Antigens for seroprevalence, Anti-glucanase antibodies and diagnostics of pneumocystis pneumonia

Abstract

The present disclosure relates generally to immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof) that can bind to glucanase, and recombinant glucanase proteins. The antibodies and proteins of the present technology are useful in methods for detecting and treating Pneumocystis jirovecii infections in a subject in need thereof.

Description

ANTIGENS FOR SEROPREVALENCE, ANTI-GLUCANASE ANTIBODIES AND DIAGNOSTICS OF PNEUMOCYSTIS PNEUMONIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 502,815, filed May 17, 2023, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present technology relates generally to the preparation of immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof) that specifically bind glucanase, recombinant glucanase antigens, and uses of the same. In particular, the present technology relates to the preparation of anti-glucanase antibodies and recombinant glucanase antigens and their use in detecting the seroprevalence of Pneumocystis jirovecii infections.STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under R01 AU20033, awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Pneumocystis jirovecii is the most common cause of fungal pneumonia in children under the age of 2. Pneumocytes is a member of the ascomycetes fungal family. Accordingly, there is an environmental form called the ascus, and an infectious form known as the troph that attaches to the lung epithelium. The troph is released from the asci and initiates infection after colonizing the lung tissue. However, the inability to culture and propagate this organism has hampered the acquisition of a fungal genome as well as the development of recombinant antigens to conduct seroprevalence studies. Currently, several seroprevalence studies have used major surface glycoprotein (MSG) antigens to detect Pneumocystis jirovecii pneumonia, because of its strong immune-stimulating properties, its ability to provide protective epitopes, and its involvement in the interactions between Pneumocystis and its mammalian host. Djawe et al., Lancet 2019;394(10200):757-79. A limitation of MSG antigens is that they are encoded by several genes and highly polymorphic. Schmid-Siegert et al, J Infect Dis. 2021;223(2):310-8.

[0006] Moreover, the fungus cannot be grown in culture, which restricts the preparations of antigens to organisms of infected host lung which may be contaminated with other co-infections. Djawe et al., Lancet 2019; 394(10200):757-79.

[0007] Accordingly, there is an urgent need for reagents to investigate the seroprevalence of Pneumocystis jirovecii in infected subjects.SUMMARY OF THE PRESENT TECHNOLOGY

[0008] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein (a) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 3, a VH-CDR2 sequence comprising SEQ ID NO: 4, and a VH-CDR3 sequence comprising SEQ ID NO: 5, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 8, a VL-CDR2 sequence comprising SEQ ID NO: 9, and a VL-CDR3 sequence comprising SEQ ID NO: 10; or (b) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 13, a VH-CDR2 sequence comprising SEQ ID NO: 14, and a VH-CDR3 sequence comprising SEQ ID NO: 15, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 18, a VL-CDR2 sequence comprising SEQ ID NO: 19, and a VL-CDR3 sequence comprising SEQ ID NO: 20; or (c) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 23, a VH-CDR2 sequence comprising SEQ ID NO: 24, and a VH-CDR3 sequence comprising SEQ ID NO: 25, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 28, a VL-CDR2 sequence comprising SEQ ID NO: 29, and a VL-CDR3 sequence comprising SEQ ID NO: 30; or (d) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 33, a VH-CDR2 sequence comprising SEQ ID NO: 34, and a VH-CDR3 sequence comprising SEQ ID NO: 35, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 38, a VL-CDR2 sequence comprising SEQ ID NO: 39, and a VL-CDR3 sequence comprising SEQ ID NO: 40; or (e) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 43, a VH-CDR2 sequence comprising SEQ ID NO: 44, and a VH-CDR3 sequence comprising SEQ ID NO: 45, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 48, a VL-CDR2 sequence comprising SEQ ID NO: 49, and a VL-CDR3 sequence comprising SEQ ID NO: 50.

[0009] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein: (a) the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32 and SEQ ID NO: 42; and / or (b) the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37 and SEQ ID NO: 47. In some embodiments, the VH amino acid sequence and the VL amino acid sequence is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 7 (8E5G9-1); SEQ ID NO: 12 and SEQ ID NO: 17 (10A12D3-1); SEQ ID NO: 22 and SEQ ID NO: 27 (13F7B7-1); SEQ ID NO: 32 and SEQ ID NO: 37 (20B12G12-1); and SEQ ID NO: 42 and SEQ ID NO: 47 (20D10D1-1), respectively.

[0010] Additionally or alternatively, in some embodiments, the antibody or antigen binding fragment thereof comprises: (a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 7, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 2; or (b) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 17, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 12; or (c) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 27, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 22; or (d) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 37, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 32; or (e) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 47, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 42.

[0011] Additionally or alternatively, in certain embodiments, the antibody or antigen binding fragment further comprises a Fc domain of an isotype selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE. In other embodiments, the antigen binding fragment is selected from the group consisting of Fab, F(ab’)2, Fab’, scFv, and Fv. The antibody may be a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0012] In any of the preceding embodiments, the antibody or antigen binding fragment binds to an epitope present in a fungal glucanase polypeptide. In some embodiments, the fungal glucanase polypeptide comprises the amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 53. The epitope can be a conformational epitope or a non-conformational epitope.

[0013] In another aspect, the present disclosure provides a composition comprising any and all embodiments of the antibody or antigen binding fragment disclosed herein and a pharmaceutically-acceptable carrier. In some embodiments, the antibody or antigen binding fragment is conjugated to an agent. Examples of suitable agents include, but are not limited to, bactrim, trimethoprim-sulfamethoxazole (TMP-SMX), rezafungin, atovaquone, trimethoprim, dapsone, primaquine, clindamycin, and pentamidine.

[0014] In one aspect, the present disclosure provides a recombinant nucleic acid encoding any and all embodiments of the antibody or antigen binding fragment described herein. In certain embodiments, the recombinant nucleic acid comprises at least one sequence selected from the group consisting of: SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, and 46. Also disclosed herein are host cells or vectors comprising the recombinant nucleic acids disclosed herein.

[0015] In yet another aspect, the present disclosure provides a kit comprising any and all embodiments of the antibody or antigen binding fragment described herein and instructions for use. In some embodiments, the antibody or antigen binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

[0016] In one aspect, the present disclosure provides a method for treating Pneumocystis pneumonia o Pneumocystis jirovecii infection in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of any and all embodiments of the antibody or antigen binding fragment disclosed herein, wherein the antibody or antigen binding fragment is optionally conjugated to at least one additional therapeutic agent. Examples of the at least one additional therapeutic agent include, but are not limited to, bactrim, trimethoprim-sulfamethoxazole (TMP-SMX), rezafungin, atovaquone, trimethoprim, dapsone, primaquine, clindamycin, and pentamidine. In some embodiments, the subject is an immunocompromised patient, a pediatric patient, a geriatric patient, a cancer patient, an autoimmune disease patient, an HIV patient, or an immunosuppressed patient.

[0017] In one aspect, the present disclosure provides a method for detecting Pneumocystis jirovecii in a biological sample comprising contacting the biological sample with any and all embodiments of the antibody or antigen binding fragment disclosed herein, and detecting Pneumocystis jirovecii by detecting binding between glucanase present in the biological sample and the antibody or antigen binding fragment. In another aspect, the present disclosure provides a method for diagnosing Pneumocystis jirovecii infection or Pneumocystis pneumonia in a subject comprising contacting a biological sample obtained from the subject with any and all embodiments of the antibody or antigen binding fragment disclosed herein, detecting binding between glucanase present in the biological sample and the antibody or antigen binding fragment, and diagnosing the subject with Pneumocystis jirovecii infection or Pneumocystis pneumonia when glucanase is detected in the biological sample. In some embodiments, binding between glucanase and the antibody or antigen binding fragment is detected via radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy or lateral flow immunoassay.

[0018] In one aspect, the present disclosure provides a P-1, 3 -endoglucanase antigenic peptide comprising an epitope having the sequence of SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 67. The antigenic peptide may further comprise a heterologous signal peptide, such as a yeast signal peptide, a mammalian signal peptide, an insect signal peptide or a synthetic signal peptide. Also provided herein are kits comprising the P-1, 3 -endoglucanase antigenic peptides disclosed herein, the anti-glucanase antibodies or antigen binding fragments disclosed herein, and instructions for use.

[0019] In one aspect, the present disclosure provides a method for determining Pneumocystis seroprevalence in a subject at risk for or suspected of having a. Pneumocystis jirovecii infection or Pneumocystis pneumonia comprising contacting a biological sample obtained from the subject with the P-1,3 endoglucanase antigenic peptide of the present technology; and detecting binding between anti-glucanase immunoglobulins present in the biological sample and the P-1,3 endoglucanase antigenic peptide. In another aspect, the present disclosure provides a method for diagnosing an active Pneumocystis infection or Pneumocystis pneumonia in a subject in needthereof comprising contacting a biological sample obtained from the subject with the P-1,3 endoglucanase antigenic peptide of the present technology; detecting binding between anti- glucanase immunoglobulins present in the biological sample and the P-1,3 endoglucanase antigenic peptide; and diagnosing the subject with an active Pneumocystis infection or Pneumocystis pneumonia when the presence of anti-glucanase immunoglobulins in the biological sample is detected. The anti-glucanase immunoglobulins may comprise anti- glucanase IgM and / or anti-glucanase IgG. In some embodiments, binding between the P-1,3 endoglucanase antigenic peptide and the anti-glucanase immunoglobulins is detected via radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy or lateral flow immunoassay.

[0020] In any of the preceding embodiments of the methods disclosed herein, the antibody or antigen binding fragment or the P-1,3 endoglucanase antigenic peptide further comprises a detectable label.

[0021] In any and all embodiments of the methods described herein, the biological sample comprises bronchoalveolar lavage fluid (BALF), sputum, throat swabs, serum, plasma, whole blood or urine. In any of the preceding embodiments of the methods disclosed herein, the subject is an immunocompromised patient, a pediatric patient, a geriatric patient, a cancer patient, an autoimmune disease patient, an HIV patient, or an immunosuppressed patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGs. 1A-1D show the identification of glucanase and recombinant glucanase production. FIG. 1A shows the sequence confirmation of P. murina protein. LC-MS / MS data were searched using the Sequest HT algorithm within Proteome Discoverer 2.4 against Mus musculus as the host and Uniprot Pneumocystis murine stain Bl 23 reference database (UP000011958) as the fungal database to obtain peptide and protein identifications. FIG. IB shows a phylogenetic analysis of Glucanase protein demonstrating conservation in Pneumocystis spp. FIG. 1C shows a CE-SDS Electropherogram of recombinant P. jirovecii glucanase protein under reducing conditions. FIG. ID shows a CE-SDS Electropherogram of recombinant P. jirovecii glucanase protein under non-reducing conditions.

[0023] FIG. 2 shows the convalescent antibody responses after P. murina infection. Anti-whole Pneumocystis antigen (PC Ag), Anti-glucanase, and anti-PNEG_01454 IgG were detected after P. murina infection in a murine model. C57BL / 6 mice were infected with 100 pl of P. murinainoculum (~2>< 105asci) via oral pharyngeal administration. 2 or 6 weeks later, sera were collected, diluted 1 :200, and IgG was measured by ELISA.

[0024] FIGs. 3A-3G show anti- . jiorvecii glucanase and anti-PNEG_01454 IgG detected in pediatric human samples measured by ELISA. Serum was diluted 1 :32, and Cohort 1 anti- glucanase IgG (FIG. 3A), Cohort 2 anti -glucanase IgG (FIG. 3B), Cohort 1 and 2 combined anti -glucanase IgG (FIG. 3C), Cohort 1 anti-PNEG_01454 IgG (FIG. 3D), Cohort 2 anti- PNEG_01454 IgG (FIG. 3E), and Cohort 1 and 2 combined anti-PNEG_01454 IgG (FIG. 3F) was measured by ELISA. FIG. 3G shows a simple linear regression of IgG Anti-glucanase titer vs. IgG Anti-PNEG_01454 titer of pediatric human samples. P-value: <0.0001.

[0025] FIGs. 4A-4C show anti- . jirovecii glucanase IgM detected in pediatric human samples measured by ELISA. Serum was diluted 1 :32, and Cohort 1 anti-glucanase IgM (FIG. 4A), Cohort 2 anti-glucanase IgM (FIG. 4B), and Cohort 1 and 2 combined anti-glucanase IgM (FIG. 4C) was measured by ELISA.

[0026] FIGs. 5A-5C show an example of epitope probability in P. jirovecii P-l,3-glucanase with presentation by HLA-DPA1*O1 :O3-DPB1*O4:OL FIG. 5A shows Antigen Processing likelihood (APL) and conformational stability by residue. FIG. 5B shows APL by peptide, predicted affinity of MHCII binding, and combined APL and MHCII binding. FIG. 5C shows the locations of predicted CD4+ epitopes in the structure of P. jirovecii P-l,3-glucanase.

[0027] FIGs. 6A-6C show levels of glucanase detected using sandwich ELISA in human bronchoalveolar lavage (BAL) samples from Pneumocystis positive and Pneumocystis negative samples. FIG. 6A shows a sandwich ELISA using Rabbit anti -Glucanase Protein A Purified capture antibody (GenScript, Bethy Laboratories, Batch 230623), followed by Biotin (Type A) Conjugation of mouse mAb 8E5G9-1 (Abeam, ab274076, Lot: GR3450691-1), and HRP- Conjugated Streptavidin (ThermoScientific, Ref N100; Lot YB364098). FIG. 6B shows a sandwich ELISA using Rabbit anti -Glucanase Protein A Purified capture antibody (GenScript Bethy Laboratories, Batch 230623), followed by HRP conjugated mouse mAb 8E5G9-1 (Abeam, ab274112, Lot: GR3425253-1). FIG. 6C shows detection of glucanase in human bronchoalveolar lavage (BAL) samples from Pneumocystis positive and Pneumocystis negative samples using the sandwich ELISA described in FIG. 6A.

[0028] FIGs. 7A-7B show the detection of glucanase in Human bronchoalveolar lavage (BAL) samples from Pneumocystis positive samples using sandwich ELISA. The sandwich ELISAused Rabbit anti-Glucanase protein affinity purified capture antibody (GenScript, Bethy Laboratories, Batch TAP185937-231128), followed by Biotin (Type A) Conjugation of mouse mAb 8E5G9-1 (Abeam, ab274076, Lot: GR3450691-1) (diluted 1 :20,000), and HRP-Conjugated Streptavidin (ThermoScientific, Ref N100; Lot YB364098), (diluted 1 :5000). Human BAL samples were added neat (25 pl per well).

[0029] FIGs. 8A-8B show the detection of glucanase in human bronchoalveolar lavage (BAL) samples from Pneumocystis negative samples via sandwich ELISA using mAb 13F7B7-1 as capture (FIG. 8A), or mAb 20B12G12-1 as capture (FIG. 8B) followed by Rabbit anti- Glucanase protein affinity purified antibody (GenScript, Bethy Laboratories, Batch TAP185937- 231128), and Goat anti-rabbit IgG-HRP (Cell Signaling 7074S). Human BAL samples were added neat (100 pl per well) and neat spiked with 250 pg / mL of glucanase.

[0030] FIGs. 9A-9E show the detection of glucanase using the different anti-glucanase antibodies of the present technology as capture antibodies. Capture antibodies 13F7B7-1 (FIG. 9A), 8E5G9-1 (FIG. 9B), 10A12D3-1 (FIG. 9C), 20B12G12-1 (FIG. 9D), and 20D10Dl-l (FIG. 9E) were used at the indicated concentrations to generate glucanase ELISA dose-response curves.

[0031] FIG. 10 shows anti-glucanase IgG responses to glucanase antigen detected in Cord Blood Samples as measured by ELISA. Serum was serially diluted 1 : 10-1 :320.

[0032] FIG. 11 shows detection of glucanase in the BAL of infected mice by direct ELISA. ELISA plates were coated with broncho-alveolar lavage of Rag 2-1- mice collected two weeks after being infected with Pneumocystis murina. Groups of C57BL / 6 mice, female, 4 to 6 weeks old were subcutaneously immunized with 10 pg glucanase in Incomplete Freund’s Adjuvant (IF A) or IF A only. Three weeks later, one group of mice were boosted with 10 pg glucanase in IF A and the other 2 groups infected with 100 pl of P. murina inoculum (2* 105cysts) via oral pharyngeal administration. 6 weeks later, all mouse sera were collected for ELISA.

[0033] FIG. 12 shows the nucleotide sequence and the amino acid sequence of the VH domain of the antibody 8E5G9-1, represented as SEQ ID NO: 1 and SEQ ID NO: 2, respectively and the nucleotide sequence and the amino acid sequence of the VL domain of the antibody 8E5G9-1, represented as SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The VH CDR1 (SEQ ID NO: 3), VH CDR2 (SEQ ID NO: 4), VH CDR3 (SEQ ID NO: 5), VL CDR1 (SEQ ID NO: 8), VL CDR2 (SEQ ID NO: 9), and VL CDR3 (SEQ ID NO: 10) are indicated in italicized font.

[0034] FIG. 13 shows the nucleotide sequence and the amino acid sequence of the VH domain of the antibody 10A12D3-1, represented as SEQ ID NO: 11 and SEQ ID NO: 12, respectively and the nucleotide sequence and the amino acid sequence of the VL domain of the antibody 10A12D3-1, represented as SEQ ID NO: 16 and SEQ ID NO: 17, respectively. The VH CDR1 (SEQ ID NO: 13), VH CDR2 (SEQ ID NO: 14), VH CDR3 (SEQ ID NO: 15), VL CDR1 (SEQ ID NO: 18), VL CDR2 (SEQ ID NO: 19), and VL CDR3 (SEQ ID NO: 20) are indicated in italicized font.

[0035] FIG. 14 shows the nucleotide sequence and the amino acid sequence of the VH domain of the antibody 13F7B7-1, represented as SEQ ID NO: 21 and SEQ ID NO: 22, respectively and the nucleotide sequence and the amino acid sequence of the VL domain of the antibody 13F7B7- 1, represented as SEQ ID NO: 26 and SEQ ID NO: 27, respectively. The VH CDR1 (SEQ ID NO: 23), VH CDR2 (SEQ ID NO: 24), VH CDR3 (SEQ ID NO: 25), VL CDR1 (SEQ ID NO: 28), VL CDR2 (SEQ ID NO: 29), and VL CDR3 (SEQ ID NO: 30) are indicated in italicized font.

[0036] FIG. 15 shows the nucleotide sequence and the amino acid sequence of the VH domain of the antibody 20B12G12-1, represented as SEQ ID NO: 31 and SEQ ID NO: 32, respectively and the nucleotide sequence and the amino acid sequence of the VL domain of the antibody 20B12G12-1, represented as SEQ ID NO: 36 and SEQ ID NO: 37, respectively. The VH CDR1 (SEQ ID NO: 33), VH CDR2 (SEQ ID NO: 34), VH CDR3 (SEQ ID NO: 35), VL CDR1 (SEQ ID NO: 38), VL CDR2 (SEQ ID NO: 39), and VL CDR3 (SEQ ID NO: 40) are indicated in italicized font.

[0037] FIG. 16 shows the nucleotide sequence and the amino acid sequence of the VH domain of the antibody 20D10D1-1, represented as SEQ ID NO: 41 and SEQ ID NO: 42, respectively and the nucleotide sequence and the amino acid sequence of the VL domain of the antibody 20D10D1-1, represented as SEQ ID NO: 46 and SEQ ID NO: 47, respectively. The VH CDR1 (SEQ ID NO: 43), VH CDR2 (SEQ ID NO: 44), VH CDR3 (SEQ ID NO: 45), VL CDR1 (SEQ ID NO: 48), VL CDR2 (SEQ ID NO: 49), and VL CDR3 (SEQ ID NO: 50) are indicated in italicized font.

[0038] FIG. 17 shows an exemplary recombinant P. jirovecii glucanase sequence (SEQ ID NO: 51). In the Examples described herein, the P. jirovecii glucanase sequence of SEQ ID NO: 51)comprises a leader sequence of METDTLLLWVLLLWVPGSTG (SEQ ID NO: 52) at its N- terminus.DETAILED DESCRIPTION

[0039] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0040] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. 1999 Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning,' Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells,' Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology . Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0041] Pneumocystis jirovecii pneumonia (PJP) is an opportunistic fungal infection that is severe in immunocompromised patients. The Pneumonia Etiology Research for Child Health (PERCH) study has shown that it is the most common fungal pneumonia in HIV-negative children under the age of five. Group PERfCHPS, Lancet. 2019;394(10200):757-79. In addition, it was amongthe top ten most common pathogens in cases of pneumonia with a positive chest x-ray and without HIV infection in Mali, Kenya, and Zambia. Id. P. jirovecii was more prevalent in children under 1 year old with pneumonia than in those 1 year or older. Id. This high incidence in the first 3-12 months of life coincides with hospitalized PJP pneumonia in children with primary immune deficiencies. Elsegeiny et al, JCI Insight. 2018;3(12); Zakrzewska et al, Dev PeriodMed. 2019;23(3):159-62. Other studies have suggested that P. jirovecii DNA can be often found in healthy infants, raising the possibility of humans being a reservoir for the infection within the community. Vargas et al, Clin Infect Dis. 2001;32(6):855-61. Recombinant antigens for detecting P. jirovecii may prove to be an effective tool for seroprevalence studies.

[0042] To determine what fungal proteins are produced during Pneumocystis pneumonia, Applicant performed proteomics on BAL fluid of mice with active Pneumocystis pneumonia to detect soluble antigens released in the lung during an active infection. A fungal [3-1,3- endoglucanase (PNEG 02407) that has been reported to be expressed in the ascus was identified. Kutty et al, J Infect Dis. 2015;211(5):719-728. RNAseq data also demonstrates expression in both asci and trophs. Additionally, PNEG 02407 is increased in vivo after antibiotic pressure by the echinocandin class of antifungals. Cushion et al, Antimicrob Agents Chemother. 2018;62(5). Glucanase was chosen for further study due to its high homology among Pneumocystis spp, but not other fungi. Applicant expressed recombinant P. jirovecii P-l,3-endoglucanase in Chinese hamster ovary (CHO) cells and used this antigen for seroprevalence studies. Evidence of maternal anti -glucanase in cord blood as well as in the first month of life in two independent pediatric cohorts was observed. A nadir in IgG titers of the two pediatric cohorts followed by an increase over the next two years, consistent with known Pneumocystis infection in the first 1-2 years of life, was also observed. Moreover, there was strong concordance with anti-P-1, 3- endoglucanase IgG with anti-PNEG_01454 IgG, another antigen previously reported. Noell et al, JCI Insight. 2022;7(17). Taken together, these data support that P. jirovecii P-1, 3- endoglucanase is a useful tool for seroprevalence and epidemiology studies of PJP. Also disclosed herein are anti -glucanase antibodies that are useful for the detection of Pneumocystis pneumonia in clinical BAL samples.Definitions

[0043] The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning-l ias commonly understood by one of ordinary skill in the art to which the present technology belongs.

[0044] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0045] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0046] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function.Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, or topically. Administration includes self-administration and the administration by another.

[0047] An “adjuvant” refers to one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens or antibodies. An adjuvant may be administered to a subject before, in combination with, or after administration of the vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B 12), Quil A (saponins), bacterial and fungal cell wall components (e.g., lipopolysaccarides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulatory factors.

[0048] As used herein, the term “amino acid” is used to refer to any organic molecule that contains at least one amino group and at least one carboxyl group. Typically, at least one amino group is at the a position relative to a carboxyl group. The term “amino acid” includes naturally- occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids. Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally-occurring amino acid, z.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally- occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0049] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, “antibodies” (includes intact immunoglobulins) and “antigen binding fragments” specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M'1greater, at least 104M'1greater or at least 105M'1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York, 1997.

[0050] More particularly, antibody refers to a polypeptide ligand comprising at least a light chain immunoglobulin variable region or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. Typically, an immunoglobulin has heavy (H)chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, Kabat el al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P- sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.

[0051] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds glucanase will have a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). “Immunoglobulin- related compositions” as used herein, refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc., as well as antibody fragments. An antibody or antigen binding fragment thereof specifically binds to an antigen.

[0052] As used herein, the term “antibody-related polypeptide” means antigen-binding antibody fragments, including single-chain antibodies, that can comprise the variable region(s) alone, or in combination, with all or part of the following polypeptide elements: hinge region, CHi, CH2, and CH3 domains of an antibody molecule. Also included in the technology are any combinations of variable region(s) and hinge region, CHi, CH2, and CH3 domains. Antibody-related molecules useful in the present methods, e.g., but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Examples include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHi domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHi domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.. Nature 341 : 544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). As such “antibody fragments” or “antigen binding fragments” can comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments or antigen binding fragments include Fab, Fab1, F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0053] As used herein, the term “conjugated” refers to the association of two molecules by any method known to those in the art. Suitable types of associations include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for instance, hydrogen bonds, dipolar interactions, van der Waal forces, electrostatic interactions, hydrophobic interactions and aromatic stacking.

[0054] As used herein, the terms “single-chain antibodies” or “single-chain Fv (scFv)” refer to an antibody fusion molecule of the two domains of the Fv fragment, VL and VH. Single-chain antibody molecules may comprise a polymer with a number of individual molecules, for example, dimer, trimer or other polymers. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv (scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA85:5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[0055] Any of the above-noted antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

[0056] As used herein, an “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen may be a polypeptide (e.g., recombinant glucanase). An antigen may also be administered to an animal to generate an immune response in the animal.

[0057] The term “antigen binding fragment” refers to a fragment of the whole immunoglobulin structure which possesses a part of a polypeptide responsible for binding to antigen. Examples of the antigen binding fragment useful in the present technology include scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2, but are not limited thereto.

[0058] By “binding affinity” is meant the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by standard methods known in the art, including those described herein. A low-affinity complex contains an antibody that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer duration.

[0059] As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid,urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a bronchoalveolar lavage (BAL) sample, a sputum sample, or a tissue sample obtained by needle biopsy or surgical biopsy.

[0060] As used herein, the term “CDR-grafted antibody” means an antibody in which at least one CDR of an “acceptor” antibody is replaced by a CDR “graft” from a “donor” antibody possessing a desirable antigen specificity.

[0061] As used herein, the term “chimeric antibody” means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced, using recombinant DNA techniques, with an Fc constant region from an antibody of another species (e.g., a human Fc constant region). See generally, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al. , Proc. Natl. Acad. Sci. USA 84: 3439- 3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47 : 999-1005, 1987; Wood et al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988.

[0062] As used herein, the term “consensus FR” means a framework (FR) antibody region in a consensus immunoglobulin sequence. The FR regions of an antibody do not contact the antigen.

[0063] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0064] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of,or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0065] An “isolated” or “purified” polypeptide or peptide is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the agent is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. For example, isolated anti-glucanase antibodies or antigen binding fragments of the present technology would be free of materials that would interfere with diagnostic or therapeutic uses of the agent. Such interfering materials may include enzymes, hormones and other proteinaceous and nonproteinaceous solutes.

[0066] As used herein, the term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. In some embodiments, an “epitope” of the glucanase protein is a region of the protein to which the anti-glucanase antibodies of the present technology specifically bind. In some embodiments, the epitope is a conformational epitope or a non- conformational epitope. To screen for anti-glucanase antibodies which bind to an epitope, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if an anti-glucanase antibody binds the same site or epitope as an anti-glucanase antibody of the present technology. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. In a different method, peptides corresponding to different regions of glucanase can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

[0067] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0068] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0069] As used herein, the term “heterologous nucleic acid molecule, peptide or polypeptide” refers to a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule), peptide or polypeptide that is either not normally expressed or is expressed at an aberrant level in a cell or sample obtained from a cell. This nucleic acid can be from another organism, or it can be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0070] As used herein, “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using thefollowing default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0071] As used herein, “humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus FR sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014); Saxena & Wu, Frontiers in immunology 7: 580 (2016).

[0072] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g,around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31- 35B (Hl), 50-65 (H2) and 95-102 (H3) in the VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a “hypervariable loop” (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (Hl), 52A-55 (H2) and 96-101 (H3) in the VH (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0073] As used herein, the terms “identical” or percent “identity”, when used in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein)), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., NCBI web site). Such sequences are then said to be “substantially identical.” This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

[0074] As used herein, the term “intact antibody” or “intact immunoglobulin” means an antibody that has at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHi, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FRi, CDRi, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0075] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0076] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, z.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, e.g., but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (See, e.g., U.S. Patent No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.

[0077] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20thedition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.).

[0078] As used herein, the term “polyclonal antibody” means a preparation of antibodies derived from at least two (2) different antibody -producing cell lines. The use of this term includes preparations of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0079] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and doublestranded regions, single- and double-stranded RNA, RNA that is mixture of single- and doublestranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0080] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, z.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.

[0081] As used herein, “prevention” or “preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0082] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0083] As used herein, the term “glucanase” refers to a fungal P-l,3-endoglucanase enzyme that breaks down large polysaccharides via hydrolysis. In some embodiments, the glucanase comprises P. jirovecii glucanase protein sequence of SEQ ID NO: 51, or the full-length wildtype P.jirovecii glucanase protein (GeneBank Accession: XP_018229679), (SEQ ID NO: 53).

[0084] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0085] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0086] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0087] As used herein, “specifically binds” refers to a molecule (e.g., an antibody or antigen binding fragment thereof) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, can be exhibited, for example, by a molecule having a KD for the molecule to which it binds to of about 104M, I O5M, 106M, 107M, 108M, 109M, 1010M, 101 1M, or 1012M. The term “specifically binds” may also refer to binding where a molecule (e.g., an antibody or antigen binding fragment thereof) binds to a particular polypeptide (e.g., a glucanase polypeptide), or an epitope on a particular polypeptide, without substantially binding to any other polypeptide, or polypeptide epitope.

[0088] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0089] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0090] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0091] Amino acid sequence modification(s) of the anti-glucanase antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an anti-glucanase antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to obtain the antibody of interest, as long as the obtained antibody possesses the desired properties. The modification also includes the change of the pattern of glycosylation of the protein. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. “Conservative substitutions” are shown in the Table below.

[0092] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites(e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage- displayed variants are then screened for their biological activity e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with similar or superior properties in one or more relevant assays may be selected for further development.Pneumocystis iirovecii

[0093] Pneumocystis jirovecii is a unicellular, obligate fungus that, has two morphological forms: the cyst, a thick-walled spherical or ovoid structure; and the trophozoite, a thin-walled excysted sporozoite. Pneumocystis was initially misclassified as a protozoan because of its atypical characteristics. It lacks chitin in its cell wall and its cell membrane contains cholesterol instead of ergosterol, making it resistant to antifungal drugs that target ergosterol, such as azoles and amphotericin B.

[0094] Pneumocystis jirovecii organisms enter the alveoli through the respiratory passages. They adhere to type 1 pneumocytes, causing diffuse alveolar damage by eroding the pneumocytes. Walzer. AIDS. 1993;7(10): 1293-1305. Normally, the host immune response eradicates the fungus, particularly by the action of alveolar macrophages. In immunocompromised patients, however, a decline in CD4 T-lymphocytes is associated with a CD8 T cell mediated immune response, which causes tissue damage and impairs gaseous exchange without eliminating the fungus. Castro et al. HIV AIDS (Auckl) 2010;2:123-134. Inhibition of phospholipid secretion by a surface antigen, known as MSG antigen, has also been implicated in pathogenesis of P. jirovecii pneumonia, producing surfactant changes and leading to impaired gas exchange. Walzer, Clin Diagn Lab Immunol. 1999;6(02): 149-155. Histological examination of lung tissue infected by Pneumocystis jirovecii shows the presence of an eosinophilic amorphous exudates in the alveolar spaces, giving the tissue a honeycomb appearance. Lymphocytic infiltration of the interstitium is also seen.Immunoglobulin-related Compositions of the Present Technology

[0095] The present technology describes methods and compositions for the generation and use of anti-glucanase immunoglobulin-related compositions (e.g., anti-glucanase antibodies or antigen binding fragments thereof). The anti-glucanase immunoglobulin-related compositions of the present disclosure may be useful in the diagnosis, or treatment of Pneumocystis pneumonia. Anti-glucanase immunoglobulin-related compositions within the scope of the present technology include, e.g., but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies and diabodies that specifically bind the target polypeptide, a homolog, derivative or a fragment thereof. The present disclosure also provides antigen binding fragments of any of the anti- glucanase antibodies disclosed herein, wherein the antigen binding fragment is selected from the group consisting of Fab, F(ab)'2, Fab’, scFv, and Fv. The anti-glucanase antibodies are also useful for selectively delivering toxic payloads (e.g., anti-fungal agents) to Pneumocystis infected cells in a subject.

[0096] FIGs. 12-16 provide the nucleotide and amino acid sequences for the VH and VL sequences as well as the corresponding CDR sequences for the antibodies disclosed herein (SEQ ID NOs: 1-50). The Table below also provides amino acid sequences for VH and VL as well as the CDR sequences for the antibodies disclosed herein.

[0097] In one aspect, the present disclosure provides an antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein (a) the VH comprises a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence selected from the group consisting of (i) SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; (ii) SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; (iii) SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively; (iv) SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively; and (v) SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; and / or (b) the VL comprises a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence selected from the group consisting of (i) SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; (ii) SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; (iii) SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; (iv) SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; and (v) SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively. In some embodiments, the antibody further comprises a Fc domain of any isotype, e.g., but are notlimited to, IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY.

[0098] Non-limiting examples of constant region sequences include:

[0099] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 54)

[0100] APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQP ATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0101] Human IgGl constant region, Uniprot: P01857 (SEQ ID NO: 55)

[0102] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0103] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 56)

[0104] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK

[0105] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 57)

[0106] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCP EPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSPGK

[0107] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 58)

[0108] GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGF PSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVS VFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGP TTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDD WNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVT GFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTC VAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0109] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 59)

[0110] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCP APEFL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0111] Human IgAl constant region, Uniprot: P01876 (SEQ ID NO: 60)

[0112] ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPS QDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPP TPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVT SILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0113] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 61)

[0114] ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVT LTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDW KKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0115] Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 62)

[0116] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0117] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or is 100% identical to SEQ ID NOS: 54-61. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or is 100% identical to SEQ ID NO: 62. In some embodiments, the immunoglobulin-related compositions of the present technology bind to a region within a glucanase polypeptide comprising the amino acid sequence of SEQ ID NO : 51 or of SEQ ID NO : 53. The epitope may be a conformational or non-conformational epitope.

[0118] In another aspect, the present disclosure provides an isolated immunoglobulin-related composition (e.g., an antibody or antigen binding fragment thereof) comprising a heavy chain immunoglobulin variable domain (VH) amino acid sequence comprising SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, or a variant thereof having one or more conservative amino acid substitutions. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain immunoglobulin variable domain (VL) amino acid sequence comprising SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, or a variant thereof having one or more conservative amino acid substitutions.

[0119] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain immunoglobulin variable domain (VH) amino acid sequence and a light chain immunoglobulin variable domain (VL) amino acid sequence selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 7 (8E5G9-1); SEQ ID NO: 12 and SEQ ID NO: 17 (10A12D3-1); SEQ ID NO: 22 and SEQ ID NO: 27 (13F7B7-1); SEQ ID NO: 32 and SEQ ID NO: 37 (20B12G12-1); and SEQ ID NO: 42 and SEQ ID NO: 47 (20D10D1-1), respectively.

[0120] In any of the above embodiments of the immunoglobulin-related compositions, the HC and LC immunoglobulin variable domain sequences form an antigen binding site that binds to glucanase. In some embodiments, the epitope is a conformational epitope or non-conformational epitope.

[0121] In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0122] In some embodiments, the immunoglobulin-related compositions of the present technology bind specifically to at least one glucanase polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology bind at least one glucanase polypeptide with a dissociation constant (KD) of about I O3M, 104M, I 05M, 106M, 107M, 108M, 109M, 1010M, 101 1M, or 1012M. In certain embodiments, the immunoglobulin- related compositions are monoclonal antibodies, chimeric antibodies, humanized antibodies, or bispecific antibodies. In some embodiments, the antibodies comprise a human antibody framework region.

[0123] In certain embodiments, the immunoglobulin-related composition includes one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 7, 17, 27, 37 or 47; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, 32 or 42. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a “conservative substitution” as defined herein.

[0124] In some aspects, the anti-glucanase immunoglobulin-related compositions described herein contain structural modifications to facilitate rapid binding and cell uptake and / or slow release. In some aspects, the anti-glucanase immunoglobulin-related composition of the present technology (e.g., an antibody) may contain a deletion in the CH2 constant heavy chain region to facilitate rapid binding and cell uptake and / or slow release. In some aspects, a Fab fragment isused to facilitate rapid binding and cell uptake and / or slow release. In some aspects, a F(ab)'2 fragment is used to facilitate rapid binding and cell uptake and / or slow release.

[0125] In one aspect, the present technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, and 46. In another aspect, the present technology provides a host cell or expression vector expressing any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0126] The immunoglobulin-related compositions of the present technology (e.g., an anti- glucanase antibody) can be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies can be specific for different epitopes of one or more glucanase polypeptides or can be specific for both the glucanase polypeptide(s) as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g.,WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; 6,106,835;Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related compositions are chimeric. In certain embodiments, the immunoglobulin-related compositions are humanized.

[0127] The immunoglobulin-related compositions of the present technology can further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.

[0128] In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen binding fragment may be optionally conjugated to an agent selected from the group consisting of isotopes, dyes, chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination thereof. Insome embodiments, the antibody or antigen binding fragment of the present technology may be combined with a pharmaceutically-acceptable carrier. For a chemical bond or physical bond, a functional group on the immunoglobulin-related composition typically associates with a functional group on the agent. Alternatively, a functional group on the agent associates with a functional group on the immunoglobulin-related composition.

[0129] The functional groups on the agent and immunoglobulin-related composition can associate directly. For example, a functional group (e.g., a sulfhydryl group) on an agent can associate with a functional group (e.g., sulfhydryl group) on an immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups can associate through a cross-linking agent (z.e., linker). Some examples of cross-linking agents are described below. The cross-linker can be attached to either the agent or the immunoglobulin-related composition. The number of agents or immunoglobulin-related compositions in a conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of agents associated with a conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin- related compositions associated with an agent depends on the number of functional groups present on the agent.

[0130] In yet another embodiment, the conjugate comprises one immunoglobulin-related composition associated to one agent. In one embodiment, a conjugate comprises at least one agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bonded to an immunoglobulin-related composition by any method known to those in the art. For example, a functional group on the agent may be directly attached to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate and hydroxyl.

[0131] The agent may also be chemically bonded to the immunoglobulin-related composition by means of cross-linking agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can, for example, be obtained from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. web-site can provide assistance. Additional cross-linking agents include the platinum cross-linking agents described in U.S. Pat. Nos. 5,580,990; 5,985,566; and 6,133,038 of Kreatech Biotechnology, B.V., Amsterdam, The Netherlands.

[0132] Alternatively, the functional group on the agent and immunoglobulin-related composition can be the same. Homobifunctional cross-linkers are typically used to cross-link identical functional groups. Examples of homobifunctional cross-linkers include EGS (z.e., ethylene glycol bisfsuccinimidylsuccinate]), DSS (z.e., disuccinimidyl suberate), DMA (z.e., dimethyl adipimidate.2HCl), DTSSP (z.e., 3,3'-dithiobis[sulfosuccinimidylpropionate])), DPDPB (z.e., 1,4- di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (z.e., bis-maleimidohexane). Such homobifunctional cross-linkers are also available from Pierce Biotechnology, Inc.

[0133] In other instances, it may be beneficial to cleave the agent from the immunoglobulin- related composition. The web-site of Pierce Biotechnology, Inc. described above can also provide assistance to one skilled in the art in choosing suitable cross-linkers which can be cleaved by, for example, enzymes in the cell. Thus the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (z.e., 4- succinimidyl oxycarbonyl -methyl-a-[2-pyridyldithio]toluene), Sulfo-LC-SPDP (z.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (z.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), Sulfo-LC-SPDP (z.e., sulfosuccinimidyl 6-(3- [2-pyridyldithio]-propionamido)hexanoate), SPDP (z.e., N-succinimidyl 3-[2-pyridyldithio]- propionamidohexanoate), and AEDP (z.e., 3-[(2-aminoethyl)dithio]propionic acid HC1).

[0134] In another embodiment, a conjugate comprises at least one agent physically bonded with at least one immunoglobulin-related composition. Any method known to those in the art can be employed to physically bond the agents with the immunoglobulin-related compositions. For example, the immunoglobulin-related compositions and agents can be mixed together by any method known to those in the art. The order of mixing is not important. For instance, agents can be physically mixed with immunoglobulin-related compositions by any method known to those in the art. For example, the immunoglobulin-related compositions and agents can be placed in a container and agitated, by for example, shaking the container, to mix the immunoglobulin-related compositions and agents.

[0135] The immunoglobulin-related compositions can be modified by any method known to those in the art. For instance, the immunoglobulin-related composition may be modified by means of cross-linking agents or functional groups, as described above.A. Methods of Preparing Anti-Glucanase Antibodies and Recombinant Glucanase Proteins of the Present Technology

[0136] General Overview. Initially, a target polypeptide is chosen to which an antibody of the present technology can be raised. For example, an antibody may be raised against the full-length glucanase protein, or to a portion of the glucanase protein. Techniques for generating antibodies directed to such target polypeptides are well known to those skilled in the art. Examples of such techniques include, for example, but are not limited to, those involving display libraries, xeno or human mice, hybridomas, and the like. Target polypeptides within the scope of the present technology include any polypeptide derived from the glucanase protein which is capable of eliciting an immune response.

[0137] It should be understood that recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, which are directed to glucanase and fragments thereof are suitable for use in accordance with the present disclosure.

[0138] Anti-glucanase antibodies that can be subjected to the techniques set forth herein include monoclonal and polyclonal antibodies, and antibody fragments such as Fab, Fab', F(ab')2, Fd, scFv, antibody light chains, antibody heavy chains and / or antibody fragments. Methods useful for the high yield production of antibody Fv-containing polypeptides, e.g., Fab' and F(ab')2 antibody fragments have been described. See U.S. Pat. No. 5,648,237.

[0139] Generally, an antibody is obtained from an originating species. More particularly, the nucleic acid or amino acid sequence of the variable portion of the light chain, heavy chain or both, of an originating species antibody having specificity for a target polypeptide antigen is obtained. An originating species is any species which was useful to generate the antibody of the present technology or library of antibodies, e.g., rat, mouse, rabbit, chicken, monkey, human, and the like.

[0140] Phage or phagemid display technologies are useful techniques to derive the antibodies of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies of the present technology, can be carried out in E. coli.

[0141] Due to the degeneracy of nucleic acid coding sequences, other sequences which encode substantially the same amino acid sequences as those of the naturally occurring proteins may be used in the practice of the present technology. These include, but are not limited to, nucleic acidsequences including all or portions of the nucleic acid sequences encoding the above polypeptides, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. It is appreciated that the nucleotide sequence of an immunoglobulin according to the present technology tolerates sequence homology variations of up to 25% as calculated by standard methods (“Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.) so long as such a variant forms an operative antibody which recognizes glucanase. For example, one or more amino acid residues within a polypeptide sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligands, etc. Additionally, an immunoglobulin encoding nucleic acid sequence can be mutated in vitro or in vivo to create and / or destroy translation, initiation, and / or termination sequences or to create variations in coding regions and / or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to in vitro site directed mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia), and the like.

[0142] Preparation of Polyclonal Antisera and Immunogens. Methods of generating antibodies or antibody fragments of the present technology typically include immunizing a subject (generally a non-human subject such as a mouse or rabbit) with a purified glucanase protein or fragment thereof or with a cell expressing the glucanase protein or fragment thereof. An appropriate immunogenic preparation can contain, e.g., a recombinantly-expressed glucanase protein or a chemically-synthesized glucanase peptide. The glucanase protein, or a portion or fragment thereof, can be used as an immunogen to generate an anti-glucanase antibody that bindsto the glucanase protein, or a portion or fragment thereof using standard techniques for polyclonal and monoclonal antibody preparation.

[0143] If needed, the immunogenicity of the glucanase protein (or fragment thereof) can be increased by fusion or conjugation to a carrier protein such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. One can also combine the glucanase protein with a conventional adjuvant such as Freund’s complete or incomplete adjuvant to increase the subject’s immune reaction to the polypeptide. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g, aluminum hydroxide), surface active substances (e.g, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0144] In describing the present technology, immune responses may be described as either “primary” or “secondary” immune responses. A primary immune response, which is also described as a “protective” immune response, refers to an immune response produced in an individual as a result of some initial exposure (e.g., the initial “immunization”) to a particular antigen, e.g., glucanase protein. In some embodiments, the immunization can occur as a result of vaccinating the individual with a vaccine containing the antigen. For example, the vaccine can be a glucanase vaccine comprising one or more glucanase protein-derived antigens. A primary immune response can become weakened or attenuated over time and can even disappear or at least become so attenuated that it cannot be detected. Accordingly, the present technology also relates to a “secondary” immune response, which is also described here as a “memory immune response.” The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has already been produced.

[0145] Thus, a secondary immune response can be elicited, e.g., to enhance an existing immune response that has become weakened or attenuated, or to recreate a previous immune response that has either disappeared or can no longer be detected. The secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells that were generated at the first presentation of the antigen. Delayed type hypersensitivity (DTH) reactions are a type of cellular secondary or memory immune response that are mediated by CD4+T cells. A first exposure to an antigen primes the immune system and additional exposure(s) results in a DTH.

[0146] Following appropriate immunization, the anti-glucanase antibody can be prepared from the subject’s serum. If desired, the antibody molecules directed against the glucanase protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography to obtain the IgG fraction.

[0147] Monoclonal Antibody. In one embodiment of the present technology, the antibody is an anti-glucanase monoclonal antibody. For example, in some embodiments, the anti-glucanase monoclonal antibody may be a human or a mouse anti-glucanase monoclonal antibody. For preparation of monoclonal antibodies directed towards the glucanase protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (See, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (See, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (See, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be utilized in the practice of the present technology and can be produced by using human hybridomas (See, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (See, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids that encode regions of antibodies can be isolated. PCR utilizing primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding portions of antibodies from the population and then DNAs encoding antibodies or fragments thereof, such as variable domains, are reconstructed from the amplified sequences. Such amplified sequences also can be fused to DNAs encoding other proteins - e.g., a bacteriophage coat, or a bacterial cell surface protein - for expression and display of the fusion polypeptides on phage or bacteria. Amplified sequences can then be expressed and further selected or isolated based, e.g., on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on the glucanase protein. Alternatively, hybridomas expressing anti-glucanase monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject’s spleen using routine methods. See, e.g., Milstein et al., (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods will produce monoclonal antibodies of varying specificity (i.e., for differentepitopes) and affinity. A selected monoclonal antibody with the desired properties, e.g., glucanase binding, can be used as expressed by the hybridoma, it can be bound to a molecule such as polyethylene glycol (PEG) to alter its properties, or a cDNA encoding it can be isolated, sequenced and manipulated in various ways. Synthetic dendromeric trees can be added to reactive amino acid side chains, e.g., lysine, to enhance the immunogenic properties of glucanase protein. Also, CPG-dinucleotide techniques can be used to enhance the immunogenic properties of the glucanase protein. Other manipulations include substituting or deleting particular amino acyl residues that contribute to instability of the antibody during storage or after administration to a subject, and affinity maturation techniques to improve affinity of the antibody of the glucanase protein.

[0148] Hybridoma Technique. In some embodiments, the antibody of the present technology is an anti -glucanase monoclonal antibody produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma techniques include those known in the art and taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et aL, Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art.

[0149] Phage Display Technique. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA and phage display technology. For example, anti -glucanase antibodies, can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them. Phages with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g., human or murine) by selecting directly with an antigen, typically an antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains that are recombinantly fused to either the phage gene III or gene VIII protein. In addition, methods can be adapted for the construction of Fab expression libraries (See, e.g., Huse, et al., Science 246: 1275-1281, 1989) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a glucanase polypeptide, e.g., a polypeptide or derivatives, fragments, analogs orhomologs thereof. Other examples of phage display methods that can be used to make the antibodies of the present technology include those disclosed in Huston et al., Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Set U.S.A., 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994;PCT / GB91 / 01134; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 96 / 06213; WO 92 / 01047 (Medical Research Council et all , WO 97 / 08320 (Morphosys); WO 92 / 01047 (CAT / MRC); WO 91 / 17271 (Affymax); and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. Methods useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides via disulfide bonds have been described by Lohning, U.S. Pat. No. 6,753,136. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0150] Generally, hybrid antibodies or hybrid antibody fragments that are cloned into a display vector can be selected against the appropriate antigen in order to identify variants that maintain good binding activity, because the antibody or antibody fragment will be present on the surface of the phage or phagemid particle. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). However, other vector formats could be used for this process, such as cloning the antibody fragment library into a lytic phage vector (modified T7 or Lambda Zap systems) for selection and / or screening.

[0151] Expression of Recombinant Anti-Glucanase Antibodies and Recombinant Glucanase Proteins. As noted above, the antibodies of the present technology can be produced through the application of recombinant DNA technology. Recombinant polynucleotide constructs encoding an anti -glucanase antibody or glucanase protein of the present technology typically include an expression control sequence operably-linked to the coding sequences of anti -glucanase antibodychains or glucanase protein, including naturally-associated or heterologous promoter regions. As such, another aspect of the technology includes vectors containing one or more nucleic acid sequences encoding an anti -glucanase antibody or glucanase protein of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, the nucleic acid containing all or a portion of the nucleotide sequence encoding the anti-glucanase antibody or glucanase protein is inserted into an appropriate cloning vector, or an expression vector (z.e., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al., U.S. Pat. Nos. 6,291,160 and 6,680,192.

[0152] In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present technology is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Such viral vectors permit infection of a subject and expression of a construct in that subject. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding the anti-glucanase antibody or glucanase protein, and the collection and purification of the anti-glucanase antibody or glucanase protein, e.g., cross-reacting anti-glucanase antibodies or glucanase proteins. See generally, U.S. 2002 / 0199213. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin-resistance or hygromycin-resi stance, to permit detection of those cells transformed with the desired DNA sequences. Vectors can also encode signal peptide, e.g., pectate lyase, useful to direct the secretion of extracellular antibody fragments. See U.S. Pat. No. 5,576,195.

[0153] The recombinant expression vectors of the present technology comprise a nucleic acid encoding a protein with glucanase binding properties or a glucanase protein in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to beused for expression that is operably-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, e.g., in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. Typical regulatory sequences useful as promoters of recombinant polypeptide expression (e.g., anti-glucanase antibody or glucanase protein), include, e.g., but are not limited to, promoters of 3 -phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. In one embodiment, a polynucleotide encoding an anti-glucanase antibody or glucanase protein of the present technology is operably-linked to an ara B promoter and expressible in a host cell. See U.S. Pat. 5,028,530. The expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides, encoded by nucleic acids as described herein (e.g., anti-glucanase antibody, glucanase protein, etc.).

[0154] Another aspect of the present technology pertains to anti-glucanase antibody-expressing host cells and glucananse protein-expressing host cells, which contain a nucleic acid encoding one or more anti-glucanase antibodies or glucanase proteins. The recombinant expression vectors of the present technology can be designed for expression of an anti-glucanase antibody or glucanase protein in prokaryotic or eukaryotic cells. For example, an anti-glucanase antibody or glucanase protein can be expressed in bacterial cells such as Escherichia co . insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated in vitro, e.g., using T7 promoter regulatory sequences and T7 polymerase. Methods useful for the preparation and screening of polypeptides having a predetermined property, e.g., anti-glucanase antibody, via expression of stochastically generated polynucleotide sequences has been previously described. See U.S. Pat. Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641.

[0155] Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or nonfusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0156] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET l id (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of distinct active peptide or protein domains to yield multifunctional polypeptides via polypeptide fusion has been described by Pack et al., U.S. Pat. Nos. 6,294,353; 6,692,935. One strategy to maximize recombinant polypeptide expression, e.g., an anti-glucanase antibody or glucanase protein, in E. coli is to express the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individualcodons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli See, e.g., Wada, et al., 1992. NucL Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the present technology can be carried out by standard DNA synthesis techniques.

[0157] In another embodiment, the anti-glucanase antibody or glucanase protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell &. 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, an anti-glucanase antibody or glucanase protein can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides, e.g., anti-glucanase antibody, in cultured insect cells e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0158] In yet another embodiment, a nucleic acid encoding an anti-glucanase antibody or glucanase protein of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, e.g., but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that are useful for expression of the anti- glucanase antibody of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0159] Another aspect of the present methods pertains to host cells into which a recombinant expression vector of the present technology has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0160] A host cell can be any prokaryotic or eukaryotic cell. For example, an anti-glucanase antibody or glucanase protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are a suitable host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986 Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. Co etal., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0161] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (See generally, Sambrook el al. , Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0162] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the anti- glucanase antibody or glucanase protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0163] A host cell that includes an anti-glucanase antibody or glucanase protein of the present technology, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) recombinant anti-glucanase antibodies or glucanase proteins. In one embodiment, the method comprises culturing the host cell (into which a recombinant expression vector encoding the anti-glucanase antibody or glucanase protein has been introduced) in a suitable medium such that the anti-glucanase antibody or glucanase protein is produced. In another embodiment, the method further comprises the step of isolating the anti-glucanase antibody or glucanase protein from the medium or the host cell. Once expressed, collections of the anti-glucanase antibody or glucanase protein, e.g., the anti-glucanase antibodies, the anti-glucanase antibody -related polypeptides, or glucanase proteins are purified from culture media and host cells. The anti- glucanase antibody or glucanase protein can be purified according to standard procedures of the art, including HPLC purification, column chromatography, gel electrophoresis and the like. In one embodiment, the anti-glucanase antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Usually, anti-glucanase antibody chains or glucanase proteins are expressed with signal sequences and are thus released to the culture media.However, if the anti-glucanase antibody chains or glucanase proteins are not naturally secreted by host cells, the anti-glucanase antibody chains or glucanase proteins can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification technique, column chromatography, ion exchange purification technique, gel electrophoresis and the like (See generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982).

[0164] Single-Chain Antibodies. In one embodiment, the anti-glucanase antibody of the present technology is a single-chain anti-glucanase antibody. According to the present technology, techniques can be adapted for the production of single-chain antibodies specific to a glucanase protein See, e.g, U.S. Pat. No. 4,946,778). Examples of techniques which can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al. , Methods in Enzymology, 203: 46-88,1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0165] Chimeric and Humanized Antibodies. In one embodiment, the anti-glucanase antibody of the present technology is a chimeric anti-glucanase antibody. In one embodiment, the anti- glucanase antibody of the present technology is a humanized anti-glucanase antibody. In other embodiements, the anti-glucanase antibody of the present technology is not humanized. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody.

[0166] Recombinant anti-glucanase antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques, and are within the scope of the present technology. For some uses, including in vivo use of the anti-glucanase antibody of the present technology in humans as well as use of these agents in in vitro detection assays, it is possible to use chimeric or humanized anti-glucanase antibodies. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, e.g., but are not limited to, methods described in International Application No.PCT / US86 / 02269; U.S. Pat. No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO 86 / 01533; U.S. Pat. Nos. 4,816,567; 5,225,539; European Patent No. 125023; Better, etal., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218;Nishimura, et al., 1987. Cancer Res. 47: 999-1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202- 1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321 : 552-525;Verhoeyan, et al., 1988. Science 239: 1534; Morrison, Science 229: 1202, 1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Pat. No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141 : 4053-4060. For example, antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400;WO 91 / 09967; U.S. Pat. No. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., Molecular Immunology, 28: 489-498,1991; Studnicka e al., Protein Engineering 7: 805-814, 1994; Roguska et aL, PNAS 91 : 969- 973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). In one embodiment, a cDNA encoding a murine anti-glucanase monoclonal antibody is digested with a restriction enzyme selected specifically to remove the sequence encoding the Fc constant region, and the equivalent portion of a cDNA encoding a human Fc constant region is substituted (See Robinson et cd., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553- 1559; U.S. Pat. No. 6,180,370; U.S. Pat. Nos. 6,300,064; 6,696,248; 6,706,484; 6,828,422.

[0167] In one embodiment, the present technology provides the construction of humanized anti- glucanase antibodies that are unlikely to induce a human anti-mouse antibody (hereinafter referred to as “HAMA”) response, while still having an effective antibody effector function. As used herein, the terms “human” and “humanized”, in relation to antibodies, relate to any antibody which is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject. In one embodiment, the present technology provides for a humanized anti- glucanase antibodies, heavy and light chain immunoglobulins.

[0168] CDR Antibodies. In some embodiments, the anti-glucanase antibody of the present technology is an anti-glucanase CDR antibody. Generally the donor and acceptor antibodies used to generate the anti-glucanase CDR antibody are monoclonal antibodies from different species; typically the acceptor antibody is a human antibody (to minimize its antigenicity in a human), in which case the resulting CDR-grafted antibody is termed a “humanized” antibody. The graft may be of a single CDR (or even a portion of a single CDR) within a single VH or VL of the acceptor antibody, or can be of multiple CDRs (or portions thereof) within one or both of the VH and VL. Frequently, all three CDRs in all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, though one needs to replace only as many as necessary to permit adequate binding of the resulting CDR-grafted antibody to glucanase protein. Methods for generating CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and Winter U.S.5,225,539; and EP 0682040. Methods useful to prepare VH and VL polypeptides are taught by Winter etal., U.S. Pat. Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142;EP 0368684; EP0451216; and EP0120694.

[0169] After selecting suitable framework region candidates from the same family and / or the same family member, either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions. Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (z.e., frameworks based on the target species and CDRs from the originating species) can be produced by oligonucleotide synthesis and / or PCR. The nucleic acid encoding CDR regions can also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes. Alternatively, the framework regions of the variable chains of the originating species antibody can be changed by site-directed mutagenesis.

[0170] Since the hybrids are constructed from choices among multiple candidates corresponding to each framework region, there exist many combinations of sequences which are amenable to construction in accordance with the principles described herein. Accordingly, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0171] This process typically does not alter the acceptor antibody’s FRs flanking the grafted CDRs. However, one skilled in the art can sometimes improve antigen binding affinity of the resulting anti-glucanase CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR See, e.g., US 5,585,089, especially columns 12-16). Or one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or a human consensus FR. Techniques for making these modifications are known in the art. Particularly if the resulting FR fits a human consensus FR for that position, or is at least 90% or more identical to such a consensus FR, doing so may not increase the antigenicity of theresulting modified anti-glucanase CDR-grafted antibody significantly compared to the same antibody with a fully human FR.

[0172] Fc Modifications. In some embodiments, the anti-glucanase antibodies of the present technology comprise a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region (or the parental Fc region), such that said molecule has an altered affinity for an Fc receptor (e.g., an FcyR), provided that said variant Fc region does not have a substitution at positions that make a direct contact with Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000).

[0173] Glycosylation Modifications . In some embodiments, anti-glucanase antibodies of the present technology have an Fc region with variant glycosylation as compared to a parent Fc region. In some embodiments, variant glycosylation includes the absence of fucose; in some embodiments, variant glycosylation results from expression in GnTl -deficient CHO cells.

[0174] In some embodiments, the antibodies of the present technology, may have a modified glycosylation site relative to an appropriate reference antibody that binds to an antigen of interest (e.g, glucanase), without altering the functionality of the antibody, e.g., binding activity to the antigen. As used herein, "glycosylation sites" include any specific amino acid sequence in an antibody to which an oligosaccharide (i.e., carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach.

[0175] Oligosaccharide side chains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine, threonine

[0176] In some embodiments, the carbohydrate content of an immunoglobulin-related composition disclosed herein is modified by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, e.g., U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US 2002 / 0028486; International Patent Application Publication WO 03 / 035835; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; U.S. Patent No.6,472,511; all of which are incorporated herein by reference in their entirety. In someembodiments, the carbohydrate content of an antibody (or relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody.

[0177] Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example N- acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application Serial No. 10 / 277,370; U.S. Patent Application Serial No. 10 / 113,929; International Patent Application Publications WO 00 / 61739A1 ; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239- 49.

[0178] Fusion Proteins. In one embodiment, the anti-glucanase antibody or glucanase protein of the present technology is a fusion protein. The anti-glucanase antibodies or glucanase proteins of the present technology, when fused to a second protein, can be used as an antigenic tag. Examples of domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but can occur through linker sequences. Moreover, fusion proteins of the present technology can also be engineered to improve characteristics of the anti-glucanase antibodies or glucanase proteins. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the anti-glucanase antibody or glucanase protein to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties can be added to an anti-glucanase antibody or glucanase protein to facilitate purification. Such regions can be removed prior to final preparation of theanti-glucanase antibody or glucanase protein. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art. The anti-glucanase antibody or glucanase protein of the present technology can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In select embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Set. USA 86: 821-824, 1989, for instance, hexahistidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0179] Thus, any of these above fusion proteins can be engineered using the polynucleotides or the polypeptides of the present technology. Also, in some embodiments, the fusion proteins described herein show an increased half-life in vivo.

[0180] Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can be more efficient in binding and neutralizing other molecules compared to the monomeric secreted protein or protein fragment alone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

[0181] Similarly, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or a fragment thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, e.g, improved pharmacokinetic properties. See EP-A 0232 262. Alternatively, deleting or modifying the Fc part after the fusion protein has been expressed, detected, and purified, may be desired. For example, the Fc portion can hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, e.g., human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0182] Labeled Anti-Glucanase antibodies. In one embodiment, the anti-glucanase antibody or glucanase protein of the present technology is coupled with a label moiety, i.e., detectable group. The particular label or detectable group conjugated to the anti-glucanase antibody or glucanase protein is not a critical aspect of the technology, so long as it does not significantly interfere with the specific binding of the anti-glucanase antibody of the present technology to the glucanaseprotein. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g, Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,3H,14C,35S,125I,121I,131I,112In,99mTc), other imaging agents such as microbubbles (for ultrasound imaging),18F,nC,150,89Zr (for Positron emission tomography), "mTc,i nIn (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and the like) beads. Patents that describe the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated herein by reference in their entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6thEd., Molecular Probes, Inc., Eugene OR.).

[0183] The label can be coupled directly or indirectly to the desired component of an assay according to methods well known in the art. As indicated above, a wide variety of labels can be used, with the choice of label depending on factors such as required sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

[0184] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an antiligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, e.g., biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally-occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., an anti-glucanase antibody.

[0185] The molecules can also be conjugated directly to signal generating compounds, e.g, by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds useful as labeling moieties, include, but are not limited to, e.g., fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties, include, but are not limited to, e.g., luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal -producing systems which can be used, see U.S. Pat. No. 4,391,904.

[0186] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels can be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0187] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies, e.g., the anti- glucanase antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.B. Identifying and Characterizing the Anti-Glucanase Antibodies of the Present Technology

[0188] Methods for identifying and / or screening the anti-glucanase antibodies of the present technology. Methods useful to identify and screen antibodies against glucanase polypeptides for those that possess the desired specificity to glucanase protein include any immunologically- mediated techniques known within the art. Components of an immune response can be detected in vitro by various methods that are well known to those of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells and the lysis of these target cells detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigens and antigen presenting cells and the synthesis and secretion of cytokines measured by standard methods (Windhagen A et al., Immunity, 2: 373-80,1995); (3) antigen presenting cells can be incubated with whole protein antigen and the presentation of that antigen on MHC detected by either T lymphocyte activation assays or biophysical methods (Harding et al.. Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells can be incubated with reagents that cross-link their Fc-epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983); and (5) enzyme- linked immunosorbent assay (ELISA).

[0189] Similarly, products of an immune response in either a model organism (e.g, mouse) or a human subject can also be detected by various methods that are well known to those of ordinary skill in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., an ELISA;(2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of skin and placing a sterile container to capture the migrating cells over scratch site (Peters et al., Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reaction can be measured using3H- thymidine; (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells together with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction of the PBMCs expressing these markers.

[0190] In one embodiment, anti-glucanase antibodies of the present technology are selected using display of glucanase peptides on the surface of replicable genetic packages. See, e.g., U.S. Pat. Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650;6,492,160; EP 585 287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844 306. Methods useful for producing / selecting a filamentous bacteriophage particle containing a phagemid genome encoding for a binding molecule with a desired specificity has been described. See, e.g, EP 774 511; US 5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0191] In some embodiments, anti-glucanase antibodies of the present technology are selected using display of glucanase peptides on the surface of a yeast host cell. Methods useful for the isolation of scFv polypeptides by yeast surface display have been described by Ki eke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0192] In some embodiments, anti-glucanase antibodies of the present technology are selected using ribosome display. Methods useful for identifying ligands in peptide libraries using ribosome display have been described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91 : 9022- 26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

[0193] In certain embodiments, anti-glucanase antibodies of the present technology are selected using tRNA display of glucanase peptides. Methods useful for in vitro selection of ligands using tRNA display have been described by Merryman et al., Chem. Biol., 9: 741-46, 2002.

[0194] In one embodiment, anti-glucanase antibodies of the present technology are selected using RNA display. Methods useful for selecting peptides and proteins using RNA display libraries have been described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Methods useful for selecting peptides and proteins using unnatural RNA display libraries have been described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0195] In some embodiments, anti-glucanase antibodies of the present technology are expressed in the periplasm of gram negative bacteria and mixed with labeled glucanase protein. See WO 02 / 34886. In clones expressing recombinant polypeptides with affinity for glucanase protein, the concentration of the labeled glucanase protein bound to the anti-glucanase antibodies is increased and allows the cells to be isolated from the rest of the library as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Pat. Publication No. 2004 / 0058403.

[0196] After selection of the desired anti-glucanase antibodies, it is contemplated that said antibodies can be produced in large volume by any technique known to those skilled in the art, e.g., prokaryotic or eukaryotic cell expression and the like. The anti-glucanase antibodies which are, e.g., but not limited to, anti-glucanase hybrid antibodies or fragments can be produced by using conventional techniques to construct an expression vector that encodes an antibody heavy chain in which the CDRs and, if necessary, a minimal portion of the variable region framework, that are required to retain original species antibody binding specificity (as engineered according to the techniques described herein) are derived from the originating species antibody and the remainder of the antibody is derived from a target species immunoglobulin which can be manipulated as described herein, thereby producing a vector for the expression of a hybrid antibody heavy chain.

[0197] Measurement of Glucanase Binding. In some embodiments, a glucanase binding assay refers to an assay format wherein glucanase protein and an anti -glucanase antibody are mixed under conditions suitable for binding between the glucanase protein and the anti-glucanase antibody and assessing the amount of binding between the glucanase protein and the anti- glucanase antibody. The amount of binding is compared with a suitable control, which can be the amount of binding in the absence of the glucanase protein, the amount of the binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assay methods include, e.g., ELISA, lateral flow immunoassays, radioimmunoassays, scintillation proximity assays, fluorescence energy transfer assays, liquid chromatography, membrane filtration assays, and the like. Biophysical assays for the direct measurement of glucanase protein binding to anti-glucanase antibody are, e.g., nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chips), biolayer interferometry, and the like. Specific binding is determined by standard assays known in the art, e.g., radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy and the like. If the specific binding of a candidate anti-glucanase antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-glucanase antibody, the candidate anti-glucanase antibody is useful as an anti-glucanase antibody of the present technology.Use of the Anti-Glucanase Antibodies of the Present Technology

[0198] General. The antibodies of the present technology are useful in methods known in the art relating to the localization and / or quantitation of glucanase polypeptides (e.g., for use in measuring levels of the glucanase protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the polypeptide, and the like). The anti-glucanase antibodies of the present technology are useful to isolate a glucanase polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-glucanase antibody of the present technology can facilitate the purification of naturally occurring glucanase proteins from biological samples, e.g., mammalian sera or cells as well as recombinantly- produced glucanase proteins expressed in a host system.

[0199] Moreover, anti-glucanase antibodies of the present technology can be used to detect an immunoreactive glucanase protein (e.g., in plasma, a cellular lysate or cell supernatant) in order to evaluate the abundance of the immunoreactive polypeptide. The anti-glucanase antibodies of the present technology can be used diagnostically to monitor immunoreactive glucanase proteinlevels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. As noted above, the detection can be facilitated by coupling (i.e., physically linking) the anti-glucanase antibodies of the present technology to a detectable substance.

[0200] In some embodiments, the anti-glucanase antibodies and recombinant glucanase proteins of the present technology can be used to detect a naturally occurring antibodies, such as an IgG produced as part of an immunological response to P. jirovecii infection. For example, the anti- glucanase antibody of the present technology may be adhered (i.e., immobilized) on a substrate, the recombinant glucanase proteins of the present technology may be bound (i.e., captured) by the immobilized anti-glucanase antibody, the naturally occurring antibody may bind the captured recombinant glucanase protein, and the bound naturally occurring antibody may be detected by a third labeled antibody (e.g., an anti-human IgG antibody) to produce a signal. The naturally occurring antibody may be isolated from a subject’s blood plasma, a cellular lysate, or a cell supernatant to evaluate the expression levels of the naturally occurring antibody. The anti- glucanase antibodies and recombinant glucanase proteins of the present technology can be used diagnostically to monitor the presence of an immune response in tissue as part of a clinical testing procedure, e.g., to determine the past or present presence of an infection.

[0201] Detection of Glucanase Protein. An exemplary method for detecting the presence or absence of a glucanase protein in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with an anti-glucanase antibody of the present technology capable of detecting a glucanase protein such that the presence of a glucanase protein is detected in the biological sample. Detection may be accomplished by means of a detectable label attached to the antibody.

[0202] The term “labeled” with regard to the anti-glucanase antibody is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled, such as a secondary antibody. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.

[0203] In some embodiments, the anti-glucanase antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, anti-glucanase antibodies may be detectably labeled by covalent or non-covalent attachment of a chromogenic, enzymatic, radioisotopic,isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent or other label.

[0204] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo- benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, A-5-steroid isomerase, yeast-alcohol dehydrogenase, a-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, P-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.

[0205] Examples of suitable radioisotopic labels include3H,i nIn,125I,1311,32P,35S,14C,51Cr,57TO,58CO,59Fe,75Se,152Eu,90Y,67Cu,217Ci,211At,212Pb,47Sc,109Pd, etc.U 1ln is an exemplary isotope where in vivo imaging is used since its avoids the problem of dehalogenation of the125I or131I-labeled glucanase-binding antibodies by the liver. In addition, this isotope has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). Examples of suitable nonradioactive isotopic labels include157Gd,55Mn,162Dy,52Tr, and56Fe.

[0206] Examples of suitable fluorescent labels include an152Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, a Green Fluorescent Protein (GFP) label, an o-phthaldehyde label, and a fluorescamine label. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0207] Examples of chemiluminescent labels include a luminol label, an isoluminol label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label. Examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and iron.

[0208] The detection method of the present technology can be used to detect a glucanase protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of a glucanase protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detection of a glucanase protein include introducing into a subject a labeled anti- glucanase antibody. For example, the anti-glucanase antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.In one embodiment, the biological sample contains glucanase protein molecules from the test subject.

[0209] Immunoassay and Imaging. An anti -glucanase antibody of the present technology can be used to assay glucanase protein levels in a biological sample (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. Jalkanen, M. et al.. J. Cell. Biol. 101 : 976-985, 1985; Jalkanen, M. etal., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes or other radioactive agent, such as iodine (125I,121I,131I), carbon (14C), sulfur (35S), tritium (3H), indium (i nIn), and technetium (99mTc), and fluorescent labels, such as fluorescein, rhodamine, and green fluorescent protein (GFP), as well as biotin.

[0210] In addition to assaying glucanase protein levels in a biological sample, the anti-glucanase antibodies of the present technology may be used for in vivo imaging of glucanase. Antibodies useful for this method include those detectable by X-radiography, NMR or ESR. For X- radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the anti-glucanase antibodies by labeling of nutrients for the relevant scFv clone.

[0211] An anti-glucanase antibody of the present technology which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (e.g.,131I,U 1ln, "mTc,18F,89Zr), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the subject. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc. The labeled anti-glucanase antibody will then accumulate at the location of cells which contain the specific target polypeptide. For example, labeled anti-glucanase antibodies of the present technology will accumulate within the subject in cells and tissues in which the glucanase protein has localized.

[0212] Thus, the present technology provides a diagnostic method of a medical condition, which involves: (a) assaying the expression of glucanase protein by measuring binding of an anti- glucanase antibody of the present technology in cells or body fluid of an individual; (b) comparing the amount of glucanase protein present in the sample with a standard reference, wherein an increase or decrease in glucanase protein levels compared to the standard is indicative of a medical condition.

[0213] Affinity Purification. The anti -glucanase antibodies of the present technology may be used to purify glucanase protein from a sample. In some embodiments, the antibodies are immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir el al.. “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al.. Meth. Enzym. 34 Academic Press, N.Y. (1974)).

[0214] The simplest method to bind the antigen to the antibody-support matrix is to collect the beads in a column and pass the antigen solution down the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using low flow rates. The immobilized antibody captures the antigen as it flows past. Alternatively, an antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or rocking the slurry, allowing maximum contact between the antigen and the immobilized antibody. After the binding reaction has been completed, the slurry is passed into a column for collection of the beads. The beads are washed using a suitable washing buffer and then the pure or substantially pure antigen is eluted.

[0215] An antibody or polypeptide of interest can be conjugated to a solid support, such as a bead. In addition, a first solid support such as a bead can also be conjugated, if desired, to a second solid support, which can be a second bead or other support, by any suitable means, including those disclosed herein for conjugation of a polypeptide to a support. Accordingly, any of the conjugation methods and means disclosed herein with reference to conjugation of a polypeptide to a solid support can also be applied for conjugation of a first support to a second support, where the first and second solid support can be the same or different.

[0216] Appropriate linkers, which can be cross-linking agents, for use for conjugating a polypeptide to a solid support include a variety of agents that can react with a functional group present on a surface of the support, or with the polypeptide, or both. Reagents useful as crosslinking agents include homo-bi-functional and, in particular, hetero-bi-functional reagents. Useful bi-functional cross-linking agents include, but are not limited to, 7V-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC and 6-HYNIC. A cross-linking agent can be selected to provide a selectively cleavable bond between a polypeptide and the solid support. For example, a photolabile cross-linker, such as 3-amino-(2-nitrophenyl)propionic acid can be employed as a means for cleaving a polypeptide from a solid support. (Brown et al., Mol. Divers, pp, 4-12 (1995); Rothschild et al, Nucl. Acids Res., 24:351-66 (1996); and US. Pat. No. 5,643,722). Other cross-linking reagents are well-known in the art. (See, e.g., Wong (1991), supra, and Hermanson (1996), supra).

[0217] An antibody or polypeptide can be immobilized on a solid support, such as a bead, through a covalent amide bond formed between a carboxyl group functionalized bead and the amino terminus of the polypeptide or, conversely, through a covalent amide bond formed between an amino group functionalized bead and the carboxyl terminus of the polypeptide. In addition, a bi-functional trityl linker can be attached to the support, e.g., to the 4-nitrophenyl active ester on a resin, such as a Wang resin, through an amino group or a carboxyl group on the resin via an amino resin. Using a bi-functional trityl approach, the solid support can require treatment with a volatile acid, such as formic acid or trifluoroacetic acid to ensure that the polypeptide is cleaved and can be removed. In such a case, the polypeptide can be deposited as a beadless patch at the bottom of a well of a solid support or on the flat surface of a solid support. After addition of a matrix solution, the polypeptide can be desorbed into a MS.

[0218] Hydrophobic trityl linkers can also be exploited as acid-labile linkers by using a volatile acid or an appropriate matrix solution, e.g., a matrix solution containing 3 -HP A, to cleave an amino linked trityl group from the polypeptide. Acid lability can also be changed. For example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can be changed to the appropriate p- substituted, or more acid-labile tritylamine derivatives, of the polypeptide, i.e., trityl ether and tritylamine bonds can be made to the polypeptide. Accordingly, a polypeptide can be removed from a hydrophobic linker, e.g., by disrupting the hydrophobic attraction or by cleaving tritylether or tritylamine bonds under acidic conditions, including, if desired, under typical MS conditions, where a matrix, such as 3 -HP A acts as an acid.

[0219] Orthogonally cleavable linkers can also be useful for binding a first solid support, e.g., a bead to a second solid support, or for binding a polypeptide of interest to a solid support. Using such linkers, a first solid support, e.g., a bead, can be selectively cleaved from a second solid support, without cleaving the polypeptide from the support; the polypeptide then can be cleaved from the bead at a later time. For example, a disulfide linker, which can be cleaved using a reducing agent, such as DTT, can be employed to bind a bead to a second solid support, and an acid cleavable bi-functional trityl group could be used to immobilize a polypeptide to the support. As desired, the linkage of the polypeptide to the solid support can be cleaved first, e.g., leaving the linkage between the first and second support intact. Trityl linkers can provide a covalent or hydrophobic conjugation and, regardless of the nature of the conjugation, the trityl group is readily cleaved in acidic conditions.

[0220] For example, a bead can be bound to a second support through a linking group which can be selected to have a length and a chemical nature such that high density binding of the beads to the solid support, or high density binding of the polypeptides to the beads, is promoted. Such a linking group can have, e.g., “tree-like” structure, thereby providing a multiplicity of functional groups per attachment site on a solid support. Examples of such linking group; include polylysine, polyglutamic acid, penta-erythrole and / / v.s-hydroxy-aminom ethane.

[0221] Noncovalent Binding Association. An antibody or polypeptide can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support, through a noncovalent interaction. For example, a magnetic bead made of a ferromagnetic material, which is capable of being magnetized, can be attracted to a magnetic solid support, and can be released from the support by removal of the magnetic field. Alternatively, the solid support can be provided with an ionic or hydrophobic moiety, which can allow the interaction of an ionic or hydrophobic moiety, respectively, with a polypeptide, e.g., a polypeptide containing an attached trityl group or with a second solid support having hydrophobic character.

[0222] A solid support can also be provided with a member of a specific binding pair and, therefore, can be conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, a bead coated with avidin or with streptavidin can be bound to a polypeptide having a biotin moiety incorporated therein, or to a second solid support coated with biotin or derivative of biotin, such as iminobiotin.

[0223] It should be recognized that any of the binding members disclosed herein or otherwise known in the art can be reversed. Thus, biotin, e.g., can be incorporated into either a polypeptide or a solid support and, conversely, avidin or other biotin binding moiety would be incorporated into the support or the polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzyme, and their substrates, a nucleotide sequence and its complementary sequence, an antibody and the antigen to which it interacts specifically, and other such pairs knows to those skilled in the art.A. Diagnostic Uses o f the Anti-Glucanase Antibodies o f the Present Technology

[0224] General. The anti-glucanase antibodies of the present technology are useful in diagnostic methods. As such, the present technology provides methods using the antibodies in the diagnosis of glucanase activity in a subject. The anti-glucanase antibodies of the present technology may be selected such that they have any level of epitope binding specificity and very high binding affinity to a glucanase polypeptide. In general, the higher the binding affinity of an antibody, the more stringent wash conditions can be performed in an immunoassay to remove nonspecifically bound material without removing target polypeptide. Accordingly, the anti- glucanase antibodies of the present technology useful in diagnostic assays usually have binding affinities of about 108M’1, 109M’1, IO10M’1, 1011M'1or 1012M’1. Further, it is desirable that anti-glucanase antibodies used as diagnostic reagents have a sufficient kinetic on-rate to reach equilibrium under standard conditions in at least 12 h, at least five (5) h, or at least one (1) hour.

[0225] Anti-glucanase antibodies can be used to detect a glucanase protein in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. See Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Biological samples can be obtained from any tissue or body fluid of a subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), BAL, and biopsied body tissue.

[0226] Immunometric or sandwich assays are one format for the diagnostic methods of the present technology. See U.S. Pat. No. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Suchassays use one antibody, e.g., the anti-glucanase antibody or a population of anti-glucanase antibodies immobilized to a solid phase, and another anti-glucanase antibody or a population of anti-glucanase antibodies in solution. Typically, the solution anti-glucanase antibody or population of anti-glucanase antibodies is labeled. If an antibody population is used, the population can contain antibodies binding to different epitope specificities within the target polypeptide. Accordingly, the same population can be used for both solid phase and solution antibody. If anti-glucanase monoclonal antibodies are used, first and second glucanase monoclonal antibodies having different binding specificities are used for the solid and solution phase. Solid phase (also referred to as “capture”) and solution (also referred to as “detection”) antibodies can be contacted with target antigen in either order or simultaneously. If the solid phase antibody is contacted first, the assay is referred to as being a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as being a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the glucanase protein with the anti-glucanase antibody, a sample is incubated for a period that usually varies from about 10 min to about 24 hr and is usually about 1 hr. A wash step is then performed to remove components of the sample not specifically bound to the anti-glucanase antibody being used as a diagnostic reagent. When solid phase and solution antibodies are bound in separate steps, a wash can be performed after either or both binding steps. After washing, binding is quantified, typically by detecting a label linked to the solid phase through binding of labeled solution antibody. Usually for a given pair of antibodies or populations of antibodies and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. Concentrations of the glucanase protein in samples being tested are then read by interpolation from the calibration curve (z.e., standard curve). Analyte can be measured either from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of the glucanase protein in a sample.

[0227] Suitable supports for use in the above methods include, e.g., nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, and also particles, such as agarose, a dextran-based gel, dipsticks, particulates, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway N.J.), and the like. Immobilization can be by absorption or by covalent attachment. Optionally, anti-glucanaseantibodies can be joined to a linker molecule, such as biotin for attachment to a surface bound linker, such as avidin.

[0228] In some embodiments, the present disclosure provides an anti-glucanase antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent may comprise a radioactive or non-radioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography or ultrasound), and the radioactive label can be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. A diagnostic agent is a molecule which is administered conjugated to an antibody moiety, z.e., antibody or antibody fragment, or subfragment, and is useful in diagnosing or detecting a disease by locating the cells containing the antigen.

[0229] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Pat. No. 6,331, 175 describes MRI technique and the preparation of antibodies conjugated to a MRI enhancing agent and is incorporated in its entirety by reference. In some embodiments, the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. In order to load an antibody component with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which are attached a multiplicity of chelating groups for binding the ions. Such a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose. Chelates may be coupled to the antibodies of the present technology using standard chemistries. The chelate is normally linked to the antibody by a group which enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes for radio-imaging. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the anti-glucanase antibodies of the present technology.

[0230] In one aspect, the present disclosure provides a method for treating Pneumocystis pneumonia o Pneumocystis jirovecii infection in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of any and all embodiments of the antibody or antigen binding fragment disclosed herein, wherein the antibody or antigen binding fragment is optionally conjugated to at least one additional therapeutic agent. Examples of the at least one additional therapeutic agent include, but are not limited to, bactrim, trimethoprimsulfamethoxazole (TMP-SMX), rezafungin, atovaquone, trimethoprim, dapsone, primaquine, clindamycin, and pentamidine. In some embodiments, the subject is an immunocompromised patient, a pediatric patient, a geriatric patient, a cancer patient, an autoimmune disease patient, an HIV patient, or an immunosuppressed patient.

[0231] In one aspect, the present disclosure provides a method for detecting Pneumocystis jirovecii in a biological sample comprising contacting the biological sample with any and all embodiments of the antibody or antigen binding fragment disclosed herein, and detecting Pneumocystis jirovecii by detecting binding between glucanase present in the biological sample and the antibody or antigen binding fragment. In another aspect, the present disclosure provides a method for diagnosing Pneumocystis jirovecii infection or Pneumocystis pneumonia in a subject comprising contacting a biological sample obtained from the subject with any and all embodiments of the antibody or antigen binding fragment disclosed herein, detecting binding between glucanase present in the biological sample and the antibody or antigen binding fragment, and diagnosing the subject with Pneumocystis jirovecii infection or Pneumocystis pneumonia when glucanase is detected in the biological sample. In some embodiments, binding between glucanase and the antibody or antigen binding fragment is detected via radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy or lateral flow immunoassay.

[0232] In one aspect, the present disclosure provides a P-l,3-endoglucanase antigenic peptide comprising an epitope having the sequence of SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 67. The antigenic peptide may further comprise a heterologous signal peptide, such as a yeast signal peptide, a mammalian signal peptide, an insect signal peptide or a synthetic signal peptide. Also provided herein are kits comprising the P-l,3-endoglucanase antigenic peptides disclosed herein, the anti-glucanase antibodies or antigen binding fragments disclosed herein, and instructions for use.

[0233] In one aspect, the present disclosure provides a method for determining Pneumocystis seroprevalence in a subject at risk for or suspected of having a. Pneumocystis jirovecii infection or Pneumocystis pneumonia comprising contacting a biological sample obtained from the subject with the P-1,3 endoglucanase antigenic peptide of the present technology; and detecting binding between anti-glucanase immunoglobulins present in the biological sample and the P-1,3 endoglucanase antigenic peptide. In another aspect, the present disclosure provides a method for diagnosing an active Pneumocystis infection or Pneumocystis pneumonia in a subject in need thereof comprising contacting a biological sample obtained from the subject with the P-1,3 endoglucanase antigenic peptide of the present technology; detecting binding between anti- glucanase immunoglobulins present in the biological sample and the P-1,3 endoglucanase antigenic peptide; and diagnosing the subject with an active Pneumocystis infection or Pneumocystis pneumonia when the presence of anti-glucanase immunoglobulins in the biological sample is detected. The anti-glucanase immunoglobulins may comprise anti- glucanase IgM and / or anti-glucanase IgG. In some embodiments, binding between the P-1,3 endoglucanase antigenic peptide and the anti-glucanase immunoglobulins is detected via radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy or lateral flow immunoassay.

[0234] In any of the preceding embodiments of the methods disclosed herein, the antibody or antigen binding fragment or the P-1,3 endoglucanase antigenic peptide further comprises a detectable label.

[0235] In any and all embodiments of the methods described herein, the biological sample comprises bronchoalveolar lavage fluid (BALF), sputum, throat swabs, serum, plasma, whole blood or urine. In any of the preceding embodiments of the methods disclosed herein, the subject is an immunocompromised patient, a pediatric patient, a geriatric patient, a cancer patient, an autoimmune disease patient, an HIV patient, or an immunosuppressed patient.B. Therapeutic Uses o f the Anti-Glucanase Antibodies o f the Present Technology

[0236] The immunoglobulin-related compositions (e.g., antibodies or antigen binding fragments thereof) of the present technology are useful for the treatment of Pneumocystis pneumonia or P. jirovecii infections. Such treatment can be used in patients identified as having pathologically high levels of the glucanase or immunoreactive proteins to the glucanase (e.g., those diagnosed by the methods described herein).

[0237] The immunoglobulin-related compositions of the present technology may be employed in conjunction with other therapeutic agents useful in the treatment of Pneumocystis pneumonia or P.jirovecii infections. For example, the antibodies of the present technology may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent, or be conjugated to at least one additional therapeutic agent, wherein the at least one additional therapeutic agent is selected from the group comprising Bactrim, trimethoprim-sulfamethoxazole (TMP-SMX), rezafungin, atovaquone, trimethoprim, dapsone, primaquine, clindamycin, pentamidine, or other drugs with prophylactic activity against P. jirovecii.

[0238] The compositions of the present technology may optionally be administered as a single bolus to a subject in need thereof. Alternatively, the dosing regimen may comprise multiple administrations performed at various times after the appearance of pneumonia symptoms.

[0239] Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intracranially, intrathecally, or topically. Administration includes self-administration and the administration by another. It is also to be appreciated that the various modes of treatment of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved.

[0240] In some embodiments, the antibodies of the present technology comprise pharmaceutical formulations which may be administered to subjects in need thereof in one or more doses. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response).

[0241] Typically, an effective amount of the antibody compositions of the present technology, sufficient for achieving a therapeutic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Typically, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For administration of anti-glucanase antibodies, the dosage ranges from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg every week, every two weeks or every three weeks, of the subject body weight. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight every week, every two weeks or every three weeks or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of antibody ranges from 0.1-10,000 micrograms per kg body weight. In one embodiment, antibody concentrations in a carrier range from 0.2 to 2000micrograms per delivered milliliter. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. Anti-glucanase antibodies may be administered on multiple occasions. Intervals between single dosages can be hourly, daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the antibody in the subject. In some methods, dosage is adjusted to achieve a serum antibody concentration in the subject of from about 75 pg / mL to about 125 pg / mL, 100 pg / mL to about 150 pg / mL, from about 125 pg / mL to about 175 pg / mL, or from about 150 pg / mL to about 200 pg / mL. Alternatively, anti-glucanase antibodies can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0242] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-glucanase antibody described herein will provide therapeutic benefit without causing substantial toxicity to the subject. Toxicity of the anti-glucanase antibody described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LDso (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the anti-glucanase antibody described herein lies within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject’s condition. See, e.g., Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0243] Formulations of Pharmaceutical Compositions. According to the methods of the present technology, the anti-glucanase antibody can be incorporated into pharmaceutical compositions suitable for administration. The pharmaceutical compositions generally comprise recombinantor substantially purified antibody and a pharmaceutically-acceptable carrier in a form suitable for administration to a subject. Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the antibody compositions (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18thed., 1990). The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0244] The terms “pharmaceutically-acceptable,” “physiologically-tolerable,” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. “Pharmaceutically-acceptable salts and esters” means salts and esters that are pharmaceutically-acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the composition are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically-acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the anti-glucanase antibody, e.g., Ci-6 alkyl esters. When there are two acidic groups present, a pharmaceutically- acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salifiedor esterified. An anti-glucanase antibody named in this technology can be present in unsalified or unesterified form, or in salified and / or esterified form, and the naming of such anti-glucanase antibody is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically-acceptable salts and esters. Also, certain embodiments of the present technology can be present in more than one stereoisomeric form, and the naming of such anti- glucanase antibody is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers. A person of ordinary skill in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present technology.

[0245] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the anti-glucanase antibody, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0246] A pharmaceutical composition of the present technology is formulated to be compatible with its intended route of administration. The anti-glucanase antibody compositions of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; or intramuscular routes, or as inhalants. The anti-glucanase antibody can optionally be administered in combination with other agents that are at least partly effective in treating various P.jirovecii infections or Pneumocystis pneumonia.

[0247] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0248] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic compounds, e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.

[0249] Sterile injectable solutions can be prepared by incorporating an anti-glucanase antibody of the present technology in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the anti-glucanase antibody into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The antibodies of the present technology can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0250] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the anti-glucanase antibody can be incorporated with excipients and used in theform of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring.

[0251] For administration by inhalation, the anti-glucanase antibody is delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0252] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the anti-glucanase antibody is formulated into ointments, salves, gels, or creams as generally known in the art.

[0253] The anti-glucanase antibody can also be prepared as pharmaceutical compositions in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0254] In one embodiment, the anti-glucanase antibody is prepared with carriers that will protect the anti-glucanase antibody against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) canalso be used as pharmaceutically-acceptable carriers. These can be prepared according to methods known to those skilled in the art, e.g., as described in U.S. Pat. No. 4,522,811.C. Kits

[0255] The present technology provides kits for the detection and / or treatment of P.jirovecii infections or Pneumocystis pneumonia, comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen binding fragment described herein), or a functional variant (e.g., substitutional variant) thereof, and / or recombinant glucanase peptides disclosed herein, and optionally instructions for use.

[0256] In one aspect, the present disclosure provides a P-l,3-endoglucanase antigenic peptide comprising an epitope having the sequence of SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 67. The antigenic peptide may further comprise a heterologous signal peptide, such as a yeast signal peptide, a mammalian signal peptide, an insect signal peptide or a synthetic signal peptide. Examples of suitable heterologous signal peptides include those described in Schutz et al., STARProtoc. 2023;4(4): 102572 and O’Neill et al., ACS Synth. Biol. 2023, 12, 2339-2352, the contents of which are incorporated by reference in their entirety.

[0257] Optionally, the above-described components of the kits of the present technology are packed in suitable containers and labeled for diagnosis and / or treatment of P.jirovecii infections or Pneumocystis pneumonia. The above-mentioned components may be stored in unit or multidose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts. The kits may optionally include instructions customarily included in commercial packages of therapeutic or diagnostic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and / or warnings concerning the use of such therapeutic or diagnostic products.

[0258] The kits are useful for detecting the presence of a glucanase protein in a biological sample, e.g., any body fluid including, but not limited to, e.g., serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascitic fluid or blood and including biopsy samples of body tissue. For example, the kit can comprise: one or more humanized, or chimeric anti- glucanase antibodies of the present technology (or antigen binding fragments thereof) capable of binding a glucanase in a biological sample; means for determining the amount of the glucanase in the sample; and means for comparing the amount of the glucanase in the sample with a standard. One or more of the anti -glucanase antibodies may be labeled. The kit components, (e.g., reagents) can be packaged in a suitable container. The kit can further comprise instructions for using the kit components to detect and / or treat P. jirovecii infections or Pneumocystis pneumonia.

[0259] For antibody -based kits, the kit can comprise, e.g., 1) a first antibody, e.g., a humanized, or chimeric anti-glucanase antibody of the present technology (or an antigen binding fragment thereof), attached to a solid support, which binds to a glucanase polypeptide; and, optionally; 2) a second, different antibody which binds to either the glucanase polypeptide or to the first antibody, and is conjugated to a detectable label.

[0260] The kit can also comprise, e.g, a buffering agent, a preservative or a protein-stabilizing agent. The kit can further comprise components necessary for detecting the detectable-label, e.g, an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, e.g., for detection of a glucanase polypeptide in vitro or in vivo, or for treatment of P. jirovecii infections or Pneumocystis pneumonia in a subject in need thereof. In certain embodiments, the use of the reagents can be according to the methods of the present technology.EXAMPLES

[0261] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The following Examples demonstrate the preparation, characterization, and use of illustrative anti-glucanase antibodies and recombinant glucanase antigenic peptides of the present technology.Example 1: Materials and Methods

[0262] Identification of Pneumocystis antigens in BAL. BAL samples were taken from C57BL / 6 mice and Rag2- / - mice infected with 100 pl of P. murina (~2* 105asci) via oral pharyngeal administration, and were collected 2-, 4-, and 6-weeks post-infection. Samples were acetone precipitated with 4X volume, vortexed and incubated at -20°C overnight. The solutions were centrifuged to acquire the pellet. Subsequently the pellet was resuspended and reduced with Dithiothreitol. This step was followed by alkylation with lodoacetamide. The mixture was dried (SpeedVac, Eppendorf) and the pellet was subsequently reconstituted in 50mM Tris-HCl and digested by trypsin at an enzyme to protein mass ratio of 1 :50 overnight at 37°C. Peptides were acidified to a final concentration of 0.1% trifluoro-acetic acid (TFA) for SDB-RPS binding, and 20 pg was loaded on double-stacked SDB-RPS discs with an 18-gauge needle and mounted in 200 pl tip as previously described. Harney et al, Mol Cell Proteomics. 2019; 18(9): 1899-915. Briefly, each tip was wetted with 50 pl of 100% acetonitrile and centrifuged at 1000g for 1 min. Following wetting, each StageTip was equilibrated with 50 pl of 30% methanol / 1% TFA with centrifugation for each at 1000g for 3 min. Each StageTip was then loaded with the equivalent of ~20 pg tryptic peptides followed by washing with 100 pl of 99% propanol and 0.1% TFA for 2 mins. The peptides were eluted with 60 pl of elution buffer (80% acetronitrile, 1% ammonium hydroxide), dried and reconstitute in 2% acetonitrile and 0.1% formic acid. For deep proteome coverage, each sample was fractionated into six fractions by the strong cation ion exchange (SCX). SCX stage tip was prepared in the similar way as the SDB-RPS tips. The tips were activated with 50 pl acetonitrile and the enriched peptides were loaded followed by their elution in 6 buffers: SCX buffer 1 : 50mM ammonium acetate, 20% acetonitrile, 0.5% formic acid; SCX buffer 2: 75mM ammonium acetate, 20% acetonitrile, 0.5% formic acid; SCX buffer 3: 125mM ammonium acetate, 20% acetonitrile, 0.5% formic acid; SCX buffer 4: 200mM ammonium acetate, 20% acetonitrile, 0.5% formic acid; SCX buffer 5: 300mM ammonium acetate, 20% acetonitrile, 0.5% formic acid; SCX buffer 6: 5% ammonium hydroxide, 80% acetonitrile. Allthe fractions were dried by vacuum centrifugation and suspended into 0.1% formic acid / 2% acetonitrile before injection. Each fraction was loaded in LC-MS.

[0263] Samples were measured using LC-MS instrumentation consisting of an Ultimate 3000 nanoLC system (Thermo Fisher Scientific) coupled via a nano-electrospray ion source (Thermo Fisher Scientific) to a QExactive HFX Orbitrap (Thermo Fisher Scien-tific). For DDA, the SCX fractions were loaded onto a 100 1m I.D. x 2.5 cm, Cl 8 trap column and PepMapTM RSLC C18 (2pm, 75pm x 25 cm) analytical column solvent A (0.1% formic acid in water). The fractions were eluted with a linear gradient from 5% to 30% buffer B over 80 min. Following the linear separation, the system was ramped up to 60% buffer B over 5 min and finally set to 95% buffer B for 8 min, which was followed by re-equilibration to 5% buffer B prior to the subsequent injection. Data were acquired using data dependent acquisition mode (DDA). After adjusting each fraction to an estimated 0.5-1.0 pg on column, the fractions were measured in a top-12 configuration with 20 s dynamic exclusion. Precursor spectra were collected from 300-1650m / z at 60,000 resolutions (AGC target of 3e6, max IT of 20 ms). The MS / MS were collected on +2H to +5H precursors achieving a minimum AGC of 2e3. MS / MS scans were collected at 15,000 resolutions (AGC target of le5, max IT of 60 ms) with an isolation width of 1.4m / z with a NCE of 27.

[0264] All LC-MS / MS data were searched using the Sequest HT algorithm within Proteome Discoverer version 2.4.1.15 (Thermo Fisher Scientific) against Mus musculus as the host and Pneumocystis murina as the fungal database to obtain peptide and protein identifications. For all searches, trypsin was specified as the enzyme for protein cleavage, allowing up to two missed cleavages. Oxidation (M) and carbamidomethylation (C) were set as dynamic and fixed modifications, respectively. For Sequest search, the precursor and fragment mass tolerances were set at 10 ppm and 0.5 Da, respectively. Both peptide spectrum match and protein false discovery rate (FDR) was set to 0.01 and determined using a percolator node.

[0265] Production of glucanase recombinant protein. Recombinant . jirovecii [3-1,3- endoglucanase was produced in Chinese Hamster Ovarian (CHO) cells by LakePharma / Curia. The recombinant glucanase amino acid sequence is shown in FIG. 17. The formulation buffer of the recombinant protein is 137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4. The protein concentration is 1.61 (mg / mL) and the physical state is frozen and stored at - 80°C.

[0266] Antigen serology ELISA. For the seroprevalence study, residual blood samples that were collected between March 18th and May 15th, 2020, from children <18 years of age from Children’s Hospital in New Orleans, Louisiana, were tested. Collection and processing have previously been described. Dietrich et al, J Clin Virol Plus. 2021;l(4): 100047.All serum samples were SARS-CoV-2 -negative and HIV-negative. Id. For the ELISA assays, Maxisorp plates (ThermoFisher Cat No. 442404) were coated with 2 pg of Pneumocystis antigen in 100 pl of IX sterile phosphate buffered saline (PBS) per well overnight at 4°C. Plates were aspirated and washed twice. Plates were blocked with 200 pl of 2X PBS with 1% Tween 20 and 10% BSA (Invitrogen, 88-50470) for two hours at room temperature. Plates were aspirated and washed twice. Plates were then stained with 50 pl sample serum diluted 1 :32 in IX PBS with 1% Tween-20 and 10% BSA, covered and incubated for 2 hours at room temperature on a microplate shaker set at 400 rpm. Plates were aspirated and washed four times. For measuring IgG, Goat anti-Human IgG HRP (SouthernBiotech, Cat No. 2040-05) was diluted 1 :4000 in IX PBS with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells. For measuring IgM, Goat anti-Human IgM HRP (SouthernBiotech, Cat No. 2020-05) was diluted 1:4000 in IX PBS with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells. Plates were covered and incubated for 1 hour at room temperature on a microplate shaker set at 400 rpm. Plates were aspirated and washed four times. 100 pL of Tetramethylbenzidine (TMB) substrate solution (Invitrogen, 88-50470) was added to each well and incubated at room temperature for 1-5 min depending on control serum. The reaction was stopped with 50 pl of 2N H2SO4 added to each well. The optical density at 450 nm (OD450) and 570 nm (OD570) was read using a BioTek Synergy HT microplate reader. The OD reported is the reading at 450 nm minus the reading at 570 nm to remove background.

[0267] For human cord blood donors (9), cord blood donors (StemCell Technologies, Vancouver, BC) were screened using the same ELISA method as previously described, except serum was serially diluted 1 : 10-1 :320 and detection antibody used was Goat anti-Human IgG HRP (SouthernBiotech, Cat No. 2040-05) diluted 1 :4000 in IX PBS with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells.

[0268] To assay convalescent titers, C57BL / 6 mice were infected with 100 pl of P. murina inoculum (~2* 105asci) via oral pharyngeal administration. Two or 6 weeks later, sera was collected and tested for IgG anti-glucanase using the same ELISA method as described above,except the sera were tested at a 1:200 dilution, and the detection antibody was Goat anti-Mouse IgG HRP (SouthemBiotech, Cat No. 1030-05) diluted 1 :4000 in IX PBS with 1% Tween-20 and 10% BSA.

[0269] Statistical analysis. The phylogenetic tree (FIG. IB) was built with the use of Blast Search in MEGA11 following the pipeline previously described, and inputting the P.jirovecii glucanase protein sequence (GeneBank Accession: XP_018229679., (SEQ ID NO: 53). Hall, Mol Biol Evol. 2013;30(5): 1229-35. Statistical analyses were conducted utilizing GraphPad Prism (version 9.5.1). The difference in antibody titers in pediatric cohort was assessed by comparing the 1 :32 OD values via the Mann-Whitney test. Any P-values that were less than 0.05 were viewed as significant.Example 2: Identification of Pneumocystis Antigens in BAL

[0270] Proteomics was performed in the BAL fluid of infected B6 mice. Several Pneumocystis proteins were identified, including several major surface glycoproteins as well as PNEG_02407, a putative endo-beta-1, 3-glucanase (MS depicted in FIG. 1A). Id. This protein was prioritized as it is highly conserved n P. jirovecii and other Pneumocystis spp., but not in environmental fungi (FIG. IB). In addition, it has been reported to be more highly expressed in trophic forms which is the form that attaches to type I pneumocytes. Cushion el al, Antimicrob Agents Chemother. 2018;62(5); Thomas et al, NatRevMicrobiol. 2007;5(4):298-308. Recombinant glucanase expressed in CHO cells was successfully expressed and purified, as demonstrated in FIGs. 1C-1DExample 3: Antigen Serology in Pneumocystis Infected Mice

[0271] IgG responses to Pneumocystis-s^Qcific, antigens were assayed by ELISA two weeks and six weeks after C57BL / 6 mice were infected with P. murina (FIG. 2). Infected mice generated significant anti-PNEG_01454 and anti -whole Pneumocystis antigen (PC antigen) IgG, as well as lower titer of anti-Glucanase IgG. PC antigen is a whole antigen preparation and thus has more reactivity. Without wishing to be bound to any particular theory, PNEG 01454 is recognized by germ line IgM and the higher titer may reflect a larger precursor pool of antigen specific B cells prior to infection. Noell et al, JCI Insight. 2022;7(17).Example 4: Antigen Serology in Pediatric Cohorts.

[0272] A total of 133 pediatric blood samples, consisting of a discovery and validation cohort, were obtained from Children’s Hospital in New Orleans from March 18th to May 15th of 2020. Subjects were HIV-negative and SARS-CoV-2 negative. The presence of anti-Glucanase IgG responses by ELISA was detected in Cohort 1 (FIG. 3A) which showed the presence of anti- Glucanase IgG responses in the first month of life (consistent with maternal IgG) followed by a nadir at 1-3 months of age. The validation cohort (Cohort 2, FIGs. 3B-3C) from the same Pediatric center, showed similar neonatal IgG levels declined reaching a nadir at 4-6 months of age. Given the fact that there was anti-glucanase IgG at birth, it was verified that there was anti- glucanase IgG detected in an independent sample of human cord blood donors and all donors tested were positive (FIG. 10).

[0273] The glucanase IgG responses in the 1-3 month of age samples from both cohorts was compared with the values from 0-30 days of age responses by Mann-Whitney U test, with a resulting P-value of 0.0007. The nadir was then followed by an increase over time and 114 of the 133 samples (85.71%) were positive (OD>0.2; 20.58% relative titer of OD). To determine if this was potentially due to Pneumocystis infection, IgG responses to a putative cell adhesion protein PNEG 01454 (FIGs. 3D-3F) was also assayed, and a high concordance of IgG was observed. Anti-glucanase IgM was also measured, and consistent with known polyreactivity of IgM, low titers were observed in the first year of life with increases in older subjects. In contrast to IgG, a nadir was not observed.Example 5: Possible CD4+ T-cell Epitopes ofP. jirovecii b-l,3-endoglucanase.

[0274] Possible CD4+ T-cell epitopes of P.jirovecii P-l,3-endoglucanase were identified on the basis of antigen processing likelihood (APL) combined with MHCII-peptide binding affinity. The APL algorithm uses a protein 3D structure and amino-acid sequence conservation to generate profiles of conformational stability, and then evaluates the likelihood that a particular peptide will be processed from the native protein and presented by an MHCII protein to CD4+ T cells. Charles et al, Biochemistry 61, 1585-1599. 10.1021 / acs.biochem.2c00237. In general, peptides within conformationally stable segments adjacent to flexible, protease-sensitive segments obtain the highest APL scores. Residue-specific APL values were averaged for a series of 15-residue peptides spanning the length of P.jirovecii glucanase in 5-residue steps. For each peptide, MHCII-peptide binding affinity [l-log50k(aff)] was predicted for several commonclass II HLA molecules (DPAl*01:03-DPBl*04:01, DRB1*15:O1, DRBl*04:01, andDRB 1*01 :01) using the NETMHCII (2.3) server, or the median binding-affinity-rank was predicted for a 7-allele HLA reference set using the IEDB server. Jensen et al, Immunology 2018;154, 394-406. 10; Bui et al, Immunogenetics 2005;57, 304-314; Paul et al, J Immunol Methods 2015;422, 28-34. Ultimately, the epitope probability of each peptide was calculated as the weighted combination of z-scores representing APL (31%) and MHCII binding (69%) for each HLA molecule or the HLA reference set.

[0275] Since no 3D structure has been reported for P. jirovecii P-l,3-endoglucanase, a homology model was created using automated functions of the Swiss-Model server. Waterhouse et al, Nucleic Acids Res 2018;46, W296-W303. Swiss-model identified a single X-ray crystal structure (PDB:4K35) that could serve as template, a P-l,3-glucanase from the fungus R. miehi. Alignment of the template and target glucanases revealed well distributed sequence identity (34% overall) spanning residues 89-785 of the 788-residue P. jirovecii glucanase. An AlphaFold of P. jirovecii P-l,3-endoglucanase was also available, but it was not selected for analysis by APL because it included segments that have poor confidence and were disordered in the R. miehi structure. We note that the well-ordered portion of the AlphaFold and the Swiss-Model were virtually identical, and backbones could be aligned with average RMSD of 1.2 angstroms.

[0276] Five highly probable CD4+ epitopes that are expected to exhibit promiscuous dominance were identified as having a combined APL / MHCII-binding score within the 80th percentile for each of the tested HLA-binding systems (FIGs. 4A-4C, FIGs. 5A-5C, and Table 1).Additionally, when APL and MHCII binding was assessed independently, these five peptides also scored in the 70th percentile of APL and 80th percentile of each HLA system. For using the peptides for diagnostics this would only work if the peptides are sufficiently different from corresponding sequences in commensal or other pathogen proteins. Peptides 26 and 109 are conserved and thus would not be suitable, but the other two segments 47-48 and 131 should be suitable (Table 1).

[0277] Table 1:Example 6: Glucanase Detection Sandwich ELISA 1.0.

[0278] Costar Assay plates, high binding clear, flat bottom (Corning Ref 9018; Lot 3012001) were coated with 100 pl per well of 3 pg / mL Rabbit anti-Glucanase Protein A Purified capture antibody (GenScript, Bethy Laboratories, Batch 230623) overnight at 4°C. Plates were aspirated and washed twice. Plates were blocked with 200 pl per well of 5% nonfat milk for one hour at room temperature. Plates were aspirated and washed twice. Plates were then stained with 100 pl per well of serially diluted glucanase (LakePharma, Lot number TP39841F) and 25 pl per well of human BAL samples, and incubated for 2 hours at room temperature on a microplate shaker set at 400 rpm. Plates were aspirated and washed four times. Biotin (Type A) Conjugation of mouse mAb 8E5G9-1 (Abeam, ab274076, Lot: GR3450691-1) was diluted 1 :20,000 in IX PBS with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells. Plates were covered and incubated for 1 hour at room temperature on shaker. Plates were aspirated and washed four times. HRP-Conjugated Streptavidin (ThermoScientific, Ref N100; Lot YB364098) was diluted 1 :5,000 with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells. Plates were covered and incubated for 1 hour at room temperature on shaker. Plates were aspirated and washed four times. 100 pL of Tetramethylbenzidine (TMB) substrate solution (Invitrogen, 88- 50470) was added to each well and incubated at room temperature for 10-30 minutes depending on the glucanase standard. The reaction was stopped with 50 pl of 2N H2SO4 added to each well. The optical density at 450 (OD450) was read using a BioTek Synergy HT microplate reader. These results are depicted in FIG. 6A and FIG. 6C.Example 7: Glucanase Detection Sandwich ELISA 2.0.

[0279] Methods are the same as Glucanase Detection Sandwich Example 6 up to the wash step after the glucanase standard and human samples are added. The capture antibody used was HRP conjugated mouse mAb 8E5G9-1 (Abeam, ab274112, Lot: GR3425253-1) diluted 1 :6,000 with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells. Plates were covered andincubated for 1 hour at room temperature on shaker. Plates were aspirated and washed four times. 100 pl of Tetramethylbenzidine (TMB) substrate solution (Invitrogen, 88-50470) was added to each well and incubated at room temperature for 10-30 minutes depending on the glucanase standard. The reaction was stopped with 50 pl of 2N H2SO4 to each well. The optical density at 450 (OD450) was read using a BioTek Synergy HT microplate reader. These results are depicted in FIG. 6B.Example 8: Glucanase Detection Sandwich ELISA 3.0.

[0280] Methods are the same Glucanase Detection Sandwich Example 6 except the protein with which the plates were initially coated. Costar Assay plates, high binding clear, flat bottom (Corning Ref 9018; Lot 3012001) were coated with 100 pl per well of 3 pg / mL Rabbit anti- Glucanase Affinity Purified capture antibody (GenScript, Bethy Laboratories, Batch TAP185937-231128) overnight at 4°C. These results are shown in FIGs. 7A-7B.Example 9: Glucanase Detection ELISA using mAh as Capture Antibody.

[0281] Costar Assay plates, high binding clear, flat bottom (Corning Ref 9018; Lot 3012001) were coated with 100 pl of 2 pg / mL mAb (13F7B7-1 or 20B12G12-1) overnight at 4°C. Plates were aspirated and washed. Plates blocked with 200 pl / well of 2X PBS with 1% Tween 20 and 10% BSA (Invitrogen, 88-50470) for 1 hour at room temperature on shaker. Plates were aspirated and washed. Plates were then stained with 100 pl per well of serially diluted glucanase (LakePharma, Lot number TP39841F) and human BAL samples were added neat 100 pl per well, and neat spiked with 250 pg / mL of glucanase (100 pl per well) and incubated for 2 hours at room temperature on shaker. Plates were aspirated and washed. Rabbit anti-Glucanase Affinity Purified antibody (GenScript, Bethy Laboratories, Batch TAP185937-231128) was diluted 1 :500 in IX PBS with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells. Plates were covered and incubated for 1 hour at room temperature on shaker. Plates were aspirated and washed four times. Goat anti-rabbit IgG-HRP (Cell Signaling 7074S) was diluted 1 : 1000 in IX PBS with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells. Plates were covered and incubated for 1 hour at room temperature on shaker. Plates were aspirated and washed four times. 100 pl of Tetramethylbenzidine (TMB) substrate solution (Invitrogen, 88-50470) was added to each well and incubated at room temperature for 10-30 minutes depending on the glucanase standard. The reaction was stopped with 50 pl of 2N H2SO4 added to each well. The optical density at 450 (OD450) was read using a BioTek Synergy HT microplate reader. Theseresults using the 13F7B7-1 and 20B12G12-1 monoclonal antibodies are shown in FIGs. 8A-8B.FIGs. 9A-9E show the detection of glucanase using the different anti-glucanase antibodies of the present technology as capture antibodies.Example 10: Antigen Detection in BAL by Direct ELISA.

[0282] Polyclonal mouse serum samples

[0283] Groups of C57BL / 6 mice, female, 4 to 6 weeks old were subcutaneously immunized with 10 pg glucanase in IFA or IFA only. Three weeks later, one group of mice were boosted with 10 pg glucanase in IFA and the other 2 groups infected with 100 pl of P. murina inoculum (2* 105cysts) via oral pharyngeal administration. 6 weeks later, all mouse sera were collected for ELISA.

[0284] Direct ELISA

[0285] Maxisorp plates (ThermoFisher Cat No. 442404) were coated with 100 ng of protein from broncho-alveolar lavage fluid (BALF) of Rag 2-1- mice which was collected two weeks after being infected with 100 pl of P. murina inoculum (2* 105cysts) via oral pharyngeal administration, in maximum of 100 pl per well of IX sterile phosphate buffered saline (PBS), overnight at 4°C. Protein concentration of Rag 2-1- mice BALF was measured using Pierce BCA Protein Assay Kit following manufacturer’s protocol (ThermoFisher Scientific Cat No. 23225). Plates were aspirated and washed twice. Plates were blocked with 200 pl of 2X PBS with 1% Tween 20 and 10% BSA (Invitrogen, 88-50470) for two hours at room temperature. Plates were aspirated and washed twice. Glucanase was detected by adding 50 pl of polyclonal mouse serum serially diluted 1 :200-1 :3200 in IX PBS with 1% Tween-20 and 10% BSA, covered and incubated for 2 hours at room temperature on a microplate shaker set at 400 rpm. Plates were aspirated and washed four times. Anti -Mouse HRP IgG (SouthernBiotech, Cat No. 1030-05) was diluted 1 :4000 in IX PBS with 1% Tween-20 and 10% BSA, and 100 pl was added to all wells. Plates were covered and incubated for 1 hour at room temperature on a microplate shaker set at 400 rpm. Plates were aspirated and washed four times. 100 pL of Tetramethylbenzidine (TMB) substrate solution (Invitrogen, 88-50470) was added to each well and incubated at room temperature for 5-10 minutes depending on control serum. The reaction was stopped with 50 pl of 2N H2SO4 to each well. The optical density at 450 (OD450) was read using a BioTek Synergy HT microplate reader. These results are shown in FIG. 11.Example 11: Examples o f Testing Inhibitory Activity o f the Antibodies.

[0286] Beta-1,3-Glucanase hydrolyzes beta-1, 3-glucosidic bonds found in beta- 1,3 -glucans such as laminarin. To assay the enzymatic activity of the glucanase enzyme, hydrolysis of beta-1, 3- glucosidic bonds to generate a reducing sugar can be measured by reaction with a dye reagent. The concentration of the reaction product is directly proportional to the enzyme activity, which can be measured by measuring the absorbance at 540 nm. An example of this assay is the Abbexa Beta- 1,3 -Glucanase Assay Kit, Catalog No.: abx097994.

[0287] This assay can be used to assay the inhibitory activity of anti-glucanase antibodies on glucanase, which may correlate with anti-fungal activity.EQUIVALENTS

[0288] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0289] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0290] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0291] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

WHAT IS CLAIMED IS:

1. An antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein(a) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 3, a VH- CDR2 sequence comprising SEQ ID NO: 4, and a VH-CDR3 sequence comprising SEQ ID NO: 5, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 8, a VL-CDR2 sequence comprising SEQ ID NO: 9, and a VL-CDR3 sequence comprising SEQ ID NO: 10; or(b) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 13, a VH- CDR2 sequence comprising SEQ ID NO: 14, and a VH-CDR3 sequence comprising SEQ ID NO: 15, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 18, a VL-CDR2 sequence comprising SEQ ID NO: 19, and a VL-CDR3 sequence comprising SEQ ID NO: 20; or(c) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 23, a VH- CDR2 sequence comprising SEQ ID NO: 24, and a VH-CDR3 sequence comprising SEQ ID NO: 25, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 28, a VL-CDR2 sequence comprising SEQ ID NO: 29, and a VL-CDR3 sequence comprising SEQ ID NO: 30; or(d) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 33, a VH- CDR2 sequence comprising SEQ ID NO: 34, and a VH-CDR3 sequence comprising SEQ ID NO: 35, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 38, a VL-CDR2 sequence comprising SEQ ID NO: 39, and a VL-CDR3 sequence comprising SEQ ID NO: 40; or(e) the VH comprises a VH-CDR1 sequence comprising SEQ ID NO: 43, a VH- CDR2 sequence comprising SEQ ID NO: 44, and a VH-CDR3 sequence comprising SEQ ID NO: 45, and the VL comprises a VL-CDR1 sequence comprising SEQ ID NO: 48, a VL-CDR2 sequence comprising SEQ ID NO: 49, and a VL-CDR3 sequence comprising SEQ ID NO: 50.

2. An antibody or antigen binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein:(a) the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32 and SEQ ID NO: 42; and / or(b) the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37 and SEQ ID NO: 47.

3. The antibody or antigen binding fragment of claim 2, wherein the VH amino acid sequence and the VL amino acid sequence is selected from the group consisting of:SEQ ID NO: 2 and SEQ ID NO: 7 (8E5G9-1);SEQ ID NO: 12 and SEQ ID NO: 17 (10A12D3-1);SEQ ID NO: 22 and SEQ ID NO: 27 (13F7B7-1);SEQ ID NO: 32 and SEQ ID NO: 37 (20B12G12-1); andSEQ ID NO: 42 and SEQ ID NO: 47 (20D10D1-1), respectively.

4. The antibody or antigen binding fragment thereof of claim 1 comprising:(a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 7, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 2; or(b) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 17, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 12; or(c) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 27, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 22; or(d) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 37, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 32; or(e) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence of SEQ ID NO: 47, and a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence of SEQ ID NO: 42.

5. The antibody or antigen binding fragment of any one of claims 1-4, further comprising a Fc domain of an isotype selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE.

6. The antibody or antigen binding fragment of any one of claims 1-4, wherein the antigen binding fragment is selected from the group consisting of Fab, F(ab’)2, Fab’, scFv, and Fv.

7. The antibody of any one of claims 1-5, wherein the antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

8. The antibody or antigen binding fragment of any one of claims 1-7, wherein the antibody or antigen binding fragment binds to an epitope present in a fungal glucanase polypeptide.

9. The antibody or antigen binding fragment of claim 8, wherein the epitope is a conformational epitope or a non-conformational epitope.

10. The antibody or antigen binding fragment of claim 8 or 9, wherein the fungal glucanase polypeptide comprises the amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 53.

11. A composition comprising the antibody or antigen binding fragment of any one of claims 1-10 and a pharmaceutically-acceptable carrier, wherein the antibody or antigen binding fragment is optionally conjugated to an agent selected from the group consisting of bactrim, trimethoprim-sulfamethoxazole (TMP-SMX), rezafungin, atovaquone, trimethoprim, dapsone, primaquine, clindamycin, and pentamidine.

12. A recombinant nucleic acid encoding the antibody or antigen binding fragment of any one of claims 1-10.

13. The recombinant nucleic acid of claim 12, comprising at least one sequence selected from the group consisting of: SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, and 46.

14. A host cell or a vector comprising the recombinant nucleic acid of claim 12 or claim 13.

15. A kit comprising the antibody or antigen binding fragment of any one of claims 1-10 and instructions for use.

16. The kit of claim 15, wherein the antibody or antigen binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label.

17. A method for treating Pneumocystis pneumonia or Pneumocystis jirovecii infection in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment of any one of claims 1-10, wherein the antibody or antigen binding fragment is conjugated to at least one additional therapeutic agent.

18. The method of claim 17, wherein the at least one additional therapeutic agent is selected from the group consisting of bactrim, trimethoprim-sulfamethoxazole (TMP-SMX), rezafungin, atovaquone, trimethoprim, dapsone, primaquine, clindamycin, and pentamidine.

19. The method of claim 17 or 18, wherein the subject is an immunocompromised patient, a pediatric patient, a geriatric patient, a cancer patient, an autoimmune disease patient, an HIV patient, or an immunosuppressed patient.

20. A method for detecting Pneumocystis jirovecii in a biological sample comprising a. contacting the biological sample with the antibody or antigen binding fragment of any one of claims 1-10, and b. detecting Pneumocystis jirovecii by detecting binding between glucanase present in the biological sample and the antibody or antigen binding fragment.

21. A method for diagnosing Pneumocystis jirovecii infection or Pneumocystis pneumonia in a subject comprising a. contacting a biological sample obtained from the subject with the antibody or antigen binding fragment of any one of claims 1-10,b. detecting binding between glucanase present in the biological sample and the antibody or antigen binding fragment, and c. diagnosing the subject with Pneumocystis jirovecii infection or Pneumocystis pneumonia when glucanase is detected in the biological sample.

22. The method of claim 20 or 21, wherein binding between glucanase and the antibody or antigen binding fragment is detected via radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy or lateral flow immunoassay.

23. A P-1, 3 -endoglucanase antigenic peptide comprising an epitope having the sequence of SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 67, optionally wherein the antigenic peptide further comprises a heterologous signal peptide.

24. The P-1, 3 -endoglucanase antigenic peptide of claim 23, wherein the heterologous signal peptide is a yeast signal peptide, a mammalian signal peptide, an insect signal peptide or a synthetic signal peptide.

25. A kit comprising the P-l,3-endoglucanase antigenic peptide of claim 23 or 24, the antibody or antigen binding fragment of any one of claims 1-10, and instructions for use.

26. A method for determining Pneumocystis seroprevalence in a subject at risk for or suspected of having Pneumocystis jirovecii infection or Pneumocystis pneumonia comprising a. contacting a biological sample obtained from the subject with the P-1,3 endoglucanase antigenic peptide of claim 23 or 24; and b. detecting binding between anti-glucanase immunoglobulins present in the biological sample and the P-1,3 endoglucanase antigenic peptide.

27. A method for diagnosing an active Pneumocystis infection or Pneumocystis pneumonia in a subject in need thereof comprising a. contacting a biological sample obtained from the subject with the P-1,3 endoglucanase antigenic peptide of claim 23 or 24;b. detecting binding between anti-glucanase immunoglobulins present in the biological sample and the P-1,3 endoglucanase antigenic peptide; and c. diagnosing the subject with an active Pneumocystis infection or Pneumocystis pneumonia when the presence of anti-glucanase immunoglobulins in the biological sample is detected.

28. The method of claim 26 or 27, wherein binding between the P-1,3 endoglucanase antigenic peptide and the anti-glucanase immunoglobulins is detected via radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy or lateral flow immunoassay.

29. The method of any one of claims 20-22 or 26-28, wherein the antibody or antigen binding fragment or the P-1,3 endoglucanase antigenic peptide further comprises a detectable label.

30. The method of any one of claims 20-22 or 26-29, wherein the biological sample comprises bronchoalveolar lavage fluid (BALF), sputum, throat swabs, serum, plasma, whole blood or urine.

31. The method of any one of claims 20-22 or 26-30, wherein the subject is an immunocompromised patient, a pediatric patient, a geriatric patient, a cancer patient, an autoimmune disease patient, an HIV patient, or an immunosuppressed patient.

32. The method of any one of claims 26-31, wherein the anti-glucanase immunoglobulins comprise anti-glucanase IgM and / or anti-glucanase IgG.

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