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.-3. (canceled)

4. An anti-Candida recombinant human antibody molecule which comprises a VH domain comprising (i) an HCDR3 having the amino acid sequence of SEQ ID NO: 6x or the sequence of SEQ ID NO: 6x with 1, 2, or 3 amino acid substitutions, deletions or insertions; and optionally (ii) an HCDR2 having the amino acid sequence of SEQ ID NO: 4x or the sequence of SEQ ID NO: 4x with 1, 2, or 3 amino acid substitutions, deletions or insertions; and optionally (iii) an HCDR1 having the amino acid sequence of SEQ ID NO: 2x or the sequence of SEQ ID NO: 2x with 1, 2 or 3 amino acid substitutions, deletions or insertions, wherein x is one letter from A to R, and said sequence is as shown in Table x herein.

5. (canceled)

6. (canceled)

7. An antibody molecule according to claim 4 wherein the antibody molecule comprises a VH domain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 2x, 4x and 6x respectively.

8. An antibody molecule according to claim 7 wherein the antibody molecule comprises a VH domain comprising one or more or all of a FW1, a FW2, a FW3 and a FW4 having the sequences of SEQ ID NOs 1x, 3x, 5x and 7x respectively.

9. An antibody molecule according to claim 4 wherein the antibody molecule comprises a VH domain having an amino acid sequence at least about 80% identical to SEQ ID NO: 15x and\or having the amino acid sequence of SEQ ID NO: 15x and\or the sequence of SEQ ID NO: 15x with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or insertions in SEQ ID NO: 15x.

10. An antibody molecule according to claim 4 wherein antibody molecule comprises a VL domain comprising LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs 9x, 11x and 13x respectively, or the sequences of SEQ ID NOs 9x, 11x and 13x respectively with, independently, 1, 2 or 3 or more amino acid substitutions, deletions or insertions.

11. An antibody molecule according to claim 10 wherein antibody molecule comprises a VL domain comprising LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs 9x, 11x and 13x respectively.

12. An antibody molecule according to claim 10 wherein the antibody molecule comprises a VL domain comprising one or more or all of a FW1, a FW2, a FW3 and a FW4 having the sequences of SEQ ID NOs 8x, 10x, 12x and 14x respectively.

13. An antibody molecule according to claim 4 wherein the antibody molecule comprises a VL domain having an amino acid sequence at least about 80% identical to SEQ ID NO: 16x and\or having the sequence of SEQ ID NO: 16x and\or the sequence of SEQ ID NO: 16x with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions in SEQ ID NO: 16x.

14. An antibody molecule according to claim 4 comprising a VH domain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ ID NOs 2x, 4x, and 6x, 17x or 18x, respectively, and a VL domain comprising a LCDR1, a LCDR2 and a LCDR3 having the sequences of SEQ ID NOs 9x, 11x and 13x, 19x or 20x, respectively.

15. An antibody molecule according to claim 4 comprising VH and VL domains having the amino acid sequences of SEQ ID NO: 15x and SEQ ID NO: 16x respectively.

16. An antibody molecule according to claim 4 which binds C. albicans with an EC.sub.50 value of 1 to 1500 ng/ml, wherein x is A-B, or H-R.

17. (canceled)

18. An antibody molecule according to claim 4 further comprising a payload which is cytotoxic.

19. A method for producing an antibody antigen-binding domain for a fungal antigen, the method comprising; (i) providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR1, HCDR2 and HCDR3, wherein the parent VH domain HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NOS: 2x, 4x and 6x respectively, a VH domain which is an amino acid sequence variant of the parent VH domain, (ii) optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and (iii) testing said VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain for the fungal antigen.

20. A method for producing an antibody molecule that specifically binds to a fungal antigen, which method comprises: providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise a HCDR1, HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or HCDR3 encoding region; combining said starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDR1, HCDR2, and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 2x, 4x and 6x respectively, such that said donor nucleic acid is or donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains; expressing the nucleic acids of said product repertoire to produce product VH domains; optionally combining said product VH domains with one or more VL domains; selecting an antibody molecule that binds the fungal antigen, which antibody molecule comprises a product VH domain and optionally a VL domain; and recovering said antibody molecule or nucleic acid encoding it.

21. An antibody molecule according to claim 4 which is a whole antibody or a scAb.

22. A method comprising contacting a Candida cell with an antibody molecule as claimed in claim 4 for the purpose of: (i) identifying or labelling the Candida cell, or the hyphae of the cell; or (ii) opsonising, or increasing the rate of opsonisation of the Candida cell; or (iii) increasing the rate of macrophage engulfment of the Candida cell; or (iv) increasing the rate of macrophage attraction to the Candida cell.

23.-25. (canceled)

26. A pharmaceutical composition comprising an antibody molecule according to claim 4 and a pharmaceutically acceptable excipient optionally including a further antifungal agent.

27.-30. (canceled)

31. A method of treatment of a fungal infection comprising administering an antibody molecule according to claim 4 to an individual in need thereof.

32. A method according to claim 31 wherein the fungal infection is a Candida infection, preferably a C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis or C. lusitaniae infection.

33. A method according to claim 32 wherein the C. albicans infection is in a hyphal or yeast phase.

34. A method according to claim 31, wherein the treatment comprises administering a second antifungal agent, wherein the second antifungal agent is optionally an azole, a polyene or an echinocandin.

35. (canceled)

36. A method for detecting the presence or absence of a fungus which is Candida spp, the method comprising (i) contacting a sample suspected of containing the fungus with an antibody molecule according to claim 4, and (ii) determining whether the antibody molecule binds to the sample, wherein binding of the antibody molecule to the sample indicates the presence of the fungus.

37. A method for diagnosing a fungal infection in an individual which is caused by Candida spp, the method comprising (i) contacting a biological sample obtained from the individual with an antibody molecule according to claim 4, and (ii) determining whether the antibody molecule binds to the biological sample, wherein binding of the antibody molecule to the biological sample indicates the presence of a fungal infection.

38. A linear flow device (LFD) for detecting 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, the or each carrier comprising an analyte-detecting means; wherein the presence of analyte produces a line in the viewing window which indicates an analyte concentration, wherein the fungal pathogen is Candida spp. and the at least one analyte-detecting means is an antibody molecule of claim 4.

39. (canceled)

40. A device as claimed in claim 38 having a plurality of analyte-detecting means capable of distinguishing between multiple fungal pathogens, wherein one of the analyte-detecting means is an antibody molecule according to claim 4.

41. A device as claimed in claim 40 wherein the multiple fungal pathogens comprise C. albicans, plus one or more or all of Aspergillus fumigatus, Cryptococcus neoformans, Pneumocystsis jirovecii, a zygomycete fungus, and a skin dermatophytic fungus.

42. (canceled)

Description

FIGURES

[0173] FIG. 1Workflow 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).

[0174] FIG. 2Representative 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.

[0175] FIG. 3Concentration 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).

[0176] FIG. 4Indirect 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 m.

[0177] FIG. 5Indirect 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 m.

[0178] FIG. 6Heat-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 (AB118-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).

[0179] FIG. 7Macrophage 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 eventsSEM (n=3). *p<0.05, **p<0.01, ****p<0.0001.

[0180] FIG. 8Macrophage 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 cellSEM (n=3) *p<0.01, ***p<0.005.

[0181] FIG. 9Macrophage engulfment of opsonized live C. albicans cells in the presence and absence of an FcR 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 FcR block. Bars represent average time taken for a macrophage to ingest a C. albicans cellSEM (n=3) *p<0.05.

[0182] FIG. 10Macrophage 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 velocitySEM (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 travelledSEM (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.

[0183] FIG. 11Assessment 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.

[0184] FIG. 12Schematic of VH, V-C and V-C 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, V-C and V-C 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, C or C 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 V-C and V-C genes, forward primers specific to human V and human V FW1 sequences were used in combination with reverse primers specific to the 3 end of the human C or human C 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, V-C and V-C genes were done in separate reactions. RT-PCRreverse transcriptase polymerase chain reaction; UTR untranslated region; Lleader sequence; V.sub.Hheavy chain variable domain; Vkappa chain variable domain; Vlambda chain variable domain; C.sub.Hheavy chain constant domain; Ckappa chain constant domain; Clambda chain constant domain.

[0185] FIG. 13Concentration 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).

[0186] FIG. 14Concentration 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).

[0187] FIG. 15Indirect 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 -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 m.

[0188] FIG. 16Human 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).

[0189] FIG. 17Counterimmunoelectrophoresis 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).

[0190] FIG. 18Immunogold 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

[0191] 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 antigenthe 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.

[0192] 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>4xbackground). Non-specific hits were identified and eliminated by performing an ELISA screen against two unrelated proteinshuman 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, V-C and V-C gene amplification via RT-PCR and nested PCR (FIGS. 2B, C). VH, V-C and V-C 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).

[0193] 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

[0194] 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).

[0195] 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).

[0196] 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

[0197] 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 hyr1 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).

[0198] 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.

[0199] 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 -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.

[0200] 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).

[0201] 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.

[0202] 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

[0203] 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 2110, 549, 225 and 682, 44.30.6 and 72 (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 FcR Binding

[0204] 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 macrophagestaking an average of 5.80.3 min to engulf opsonised hyphae compared to 8.80.8 min for the control (FIG. 8E).

[0205] Similar observations were obtained using human monocyte-derived macrophages (FIG. 16).

[0206] Blocking FcRs 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 FcRs.

EXAMPLE 6

Macrophages Migrate Further, Faster and More Direct Towards Anti-Candida mAb Bound C. Albicans Cells

[0207] 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. 10A,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

[0208] 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

[0209] 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.

[0210] 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 proteina 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.

[0211] 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.

[0212] 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 FcR. 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.

[0213] 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 hIgG1 so this isotype has been routinely tested pre-clinically in murine models of disease (47). Furthermore, the literature shows that hIgG1 binds to all activating mFcRs with a similar profile to the most potent IgG isotype in mice, mIgG2a, validating the use of mouse models to assess Fc-mediated effects of hIgG1 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.

[0214] 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.

[0215] 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

[0216] 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

[0217] 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.

General Methods

Candida Strains and Growth Conditions

[0218] C. albicans serotype A strain CAl4+Clp10 (NGY152) was used as a control and its parent strain CAl4, used to construct the hyr1 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 110.sup.8 spores/ml. Swollen spores were generated by incubation in RPMI media for 4 h at 37 C.

[0219] 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 m Nylon Cell Strainer (BD Biosciences). Cryptococcus neoformans KN99 and Cryptococcus gattii R265 were grown in YPD overnight, washed in PBS and 110.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.

Generation of Recombinant Hyr1 N-Protein

[0220] The recombinant N-terminus of the Hyr1 protein (amino acids 63 to 350Table S2) incorporating an N-terminal 6xHis 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).

Identification of Human Anti-Hyri and Anti-Whole Cell mAbs From Donor B Cells PBMC Isolation

[0221] 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 250g for 10 min three times before final resuspension at a concentration of 110.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.

Purification of Donor Plasma

[0222] 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).

Circulating IgG Enzyme-Linked Immunosorbent Assay (ELISA) To Identify Donors With B Cells To Take Forward

[0223] 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 g/ml purified, recombinant N-terminus hyr1 protein antigen in 1PBS and incubated at 4 C. overnight. The next day, wells were washed three times with wash buffer (1PBS+0.05% Tween) using a Zoom Microplate Washer (Titertek). Wells were then blocked with block buffer (1PBS+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.

Isolation of Class Switched Memory B Cells

[0224] 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, 510.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 lysed cells and their components didn't interfere with the live cells), and the cells were centrifuged at 300g 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.

[0225] 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.

Activation of CSM B Cells

[0226] 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 l/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 l/well of supernatant was removed and replaced with 30 l 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.

B Cell Supernatant Screen Against Target Antigens via ELISA

[0227] 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 g/ml purified, recombinant N-terminus hyr1 protein antigen in 1PBS 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 450nm on an Envision plate reader (PerkinElmer).

[0228] Positive hits were defined as wells with an OD.sub.450 reading >4xbackground. B cells in positive hit wells were resuspended in lysis buffer (ml DEPC-treated H2O (Life Technologies), 10 l 1 M Tris pH 8, 25 l RNAsin Plus RNAse Inhibitor (Promega)) and stored at 80 C.

Generation of Recombinant Anti-Hyr1 and Anti-Whole Cell IgG1 mAbs: Amplification of VH, V-C and V-C GenescDNA Synthesis and PCR

[0229] 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 V-C genes, three forward primers specific to the leader sequences for the different human V germline families (V1-4) were used with a reverse primer specific to the human kappa constant region (C) 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 V genes, 7 forward primers capturing the leader sequences for the different human V germline families (V1-8) 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 (C).

[0230] 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 M) (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 (V-C) and the variable and constant domains of human Ig lambda light chain genes (V-C), 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 M), 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 l/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.

Amplification of VH, V-C and V-C GenesNested PCR Reaction

[0231] 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 V-C gene, a mixture of 18 forward primers specific for human V FW1 sequence were used with a reverse primer specific to the human kappa constant region 3 end. For amplification of the V-C gene, a mixture of 31 forward primers specific for human V 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 M) and gene specific reverse primer mix (10 M) was added to wells in a 96-well PCR plate before addition of cDNA template. Amplification of VH genes, V-C genes and V-C 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.

pTT5 Mammalian Expression Vector Preparation

[0232] 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 SalI 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 -C or V-C. 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).

In-Fusion Cloning

[0233] The In-Fusion HD Cloning Kit (Clontech, USA) was used to clone the IgG VH, V-C and V-C 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 l 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 l 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 g/ml ampicillin. Plates were incubated at 37 C. overnight and single colonies picked the next day.

Plasmid DNA Generation for Transfection

[0234] Following transformation, 8-16 single colonies per initial hit well for VH, Vand V were picked and used to inoculate 2TY media containing 100 g/ml ampicillin in a Greiner deep well, 96-well plate (Sigma). VH, V and V plates were set up separately with the same plate layout to facilitate visual screening. Cells were grown at 37 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.

Small Scale Expression of Recombinant mAbs

[0235] Following VH, V and V gene sequencing, a file was generated containing all possible VH and V/V 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 g of HC plasmid DNA and 1.5 g 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 2.510.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, Calif., USA) for identification of mAbs to upscale.

Large Scale Expression, Purification and QC of Recombinant mAbs

[0236] 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 g/l.

[0237] For large scale mAb expression, 100 g of total DNA (50 g of HC plasmid DNA and 50 g LC plasmid DNA) was used to transiently transfect 100 ml of suspension cultured Expi293F cells (Life Technologies, USA) at a density of 2.510.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 KTA (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.

ELISA with Purified Recombinant mAbs

[0238] 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.

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

[0239] 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 g/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 g/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 mm20 mm coverslip to the slide. Cells were imaged in 3D on an UltraVIEW VoX spinning disk confocal microscope (Nikon, Surrey, UK).

Preparation of Human Monocyte-Derived Macrophages

[0240] 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/mI 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 1.210.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 M 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.

Preparation of J774.1 Mouse Macrophage Cell Line

[0241] 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/mI 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 l supplemented DMEM at a density of 110.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 l pre-warmed supplemented CO.sub.2-independent media (Gibco, Invitrogen, Paisley, UK) containing 1 M LysoTracker Red DND-99 (Invitrogen, Paisley, UK).

Preparation of Fluorescein Isothiocyanate (FITC)-Stained C. Albicans

[0242] 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 1PBS to remove any residual FITC and finally re-suspended in 1PBS or 1PBS containing purified anti-Candida mAb at 1-50 g/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 g/ml and incubated at 37 C. with gentle shaking for 45 min.

Live Cell Video Microscopy Phagocytosis Assays

[0243] 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 (CA14-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 uptakedefined as the number of C. albicans cells taken up by an individual phagocyte over the 3 h period; C. albicans rate of engulfmentdefined 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.

[0244] Mean values and standard deviations were calculated. One- or two-way ANOVA followed by Bonferroni multiple comparison tests or unpaired, two-tailed t tests were used to determine statistical significance.

Systemic Candidiasis Infection Model

[0245] 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, 3.210.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.

Enzymatic Modification of Candida Albicans Cell Wall

[0246] 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 g/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/l; 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 g/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).

Preparation of Human Monocyte-Derived Macrophages

[0247] 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 610.sup.5 in 300 l/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 M LysoTracker Red DND-99 (Invitrogen) immediately prior to phagocytosis experiments.

Counterimmunoelectrophoresis

[0248] 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 l 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.

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

[0249] 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 min3. Sections were then incubated with anti-Candida mAb (5 g/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.

TABLE-US-00002 TABLE S1 Clinical isolates and strains Strain name Genotype Reference CAl4 + Clp10 (NGY152) ura3::imm434/ura3::imm434 Brand et al. 2004 RPS1/rps1::URA3 hyr1 hyr1::hisG/hyr1:hisG-URA-3-hisG Bailey et al. 1996 hyr1 + HYR1 hyr1::hisG/hyr1::hisG/RPS1/rps1::HYR1 Belmonte (unpublished) tup1 tup1::hisG/tup1::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 KN99 H99 mating type 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

TABLE-US-00003 TABLES2 RecombinantHyr1proteinaminoacidsequence.The leadersequenceisunderlinedandthe6xHistagis initalics,andisfollowedbythelinkerG. Hyr1proteinaminoacids63-350makeupthe remainderofthesequence. Recombinantprotein Aminoacidsequence antigenname (aminoacids63-350) RecombinantHyr1 METDTLLLWVLLLWVPGSTGGSGHHHHHH N-terminusfragment GEVEKGASLFIKSDNGPVLALNVALSTLV RPVINNGVISLNSKSSTSFSNFDIGGSSF TNNGEIYLASSGLVKSTAYLYAREWTNNG LIVAYQNQKAAGNIAFGTAYQTITNNGQI CLRHQDFVPATKIKGTGCVTADEDTWIKL GNTILSVEPTHNFYLKDSKSSLIVHAVSS NQTFTVHGFGNGNKLGLTLPLTGNRDHFR FEYYPDTGILQLRAAALPQYFKIGKGYDS KLFRIVNSRGLKNAVTYDGPVPNNEIPAV CLIPCTNGPSAPESESDLNTPTTSSIGT

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

TABLE-US-00005 TABLEVH SEQ AB VH ID name VHFW1 CDR1 VHFW2 VHCDR2 VHFW3 VHCDR3 VHFW4 NO 06- VH3 QVTLKESGGGLVQPG RTY WVRQDPG RLDEVGRLT RFTISRDNAKNILYLQMN DLSGSADY WGQGTLV 15A AB- GSLRLSCVASGFTF WMH KGLVWVS SYADSVNG SLRAEDTGVYYCAR TVSS 119 06- VH3 EVQLVESGGGLVQPG SNY WVRQVPG RINEDGSVT RFTISRDNAKNTLYLQM DLCGERDD WGQGTLV 15B AB- GSLRLSCSASQFIL WVH EGLVWVS SYADSVKG NSLRVDDTAVYYCVR SVSS 118 06- VH3 EVQLVQSGGGLVQPG TSY WVRQAPG VITGNVGTS RFTISRDNSKKTVSLQM TRYDFSSGYY WGQGTLV 15C AB- GSLGLSCAASGFIF AMT KGLEWVS YYADSVKG NSLRAEDTAIYYCVK FDD SVSS 120 06- VH3 EVQLVESGGTLVQPG SDY WVRQAPG NIKQDGSEK RVTISRDNAQNSVFLQM DGYTFGPATT WGRGTLV 15D AB- GSLRLSCAASGFTF WMN KGLEWVA YYVDSLRG HSLSVEDTAVYYCAR ELDH SVSS 121 06- VH3 EVQLVQSGGGLAQPG DDF WVRQPPG GLIVVNGGSI RFTISRDNAKNSLFLQM GLSGGTMAPF WGQGTMV 15E AB- RSLRLSCAASGFGF AMH KGLEWVS DYAGSVRG NSLRAEDTALYYCAK DI SVSS 122 06- VH3 EVQLLESGGGVVQPG SNY WVRQAPG VVWFDGSY RFTISRDNSKSTLYLQM PIMTSAFDI WGPGTMV 15F AB- RSLRLSCAASGFTF GMH KGLEWVA KYYTDSVKG NSLRAEDTAVYYCVS SVSS 123 06- VH3 EVQLVESGGGVVQPG SNY WVRQAPG VVWLDGSY RFTISRDNSKSTLYLQM PIMTSAFDI WGPGTMV 15G AB- RSLRLSCAASGFTF GMH KGLEWVA KYYTGSVKG NSLRAEDTAAYYCVS TVSS 124 06- VH3 EVQLVESGGGLAQPG AGN WVRQAPG AIGGSDDRT RFTISRDKSKNTLSLQM DIWRWAFDY WGQGTLV 15H AB- GSLRLSCEASGFHL AMA KGLEWVA DYADSVKG NSLRVEDTAVYYCAK SVSS 126 06- VH3 EVQLVESGGGLVNPG SNY WVRQAPG SISRSGDYIY RSTISRDNAKNSLFLQM DWGRLGYCSS WGQGTRV 15I AB- GSLRLSCAASGFTF AMN KGLEWVS YADSLKG NSLRAEDSAVYYCAR NNCPDAFDV SVSS 127 06- VH3 QVQLVESGGGLVQPG SNY WVRQVPG RINEDGSVT RFTISRDNAKNTLYLQM DLCGERDD WGQGTLV 15J AB- GSLRLSCSASQFIL WVH EGLVWVS SYADSVKG NSLRVDDTAVYYCVR TVSS 129 06- VH3 QLQLQESGGGLVQPG SNY WVRQVPG RINEDGSVT RFTISRDNAKNTLYLQM DLCWERDD WGQGTLV 15K AB- GSLRLSCSASQFIL WVH EGLVWVS SYADSVKG NSLRVDDTAVYYCVR SVSS 130 06- VH3 QVQLVQSGGGVVQPG KISI WVRQAPG AMSYDGFSK RLTISRDSSTNTLYLEMN EAYTSGRAGC WGQGVLV 15L AB- GSLRLSCAASPFTF LH KGLEWVS YYADSVKG SLRFEDTALYFCAR FNP SVSS 131 06- VH3 QVLKESGGGVVQPGG ETSI WVRQAPG AMSYDGFSK RLTISRDSSTNTLYLEMN EAYTSGRAGC WGQGVLV 15M AB- SLRLSCAASPFTF LH KGLEWVS YYADSVKG SLRFEDTALYFCAR FDP SVSS 132 06- VH3 EVQLVESGGGLVQPG NTY WVRQAPG RINEDGTTIS RFTISRDNAENTLYLQM DFTGPFDS WGQGTLV 15N AB- GSLRVSCAASGFTL WMH KGLVWVS YADSVRG HSLRAEDTGVYYCAR SVSS 133 06- VH3 QLQLQESGGGLVQPG SSH WVRQAPG SISISGGDTF RFTIFRDNSKNTVYLQM ETSPNDY WGQGTLV 15O AB- GSLRLSCVVSGFTF AMS KGLEWVS YADSVRG NSLRAEDTAVYYCAT SVSS 134 06- VH3 EVQLVETGGGLVQPG SSH WVRQAPG SISISGGDTF RFTIFRDNSKNTVYLQM ETSPNDY WGQGTLV 15P AB- GSLRLSCVVSGFTF AMS KGLEWVS YADSVRG NSLRAEDTAVYYCAT TVSS 135 06- VH3 EVQLVESGGGLVQPG NTY WVRQAPG RINEDGTTIS RFTISRDNAENTLYLQM DFTGPFDS WGQGTLV 15Q AB- GSLRVSCAASGFTL WMH KGLVWVS YADSVRG HSLRAEDTGVYYCAR SVSS 139 06- VH3 EVQLVESGGGLVQPG NTY WVRQAPG RINEDGTTIS RFTISRDNAENTLYLQM DFTGPFDS WGQGTLV 15R AB- GSLRVSCAASGFTL WMH KGLVWVS YADSVRG HSLRAEDTGVYYCAR SVSS 140

TABLE-US-00006 TABLEVL SEQ AB VL VL ID name VLFW1 CDR1 VLFW2 CDR2 VLFW3 VLCDR3 VLFW4 NO 06-AB- VK2 DVVLTQSPLFLPVT RSSQSLLHS WYLQKPGQS SVFN GVPDRFSGSGSGTDFTL MQALEPPYT FGQGTKLE 16A 119 PGEPASISC RGHTSLH PHLLIY RAS KISRVEAEDVGVYYC IK 06-AB- VK2 DIVMTQSPLSLPVT RSSQSLLHR WYLQKPGQS LGSN GVPDRFSGSGSGTDFTL MQGLQTPY FGQGTKLE 16B 118 PGEAASISC NGKTFFA PQILIY RAS KISRVEAEDVGIYYC T IK 06-AB- VK2 DIVMTQSPSSVSAS RASQGISRW WYQQKPGEA AASS GVPSRFSGSGSGTDFTL QQANSFPIT FGQGTRL 16C 120 VGDKVTITC LA PELLIY LOS TISSLQPEDFATYYC QIK 06-AB- VL3 QLVLTQPPSVSVSP SGDELRNKY WYQQKSGQS QDNN GIPERFSGSQSGDTATL QAWVSQTL FGGGTKLT 16D 121 GQTASITC TS PVLVIY RPS TISGTQAVDEADYYC V VL 06-AB- VL3 QAGLTQPPSVSVA GGNNIGSKH WYQQKPGQA DDSD GVPERFSGSNSGNTATL QVWDRSSD FGGGTRLT 16E 122 PGQTATIPC VH PVAVVY RPS TISSVEAGDEADYYC HFWL VL 06-AB- VL2 QLVLTQPPSASGS TGTSSDVGG WYQHHPGKA EVSQ GVPDRFSGSKSGNTASL SSYAGSVVL FGGGTKLT 16F 123 PGQSVTISC SNFVS PKLMIY RPS TVSGLQADDEADYYC VL 06-AB- VL2 QLVLTQPPSASGS TGTSSDVGG WYQHHPGKA EVSQ GVPDRFSGSKSGNTASL SSYAGSVVL FGGGTKLT 16G 124 PGQSVTISC SNFVS PKLMIY RPS TVSGLQADDEADYYC VL 06-AB- VK3 DIVMTQSPATLSLS WASQYINTY WYQHKPGQA DASK GIPARFSGSGSGTDFTL QQGSNWPL FGQGTRL 16H 126 PGERATLSC VN PRLLIY RAT TISSLEPEDFAVYYC T EIK 06-AB- VK1 EIVMTQSPSFVSAS RASQDISNW WYQQKPGKA ASSN GVPSRFSGSGSGTDFAL QQENSFPY FGQGTKLE 16I 127 VGDRVTITC LV PKLLIY LOS TIISLQPEDFATYYC T IK 06-AB- VK2 VIWMTQSPLSLPVT RSSQSLLHR WYLQKPGQS LGSN GVPDRFSGSGSGTDFTL MQGLQTPY FGQGTKLE 16J 129 PGEAASISC NGRTFFA PQILIY RAF KISRVEAEDVGIYYC T IK 06-AB- VK2 VIWMTQSPLSLPVT RSSQSLLHR WYLQKPGQS LGSN GVPDRFSGSGSGTDFTL MQGLQTPY FGQGTKLE 16K 130 PGEAASISC NGRTFFA PQILIY RAF KISRVEAEDVGIYYC T IK 06-AB- VK1 DIVMTQTPSTQSAS RASQSISIWL WYQQKPGKA DAST GVPSRFSGSGSGTEFTL QRYNDYPP FGPGTKVE 16L 131 VGDRVTITC A PKLLIH LES TISSLQPDDSATYYC T IK 06-AB- VK1 EIVMTQSPSTQSAS RASQSISIWL WYQQKPGKA DAST GVPSRFSGSGSGTEFTL QRYNDYPP FGPGTKVE 16M 132 VGDRVTITC A PKLLIH LES TISSLQPDDSATYYC T IK 06-AB- VL1 QSVLTQPPSVSGT SGSNSNAG WYQQVPGTA KNNQ GVPDRFSGSKSGTSASL IVWDGSLSG FGTGTKVT 16N 133 PGQRVTISC RDYVS PKLLIY RPS AISGLRSEDDGDYYC YV VL 06-AB- VL7 SYELTQPSSLTVSP GLSSGAVTS WFQQKPGQA DTSR WTPARFSGSLLGGKAAL LLACNGACV FGGGTKLT 16O 134 GGTVTLTC GHYPY PKTLIF KHS TLSGAQPEDDADYYC VL 06-AB- VL7 SYELTQPSSLTVSP GLSSGAVTS WFQQKPGQA DTSR WTPARFSGSLLGGKAAL LLACNGACV FGGGTKLT 16P 135 GGTVTLTC GHYPY PKTLIF KHS TLSGAQPEDDADYYC VL 06-AB- VL1 QSVLTQPPSVSGT SGSNSNVG WYQQVPGTA KNNR GVPDRFSGSKSGTSASL IVWDGSLSG FGTGTKVT 16Q 139 PGQRVTISC RDYVS PKLLIY RPS AISGLRSEDDGDYYC YV VL 06-AB- VL1 QLVLTQPPSVSGT SGSNSNVG WYQQVPGTA KNNQ GVPDRFSGSKSGTSASL IVWDGSLSG FGTGTKVT 16R 140 PGQRVTISC RDYVS PKLLIY RPS AISGLRSEDDGDYYC YV VL

Antibody Sequences and Seq ID No.s

[0250]

TABLE-US-00007 TABLEA AntibodyAB119 06-AB- Seq. 119 Sequence IDNo. VHFW1 QVTLKESGGGLVQPGGSLRLSCVASGFTF 1A VHCDR1 RTYWMH 2A VHFW2 WVRQDPGKGLVWVS 3A VHCDR2 RLDEVGRLTSYADSVNG 4A VHFW3 RFTISRDNAKNILYLQMNSLRAEDTGVYYCAR 5A VHCDR3 DLSGSADY 6A VHFW4 WGQGTLVTVSS 7A VLFW1 DVVLTQSPLFLPVTPGEPASISC 8A VLCDR1 RSSQSLLHSRGHTSLH 9A VLFW2 WYLQKPGQSPHLLIY 10A VLCDR2 SVFNRAS 11A VLFW3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 12A VLCDR3 MQALEPPYT 13A VLFW4 FGQGTKLEIK 14A

TABLE-US-00008 TABLEB AntibodyAB118 06-AB- Seq. 118 Sequence IDNo. VHFW1 EVQLVESGGGLVQPGGSLRLSCSASQFIL 1B VHCDR1 SNYWVH 2B VHFW2 WVRQVPGEGLVWVS 3B VHCDR2 RINEDGSVTSYADSVKG 4B VHFW3 RFTISRDNAKNTLYLQMNSLRVDDTAVYYCVR 5B VHCDR3 DLCGERDD 6B VHFW4 WGQGTLVSVSS 7B VLFW1 DIVMTQSPLSLPVTPGEAASISC 8B VLCDR1 RSSQSLLHRNGKTFFA 9B VLFW2 WYLQKPGQSPQILIY 10B VLCDR2 LGSNRAS 11B VLFW3 GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC 12B VLCDR3 MQGLQTPYT 13B VLFW4 FGQGTKLEIK 14B

TABLE-US-00009 TABLEC AntibodyAB120 06-AB- Seq. 120 Sequence IDNo. VHFW1 EVQLVQSGGGLVQPGGSLGLSCAASGFIF 1C VHCDR1 TSYAMT 2C VHFW2 WVRQAPGKGLEWVS 3C VHCDR2 VITGNVGTSYYADSVKG 4C VHFW3 RFTISRDNSKKTVSLQMNSLRAEDTAIYYCVK 5C VHCDR3 TRYDFSSGYYFDD 6C VHFW4 WGQGTLVSVSS 7C VLFW1 DIVMTQSPSSVSASVGDKVTITC 8C VLCDR1 RASQGISRWLA 9C VLFW2 WYQQKPGEAPELLIY 10C VLCDR2 AASSLQS 11C VLFW3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 12C VLCDR3 QQANSFPIT 13C VLFW4 FGQGTRLQIK 14C

TABLE-US-00010 TABLED AntibodyAB121 06-AB- Seq. 121 Sequence IDNo. VHFW1 EVQLVESGGTLVQPGGSLRLSCAASGFTF 1D VHCDR1 SDYWMN 2D VHFW2 WVRQAPGKGLEWVA 3D VHCDR2 NIKQDGSEKYYVDSLRG 4D VHFW3 RVTISRDNAQNSVFLQMHSLSVEDTAVYYCAR 5D VHCDR3 DGYTFGPATTELDH 6D VHFW4 WGRGTLVSVSS 7D VLFW1 QLVLTQPPSVSVSPGQTASITC 8D VLCDR1 SGDELRNKYTS 9D VLFW2 WYQQKSGQSPVLVIY 10D VLCDR2 QDNNRPS 11D VLFW3 GIPERFSGSQSGDTATLTISGTQAVDEADYYC 12D VLCDR3 QAWVSQTLV 13D VLFW4 FGGGTKLTVL 14D

TABLE-US-00011 TABLEE AntibodyAB122 06-AB- Seq. 122 Sequence IDNo. VHFW1 EVQLVQSGGGLAQPGRSLRLSCAASGFGF 1E VHCDR1 DDFAMH 2E VHFW2 WVRQPPGKGLEWVS 3E VHCDR2 GLTWNGGSIDYAGSVRG 4E VHFW3 RFTISRDNAKNSLFLQMNSLRAEDTALYYCAK 5E VHCDR3 GLSGGTMAPFDI 6E VHFW4 WGQGTMVSVSS 7E VLFW1 QAGLTQPPSVSVAPGQTATIPC 8E VLCDR1 GGNNIGSKHVH 9E VLFW2 WYQQKPGQAPVAVVY 10E VLCDR2 DDSDRPS 11E VLFW3 GVPERFSGSNSGNTATLTISSVEAGDEADYYC 12E VLCDR3 QVWDRSSDHFWL 13E VLFW4 FGGGTRLTVL 14E

TABLE-US-00012 TABLEF AntibodyAB123 Seq. 06-AB-123 Sequence IDNo. VHFW1 EVQLLESGGGVVQPGRSLRLSCAASGFTF 1F VHCDR1 SNYGMH 2F VHFW2 WVRQAPGKGLEWVA 3F VHCDR2 VVWFDGSYKYYTDSVKG 4F VHFW3 RFTISRDNSKSTLYLQMNSLRAEDTAVYYCVS 5F VHCDR3 PIMTSAFDI 6F VHFW4 WGPGTMVSVSS 7F VLFW1 QLVLTQPPSASGSPGQSVTISC 8F VLCDR1 TGTSSDVGGSNFVS 9F VLFW2 WYQHHPGKAPKLMIY 10F VLCDR2 EVSQRPS 11F VLFW3 GVPDRFSGSKSGNTASLTVSGLQADDEADYYC 12F VLCDR3 SSYAGSVVL 13F VLFW4 FGGGTKLTVL 14F

TABLE-US-00013 TABLEG AntibodyAB124 Seq. 06-AB-124 Sequence IDNo. VHFW1 EVQLVESGGGVVQPGRSLRLSCAASGFTF 1G VHCDR1 SNYGMH 2G VHFW2 WVRQAPGKGLEWVA 3G VHCDR2 VVWLDGSYKYYTGSVKG 4G VHFW3 RFTISRDNSKSTLYLQMNSLRAEDTAAYYCVS 5G VHCDR3 PIMTSAFDI 6G VHFW4 WGPGTMVTVSS 7G VLFW1 QLVLTQPPSASGSPGQSVTISC 8G VLCDR1 TGTSSDVGGSNFVS 9G VLFW2 WYQHHPGKAPKLMIY 10G VLCDR2 EVSQRPS 11G VLFW3 GVPDRFSGSKSGNTASLTVSGLQADDEADYYC 12G VLCDR3 SSYAGSVVL 13G VLFW4 FGGGTKLTVL 14G

TABLE-US-00014 TABLEH AntibodyAB126 Seq. 06-AB-126 Sequence IDNo. VHFW1 EVQLVESGGGLAQPGGSLRLSCEASGFHL 1H VHCDR1 AGNAMA 2H VHFW2 WVRQAPGKGLEWVA 3H VHCDR2 AIGGSDDRTDYADSVKG 4H VHFW3 RFTISRDKSKNTLSLQMNSLRVEDTAVYYCAK 5H VHCDR3 DIWRWAFDY 6H VHFW4 WGQGTLVSVSS 7H VLFW1 DIVMTQSPATLSLSPGERATLSC 8H VLCDR1 WASQYINTYVN 9H VLFW2 WYQHKPGQAPRLLIY 10H VLCDR2 DASKRAT 11H VLFW3 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC 12H VLCDR3 QQGSNWPLT 13H VLFW4 FGQGTRLEIK 14H

TABLE-US-00015 TABLEI AntibodyAB127 Seq. 06-AB-127 Sequence IDNo. VHFW1 EVQLVESGGGLVNPGGSLRLSCAASGFTF 1I VHCDR1 SNYAMN 2I VHFW2 WVRQAPGKGLEWVS 3I VHCDR2 SISRSGDYIYYADSLKG 4I VHFW3 RSTISRDNAKNSLFLQMNSLRAEDSAVYYCAR 5I VHCDR3 DWGRLGYCSSNNCPDAFDV 6I VHFW4 WGQGTRVSVSS 7I VLFW1 EIVMTQSPSFVSASVGDRVTITC 8I VLCDR1 RASQDISNWLV 9I VLFW2 WYQQKPGKAPKLLIY 10I VLCDR2 ASSNLQS 11I VLFW3 GVPSRFSGSGSGTDFALTIISLQPEDFATYYC 12I VLCDR3 QQENSFPYT 13I VLFW4 FGQGTKLEIK 14I

TABLE-US-00016 TABLEJ AntibodyAB129 Seq. 06-AB-129 Sequence IDNo. VHFW1 QVQLVESGGGLVQPGGSLRLSCSASQFIL 1J VHCDR1 SNYWVH 2J VHFW2 WVRQVPGEGLVWVS 3J VHCDR2 RINEDGSVTSYADSVKG 4J VHFW3 RFTISRDNAKNTLYLQMNSLRVDDTAVYYCVR 5J VHCDR3 DLCGERDD 6J VHFW4 WGQGTLVTVSS 7J VLFW1 VIWMTQSPLSLPVTPGEAASISC 8J VLCDR1 RSSQSLLHRNGRTFFA 9J VLFW2 WYLQKPGQSPQILIY 10J VLCDR2 LGSNRAF 11J VLFW3 GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC 12J VLCDR3 MQGLQTPYT 13J VLFW4 FGQGTKLEIK 14J

TABLE-US-00017 TABLEK AntibodyAB130 Seq. 06-AB-130 Sequence IDNo. VHFW1 QLQLQESGGGLVQPGGSLRLSCSASQFIL 1K VHCDR1 SNYWVH 2K VHFW2 WVRQVPGEGLVWVS 3K VHCDR2 RINEDGSVTSYADSVKG 4K VHFW3 RFTISRDNAKNTLYLQMNSLRVDDTAVYYCVR 5K VHCDR3 DLCWERDD 6K VHFW4 WGQGTLVSVSS 7K VLFW1 VIWMTQSPLSLPVTPGEAASISC 8K VLCDR1 RSSQSLLHRNGRTFFA 9K VLFW2 WYLQKPGQSPQILIY 10K VLCDR2 LGSNRAF 11K VLFW3 GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYC 12K VLCDR3 MQGLQTPYT 13K VLFW4 FGQGTKLEIK 14K

TABLE-US-00018 TABLEL AntibodyAB131 Seq. 06-AB-131 Sequence IDNo. VHFW1 QVQLVQSGGGVVQPGGSLRLSCAASPFTF 1L VHCDR1 KTSILH 2L VHFW2 WVRQAPGKGLEWVS 3L VHCDR2 AMSYDGFSKYYADSVKG 4L VHFW3 RLTISRDSSTNTLYLEMNSLRFEDTALYFCAR 5L VHCDR3 EAYTSGRAGCFNP 6L VHFW4 WGQGVLVSVSS 7L VLFW1 DIVMTQTPSTQSASVGDRVTITC 8L VLCDR1 RASQSISIWLA 9L VLFW2 WYQQKPGKAPKLLIH 10L VLCDR2 DASTLES 11L VLFW3 GVPSRFSGSGSGTEFTLTISSLQPDDSATYYC 12L VLCDR3 QRYNDYPPT 13L VLFW4 FGPGTKVEIK 14L

TABLE-US-00019 TABLEM AntibodyAB132 Seq. 06-AB-132 Sequence IDNo. VHFW1 QVLKESGGGVVQPGGSLRLSCAASPFTF 1M VHCDR1 ETSILH 2M VHFW2 WVRQAPGKGLEWVS 3M VHCDR2 AMSYDGFSKYYADSVKG 4M VHFW3 RLTISRDSSTNTLYLEMNSLRFEDTALYFCAR 5M VHCDR3 EAYTSGRAGCFDP 6M VHFW4 WGQGVLVSVSS 7M VLFW1 EIVMTQSPSTQSASVGDRVTITC 8M VLCDR1 RASQSISIWLA 9M VLFW2 WYQQKPGKAPKLLIH 10M VLCDR2 DASTLES 11M VLFW3 GVPSRFSGSGSGTEFTLTISSLQPDDSATYYC 12M VLCDR3 QRYNDYPPT 13M VLFW4 FGPGTKVEIK 14M

TABLE-US-00020 TABLEN AntibodyAB133 Seq. 06-AB-133 Sequence IDNo. VHFW1 EVQLVESGGGLVQPGGSLRVSCAASGFTL 1N VHCDR1 NTYWMH 2N VHFW2 WVRQAPGKGLVWVS 3N VHCDR2 RINEDGTTISYADSVRG 4N VHFW3 RFTISRDNAENTLYLQMHSLRAEDTGVYYCAR 5N VHCDR3 DFTGPFDS 6N VHFW4 WGQGTLVSVSS 7N VLFW1 QSVLTQPPSVSGTPGQRVTISC 8N VLCDR1 SGSNSNAGRDYVS 9N VLFW2 WYQQVPGTAPKLLIY 10N VLCDR2 KNNQRPS 11N VLFW3 GVPDRFSGSKSGTSASLAISGLRSEDDGDYYC 12N VLCDR3 IVWDGSLSGYV 13N VLFW4 FGTGTKVTVL 14N

TABLE-US-00021 TABLEO AntibodyAB134 Seq. 06-AB-134 Sequence IDNo. VHFW1 QLQLQESGGGLVQPGGSLRLSCVVSGFTF 1O VHCDR1 SSHAMS 2O VHFW2 WVRQAPGKGLEWVS 3O VHCDR2 SISISGGDTFYADSVRG 4O VHFW3 RFTIFRDNSKNTVYLQMNSLRAEDTAVYYCAT 5O VHCDR3 ETSPNDY 6O VHFW4 WGQGTLVSVSS 7O VLFW1 SYELTQPSSLTVSPGGTVTLTC 8O VLCDR1 GLSSGAVTSGHYPY 9O VLFW2 WFQQKPGQAPKTLIF 10O VLCDR2 DTSRKHS 11O VLFW3 WTPARFSGSLLGGKAALTLSGAQPEDDADYYC 12O VLCDR3 LLACNGACV 13O VLFW4 FGGGTKLTVL 14O

TABLE-US-00022 TABLEP AntibodyAB135 Seq. 06-AB-135 Sequence IDNo. VHFW1 EVQLVETGGGLVQPGGSLRLSCVVSGFTF 1P VHCDR1 SSHAMS 2P VHFW2 WVRQAPGKGLEWVS 3P VHCDR2 SISISGGDTFYADSVRG 4P VHFW3 RFTIFRDNSKNTVYLQMNSLRAEDTAVYYCAT 5P VHCDR3 ETSPNDY 6P VHFW4 WGQGTLVTVSS 7P VLFW1 SYELTQPSSLTVSPGGTVTLTC 8P VLCDR1 GLSSGAVTSGHYPY 9P VLFW2 WFQQKPGQAPKTLIF 10p VLCDR2 DTSRKHS 11P VLFW3 WTPARFSGSLLGGKAALTLSGAQPEDDADYYC 12P VLCDR3 LLACNGACV 13P VLFW4 FGGGTKLTVL 14P

TABLE-US-00023 TABLEQ AntibodyAB139 Seq. 06-AB-139 Sequence IDNo. VHFW1 EVQLVESGGGLVQPGGSLRVSCAASGFTL 1Q VHCDR1 NTYWMH 2Q VHFW2 WVRQAPGKGLVWVS 3Q VHCDR2 RINEDGTTISYADSVRG 4Q VHFW3 RFTISRDNAENTLYLQMHSLRAEDTGVYYCAR 5Q VHCDR3 DFTGPFDS 6Q VHFW4 WGQGTLVSVSS 7Q VLFW1 QSVLTQPPSVSGTPGQRVTISC 8Q VLCDR1 SGSNSNVGRDYVS 9Q VLFW2 WYQQVPGTAPKLLIY 10Q VLCDR2 KNNRRPS 11Q VLFW3 GVPDRFSGSKSGTSASLAISGLRSEDDGDYYC 12Q VLCDR3 IVWDGSLSGYV 13Q VLFW4 FGTGTKVTVL 14Q

TABLE-US-00024 TABLER AntibodyAB140 Seq. 06-AB-140 Sequence IDNo. VHFW1 EVQLVESGGGLVQPGGSLRVSCAASGFTL 1R VHCDR1 NTYWMH 2R VHFW2 WVRQAPGKGLVWVS 3R VHCDR2 RINEDGTTISYADSVRG 4R VHFW3 RFTISRDNAENTLYLQMHSLRAEDTGVYYCAR 5R VHCDR3 DFTGPFDS 6R VHFW4 WGQGTLVSVSS 7R VLFW1 QLVLTQPPSVSGTPGQRVTISC 8R VLCDR1 SGSNSNVGRDYVS 9R VLFW2 WYQQVPGTAPKLLIY 10R VLCDR2 KNNQRPS 11R VLFW3 GVPDRFSGSKSGTSASLAISGLRSEDDGDYYC 12R VLCDR3 IVWDGSLSGYV 13R VLFW4 FGTGTKVTVL 14R

TABLE-US-00025 TABLEVH-CDR3-MOD SEQ (Variant Lightor ID ofSEQ HeavyCDR3 NO IDNO) 06-AB-118.Heavy DLAGERDD 17B 6B C101A 06-AB-118.Heavy DLSGERDD 18B 6B C101S 06-AB-127.HeavyWY DWGRLGYWSSNNY 17I 6I PDAFDV 06-AB-127.HeavyAA DWGRLGYASSNNA 18I 6I PDAFDV 06-AB-131.HeavyW EAYTSGRAGWFNP 17L 6L 06-AB-131.HeavyA EAYTSGRAGAFNP 18L 6L 06-AB-132.HeavyW EAYTSGRAGWFDP 17M 6M 06-AB-132.HeavyA EAYTSGRAGAFDP 18M 6M 06-AB-129.HeavyW DLWGERDD 17J 6J 06-AB-129.HeavyA DLAGERDD 18J 6J

TABLE-US-00026 TABLEVL-CDR3-MOD 06-AB-134.LightYW LLAYNGAWV 19O 13O 06-AB-134.LightAA LLAANGAAV 20O 13O 06-AB-135.LightYW LLAYNGAWV 19P 13P 06-AB-135.LightAA LLAANGAAV 20P 13P

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