-binding fragment thereof, is human, humanized, or chimeric. In a further aspect, the invention concerns a bispecific binding compound comprising two polypeptide chains each comprising a binding sequence of a fungal polysaccharide binding protein, such as a Dectin (including Dectins-1 and -2). Preferably, the bispecific binding compound comprises two polypeptide chains, each comprising a Dectin extracellular domain sequence fused to an antibody heavy chain constant region sequence and a binding moiety having binding affinity to a T-cell antigen, linked to the C-terminus of each of the polypeptide chains.

In one embodiment, the Dectin is Dectin-1.

In another embodiment, the antibody heavy chain constant region sequence is an Fc region.

In yet another embodiment, the T-cell antigen is a component of the CD3 complex, preferably CD3s.

In a further embodiment, the binding moiety linked to the C-terminus of each of the polypeptide chains is a single-chain Fv (scFv) antibody having binding affinity to CD3s.

In a still further embodiment, the binding moiety is linked to the antibody heavy chain constant region sequence through a polypeptide linker, such as a BGL2 linker (SEQ ID NO: 13).

In a further aspect, the invention concerns a pharmaceutical composition comprising an effective amount of any of the binding compounds or antibodies of the present invention, optionally in admixture with a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.

In yet another aspect, the invention concerns a method for the treatment of a microbial disease or condition, comprising administering to a subject in need an effective amount of any of the binding compounds or antibodies of the present invention.

In one embodiment, the microbial disease or condition is a fungal or bacterial disease or condition

In another embodiment, the microbial disease is a fungal disease or condition.

In yet another embodiment, the fungal disease or condition is a systemic fungal infection. In a still further embodiment, the fungal disease or condition is a fungal infection, preferably a systemic fungal infection, caused by a Candida or Aspergillus species, such as, for example, Candida albicans, C. parapsilosis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. tropicalis, Aspergillus fumigatus, A. flavus, A. terreus, A. niger, A. candidus, A. clavatus, or A. ochraceus.

In another aspect, the invention concerns the use of a binding compound or antibody herein, or a pharmaceutical composition comprising one or more of the binding compounds or antibodies herein, in the preparation of a medicament for the treatment of a microbial disease or condition, such as a fungal disease or condition, e.g., a fungal infection, including, without limitation, fungal infections caused by a Candida or Aspergillus species, such as, for example, Candida albicans, C. parapsilosis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. tropicalis, Aspergillus fumigatus, A. flavus, A. terreus, A. niger, A. candidus, A. clavatus, or A. ochraceus.

In various embodiments, the multi-specific binding compounds of the present invention can be combined with, used or administered in combination with, one or more further anti-fungal agent.

In one embodiment, the further antifungal agent is from the Echinocandin class of antifungal compounds, and may, for example, be selected from the group consisting of caspofungin, echinocandin B, anidulafungin, pneumocandin B₀, aculeacin Α_γ, micafungin, and their derivatives.

In another embodiment, the further antifungal agent is an azole-type antifungal agent, and may, for example, be selected from the group consisting of voriconazole, clotrimazole, ravuconazole, posaconazole, econazole, fluconazole, itraconazole, tebuconazole, propiconazole, enilaconazole, miconazole, oxiconazole, sulconazole, and tioconazole.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a schematic representation of a multi-specific binding molecule having binding specificity to a fungal cell wall polysaccharide and a T-cell. As shown, the bispecific binding molecule is a bispecific antibody in a bispecific T-cell engaging format (referred to herein as a "BiTE" format) comprising a first antigen-binding fragment (2G8 scFv) having binding affinity to a fungal cell wall polysaccharide, P-l,3-glucan and a second antigen-binding fragment (anti- CD3 scFv) having binding affinity to the CD3 receptor complex on T-cells, wherein the first and second antigen-binding fragments are linked to each other through a polypeptide linker.

FIG. 2A is a schematic representation of a Dectin-l-Fc-anti-CD3 construct (in this example, CTP-14). The molecule comprises a Dectin extracellular domain sequence (mDectin-1 extracellular domain or h-Dectin-la extracellular domain) linked to the CH2-CH3 constant regions of an immunoglobulin (e.g., a murine IgG2a immunoglobulin, or a murine IgG2ael immunogloblin, or a human IgGl immunoglobulin) linked, through a polypeptide linker to the scFv sequence of the 2C11 mouse anti-CD3 antibody, or to the scFv sequence of the 500A2 mouse anti-CD3 antibody, or to the scFv sequence of the KT3 mouse anti-CD3 antibody, or to the scFv sequence of the UCHT1 human anti-CD3 antibody.

FIG. 2B is a schematic representation of a Dectin- 1-Fc construct (in this example, CTP- 16). The molecule comprises a Dectin-1 extracellular domain sequence (mDectin-1 extracellular domain or h-Dectin-la extracellular domain) linked to the CH2-CH3 constant regions of a murine IgG2a immunoglobulin or of a murine IgG2ael immunogloblin.

FIG. 3 is a schematic representation of a tetravalent bispecific binding compound having binding affinity to P-l,3-glucan and a T-cell. The illustrated tetravalent structure comprises two heavy and light chain pairs of a mouse anti- P-l,3-glucan antibody, 2G8, and an scFv sequence of the 2C11 mouse anti-CD3 antibody, or to the scFv sequence of the 500A2 mouse anti-CD3 antibody, or to the scFv sequence of the KT3 mouse anti-CD3 antibody, which is linked to the C- terminus of the 2G8 antibody light chain through a polypeptide linker.

FIG. 4A is a schematic illustration of construct CTP-014.

FIG. 4B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 5A is a schematic illustration of construct CTP-052.

FIG. 5B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 6A is a schematic illustration of construct CTP-054.

FIG. 6B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 7A is a schematic illustration of construct CTP-060. FIG. 7B is an image of a gel analysis of this constmct under non-reducing (NR) conditions.

FIG. 8A is a schematic illustration of construct CTP-064.

FIG. 8B is an image of a gel analysis of this construct under non-reducing (NR) conditions.

FIG. 9A is a schematic illustration of construct CTP-067.

FIG. 9B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 10A is a schematic illustration of construct CTP-076.

FIG. 10B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 11A is a schematic illustration of construct CTP-077.

FIG. 11B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 12A is a schematic illustration of construct CTP-029/030.

FIG. 12B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 13A is a schematic illustration of construct CTP-029/057.

FIG. 13B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 14A is a schematic illustration of construct CTP-029/058.

FIG. 14B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 15A is a schematic illustration of construct CTP-054B.

FIG. 15B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIG. 16A is a schematic illustration of construct CTP-054C.

FIG. 16B is an image of a gel analysis of this construct under non-reducing (NR) and reducing (R) conditions.

FIGS. 17A, 17B and 17C are graphs showing laminarin, pustulan and BSA binding

(measured by ELISA) for construct CTP-029/030. FIGS. 18A, 18B and 18C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-052.

FIGS. 19A, 19B and 19C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-054.

FIGS. 20A, 20B and 20C are graphs showing laminarin, pustulan and BSA binding

(measured by ELISA) for construct CTP-054B.

FIGS. 21A, 21B and 21C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-054C.

FIGS. 22A, 22B and 22C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-014.

FIGS. 23 A, 23B and 23C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-016.

FIGS. 24A, 24B and 24C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-067.

FIGS. 25A, 25B and 25C are graphs showing laminarin, pustulan and BSA binding

(measured by ELISA) for construct CTP-076.

FIGS. 26A, 26B and 26C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-077.

FIGS. 27A, 27B and 27C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-029/057.

FIGS. 28A, 28B and 28C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-029/058.

FIGS. 29A, 29B and 29C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-060.

FIGS. 30A, 30B and 30C are graphs showing laminarin, pustulan and BSA binding

(measured by ELISA) for construct CTP-063.

FIGS. 31A, 31B and 31C are graphs showing laminarin, pustulan and BSA binding (measured by ELISA) for construct CTP-064.

FIGS. 32A and 32B are graphs showing flow cytometry data for CD4+ and CD8+ T- cells contacted with Dectin-l-Fc-anti-CD3 (CTP-14), Dectin-l-Fc (CTP-16), or isotype control. FIG. 33 is a collection of graphs showing binding of the indicated constructs to mouse T- cells. Open black histograms represent binding of CD3 -containing constructs to mouse T-cells. Gray, filled histograms represent binding of the isotype (negative) control. CTP-016 has no CD3 binding domain, and also serves as a negative control.

FIG. 34 is a collection of graphs showing binding of the indicated constructs to human T- cells. Open black histograms represent binding of CD3 -containing constructs to human T-cells. Gray, filled histograms represent binding of the isotype (negative) control.

FIG. 35 is a collection of graphs showing binding of the indicated constructs to mouse T- cells. Open black histograms represent binding of CD3 -containing constructs to human T-cells. Gray, filled histograms represent binding of the isotype (negative) control.

FIG. 36 is a collection of graphs showing binding of the indicated constructs to human T- cells. Open black histograms represent binding of CD3 -containing constructs to human T-cells. Gray, filled histograms represent binding of the isotype (negative) control.

FIGS. 37A and 37B are graphs showing flow cytometry data for CD4+ and CD8+ T- cells contacted with a commercially-available anti-CD3 antibody (open black histograms), or isotype (negative) control (filled gray histograms).

FIGS. 38A and 38B are graphs showing induction of IFN-gamma production in mouse splenocytes by various constructs and controls.

FIG. 39 is a collection of bright field and fluorescent microscopy images showing Dectin-l-Fc-anti-CD3 (CTP-14) and isotype control binding to A. fumigatus.

FIG. 40 is a collection of bright field and fluorescent microscopy images showing Dectin-l-Fc (CTP-16) and isotype control binding to A. fumigatus.

FIG. 41 is a collection of bright field and fluorescent microscopy images showing Dectin-l-Fc (CTP-16) and isotype control binding to caspofungin-treated A. fumigatus.

FIG. 42 is a collection of fluorescent microscopy images showing Dectin-l-Fc (CTP-16) binding to A. fumigatus in the presence of varying levels of fetal bovine serum (FBS).

FIG. 43 is a collection of fluorescent microscopy images showing binding of construct CTP-016 to a subset of resting A. fumigatus conidia.

FIG. 44 is a collection of fluorescent microscopy images showing binding of construct CTP-029/030 to a subset of resting A. fumigatus conidia. FIG. 45 is a collection of fluorescent microscopy images showing increased binding of construct CTP-016 to swollen A. fumigatus conidia.

FIG. 46 is a collection of fluorescent microscopy images showing increased binding of construct CTP-029/030 to swollen A. fumigatus conidia.

FIGS. 47-52 provide a collection of fluorescent microscopy images showing binding of the indicated constructs to fungal hyphae.

FIG. 53 is a collection of graphs showing inhibition of A. fumigatus germination by a human immune cell preparation. Black bars represent inhibition mediated by the indicated molecule at 10, 1 or 0.1 uM. White bars represent inhibition by human immune cells alone, and open triangles wells contain fungus alone.

FIG. 54 is a collection of graphs showing inhibition of A. fumigatus germination by a human immune cell preparation, or by a T-cell-depleted human immune cell preparation in the presence or absence of human Dectin-1 -based constructs. Black bars represent inhibition mediated by mock-depleted immune cells in the presence of Dectin-1 -based constructs, gray bars inhibition mediated by CD3 (T-cell)-depleted immune cells in the presence of Dectin-1 -based constructs, open bars with open circles inhibition mediated by mock-depleted immune cells in the absence of Dectin-1 -based constructs, open bars with open squares inhibition mediated by CD3 (T-cells)-depleted immune cells in the absence of Dectin-1 -based constructs and open triangles wells that contain fungus alone.

FIG. 55 is a table showing % human immune cells and % T-cells for a mock-depleted immune cell preparation and a CD3 -depleted immune cell preparation. CD3 depletion resulted in a reduction in the percentage of T-cells in the immune cell preparation from 1.27% to 0.027%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns multi-specific binding molecules that target and kill microbial pathogens, such as fungal pathogens, via recruitment and activation of immune cells of the host. In particular, the invention concerns bispecific binding molecules, such as bispecific antibodies, which bind to a fungal cell wall polysaccharide and an immune cell (e.g., a T-cell). Without being bound by any theory, such binding molecules exhibit superior antimicrobial activity, combining the effect of direct inhibition of microbial growth by binding to the microbial target, such as a fungus, and the killing mechanism resulting from specific recruitment of the host's immune cells (e.g., the host's T-cells).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the inventions described herein belong. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.

The term "antibody" is used herein in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, and any fragment, variant or derivative thereof so long as they exhibit the desired biological activity (e.g., epitope binding activity, or affinity to a desired target). See, e.g., Miller et al. Jour, of Immunology 170:4854-4861 (2003). Examples of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, antibody analogs, and antibody fragments, and antibody constructs, specifically including, without limitation multi-specific, e.g. bispecific antibodies, such as bispecific T-cell engaging (BiTE) antibody constructs; multi-valent, e.g. tetravalent antibodies, and antigen-bindig (target-binding) fragments thereof. Antibodies may be murine, human, humanized, chimeric, or derived from other species.

Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1- 113 of the heavy chain) {e.g., Kabat et al, Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system " or "EU index " is generally used when referring to a residue in an immunoglobulin heavy chain constant region {e.g., the EU index reported in Kabat et al, supra). The "EU index as in Kabat " refers to the residue numbering of the human IgGl EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies mean residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies mean residue numbering by the EU numbering system.

The term "multi-specific antibody" is used herein in the broadest sense and specifically covers an antibody that has polyepitopic specificity. Multi specific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VH-VL unit has polyepitopic specificity, antibodies having two or more VL and VH domains where each VH-VL unit binds to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, full length antibodies, and antibodies comprising one or more antibody fragments as well as antibodies comprising antibody fragments that have been linked covalently or non-covalently. The term specifically includes multivalent, e.g., tetravalent antibodies and antibody fragments. According to one embodiment the multispecific antibody is a bispecific IgG antibody that binds to each binding target (target antigen) with an affinity of 5 μΜ to 0.001 pM, 3 μ M to 0.001 pM, 1 μΜ to 0.001 pM, 0.5 μΜ to 0.001 pM, or 0.1 μΜ to 0.001 pM. "Multi-specific " binding compounds and "multi-specific binding molecules, " which are used interchangeably, are defined in an analogous manner.

The term "multi-specific " specifically includes "bispecific " and "tri specific, " including, without limitation, bispecific T-cell engaging antibodies (BiTE antibodies). The term "multi- specific " also includes higher-order independent specific binding affinities, such as higher-order polyeptopic specificity, as well as tetravalent antibodies.

"Antibody fragments" comprise only a portion of an intact antibody, where the portion retains at least one, and may retain most or all, of the functions normally associated with that portion when present in an intact antibody. An antibody fragment of the invention may comprise a sufficient portion of the constant region to permit dimerization (or multimerization) of heavy chains that have reduced disulfide linkage capability, for example where at least one of the hinge cysteines normally involved in inter-heavy chain disulfide linkage is altered as described herein. In one embodiment, an antibody fragment comprises an antigen binding site or variable domains of the intact antibody and thus retains the ability to bind antigen (i.e., is an antigen-binding fragment). In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function, and/or complement binding (for example, where the antibody has a glycosylation profile necessary for ADCC function or complement binding). Examples of antibody fragments include, without limitation, linear antibodies; single-chain antibody molecules (scFvs); and multispecific antibodies formed from antibody fragments, and any and all of the antigen-binding fragments listed below.

The term "antigen-binding fragment ", as used herein, refers to one or more fragments of an antibody that retain the ability to bind to a target antigen. Examples of antigen-binding fragments include, without limitation, Fab, Fab', F(ab')₂, Fv, diabodies (Db); tandem diabodies (taDb), linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al, Protein Eng. 8(10): 1057- 1062, 1995); one-armed antibodies, single variable domain antibodies, minibodies (Olafsen et al., Protein Eng. Design & Sel. 17(4):315-323, 2004), single-chain antibody molecules (scFvs), fragments produced by a Fab expression library, anti -idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments.

The term "Fab " refers to an antibody fragment that consists of an entire L chain (V_L and C_L) along with the variable region domain of the H chain (V_H), and the first constant domain of one heavy chain (CHI). Papain digestion of an intact antibody can be used to produce two Fab fragments, each of which contains a single antigen-binding site. Typically, the L chain and H chain fragment of the Fab produced by papain digestion are linked by an interchain disulfide bond.

The term "Fc " refers to an antibody fragment that comprises the carboxy-terminal portions of both H chains (CH2 and CH3) and a portion of the hinge region held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region; this region is also the part recognized by Fc receptors (FcR) found on certain types of cells. One Fc fragment can be obtained by papain digestion of an intact antibody.

The term "F(ab') ₂ " refers to an antibody fragment produced by pepsin digestion of an intact antibody. F(ab')₂ fragments contain two Fab fragments and a portion of the hinge region held together by disulfide bonds. F(ab')₂ fragments have divalent antigen-binding activity and are capable of cross-linking antigen.

The term Fab' refers to an antibody fragment that is the product of reduction of an F(ab')₂ fragment. Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.

The term "hinge region " refers to the portion of an antibody stretching from Glu216 to Pro230 of human IgGl (Burton, Molec. Immunol. 22: 161-206, 1985). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain S--S bonds in the same positions.

The term "Fv " refers to an antibody fragment that consists of a dimer of one heavy-chain variable region and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although often at a lower affinity than the entire binding site.

The term "Single-chain Fv " also abbreviated as "sFv " or "scFv" refer to antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); and Malmborg et al, J. Immunol. Methods 183 :7-13, 1995.

The term "diabodies " refers to small antibody fragments prepared by constructing scFv fragments with short linkers (typically about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Exemplary diabodies are described in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444- 6448 (1993).

The term "one-armed antibody " refers to an antibody that comprises (1) a variable domain joined by a peptide bond to a polypeptide comprising a CH2 domain, a CH3 domain or a CH2-CH3 domain and (2) a second CH2, CH3 or CH2-CH3 domain lacking a variable domain. One-armed antibodies may comprise 3 polypeptides (1) a first polypeptide comprising a variable domain (e.g., VH), CHI, CH2 and CH3, (2) a second polypeptide comprising a variable domain (e.g., VL) and a CL domain, and (3) a third polypeptide comprising a CH2 and CH3 domain. One-armed antibodies may have a partial hinge region containing the two cysteine residues which form disulphide bonds linking the constant heavy chains. Typically, the variable domains of the one armed antibody form an antigen binding region. In certain instances, the variable domains of the one armed antibody are single variable domains, wherein each single variable domain is an antigen binding region.

The term "single domain antibodies " (sdAbs) or "single variable domain (SVD) antibodies " refers to antibodies in which a single variable domain (VH or VL) confers antigen binding. In other words, the single variable domain does not need to interact with another variable domain to recognize and bind the target antigen. Examples of single domain antibodies include those derived from camelids (lamas and camels) and cartilaginous fish (e.g., nurse sharks) and those derived from recombinant methods from humans and mouse antibodies (Nature (1989) 341 :544-546; Dcv Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21 :484-490; WO 2005/035572; WO 03/035694; Febs Lett (1994) 339:285-290; WO00/29004; WO 02/051870).

The term "linear antibodies " refers to the antibodies described in Zapata et al, Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

The term "monoclonal antibody " refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal " indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see for example: U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,807,715). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al., J. Mol. Biol, 222:581-597, 1991.

An "intact antibody " refers to an antibody comprising VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains {e.g., human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more "effector functions " which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor.

The term "native antibody " refers to a naturally occurring basic four-chain antibody unit that is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called J chain, and therefore contains 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has, at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the alpha and gamma chains and four CH domains for mu and epsilon isotypes. Each L chain has, at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

The term "variable " refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term "hypervariable region, " "HVR, " or "HV, " refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al, Immunity 13 :37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al, Nature 363 :446-448 (1993); Sheriff et al, Nature Struct. Biol. 3 :733-736 (1996). "Framework regions " (FR) are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FRl, FR2, FR3, and FR4. If the CDRs are defined according to Kabat, the light chain FR residues are positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are positioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the CDRs comprise amino acid residues from hypervariable loops, the light chain FR residues are positioned about at residues 1- 25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain and the heavy chain FR residues are positioned about at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain residues. In some instances, when the CDR comprises amino acids from both a CDR as defined by Kabat and those of a hypervariable loop, the FR residues will be adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, the heavy chain FRl residues are at positions 1-25 and the FR2 residues are at positions 36-49.

A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat. In certain instances, for the VL, the subgroup is subgroup kappa I as in Kabat. In certain instances, for the VH, the subgroup is subgroup III as in Kabat.

"Chimeric antibodies " refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies include primatized antibodies comprising variable domain antigen-binding sequences derived from a non-human primate and human constant region sequences.

"Humanized " forms of non-human {e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term "BiTE" is used herein to refer to Bispecific T-cell engaging monoclonal antibodies. BiTEs are fusion proteins consisting of two single-chain variable region fragments (scFvs) on a single peptide chain, wherein one of the scFvs binds to the CD3 receptor on T-cells, while the other to a different target antigen.

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc constant region of an antibody. Typically, FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc. gamma. RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITEVI) in its cytoplasmic domain. (See review M. in Daeron, Annu Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu Rev. Immunol., 9:457-92 (1991); Capel et al (1994) Immunomethods 4:25-34; and de Haas et al (1995) J. Lab. Clin. Med. 126:330-41. Other FcRs are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al (1976) J. Immunol, 117:587 and Kim et al (1994) J Immunol. 24:249).

As used herein, the term "immunoadhesin " designates antibody-like molecules that combine the "binding domain " of a heterologous "adhesin " protein (for example, a receptor, ligand, or enzyme) with the effector functions of an immunoglobulin constant domain. Structurally, the immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity that is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e. is "heterologous ") and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence in the immunoadhesin is preferably derived from γΐ, γ2, or γ4 heavy chains, since immunoadhesins comprising these regions can be purified by Protein A chromatography. See, for example, Lindmark et al, 1983, J. Immunol. Meth. 62: 1-13.

"Complement dependent cytotoxicity" or "CDC " refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule {e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al J. Immunol. Methods, 202: 163 (1996), may be performed.

"Binding affinity " refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule {e.g., an antibody) and its binding partner {e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity " refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair {e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). For example, the Kd can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or stronger. Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain.

As used herein, the "Kd " or "Kd value " refers to a dissociation constant measured by using surface plasm on resonance assays, for example, using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 °C. with immobilized antigen CM5 chips at .about.10 response units (RU). For further details see, e.g., Chen et al, J. Mol. Biol. 293 :865-881 (1999).

An "epitope " is the site on the surface of an antigen molecule to which a single antibody molecule binds. Generally an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

An antibody binds "essentially the same epitope " as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody. Usually, the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.

"Epitope mapping" is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure.

"Epitope binning ", as defined herein, is the process of grouping antibodies based on the epitopes they recognize. More particularly, epitope binning comprises methods and systems for discriminating the epitope recognition properties of different antibodies, combined with computational processes for clustering antibodies based on their epitope recognition properties and identifying antibodies having distinct binding specificities.

The terms "treat" or "treatment " refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival, as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

Administration "in combination with " one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

The term "bioavailability, " as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increases in bioavailability refer to increasing the rate and extent a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. By way of example, an increase in bioavailability may be indicated as an increase in concentration of the substance or its active moiety in the blood when compared to other substances or active moieties. A non-limiting example of a method to evaluate increases in bioavailability is given in examples 21-25. This method may be used for evaluating the bioavailability of any polypeptide.

By "modulating biological activity" is meant increasing or decreasing the reactivity of an antibody, antibody fragment, or polypeptide, altering the selectivity of the antibody, antibody fragment, or polypeptide, enhancing or decreasing the substrate selectivity.

The term "identical, " as used herein, refers to two or more sequences or subsequences that are the same. In addition, the term "substantially identical, " as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using comparison algorithms or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical " if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages to describe the "percent identity " of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are the same, while two or more polypeptide sequences are "substantially identical " if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 amino acids in length, over a region that is about 50 amino acids in length, or, where not specified, across the entire sequence of a polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are the same, while two or more polynucleotide sequences are "substantially identical " if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, where not specified, across the entire sequence of a polynucleotide sequence.

The term "pharmaceutically acceptable ", as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term "polymer, " as used herein, refers to a molecule composed of repeated subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides, or polysaccharides or polyalkylene glycols.

The term "polysaccharide, " is used herein in the broadest sense including, without limitation, saccharides comprising a plurality of repeating units. Polysaccharide structures may be linear or branched, a characteristic that is observed when a monosaccharide constituent of a polysaccharide is involved in more than two glycosidic bonds. Polysaccharides can be classified as homopolymers, indicating that the polymer composed of identical monosaccharides, or heteropolymers, a term used for classification of polysaccharides composed of two or more types of monosaccharides. The polysaccharides can be from any source, for example, they can be derived from naturally-occurring fungi or yeast cells, genetically engineered bacteria, or can be produced synthetically.

The term "fungal polysaccharide," as used herein, encompasses all polysaccharides present in fungi, whether in the cell wall or otherwise, including, without limitation, polysaccharides present in the following species: pathogenic and/or opportunistic fungi, such as, for example, fungal infections (or mycoses) involving, Candida species, such as C. albicans, C. krusei, C. glabrata; Aspergillus species, such as Aspergillus fumigatus, Cryptococcus species, such as Cryptococcus neoformans, Pneumocystis species, such as Pneumocystis carinii, Penicillium species, such as Penicillium marneffei; Histoplasma capsulatum, and Coccidioides immitis; pathogens of Epidermophyton spp., Exophiala spp., Microsporum spp., Trichophyton spp. (e.g., T. rubrum and T. inter digitale), Tinea spp., Blastomyces spp., Blastoschizomyces spp., Coccidioides spp., Histoplasma spp., Paracoccidiomyces spp., Sporotrix spp., Absidia spp., Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia spp., Arthrogr aphis spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveria spp., Chrysosporium spp., Conidiobolus spp., Cunninghamella spp., Fusarium spp., Geotrichum spp., Graphium spp., Leptosphaeria spp., Malassezia spp., Mucor spp., Neotestudina spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Phoma spp., Piedraia spp., Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsisspp., Sporobolomyces spp., Syncephalastrum spp., Trichoderma spp., Trichosporon spp., Ulocladium spp., Ustilago spp., Verticillium spp. or, Wangiella spp., and the like.

The term "fungal cell wall polysaccharide" includes polysaccharide components of fungal cell walls, including glucans, glycogen-like compounds, mannans (polymers consisting of repeating units of, respectively, glucose, N-acetylglucosamine, and mannose), chitosan (glucosamine polymers), galactans (galactose polymers), fucose, rhamnose, xylose, and uronic acids. The term specifically includes, without limitation, glucans, mannans and chitosans, preferably glucans, such as a P-l,3-glucan or a β-Ι,ό-glucan.

As used herein, the term "glucan ", refers to a polysaccharide containing glucose monomers such as cellulose. The term is used in reference to a large group of D-glucose polymers with glycosidic bonds. Beta-glucan is a naturally occurring class of glucans that can be extracted from fungi, Baker's yeast, other yeast species, mushrooms, plants and some bacterial, lichen and algal species (reviewed in Chemistry and Biology of (1→3)-P-Glucans, B. A. Stone and A. E. Clarke, 1992, La Trobe University Press, Australia).

As used herein, the term "fungal polysaccharide binding protein" refers to polypeptides with binding affinity to a fungal polysaccharide, such as a fungal cell wall polysaccharide. The term specifically includes immunoadhesins such as C-type lectins having binding affinity to a fungal cell wall polysaccharide, such as Dectin (including Dectins-1 and -2). The term also includes, without limitation, antibodies and antibody fragments.

As used herein, the term "water soluble polymer" refers to any polymer that is soluble in aqueous solvents. Such water soluble polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, mono Ci-Cio alkoxy or aryloxy derivatives thereof (described in U.S. Patent No. 5,252,714 which is incorporated by reference herein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-DL- aspartamide, and the like, or mixtures thereof. By way of example only, coupling of such water soluble polymers may result in changes including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, modulated biological activity, extended circulation time, modulated immunogenicity, modulated physical association characteristics including, but not limited to, aggregation and multimer formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. In addition, such water soluble polymers may or may not have their own biological activity.

The term "polyalkylene glycol, " as used herein, refers to linear or branched polymeric polyether polyols. Such polyalkylene glycols, including, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications " (2001). By way of example only, such polymeric polyether polyols have average molecular weights between about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some embodiments molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 2,000 to about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched chain PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da. In other embodiments, the molecular weight of the branched chain PEG is between about 2,000 to about 50,000 Da. The term "antimicrobial " is used herein to encompass any compound exhibiting antifungal, antibacterial and/or antiviral activities. In a preferred embodiment, the antimicrobial activity is antifungal activity.

The terms "fungal infection, " "fungal disease, " and "fungal condition " are used interchangeably and include any disease or condition the symptoms of which are caused or contributed to, by a fungus. Fungal infections, and particularly those referred to as systemic fungal infections or systemic mycoses, may be caused by pathogenic and/or opportunistic fungi, such as, for example, fungal infections (or mycoses) involving, Candida species, such as C. albicans, C. krusei, C. glabrata; Aspergillus species, such as Aspergillus fumigatus, Cryptococcus species, such as Cryptococcus neoformans, Pneumocystis species, such as Pneumocystis carinii, Penicillium species, such as Penicillium marneffei; Histoplasma capsulatum, and Coccidioides immitis. The fungal pathogen may be further derived from other fungal pathogens, including pathoges of Epidermophyton spp., Exophiala spp., Microsporum spp., Trichophyton spp. (e.g., T. rubrum and T. inter digitale), Tinea spp., Blastomyces spp., Blastoschizomyces spp., Coccidioides spp., Histoplasma spp., Paracoccidiomyces spp., Sporotrix spp., Absidia spp., Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia spp., Arthrographis spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveria spp., Chrysosporium spp., Conidiobolus spp., Cunninghamella spp., Fusarium spp., Geotrichum spp., Graphium spp., Leptosphaeria spp., Malassezia spp., Mucor spp., Neotestudina spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Phoma spp., Piedraia spp., Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsisspp., Sporobolomyces spp., Syncephalastrum spp., Trichoderma spp., Trichosporon spp., Ulocladium spp., Ustilago spp., Verticillium spp. or, Wangiella spp. Specifically included within the term "fungal infection " infections selected from tinea capitis, tinea corporis, tinea pedis, onychomycosis, perionychomycosis, pityriasis versicolor, oral thrush, vaginal candidosis, respiratory tract candidosis, biliary candidosis, eosophageal candidosis, urinary tract candidosis, systemic candidosis, mucocutaneous candidosis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis, fungal sinusitis, or chronic sinusitis. For example, the infection being treated can be an infection by Candida albicans, C. parapsilosis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. tropicalis, Aspergillus fumigatus, A. flavus, A. terreus, A. niger, A. candidus, A. carneus, A. deflectus, A. fischeri, A. flavipes, A. glaucus, A. nidulans, A. oryzae, Rhizopus oryzae, A. clavatus, or A. ochraceus.

The term "recombinant host cell, " also referred to as "host cell, " refers to a cell which includes an exogenous polynucleotide, wherein the methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. By way of example only, such exogenous polynucleotide may be a nonintegrated vector, including but not limited to a plasmid, or may be integrated into the host genome.

The term "redox-active agent, " as used herein, refers to a molecule which oxidizes or reduces another molecule, whereby the redox active agent becomes reduced or oxidized.

Examples of redox active agent include, but are not limited to, ferrocene, quinones, Ru^2+/3+ complexes, Co^2+/3+ complexes, and Os^2+/3+ complexes.

The term "subject, " or "patient", as used herein, refers to a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals (such as cows), sport animals, and pets (such as cats, dogs, and horses). In certain embodiments, a mammal is a human.

The term "therapeutically effective amount, " as used herein, refers to the amount of a composition containing a multi-specific molecule, e.g., antibody of the present invention administered to a subject, e.g., a human patient, already suffering from a microbial, e.g., fungal disease, condition or disorder, sufficient to cure or at least partially arrest, or relieve to some extent one or more of the symptoms of the disease, disorder or condition being treated. The effectiveness of such compositions depend conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.

Modes o f Carrying Out the Invention

As discussed above, in one aspect the present invention is directed to multi-specific binding molecules having binding specificity to a fungal cell wall polysaccharide and an immune cell (e.g., a T-cell). In one preferred embodiment, the binding molecule is bispecific. In another preferred embodiment, the bispecific binding molecule is a bispecific antibody which binds to the fungal cell wall glycoprotein, P-l,3-glucan and the CD3 receptor complex on T-cells. The particular format of such bispecific antibody is not narrowly critical and can include, for example and without limitation, a BiTE format (FIG. 1) or a tetravalent format (FIG. 3). In another preferred embodiment, the bispecific binding molecule comprises a Dectin-1 extracellular domain (ECD) sequence fused to an immunoglobulin (Ig) sequence (also referred to as an "immunoadhesin "), which provides binding specificity to the fungal cell wall, and a T-cell binding region, e.g., a CD3 binding region, of an antibody (FIG. 2A).

Antibodies

Many techniques for the production of antibodies have been described which include the traditional hybridoma method for making monoclonal antibodies, recombinant techniques for making antibodies (including chimeric antibodies, e.g., humanized antibodies), antibody production in transgenic animals and phage display technology for preparing "fully human " antibodies.

Monoclonal antibodies may be obtained from a population of substantially homogeneous antibodies using the hybridoma method first described by Kohler and Milstein, Nature 256:495 (1975) or may be made by recombinant DNA methods (Cabilly et al, U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as hamster, is immunized as hereinabove described to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59 103 (Academic Press, 1986)). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133 :3001 (1984); and Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51 63, Marcel Dekker, Inc., New York, 1987). See, also, Boerner et al, J. Immunol, 147(1):86 95 (1991) and WO 91/17769, published Nov. 28, 1991, for techniques for the production of human monoclonal antibodies. Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen of interest. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. 107:220 (1980). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Goding, Monoclonal Antibodies: Principles and Practice, pp.59 104 (Academic Press, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Alternatively, it is possible to produce transgenic animals {e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J_H) gene in chimeric and germ- line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA 90:2551 255 (1993); Jakobovits et al., Nature 362:255 258 (1993); Fishwild, D. M., et al. (1996) Nat. Biotech 14:845 851; and Mendez, M. J., et al. (1997) Nat. Genetics 15: 146 156).

In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552 554 (1990), using the antigen of interest to select for a suitable antibody or antibody fragment. Clackson et al, Nature, 352:624 628 (1991) and Marks et al, J. Mol. Biol, 222:581 597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Mark et al, Bio/Technol. 10:779 783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al, Nuc. Acids Res., 21 :2265 2266 (1993); Griffiths, A. D., et al. (1994) EMBO J. 13 :3245 3260; and Vaughan, et al. (1996) supra).

DNA encoding the antibodies of the invention is readily isolated and sequenced using conventional procedures {e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al, Proc. Nat. Acad. Sci. 81 :6851 (1984). In that manner, "chimeric " antibodies are prepared that have the binding specificity of an anti-antigen monoclonal antibody herein.

Monoclonal antibodies with binding affinity to the fungal cell wall glycoprotein, β-1,3- glucan are known in the art. See, for example, Rachini et al, Infection and Immunity 2007, 75(l l):50-85-5094; Torosantucci et al, PLoS ONE 4(4): e5392 doi: 10.1371/journal.pone.0005392; and U.S. Patent No. 8,414,889 for the disclosure of MAb 2G8. Monoclonal antibodies with binding affinity to T-cell antigens, such as the CD3 complex are also known, and are commercially available. See, for example mAbs 2C11 (Abeam, Pierce), 500A2 (Pierce), and KT3 (Abeam, Serotec). Multi-specific Antibodies

Multi specific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein. A schematic respresentation of a tetravalent antibody if provided in FIG. 3

Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al, 1983, Nature, 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, 1991, EMBO J., 10:3655-3659.

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion can be with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three antibody fragments in embodiments when unequal ratios of the three antibody chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three antibody chains in one expression vector when the expression of at least two antibody chains in equal ratios results in high yields or when the ratios are of no particular significance.

In another embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile method of separation. This approach is disclosed in WO 94/04690. For further details of methods for generating multi-specific, e.g., bispecific antibodies, see, for example, Segal, David M., and Bert JEG Bast. "Production of bispecific antibodies. " Current protocols in immunology (1995): 2-13. Trispecific antibodies can be prepared, e.g., according to Tutt et al, 1991, J. Immunol. 147: 60.

Exemplary bispecific antibodies of the present invention include single chain antibody molecules, comprising a first binding domain for beta-l,3-glucan (B13G) linked through a linker (L) to a second binding domain for human CD3 (CD3), each of the first and second binding domains comprising a heavy chain variable region (VH) and a light chain variable region (VL), the corresponding VH and VL regions being arranged, from N-terminus to C- terminus, in an order selected from

VH(B 13 G)-VL(B 13G)-L-VH(CD3)-VL(CD3),

VH(CD3)-VL(CD3)-L-VL(B 13G)-VH(B 13 G)

VH(CD3)-VL(CD3)-L-VH(B 13G)-VL(B 13 G) or

VL(CD3)-VH(CD3)-L-VH(B 13G)-VL(B 13 G).

VH(CD3)-VL(CD3)-L-VH(B 13G)-VL(B 13 G)

VH(CD3)-VL(CD3)-L-VL(B 13G)-VH(B 13 G)

VL(CD3)-VH(CD3)-L-VH(B 13G)-VL(B 13 G)

VL(CD3)-VH(CD3)-L-VL(B 13 G)-VH(B 13 G

In general, "L " can be any linking moiety covalently bonded to each of a first binding domain for B13G and a second binding domain for an antigen on an immune cell (e.g., human CD3 on a T-cell). Suitable linkers include cleavable and non-cleavable linkers, i.e., linkers susceptible or resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and/or disulfide bond cleavage, under conditions to which the molecule is subjected. In a preferred embodiment, the linker is a short, flexible peptide selected to assure that the proper three-dimensional folding of the (VL) and (VH) domains occurs once they are linked so as to maintain binding-specificities to the target antigens. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker. Other suitable linkers include linkers hydrolyzable at a pH of less than 5.5, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers. Suitable non-peptide linkers include, for example, N-succinimidyl 3-(2- pyridyldithio)propionate (SPDP) (see, e.g., Carlsson et al, Biochem. J., 173, 723-737 (1978)), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No. 4,563,304), N- succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see, e.g., CAS Registry number 341498-08- 6), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) (see, e.g., Yoshitake et al, Eur. J. Biochem., 101, 395-399 (1979)), and N-succinimidyl 4-methyl-4-[2-(5- nitro-pyridyl)-dithio]pentanoate (SMNP) (see, e.g., U.S. Pat. No. 4,563,304).

For the purposes of the present invention, peptide linkers are preferred. A peptide linker preferably is about 2 to about 50 residues, preferably about 4 to about 40 residues, more preferably about 5 to about 30 residues in length. Preferred peptide linkers include, without limitation, (G4S)n linkers, (where n = 1-9), preferably (G4S)3, and a BGL1 linker (SEQ ID NO: 16) and BGL2 linker (SEQ ID NO: 13), as disclosed herein.

In a particular embodiment, the bispecific antibody has the following structure:

VH(CD3) - (G4S)3 linker - VL(CD3) - Tandem linker - VH(2G8) - (G4S)3 linker -

VL(2G8).

VH(CD3) and VL(CD3) are the heavy chain and light chain sequences of the CD3 receptor complex -binding scFv antbody fragment. In some embodiments, the position of the VH and VL domains, proceeding in the direction from N-terminus to C-terminus along a peptide, can be freely interchanged without impacting the functional characteristics of the binding molecule. For example, in a bispecific antibody construct having the structure: VH(CD3) - (G4S)3 linker - VL(CD3) - Tandem linker - VH(2G8) - (G4S)3 linker - VL(2G8), the position of the VH(CD3) and VL(CD3) domains, and/or the VH(2G8) and VL(2G8) domains, can be reversed without impacting the ability of the molecule to bind CD3 or β-glucan, respecitively. As discussed above, anti-CD3 antibodies are known in the art and are commercially available. The bispecific antibodies of the present invention may comprise the antigen-binding region, such as the heavy and light chain variable region sequences or Complementarity Determining Regions (CDRs), of any known anti-CD3 antibody, such as KT3 (US 7,635,472 B2; Micromet); 2C11 (Pierce); 500A2 (Life Technologies); or OKT3 (Pierce), or variants of such variable region sequences.

In one embodiment, the bispecific antibody has the following structure:

2G8(VL-linker(3)-VH)-Tandem linker-2Cl l(VH-linker(3)-VL)-His tag (CTP-060, SEQ

ID NO: 50). In some embodiments, a bispecific antibody comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 50.

2G8 VL and VH are the heavy chain and light chain sequences of antibody 2G8, which binds to the fungal cell wall glycoprotein, P-l,3-glucan. 2C11 VH and VL are the heavy chain and light chain sequences of the CD3 receptor complex-binding scFv antibody fragment.

In one embodiment, the bispecific antibody has the following structure:

UCHTl(VL-linker(3)-VH) - Tandem linker-029/030 scFv (VL-linker(3)-VH) - His tag (CTP-063, SEQ ID NO: 51). In some embodiments, a bispecific antibody comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%), or 100%) identical to the peptide sequence provided in SEQ ID NO: 51.

UCHT1 VL and VH are the heavy chain and light chain sequences of the CD3 receptor complex-binding scFv antibody fragment. 029/030 are the VH and VL of a murine antibody that binds to the fungal cell wall glycoprotein, P-l,3-glucan.

In one embodiment, the bispecific antibody has the following structure:

UCHTl(VL-linker(3)-VH) - Tandem linker-2G8 scFv (VL-linker(3)-VH) - His tag (CTP-064, SEQ ID NO: 52). In some embodiments, a bispecific antibody comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 52.

UCHT1 VL and VH are the heavy chain and light chain sequences of the CD3 receptor complex-binding scFv antibody fragment. 2G8 VL and VH are the heavy chain and light chain sequences of antibody 2G8, which binds to the fungal cell wall glycoprotein, P-l,3-glucan.

In one embodiment, the bispecific antibody is composed of two separate peptide chains, and has the following structure:

Chain 1 :VH (IgGl) (CTP-029, SEQ ID NO: 53); Chain 2: CTP-030-VL(IgGl) - linker(T) - 2C11 (VL-linker(3)-VH) (CTP-057, SEQ ID NO: 54).

In some embodiments, a bispecific antibody comprises a peptide chain having a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%), or 100% identical to peptide sequence provided in SEQ ID NO: 53, and a peptide chain having a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 54.

VH (IgGl) is a heavy chain of a murine P-l,3-glucan binding antibody; CTP-030- VL(IgGl) is a light chain region of a murine P-l,3-glucan binding antibody, and 2C11 VH and VL are the heavy chain and light chain sequences of the CD3 receptor complex -binding scFv murine antibody fragment.

In one embodiment, the bispecific antibody is composed of two separate peptide chains, and has the following structure:

Chain 1 :VH (IgGl) (CTP-029, SEQ ID NO: 53);

Chain 2: CTP-030-VL(IgGl) - linker(T) - UCHT1 (VL-linker(3)-VH) (CTP-058, SEQ

ID NO: 55). In some embodiments, a bispecific antibody comprises a peptide chain having a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 53, and a peptide chain having a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to peptide sequence provided in SEQ ID NO: 55.

VH (IgGl) is a heavy chain of a murine P-l,3-glucan binding antibody; CTP-030- VL(IgGl) is a light chain region of a murine P-l,3-glucan binding antibody, and UCHT1 VL and VH are the heavy chain and light chain sequences of the CD3 receptor complex-binding scFv human antibody fragment.

(G4S)3 is a flexible linker to join the VH and VL polypeptides, and may, for example, have the following sequence:

GGGGSGGGGSGGGGS (SEQ ID NO: 15).

Linker(T) is a flexible linker to join the VH and VL polypeptides, and may, for example, have the following sequence:

GTGGGGSGGGGS (SEQ ID NO: 14). The "Tandem linker" is a flexible linker used to fuse the anti-CD3 antibody and anti-β- 1,3 glucan antibody fragments together, and may, for example, have the following sequence:

ASTKGPSVFPLAPSSSGSG (SEQ ID NO: 16).

VH(2G8) and VL(2G8) represent the heavy and light chain sequences of the β-1,3- glucan-binding scFv (SEQ ID NOS: 1 and 2, respectively).

Optionally, the construct may comprise a periplasmic leader sequence for E, coli expression, to transport the recombinant protein into the periplasmic space, to facilitate correct protein folding. An exemplary periplasmic leader peptide has the sequence: MKKNIFLL ASMF VF SI ATNAYA (SEQ ID NO: 65). Other leader sequences, such as pelB, may also be used. The leader sequence is cleaved after export to the periplasm and is not present in the final bispecific antibody molecule. In some embodiments, a can be expressed in and purified from a eukaryotic host cell. In such embodiments, a leader peptide sequence can be used to facilitate extracellular transport of a recombinantly expressed protein. Exemplary eukaryotic leader sequences include, but are not limited to: interleukin-2 (IL-2) signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 66) and Human albumin signal peptide: MKW VTFISLLFLF S SAYS (SEQ ID NO: 67). In certain embodimets, a leader sequence is cleaved from, and is therefore absent from, the mature polypeptide.

The constructs may also contain an affinity tag to facilitate purification. Humanization

A humanized antibody has one or more amino acid residues from a source that is non- human. The non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. Humanization can be performed generally following the method of Winter and co-workers (Jones et al, 1986, Nature, 321 :522-525; Riechmann et al, 1988, Nature, 332:323-327; Verhoeyen et al, 1988, Science, 239: 1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized " antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in non-human, for example, rodent antibodies. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best- fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., 1987, J. Immunol., 151 :2296; Chothia et al, 1987, J. Mol. Biol., 196:901). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al, 1992, Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al., 1993, J. Immunol, 151 :2623).

It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

Immunoadhesins

Immunoadhesins (la's) are antibody-like molecules which combine the binding domain of a protein such as a cell-surface receptor or a ligand (an "adhesin ") with the effector functions of an immunoglobulin constant domain. Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate human immunoglobulin hinge and constant domain (Fc) sequence, the binding specificity of interest can be achieved using entirely human components. Such immunoadhesins are minimally immunogenic to the patient, and are safe for chronic or repeated use.

Antibody-immunoadhesin (Ab/Ia) chimeras have also been described in the literature. These molecules combine the binding region of an immunoadhesin with the binding domain of an antibody. Berg et al, PNAS (USA) 88:4723 4727 (1991) made a bispecific antibody- immunoadhesin chimera which was derived from murine CD4-IgG.

Antibody-immunoadhesin constructs comprising the extracellular domain of Dectin-1 receptor fused to an immunoglobulin (Ig) heavy chain constant region sequence in one arm, and an antibody, or antigen-binding fragment thereof, with binding affinity for an immune cell, e.g., a T-cell antigen, e.g., CD3s, can be prepared in an analogous manner. Dectin-1 -Fc-anti-CD3 constructs are illustrated in FIG. 2A. In these structures, a Dectin extracellular domain sequence (mDectin-1 extracellular domain (DCml, SEQ ID NO: 18) or h-Dectin-la extracellular domain (DChl, SEQ ID NO: 68) is fused to the CH2-CH3 constant regions of a murine IgG2a immunoglobulin (FCml, SEQ ID NO: 22) or of a murine IgG2a immunogloblin (FCm2, SEQ ID NO: 23), linked, through a polypeptide linker (BGL2, SEQ ID NO: 13), to the scFv sequence of the 2C11 mouse anti-CD3 antibody, or to the scFv sequence of the 500A2 mouse anti-CD3 antibody, or to the scFv sequence of the KT3 mouse anti-CD3 antibody, or to the scFv seqence of the UCHT1 human anti-CD3 antibody. FIG. 2B provides an illustration of a structure wherein a Dectin extracellular domain sequence (mDectin-1 extracellular domain (DCml, SEQ ID NO: 18) or h-Dectin-la extracellular domain (DChl, SEQ ID NO: 68) is fused to the CH2-CH3 constant regions of a murine IgG2a immunoglobulin (FCml, SEQ ID NO: 22) or of a murine IgG2a immunogloblin (FCm2, SEQ ID NO: 23).

In one embodiment, the immunoadhesin has the following structure:

Dectin-1 - linker(l) - IgG2a - linker(2) - 2C11 (VH-linker(3)-VL) (CTP-014, SEQ ID

NO: 57). In some embodiments, an immunoadhesin comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 57.

Dectin-1 is a fungal cell wall polysaccharide binding protein, IgG2a is a murine immunoglobulin sequence, and 2C11 VH and VL are the heavy chain and light chain sequences of the CD3 receptor complex-binding scFv murine antibody fragment. In one embodiment, the immunoadhesin has the following structure:

UCHT1 (VL-linker(3)-VH scFv) - linker(l)-Fc-IgGl - linker(2) - Dectin-1 (CTP-052, SEQ ID NO: 58). In some embodiments, an immunoadhesin comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 58.

UCHT1 VL and VH are the light chain and heavy chain sequences of the CD3 receptor complex-binding scFv human antibody fragment, Fc - IgGl is a human immunoglobulin sequence, and Dectin-1 is a fungal cell wall polysaccharide binding protein.

In one embodiment, the immunoadhesin has the following structure:

UCHT1 (VL-linker(3)-VH scFv) - linker(l) - Fc-IgGl - linker(2) - Dectin stalk - Dectin-

1 (CTP-054, SEQ ID NO: 59). In some embodiments, an immunoadhesin comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 59.

UCHT1 VL and VH are the light chain and heavy chain sequences of the CD3 receptor complex-binding scFv human antibody fragment, Fc - IgGl is a human immunoglobulin sequence, stalk is an extracellular sequence that separates a Dectin receptor from the cell surface, and Dectin-1 is a fungal cell wall polysaccharide binding protein.

In one embodiment, the immunoadhesin has the following structure:

UCHT1 (VL-linker(3)-VH scFv) - linker(l) - Fc-IgGl - linker(2) - Dectin stalk - Dectin- 1 (S239D, A330L, I332E) (CTP-054B, SEQ ID NO: 60). In some embodiments, an immunoadhesin comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 60.

UCHT1 VL and VH are the light chain and heavy chain sequences of the CD3 receptor complex-binding scFv human antibody fragment, Fc - IgGl is a human immunoglobulin sequence, stalk is an extracellular sequence that separates a Dectin receptor from the cell surface, and Dectin-1 (S239D, A330L, I332E) is a fungal cell wall polysaccharide binding protein with the following mutations: S to D at position 239; A to L at position 330; I to E at position 332.

In one embodiment, the immunoadhesin has the following structure:

UCHT1 (VL-linker(3)-VH scFv) - linker(l) - Fc-IgGl - linker(2) - Dectin stalk - Dectin-

1 (K326W, E333S) (CTP-054C, SEQ ID NO: 61). In some embodiments, an immunoadhesin comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 61.

UCHT1 VL and VH are the light chain and heavy chain sequences of the CD3 receptor complex-binding scFv human antibody fragment, Fc - IgGl is a human immunoglobulin sequence, stalk is an extracellular sequence that separates a Dectin receptor from the cell surface, and Dectin-1 (K326W, E333S) is a fungal cell wall polysaccharide binding protein with the following mutations: K to W at position 326; E to S at position 333.

In one embodiment, the immunoadhesin has the following structure:

2C11 (VH-linker(5)-VL) - linker(l) - Fc-IgG2a - linker(3) - mDectin-1 (CTP-067, SEQ ID NO: 62). In some embodiments, an immunoadhesin comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 62.

2C11 VH and VL are the heavy chain and light chain sequences of the CD3 receptor complex-binding scFv murine antibody fragment, Fc - IgG2a is a murine immunoglobulin sequence, and mDectin-1 is a fungal cell wall polysaccharide binding protein.

In one embodiment, the immunoadhesin has the following structure:

500A2 (VH-linker(5)-VL) - linker(l) - Fc-IgG2a - linker(3) - mDectin-1 (CTP-076, SEQ ID NO: 63) In some embodiments, an immunoadhesin comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 63.

500A2 VH and VL are the heavy chain and light chain sequences of the CD3 receptor complex-binding scFv murine antibody fragment, Fc - IgG2a is a murine immunoglobulin sequence, and mDectin-1 is a fungal cell wall polysaccharide binding protein.

In one embodiment, the immunoadhesin has the following structure:

500A2 (VL-linker(5)-VH) - linker(l) - Fc-IgG2a - linker(3) - mDectin-1 (CTP-077, SEQ

ID NO: 64). In some embodiments, an immunoadhesin comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the peptide sequence provided in SEQ ID NO: 64.

500A2 VL and VH are the light chain and heavy chain sequences of the CD3 receptor complex-binding scFv murine antibody fragment, Fc - IgG2a is a murine immunoglobulin sequence, and mDectin-1 is a fungal cell wall polysaccharide binding protein. Pharmaceutical Compositions and Their Uses

In one aspect, the present invention provides pharmaceutical compositions comprising at least one of the multi-specific binding compounds of the invention, or pharmaceutical compositions comprising two or more different multi-specific binding compounds of the invention. Pharmaceutical compositions may comprise an effective amount of any of the multi- specific binding compounds, multi-specific single chain antibody molecules or multi-specific antibodies of the present invention, optionally in admixture with a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers as used herein are exemplified, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof. Therapeutic formulations of the multi-specific binding compounds used in accordance with the present invention are prepared for storage by mixing multi-specific binding compounds having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), such as in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes {e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Pharmaceutical compositions of the invention may also be used prophylactically, e.g., in a situation where contact with microbes is expected and where establishment of infection is to be prevented. For instance, the composition may be administered prior to surgery. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the multi-specific binding compound, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactide, degradable lactic acid-glycolic acid copolymers, and poly-D-(- )-3-hydroxybutyric acid.

Microbial infections affect various areas of the body and so the compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared for reconstitution with sterile water, optionally containing a preservative. The composition may be lyophilised and reconstituted for use, or may be stable liquid formulations with long term storage stability.

Included in the present invention are formulations comprising a multi-specific binding compound of the invention that are further resistant to aggregation, degradation or fragmentation under given manufacture, preparation, transportation and storage conditions. Further provided are multi-specific binding compounds that retain biological activity under given formulation manufacture, preparation, transportation and storage conditions.

Compositions of the invention may be used in conjunction with known anti-fungals. Suitable anti-fungals include, but are not limited to, azoles (e.g., fluconazole, itraconazole), polyenes (e.g., amphotericin B), flucytosine, and squalene epoxidase inhibitors (e.g., terbinafine). Compositions may also be used in conjunction with known antivirals e.g. HIV protease inhibitors, a 2',3 '-dideoxynucleoside (e-g, DDC, DDI), 3 '-azido-2',3 '- dideoxynucleosides (AZT), 3 '-fluoro-2',3 '-dideoxynucleosides (FLT), 2',3 '-didehydro-2',3 '- dideoxynucleosides (e.g., D4C, D4T) and carbocyclic derivatives thereof (e.g., carbovir), 2'- fluoro-ara-2',3 '-dideoxynucleosides, 1,3-dioxolane derivatives (e-g-, 2',3 '-dideoxyl-3 '- thiacytidine), oxetanocin analogues and carbocyclic derivatives thereof (e.g., cyclobut-G) and the 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2- phosphonylmethoxypropyl)adenine (FPMPA) derivatives, tetrahydro-imidazo[4,5, l-jk][l,4]- benzodiazepin-2(lH)one (TIBO), 1 -[(2 -hydroxy ethoxy)-methyl]-6-(phenylthio)thymine (HEPT), dipyrido[3,2-b:2',3 '-e]-[l,4]diazepin-6-one (nevirapine) and pyridin-2(lH)one derivatives, 3TC, etc. Medical Treatments and Uses

The multi-specific binding compounds of the invention can be used to eradicate, alleviate, or prevent a fungal infection in a subject. The multi-specific binding compounds of the invention can be used for protection against microbial infection and/or disease.

Thus, the invention provides a multi-specific binding compound of the invention for use as a medicament. The invention also provides a method for protecting a patient from a microbial infection, comprising administering to the patient a pharmaceutical composition of the invention. The invention also provides the use of multi-specific binding compounds of the invention in the manufacture of a medicament for the prevention of microbial infection and/or disease.

The multi-specific binding compound pharmaceutical composition of the invention also provides a method for treating a patient suffering from a microbial infection, comprising administering to the patient a pharmaceutical composition of the invention. The invention also provides the use of multi-specific binding compounds of the invention in the manufacture of a medicament for treating a patient.

The multi-specific binding compounds of the invention are particularly useful for treating microbial infections in patients who are immunocompromised/immunosuppressed; pregnant; or undergoing antibiotic therapy or chemotherapy. The multi-specific binding compounds of the invention are also useful for treating microbial infection in patients who have systemic microbial infection; indwelling intravascular catheters; HIV; AIDS; neutropenia; previous fungal colonisation; diabetes; leukaemia; lymphoma; burns; maceration; oral cavity infections and patients who have had prior hemodialysis or who have undergone organ transplants.

The uses and methods are particularly useful for treating diseases including, but not limited to: candidosis, aspergillosis, cryptococcosis, dermatomycoses, sporothrychosis and other subcutaneous mycoses, AIDS-related conditions, blastomycosis, histoplasmosis, coccidiomycosis, paracoccidiomycosis, pneumocystosis, thrush, tuberculosis, mycobacteriosis, respiratory infections, scarlet fever, pneumonia, impetigo, rheumatic fever, sepsis, septicaemia, cutaneous and visceral leishmaniasis, corneal acanthamoebiasis, keratitis, cystic fibrosis, typhoid fever, gastroenteritis and hemolytic-uremic syndrome.

Efficacy of treatment can be tested by monitoring microbial infection after administration of the pharmaceutical composition of the invention.

Compositions of the invention will generally be administered directly to a patient. Direct delivery may, for example, be accomplished by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue). Injection is preferred. It will be appreciated that the active ingredient in the composition will be an antibody compound. As such, it might be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition might need to contain agents which protect the antibody from degradation but which release the antibody compound once it has been absorbed from the gastrointestinal tract. Dosage treatment can be a single dose schedule or a multiple dose schedule.

Methods to treat human subjects, including but not limited to patients with a systemic fungal infection, with the multi-specific binding compounds herein are specifically within the scope of the present invention. In another aspect, the invention provides the use of multi-specific binding compounds for the preparation of a medicament for use in the treatment or diagnosis of a disease or disorder in a human or animal subject. In certain embodiments, the multi-specific binding compounds of the invention are particularly useful for treating infections of the microbial species listed above. A particular group of such species includes: Candida species, such as C. albicans; Cryptococcus species, such as C. neoformans; Enterococcus species, such as E. faecalis; Streptococcus species, such as S.pneumoniae, S.mutans, S.agalactiae and S.pyogenes; Leishmania species, such as L.major and L.infantum; Acanthamoeba species, such as A.castellani; Aspergillus species, such as A.fumigatus and A. flavus; Pneumocystis species, such as P.carinii; Mycobacterium species, such as M. tuberculosis; Pseudomonas species, such as P. aeruginosa; Staphylococcus species, such as S. aureus; Salmonella species, such as S.typhimurium; Coccidioides species such as C. immitis; Trichophyton species such as T.verrucosum; Blastomyces species such as B.dermatidis; Histoplasma species such as H. capsulatum; Paracoccidioides species such as P.brasiliensis; Pythiumn species such as P.insidiosum; and Escherichia species, such as E. coli. In yet another aspect, the invention concerns a method for the treatment of a microbial disease or condition, comprising administering to a subject in need an effective amount of any of the multi-specific binding compounds, multi-specific single chain antibody molecules or multi- specific antibodies of the present invention.

In one embodiment, the microbial disease or condition is a fungal or bacterial disease or condition. In other embodiments, the "fungal condition " can include any disease or condition the symptoms of which are caused or contributed to, by a fungus. Fungal infections, and particularly those referred to as systemic fungal infections or systemic mycoses, may be caused by pathogenic and/or opportunistic fungi, such as, for example, fungal infections (or mycoses) involving, Candida species, such as C. albicans, C. krusei, C. glabrata; Aspergillus species, such as Aspergillus fumigatus, Cryptococcus species, such as Cryptococcus neoformans, Pneumocystis species, such as Pneumocystis carinii, Penicillium species, such as Penicillium marneffei; Histoplasma capsulatum, and Coccidioides immitis. The fungal pathogen may be derived from a fungal pathogen which is of the genus Candida spp., (e.g., C. albicans), Epidermophyton spp., Exophiala spp., Microsporum spp., Trichophyton spp., (e.g., T. rubrum and T. inter digitale), Tinea spp., Aspergillus spp., Blastomyces spp., Blastoschizomyces spp., Coccidioides spp., Cryptococcus spp., Histoplasma spp., Paracoccidiomyces spp., Sporotrix spp., Absidia spp., Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia spp., Arthrographis spp., Acremonium spp., Actinomadura spp., Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveria spp., Chrysosporium spp., Conidiobolus spp., Cunninghamella spp., Fusarium spp., Geotrichum spp., Graphium spp., Leptosphaeria spp., Malassezia spp., Mucor spp., Neotestudina spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Phoma spp., Piedraia spp., Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsisspp., Sporobolomyces spp., Syncephalastrum spp., Trichoderma spp., Trichosporon spp., Ulocladium spp., Ustilago spp., Verticillium spp. or, Wangiella spp. Specifically included within the term "fungal infection " infections selected from tinea capitis, tinea corporis, tinea pedis, onychomycosis, perionychomycosis, pityriasis versicolor, oral thrush, vaginal candidosis, respiratory tract candidosis, biliary candidosis, eosophageal candidosis, urinary tract candidosis, systemic candidosis, mucocutaneous candidosis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis, fungal sinusitis, or chronic sinusitis. For example, the infection being treated can be an infection by Candida albicans, C. parapsilosis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. tropicalis, Aspergillus fumigatus, A. flavus, A. terreus, A. niger, A. candidus, A. carneus, A. deflectus, A. fischeri, A. flavipes, A. glaucus, A. nidulans, A. oryzae, Rhizopus oryzae, A. clavatus, or A. ochraceus.

The binding compounds and compositions of the present invention can be used in combination with one or more further antifungal agents, e.g. antofungal agents which are effective for the particular causative species of fungus.

Thus, for example, the multi-specific binding compounds (e.g. antibodies) herein can be used in combination with Echinocandin class antifungal compounds, including, without limitation, caspofungin, echinocandin B, anidulafungin, pneumocandin B₀, aculeacin Α_γ, micafungin, and their derivatives. Echinocandin class compounds can be synthesized, for example, by coupling functionalized or unfunctionalized echinocandin class compounds with the appropriate acyl, alkyl, hydroxyl, and/or amino groups under standard reaction conditions (see PCT Publication No. WO 2011/025875, and U.S. provisional Ser. No. 61/448,807, herein incorporated by reference). See also, U.S. Patent Application Publication No. 20150087583.

The multi-specific binding compounds (e.g. antibodies) herein can also be used in combination with azole-type antifungal agents, including, without limitation, voriconazole, clotrimazole, ravuconazole, posaconazole, econazole, fluconazole, itraconazole, tebuconazole, propiconazole, enilaconazole, miconazole, oxiconazole, sulconazole, and tioconazole.

TABLE 1

SEQUENCES

>VH_2G8

QVQLQQSGAELMKPGASVKISCKATGYTLSSYWLEWVKQRPGHGLEWIGEILPGSGSTN YNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCAREGWYFDVWGAGTTVTVSS

(SEQ ID NO: 1)

>VL_2G8

DIVMTQSPLTLSVTIGQPASISCKSSQSLLYSNGNTHLNWLLQRPGQSPKRLIYLVSKL DSGVPDRFTGSGSGTDFTLKISRVEAEDLGFYYCVQGTHFPYTFGGGTKLEIK

(SEQ ID NO: 2)

>VH_Aus

EVKLEVSGGGLVRPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGIEWVAEIRLRSNNYA THYAESVKGRFTISRDDSKSSLYLQMNNLRAEDTGIYYCTPPTMVTRFPYWGQGTLVTV SA

(SEQ ID NO: 3) >VL_Aus

DIQMTQSPSSLSASLGGKVTITCKASQDINKSIAWFQHKPGKGPRLLILYTSTLQPGIP

SRFSGSGSGRDYSFSISNLEPEDIATYYCLQYNYLWTFGGGTKLEIK

(SEQ ID NO: 4)

>VH_2C11

EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIK YADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS

(SEQ ID NO: 5)

>VL_2C11

DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVP

SRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK

(SEQ ID NO: 6)

>VH_500_A2

QVKLQQSGSELGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTS YNQKFQGKATLTVDKISSTAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGIQVTV SS

(SEQ ID NO: 7)

>VL_500_A2

DIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPRALIKYASQSIPGIPS RFSGSGSETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLEIK

(SEQ ID NO: 8)

>CDhl_UCHTl_VL

DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRT

(SEQ ID NO: 9)

>CDhl_UCHTl_VH

EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTY NQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SS

(SEQ ID NO: 10)

>Linker 1

GGGGS

(SEQ ID NO: 11)

>Linker 2

GGGGSMVRS

(SEQ ID NO: 12)

>Linker_3 (BGL2 linker)

GGGGSGGGGS

(SEQ ID NO: 13)

>Linker 4

GTGGGGSGGGGS

(SEQ ID NO: 14)

>Linker 5

GGGGSGGGGSGGGGS

(SEQ ID NO: 15) >BGL1 (linker between scFv's on BiTE constructs) ASTKGPSVFPLAPSSSGSG

(SEQ ID NO: 16)

>T1

GGHHHHHH

(SEQ ID NO: 17)

>mDectin-l extracellular domain including stalk

WRHNSGRNPEEKDSFLSRNKENHKPTESSLDEKVAPSKASQTTGGFSQSCLPNWIMHGK SCYLFSFSGNSWYGSKRHCSQLGAHLLKIDNSKEFEFIESQTSSHRINAFWIGLSRNQS EGPWFWEDGSAFFPNSFQVRNAVPQESLLHNCVWIHGSEVYNQICNTSSYSICEKEL

(SEQ ID NO: 18)

>hDectin-l stalk

TMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLEDSVTPTKAVK

(SEQ ID NO: 19)

>hDectin-l

TTGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQ VSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIY DQLCSVPSYSICEKKFSM

(SEQ ID NO: 20)

>IgGlml (CH2 CH3)

VPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVWDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCR SAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPI MDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

(SEQ ID NO: 21)

>FCml (CH2 CH3 mIgG2a)

PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPDVQ ISWF NVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCK NKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYK NTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGK

(SEQ ID NO: 22)

>FCm2 (CH2 CH3 mIgG2ael)

PRGPTIKPCPPCKCPAPNLEGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPDVQ ISWF NVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKAFACA NKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYK NTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGK

(SEQ ID NO: 23)

>FChl (Human-CH2-CH3-IgGl)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 24)

>FChl-A

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIAKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 25)

>FChl-B

DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 26)

>FChl-C

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNWALPAPISKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 27)

>hKappa

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO: 28)

>FCh2 (Human-CH2-CH3-IgGl E233P, L234V, L235A)

DKTHTCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI

SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 29)

>hCHl

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSWTVPSSSLGTQTYICN HKPSNTKVDKKVEPKSC

(SEQ ID NO: 30)

>mCLl

RADAAPTVSIFPPSSEQLTSGGASVLCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD QDSKDSTYSMSSTLTLTKDEYERHNTYTCEATHKTSTSPIVKSFNRNEC

(SEQ ID NO: 31)

>mkappal

RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD QDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

(SEQ ID NO: 32)

>mCH-l

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQ

SDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKI

(SEQ ID NO: 33)

>mCH-2

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQ

SDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIE

(SEQ ID NO: 34)

>mCH-3

NTAYMQLSSLTSEDSAVYYCAREGWYFDVWGAGTTVTVSSAKTTAPSVYPLAPVCGDT TGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWP

SQSITCNVAHPASSTKVDKKIE

(SEQ ID NO: 35)

TABLE 2

2G8 Sequences

2G8 Light chain variable region polypeptide (SEQ ID NO: 36)

DIVMTQSPLTLSVTIGQPASISCKSSQSLLYSNGNTHLNWLLQRPGQSPKRLIY

LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGFYYCVQGTHFPYTFGGGTKLEIK

2G8 Heavy chain variable region polypeptide (SEQ ID NO: 37)

QVQLQQSGAELMKPGASVKISCKATGYTLSSYWLEWVKQRPGHGLEWIGEILPGSGSTNY NEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCAREGWYFDVWGAGTTVTVSS

2G8 Heavy chain CDR1 nucleic acid (SEQ ID NO: 38)

ggctacacac tcagtagcta ctgg

2G8 Heavy chain CDRlpolypeptide (SEQ ID NO: 39)

GYTLSSYW

2G8 Heavy chain CDR2 nucleic acid (SEQ ID NO: 40)

attttacctg gaagtggtag tact

2G8 Heavy chain CDR2 polypeptide (SEQ ID NO: 41)

ILPGSGST

2G8 Heavy chain CDR3 nucleic acid (SEQ ID NO: 42)

gcaagagagg gttggtactt cgatgtc

2G8 Heavy chain CDR3 polypeptide (SEQ ID NO: 43)

AREGWYFDV

2G8 Light chain CDR1 nucleic acid (SEQ ID NO: 44)

cagagcctct tatatagtaa tggaaacacc cat

2G8 Light chain CDRlpolypeptide (SEQ ID NO: 45)

QSLLYSNGNTH

2G8 Light chain CDR2nucleic acid (SEQ ID NO: 46)

ctggtgtct

2G8 Light chain CDR2 polypeptide (SEQ ID NO: 47)

LVS 2G8 Light chain CDR3 nucleic acid (SEQ ID NO: 48) gtgcaaggta cacattttcc gtacagg

2G8 Light chain CDR3 polypeptide (SEQ ID NO: 49)

VQGTHFPYT

VLCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNTYTCEA THKTSTSPIVKSFNRNECGTGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNW YQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKVEIKRTGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSD WYFDVWGQGTLVTVSS (SEQ ID NO: 55)

CTP-030 DIQMTQSPSSLSASLGGKVTITCKASQDINKSIAWFQHKPGKGPRLLILYTSTLQPGIPSRFSGS

GSGRDYSFSISNLEPEDIATYYCLQYNYLWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGAS VLCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNTYTCEA THKTSTSPIVKSFNRNEC (SEQ ID NO: 56)

Table 4: Immunoadhesin Construct Sequences

VKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLC SVPSYSICEKKFSM (SEQ ID NO: 60)

CTP-054C DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSG

SGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTGGGGSGGGGSGGGGSEVQLVESG GGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDK SKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSMVRSDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRWSVLTVLHQDWLNGKEYKCKVSNWALPAPISKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSTMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLE DSVTPTKAVKTTGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFI VKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLC SVPSYSICEKKFSM (SEQ ID NO: 61)

CTP-067 EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKG

RFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSGGGGSGGGGSGGGGSD IQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGS GRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKGGGGSPRGPTIKPCPPCKCPAPNLLG GPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPDVQISWF NVEVHTAQTQTHREDYNSTLRV VSALPIQHQDWMSGKEFKCK NKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCM VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHN HHTTKSFSRTPGKGGGGSGGGGSWRHNSGRNPEEKDSFLSRNKENHKPTESSLDEKVAPSKASQTT GGFSQSCLPNWIMHGKSCYLFSFSGNSWYGSKRHCSQLGAHLLKIDNSKEFEFIESQTSSHRINAF WIGLSRNQSEGPWFWEDGSAFFPNSFQVRNAVPQESLLHNCVWIHGSEVYNQICNTSSYSICEKEL

(SEQ ID NO: 62)

CTP-076 QVKLQQSGSELGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTSY

NQKFQGKATLTVDKISSTAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGIQVTVSS GGGGSGGGGSGGGGSDIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPR ALIKYASQSIPGIPSRFSGSGSETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLE IKGGGGSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSE DDPDVQISWF NVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCK NKDL PAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE LNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGKG GGGSGGGGSWRHNSGRNPEEKDSFLSRNKENHKPTESSLDEKVAPSKASQTTGGFSQSCL PNWIMHGKSCYLFSFSGNSWYGSKRHCSELGAHLLKIDNSKEFEFIESQTSSHRINAFWI GLSRNQSEGPWFWEDGSAFFPNSFQVRNAVPQESLLHNCVWIHGSEVYNQICNTSSYSIC EKEL (SEQ ID NO: 63)

CTP-077 DIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPRALIKYASQSIPGIPSRFSGSG

SETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLEIKGGGGSGGGGSGGGGSQVKLQQSGSE LGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTSYNQKFQGKATLTVDKIS STAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGIQVTVSSGGGGSPRGPTIKPCPPCKCPAP NLLGGPSVFIFPPKIKDVLMISLSPIVTCVWDVSEDDPDVQISWF NVEVHTAQTQTHREDYNS TLRWSALPIQHQDWMSGKEFKCK NKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHE GLHNHHTTKSFSRTPGKGGGGSGGGGSWRHNSGRNPEEKDSFLSRNKENHKPTESSLDEKVAPSKA SQTTGGFSQSCLPNWIMHGKSCYLFSFSGNSWYGSKRHCSELGAHLLKIDNSKEFEFIESQTSSHR INAFWIGLSRNQSEGPWFWEDGSAFFPNSFQVRNAVPQESLLHNCVWIHGSEVYNQICNTSSYSIC EKEL (SEQ ID NO: 64)

Table 5: Additional Sequences

signal sequence

DChl (hDectin-la) MEYHPDLENLDEDGYTQLHFDSQSNTRIAWSEKGSCAASPPWRLIAVILGILCLVILV

IAWLGTMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLEDSVTPTKAVKTTGVLSS PCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDN SFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVP SYSICEKKFSM (SEQ ID NO: 68)

All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications, which might be used in connection with the presently described inventions. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

The present invention is explained in more detail by the following non-limiting examples.

EXAMPLES Example 1

Preparation of a BiTE aL2G8-VH2G8-VHCD3-VLCD3-8His tas) multi-specific antibody construct

cDNA encoding an anti-beta-glucan x anti-CD3 multi-specific antibody with an anti- beta-glucan VL and VH portion of the molecule and an anti-CD3 VL and VH portion of the molecule was cloned.

Constructs: Reverse translations of the variable regions of the light VL and heavy VH chains of IgG mAb 2G8 and anti-CD3 mAb were synthesized by solid-phase synthesis and cloned into pD451-SR (DNA 2.0 Menlo Park, Ca). The two scFvs were transcriptionally fused with a linker encoding a 15 residue long flexible peptide (Gly4Ser)3. The DNA fragment encoded the bispecific single-chain antibody 2G8-CD3, with the domain arrangement VL2G8- VH2G8-VHCD3-VLCD3-8His tag. Sequence specific primers with restriction sites were designed to facilitate sub-cloning into different expression vectors. Expression constructs were generated by cloning the 2G8 anti-CD3 DNA construct into pET26b(+) (EMD/Millipore) for both cytosolic and signal sequence mediated translocation of cytoplasmic protein to the periplasm. For mammalian expression, the template was cloned into pcDNA3.1 (LifeTech, Carlsbad, CA, USA) at the cloning sites BamHI and Xhol (New England Biolabs, Ipswich, MA, USA) and included a signal sequence derived from the human light chain IgGl .

Antibody expression and purification: E.coli expression: Plasmids were transformed into E. coli BL21(DE3). Fresh cultures were grown to mid-log (A600 0.6-0.8) and induced by adding IPTG to 1 mM, and grown for an additional 4-8 hours. Cells were harvested by centrifugation. Target protein was purified from inclusion bodies by first solubilizing in 6M Guanidine Chloride followed by metal affinity chromatography. 2G8-CD3 was refolded by slow dilution until ultimately free of denaturant. Secreted protein was harvested from the periplasmic fraction using osmotic shock and followed by metal affinity chromatography.

Mammalian expression: The 2G8-CD3/ pcDNA3.1, plasmids were prepared using the Endofree Plasmid Maxi Kit (Qiagen). The plasmid DNA was delivered with Lipofectamine 2000 (Life Technologies) DNA transfection reagent into CHO and/or HEK-293 cells per the manufacturer's protocol. The supernatant was purified using metal affinity chromatography.

Example 2

Preparation of a Dectin-l-Fc-anti-CD3 multi-specific antibody construct cDNA encoding a fusion protein of Dectin-l-Fc-anti-CD3 functioning as multi-specific antibody with a beta-glucan binding Dectin-1 domain of the molecule fused to an antibody Fc domain (immunoadhesin), and an anti-CD3 VL and VH portion of the molecule was cloned.

Constructs: The DNA encoding amino acids 67-247 of Dectin-1 were synthesized by solid-phase synthesis and cloned into pD451-SR (DNA 2.0 Menlo Park, Ca). Sequence specific primers were used to engineer a Dectin-1 -IgGl -Fc transcriptional fusion by cloning into the EcoRI and Bglll (New England Biolabs, Ipswich, MA, USA) sites of the vector pFUSE-hlgGl- Fc2 (Invivogen, San Diego, CA, USA). This construct served as a template for site directed mutagenesis to remove the stop codon from the IgGl-Fc and simultaneously add an Agel restriction site. Primers specific for the anti-CD3 scFv portion from the 2G8-anti-CD3 DNA template were used to amplify and clone the scFv to the 3' end of the Fc domain.

Expression and Purification: The Dectin-1 -Fc-anti-CD3 plasmids were prepared using the Endofree Plasmid Maxi Kit (Qiagen). The plasmid DNA was delivered with Lipofectamine 2000 (Life Technologies) DNA transfection reagent into CHO and HEK-293 cells according to the manufacturer's protocol. The protein was purified from the culture supernatant using a protein-A affinity column.

Example 3

Preparation of a Tetravalent beta-glucan x anti-CD3 multi-specific antibody construct

DNA encoding a tetravalent multi-specific antibody incorporating an anti -beta-glucan VL and VH portion of the molecule and an anti-CD3 VL and VH portion of the molecule were cloned.

Constructs: Tetravalent constructs require two polypeptides to form a multimeric protein. For the heavy chain, a reverse translation of the variable region of the heavy chain (VH) of 2G8 mAb fused to the CH1-CH2-CH3 of murine IgG2a were synthesized by solid-phase synthesis and cloned into pD451-SR (DNA 2.0 Menlo Park, Ca). For the light chain, the variable light (VL) of mAb 2G8 were transcriptionally fused to the murine CL Ig-Mk domain followed by a DNA linker encoding (Gly4Ser)2 followed by a transcriptional fusion of a murine Anti-CD3 scFv. These constructs were also generated by solid-phase synthesis.

Mammalian expression: The heavy and light chain template DNA were sub-cloned into pcDNA3.1 (LifeTech, Carlsbad, CA, USA) at the cloning sites BamHI and Xhol (New England Biolabs, Ipswich, MA, USA).

The heavy and light pcDNA3.1, plasmids were prepared using the Endofree Plasmid Maxi Kit (Qiagen). The plasmid DNA was delivered with Lipofectamine 2000 (Life Technologies) DNA transfection reagent into CHO and/or HEK-293 cells per the manufacturer's protocol. The supernatant was purified using Protein A and ion-exchange chromatography.

Example 4

Preparation and Expression of Antibody and Immunoadhesin Constructs

Antibody and immunoadhesin constructs were prepared and expressed as described above in Examples 1-3. Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 ug of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards. Reduced and non-reduced lanes are denotes by "R" and "NR". An illustration of each construct, along with the results of the gel analyses, are provided in FIGS. 4A-16B.

Example 5

Binding to beta-glucan

Enzyme-linked immunosorbent assay (ELISA).

Binding of beta-glucan was measured through ELISA. 96 well ELISA plates were coated overnight at 4^°C with 2 ug/well with either 1) laminarin (beta-l,3-glucan), 2) pustulan (beta-1,6- glucan), or 3) controls (BSA), and blocked with 1% BSA in PBS. Human and murine Dectin-1- Fc-anti-CD3, human and murine Dectin-l-Fc, human and murine BITE constructs and human and murine monoclonal Ab (mAb) and tetravalent mAb constructs were added in 100 μΐ. per well. Serial dilutions were made of each composition, from 2 to 0.001 μg/mL. Binding was compared to positive control antibody (monoclonal antibody to beta-l,3-glucan, Biosupplies, Australia Catalog No. 400-2). For detection, HRP -labeled secondary anti-mouse IgG (anti- Mouse IgG HRP, Cat#: NA931V, Vendor: GE Healthcare, anti-human IgG (Anti-Human IgG HRP, Cat#: NA933V, Vendor: GE Healthcare) or anti-His tag Ab Mouse monoclonal anti-poly Histidine-peroxidase antibody, clone HIS-1, Cat#: A7058, Vendor: Sigma-Aldrich (for BITE constructs) was used. Absorbance was measured at 450 nm with a 96-well microplate reader. The data are provided in FIGS. 17A-31C. The results demonstrate that the constructs bind to laminarin, and pustulan, but not to BSA (negative control). Furthermore, Dectin-1 based constructs (both murine and human) generally show stronger binding to β 1, 6 glucan (as measured by binding to pustulan) than mAb constructs.

Example 6

Binding to CD3s on T-Cells

Flow Cytometry: Binding of murine Dectin-1 -Fc-anti-CD3 (CTP-014) and Dectin-l-Fc

(CTP-016) constructs to CD3 expressed on T-cells was measured through flow cytometry.

Mouse splenoctye single cell suspensions were prepared from a C57BL/6 spleen. Red blood cells were lysed and remaining splenocytes were stained for flow cytometry as follows: 10⁶ cells were stained with Fc-block at 1 : 100 (BD Biosciences, Catalog No. 553142) for 10 minutes on ice.

Cells were washed and stained with a cocktail containing anti-mouse B220 Alexa 488 at 1 : 100 (BD Biosciences, Catalog No. 557669), anti-mouse CD8 V450 at 1 :400 (BD Biosciences, Catalog No. 560569), anti-mouse CD4 APC at 1 :800 (BD Biosciences, Catalog No. 553051), and either CTP-014, CTP-016, or a mouse IgG2a Isotype control (MOPC-173, BioLegend, Catalog. No. 401503) at 4ug/ml for 30 minutes on ice. Cells were washed 2x and stained with an anti-mouse IgG2a-PE secondary antibody (BioLegend, Catalog No. 1670168) at 0.5ug/ml for 30 minutes on ice. Cells were washed 2x again and fixed in 2% PFA (in PBS) on ice for 10 minutes before being washed 2x and resuspended in staining buffer. Acquisition and analysis was performed using a BD FACSCanto II or a BD FACS Calibur. For analysis, lymphocytes and single cells were gated based on their SSC VS FSC properties. B200+ cells (B cells) were gated out and the remaining CD4+ and/or CD8+ cells were analyzed for fluorescence in the PE channel. The data are provided in FIGS. 32A-36. The results demonstrate that the constructs bind to CD3 on both CD4 and CD 8 positive T-cells.

Positive control staining was conducted using a commercially-available anti-CD3 antibody. The method was performed as described above, using a hamster anti-mouse CD3 antibody (clone 145-2C11) (BD Biosciences, Catalog No. BDB550275). The data are provided in FIGS. 37A and 37B.

Verification of CD3 binding of subsequent murine Dectin-l-Fc constructs was performed as described above. The data are provided in FIG. 33 and show CD3 -specific binding of all murine CD3 -targeting constructs (CTP-014, CTP-067, CTP-076 and CTP-077). As expected, constructs without a CD3 binding domain (CTP-016) did not bind to T-cells.

For testing of binding of human Dectin-l-Fc constructs to T-cells, human PBMCs were isolated from heparinized human blood using Ficol®Paque Plus. 10⁶ cells/sample were stained for flow cytometry as follows: Fc receptors were blocked by incubation with 7.5 ug human Fc- block (BD 564220) for 10 minutes on ice. Cells were washed and stained with an antibody cocktail containing anti-human CD4 FITC at 20ul per 106 cells (BD 555346), anti-human CD8 APC at 20ul per 10⁶ cells (BD 555369), anti-human CD15 & CD16 V450 at 5ul per 10⁶ cells (BD 561584/561310), anti-human CD19 at 5ul per 10⁶ cells (BD 561295) human Dectin-l-Fc constructs CTP-052 and CTP-054 or a negative control (hlgGl, Crown Biosciences Inc. Cat# COOOl) at ΙΟηΜ for 30 minutes on ice. Cells were washed 2x and stained with an anti-hlgG Fc PE secondary antibody (BioLegend 409304) at 5ul per sample for 30 minutes on ice. Cells were washed 2x again and fixed in 2% PFA (in PBS) on ice for 10 minutes before being washed 2x and resuspended in staining buffer. Acquisition and analysis was performed using a BD FACS Calibur. For analysis, lymphocytes and single cells were gated based on their SSC VS FSC properties. B cells were gated out and the remaining CD 19- cells were analyzed for fluorescence in the PE channel. The results are provided in FIG. 34. These results demonstrate binding of CD3 on human T-cells by Dectin-1-Fc-CD3 constructs CTP-052 and CTP-054.

For testing of binding of murine BITE constructs to T-cells, single cell suspensions were prepared from a C57BL/6 spleen by crushing spleen between two sterile, frosted glass slides. Cells were then filtered, pelleted and red blood cells were lysed using ACK buffer. The resulting cell suspension was counted and 10⁶ cell were stained for flow cytometry as follows: Fc receptors were blocked with murine Fc-block at 1 : 100 (BD 553142) for 10 minutes on ice. Cells were washed and stained with an antibody cocktail containing anti-mouse B220 Alexa488 at 1 : 100 (BD 557669), anti-mouse CD4 V450 at 1 :400 (BD 560468), anti-mouse CD8a APC at 1 :800 (BD 553035), and mouse BITE constructs CTP-059, CTP-060 or a negative control (human BITe construct CTP-064) at 10 nM for 30 minutes on ice. Cells were washed 2x and stained with an anti-HIS tag PE (BioLegend 362603) secondary antibody at 5ul per sample for 30 minutes on ice. Cells were washed 2x again and fixed in 2% PFA (in PBS) on ice for 10 minutes before being washed 2x and resuspended in staining buffer. Acquisition and analysis was performed using a BD FACSCalibur. For analysis, lymphocytes and single cells were gated based on their SSC VS FSC properties. B200+ cells (B cells) were gated out and the remaining lymphocytes were analyzed for fluorescence in the PE channel. The results are provided in FIG. 35. These results demonstrate binding of CD3 by murine BITE constructs CTP-059 and CTP- 060.

For testing of binding of human BITE constructs to T-cells, human PBMCs were isolated from heparinized human blood using Ficol®Paque Plus. 10⁶ cells/sample were stained for flow cytometry as follows: Fc receptors were blocked by incubation with 7.5 ug human Fc-block (BD 564220) for 10 minutes on ice. Cells were washed and stained with an antibody cocktail containing anti-human CD4 FITC at 20ul per 10⁶ cells (BD 555346), anti-human CD8 APC at 20ul per 10⁶ cells (BD 555369), anti-human CD15 & CD16 V450 at 5ul per 10⁶ cells (BD 561584/561310), anti-human CD19 at 5ul per 10⁶ cells (BD 561295) and human BITE constructs CTP-063, CTP-064 or a negative control (mouse CTP-060 BITE construct) at ΙΟηΜ for 30 minutes on ice. Cells were washed 2x and stained with an anti-HIS tag PE secondary antibody (BioLegend 362603) at 5ul per sample for 30 minutes on ice. Cells were washed 2x again and fixed in 2% PFA (in PBS) on ice for 10 minutes before being washed 2x and resuspended in staining buffer. Acquisition and analysis was performed using a BD FACS Calibur. For analysis, lymphocytes and single cells were gated based on their SSC VS FSC properties. B cells were gated out and the remaining CD 19- cells were analyzed for fluorescence in the PE channel. Data are shown in FIG. 36. These results demonstrate binding of CD3 by human BITE constructs CTP-063 and CTP-064.

Example 7

Antibody construct-specific IFN-gamma Production (glucan coated plates)

T-cell activation assay: Induction of IFN-gamma production triggered by Dectin-l-Fc- anti-CD3 (CTP-14), Dectin-l-Fc (CTP-16), anti-mouse CD3s mAb (clone 145-2C11; BD Biosciences Catalog No. BDB550275), and concanavalin A (ConA, Aniara Diagnostica LLC, Fisher Scientific, Catalog No. 50-100-8770) positive control was measured as compared to media-only and splenocytes-only controls. Plates were coated with laminarin (beta- 1,3 -glucan) or BSA, washed, blocked with 1% BSA-PBS, washed, then incubated for 2 h at 37°C with 10- fold serial dilutions of molecules (5.0 to 0.005 ug/ml for Dectin-l-Fc-anti-CD3 (CTP-14), Dectin-l-Fc (CTP-16) and anti-mouse CD3s; or 20 to 0.02 ug/ml for ConA), prior to adding 100 ul (2E5) of freshly prepared mouse splenocytes to each well. Mouse splenocytes were incubated at 37°C +5% C0₂ for 36 h. Tissue culture supematants were harvested and the amount of IFN-gamma produced by the splenocytes was determined using a mouse IFN-gamma ELISA kit (BD Biosciences; Catalog No. 555138). The data are provided in FIGS. 38A and 38B. The results demonstrate that Dectin-l-Fc-anti-CD3 (CTP-14), but not Dectin-l-Fc (CTP-16), induces antigen-specific IFN-gamma production in mouse splenocytes.

Example 8

Antibody construct-specific binding to Aspergillus fumigatus resting conidia, swollen conidia and hyphae

The fungal cell wall composition varies during the process of germination and hyphal growth. Binding to different A. fumigatus morphotypes was tested as described below. Aspergillus fumigatus (A. fumigatus strain 13073) hyphae were plated and stained in groups treated with murine Dectin-l-Fc-anti-CD3 (CTP-14), murine Dectin-l-Fc (CTP-16), and controls. lxlO⁴ A. fumigatus were plated in 8-well (permanox plastic) chamber slides and incubated overnight +/- the presence of 0.03 ug/ml caspofungin. Cells were washed 2x with PBS, slides were blocked using 3% BSA for 1 hr and stained with 1 ug Dectin-l-Fc-anti-CD3 (CTP- 14), Dectin-l-Fc (CTP-16), or mIgG2a isotype control (MOPC-173, BioLegend, Catalog No. 401503) for lhr. Subsequently, cells were washed and incubated with anti-mIgG2a-PE, washed again and mounted using Vectra shield.

The data are provided in FIGS. 39-41. All images are presented at 40X magnification. The results demonstrate that Dectin-l-Fc-anti-CD3 (CTP-14) and Dectin-l-Fc (CTP-16) bind to A. fumigatus hyphae in presence and absence of Caspofungin.

To test for binding in the presence of fetal bovine serum (FBS), lxlO⁴ A. fumigatus were plated in 8-well (permanox plastic) chamber slides and incubated overnight. Cells were washed 2x with PBS, slides were blocked using 3% BSA for lhr and stained with 1 ug Dectin-l-Fc (CTP-16, batch 2 A) in the presence of the indicated % of fetal bovine serum (FBS) for lhr. Subsequently, cells were washed and incubated with anti-mIgG2a-PE, washed again and mounted using Vectra shield. These data are provided in FIG. 42. The results demonstrate that Dectin-l-Fc (CTP-16) binds to A. fumigatus hyphae, and in the presence of the indicated concentrations of FBS.

In a separate set of experiments, the binding of Dectin-l-Fc (CTP-16) and the β-glucan specific mAb CTP-029/030 to resting and swollen conidia was tested. Swollen conidia were generated by shaking resting A. fumigatus conidia for 5 hrs at 220 rpm at 25^°C. Staining was performed as described above. The data are provided in FIGS. 43-46. The results demonstrate that resting conidia are stained poorly indicating low β-glucan exposure (this finding is in accordance with the literature). When conidia start germinating (characterized by swelling), binding of CTP-016 and CTP-029/030 is increased.

Subsequent routine testing for compound binding to A. fumigatus was performed on hyphae. For this, 7.5 x 10³ A. fumigatus (strain 293) conidia per test article were grown at 37°C overnight in an 8-chamber glass slide (EMD Millipore, PEZGS0896) in RPMI with 10% FCS. This results in hyphae formation. The fungus was stained with lOOnM of the test article or Isotype Control (CrownBio C0001-2) for 1 hour, rinsed 2x with PBS and stained with the appropriate secondary antibody (for human constructs: PE anti -human IgG Fc (Biolegend 409304); for murine constructs: for BITEs : anti-His Ab (Thermo Fisher MA1-21315-D550)) for 1 hour. The fungus was rinsed 2x with PBS then fixed in 2% PFA for 10 mins. Each well is stained with the mounting media (Mol Probes P36966) and left to cure at RT. Slides were imaged following a 24-hr curing period. The data are provided in FIGS. 47-52.

Example 9

Construct-specific immune cell-mediated killing/inhibition of fungi

Inhibition of A. fumigatus germination was assessed by measuring the fungal metabolic activity using a standard XTT assay. A. fumigatus strain 293 was streaked on a SDA plate and incubated for 48 hours. After 48 hours, 1 mL of 0.85% NaCl + 0.01 mL of Tween 20 was placed on the sporulating colonies. The tip of a pipet was used to gently probe the colonies, resulting in a mixture of conidia and hyphal fragments. This mixture was transferred to a sterile test tube and heavy particles were allowed to settle for five minutes. After five minutes, the upper homogenous suspension was transferred to a sterile test tube and the OD530 was adjusted to 1.0 in PBS. The suspension was filtered using a 50ml conical tube cell strainer (40um), centrifuged, resuspended in media and counted using a hemocytometer. 10⁵ conidia/well were plated in flat- bottom, tissue culture-treated plates. Dectin-l-Fc, Dectin-1-Fc-CD3, or appropriate isotype controls were incuabted with condia at 37 ° C for lhr. An immune cell preparation was enriched from heparinized human blood using POLYMORPHPREP™ (Fisher Cat# NC9189798) and 2 x 10⁵/well (E:T 2: 1) immune cells/well were incuabted with conidia overnight in a humidified incubator at 37C. The next morning, the immune cells were lysed with high pH water and remaining fungus was washed. Fungal metaboloic activity was measured using a standard XTT assay kit (Cell Signaling Cat# 9095). The % inhibition of germination was calculated as follows: % inhibition= (1-X/C)* 100. Where X is the OD 450nm of a test well and C is the average OD 450nm of fungus only wells.

Human Dectin-l-Fc (CTP-053 and CTP-072) and human Dectin-1-Fc-CD3 constructs (CTP-052 and CTP-054) or isotype control (Crown Bioscience Inc. Cat# COOOl, data not shown) were tested for their ability to enhance human immune cell-mediated fungal damage/inhibition. The data are provided in FIG. 53 and show that the Dectin-1-Fc-CD3 constructs promote enhanced fungal damage/inhibition mediated by immune cells as compared to immune cells alone. Those human Dectin-l-Fc constructs that contain the CD3 -targeting motif, namely CTP- 052 and CTP-054 (including CTP054B and C, data not shown), reproducibly performed better in the fungal damage/inhibition assay than their non-CD3-containig counterparts (CTP-053, CTP- 072) when T-cells are present in the immune cell preparation.

To address the role of T-cells in this assay, the inhibition of germination assay was performed with immune cell preparations that have been depleted of T-cells. Controls were mock treated. Briefly, lx 10⁵ A. Fumigatus 293 conidia/well were plated in a TC-treated 96-well, flat- bottom plates. Human Dectin-1 -based constructs CTP-052, CTP-053, CTP-054 and CTP-072 were added at the indicated concentrations and allowed to incubate with conidia for 1 hour at 37°C. Immune cells were isolated from heparinized human blood using POLYMORPHPREP™. Immune cells were then subjected to a mock depletion or depletion of CD3⁺ cells using magnetic beads (Miltenyi Cat#130-050-101). The resulting T-cell-depleted immune cell preparations (containing <0.03% CD3⁺ cells, data shown in FIG. 55) were added to the conidia at an effector: target ratio of 2: 1. Plates were incubated overnight at 37°C. Following lysis of immune cells with high pH water, the metabolic activity of the remaining A. fumigatus was measuring using a standard XTT assay kit (Cell Signaling Cat#9095). Data are provided in FIG. 54. In the absence of T-cells, human Dectin-l-Fc constructs that contain the CD3-targeting motif were only able to enhance fungal damage/inhibition to the same extent as the non-CD3 -containing human Dectin- l-Fc constructs (FIG. 54). These data demonstrate that CD3 -containing constructs CTP-052 and CTP-054 are able to engage human T-cells, resulting in enhanced fungal damage/inhibition.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A multi-specific binding compound comprising a first polypeptide having binding affinity to a microbial polysaccharide linked to a second polypeptide having binding affinity to an immune cell.

2. The multi-specific binding compound of claim 1, wherein said microbial polysaccharide is a fungal polysaccharide.

3. The multi-specific binding compound of claim 2, wherein said fungal

polysaccharide is a fungal cell wall polysaccharide.

4. The multi-specific binding compound of claim 3, wherein the fungal cell wall polysaccharide is a glucan, a chitin, or a mannan.

5. The multi-specific binding compound of claim 4, wherein the fungal cell wall polysaccharide is a glucan.

6. The multi-specific binding compound of claim 5, wherein the glucan is a β- glucan.

7. The multi-specific binding compound of claim 6, wherein the β-glucan is a β-1,3- glucan or a β-Ι,ό-glucan.

8. The multi-specific binding compound of claim 7, wherein the β-glucan is a β-1,3- glucan.

9. The multi-specific binding compound of any one of claims 1 to 3, wherein

(a) said first polypeptide is an antibody, an antigen-binding fragment thereof, an antibody-like molecule, or an immunoadhesin; or

(b) said second polypeptide is an antibody, an antigen-binding fragment thereof, or an antibody-like molecule; or

(c) said first polypeptide is an antibody, an antigen-binding fragment thereof, an antibody-like molecule, or an immunoadhesin, and said second polypeptide is an antibody, an antigen-binding fragment thereof, or an antibody-like molecule.

10. The multi-specific binding compound of claim 9, wherein said first polypeptide is an antibody, or an antigen-binding fragment thereof.

11. The multi-specific binding compound of claim 9, wherein said second polypeptide is an antibody, or an antigen-binding fragment thereof.

12. The multi-specific binding compound of claim 9, wherein each of said first and second polypeptides is an antibody, or an antigen-binding fragment thereof.

13. The multi-specific binding compound of any one of claims 9 to 12, wherein said antigen-binding fragment is selected from the group consisting of a single-domain antibody, Fab, Fab', F(ab')₂, scFv, and (scFv)₂ fragments.

14. The multi-specific binding compound of claim 9, wherein at least one of said first and second polypeptides is an antibody-like molecule.

15. The multi-specific binding compound of claim 14, wherein said antibody-like molecule is selected from the group consisting of a human domain antibody (dAb), Dual-Affinity Re-Targeting (DART) molecule, a diabody, a di-diabody, dual-variable domain antibody, a Stacked Variable Domain antibody, a Small Modular ImmunoPharmaceutical (SMTP), a

Surrobody, a strand-exchange engineered domain (SEED)-body, and TandAb.

16. The multi-specific binding compound of any one of claims 9 to 15, wherein said fungal polysaccharide is a fungal cell wall polysaccharide.

17. The multi-specific binding compound of claim 16, wherein the fungal cell wall polysaccharide is a glucan, a chitin, or a mannan.

18. The multi-specific binding compound of claim 17, wherein the fungal cell wall polysaccharide is a glucan.

19. The multi-specific binding compound of claim 18, wherein the glucan is a β- glucan.

20. The multi-specific binding compound of claim 19, wherein the β-glucan is a β- 1,3-glucan or a β-Ι,ό-glucan.

21. The multi-specific binding compound of claim 20, wherein the β-glucan is a β- 1,3-glucan.

22. The multi-specific binding compound of claim 9, wherein said first polypeptide is an immunoadhesin.

23. The multi-specific binding compound of claim 22, wherein said immunoadhesin comprises a binding sequence of a fungal polysaccharide binding protein.

24. The multi-specific binding compound of claim 23, wherein said fungal polysaccharaide binding protein is selected from the group consisting of Dectin-1, and Dectin-2.

25. The multi-specific binding compound of claim 24, wherein said fungal polysaccharide binding protein is Dectin-1.

26. The multi-specific binding compound of claim 24, wherein said polysaccharide binding protein is an extracellular domain (ECD) sequence of a Dectin receptor fused to an immunoglobulin (Ig) heavy chain constant region sequence.

27. The multi-specific binding compound of claim 15, wherein the Dectin receptor is a Dectin-1 receptor.

28. The multi-specific binding compound of claim 27, wherein the ECD domain of the Dectin-1 receptor is fused to an Fc region of an immunoglobulin.

29. The multi-specific binding compound of any one of claims 1 to 28, wherein said antibody or immunoglobulin is an IgG.

30. The multi-specific binding compound of claim 29, wherein said IgG is IgGl, I_gG2, IgG3, or IgG4.

31. The multi-specific binding compound of claim 30, wherein said IgG is IgGl .

32. The multi-specific binding compound of any one of claims 1 to 31, wherein said immune cell is a T-cell.

33. The multi-specific binding compound of claim 32, wherein said second polypeptide binds to a T-cell antigen.

34. The multi-specific binding compound of claim 33, wherein said T-cell antigen is a component of the CD3 complex.

35. The multi-specific binding compound of claim 34, wherein said T-cell antigen is

CD38.

36. The multi-specific binding compound of any claims 1-35, wherein at least one of the first or second polypeptides is linked to one or more water soluble polymers.

37. The multi-specific binding compound of any of claims 1-35, wherein at least one of the first or second polypeptides is linked to one or more polyethylene glycol molecules.

38. The multi-specific binding compound of any of claims 1-35, wherein at least one of the first or second polypeptides is coupled with at least one half-life extending moiety.

39. The multi-specific binding compound according to claim 38, wherein the half-life extending moiety is selected from the group consisting of biocompatible fatty acids and derivatives thereof, hydroxy alkyl starch (HAS), hydroxy ethyl starch (HES), polyethelene glycol (PEG), hyaluronic acid (HA), fleximers, dextran, poly-sialic acids (PSA), Fc domains, transferrin, albumin, elastin-like (ELP) peptides, XTEN polymers, albumin binding peptides and combinations thereof.

40. A multi-specific binding compound, comprising a first antibody, or an antigen- binding fragment thereof, comprising a first binding domain to a yeast cell wall polysaccharide, and a second antibody, or an antigen-binding fragment thereof, comprising a second binding domain to a T-cell antigen.

41. The multi-specific binding compound of claim 40, wherein said fungal cell wall polysaccharide is a glucan, a chitin, or a mannan.

42. The multi-specific binding compound of claim 41, wherein the fungal cell wall polysaccharide is a glucan.

43. The multi-specific binding compound of claim 43, wherein the glucan is a β- glucan.

44. The multi-specific binding compound of claim 43, wherein the β-glucan is a β- 1,3-glucan or a β-Ι,ό-glucan.

45. The multi-specific binding compound of claim 44, wherein the β-glucan is a β- 1,3-glucan.

46. The multi-specific binding compound of any one of claims 40 to 45, wherein said T-cell antigen is a component of the CD3 complex.

47. The multi-specific binding compound of claim 46, wherein said T-cell antigen is

CD38.

48. The multi-specific binding compound of any one of claims 40 to 47, wherein the first antibody, or antigen-binding fragment, is human, humanized, or chimeric.

49. The multi-specific binding compound of any one of claims 40 to 48, wherein the second antibody, or antigen-binding fragment, is human, humanized, or chimeric.

50. The multi-specific binding compound of any one of claims 40 to 49, wherein the first antibody, or antigen-binding fragment, is cross-species reactive.

51. The multi-specific binding compound of any one of claims 40 to 50, wherein the second antibody, or antigen-binding fragment, is cross-species reactive.

52. The multi-specific binding compound of any one of claims 40 to 51, wherein the first antibody, or antigen-binding fragment, has cidal activity against a microbial pathogen.

53. The multi-specific binding compound of any one of claims 40 to 52, wherein said antigen-binding fragment is selected from the group consisting of a single-domain antibody, Fab, Fab', F(ab')₂, scFv, and (scFv)₂ fragments.

54. The multi-specific binding compound of any one of claims 40 to 53, comprising a first antibody, or antigen-binding fragment thereof, linked to a second antibody or antigen- binding fragment thereof.

55. The multi-specific binding compound of claim 54, wherein the linkage is through direct fusion.

56. The multi-specific binding compound of claim 54, wherein the linkage is through a linker.

57. The multi-specific binding compound of claim 56, wherein the linker is a peptide or polypeptide linker.

58. The multi-specific binding compound of claim 57 wherein the linker is a peptide linker of about 2 to about 50 residues, or about 4 to about 40 residues, or about 5 to about 30, or about 10 to about 25 residues.

59. The multi-specific binding compound of claim 58, wherein the linker is a (G4S)n linker, where n = 1-9.

60. The multi-specific binding compound of claim 59, wherein the linker is a (G4S)3 linker (SEQ ID NO: 15).

61. The multi-specific binding compound of claim 58, wherein the linker is a tandem linker of SEQ ID NO: 16.

62. The multi-specific binding compound of claim 58, wherein the linker is BGL1 (SEQ ID NO: 16) or BGL2 (SEQ ID NO: 13).

63. A multi-specific single chain antibody, comprising a first binding domain for beta-1,3 glucan (B13G) linked through a linker (L) to a second binding domain for human CD3 (CD3), each of the first and second binding domains comprising a heavy chain variable region (VH) and a light chain variable region (VL), the corresponding VH and VL regions being arranged, from N-terminus to C- terminus, in an order selected from

VH(B 13 G)-VL(B 13G)-L-VH(CD3)-VL(CD3),

VH(CD3)-VL(CD3)-L-VL(B 13G)-VH(B 13 G)

VH(CD3)-VL(CD3)-L-VH(B 13G)-VL(B 13 G)

VL(CD3)-VH(CD3)-L-VH(B 13G)-VL(B 13 G)

VH(CD3)-VL(CD3)-L-VH(B 13G)-VL(B 13 G)

VH(CD3)-VL(CD3)-L-VL(B 13G)-VH(B 13 G)

VL(CD3)-VH(CD3)-L-VH(B 13G)-VL(B 13 G) or

VL(CD3)-VH(CD3)-L-VL(B 13 G)-VH(B 13 G.

64. A multi-specific antibody comprising:

i) a first light chain Complementary Determining Region (Li-CDR) comprising a Li-CDRl, a Li-CDR2, or a L CDR3, wherein Li-CDRl, Li-CDR2 and L CDR3 are at least 90%, or at least 92%, or at least 95%, or at least 98%, or at least 99% homologous, or are substantially 100% identical, to parental OKT3 antibody Li-CDRl, L CDR2 and L CDR3, respectively;

ii) a first heavy chain Complementary Determining Region (H-CDR) comprising a Hi-CDRl, a Hi-CDR2, or a Hi-CDR3, wherein Hi-CDRl, Hi-CDR2 and Hi-CDR3 are at least 90% homologous to parental OKT3 antibody Hi-CDRl, Hi-CDR2 and Hi-CDR3, respectively, iii) a second light chain Complementary Determining Region (L₂-CDR) comprising a L₂-CDRI, a L₂-CDR2, or a L₂-CDR3, wherein L₂-CDR1, L₂-CDR2 and L₂-CDR3 are at least 90%, or at least 92%, or at least 95%, or at least 98%, or at least 99% homologous, or are substantally 100% identical, to parental 2G8 antibody L₂-CDR1, L₂-CDR2 and L₂-CDR3, respectively; and

iv) a second heavy chain Complementary Determining Region (H₂-CDR) comprising a H₂-CDR1, a H₂-CDR2, or a H₂-CDR3, wherein H₂-CDR1, H₂-CDR2 and H₂-CDR3 are at least 90%), or at least 92%, or at least 95%, or at least 98%, or at least 99% homologous, or are substantially 100% identical, to parental 2G8 antibody H₂-CDR1, H₂-CDR2 and H-CDR3, respectively,

or an antigen-binding fragment thereof.

65. The multi-specific antibody of claim 64, wherein said antigen-binding fragment is selected from the group consisting of a single-domain antibody, Fab, Fab', F(ab')₂, scFv, and (scFv)₂ fragments.

66. The multi-specific antibody, or antigen-binding fragment, of any one of claims 63 to 65, which is a human, humanized, or chimeric antibody, or an antigen-binding fragment thereof.

67. A tetravalent bispecific binding compound comprising two antibody heavy and light chain pairs having binding affinity to a fungal cell wall polysaccharide and a binding moiety linked to the C-terminus of each of the antibody light chains, having binfing affinity to a T-cell antigen.

68. The tetravalent bispecific compound of claim 67, wherein the fungal cell wall protein is a glucan, a chitin, or a mannan.

69. The multi-specific binding compound of claim 68, wherein the fungal cell wall polysaccharide is a glucan.

70. The multi-specific binding compound of claim 69, wherein the glucan is a β- glucan.

71. The multi-specific binding compound of claim 70, wherein the β-glucan is a β- 1,3 -glucan or a β-Ι,ό-glucan.

72. The multi-specific binding compound of claim 71, wherein the β-glucan is a β- 1,3-glucan.

73. The tetravalent bispecific binding compound of claim 72, wherein the fungal cell wall polysaccharide is P-l,3-glucan.

74. The tetravalent bispecific compound of any one of claims 67 to 73, wherein the T- cell antigen is a component of the CD3 complex.

75. The tetravalent bispecific ompound of claim 74, wherein the T-cell antigen is

CD38.

76. The tetravalent bispecific binding compound of claim 75, wherein the binding moiety linked to the C-terminus of each of the antibody light chains is a single-chain Fv (scFv) antibody having binding affinity to CD3s.

77. The tetravalent bispecific binding compound of any one of claims 67 to 76, wherein the T-cell antigen binding moiety is linked through a peptide linker.

78. The tetravalent bispecific binding compound of claim 77, wherein the peptide linker is BGL2 (SEQ ID NO: 13).

79. A bispecific binding compound comprising two polypeptide chains each comprising a binding sequence of a fungal polysaccharide binding protein fused to an antibody heavy chain constant region sequence and a binding moiety having binding affinity to a T-cell antigen, linked to the C-terminus of each of said polypeptide chains.

80. The bispecific binding compound of claim 79, wherein the fungal polysaccharide binding protein is Dectin-1 or Dectin-2.

81. The bispecific binding compound of claim 80, wherein the fungal polysaccharide binding protein is Dectin-1.

82. The bispecific binding compound of claim 81, wherein said binding sequence is a Dectin-1 extracellular domain sequence.

83. The bispecific binding compound of any one of claims 79 to 82, wherein the the antibody heavy chain constant region sequence is an Fc region sequence.

84. The bispecific binding compound of any one of claims 79 to 83, wherein the T- cell antigen is a component of the CD3 complex.

85. The bispecific binding compound of claim 84, wherein the T-cell antigen is

CD38.

86. The bispecific binding compound of claim 85, wherein the binding moiety linked to the C-terminus of each of said polypeptide chains is a single-chain Fv (scFv) antibody having binding affinity to CD3s.

87. The bispecific binding compound of any one of claims 79 to 87, wherein the T- cell antigen binding moiety is linked through a peptide linker.

88. The bispecific binding compound of claim 87, wherein the peptide linker is BGL2 (SEQ ID NO: 13).

89. A pharmaceutical composition comprising an effective amount of the binding compound or antibody of any one of claims 1-88.

90. The pharmaceutical composition of claim 89, further comprising a

pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient or a

pharmaceutically acceptable carrier.

91. A method for the treatment of a microbial disease or condition, comprising administering to a subject in need an effective amount of the binding compound or antibody of any one of claims 1 to 88 or a pharmaceutical composition of claim 89 or claim 90.

92. The method of claim 91, wherein said microbial disease or condition is a fungal disease or condition.

93. The method of claim 92, wherein said fungal disease or condition is a fungal infection.

94. The method of claim 93, wherein said fungal infection is a systemic fungal infection.

95. The method of claim 94, wherein said systemic fungal infection is caused by a Candida or an Aspergillus species.

96. The method of claim 95, wherein said systemic fungal infection is caused by Candida albicans, C. parapsilosis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. tropicalis, Aspergillus fumigatus, A.flavus, A. terreus, A. niger, A. candidus, A. clavatus, or A.

97. The method of any one of claims 91 to 96, further comprising administration of a further antifungal agent.

98. The method of claim 97, wherein the further antifungal agent is from the

Echinocandin class of antifungal compounds.

99. The method of claim 98,wherein the further antifungal compound is selected from the group consisting of caspofungin, echinocandin B, anidulafungin, pneumocandin B₀,

aculeacin Α_γ, micafungin, and their derivatives.

100. The method of claim 97, wherein the further antifungal agent is an azole-type antifungal agent.

101. The method of claim 100, wherein the azole-type antifungal agent is selected from the group consisting of voriconazole, clotrimazole, ravuconazole, posacoiiazole, ecoiiazole, fluconazole, itraconazole, tebuconazole, propiconazole, enilaconazole, miconazole, oxiconazole, sulconazole, and tioconazole.

102. Use of a binding compound or antibody of any one of claims 1 to 88 or a pharmaceutical composition of claim 89 or claim 90, in the preparation of a medicament for the treatment of a microbial disease or condition.

103. The use of claim 102, wherein said microbial disease or condition is a fungal disease or condition.

104. The use of claim 103, wherein said fungal disease or condition is a fungal infection.

105. The use of claim 104, wherein said fungal infection is a systemic fungal infection.

106. The use of claim 105, wherein said systemic fungal infection is caused by a Candida or an Aspergillus species.

107. The use of claim 106, wherein said systemic fungal infection is caused by Candida albicans, C. parapsilosis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. tropicalis, Aspergillus fumigatus, A.flavus, A. terreus, A. niger, A. candidus, A. clavatus, or A. ochraceus.

108. The use of any one of claims 102 to 107, further comprising administration of a further antifungal agent.

109. The use of claim 108, wherein the further antifungal agent is from the

Echinocandin class of antifungal compounds.

110. The use of claim 109, wherein the further antifungal compound is selected from the group consisting of caspofungin, echinocandin B, anidulafungin, pneumocandin B₀,

aculeacin Α_γ, micafungin, and their derivatives.

111. The use of claim 108, wherein the further antifungal agent is an azole-type antifungal agent.

112. The use of claim 111, wherein the azole-type antifungal agent is selected from the group consisting of voriconazole, clotrimazole, ravuconazole, posaconazole, econazole, fluconazole, itraconazole, tebuconazole, propiconazole, enilaconazole, miconazole, oxiconazole, sulconazole, and tioconazole.