Antibody molecules and uses thereof

Abstract

This invention relates to recombinant human antibody molecules. The antibodies bind fungal antigens, for example from Candida spp. Human antibody encoding genes targeting clinically relevant Candida epitopes have been isolated from single B cells from carefully selected donors and screened with specified types of protein or cell wall extract. The panel of purified, fully human recombinant IgG1 mAbs generated displayed a diverse range of specific binding profiles and demonstrated efficacy in a disease model. The fully human mAbs and derivatives thereof have utility in the generation of diagnostics, therapeutics and vaccines.

Claims

1. A method comprising contacting a Candida cell with an anti-Candida antibody molecule, wherein the anti-Candida antibody molecule is a whole antibody comprising a VH domain comprising: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 39; (ii) a HCDR2 having the amino acid sequence of SEQ ID NO: 41; and (iii) a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and a VL domain comprising: (i) a LCDR1 having the amino acid sequence of SEQ ID NO: 46; (ii) a LCDR2 having the amino acid sequence of SEQ ID NO: 48; and (iii) a LCDR3 having the amino acid sequence of SEQ ID NO: 50, wherein the binding of the anti-Candida antibody to the Candida cell (i) opsonises, or increases the rate of opsonisation of the Candida cell; or (ii) increases the rate of macrophage engulfment of the Candida cell; or (iii]) increases the rate of macrophage attraction to the Candida cell.

2. A method of treatment of a Candida infection comprising administering an anti-Candida antibody molecule to an individual in need thereof, wherein the antibody molecule is a whole antibody comprising a VH domain comprising: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 39; (ii) a HCDR2 having the amino acid sequence of SEQ ID NO: 41; and (iii) a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and a VL domain comprising: (i) a LCDR1 having the amino acid sequence of SEQ ID NO: 46; (ii) a LCDR2 having the amino acid sequence of SEQ ID NO: 48; and (iii) a LCDR3 having the amino acid sequence of SEQ ID NO: 50.

3. A method according to claim 2 wherein the fungal infection is caused by C. albicans and wherein the infection is in a hyphal or yeast phase.

4. A method according to claim 2, wherein the treatment further comprises administering an additional antifungal agent.

5. A method for detecting the presence or absence of a fungus which is a Candida spp, the method comprising (i) contacting a sample suspected of containing the fungus with an anti-Candida antibody molecule, and (ii) determining whether the anti-Candida antibody molecule binds to the sample, binding of the antibody molecule to the sample indicates the presence of the fungus, and wherein the anti-Candida antibody molecule comprises a VH domain comprising: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 39; (ii) a HCDR2 having the amino acid sequence of SEQ ID NO: 41; and (iii) a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and a VL domain comprising (i) a LCDR1 having the amino acid sequence of SEQ ID NO: 46; (ii) a LCDR2 having the amino acid sequence of SEQ ID NO: 48; and (iii) a LCDR3 having the amino acid sequence of SEQ ID NO: 50.

6. A lateral flow device (LFD) for detecting the presence of an analyte which is a fungal pathogen in a sample fluid, wherein said LFD comprises: (i) a housing, and ii) at least one flow path leading from a sample well to a viewing window, wherein said flow path comprises one or more carriers along which the sample fluid is capable of flowing by capillary action, and wherein the one or more carriers comprise an analyte-detecting means; wherein the presence of analyte produces a line in the viewing window which indicates the presence of the fungal pathogen, wherein the fungal pathogen is a Candida spp. and the at least one analyte-detecting means is an anti-Candida antibody molecule comprising a VH domain comprising: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 39; (ii) a HCDR2 having the amino acid sequence of SEQ ID NO: 41; and (iii) a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and a VL domain comprising: (i) a LCDR1 having the amino acid sequence of SEQ ID NO: 46; (ii) a LCDR2 having the amino acid sequence of SEQ ID NO: 48; and (iii) a LCDR3 having the amino acid sequence of SEQ ID NO: 50.

7. The device as claimed in claim 6, wherein the one or more carriers comprises a plurality of analyte-detecting means, each analyte-detecting means is specific for a different fungal pathogen, and wherein the plurality of analyte-detecting means are capable of distinguishing between multiple fungal pathogens.

8. The device as claimed in claim 7 wherein the multiple fungal pathogens comprise C. albicans, and at least one fungus selected from the group consisting of Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystis jirovecii, a zygomycete fungus, and a skin dermatophytic fungus.

9. The method according to claim 2, wherein the VH domain comprises at least one sequence selected from the following: (i) a FW1 having the amino acid sequence of SEQ ID NO: 38; (ii) a FW2 having the amino acid sequence of SEQ ID NO: 40; (iii) a FW3 having the amino acid sequence of SEQ ID NO: 42; and (iv) a FW4 having the amino acid sequence of SEQ ID NO: 44.

10. The method according to claim 2, wherein the VL domain comprises at least one sequence selected from the following: (i) a FW1 having the amino acid sequence of SEQ ID NO: 45; (ii) a FW2 having the amino acid sequence of SEQ ID NO: 47; (iii) a FW3 having the amino acid sequence of SEQ ID NO: 49; and (iv) a FW4 having the amino acid sequence of SEQ ID NO: 51.

11. The method according to claim 2, wherein the VH domain and the VL domain have amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 20, respectively.

12. The method of claim 2, wherein the fungal infection is caused by a species selected from the group consisting of C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. lusitaniae.

13. The method of claim 4, wherein the additional antifungal agent is an azole, a polyene or an echinocandin.

14. A method comprising contacting a Candida cell with an anti-Candida antibody molecule, wherein the anti-Candida antibody molecule comprises a VH domain comprising: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 39; (ii) a HCDR2 having the amino acid sequence of SEQ ID NO: 41; and (iii) a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and a VL domain comprising: (i) a LCDR1 having the amino acid sequence of SEQ ID NO: 46; (ii) a LCDR2 having the amino acid sequence of SEQ ID NO: 48; and (iii) a LCDR3 having the amino acid sequence of SEQ ID NO: 50, wherein the anti-Candida antibody molecule binds to the Candida cell or a hyphae of the cell, and wherein the method further comprises detecting the bound anti-Candida antibody to identify or detect the Candida cell or a hyphae of the Candida cell.

Description

FIGURES

(1) FIG. 1—Workflow for the generation of human monoclonal antibodies from single B cells. Class-switched memory B cells were isolated from individuals and microcultured in activating media to promote IgG secretion for screening against target antigens. VH and VL genes from B cells positive for the target were amplified and cloned into a mammalian expression vector for expression and purification via fast protein liquid chromatography. Following QC, recombinant mAbs were assessed for functional activity in vitro and in vivo. Adapted from Huang et al. 2013 (38).

(2) FIG. 2—Representative images from the process employed to generate fully human anti-Candida mAbs. FIG. 2A shows the ELISA screening of purified donor circulating IgG against the target antigens C. albicans ‘whole cell’ yeast and hyphae, and purified Hyr1 protein, to select the donors to take forward for B cell isolation. FIGS. 2B and 2C are representative agarose gel images following RT-PCR and nested PCR of VH and Vk-Ck genes respectively. FIGS. 2D and 2E are analytical mass spectrometry and analytical SEC traces of one of the purified recombinant IgG1 mAbs. Further quality control was carried out by SDS-PAGE gel analysis under non-reducing and reducing conditions as shown in FIGS. 2F and 2G.

(3) FIG. 3—Concentration response curves showing anti-Candida mAbs binding to target antigens. FIG. 3A shows purified anti-Hyr1 mAbs binding to purified recombinant Hyr1 protein in a concentration-dependent manner via ELISA. Binding of purified anti-‘whole cell’ Candida mAbs to C. albicans yeast (FIGS. 3B and 3C) and hyphal cells (FIGS. 3D and 3E) via ELISA are also shown. Values represent mean±SEM (n=2-4).

(4) FIG. 4—Indirect immunofluorescence of AB120 binding to Hyr1 protein expressed on C. albicans hyphal cells. Indirect immunofluorescence with anti-Hyr1 mAb AB120 against WT CAl4-Clp10 (A), Hyr1 null mutant (B) and a Hyr1 re-integrant strain (C). A fluorescently conjugated secondary goat anti-human IgG antibody was used to detect anti-Hyr1 mAb binding. Scale bars represent 15 μm.

(5) FIG. 5—Indirect immunofluorescence of anti-whole cell mAbs binding to WT CAl4-Clp10. Indirect immunofluorescence demonstrating the distinct binding patterns of the panel of anti-Candida mAbs. Shown are representative images of mAbs binding strongly to targets expressed on both CAl4-Clp10 yeast and hyphae (A), mAbs binding primarily to targets expressed on hyphae but with some binding to yeast (B), mAbs binding specifically to hyphae (C) and mAbs binding to yeast and the growing hyphal tip (D). A fluorescently conjugated secondary goat anti-human IgG antibody was used to detect anti-Candida mAb binding. Scale bars represent 19 μm.

(6) FIG. 6—Heat-map of anti-Candida mAbs binding to Candida species and other pathogenic fungi. Immunofluorescence microscopy analysis of (a) anti-Hyr1 mAbs (AB120-AB123) and (b) cell wall mAbs (AB1 18-AB140) binding to C. albicans and other clinically relevant fungal species depicted in a heat map. Binding was graded from red (high) to yellow (none).

(7) FIG. 7—Macrophage uptake of live C. albicans cells pre-incubated with saline, isotype control mAb or anti-Candida mAb. FIG. 7A shows the time at which an uptake event occurred over the first 90 min of the assay following C. albicans pre-incubation with saline, an IgG1 control antibody, an anti-whole cell mAb (AB118, AB119 and AB140) or an anti-Hyr1 mAb (AB120). FIG. 7B shows the morphology of C. albicans cells during each uptake event over the first 90 min of the assay following C. albicans pre-incubation with saline, an IgG1 control antibody, an anti-whole cell mAb (AB118, AB119 and AB140) or an anti-Hyr1 mAb (AB120). An uptake event was defined as the complete engulfment of a C. albicans cell by a macrophage. Bars represent percentage of uptake events ±SEM (n=3). *p<0.05, **p<0.01, ****p<0.0001.

(8) FIG. 8—Macrophage engulfment of live C. albicans cells pre-incubated with saline, isotype control mAb or anti-Candida mAb. FIGS. 8A-8C are snapshots taken from live cell video microscopy capturing the stages of C. albicans engulfment by J774.1 macrophages. FIG. 8A shows the macrophage (red, *) and C. albicans (green) prior to cell-cell contact, FIG. 8B shows the cells once cell-cell contact has been established and FIG. 8C shows the C. albicans within the phagocyte following ingestion. FIG. 8D shows the average time taken for a macrophage to engulf a live C. albicans cell following pre-incubation with saline, an IgG1 control antibody, an anti-whole cell mAb (AB118, AB119 and AB140) or an anti-Hyr1 mAb (AB120). FIG. 8E shows the time taken for a macrophage to ingest a filamentous C. albicans cell following pre-incubation of AB120 with hyphal C. albicans. Rate of engulfment was defined as the time taken from cell-cell contact to complete ingestion of the C. albicans cell inside the macrophage resulting in a loss of green fluorescence. Bars represent average time taken for a macrophage to ingest a C. albicans cell ±SEM (n=3) *p<0.01, ***p<0.005.

(9) FIG. 9—Macrophage engulfment of opsonized live C. albicans cells in the presence and absence of an FcγR blocker. The average time taken for a macrophage to ingest a live C. albicans cell following pre-incubation with saline or an anti-whole cell mAb (AB140) in the presence or absence of an FcγR block. Bars represent average time taken for a macrophage to ingest a C. albicans cell ±SEM (n=3) *p<0.05.

(10) FIG. 10—Macrophage migration towards C. albicans cells following pre-incubation with saline, an isotype control mAb or anti-Candida mAb. FIG. 10A shows mean velocity of macrophages as they migrate towards C. albicans cells following pre-incubation with saline, an IgG1 control mAb, or an anti-whole cell mAb (AB140). Bars represent macrophage mean track velocity ±SEM (n=3). FIG. 10B shows average distance travelled by a macrophage to engulf a C. albicans cell following pre-incubation with saline, an IgG1 control mAb, or an anti-whole cell mAb (AB140). Bars represent average distance travelled ±SEM (n=3). FIGS. 10C, 10D and 10E are tracking diagrams representing macrophage migration towards C. albicans cells pre-incubated with saline (blue), AB140 (pink) or IgG1 control mAb (green). Tracks represent the movement of individual macrophages relative to their starting position, up until the first uptake event. *p<0.05, **p <0.01, ***p<0.005.

(11) FIG. 11—Assessment of anti-Candida mAbs in an in vivo model of disseminated candidiasis. C. albicans SC5314 was pre-incubated with saline, IgG1 control, anti-whole cell mAb (AB119) or anti-Hyr1 mAb (AB120) and then injected iv into the tail vein of BALB/c mice (n=6 per group). Kidney fungal burdens from each group were determined on day 3 post infection (FIG. 11A) and combined with the change in animal weight during the course of the infection to give an overall outcome score for disease progression (FIG. 11B). Dots represent individual animals; horizontal lines represent mean, *p<0.05, **p<0.01.

(12) FIG. 12—Schematic of VH, Vκ-Cκ and Vλ-Cλ cloning into pTT5 expression vector. B cells positive for antigen binding in the initial ELISA screen were lysed. mRNA in B cell lysate was used as a template for VH, Vκ-Cκ and Vλ-Cλ gene amplification via RT-PCR. RT-PCR was carried out using forward primers specific to human V domain leader sequences and reverse primers specific for human IgCH1, Cκ or Cλ regions or light chain UTR. To increase the specificity of gene amplification, nested PCR was carried out using RT-PCR products as the template. Forward primers specific for human VH FW1 sequences and reverse primers specific for human VH FW4 sequences were used to amplify VH genes. To capture Vκ-Cκ and Vλ-Cλ genes, forward primers specific to human Vκ and human Vλ FW1 sequences were used in combination with reverse primers specific to the 3′ end of the human Cκ or human Cλ regions. Primers used in nested PCR reactions contained 15 bp extensions which were complementary to the pTT5 expression vector to facilitate downstream Infusion cloning. Amplification of VH, Vκ-Cκ and Vλ-Cλ genes were done in separate reactions. RT-PCR—reverse transcriptase polymerase chain reaction; UTR untranslated region; L—leader sequence; V.sub.H—heavy chain variable domain; Vκ—kappa chain variable domain; Vλ—lambda chain variable domain; C.sub.H— heavy chain constant domain; Cκ—kappa chain constant domain; Cλ—lambda chain constant domain.

(13) FIG. 13—Concentration response curves of purified anti-Hyr1 mAbs screened for binding to unrelated proteins. (a, b) Purified anti-Hyr1 mAbs screened against HSA and HEK NA respectively via ELISA. Values represent mean (n=2-4).

(14) FIG. 14—Concentration response curves showing anti-whole cell mAbs screened for binding to unrelated proteins. (a, b) Purified cell wall mAbs screened against HSA. (c, d) the same mAbs screened against HEK NA via ELISA. Values represent mean (n=2-4).

(15) FIG. 15—Indirect immunofluorescence of mAbs binding to WT CAl4-Clp10 before and after enzymatic modification of the cell wall. Proteinase K treatment was used to reduce protein residues; Zymolyase 20T enzyme was used to digest B-1,3-glucans; Endoglycosidase H treatment reduced N-linked glycans on the CAl4-Clp10 cell wall. Decrease in indirect immunofluorescence after enzymatic treatments suggested the nature of the mAb epitopes. A fluorescently conjugated secondary goat anti-human IgG antibody was used to detect anti-Candida mAb binding. Scale bars represent 4 μm.

(16) FIG. 16—Human monocyte-derived macrophage phagocytosis of live C. albicans cells pre-incubated with saline, isotype control mAb or anti-Candida mAb. (a) Time at which an uptake event occurred over the first 90 min of the assay following C. albicans pre-incubation with saline, an IgG1 control antibody, an anti-whole cell reactive mAb (AB119 and AB140) or an anti-Hyr1 mAb (AB120). Bars represent percentage of uptake events (n=2). (b) Percentage of these uptake events that occurred within the first 30 min of the assay. Dots represent average from individual experiments, line represents average (n=2) and (c) average time taken for a macrophage to engulf a live C. albicans cell following pre-incubation with saline, an IgG1 control antibody, an anti-whole cell mAb (AB119 and AB140) or an anti-Hyr1 mAb (AB120) at a MOI of 3 (n=2).

(17) FIG. 17—Counterimmunoelectrophoresis of anti-Candida mAbs with C. albicans. Purified anti-Candida mAbs AB119, AB140 and AB118C101S reacted with yeast supernatant antigenic preparation and a crude yeast extract (a). AB119, AB140, AB118C101S and AB135 reacted with hyphal supernatant antigenic preparation and a crude hyphal extract (b).

(18) FIG. 18—Immunogold localization of anti-Candida mAbs to the cell wall of C. albicans yeast (top panel) and hyphal (bottom panel) cell walls.

EXAMPLE 1—GENERATION OF FULLY HUMAN ANTI-CANDIDA MABS BY SINGLE B CELL CLONING

(19) The generation of recombinant mAbs through direct amplification of VH and VL genes from single B cells produces fully human, affinity matured mAbs with the native antibody heavy and light chain pairing intact (14). We employed this technology to generate human recombinant anti-Candida mAbs to a defined C. albicans antigen—the morphogenesis-regulated protein 1 (Hyr1) protein expressed only in the hyphal cell wall (40), and to C. albicans whole cell wall preparations. Hyr1 protein was selected based on its role in proposed role in resisting phagocyte killing and pre-clinical data demonstrating that a recombinant N-terminal fragment of Hyr1 confers protection in a murine model of disseminated candidiasis (23, 29, 41). Furthermore, because Hyr1 is expressed solely on C. albicans hyphal cells so mAbs generated against this protein would serve as C. albicans-specific markers. In addition we used C. albicans whole cell wall extracts as a target to screen against allows for the isolation of mAbs that bind to an array of different antigens, anticipating that some of the resulting mAbs would be pan fungal and therefore possess a broad spectrum of therapeutic activity and pan-Candida diagnostic specificity.

(20) To enhance the likelihood of isolating Candida-related antibodies, the class switched memory (CSM) B cells used in this study were isolated from the blood of individuals who had recovered from a superficial Candida infection within a year of sampling. Donors were selected from a panel of volunteers and the levels of target-specific circulating IgG in the donor plasma was assessed via ELISA. In this screen, donor 85 demonstrated the greatest IgG activity against C. albicans whole cell and donor 23 had the highest IgG titre against Hyr1 (FIG. 2A). These donors were selected to provide the source of B cells to use for the generation of Candida-specific recombinant antibodies. After the isolation of CSM B cells from a donor, approximately 80000-150000 cells were plated out at 5 cells/well and activated with a cocktail of cytokines and supplements to promote secretion of IgG into the supernatant. A high throughput screening platform was then employed to facilitate the detection of IgG in the B cell supernatant against target antigens by ELISA. Positive ELISA hits enabled identification of wells containing B cells secreting antigen-specific IgG into the supernatant. Typically, approximately 0.05% wells/screen were positive (OD>4×background). Non-specific hits were identified and eliminated by performing an ELISA screen against two unrelated proteins—human serum albumin (HSA) and human embryonic kidney nuclear antigen (HEK NA). CSM B cells from wells that were positive for the antigen screen and negative for the unrelated protein screen were then lysed and used as the source for VH, Vκ-Cκ and Vλ-Cλ gene amplification via RT-PCR and nested PCR (FIGS. 2B, C). VH, Vκ-Cκ and Vλ-Cλ genes were sub cloned into the pTT5 mammalian expression vector and the sequences analysed (data not shown). Corresponding heavy and light chains originating from the same hit well were co-transfected into Expi293F cells for small scale whole IgG1 expression. From these co-transfections, recombinant mAbs that demonstrated binding to the original target were selected for large scale recombinant expression. These were then purified via affinity-based FPLC using a protein A resin and quality control checked via analytical mass spectrometry, SDS-PAGE gel analysis and analytical SEC (FIGS. 2D-G).

(21) In total, 18 purified recombinant IgG1 mAbs were generated using the single B cell technology described above. Five of these mAbs bound to purified Hyr1 protein and 13 bound to C. albicans whole cells (Table S3).

EXAMPLE 2—PURIFIED RECOMBINANT ANTI-CANDIDA MABS EXHIBIT SPECIFIC TARGET BINDING

(22) Purified anti-Hyr1 mAbs were primarily assessed for functionality through binding to the purified recombinant N-terminus of Hyr1 protein via ELISA. Four of the five mAbs demonstrated strong binding to the purified antigen with EC.sub.50 values of 104 ng/ml, 76.5 ng/ml, 49.6 ng/ml and 53.3 ng/ml for AB120, AB121, AB122 and AB123 (FIG. 3A) respectively. AB124 bound to Hyr1 with a lower affinity with an EC.sub.50 value of 1050 ng/ml. To examine the specificity of these mAbs for the target protein, all five were tested against the unrelated antigens HSA and HEK nuclear antigen as negative controls and demonstrated no binding (FIG. 13).

(23) The purified recombinant anti-whole cell mAbs were originally screened and isolated against C. albicans overnight culture. As such, the initial QC of these mAbs was to assess their binding to C. albicans whole cells via ELISA. The majority of purified anti-whole cell mAbs bound C. albicans yeast cells with high affinity with EC.sub.50 values ranging from 2.8 to 31.1 ng/ml (FIGS. 3B, C). AB134 and AB135, which have similar amino acid sequences, both demonstrated slightly lower affinity for the target with EC.sub.50 values of 1060 and 224 ng/ml respectively (FIG. 3C).

(24) Purified anti-whole cell mAbs exhibited a variety of affinities when binding to C. albicans cells where both yeast and hyphal morphologies were present (FIGS. 3D, E). The majority bound these cells with high affinity with EC.sub.50 values ranging between 3 and 50 ng/ml. As observed with C. albicans yeast cell binding, AB134 and AB135 demonstrated slightly lower affinities with EC.sub.50 values of 684 and 69.4 ng/ml. EC.sub.50 values were used here as a simple comparison to demonstrate the variability in anti-whole cell mAbs binding to C. albicans cell surface antigens. Therefore this methodology generated a panel of mAbs which bound to a variety of specific cell targets. Specificity of the anti-whole cell mAbs for a target C. albicans antigen was assessed through binding to the two unrelated antigens HSA and HEK NA. All mAbs demonstrated no binding to these antigens confirming their specificity for the fungal cells (FIG. 14).

EXAMPLE 3—PURIFIED RECOMBINANT ANTI-CANDIDA MABS SHOW DISTINCT BINDING PATTERNS TO C. albicans and Other Fungal Species

(25) The recombinant anti-Hyr1 mAbs generated by single B cell technology were initially isolated by screening against N-terminus of Hyr1 protein and, following purification, demonstrated binding to this recombinant antigen (above). We then visualized binding of these mAbs to Hyr1 protein expressed on the C. albicans cell surface by immunofluorescent staining using a fluorescently labelled secondary anti-human IgG mAb for detection. It was observed that the anti-Hyr1 mAbs bound to the predicted cellular location on the hyphae, and not the WT C. albicans yeast cells grown in different culture conditions (FIG. 4A). We verified that the anti-Hyr1 mAbs did not bind to hyphae of a Δhyr1 null mutant (FIG. 4B) and that binding was restored in a C. albicans strain containing a single reintegrated copy of the deleted HYR1 gene (FIG. 4C).

(26) Next we visualised binding to WT C. albicans for the anti-whole cell mAbs via indirect immunofluorescent staining. The anti-whole cell mAbs demonstrated a range of binding profiles to WT C. albicans (FIG. 5). mAbs AB118, AB119, AB129, AB130, AB133, AB134, AB135, AB139, AB140 bound strongly to both C. albicans yeast and hyphae (FIG. 5A). AB132 bound to both yeast and hyphae but exhibited stronger binding to hyphae (FIG. 5B). AB126 and AB131 appeared to be hypha-specific (FIG. 5C) and AB127 stained the mother yeast cell and the tip of the growing hyphae (FIG. 5D). Therefore the panel of antibodies apparently detected both morphology specific and morphology-independent epitopes.

(27) C. albicans cells were enzymatically treated with proteinase K, endoglycosidase H (endo-H) and zymolyase 20T and assessed for mAb binding. Proteinase K treatment reduced AB120 (anti-Hyr1) but not anti-whole cell mAbs binding to C. albicans confirming that anti-Hyr1 antibody recognised a protein epitope (FIG. 15a). Following zymolyase 20T and endo-H treatments, binding of other anti-whole cell mAbs decreased suggesting that the cognate epitopes might be β-glucan or N-mannan respectively (FIG. 15b, c). Some anti-whole cell mAbs demonstrated increased fluorescence after enzymatic treatment suggesting that their epitopes might be located deeper in the cell wall.

(28) Commensurate with the C. albicans-specific nature of HYR1, anti-Hyr1 mAbs only bound to C. albicans and not to a range of other Candida species (FIG. 6a). In contrast, a range of binding patterns were observed for the binding of anti-whole cell mAbs to other pathogenic fungal species. The majority of mAbs bound strongly to the closely related species C. dubliniensis, C. tropicalis, C. parapsilosis and C. lusitaniae. There was little binding of mAbs to the more distantly related C. glabrata and C. krusei. Only the homologous AB131 and AB132 antibodies demonstrated some weak binding to C. krusei(FIG. 6b).

(29) To assess for pan-fungal binding activity, all the anti-whole cell mAbs were tested against A. fumigatus. C. neoformans, C. gattii, P. carinii, M. circinelloides and M. dermatis but no binding was observed (FIG. 6b). Therefore the anti-Hyr1 mAbs are C. albicans-specific and the anti-whole cell mAbs demonstrate a variety of binding patterns to WT C. albicans and other pathogenic Candida species, indicating that they target a range of different antigens and the expression levels of these antigens varies from species to species.

(30) In conclusion, all purified recombinant mAbs generated by this single B cell technology bound specifically to their target antigens with high affinity. As expected, the anti-whole cell mAbs demonstrated distinct binding patterns to WT C. albicans and other pathogenic fungi, indicating that they target a range of different antigens and the expression levels of these antigens varies from species to species.

EXAMPLE 4—PURIFIED RECOMBINANT ANTI-CANDIDA MABS OPSONISE C. ALBICANS FOR PHAGOCYTOSIS BY MACROPHAGES

(31) Phagocytic cells of the innate immune system are the first line of defence against fungal pathogens. Antibody binding enhances phagocytic clearance of pathogens. We utilised a live cell phagocytosis assay to examine whether the anti-Candida mAbs generated in this study opsonized C. albicans for phagocytosis by J774.1 macrophages and human monocyte-derived macrophages. The macrophages were challenged with live, C. albicans CAl4-Clp10 which had been pre-incubated with an anti-Candida mAb, an isotype control mAb or saline for 1 h. Live cell video microscopy using our standard phagocytosis assay (42, 43) was employed to determine the degree of opsonisation. No significant difference was observed between the saline control and anti-Candida mAb groups in terms of the overall number of C. albicans cells taken up during the 3 h by macrophages. However, there was a difference in the time by which the majority of uptake events had occurred (FIG. 7A). C. albicans cells that had been pre-incubated with either AB118, AB119 or AB140 (anti-whole cell mAbs) were taken more rapidly compared to the saline control-treated fungal cells, the IgG1 control pre-incubated fungal cells or AB120 pre-incubated fungal cells. The percentage of uptake events occurring by 20 min was 21±10, 54±9, 22±5 and 68 2, 44.3±0.6 and 7±2 (mean±SD) for saline control, AB118, AB120, AB140, AB119 and isotype control respectively (FIG. 7A). A majority of C. albicans cells pre-incubated with AB118, AB119 or AB140 were taken up as yeast cells and the majority of cells taken up by the saline control group, AB120 group and isotype control group, were hyphal cells (FIG. 7B).

EXAMPLE 5—MACROPHAGES RAPIDLY ENGULF MAB-BOUND C. ALBICANS CELLS THROUGH FCγR BINDING

(32) Next we used live cell video microscopy and image analysis to examine whether there was any difference in the rate of engulfment between C. albicans cells pre-incubated with saline compared to C. albicans cells pre-incubated with selected anti-Candida mAbs. As shown previously we defined the rate of engulfment as the time taken from establishment of cell-cell contact to the time at which a C. albicans cell had been completely engulfed by a macrophage as indicated by its loss of FITC green fluorescence (42, 43) (FIGS. 8A-C). When C. albicans yeast cells were pre-incubated with AB120 (anti-Hyr1 mAb) there was no difference in the rate of engulfment from the saline control or IgG1 control mAb however, in the presence of either AB118, AB119 or AB140 (anti-whole cell mAbs), fungal cells were engulfed at a significantly faster rate compared to the saline control and IgG1 control mAb, (FIG. 8D). The hypha-specific mAb AB120 stimulated faster macrophage engulfment of C. albicans hyphal cells by macrophages—taking an average of 5.8±0.3 min to engulf opsonised hyphae compared to 8.8±0.8 min for the control (FIG. 8E).

(33) Similar observations were obtained using human monocyte-derived macrophages (FIG. 16).

(34) Blocking FcγRs on the surface of the macrophage decreased the rate of engulfment of AB140-bound C. albicans compared to that of the saline control (FIG. 9) indicating that the increased rate of engulfment of mAb-bound Candida cells is, at least in part, due to uptake through the FcγRs.

EXAMPLE 6—MACROPHAGES MIGRATE FURTHER, FASTER AND MORE DIRECT TOWARDS ANTI-CANDIDA MAB BOUND C. ALBICANS CELLS

(35) We showed that antibody-bound C. albicans cells were cleared earlier by macrophages than control cells. To determine the effect of antibody binding on uptake dynamics, we used imaging analysis to digitise the migration of macrophages until their first uptake event, measuring the distance travelled, directionality and velocity of the macrophage towards control or antibody-bound fungal cells. Macrophages travelled further and at a greater velocity towards C. albicans yeast cells that had been pre-incubated with a whole-cell mAb (AB140) compared to control fungal cells or those pre-incubated with IgG1 control mAb (FIG. 10 A,B). Furthermore we observed that macrophages moved in a more directional manner towards antibody-bound C. albicans cells compared to control cells or those pre-incubated with IgG1 control mAb (FIGS. 10 C, D and E).

EXAMPLE 7—ANTI-WHOLE CELL MAB REDUCES FUNGAL BURDEN IN A MODEL OF DISSEMINATED CANDIDIASIS

(36) To determine whether the anti-Candida mAbs possessed therapeutic potential in vivo, their action was assessed in a murine model of systemic candidiasis (44). C. albicans SC5314 yeast cells were pre-incubated for 1 h with either saline, an IgG1 isotype control mAb, AB119 (anti-whole cell) or AB120 (anti-Hyr1) before iv injection into the mouse lateral tail vein. Disease progression was monitored by weight change and kidney fungal burdens at day 3 which together generated an overall outcome score for disease progression (44). When SC5314 was pre-incubated with AB120 there was no decrease in fungal burden compared to the saline control or the IgG1 control mAb (FIG. 11A). However, when AB119 was pre-incubated with SC5314, there was a significant decrease in kidney fungal burden compared to the saline control (FIG. 11A, p<0.01). This was also considerably less than the kidney fungal burden for the IgG1 isotype control. By weight change there was no significant difference in disease outcome score between AB120 and the saline control and isotype control (FIG. 11B). However, mice that had been injected with SC5314 pre-incubated with AB119 had a significantly lower disease outcome score than both the saline control group (p<0.01) and the isotype control group (p<0.05) indicating that when AB119 is present, the mice are able to clear infection more quickly and disease progression is limited (FIG. 11B). Therefore exposure to antibody improved the survival of mice in a systemic disease model.

EXAMPLE 8—DISCUSSION OF EXAMPLES 1-7

(37) Monoclonal antibodies (mAbs) have the potential to be used in multiple fungal therapy and disease management situations. Here we describe and use for the first time a novel technology facilitating the isolation of fully human anti-Candida mAbs against whole cells and a specific cellular target. These mAbs were derived directly from single B cells from donors with a history of mucosal Candida infection and demonstrated distinct binding profiles to C. albicans and other pathogenic fungi, as well as the ability to opsonise fungal cells and to enhance phagocytosis and show partial protection in a murine model of disseminated candidiasis.

(38) mAbs-based agents have been identified as an alternative strategy to complement the medical gaps associated with current antifungal treatments and diagnostics (13, 45, 46). In this study we generated 18 fully human recombinant anti-Candida mAbs through the direct amplification of mRNA isolated from VH and VL antibody genes produced naturally in vivo in response to a Candida infection. By employing this method, the purified, affinity matured recombinant mAbs generated were less likely to be immunogenic, had importantly retained their native antibody heavy and light chain pairings, and therefore are more likely to be of therapeutic benefit (35). IgG1 was selected as the antibody scaffold because this isotype makes up the majority of mAbs in the clinic and so is the best characterised in terms of drug development (47, 48). Thirteen of the mAbs generated bound to C. albicans whole cell and 5 bound to recombinant purified Hyr1 protein—a protein which is considered to be important for C. albicans resistance to phagocytosis and is currently in development as an experimental vaccine (29, 41) demonstrating that this novel technology can be utilised for screening against a wide range of specific antigens.

(39) An antibody that recognises an antigen expressed across different fungal species could be highly beneficial as a pan-fungal therapeutic. At the same time, one of the major contributors to poor prognosis in the clinic is the lack of accurate and timely diagnostics with a knock on delay in appropriate treatment (6, 7, 49). In this case, it would be more beneficial to have a species-specific antibody which recognises an antigen only expressed on one species. As such, we assessed binding of our panel of mAbs to a number of emerging and resistant pathogenic fungi. We observed that anti-Hyr1 mAbs bound solely to C. albicans hyphae, correlating with findings that have reported that Hyr1 is only expressed on C. albicans hyphal cells (29, 40, 50). The binding pattern of anti-whole cell mAbs was more varied with the majority of mAbs binding strongly to the species that are closely related to C. albicans such as the emerging pathogens C. tropicalis and C. parapsilosis (51). As expected, little or no binding was observed to the more evolutionarily distinct species C. glabrata and C. krusei. Altogether this demonstrates that the novel technology employed here can be utilised to generate species-specific as well as pan fungal mAbs, which has great implications in terms of anti-fungal drug discovery and diagnostics. Furthermore, these mAbs could be utilised to isolate and identify protective antigens for development as fungal vaccines.

(40) One of the many ways mAbs exert their protective effects is through opsonizing cells for phagocytosis (15). We have shown previously that by employing live cell imaging we can breakdown this process down into its component parts, thus allowing us to do a more in-depth analysis on the effect of mAbs on the individual stages of phagocytosis (42, 43). Here we observed that when yeast and hyphal cells were coated with an anti-whole cell mAb or a hyphal cell was coated with an anti-Hyr1 mAb, cells were engulfed at a significantly faster rate compared to unopsonized cells, and this was through engagement of the FcγR. Furthermore, macrophages migrated further, faster and in a more direct manner towards opsonized C. albicans cells and this contributed to earlier clearance of fungal cells.

(41) A number of invasive infections occur in the immunocompetent patient population as a consequence of severe trauma, and in these situations opsonizing mAbs could be a viable treatment option. The majority of antibody therapeutics in the clinic are hlgG1 so this isotype has been routinely tested pre-clinically in murine models of disease (47). Furthermore, the literature shows that hlgG1 binds to all activating mFcγRs with a similar profile to the most potent IgG isotype in mice, mlgG2a, validating the use of mouse models to assess Fc-mediated effects of hlgG1 mAbs (47). As such, we utilised an established three-day murine model of disseminated candidiasis (44, 52) to assess the efficacy of anti-Candida mAbs in vivo and observed a significant decrease in kidney fungal burden and overall disease outcome score when C. albicans was pre-incubated with an anti-whole cell mAb.

(42) We have generated fully human antibodies from single B-cells to create reagents that have high specificity for targets with utility in the antifungal diagnostic and therapeutic markets. The antibodies are of high affinity and are and can be synthesised in milligram quantities under defined conditions for heterologous protein expression.

(43) The relative by which these antibodies can be produced means that they could be used singly or in multiplex formats to create novel polyvalent diagnostic tests, as vaccine Candidates or as therapeutic delivery systems to target toxic molecules to specific microbial or cellular targets.

EXAMPLE 9—CIE ANALYSIS

(44) FIG. 17 shows the results of counterimmunoelectrophoresis (CIE) analysis. This shows selected mAbs were able to detect C. albicans antigens in a format commonly used for the diagnosis of patients with a Candida infection.

EXAMPLE 10—TEM ANALYSIS

(45) FIG. 18 shows transmitting electron microscopy (TEM) images illustrating the binding of a select panel (one mAb from each CDR3 amino acid sequence cluster) of the anti-whole cell mAbs to C. albicans yeast and hyphal cell walls via immunogold labelling. The images show that the mAbs are very specific to the cell wall and that there are a variety of binding targets, for example AB126, AB127 and AB131 appear mainly to bind to hypha, whereas AB118C101S, AB119, AB140 and AB135 appear to bind to more abundantly expressed targets in both yeast and hyphal cells.

(46) General Methods

(47) Candida Strains and Growth Conditions

(48) C. albicans serotype A strain CA14+Clp10 (NGY152) was used as a control and its parent strain CA14, used to construct the Δhyr1 null mutant C. albicans strain (40) and the hyr1 re-integrant strain (unpublished). The clinical isolates C. albicans SC5314, C. glabrata SC571182B, C. tropicalis AM2005/0546, C. parapsilosis ATCC22019, C. lusitaniae SC5211362H, C. krusei SC571987M, C. dubliniensis CD36 are shown in Table S1. All strains were obtained from glycerol stocks stored at −80° C. and plated onto YPD plates (2% (w/v) mycological peptone (Oxoid, Cambridge, UK), 1% (w/v) yeast extract (Oxoid), 2% (w/v) glucose (Fisher Scientific, Leicestershire, UK) and 2% (w/v) technical agar (Oxoid)). Candida strains tested were routinely grown in YPD (see above without the technical agar) except in the in vivo experiments where strains were grown in NGY medium (0.1% (w/v) Neopeptone (BD Biosciences), 0.4% (w/v) glucose (Fisher Scientific), 0.1% (w/v) yeast extract (Oxoid). Aspergillus fumigatus clinical isolate V05-27 was cultured on Potato Dextrose Agar slants for seven days before the spores were harvested by gentle shaking with sterile 0.1% Tween 20 in PBS. Harvested spores were purified, counted and re-suspended at a concentration of 1×10.sup.8 spores/ml. Swollen spores were generated by incubation in RPMI media for 4 h at 37° C.

(49) Malassezia dermatis CBS9169 was cultured on Modified Dixon agar (3.6% (w/v) Malt extract (Oxoid), 1% (w/v) Bacto peptone (BD Biosciences), 2% (w/v) Bile salts (Oxoid), 1% (w/v) Tween40 (Sigma), 0.2% (w/v) Glycerol (Acros Organics), 0.2% (w/v) Oleic acid (Fisher Scientific), 1.5% technical Agar (Oxoid)) supplemented with chloramphenicol (0.05% (w/v) Sigma) and cycloheximide (0.05% (w/v) Sigma)). Overnight culture of M. dermatis was grown in Modified Dixon Medium. Mucor circinelloides CBS277.49 was grown on Potato Dextrose Agar for 7 days before spores were harvested in PBS and filtered through 40 μm Nylon Cell Strainer (BD Biosciences). Cryptococcus neoformans KN99a and Cryptococcus gattii R265 were grown in YPD overnight, washed in PBS and 1×10.sup.7 cells were added to 6 ml RPMI+10% FCS in 6 well-plates. Plates were incubated at 37′C+5% CO.sub.2 for 5 days to induce capsule formation. Harvested cells were washed in PBS. Rat lung tissue isolates of Pneumocystis carinii M167-6 were washed in PBS and immunostained.

(50) Generation of Recombinant Hyr1 N— Protein

(51) The recombinant N-terminus of the Hyr1 protein (amino acids 63 to 350—Table S2) incorporating an N-terminal 6×His tag was expressed in HEK293F cells and purified by nickel-based affinity chromatography using a nickel NTA superflow column (QIAGEN, USA). Fractions containing the recombinant N-terminus of the Hyr1 protein were pooled and further purified via Analytical Superdex 200 gel filtration chromatography (GE Healthcare, USA) in PBS. QC of the recombinant protein via SDS-PAGE gel analysis, analytical size exclusion chromatography (SEC) and Western blot (using an anti-His antibody for detection) confirmed a protein of 32 kDa (data not shown).

(52) Identification of Human Anti-Hyr1 and Anti-Whole Cell mAbs from Donor B Cells PBMC Isolation

(53) In brief, peripheral venous blood from donors who had recovered from a Candida infection within the last year was collected in EDTA-coated vacutainers tubes and pooled. PBMCs and plasma were separated from the whole blood suspension via density gradient separation using Accuspin System-Histopaque-1077 kits (Sigma-Aldrich) according to manufacturer's instructions. Following separation, the plasma layer was aspirated and stored at 4° C. for later analysis of antibody titre and the PBMC layer was aspirated and washed in PBS and centrifugation at 250×g for 10 min three times before final resuspension at a concentration of 1×10.sup.7 cells/ml in R10 media (RPMI 1640 (Gibco, Life Technologies), 10% FCS, 1 mM sodium pyruvate (Sigma), 10 mM HEPES (Gibco, Life Technologies), 4 mM L-glutamine (Sigma), 1×penicillin/streptomycin (Sigma)) containing additional 10% FCS and 10% DMSO. PBMCs were split into 1 ml aliquots and stored in liquid nitrogen until they were required.

(54) Purification of Donor Plasma

(55) IgG was purified from donor plasma using VivaPure MaxiPrepG Spin columns (Sartorius Stedman) according to manufacturer's instructions. In brief, plasma sample was applied to the spin column to facilitate IgG binding. The column was washed twice in PBS and then bound IgG was eluted in an amine buffer, pH 2.5 and neutralized with 1 M Tris buffer, pH8. Eluted IgG concentration was measured by absorbance at 280 nm using a NanoVue Plus Spectrophotometer (GE Healthcare).

(56) Circulating IgG Enzyme-Linked Immunosorbent Assay (ELISA) to Identify Donors with B Cells to Take Forward

(57) To identify the donor to use for subsequent class switched memory (CSM) B cell isolation and activation, ELISAs were carried out against the target antigens using IgG purified from donor plasma. NUNC maxisorp 384-well plates (Sigma) were coated with C. albicans overnight culture (whole cell) or 1 μg/ml purified, recombinant N-terminus hyr1 protein antigen in 1×PBS and incubated at 4° C. overnight. The next day, wells were washed three times with wash buffer (ixPBS+0.05% Tween) using a Zoom Microplate Washer (Titertek). Wells were then blocked with block buffer (1×PBS+0.05% Tween+0.5% BSA) for 1 h at room temperature with gentle shaking to inhibit non-specific binding. After three washes (as above), titrated purified IgG or IVIG in block buffer was added in duplicate, and the plates were incubated for 2 h at room temperature with gentle shaking. Wells were washed with wash buffer as above before addition of goat anti-human IgG, HRP conjugated (ThermoScientific) secondary antibody at 1:5000 dilution in blocking buffer. Plates were incubated for 45 min at room temperature with gentle shaking. To develop the ELISA, wells were washed three times with wash buffer (as above) before the addition of TMB (Thermo Scientific). Plates were incubated at room temperature for 5 min to allow the blue colour to develop and the reaction was quenched by the addition of 0.18 M sulphuric acid. The plates were then read at an OD of 450 nm on an Envision plate reader (PerkinElmer). Labstats software in Microsoft Excel was used to generate concentration-response curves for EC.sub.50 determination and donor selection for subsequent CSM B cell isolation and activation.

(58) Isolation of Class Switched Memory B Cells

(59) The PBMCs from donors who displayed a strong IgG response to the antigen of interest in the screening ELISA were taken forward for CSM B cell isolation and activation. The process of generating recombinant mAbs from a single donor's B cells to one particular antigen, beginning with the isolation of CSM B cells all the way through to expression and purification of recombinant mAbs, was termed an ‘Activation’. For each Activation, 5×10.sup.7 PBMCs were removed from the liquid nitrogen store and thawed by adding pre-warmed R10 media drop wise to the cells. The diluted cell suspension was then transferred into a fresh polypropylene tube containing pre-warmed R10, resulting in a final cell dilution of approximately 1:10. Benzonase nuclease HC, purity >99% (Novagen) was added at a 1:10000 dilution (to ensure any Iysed cells and their components didn't interfere with the live cells), and the cells were centrifuged at 300×g for 10 min at room temperature and the supernatant removed. PBMCs were then washed again in R10 before final resuspension in 1 ml R10 for PBMC cell number and viability determination.

(60) Isolation of class switched memory B cells from PBMCs was carried out by magnetic bead separation using a Switched Memory B cell isolation kit with Pre-Separation Filters and LS columns (MACS Miltenyi Biotec) according to manufacturer's instructions. In brief, counted PBMCs were incubated with a cocktail of biotin-conjugated antibodies against CD2, CD14, CD16, CD36, CD43, CD235a (glycophorin A), IgM and IgD. Cells were then washed and incubated with anti-biotin microbeads. Following another wash step, the suspension was passed through a Pre-Separation Filter (to remove cell aggregates) before applying it to an LS column where the magnetically labelled cells were retained in the column and the unlabelled CSM B cells passed through and could be collected in the flow-through for determination of cell number and viability.

(61) Activation of CSM B Cells

(62) To activate CSM B cells and promote antibody secretion into the supernatant, a mixture of cytokines, mAb, TLR agonist and a supplement were added to the R10 media (see above) to make complete R10 media. CSM B cells were resuspended in complete R10 media at 56 cells/ml and then plated out at 90 μl/well (5 cells/well) in ThermoFisher Matrix 384 well plates using a Biomek FX (Beckman Coulter). Cells were incubated at 37° C., 5% CO.sub.2 for seven days. On day 7, 30 μl/well of supernatant was removed and replaced with 30 μl fresh complete R10. On day 13, all the supernatant was harvested from all plates and screened against the antigen of interest via ELISA. B cell activation and culturing was monitored by measuring IgG1 concentrations in B cell supernatants at day 7 and day 13.

(63) B Cell Supernatant Screen Against Target Antigens Via ELISA

(64) For B cell supernatant screening against target antigens, NUNC maxisorp 384-well plates (Sigma) were coated with C. albicans overnight culture (whole cell) or 1 μg/ml purified, recombinant N-terminus hyr1 protein antigen in 1×PBS and incubated at 4° C. overnight. Wells were washed three times with wash buffer using a Zoom Microplate Washer (Titertek) as above before incubation with blocking buffer for 1 h at room temperature with gentle shaking. After another three washes (as above), B cell supernatant was added and the plates incubated for 2 h at room temperature with gentle shaking. Wells were washed with wash buffer as above before addition of goat anti-human IgG, HRP conjugated (ThermoScientific) secondary antibody at 1:5000 dilution in blocking buffer and incubation for 45 min at room temperature with gentle shaking. ELISAs were developed and plates read at an OD of 450 nm on an Envision plate reader (PerkinElmer).

(65) Positive hits were defined as wells with an OD.sub.450 reading >4× background. B cells in ‘positive hit’ wells were resuspended in lysis buffer (ml DEPC-treated H2O (Life Technologies), 10 μl 1 M Tris pH 8, 25 μl RNAsin Plus RNAse Inhibitor (Promega)) and stored at −80° C.

(66) Generation of Recombinant Anti-Hyr1 and Anti-Whole Cell IgG1 mAbs: Amplification of VH, Vκ-Cκ and Vλ-Cλ Genes—cDNA Synthesis and PCR

(67) A schematic of the cloning protocol is shown in FIG. 12. Primers used for the RT-PCR reaction were based on those used by Smith et. al., (36). To ensure all possible VH germline families were captured during the amplification, four forward primers specific to the leader sequences encompassing the different human VH germline families (VH1-7) were used in combination with two reverse primers; both placed in the human CgCH1 region. For the RT-PCR of human Vκ-Cκ genes, three forward primers specific to the leader sequences for the different human Vκ germline families (Vκ1-4) were used with a reverse primer specific to the human kappa constant region (Cκ) and two further reverse primers which were specific to the C- and N-terminal ends of the 3′ untranslated region (UTR). To capture the repertoire of human Vλ genes, 7 forward primers capturing the leader sequences for the different human Vλ germline families (Vλ1-8) were used in a mixture with two reverse primers which were complementary to the C- and N-terminal ends of the 3′ UTR and another reverse primer specific to the human lambda constant region (Cλ).

(68) Prior to cDNA synthesis, B cell lysates were thawed and diluted 1:5, 1:15 and 1:25 in nuclease-free H.sub.2O (Life Technologies) before addition of oligodT.sub.20 (50 μM) (Invitrogen, Life Technologies) and incubation at 70° C. for 5 min. Reverse transcription and the first PCR reaction (RT-PCR) were done sequentially using the QIAGEN OneStep RT-PCR kit according to manufacturer's instructions. For this step and the subsequent nested PCR step, amplification of the variable domain of human Ig heavy chain genes (VH), the variable and constant domains of human Ig kappa light chain genes (Vκ-Cκ) and the variable and constant domains of human Ig lambda light chain genes (Vλ-Cλ), were done in separate reactions. In brief, a reaction mixture was prepared containing QIAGEN OneStep RT-PCR Buffer 5×, dNTPs (10 mM), gene-specific forward and reverse primer mixes (10 μM), QIAGEN OneStep RT-PCR Enzyme Mix and nuclease-free H.sub.2O. Reaction mixture was then added to wells of a 96-well PCR plate before addition of neat or diluted (1:5, 1:15, 1:25) B cell lysate as the template, resulting in a final reaction volume of 50 μl/well. The following cycling conditions were used for the RT-PCR reaction; 50° C. for 30 min, 95° C. for 15 min then 35-40 cycles of (94° C. for 1 min, 55° C. for 1 min and 72° C. for 1 min) with a final extension at 72° C. for 10 min.

(69) Amplification of VH, Vκ-Cκ and Vλ-Cλ Genes—Nested PCR Reaction

(70) Nested PCR reactions were carried out using the PCR products from the RT-PCR reaction as the template, nested gene-specific primers based on Smith et al. (36) and Platinum PCR SuperMix High-Fidelity (Invitrogen, Life Technologies). A total of 27 forward primers specific for the VH framework 1 (FW1) sequence were used together with two reverse primers specific for the framework 4 (FW4) region of the VH gene. For nested PCR of the Vκ-Cκ gene, a mixture of 18 forward primers specific for human Vκ FW1 sequence were used with a reverse primer specific to the human kappa constant region 3′ end. For amplification of the Vλ-Cλ gene, a mixture of 31 forward primers specific for human Vλ FW1 sequences were used together with a reverse primer that was placed at the 3′ end of the human lambda constant region. The primers used to generate the PCR fragments in these nested PCR reactions contained 15 bp extensions which were complementary to the target downstream pTT5 expression vector. Reaction mixtures containing Platinum PCR SuperMix High Fidelity, gene-specific forward primer mix (10 μM) and gene specific reverse primer mix (10 μM) was added to wells in a 96-well PCR plate before addition of cDNA template. Amplification of VH genes, Vκ-Cκ genes and Vλ-Cλ genes, were done in separate reactions. After the nested PCR reaction, samples were analysed via agarose gel electrophoresis and positive hits identified and taken forward for downstream InFusion cloning with pTT5 mammalian expression vector.

(71) pTT5 Mammalian Expression Vector Preparation

(72) The pTT5mammalian expression used for mAb expression (licensed from the National Research Council of Canada (NRCC)) (53). The pTT5 vector plasmid contained an IgG1 heavy chain gene in the multiple cloning site so digestion to generate the heavy chain (HC) backbone for downstream sub cloning of VH was done by double digestion using FastDigest Restriction enzymes (Thermo Scientific) with BssHII before the leader sequence of the VH region and SaA restriction after the FW4 of the VH domain. This yielded the heavy chain constant region in the vector backbone. For double digestion of the vector to generate the light chain (LC) backbone, the whole IgG1 heavy chain gene was with BssHII and BamHI astDigest Restriction enzymes (Thermo Scientific) to generate the vector ready for insertion of either κ-Cκ or Vλ-Cλ. Digestion reactions to generate HC and LC backbones were carried out separately. Following confirmation of digestion, samples were run on a 1% agarose gel and bands were excised from the gel and purified using the QIAquick Gel Extraction kit (QIAGEN). DNA was quantified on a NanoVue Plus Spectrophotometer (GE Healthcare). To prevent vector self-ligation, the 3′- and 5′-termini of the linearized plasmids were dephosphorylated using FastAP Thermosensitive Alkaline phosphatase (Thermo Scientific). Reaction mixtures were cleaned up using the MinElute Reaction Cleanup Kit (QIAGEN) and then run on a 1% agarose gel. Bands corresponding to dephosphorylated HC and LC backbones were excised from the gel and purified using the QIAQuick Gel Extraction kit (QIAGEN) as above. Dephosphorylated linearized vector DNA was quantified on a NanoVue Plus spectrophotometer (GE Healthcare).

(73) In-Fusion Cloning

(74) The In-Fusion HD Cloning Kit (Clontech, USA) was used to clone the IgG VH, Vκ-Cκ and Vλ-Cλ genes into a pTT5 mammalian expression vector. To avoid the need for nested PCR product purification before cloning, cloning enhancer (Clontech, USA) was added to each nested PCR product in a 96-well PCR plate and incubated at 37° C. for 15 min, then 80° C. for 15 min. The cloning enhancer-treated PCR product was then added to the In-Fusion Enzyme Premix and linearized vector DNA (˜5-10 ng). Reactions were made up to 10 μl with nuclease-free H.sub.2O and incubated for 15 min at 50° C. Samples were then either stored at −20° C. or placed on ice before transformation of Stellar Competent cells (Clontech). For transformation, 2 μl of each In-Fusion reaction mixture was added to cells in a 96-well plate format, and left on ice for 30 min before heat shock at 42° C. for 40 sec and then returning to ice for 2 min. Cells were then recovered in SOC medium (Clontech, USA) with gentle shaking at 37° C. for 45-60 min before plating out onto LB agar plates (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, 1.5% (w/v) agar) containing 100 μg/ml ampicillin. Plates were incubated at 37° C. overnight and single colonies picked the next day.

(75) Plasmid DNA Generation for Transfection

(76) Following transformation, 8-16 single colonies per initial hit well for VH, Vκ and Vλ were picked and used to inoculate 2×TY media containing 100 μg/ml ampicillin in a Greiner deep well, 96-well plate (Sigma). VH, Vκ and Vλ plates were set up separately with the same plate layout to facilitate visual screening. Cells were grown at 370° C., 200 rpm overnight, and glycerol stocks were made the following day and stored at −80° C. To ensure accurate tracking of DNA sequences for downstream sequencing and transfections, each well inoculated by a single colony was given a unique ID based on the colony's original hit well and its position in the deep well 96 well plate following transformations. To obtain plasmid DNA for gene sequencing and small scale mammalian transfections, DNA minipreps from the overnight cultures were carried out in a 96-well plate format using the EPmotion (Eppendorf), according to manufacturer's instructions. DNA not taken for gene sequencing was stored at −20° C. until required for small scale transfections. Sequence data was analysed for CDR diversity and comparisons to germline sequences and used to identify clones to take forward for small scale transfection.

(77) Small Scale Expression of Recombinant mAbs

(78) Following VH, Vκ and Vλ gene sequencing, a file was generated containing all possible VH and Vκ/Vλ combinations resulting from the original hit wells from the primary ELISA screen. Automated mixing of the native heavy and light chain DNA pairing combinations (1.5 μg of HC plasmid DNA and 1.5 μg of LC plasmid DNA) into a new 96-well plate was facilitated through a HAMILTON MICROLAB® Starline liquid handling platform (Life Science robotics, Hamilton Robotics). Subsequent mixed DNA was used for small scale transient transfection of 3 ml of suspension cultured Expi293F cells (Life Technologies, USA) at a density of 2.5×10.sup.6 cells/ml in 24-well tissue culture plates using the Expifectamine 293 Transfection kit (Life Technologies, USA) in accordance with manufacturer's instructions. Expi293F cells were maintained in pre-warmed (37° C.) sterile Expi293 expression media (Invitrogen) without antibiotics at 37° C., 7% CO.sub.2, 120 rpm shaking. Supernatants were harvested on day 6 and recombinant mAb expression was quantified using anti-human IgG Fc sensors on an Octet QK.sup.e (ForteBio, Cλ, USA) for identification of mAbs to upscale.

(79) Large Scale Expression, Purification and QC of Recombinant mAbs

(80) For downstream large scale mammalian transfections, where a greater amount of DNA was required, DNA was prepared using a QIAGEN Plasmid Maxi Kit (QIAGEN, USA) according to manufacturer's instructions with typical yields of 1.5 μg/μl.

(81) For large scale mAb expression, 100 μg of total DNA (50 μg of HC plasmid DNA and 50 μg LC plasmid DNA) was used to transiently transfect 100 ml of suspension cultured Expi293F cells (Life Technologies, USA) at a density of 2.5×10.sup.6 cells/ml using the Expifectamine 293 Transfection Kit (Life Technologies, USA) in accordance with the manufacturer's instructions. Supernatants were harvested on day 6 and recombinant mAb expression was quantified as above using an Octet QK.sup.e (ForteBio). Recombinant mAbs were purified via affinity based Fast Protein Liquid Chromatography using HiTrap Protein A HP columns on an ÄKTA (GE Healthcare) and eluted in 20 mM citric acid, 150 nM NaCl (pH2.5) before neutralisation with 1 M Tris buffer (pH8). Purified mAbs were dialysed in PBS overnight and IgG concentration was quantified on a NanoVue Spectrophotometer (GE Healthcare). All purified recombinant mAbs were quality control checked via SDS-PAGE gel analysis using 4-12% Bis-Tris SDS-PAGE gels under reducing and non-reducing conditions to confirm mass, analytical size exclusion chromatography (SEC) to check for protein aggregation/degradation and analytical mass spectrometry to confirm the amino acid sequence identity of each mAb. Purified recombinant mAbs were also tested for functionality by binding to target antigen/whole cell via ELISA.

(82) ELISA with Purified Recombinant mAbs

(83) For confirmation of binding to target as purified recombinant mAbs an ELISA was carried out using the protocol for B cell supernatant screen. The only change was that titrated purified recombinant mAb was added in place of B cell supernatant.

(84) Immunofluorescence Imaging of Anti-Hyr1 and Anti-Whole Cell mAbs Binding to Fungal Cells

(85) Indirect immunofluorescence was performed using purified recombinant mAbs. A single Candida colony was used to inoculate 10 ml YPD medium and incubated at 30° C., 200 rpm overnight. Overnight cultures were diluted 1:1333 in milliQ water and then added to a poly-L-lysine coated glass slide (Thermo Scientific, Menzel-Glaser) and incubated for 30 min at room temperature to allow for adherence of yeast cells to the slide. To induce filamentation, cells were incubated in pre-warmed RPMI+10% FCS at 37° C. for 90 min-2 h (this step was omitted for staining of yeast cells), after which they were washed in Dulbecco's Phosphate Buffered Saline (DPBS) and fixed with 4% paraformaldehyde. Cells were washed again and blocked with 1.5% normal goat serum (Life Technologies) before staining with an anti-Candida mAb at 1-10 μg/ml for 1 h at room temperature. After three PBS washes, cells were stained with Alexa Fluor@ 488 goat anti-human IgG antibody (Life Technologies) at a 1:400 dilution and incubated at room temperature for 1 h in the dark. For additional staining of fungal cell wall chitin, Calcofluor White (CFW) was added at 25 μg/ml and cells were incubated for 10 min at room temperature in the dark and washed with DPBS. Slides were left to air dry before adding one drop of Vectashield mounting medium (Vector Labs) and applying a 20 mm×20 mm coverslip to the slide. Cells were imaged in 3D on an UltraVIEW® VoX spinning disk confocal microscope (Nikon, Surrey, UK).

(86) Preparation of Human Monocyte-Derived Macrophages

(87) Human monocyte-derived macrophages were isolated from the blood of healthy volunteers. In brief, the PBMC layer was isolated as described above and was then washed and re-suspended in DMEM medium (Lonza, Slough, UK) supplemented with 200 U/ml penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK) and 2 mM L-glutamine (Invitrogen, Paisley, UK). Serum was separated from blood using standard methods and heat-inactivated at 56° C. for 20 min before use. Monocytes were isolated from PBMCs via positive selection using CD14 microbeads (MACS, Miltenyi Biotec) according to manufacturer's instructions. PBMCs were incubated with MicroBeads conjugated to monoclonal anti-human CD14 antibodies. Cells were then washed and run through an LS column in a magnetic field causing the CD14.sup.+ cells to be retained in the column and the unlabelled cells to run through. The CD14.sup.+ cells were then eluted and resuspended in supplemented DMEM containing 10% donor-specific serum, for determination of cell count and viability. Monocytes were then plated out at a density of 1.2×10.sup.5 cells/well in an 8-well glass based imaging dish (Ibidi, Munich, Germany) and incubated at 37°, 5% CO.sub.2 for 7 days. Cells were used in imaging experiments on day 7. Immediately prior to phagocytosis experiments, supplemented DMEM was replaced with pre-warmed supplemented CO.sub.2-independent media (Gibco, Invitrogen, Paisley, UK) containing 1 μM LysoTracker Red DND-99 (Invitrogen, Paisley, UK). LysoTracker Red is a fluorescent dye that stains acidic compartments in live cells, enabling tracking of these cells during phagocytosis and phagolysosome maturation.

(88) Preparation of J774.1 Mouse Macrophage Cell Line

(89) J774.1 macrophages (ECACC, HPA, Salisbury, UK) were maintained in tissue culture flasks in DMEM medium (Lonza, Slough, UK) supplemented with 10% (v/v) FCS (Biosera, Ringmer, UK), 200 U/ml penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK) and 2 mM L-glutamine (Invitrogen, Paisley, UK) and incubated at 37° C., 5% CO.sub.2. For phagocytosis assays, macrophages were seeded in 300 μl supplemented DMEM at a density of 1×10.sup.5 cells/well in an 8-well glass based imaging dish (Ibidi, Munich, Germany) and incubated overnight at 37° C., 5% CO.sub.2. Immediately prior to phagocytosis experiments, supplemented DMEM was replaced with 300 μl pre-warmed supplemented CO.sub.2-independent media (Gibco, Invitrogen, Paisley, UK) containing 1 μM LysoTracker Red DND-99 (Invitrogen, Paisley, UK).

(90) Preparation of Fluorescein Isothiocyanate (FITC)-Stained C. albicans

(91) C. albicans colonies were grown in YPD medium and incubated at 30′C, 200 rpm overnight. Live C. albicans cells were stained for 10 min at room temperature in the dark with 1 mg/ml FITC (Sigma, Dorset, UK) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) (BDH Chemicals, VWR International, Leicestershire, UK). Following the 10 min incubation, in phagocytosis assays using C. albicans FITC-labelled yeast, the cells were washed three times in 1×PBS to remove any residual FITC and finally re-suspended in 1×PBS or 1×PBS containing purified anti-Candida mAb at 1-50 μg/ml. For assays where pre-germinated C. albicans was to be added to immune cells, cells were washed and re-suspended in supplemented CO.sub.2-independent media with or without anti-Candida mAb at 1-50 ag/ml and incubated at 37° C. with gentle shaking for 45 min.

(92) Live Cell Video Microscopy Phagocytosis Assays

(93) Phagocytosis assays were performed using our standard protocol with modifications (42, 43, 54). Following pre-incubation with/without anti-Candida mAb, live FITC-stained wild type C. albicans (Cλ14-Clp10) yeast or hyphal cells were added to LysoTracker Red DND-99-stained J774.1 murine macrophages or human monocyte-derived macrophages in an 8-well glass based imaging dish (Ibidi) at a multiplicity of infection (MOI) of 3. Video microscopy was performed using an UltraVIEW® VoX spinning disk confocal microscope (Nikon, Surrey, UK) in a 37° C. chamber and images were captured at 1 min intervals over a 3 h period. At least three independent experiments were performed for each antibody and at least 2 videos were analysed from each experiment using Volocity 6.3 imaging analysis software (Improvision, PerkinElmer, Coventry, UK). Twenty five macrophages were selected at random from each experiment and analysed individually at 1 min intervals over a 3 h period. Measurements taken included: C. albicans uptake—defined as the number of C. albicans cells taken up by an individual phagocyte over the 3 h period; C. albicans rate of engulfment—defined as the time point at which cell-cell contact was established until the time point at which C. albicans was fully engulfed (a fungal cell was considered to have been fully ingested when its FITC-fluorescent signal was lost, indicating that the fungal cell was now inside the phagocyte and not merely bound to the phagocyte cell surface) and finally Volocity 6.3 imaging analysis software was used to measure the distance travelled, directionality and velocity of macrophages at 1 min intervals during the first hour of the assay which provided a detailed overview of macrophage migration towards C. albicans cells.

(94) Mean values and standard deviations were calculated. One- or two-way ANOVλ followed by Bonferroni multiple comparison tests or unpaired, two-tailed t tests were used to determine statistical significance.

(95) Systemic Candidiasis Infection Model

(96) A well-established three-day model of disseminated candidiasis was employed to assess the efficacy of anti-Candida mAbs in vivo (44, 52). On day 0, ˜3.2×10.sup.5 C. albicans SC5314 yeast cells were pre-incubated at RT with 7.5 mg/kg purified recombinant anti-Candida mAb for 60 min to allow binding of the antibody to the Candida cell surface before administration intravenously via the lateral tail vein. Assessment of disease progression was carried out by observation and weighing on successive days from day 0 up to and including day 3, at which point the animals were culled and the kidneys harvested for analysis of fungal burden. Fungal burdens were quantitated by homogenising the organ, and plating out serial dilutions on Sabouraud dextrose agar plates (1% mycological peptone (w/v), 4% glucose (w/v), 2% agar (w/v)) before incubation at 35° C. overnight. Colonies were counted the next day and fungal burden expressed as log CFU per gram of infected organ. An overall disease outcome score devised from the combination of 3-day weight loss and kidney burden data was also generated to assess disease progression.

(97) Enzymatic Modification of Candida albicans Cell Wall

(98) For proteinase K treatment, single colonies of Candida were inoculated into 10 ml YPD medium and incubated at 30° C., 200 rpm overnight. Cultures were diluted in milliQ water and then adhered on poly-L-lysine coated glass slides. To induce filamentation, cells were incubated in pre-warmed RPMI+10% FCS at 37° C. for 90 min-2 h. Slides were washed with DPBS and cells were treated with 50 μg/ml proteinase K at 37° C. for 1 h. For Endo-H and zymolyase 20T treatments, C. albicans overnight yeast cells were washed and resuspended in DPBS. Filamentous cells were induced as above. Cells were washed in DPBS and resuspended in Glycobuffer and Endoglycosidase H (10 U/μl; NEB) or Buffer S and Zymolyase 20T (50 U/g wet cells; MPBIO) at 37° C. for 2 h. Cells were then washed in DPBS and fixed with 4% paraformaldehyde, washed and blocked with 1.5% normal goat serum (Life Technologies) before staining with an anti-Candida mAb at 1 μg/ml for 1 h at room temperature. After 3 washes with DPBS, cells were stained with Alexa Fluor® 488 goat anti-human IgG antibody (Life Technologies) at a 1:400 dilution and incubated at room temperature for 1 h prior to imaging in 3D on an UltraVIEW® VoX spinning disk confocal microscope (Nikon, Surrey, UK).

(99) Preparation of Human Monocyte-Derived Macrophages

(100) Human macrophages were derived from monocytes isolated from the blood of healthy volunteers. PBMCs were resuspended in Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Slough, UK) supplemented with 200 U/ml penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK) and 2 mM L-glutamine (Invitrogen, Paisley, UK). Serum isolated from blood was heat inactivated for 20 min at 56° C. PBMCs were seeded at 6×10.sup.5 in 300 μl/well supplemented DMEM medium containing 10% autologous human serum, onto an 8-well glass based imaging dish (Ibidi, Munich, Germany) and incubated at 37° C. with 5% CO.sub.2 for 1 h 45 min to facilitate monocyte adherence to the glass surface. Floating lymphocytes in the supernatant were aspirated and the same volume of fresh pre-warmed supplemented DMEM containing 10% autologous human serum added to the well. Cells were incubated at 37° C., 5% CO.sub.2 for 7 days with media changed on days 3 and 6. Cells were used in imaging experiments on day 7. Supplemented DMEM was replaced with pre-warmed supplemented CO.sub.2-independent media containing 1 μM LysoTracker Red DND-99 (Invitrogen) immediately prior to phagocytosis experiments.

(101) Counterimmunoelectrophoresis

(102) Agar gels were prepared (Veronal buffer+0.5% (w/v) purified agar+0.5% (w/v) LSA agarose+0.05% (w/v) sodium azide, pH 8.2) and wells were cut out using a cutter. Into one column of wells, 10 μl of neat anti-Candida mAb was added. The same volume of antigen (crude C. albicans yeast or hyphal preparation (following glass bead disruption of cells and 1 min centrifugation at 13000 rpm to generate disrupted cell wall/glass bead slurry and cell supernatant antigenic preparations)) was added to the second column of wells and gels were placed into an electrophoresis tank containing veronal buffer. Gels were oriented so that the antibody wells were lined up alongside the anode and the antigen wells alongside the cathode due to antibody migration towards the cathode via electroendosmosis and antigen migration towards the anode due to lower isoelectric points than the buffer pH. The gels were run at 100V for 90 min before removal and immersion in saline-trisodium citrate overnight. The following day the gels were rinsed with water and covered with moistened filter paper and left to dry in an oven for 2 h. Once dried, the filter paper was moistened and removed and the gels put back into the oven for a further 15 min to dry completely. Gels were then immersed in Buffalo black solution (0.05% (v/v) Buffalo black, 50% (v/v) distilled water, 40% (v/v) methylated spirit, 10% (v/v) acetic acid) for 10 min before destaining in destaining solution (45% (v/v) industrial methylated spirits, 10% (v/v) acetic acid, 45% (v/v) distilled water) for 10 min. Gels were then dried and examined for the formation of precipitin lines. The results are shown in FIG. 17.

(103) High-Pressure Freezing (HPF) of Samples for Immunogold Labelling of C. albicans Cells with Anti-Candida mAbs for Transmission Electron Microscopy (TEM).

(104) C. albicans yeast and hyphal cell samples were prepared by high-pressure freezing using an EMPACT2 high-pressure freezer and rapid transport system (Leica Microsystems Ltd., Milton Keynes, United Kingdom). Using a Leica EMAFS2, cells were freeze-substituted in substitution reagent (1% (w/v) OsO4 in acetone) before embedding in Spurr resin and polymerizing at 60° C. for 48 h. A Diatome diamond knife on a Leica UC6 ultramicrotome was used to cut ultrathin sections which were then mounted onto nickel grids. Sections on nickel grids were blocked in blocking buffer (PBS+1% (w/v) BSA and 0.5% (v/v) Tween20) for 20 min before incubation in incubation buffer (PBS+0.1% (w/v) BSA) for 5 min×3. Sections were then incubated with anti-Candida mAb (5 μg/ml) for 90 min before incubation in incubation buffer for 5 min a total of 6 times. mAb binding was detected by incubation with Protein A gold 10 nm conjugate (Aurion) (diluted 1:40 in incubation buffer) for 60 min before another six 5 min washes in incubation buffer followed by three 5 min washes in PBS and three 5 min washes in water. Sections were then stained with uranyl acetate for 1 min before three 2 min washes in water and then left to dry. TEM images were taken using a JEM-1400 Plus using an AMT UltraVUE camera. The results are shown in FIG. 18.

(105) TABLE-US-00002 TABLE S1 Clinical isolates and strains Strain name Genotype Reference CAl4 + Clp10 (NGY152) ura3Δ::λimm434/ura3Δ::λimm434 Brand et al. 2004 RPS1/rps1::URA3 hyr1Δ hyr1Δ::hisG/hyr1Δ:hisG-URA-3-hisG Bailey et al. 1996 hyr1Δ + HYR1 hyr1::hisG/hyr1::hisG/RPS1/rps1::HYR1 Belmonte (unpublished) tup1Δ tup1Δ::hisG/tup1Δ::hisG-URA3-hisG Fonzi & Irwin 1993 C. albicans SC5314 Clinical isolate Gillum et al. 1984 C. glabrata SCS71182B Clinical isolate Odds et al. 2007 C. tropicalis AM2005/0546 Clinical isolate Clinical isolate from Aberdeen C. lusitaniae Clinical isolate Odds et al. 2007 SCS211362H C. krusei SCS71987M Clinical isolate Odds et al. 2007 C. parapsilosis Clinical isolate Rudek 1978 ATCC22019 C. dubliniensis CD36 Clinical isolate Moran et al. 1998 A. fumigatus V05-27 Clinical isolate Netea et al. 2003 C. auris CBS 10913T Clinical isolate Satoh et al. 2009 C. haemulonii CBS 5149T Clinical isolate Khan et al. 2007 C. neoformans KN99α H99 mating type α Nielsen et al. 2003 C. gattii R265 Clinical isolate Fyfe et al. 2002 P. carinii M167-6 Isolated from rat lung tissue — M. dermatis CBS 9169 CBS Sugita et al. 2002 M. circinefioides CBS CBS Li et al. 2011 277.49

(106) TABLE-US-00003 TABLE S2 Recombinant Hyr1 protein amino acid sequence. The leader sequence is underlined and the 6xHis tag is in italics, and is followed by the linker ′G′. Hyr1 protein amino acids 63-350 make up the remainder of the sequence. Recombinant  Amino acid sequence SEQ protein ID antigen name (amino acids 63-350) NO: Recombinant  METDTLLLWVLLLWVPGSTGGSGHHHHHH 1 Hyr1 N-terminus GEVEKGASLFIKSDNGPVLALNVALSTLV fragment RPVINNGVISLNSKSSTSFSNFDIGGSSF TNNGEIYLASSGLVKSTAYLYAREWTNNG LIVAYQNQKAAGNIAFGTAYQTITNNGQI CLRHQDFVPATKIKGTGCVTADEDTWIKL GNTILSVEPTHNFYLKDSKSSLIVHAVSS NQTFTVHGFGNGNKLGLTLPLTGNRDHFR FEYYPDTGILQLRAAALPQYFKIGKGYDS KLFRIVNSRGLKNAVTYDGPVPNNEIPAV CLIPCTNGPSAPESESDLNTPTTSSIGT

(107) TABLE-US-00004 TABLE S3 Purified recombinant human IgG1 mAbs generated using the single B cell technology. Antibody Yield (mg) Target AB-120 12 Hyr1 protein AB-121 28.5 Hyr1 protein AB-122 67.9 Hyr1 protein AB-123 67.3 Hyr1 protein AB-124 38.9 Hyr1 protein AB-118 7.5 C. albicans ‘whole cell’ AB-119 13.5 C. albicans ‘whole cell’ AB-126 60.9 C. albicans ‘whole cell’ AB-127 24.5 C. albicans ‘whole cell’ AB-129 2.3 C. albicans ‘whole cell’ AB-130 1.1 C. albicans ‘whole cell’ AB-131 24.1 C. albicans ‘whole cell’ AB-132 9.3 C. albicans ‘whole cell’ AB-133 19 C. albicans ‘whole cell’ AB-134 7.7 C. albicans ‘whole cell’ AB-135 16.5 C. albicans ‘whole cell’ AB-139 12.2 C. albicans ‘whole cell’ AB-140 19.5 C. albicans ‘whole cell’

(108) TABLE-US-00005 TABLE VH SEQ AB VH ID name VH FW1 CDR1 VH FW2 VH CDR2 VH FW3 VH CDR3 VH FW4 NO: 06- VH3 QVTLKESGGGLVQPG RTY WVRQDPG RLDEVGRLT RFTISRDNAKNILYLQMN DLSGSADY WGQGTLV 2 AB- GSLRLSCVASGFTF WMH KGLVWVS SYADSVNG SLRAEDTGVYYCAR TVSS 119 06- VH3 EVQLVESGGGLVQPG SNY WVRQVPG RINEDGSVT RFTISRDNAKNTLYLQM DLCGERDD WGQGTLV 3 AB- GSLRLSCSASQFIL WVH EGLVWVS SYADSVKG NSLRVDDTAVYYCVR SVSS 118 06- VH3 EVQLVQSGGGLVQPG TSY WVRQAPG VITGNVGTS RFTISRDNSKKTVSLQM TRYDFSSGYY WGQGTLV 4 AB- GSLGLSCAASGFIF AMT KGLEWVS YYADSVKG NSLRAEDTAIYYCVK FDD SVSS 120 06- VH3 EVQLVESGGTLVQPG SDY WVRQAPG NIKQDGSEK RVTISRDNAQNSVFLQM DGYTFGPATT WGRGTLV 5 AB- GSLRLSCAASGFTF WMN KGLEWVA YYVDSLRG HSLSVEDTAVYYCAR ELDH SVSS 121 06- VH3 EVQLVQSGGGLAQPG DDF WVRQPPG GLIVVNGGSI RFTISRDNAKNSLFLQM GLSGGTMAPF WGQGTMV 6 AB- RSLRLSCAASGFGF AMH KGLEWVS DYAGSVRG NSLRAEDTALYYCAK DI SVSS 122 06- VH3 EVQLLESGGGVVQPG SNY WVRQAPG VVWFDGSY RFTISRDNSKSTLYLQM PIMTSAFDI WGPGTMV 7 AB- RSLRLSCAASGFTF GMH KGLEWVA KYYTDSVKG NSLRAEDTAVYYCVS SVSS 123 06- VH3 EVQLVESGGGVVQPG SNY WVRQAPG VVWLDGSY RFTISRDNSKSTLYLQM PIMTSAFDI WGPGTMV 8 AB- RSLRLSCAASGFTF GMH KGLEWVA KYYTGSVKG NSLRAEDTAAYYCVS TVSS 124 06- VH3 EVQLVESGGGLAQPG AGN WVRQAPG AIGGSDDRT RFTISRDKSKNTLSLQM DIWRWAFDY WGQGTLV 9 AB- GSLRLSCEASGFHL AMA KGLEWVA DYADSVKG NSLRVEDTAVYYCAK SVSS 126 06- VH3 EVQLVESGGGLVNPG SNY WVRQAPG SISRSGDYIY RSTISRDNAKNSLFLQM DWGRLGYCSS WGQGTRV 10 AB- GSLRLSCAASGFTF AMN KGLEWVS YADSLKG NSLRAEDSAVYYCAR NNCPDAFDV SVSS 127 06- VH3 QVQLVESGGGLVQPG SNY WVRQVPG RINEDGSVT RFTISRDNAKNTLYLQM DLCGERDD WGQGTLV 11 AB- GSLRLSCSASQFIL WVH EGLVWVS SYADSVKG NSLRVDDTAVYYCVR TVSS 129 06- VH3 QLQLQESGGGLVQPG SNY WVRQVPG RINEDGSVT RFTISRDNAKNTLYLQM DLCWERDD WGQGTLV 12 AB- GSLRLSCSASQFIL WVH EGLVWVS SYADSVKG NSLRVDDTAVYYCVR SVSS 130 06- VH3 QVQLVQSGGGVVQPG KISI WVRQAPG AMSYDGFSK RLTISRDSSTNTLYLEMN EAYTSGRAGC WGQGVLV 13 AB- GSLRLSCAASPFTF LH KGLEWVS YYADSVKG SLRFEDTALYFCAR FNP SVSS 131 06- VH3 QVLKESGGGVVQPGG ETSI WVRQAPG AMSYDGFSK RLTISRDSSTNTLYLEMN EAYTSGRAGC WGQGVLV 14 AB- SLRLSCAASPFTF LH KGLEWVS YYADSVKG SLRFEDTALYFCAR FDP SVSS 132 06- VH3 EVQLVESGGGLVQPG NTY WVRQAPG RINEDGTTIS RFTISRDNAENTLYLQM DFTGPFDS WGQGTLV 15 AB- GSLRVSCAASGFTL WMH KGLVWVS YADSVRG HSLRAEDTGVYYCAR SVSS 133 06- VH3 QLQLQESGGGLVQPG SSH WVRQAPG SISISGGDTF RFTIFRDNSKNTVYLQM ETSPNDY WGQGTLV 16 AB- GSLRLSCVVSGFTF AMS KGLEWVS YADSVRG NSLRAEDTAVYYCAT SVSS 134 06- VH3 EVQLVETGGGLVQPG SSH WVRQAPG SISISGGDTF RFTIFRDNSKNTVYLQM ETSPNDY WGQGTLV 17 AB- GSLRLSCVVSGFTF AMS KGLEWVS YADSVRG NSLRAEDTAVYYCAT TVSS 135 06- VH3 EVQLVESGGGLVQPG NTY WVRQAPG RINEDGTTIS RFTISRDNAENTLYLQM DFTGPFDS WGQGTLV 18 AB- GSLRVSCAASGFTL WMH KGLVWVS YADSVRG HSLRAEDTGVYYCAR SVSS 139 06- VH3 EVQLVESGGGLVQPG NTY WVRQAPG RINEDGTTIS RFTISRDNAENTLYLQM DFTGPFDS WGQGTLV 19 AB- GSLRVSCAASGFTL WMH KGLVWVS YADSVRG HSLRAEDTGVYYCAR SVSS 140

(109) TABLE-US-00006 TABLE VL SEQ AB VL VL ID name VL FW1 CDR1 VL FW2 CDR2 VL FW3 VL CDR3 VL FW4 NO: 06-AB- VK2 DVVLTQSPLFLPVT RSSQSLLHS WYLQKPGQS SVFN GVPDRFSGSGSGTDFTL MQALEPPYT FGQGTKLE 20 119 PGEPASISC RGHTSLH PHLLIY RAS KISRVEAEDVGVYYC IK 06-AB- VK2 DIVMTQSPLSLPVT RSSQSLLHR WYLQKPGQS LGSN GVPDRFSGSGSGTDFTL MQGLQTPY FGQGTKLE 21 118 PGEAASISC NGKTFFA PQILIY RAS KISRVEAEDVGIYYC T IK 06-AB- VK2 DIVMTQSPSSVSAS RASQGISRW WYQQKPGEA AASS GVPSRFSGSGSGTDFTL QQANSFPIT FGQGTRL 22 120 VGDKVTITC LA PELLIY LOS TISSLQPEDFATYYC QIK 06-AB- VL3 QLVLTQPPSVSVSP SGDELRNKY WYQQKSGQS QDNN GIPERFSGSQSGDTATL QAWVSQTL FGGGTKLT 23 121 GQTASITC TS PVLVIY RPS TISGTQAVDEADYYC V VL 06-AB- VL3 QAGLTQPPSVSVA GGNNIGSKH WYQQKPGQA DDSD GVPERFSGSNSGNTATL QVWDRSSD FGGGTRLT 24 122 PGQTATIPC VH PVAVVY RPS TISSVEAGDEADYYC HFWL VL 06-AB- VL2 QLVLTQPPSASGS TGTSSDVGG WYQHHPGKA EVSQ GVPDRFSGSKSGNTASL SSYAGSVVL FGGGTKLT 25 123 PGQSVTISC SNFVS PKLMIY RPS TVSGLQADDEADYYC VL 06-AB- VL2 QLVLTQPPSASGS TGTSSDVGG WYQHHPGKA EVSQ GVPDRFSGSKSGNTASL SSYAGSVVL FGGGTKLT 26 124 PGQSVTISC SNFVS PKLMIY RPS TVSGLQADDEADYYC VL 06-AB- VK3 DIVMTQSPATLSLS WASQYINTY WYQHKPGQA DASK GIPARFSGSGSGTDFTL QQGSNWPL FGQGTRL 27 126 PGERATLSC VN PRLLIY RAT TISSLEPEDFAVYYC T EIK 06-AB- VK1 EIVMTQSPSFVSAS RASQDISNW WYQQKPGKA ASSN GVPSRFSGSGSGTDFAL QQENSFPY FGQGTKLE 28 127 VGDRVTITC LV PKLLIY LOS TIISLQPEDFATYYC T IK 06-AB- VK2 VIWMTQSPLSLPVT RSSQSLLHR WYLQKPGQS LGSN GVPDRFSGSGSGTDFTL MQGLQTPY FGQGTKLE 29 129 PGEAASISC NGRTFFA PQILIY RAF KISRVEAEDVGIYYC T IK 06-AB- VK2 VIWMTQSPLSLPVT RSSQSLLHR WYLQKPGQS LGSN GVPDRFSGSGSGTDFTL MQGLQTPY FGQGTKLE 30 130 PGEAASISC NGRTFFA PQILIY RAF KISRVEAEDVGIYYC T IK 06-AB- VK1 DIVMTQTPSTQSAS RASQSISIWL WYQQKPGKA DAST GVPSRFSGSGSGTEFTL QRYNDYPP FGPGTKVE 31 131 VGDRVTITC A PKLLIH LES TISSLQPDDSATYYC T IK 06-AB- VK1 EIVMTQSPSTQSAS RASQSISIWL WYQQKPGKA DAST GVPSRFSGSGSGTEFTL QRYNDYPP FGPGTKVE 32 132 VGDRVTITC A PKLLIH LES TISSLQPDDSATYYC T IK 06-AB- VL1 QSVLTQPPSVSGT SGSNSNAG WYQQVPGTA KNNQ GVPDRFSGSKSGTSASL IVWDGSLSG FGTGTKVT 33 133 PGQRVTISC RDYVS PKLLIY RPS AISGLRSEDDGDYYC YV VL 06-AB- VL7 SYELTQPSSLTVSP GLSSGAVTS WFQQKPGQA DTSR WTPARFSGSLLGGKAAL LLACNGACV FGGGTKLT 34 134 GGTVTLTC GHYPY PKTLIF KHS TLSGAQPEDDADYYC VL 06-AB- VL7 SYELTQPSSLTVSP GLSSGAVTS WFQQKPGQA DTSR WTPARFSGSLLGGKAAL LLACNGACV FGGGTKLT 35 135 GGTVTLTC GHYPY PKTLIF KHS TLSGAQPEDDADYYC VL 06-AB- VL1 QSVLTQPPSVSGT SGSNSNVG WYQQVPGTA KNNR GVPDRFSGSKSGTSASL IVWDGSLSG FGTGTKVT 36 139 PGQRVTISC RDYVS PKLLIY RPS AISGLRSEDDGDYYC YV VL 06-AB- VL1 QLVLTQPPSVSGT SGSNSNVG WYQQVPGTA KNNQ GVPDRFSGSKSGTSASL IVWDGSLSG FGTGTKVT 37 140 PGQRVTISC RDYVS PKLLIY RPS AISGLRSEDDGDYYC YV VL

(110) Antibody Sequences and Seg ID No.s

(111) TABLE-US-00007 TABLE A Antibody AB119 06-AB- SEQ 119 Sequence ID NO: VH FW1 QVTLKESGGGLVQPGGSLRLSCVASGFTF 38 VH CDR1 RTYWMH 39 VH FW2 WVRQDPGKGLVWVS 40 VH CDR2 RLDEVGRLTSYADSVNG 41 VH FW3 RFTISRDNAKNILYLQMNSLRAEDTGVYYCAR 42 VH CDR3 DLSGSADY 43 VH FW4 WGQGTLVTVSS 44 VL FW1 DVVLTQSPLFLPVTPGEPASISC 45 VL CDR1 RSSQSLLHSRGHTSLH 46 VL FW2 WYLQKPGQSPHLLIY 47 VL CDR2 SVFNRAS 48 VL FW3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 49 VL CDR3 MQALEPPYT 50 VL FW4 FGQGTKLEIK 51

(112) TABLE-US-00008 TABLE B Antibody AB118 06-AB- SEQ 118 Sequence ID NO: VH FW1 EVQLVESGGGLVQPGGSLRLSCSASQFIL 52 VH CDR1 SNYWVH 53 VH FW2 WVRQVPGEGLVWVS 54 VH CDR2 RINEDGSVTSYADSVKG 55 VH FW3 RFTISRDNAKNTLYLQMNSLRVDDTAVYYCVR 56 VH CDR3 DLCGERDD 57 VH FW4 WGQGTLVSVSS 58 VL FW1 DIVMTQSPLSLPVTPGEAASISC 59 VL CDR1 RSSQSLLHRNGKTFFA 60 VL FW2 WYLQKPGQSPQILIY 61 VL CDR2 LGSNRAS 62 VL FW3 GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC 63 VL CDR3 MQGLQTPYT 64 VL FW4 FGQGTKLEIK 65

(113) TABLE-US-00009 TABLE C Antibody AB120 06-AB- SEQ 120 Sequence ID NO: VH FW1 EVQLVQSGGGLVQPGGSLGLSCAASGFIF 66 VH CDR1 TSYAMT 67 VH FW2 WVRQAPGKGLEWVS 68 VH CDR2 VITGNVGTSYYADSVKG 69 VH FW3 RFTISRDNSKKTVSLQMNSLRAEDTAIYYCVK 70 VH CDR3 TRYDFSSGYYFDD 71 VH FW4 WGQGTLVSVSS 72 VL FW1 DIVMTQSPSSVSASVGDKVTITC 73 VL CDR1 RASQGISRWLA 74 VL FW2 WYQQKPGEAPELLIY 75 VL CDR2 AASSLQS 76 VL FW3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 77 VL CDR3 QQANSFPIT 78 VL FW4 FGQGTRLQIK 79

(114) TABLE-US-00010 TABLE D Antibody AB121 06-AB- SEQ 121 Sequence ID NO: VH FW1 EVQLVESGGTLVQPGGSLRLSCAASGFTF 80 VH CDR1 SDYWMN 81 VH FW2 WVRQAPGKGLEWVA 82 VH CDR2 NIKQDGSEKYYVDSLRG 83 VH FW3 RVTISRDNAQNSVFLQMHSLSVEDTAVYYCAR 84 VH CDR3 DGYTFGPATTELDH 85 VH FW4 WGRGTLVSVSS 86 VL FW1 QLVLTQPPSVSVSPGQTASITC 87 VL CDR1 SGDELRNKYTS 88 VL FW2 WYQQKSGQSPVLVIY 89 VL CDR2 QDNNRPS 90 VL FW3 GIPERFSGSQSGDTATLTISGTQAVDEADYYC 91 VL CDR3 QAWVSQTLV 92 VL FW4 FGGGTKLTVL 93

(115) TABLE-US-00011 TABLE E Antibody AB122 06-AB- SEQ 122 Sequence ID NO: VH FW1 EVQLVQSGGGLAQPGRSLRLSCAASGFGF 94 VH CDR1 DDFAMH 95 VH FW2 WVRQPPGKGLEWVS 96 VH CDR2 GLTWNGGSIDYAGSVRG 97 VH FW3 RFTISRDNAKNSLFLQMNSLRAEDTALYYCAK 98 VH CDR3 GLSGGTMAPFDI 99 VH FW4 WGQGTMVSVSS 100 VL FW1 QAGLTQPPSVSVAPGQTATIPC 101 VL CDR1 GGNNIGSKHVH 102 VL FW2 WYQQKPGQAPVAVVY 103 VL CDR2 DDSDRPS 104 VL FW3 GVPERFSGSNSGNTATLTISSVEAGDEADYYC 105 VL CDR3 QVWDRSSDHFWL 106 VL FW4 FGGGTRLTVL 107

(116) TABLE-US-00012 TABLE F Antibody AB123 SEQ 06-AB-123 Sequence ID NO: VH FW1 EVQLLESGGGVVQPGRSLRLSCAASGFTF 108 VH CDR1 SNYGMH 109 VH FW2 WVRQAPGKGLEWVA 110 VH CDR2 VVWFDGSYKYYTDSVKG 111 VH FW3 RFTISRDNSKSTLYLQMNSLRAEDTAVYYCVS 112 VH CDR3 PIMTSAFDI 113 VH FW4 WGPGTMVSVSS 114 VL FW1 QLVLTQPPSASGSPGQSVTISC 115 VL CDR1 TGTSSDVGGSNFVS 116 VL FW2 WYQHHPGKAPKLMIY 117 VL CDR2 EVSQRPS 118 VL FW3 GVPDRFSGSKSGNTASLTVSGLQADDEADYYC 119 VL CDR3 SSYAGSVVL 120 VL FW4 FGGGTKLTVL 121

(117) TABLE-US-00013 TABLE G Antibody AB124 SEQ 06-AB-124 Sequence ID NO: VH FW1 EVQLVESGGGVVQPGRSLRLSCAASGFTF 122 VH CDR1 SNYGMH 123 VH FW2 WVRQAPGKGLEWVA 124 VH CDR2 VVWLDGSYKYYTGSVKG 125 VH FW3 RFTISRDNSKSTLYLQMNSLRAEDTAAYYCVS 126 VH CDR3 PIMTSAFDI 127 VH FW4 WGPGTMVTVSS 128 VL FW1 QLVLTQPPSASGSPGQSVTISC 129 VL CDR1 TGTSSDVGGSNFVS 130 VL FW2 WYQHHPGKAPKLMIY 131 VL CDR2 EVSQRPS 132 VL FW3 GVPDRFSGSKSGNTASLTVSGLQADDEADYYC 133 VL CDR3 SSYAGSVVL 134 VL FW4 FGGGTKLTVL 135

(118) TABLE-US-00014 TABLE H Antibody AB126 SEQ 06-AB-126 Sequence ID NO: VH FW1 EVQLVESGGGLAQPGGSLRLSCEASGFHL 136 VH CDR1 AGNAMA 137 VH FW2 WVRQAPGKGLEWVA 138 VH CDR2 AIGGSDDRTDYADSVKG 139 VH FW3 RFTISRDKSKNTLSLQMNSLRVEDTAVYYCAK 140 VH CDR3 DIWRWAFDY 141 VH FW4 WGQGTLVSVSS 142 VL FW1 DIVMTQSPATLSLSPGERATLSC 143 VL CDR1 WASQYINTYVN 144 VL FW2 WYQHKPGQAPRLLIY 145 VL CDR2 DASKRAT 146 VL FW3 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC 147 VL CDR3 QQGSNWPLT 148 VL FW4 FGQGTRLEIK 149

(119) TABLE-US-00015 TABLE I Antibody AB127 SEQ 06-AB-127 Sequence ID NO: VH FW1 EVQLVESGGGLVNPGGSLRLSCAASGFTF 150 VH CDR1 SNYAMN 151 VH FW2 WVRQAPGKGLEWVS 152 VH CDR2 SISRSGDYIYYADSLKG 153 VH FW3 RSTISRDNAKNSLFLQMNSLRAEDSAVYYCAR 154 VH CDR3 DWGRLGYCSSNNCPDAFDV 155 VH FW4 WGQGTRVSVSS 156 VL FW1 EIVMTQSPSFVSASVGDRVTITC 157 VL CDR1 RASQDISNWLV 158 VL FW2 WYQQKPGKAPKLLIY 159 VL CDR2 ASSNLQS 160 VL FW3 GVPSRFSGSGSGTDFALTIISLQPEDFATYYC 161 VL CDR3 QQENSFPYT 162 VL FW4 FGQGTKLEIK 163

(120) TABLE-US-00016 TABLE J Antibody AB129 SEQ 06-AB-129 Sequence ID NO: VH FW1 QVQLVESGGGLVQPGGSLRLSCSASQFIL 164 VH CDR1 SNYWVH 165 VH FW2 WVRQVPGEGLVWVS 166 VH CDR2 RINEDGSVTSYADSVKG 167 VH FW3 RFTISRDNAKNTLYLQMNSLRVDDTAVYYCVR 168 VH CDR3 DLCGERDD 169 VH FW4 WGQGTLVTVSS 170 VL FW1 VIWMTQSPLSLPVTPGEAASISC 171 VL CDR1 RSSQSLLHRNGRTFFA 172 VL FW2 WYLQKPGQSPQILIY 173 VL CDR2 LGSNRAF 174 VL FW3 GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC 175 VL CDR3 MQGLQTPYT 176 VL FW4 FGQGTKLEIK 177

(121) TABLE-US-00017 TABLE K Antibody AB130 SEQ 06-AB-130 Sequence ID NO: VH FW1 QLQLQESGGGLVQPGGSLRLSCSASQFIL 178 VH CDR1 SNYWVH 179 VH FW2 WVRQVPGEGLVWVS 180 VH CDR2 RINEDGSVTSYADSVKG 181 VH FW3 RFTISRDNAKNTLYLQMNSLRVDDTAVYYCVR 182 VH CDR3 DLCWERDD 183 VH FW4 WGQGTLVSVSS 184 VL FW1 VIWMTQSPLSLPVTPGEAASISC 185 VL CDR1 RSSQSLLHRNGRTFFA 186 VL FW2 WYLQKPGQSPQILIY 187 VL CDR2 LGSNRAF 188 VL FW3 GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC 189 VL CDR3 MQGLQTPYT 190 VL FW4 FGQGTKLEIK 191

(122) TABLE-US-00018 TABLE L Antibody AB131 SEQ 06-AB-131 Sequence ID NO: VH FW1 QVQLVQSGGGVVQPGGSLRLSCAASPFTF 192 VH CDR1 KTSILH 193 VH FW2 WVRQAPGKGLEWVS 194 VH CDR2 AMSYDGFSKYYADSVKG 195 VH FW3 RLTISRDSSTNTLYLEMNSLRFEDTALYFCAR 196 VH CDR3 EAYTSGRAGCFNP 197 VH FW4 WGQGVLVSVSS 198 VL FW1 DIVMTQTPSTQSASVGDRVTITC 199 VL CDR1 RASQSISIWLA 200 VL FW2 WYQQKPGKAPKLLIH 201 VL CDR2 DASTLES 202 VL FW3 GVPSRFSGSGSGTEFTLTISSLQPDDSATYYC 203 VL CDR3 QRYNDYPPT 204 VL FW4 FGPGTKVEIK 205

(123) TABLE-US-00019 TABLE M Antibody AB132 SEQ 06-AB-132 Sequence ID NO: VH FW1 QVLKESGGGVVQPGGSLRLSCAASPFTF 206 VH CDR1 ETSILH 207 VH FW2 WVRQAPGKGLEWVS 208 VH CDR2 AMSYDGFSKYYADSVKG 209 VH FW3 RLTISRDSSTNTLYLEMNSLRFEDTALYFCAR 210 VH CDR3 EAYTSGRAGCFDP 211 VH FW4 WGQGVLVSVSS 212 VL FW1 EIVMTQSPSTQSASVGDRVTITC 213 VL CDR1 RASQSISIWLA 214 VL FW2 WYQQKPGKAPKLLIH 215 VL CDR2 DASTLES 216 VL FW3 GVPSRFSGSGSGTEFTLTISSLQPDDSATYYC 217 VL CDR3 QRYNDYPPT 218 VL FW4 FGPGTKVEIK 219

(124) TABLE-US-00020 TABLE N Antibody AB133 SEQ 06-AB-133 Sequence ID NO: VH FW1 EVQLVESGGGLVQPGGSLRVSCAASGFTL 220 VH CDR1 NTYWMH 221 VH FW2 WVRQAPGKGLVWVS 222 VH CDR2 RINEDGTTISYADSVRG 223 VH FW3 RFTISRDNAENTLYLQMHSLRAEDTGVYYCAR 224 VH CDR3 DFTGPFDS 225 VH FW4 WGQGTLVSVSS 226 VL FW1 QSVLTQPPSVSGTPGQRVTISC 227 VL CDR1 SGSNSNAGRDYVS 228 VL FW2 WYQQVPGTAPKLLIY 229 VL CDR2 KNNQRPS 230 VL FW3 GVPDRFSGSKSGTSASLAISGLRSEDDGDYYC 231 VL CDR3 IVWDGSLSGYV 232 VL FW4 FGTGTKVTVL 233

(125) TABLE-US-00021 TABLE O Antibody AB134 SEQ 06-AB-134 Sequence ID NO: VH FW1 QLQLQESGGGLVQPGGSLRLSCVVSGFTF 234 VH CDR1 SSHAMS 235 VH FW2 WVRQAPGKGLEWVS 236 VH CDR2 SISISGGDTFYADSVRG 237 VH FW3 RFTIFRDNSKNTVYLQMNSLRAEDTAVYYCAT 238 VH CDR3 ETSPNDY 239 VH FW4 WGQGTLVSVSS 240 VL FW1 SYELTQPSSLTVSPGGTVTLTC 241 VL CDR1 GLSSGAVTSGHYPY 242 VL FW2 WFQQKPGQAPKTLIF 243 VL CDR2 DTSRKHS 244 VL FW3 WTPARFSGSLLGGKAALTLSGAQPEDDADYYC 245 VL CDR3 LLACNGACV 246 VL FW4 FGGGTKLTVL 247

(126) TABLE-US-00022 TABLE P Antibody AB135 SEQ 06-AB-135 Sequence ID NO: VH FW1 EVQLVETGGGLVQPGGSLRLSCVVSGFTF 248 VH CDR1 SSHAMS 249 VH FW2 WVRQAPGKGLEWVS 250 VH CDR2 SISISGGDTFYADSVRG 251 VH FW3 RFTIFRDNSKNTVYLQMNSLRAEDTAVYYCAT 252 VH CDR3 ETSPNDY 253 VH FW4 WGQGTLVTVSS 254 VL FW1 SYELTQPSSLTVSPGGTVTLTC 255 VL CDR1 GLSSGAVTSGHYPY 256 VL FW2 WFQQKPGQAPKTLIF 257 VL CDR2 DTSRKHS 258 VL FW3 WTPARFSGSLLGGKAALTLSGAQPEDDADYYC 259 VL CDR3 LLACNGACV 260 VL FW4 FGGGTKLTVL 261

(127) TABLE-US-00023 TABLE Q Antibody AB139 SEQ 06-AB-139 Sequence ID NO: VH FW1 EVQLVESGGGLVQPGGSLRVSCAASGFTL 262 VH CDR1 NTYWMH 263 VH FW2 WVRQAPGKGLVWVS 264 VH CDR2 RINEDGTTISYADSVRG 265 VH FW3 RFTISRDNAENTLYLQMHSLRAEDTGVYYCAR 266 VH CDR3 DFTGPFDS 267 VH FW4 WGQGTLVSVSS 268 VL FW1 QSVLTQPPSVSGTPGQRVTISC 269 VL CDR1 SGSNSNVGRDYVS 270 VL FW2 WYQQVPGTAPKLLIY 271 VL CDR2 KNNRRPS 272 VL FW3 GVPDRFSGSKSGTSASLAISGLRSEDDGDYYC 273 VL CDR3 IVWDGSLSGYV 274 VL FW4 FGTGTKVTVL 275

(128) TABLE-US-00024 TABLE R Antibody AB140 SEQ 06-AB-140 Sequence ID NO: VH FW1 EVQLVESGGGLVQPGGSLRVSCAASGFTL 276 VH CDR1 NTYWMH 277 VH FW2 WVRQAPGKGLVWVS 278 VH CDR2 RINEDGTTISYADSVRG 279 VH FW3 RFTISRDNAENTLYLQMHSLRAEDTGVYYCAR 280 VH CDR3 DFTGPFDS 281 VH FW4 WGQGTLVSVSS 282 VL FW1 QLVLTQPPSVSGTPGQRVTISC 283 VL CDR1 SGSNSNVGRDYVS 284 VL FW2 WYQQVPGTAPKLLIY 285 VL CDR2 KNNQRPS 286 VL FW3 GVPDRFSGSKSGTSASLAISGLRSEDDGDYYC 287 VL CDR3 IVWDGSLSGYV 288 VL FW4 FGTGTKVTVL 289

(129) TABLE-US-00025 TABLE VH-CDR3-mod SEQ (Variant Light or ID of SEQ Heavy CDR3 NO: ID NO:) 06-AB-118.Heavy DLAGERDD 290 57 C101A 06-AB-118.Heavy DLSGERDD 291 57 C101S 06-AB-127.HeavyWY DWGRLGYWSSNNY 292 155 PDAFDV 06-AB-127.HeavyAA DWGRLGYASSNNA 293 155 PDAFDV 06-AB-131.HeavyW EAYTSGRAGWFNP 294 197 06-AB-131.HeavyA EAYTSGRAGAFNP 295 197 06-AB-132.HeavyW EAYTSGRAGWFDP 296 211 06-AB-132.HeavyA EAYTSGRAGAFDP 297 211 06-AB-129.HeavyW DLWGERDD 298 169 06-AB-129.HeavyA DLAGERDD 299 169

(130) TABLE-US-00026 TABLE VL-CDR3-mod 06-AB-134.LightYW LLAYNGAWV 300 246 06-AB-134.LightAA LLAANGAAV 301 246 06-AB-135.LightYW LLAYNGAWV 302 260 06-AB-135.LightAA LLAANGAAV 303 260

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ADDITIONAL REFERENCES FOR TABLE S1

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