Novel Anti-LAM and Anti-PIM6 / LAM monoclonal antibodies for diagnosis and treatment of mycobacterium tuberculosis infections

Abstract

To provide monoclonal antibodies specific to epitopes found within lipoarabinomannan (LAM) and phosphatidyl-myo-inositol mannoside 6 (PIM6) for the diagnosis and treatment of Mycobacterium tuberculosis infections.SOLUTION: The present invention provides a monoclonal anti-PIM6 / LAM antibody, or an antigen-binding portion thereof, that specifically binds to an epitope present in LAM and PIM6, the epitope including at least one polymannose structure. The anti-PIM6 / LAM antibody includes CDR1 light chain variable region, CDR2 light chain variable region, CDR3 light chain variable region, CDR1 heavy chain variable region, CDR2 heavy chain variable region, and CDR3 heavy chain variable region, each including a specific amino acid sequence.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 293,406, filed February 10, 2016, which is incorporated by reference in its entirety.

[0002] Described herein are compositions, kits, vectors, and methods comprising antibodies specific for epitopes found in lipoarabinomannan (LAM), lipomannan (LM), and phosphatidyl-myo-inositol mannoside 6 (PIM6) for the diagnosis, prevention, and treatment of Mycobacterium tuberculosis infection.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-WEB, and is hereby incorporated by reference in its entirety. The ASCII copy, created on February 1, 2017, is named 096747.00337_ST25.txt and is 29,097 bytes in size. [Background technology]

[0004] A. Mycobacterium Tuberculosis Tuberculosis (TB) remains one of the deadliest infectious diseases in global history, currently infecting approximately one-third of the world's population. According to the WHO Global Tuberculosis Report, 2014:Tuberculosis, in 2013, an estimated 9 million people developed TB and 15 million died from the disease. Currently, effective drugs against TB are available, but these require long-term treatment with multiple antibiotics and have increased susceptibility due to the emergence of multi-drug resistant (MDR-TB) strains, which are currently responsible for approximately 3.5% of recent infections. These strains have become much more difficult to treat and have significantly reduced cure rates. Also, extensively drug resistant TB (XDR-TB) strains have spread, which are even more expensive and difficult to treat than MDR-TB strains and have now been reported in 100 countries worldwide. As a result, new approaches for earlier diagnosis and treatment of TB infections are needed.

[0005] B. Lipoarabinomannan (LAM) The glycolipid lipoarabinomannan (LAM) is a major structural and antigenic component of the cell wall of members of the Mycobacterium tuberculosis complex, where it mediates several important functions that promote productive infection and disease development. LAM is also an important immunodiagnostic target for detecting active infection with TB, especially in patients co-infected with HIV-1, and a promising vaccine target. Despite the importance of LAM as an immunodiagnostic target and its significant role in the physiology and pathogenesis of M.tb infection, surprisingly little is known about the nature of the human humoral response towards this antigen. Previously available monoclonal antibodies specific for LAM were obtained from mice immunized with LAM purified from either Mycobacterium leprae or Mycobacterium tuberculosis, and no human monoclonal antibodies against LAM that have been induced in response to either immunization or infection with Mycobacterium tuberculosis have been described.

[0006] Lipomannan (LM) is the immediate precursor to LAM and has a phosphatidyl-myo-inositol domain modified by a mannan domain consisting of an α(1→6)-linked Manp backbone substituted with short α(1→2)-mannopyranosyl side chains rather than arabinose side chains.

[0007] C. Phosphatidyl-myo-inositol mannoside 6 (PIM6) PIM6 is the product of PIM2, a common precursor to LMs and LAMs. The core of these molecules is a myo-inositol structure glycosylated with Manp units at positions 2 and 6. In PIM6, the Manp unit at position 6 is further replaced by two terminal α-Manp(1→2)-linked sugars that match the mannose cap on ManLAM. These molecules are acylated with as many as four fatty acid chains attached to the inositol head group and the core Man residue, non-covalently anchoring them to the inner and outer membranes of the cell envelope. PIM6 was reported to be a potent TLR2 agonist and enhancer of HIV replication that binds to C-type lectins and DC-SIGN (the major receptor on dendritic cells) and has potent anti-inflammatory activity. Summary of the Invention

[0008] Described herein are novel anti-LAM and anti-PIM6 / LAM monoclonal antibodies (mAbs) for the diagnosis and treatment of Mycobacterium tuberculosis infection. The isolation and characterization of these novel human antibodies specific for Mycobacterium tuberculosis glycolipids, including a human mAb specific for a LAM epitope and a human mAb specific for an epitope shared by LAM and PIM6, are described below.

[0009] Thus, described herein is a human monoclonal anti-lipoarabinomannan (anti-LAM) antibody, or an antigen-binding portion thereof, that specifically binds to a LAM epitope comprising an Ara4 structure, an Ara6 structure, or a combination thereof, wherein the anti-LAM antibody comprises a CDR1 variable light region having at least 80% identity with SEQ ID NO:1, or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:2, or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:26, or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:4, or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:5, or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:6 or SEQ ID NO:23, or an antigenic fragment thereof. The human monoclonal anti-LAM antibody or antigen-binding portion thereof may comprise a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:23, and a light chain variable region comprising the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:26. The anti-LAM antibody may be an scFv-IgG, and an IgGa or IgM antibody. An example of an anti-LAM antibody is A194.

[0010] Further described herein is a human monoclonal anti-LAM antibody or antigen-binding portion thereof that specifically binds to a LAM epitope comprising at least one of a mannose-capped Ara4 structure and a mannose-capped Ara6 structure. The anti-LAM antibody may comprise a CDR1 variable light region having at least 80% identity with SEQ ID NO:7 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:8 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:9 or SEQ ID NO:32 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:10 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:11 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:12 or SEQ ID NO:29 or an antigenic fragment thereof. The antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:43 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:44. The anti-LAM antibody can be, for example, an IgM or IgA antibody. An example of an anti-LAM antibody is P3B09.

[0011] Further described herein is a human monoclonal anti-LAM antibody, or antigen-binding portion thereof, that specifically binds to a LAM epitope comprising an α-Manp(1→2) linkage structure attached at the non-reducing end of Ara4 or Ara6, wherein the anti-LAM antibody comprises a CDR1 variable light region having at least 80% identity with SEQ ID NO:7 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:8 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:9 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:10 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:11 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:12 or an antigenic fragment thereof. The anti-LAM antibody (e.g., P3B09) can be, for example, an IgM or IgA antibody.

[0012] Further described herein is a human monoclonal anti-PIM6 / LAM antibody, or an antigen-binding portion thereof, that specifically binds to an epitope present in LAM and PIM6, wherein the epitope comprises at least one polymannose structure. The epitope is present within the PIM6 mannan domain and is also present in mycobacterial lipomannan (LM). The anti-PIM6 / LAM antibody may comprise a CDR1 variable light region having at least 80% identity with SEQ ID NO: 13 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO: 14 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO: 15 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO: 16 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO: 17 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO: 18 or an antigenic fragment thereof. The antibody may, for example, comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 48. The anti-PIM6 / LAM antibody may, for example, be an IgM, IgA or IgG antibody. An example of an anti-PIM6 / LAM antibody is P95C1.

[0013] Further described herein is a kit for detecting at least one LAM epitope.The kit comprises: (a) at least a first anti-LAM antibody that specifically binds to the LAM epitope; (b) a support to which the at least a first anti-LAM antibody is bound; (c) a detection antibody that specifically binds to LAM or specifically binds to at least the first anti-LAM antibody and is labeled with a reporter molecule; and (d) a buffer.The at least a first anti-LAM antibody is, for example, a human monoclonal anti-LAM antibody as described herein.The detection antibody can be, for example, a second anti-LAM antibody that specifically binds to LAM. In some embodiments, at least one of the first and second anti-LAM antibodies is an scFv-IgG or IgM antibody and comprises a CDR1 variable light region having at least 80% identity with SEQ ID NO:1 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:2 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:26 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:4 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:5 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:6 or SEQ ID NO:23 or an antigenic fragment thereof. In some embodiments of the kit, at least one of the first and second anti-LAM antibodies comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:23, and a light chain variable region comprising the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:26.

[0014] Further described herein is a method of diagnosing an active tuberculosis infection in an individual, comprising: (a) obtaining a sample from an individual containing or suspected of containing LAM; (b) treating the sample to expose it to a distinct LAM epitope; (c) contacting the sample with at least a first antibody that specifically binds to a first epitope on the LAM; (d) contacting the sample with a detection antibody that specifically binds to LAM or specifically binds to at least the first antibody; (e) detecting binding of the at least first antibody to the first epitope on LAM; and (f) diagnosing the patient as having an active tuberculosis infection, wherein binding of the at least first antibody to the first epitope on LAM is indicative of an active tuberculosis infection. The at least first antibody is, for example, a human monoclonal anti-LAM antibody or a human monoclonal anti-PIM6 / LAM antibody as described herein. The detection antibody can be, for example, an anti-LAM antibody that specifically binds to LAM. In some embodiments of the method, at least the first antibody and the detection antibody each comprise a CDR1 variable light region having at least 80% identity with SEQ ID NO:1 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:2 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:26 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:4 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:5 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:6 or SEQ ID NO:23 or an antigenic fragment thereof.In some embodiments of the method, at least one of the primary antibody and the detection antibody is an scFv-IgG or IgM antibody and comprises a CDR1 region having a variable light region having at least 80% identity with SEQ ID NO:1 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:2 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:26 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:4 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:5 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:6 or SEQ ID NO:23 or an antigenic fragment thereof. In some embodiments, the individual is a human.

[0015] Further described herein is a method of treating tuberculosis infection in an individual (e.g., a human). The method comprises administering to the individual at least one human monoclonal anti-LAM antibody or human monoclonal anti-PIM6 / LAM antibody as described herein in a therapeutically effective amount. The method may further comprise administering to the individual at least one antibiotic in a therapeutically effective amount. The tuberculosis infection may be a multi-drug resistant (MDR-TB) tuberculosis infection.

[0016] Further described herein are nucleotide sequences encoding the heavy and light chains (including the variable regions) of the antibodies described herein.

[0017] A. Anti-LAM and anti-PIM6 / LAM antibodies In some embodiments, the invention provides an anti-LAM antibody, or an antigen-binding portion thereof. In some embodiments, the invention provides an anti-PIM6 / LAM antibody, or an antigen-binding portion thereof. An anti-LAM antibody (or an antigen-binding portion thereof) as described herein specifically binds to a LAM epitope. An anti-PIM6 / LAM antibody (or an antigen-binding portion thereof) as described herein specifically binds to both a LAM epitope and a PIM6 epitope. In some embodiments, the LAM and PIM6 epitopes are derived from various mycobacterial species. In further embodiments, the various mycobacterial species are virulent members of the Mycobacterium tuberculosis complex. In further embodiments, the mycobacterial species is Mycobacterium tuberculosis. In some embodiments, the anti-LAM antibody or anti-PIM6 / LAM antibody is a monoclonal antibody (mAb). In further embodiments, the anti-LAM or anti-PIM6 / LAM antibody is a human monoclonal anti-LAM or human monoclonal anti-PIM6 / LAM antibody, respectively. In other embodiments, the anti-LAM or anti-PIM6 / LAM antibody is a humanized monoclonal anti-LAM or anti-PIM6 / LAM antibody, respectively. In some embodiments, the anti-LAM antibody binds to the Ara4 and Ara6 structures.

[0018] In some embodiments, the LAM epitope is an uncapped arabinose chain. In some embodiments, the LAM epitope is an uncapped or single mannose capped arabinose chain, with or without terminal MTX substitution.

[0019] In some embodiments, the LAM epitope is a mannose-capped Ara4 structure and a mannose-capped Ara6 structure. In other embodiments, the anti-LAM antibody specifically binds to an α(1→2)-linked dimannose structure, which can be linked to either an Ara4 / Ara6 structure or a polymannose structure (FIG. 8). In some embodiments, the PIM6 epitope comprises at least one polymannose structure that is also present in mycobacterial lipomannan (LM). In some embodiments, the anti-PIM6 / LAM antibody specifically binds to a PIM6 epitope that comprises at least one polymannose structure within the PIM6 mannan domain. In some embodiments, the LAM epitope comprises at least one methylthioxylose (MTX) or methylsylfinylxylofuranosyl (MSX) substitution. In some embodiments, the LAM epitope comprises at least one phosphatidyl-myo-inositol substitution (PILAM). In some embodiments, the LAM epitope is an arabinose chain capped with at least one mannose, i.e., a mannosylated Man-LAM epitope. In further embodiments, the capped arabinose chain comprises an Ara4 and / or Ara6 structure. In some embodiments, the Man-LAM epitope comprises a monomannose-substituted arabinose chain, a dimannose-substituted arabinose chain, a trimannose-substituted arabinose chain, or a combination thereof. In some embodiments, the Man-LAM epitope comprises a dimannose- or trimannose-capped Ara4 and / or Ara6 structure. In some embodiments, the Man-LAM epitope is a dimannose-capped Ara6. In some embodiments, the anti-LAM or anti-PIM6 / LAM antibody comprises an IgG antibody. In further embodiments, the IgG anti-LAM or anti-PIM6 / LAM antibody comprises an IgG1, IgG2, or IgG3 subclass. In some embodiments, the anti-LAM or anti-PIM6 / LAM antibody is not an IgG antibody, hi other embodiments, the anti-LAM or anti-PIM6 / LAM antibody comprises an IgA antibody.In other embodiments, the anti-LAM or anti-PIM6 / LAM antibody comprises an IgM antibody. In some embodiments, the anti-LAM or anti-PIM6 / LAM antibody comprises a second isotype that has been switched from the isotype in which it was originally isolated. In some embodiments, the anti-LAM or anti-PIM6 / LAM antibody comprises a recombinant antibody. In some embodiments, the recombinant antibody comprises a multivalent IgM antibody. In further embodiments, the recombinant antibody comprises a pentavalent IgM antibody. In other embodiments, the recombinant antibody comprises an ScFv-IgG antibody, in which a single chain Fv fragment of one antibody is linked to the N-terminus of the heavy chain of the or a different anti-LAM mAb. In further embodiments, the recombinant antibody comprises a multivalent ScFv-IgG antibody. In further embodiments, the recombinant antibody comprises a homologous tetravalent ScFv-IgG antibody, in which the scFv domains are derived from the variable regions of the IgG present in the construct. In further embodiments, the recombinant antibody comprises a heterogeneous tetrameric scFv-IgG antibody, in which the scFv region is derived from a different anti-LAM or anti-PIM6 / LAM antibody than the IgG region comprised therein. In some embodiments, the scFv domain comprises a leader sequence linked to the variable heavy (VH) region of a second anti-LAM or anti-PIM6 / LAM antibody linked to the variable light (VL) domain of said anti-LAM or anti-PIM6 / LAM antibody. In other embodiments, the scFv domain comprises a leader sequence linked to the variable light chain region of a first anti-LAM or anti-PIM6 / LAM antibody linked to the variable heavy (VH) region of a second anti-LAM or anti-PIM6 / LAM antibody. In some embodiments, the anti-LAM antibody is an isolated anti-LAM antibody that specifically binds to a LAM epitope (e.g., an α(1→2)-linked dimannose structure that may be linked to either Ara4 and Ara6 or one of their combinations, an Ara4 / Ara6 structure, or a polymannose structure). In some embodiments, the anti-LAM antibody does not compete with CS-35 and FIND25. In some embodiments, the anti-PIM6 / LAM antibody is an isolated anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure in mycobacterial lipomannan (LM).

[0020] In some embodiments, the anti-LAM or anti-PIM6 / LAM antibody comprises a flexible linker. In some embodiments, the flexible linker links the corresponding heavy and light chain domains into a single chain molecule. In some embodiments, the flexible linker connects the immunoglobulin light chain (IgVL) to the immunoglobulin heavy chain (IgVH). In further embodiments, the flexible linker has the formula (GGSGG) n (SEQ ID NO: 19) (wherein n is any positive integer in the range of 1 to 200, for example, 1 to 5, 1 to 10, 1 to 15, 1 to 25, 1 to 50, 5 to 10, 5 to 25, 10 to 25, 10 to 50, 1 to 100, 1 to 150, and any intervening range).

[0021] In some embodiments, the anti-LAM antibody (e.g., P30B9, A194-01) has at least one (e.g., one, two, three) complementarity determining regions (CDRs) (e.g., CDR1, CDR2, CDR3). In some embodiments, the variable light region of CDR1 consists essentially of SEQ ID NO:1 or an antigenic fragment thereof. In other embodiments, the variable light region of the CDR1 region consists essentially of SEQ ID NO:7 or an antigenic fragment thereof. In other embodiments, the variable light region of the CDR1 region consists essentially of SEQ ID NO:13 or an antigenic fragment thereof. In some embodiments, the variable heavy region of the CDR1 region consists essentially of SEQ ID NO:4 or an antigenic fragment thereof. In other embodiments, the variable heavy region of the CDR1 region consists essentially of SEQ ID NO:10 or an antigenic fragment thereof. In other embodiments, the variable heavy region of the CDR1 region consists essentially of SEQ ID NO:16 or an antigenic fragment thereof.

[0022] In some embodiments, the variable light region of CDR2 consists essentially of SEQ ID NO:2 or an antigenic fragment thereof. In other embodiments, the variable light region of CDR2 consists essentially of SEQ ID NO:8 or an antigenic fragment thereof. In other embodiments, the variable light region of CDR2 consists essentially of SEQ ID NO:14 or an antigenic fragment thereof. In some embodiments, the variable heavy region of CDR2 consists essentially of SEQ ID NO:5 or an antigenic fragment thereof. In other embodiments, the variable heavy region of CDR2 consists essentially of SEQ ID NO:11 or an antigenic fragment thereof. In other embodiments, the variable heavy region of CDR2 consists essentially of SEQ ID NO:17 or an antigenic fragment thereof.

[0023] In some embodiments, the variable light region of the CDR3 consists essentially of SEQ ID NO:3 or an antigenic fragment thereof. In other embodiments, the variable light region of the CDR3 consists essentially of SEQ ID NO:9 or an antigenic fragment thereof. In other embodiments, the variable light region of the CDR3 consists essentially of SEQ ID NO:15 or an antigenic fragment thereof. In some embodiments, the variable heavy region of the CDR3 consists essentially of SEQ ID NO:6 or an antigenic fragment thereof. In other embodiments, the variable heavy region of the CDR3 consists essentially of SEQ ID NO:12 or an antigenic fragment thereof. In other embodiments, the variable heavy region of the CDR3 consists essentially of SEQ ID NO:18 or an antigenic fragment thereof.

[0024] In some embodiments, an anti-PIM6 / LAM antibody (e.g., P95C1) has at least one (e.g., one, two, three) CDRs (e.g., CDR1, CDR2, CDR3). In some embodiments, the variable light region of CDR1 consists essentially of SEQ ID NO: 13, or an antigenic fragment thereof. In some embodiments, the variable heavy region of CDR1 consists essentially of SEQ ID NO: 16, or an antigenic fragment thereof. In some embodiments, the variable light region of CDR2 consists essentially of SEQ ID NO: 14, or an antigenic fragment thereof. In some embodiments, the variable heavy region of CDR2 consists essentially of SEQ ID NO: 17, or an antigenic fragment thereof. In some embodiments, the variable light region of CDR3 consists essentially of SEQ ID NO: 15, or an antigenic fragment thereof. In some embodiments, the variable heavy region of CDR3 consists essentially of SEQ ID NO: 18, or an antigenic fragment thereof.

[0025] B. Diagnostic Kits and Methods In some embodiments, the present invention provides a kit for detecting the presence of LAM and / or PIM6 in biological fluids of human subjects. In some embodiments, the components of the assay are assembled in a lateral flow device (see World Health Organization 2015, The use of lateral flow urine lipoarabinomannan assay (LF-LAM) for the diagnosis and screening of active tuberculosis in people living with HIV). In some embodiments, the kit includes a first anti-LAM (e.g., A194-01, P30B9) or anti-PIM6 / LAM (e.g., P95C1) capture antibody, a second anti-LAM or anti-PIM6 / LAM detector (detection) antibody labeled with a reporter molecule, a support to which the first anti-LAM or anti-PIM6 / LAM antibody is bound, and a buffer. In some embodiments, at least one of the first anti-LAM or anti-PIM6 / LAM antibody and the second anti-LAM or anti-PIM6 / LAM antibody is a human monoclonal anti-LAM antibody that specifically binds to Ara4 and Ara6 or one of their combinations, or a human monoclonal anti-PIM6 / LAM antibody that specifically binds to the mannan domain of LAM (and lipomannan (LM)). In some embodiments, the first anti-LAM antibody and the second anti-LAM antibody bind to the same LAM epitope present in multiple copies on a single LAM molecule. In other embodiments, the first anti-LAM antibody and the second anti-LAM antibody bind to different epitopes present on a single LAM molecule. The LAM and PIM6 epitopes may be any of the LAM and PIM6 epitopes described herein. In other embodiments, a third detector (detection) antibody is included that binds to a non-competing site on the second antibody. In some embodiments, the first and second antibodies are of the same isotype. In other embodiments, the first and second antibodies are of different isotypes.In some embodiments of the capture assay, simply either the capture antibody or the detection antibody is an anti-LAM antibody (eg, A194-01, P30B9) or an anti-PIM6 / LAM antibody (eg, P95C1) as described herein.

[0026] The antibodies described herein can be used for further detection and diagnostic applications. For example, in one diagnostic assay, one or more of the antibodies described herein (e.g., A194-01, P30B9, P95C1) can be used to stain tissues obtained from patients to detect the presence of LAM in lesions (e.g., granulomas) suspected of containing TB or TB-infected cells. This can be done, for example, using a single antibody as described herein (e.g., A194-01, P30B9, P95C1) conjugated with a label that allows for sensitive detection. In such a method or assay, detection of PIM6 or related molecules by P95C1 can be done in infected tissue. In another example, P95C1 can be used in a PIM6 competition assay in which capture of a labeled form of PIM6 by immobilized P95C1 is competed by soluble PIM6 present in the suspect's biological fluid (e.g., blood or urine). In the absence of soluble PIM6, this would result in signal capture that is competed for by the presence of soluble PIM6 (see World Health Organization 2015, Policy Guidance-The use of lateral flow urine lipoarabinomannan assay (LF-LAM) for the diagnosis and screening of active tuberculosis in people living with HIV).

[0027] In some embodiments, the present invention provides a kit for distinguishing between pathogenic members of the Mycobacterium tuberculosis complex and non-pathogenic members of the Mycobacterium tuberculosis complex. In some embodiments, the anti-LAM antibody is a human monoclonal anti-LAM antibody that specifically binds to one of the Ara4 and Ara6 structures or a combination thereof, with or without Man or MTX-Man substitution, or an anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure in PIM6 or in the LAM mannan domain. In some embodiments, the anti-LAM antibody specifically binds to a Man-LAM epitope, such as a dimannose-substituted side chain, a trimannose-substituted side chain, or a combination thereto. In further embodiments, the anti-LAM antibody specifically binds to a Man-LAM epitope, such as an Ara4 and / or Ara6 structure capped with dimannose or trimannose. In a further embodiment, the anti-LAM antibody specifically binds to dimannose-capped Ara6 structures.

[0028] In some embodiments, the present invention provides methods for diagnosing active tuberculosis infection in an individual. In some embodiments, anti-LAM or anti-PIM6 / LAM antibodies can be modified with a high sensitivity tag and used to identify mycobacterial PIM6 or LAM associated material in tissue samples as a diagnostic for TB infection and localization. In some embodiments, the method involves capturing soluble LAM and includes the steps of: (a) obtaining a sample from an individual containing LAM; (b) processing the sample to isolate or expose the LAM; (c) capturing the isolated or exposed LAM using a first anti-LAM antibody that binds to a first epitope on the LAM; (d) contacting the isolated or exposed LAM with a second anti-LAM antibody that binds to a second epitope on the LAM; (e) detecting binding of the first anti-LAM antibody and the second anti-LAM antibody to at least one of the LAM; and (f) diagnosing the patient as having an active tuberculosis infection, wherein the presence of binding of the first anti-LAM antibody and the second anti-LAM antibody to at least one of the LAM is indicative of an active tuberculosis infection. In some embodiments, at least one of the first and second anti-LAM antibodies is a human monoclonal anti-LAM antibody that specifically binds to Ara4 and Ara6 or one of their combinations. In some embodiments, at least one of the first and second antibodies is a human monoclonal anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure in the LAM mannan domain. In further embodiments, the first and second antibodies are of different isotypes. In some embodiments, at least one of the first and second antibodies is a recombinant antibody. In other embodiments, neither the first nor the second antibody is a recombinant antibody. In still other embodiments, both the first and the second antibodies are recombinant antibodies.

[0029] In some embodiments, the present invention provides a method for quantifying the amount of LAM and / or PIM6 present in a sample. In some embodiments, the method includes: (a) obtaining a sample containing LAM and / or PIM6; (b) contacting the sample with an anti-LAM antibody and / or an anti-PIM6 antibody; (c) detecting the presence of specific binding of the anti-LAM antibody to the LAM and / or binding of the anti-PIM6 / LAM antibody to the LAM or PIM6; and (d) quantifying the amount of LAM or PIM6 in the sample. In some embodiments, the anti-LAM antibody is a human monoclonal anti-LAM antibody that specifically binds to Ara4 and Ara6 or one of their combinations. In some embodiments, the anti-PIM6 / LAM antibody is a human monoclonal anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure in the PIM6 mannan domain (e.g., at least one polymannose structure in mycobacterial lipomannan (LM)). In some embodiments, quantification of the amount of LAM and / or PIM6 is achieved by comparing the signal intensity with the signal intensity of serially diluted control samples having known concentrations of LAM and / or PIM6.

[0030] In some embodiments, the present invention provides a method for diagnosing an individual as infected with Mycobacterium tuberculosis. In some embodiments, the method comprises: (a) obtaining a sample containing LAM or PIM6; (b) contacting the sample with an anti-LAM antibody and / or an anti-PIM6 antibody; and (c) detecting the presence of specific binding of the anti-LAM antibody to the Man-LAM and / or the presence of specific binding of the anti-PIM6 / LAM antibody to the PIM6, where the anti-LAM antibody specifically binds to a LAM epitope comprising Man-LAM with at least one 5-deoxy-5-methylthiopentofuranosyl (MTX) substitution, and the anti-PIM6 / LAM antibody specifically binds to an epitope comprising at least one polymannose structure within the LAM mannan domain. In some embodiments, the anti-LAM antibody is a human monoclonal anti-LAM antibody that specifically binds to one of Ara4 and Ara6 or a combination thereof. In some embodiments, the anti-PIM6 / LAM antibody is a human monoclonal anti-PIM6 / LAM antibody (eg, P95C1) that specifically binds to at least one polymannose structure within the PIM6 mannan domain.

[0031] In some embodiments, the method includes an amplification step that increases the sensitivity of the detection method. Examples include generating additional target sites using the Tyramide Signal Amplification kit (Perkin-Elmer) or amplifying the initial signal using the ELISA Amplification System (Thermo Fisher).

[0032] In some embodiments, the invention provides a method for distinguishing between a pathogenic member of the Mycobacterium tuberculosis complex and a non-pathogenic member of the Mycobacterium tuberculosis complex, the method comprising: (a) obtaining a sample comprising LAM and / or PIM6; (b) contacting the sample with an anti-LAM antibody that specifically binds to a Man-LAM epitope comprising a dimannose-substituted side chain, a trimannose-substituted side chain, or a combination thereof, or an anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure within the PIM6 mannan domain; and (c) detecting the presence of specific binding of the anti-LAM antibody to the Man-LAM or the presence of specific binding of the anti-PIM6 / LAM antibody to the at least one polymannose structure within the PIM6 mannan domain, wherein the presence of the specific binding indicates the presence of a pathogenic member of the Mycobacterium tuberculosis complex. In some embodiments, the anti-LAM antibody is a human monoclonal anti-LAM antibody that specifically binds to Ara4 and Ara6 or one of their combinations. In further embodiments, the Man-LAM epitope comprises a dimannose- or trimannose-capped Ara4 and / or Ara6 structure. In further embodiments, the Man-LAM epitope is a dimannose-capped Ara6. In some embodiments, the anti-PIM6 / LAM antibody is a human monoclonal anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure within the PIM6 mannan domain.

[0033] C. Therapeutic Compositions, Methods, Vaccines, and Vectors In some embodiments, the present invention provides methods for treating an infection with a virulent member of the Mycobacterium tuberculosis complex in an individual. In some embodiments, the method comprises administering at least one anti-LAM antibody or anti-PIM6 / LAM antibody in a therapeutically effective amount to an individual exposed to infectious Mycobacterium tuberculosis (M.tb). In further embodiments, the method comprises administration of at least one antibiotic. In some embodiments, the TB infection is active. In other embodiments, the TB infection is latent. In some embodiments, the infection involves a multi-drug resistant (MDR) strain of tuberculosis. In other embodiments, the infection involves an extensively drug resistant (XDR) strain of tuberculosis.

[0034] In some embodiments, the present invention provides a combination therapy for treating infection with virulent members of the Mycobacterium tuberculosis complex in an individual. In some embodiments, the method comprises administering a therapeutically effective amount of a first anti-LAM antibody that specifically binds to a first LAM epitope comprising at least one of unsubstituted LAM, monomannosylated Man-LAM, MSX-substituted LAM, and combinations thereof, or a first anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure in PIM6 and LAM mannan domains; and administering a therapeutically effective amount of a second anti-LAM antibody that specifically binds to a second LAM epitope comprising at least one of dimannose-substituted Man-LAM, trimannose-substituted Man-LAM, and combinations thereof. In some embodiments, the first antibody and the second antibody are administered simultaneously (e.g., in a single composition or in two compositions administered simultaneously). In other embodiments, the first antibody and the second antibody are administered at different times. In some embodiments, at least one of the first anti-LAM antibody and the second anti-LAM antibody is a human monoclonal anti-LAM antibody that specifically binds to Ara4 and Ara6 or one of their combinations. In some embodiments, the anti-PIM6 / LAM antibody is a human monoclonal anti-PIM6 antibody that specifically binds to at least one polymannose structure in PIM6 and / or within the PIM6 cross-reactive mannan domain of LAM. In some embodiments, the first anti-LAM antibody and the second anti-LAM antibody, or the anti-PIM6 / LAM antibody, are of different isotypes. In some embodiments, at least one of the first anti-LAM antibody and the second anti-LAM antibody, and the anti-PIM6 / LAM antibody are recombinant antibodies. In other embodiments, none of the first anti-LAM antibody, the second anti-LAM antibody, or the anti-PIM6 / LAM antibody is a recombinant antibody. In yet other embodiments, both the first anti-LAM antibody and the second anti-LAM antibody, or the anti-PIM6 / LAM antibody, are recombinant antibodies. In further embodiments, the method includes administration of at least one antibiotic.In such embodiments, the at least one antibiotic may be administered simultaneously (e.g., co-administered) with the first and second antibodies, or the at least one antibiotic may be administered at a time different from the time of administration of the first and second antibodies. In some embodiments, the infection is active. In other embodiments, the infection is latent. In some embodiments, the infection is a multi-drug resistant (MDR) tuberculosis infection. In other embodiments, the infection is an extensively drug resistant (XDR) tuberculosis infection.

[0035] In some embodiments, the present invention provides a vaccine or pharmaceutical composition for preventing infection with virulent members of the Mycobacterium tuberculosis complex. In some embodiments, the present invention provides a method for stimulating a host immune response in a patient, comprising administering to said patient a therapeutically effective amount of a LAM antigen and / or a PIM6 antigen. In some embodiments, these antigens are conjugated to an immunogenic protein carrier and / or co-administered with an adjuvant that potently stimulates an immune response to the glycolipid antigen. In some embodiments, the vaccine or pharmaceutical composition induces anti-LAM antibodies that specifically bind to the Man-LAM epitope and / or anti-PIM6 / LAM antibodies that specifically bind to at least one polymannose structure within the PIM6 mannan domain. In further embodiments, the Man-LAM epitope present in the vaccine or pharmaceutical composition comprises a dimannose or trimannose capped Ara4 and / or Ara6 structure. In further embodiments, the Man-LAM epitope is a dimannose capped Ara6. In some embodiments, the Man-LAM epitope has at least one MTX substitution. In some embodiments, the anti-LAM antibody and / or the anti-PIM6 / LAM antibody is an IgM antibody. In other embodiments, the anti-LAM antibody and / or the anti-PIM6 / LAM antibody is a recombinant antibody.

[0036] In some embodiments, the present invention provides a method for preventing infection by virulent members of the Mycobacterium tuberculosis complex in an individual by passive administration of protective antibodies. In some embodiments, the anti-LAM antibody is a human monoclonal anti-LAM antibody that specifically binds to Ara4 and Ara6 or one of their combinations. In some embodiments, the anti-PIM6 / LAM antibody is a human monoclonal anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure in the PIM6 and LAM mannan domains. In some embodiments, the method comprises administering to the individual a therapeutically effective amount of an anti-LAM antibody that specifically binds to a Man-LAM epitope and / or an anti-PIM6 antibody that specifically binds to a PIM6 epitope (e.g., an epitope shared by PIM6 and LAM). In further embodiments, the targeted ManLAM epitope comprises an Ara4 and / or Ara6 structure capped with dimannose or trimannose. In further embodiments, the ManLAM epitope is Ara6 capped with dimannose. In some embodiments, the ManLAM epitope has at least one MTX substitution. In some embodiments, the anti-LAM antibody or anti-PIM6 / LAM antibody is an IgM antibody. In other embodiments, the anti-LAM antibody or anti-PIM6 / LAM antibody is a recombinant antibody.

[0037] In some embodiments, the invention provides passive administration of protective antibodies via a recombinant vector. In some embodiments, the recombinant vector comprises a first nucleic acid encoding an IgVL of an anti-LAM antibody and a second nucleic acid encoding an IgVH of an anti-LAM antibody, where each of the nucleic acids is operably linked to a promoter region. In some embodiments, at least one of the IgVL and IgVH is derived from a human monoclonal anti-LAM antibody that specifically binds to one of Ara4 and Ara6 or a combination thereof. In other embodiments, the recombinant vector comprises a first nucleic acid encoding an IgVL of an anti-PIM6 / LAM antibody and a second nucleic acid encoding an IgVH of an anti-PIM6 / LAM antibody, where each of the nucleic acids is operably linked to a promoter region. In some embodiments, the recombinant vector comprises additional transcriptional regulatory elements. In some embodiments, at least one of the first and second nucleic acid sequences is organized in an operon. In some embodiments, at least one of the first and second nucleic acid sequences is organized in an expression cassette. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are organized into a single expression cassette. In some embodiments, the first nucleic acid and the second nucleic acid are located in the same cloning vector. In other embodiments, the first nucleic acid and the second nucleic acid are located in different cloning vectors. In some embodiments, the expression of the first nucleic acid and the second nucleic acid can be simultaneous. In other embodiments, the expression of the first nucleic acid and the second nucleic acid can be separately inducible. In some embodiments, the expression of the first nucleic acid can be separated in time from the expression of the second nucleic acid. In some embodiments, the recombinant vector is a plasmid. In other embodiments, the recombinant vector is a non-replicating virus. In further embodiments, the recombinant vector is an adeno-associated virus.

[0038] In some embodiments, the present invention provides a method for treating an infection by a virulent member of the Mycobacterium tuberculosis complex in an individual. In some embodiments, the method comprises administering to the individual a first nucleic acid encoding an IgVH of an anti-LAM antibody and a second nucleic acid encoding an IgVL of an anti-LAM antibody, where each of the nucleic acids is operably linked to a promoter region. In other embodiments, the method comprises administering to the individual a first nucleic acid encoding an IgVH of an anti-PIM6 / LAM antibody and a second nucleic acid encoding an IgVL of an anti-PIM6 / LAM antibody, where each of the nucleic acids is operably linked to a promoter region. In some embodiments, at least one of the IgVL and IgVH is derived from a human monoclonal anti-LAM antibody that specifically binds to one of Ara4 and Ara6 or a combination thereof, or from a human monoclonal anti-PIM6 / LAM antibody that specifically binds to at least one polymannose structure in the PIM6 mannan domain. In some embodiments, the first nucleic acid and the second nucleic acid are located in the same cloning vector. In other embodiments, the first nucleic acid and the second nucleic acid are located in different cloning vectors. In some embodiments, the recombinant vector is a plasmid. In other embodiments, the recombinant vector is a non-replicating virus. In further embodiments, the recombinant vector is an adeno-associated virus.

[0039] Further embodiments, features and advantages will be readily apparent to those skilled in the art based on the disclosure provided herein. Other features will be fully understood by those skilled in the art to which the package pertains from the following description and claims. Although similar or equivalent antibodies, compositions, kits and methods as described herein can be used in the practice or testing of the present invention, preferred antibodies, compositions, kits and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The specific embodiments discussed below are merely illustrative and are not intended to be limiting. [Brief description of the drawings]

[0040] [Figure 1] Figure 1A is a model of the IgG forms of A194-01 and fragments used in the binding competition assay. These included monovalent scFv and Fab structures, as well as bivalent scFv dimers and native IgG. Figure 1B is a competition curve showing that the monovalent form of A194-01 did not compete as effectively as the bivalent form. Figure 1C is a structure of a higher valency form of A194-01. This represents a homologous tetravalent A194-01 scFv-IgG, with the A194-01 scFv domain linked to the N-terminus of each of the normal heavy chains. [Diagram 2]Figure 2A is the binding activity of P30B9 IgG and IgM versions and IgMs in which a six amino acid insertion in the VH region was deleted or nine somatic mutations in the VH region were reverted to the closest germline sequence, ManLAM from Mycobacterium tuberculosis. The six amino acid insertions contributed to the reactivity to a greater extent than the nine somatic mutations. Figures 2B and 2C compare the reactivity of P30B9 IgM and IgG versions and mutations with six amino acid deletions in the heavy chain to ManLAM from Mycobacterium tuberculosis (B) and PILAM from Mycobacterium smegmatis (C). Unlike the IgG type, the IgM type reacted specifically with ManLAM (2B) derived from Mycobacterium tuberculosis, but did not react specifically with PILAM (2C). The reactivity of the Δ6 amino acid mutant ManLAM was greatly reduced and the IgM type was negative for PILAM. [Diagram 3] Figure 3 compares the reactivity of two human and four murine mAbs to PILAM on the left panel and to ManLAM isolated from the H37Rv strain of Mycobacterium tuberculosis on the right panel. Curves were plotted using molar concentrations of antibody to control for the different molecular weights of these reagents. [Figure 4A] Figure 4A shows the structures of 25 synthetic oligosaccharides representing microbial glycan structures related to motifs present in LAMs. These structures were coupled to the BSA carrier protein and used to probe epitope specificity. [Figure 4B] Figure 4B shows the binding characteristics of six LAM-specific monoclonal antibodies to a panel of 25 synthetic oligosaccharides. Binding results are shown for three concentrations, and the titration patterns indicate the relative affinities of the antibodies to these antigens. [Diagram 5]Figure 5. The left panel shows the structures of IgA1 (A), IgA2 (B) and the dimeric IgA1-J dimer complex (C). The right panel shows an SDS-PAGE gel of purified P30B9 IgA1, IgA2 and IgA3 proteins both before and after reduction with DTT. P30B9 IgA3 was later shown to be a PCR artifact with a longer hinge region. [Figure 6] FIG. 6 shows the binding curves of different isotypes of P30B9 to ManLAM, which shows the highest activity against IgM and then IgA, and has no reactivity against IgG. [Figure 7] FIG. 7 is a comparison of the efficiency of biotinylated monoclonal antibody probes in detecting soluble ManLAM in a CS-35 capture assay, in which the indicated concentrations of ManLAM were captured by CS-35 and detected with the indicated mAbs labeled with biotin. [Figure 8] Figure 8 shows the binding curves of P30B9 to various mannose-capped Ara4 structures, or tetra- and pentamannose structures. Preferential binding was seen for structures 3 (dimannose-Ara4) and 59, which have related α-Manp(1→2)-Manp linkages. [Figure 9] Figure 9: Titration of monoclonal anti-LAM antibodies against various uncapped LAM-related glycoconjugates to determine the structural requirements for reactivity. Figure 9A: Analysis of the importance of Ara-α(1→5)-Ara bond at the penultimate position from the non-reducing end of the Ara4 sequence. Figure 9B: Analysis of the dependency of Ara-β(1→2)-Ara bond at the terminal position of the Ara4 sequence. [Figure 10] FIG. 10 shows binding curves of A194-01 IgG and three mouse anti-LAM antibodies to various Ara6-bearing glycoconjugates showing the effect of different capping motifs on antibody reactivity. [Figure 11]Figure 11 is a binding competition study to measure the ability of individual anti-LAM antibodies to compete for the binding of the probe antibody to the ManLAM antigen. The antibodies were biotinylated when tested against antibodies from the same species. Note that A194-01 cannot compete for biotinylated P30B9. [Figure 12] Figure 12: Competition of anti-LAM monoclonal antibodies for binding to LAM from Mycobacterium tuberculosis (ManLAM) and LAM from Mycobacterium smegmatis (PILAM). Efficient competition between FIND25 and P30B9 for ManLAM is consistent with dominance of the dimannose-substituted Ara6, while the lack of competition of these two mAbs with A194 is consistent with its low reactivity with the dimannose-capped structure. Efficient competition of A194 for FIND25 versus PILAM is consistent with the absence of dimannose capping in this structure. [Figure 13] Figure 13 shows the binding competition of biotinylated probe monoclonal antibody with redundant unmodified antibodies against native LAM antigens and selected glycoconjugates. Figure 13A shows the competition of binding of biotinylated A194-01 IgG, CS-35 and FIND25 to MAnLAM by four mAbs; Figure 13B shows the competition of binding of FIND25 to both ManLAM and PILAM by three mAbs; Figure 13C shows the competition of binding of P30B9 IgM to MAnLAM and two synthetic glycoconjugate antigens by four mAbs. [Figure 14] Engineered variants and / or derivatives of A194-01 react with a broader range of glycoconjugates, including di- and trimannose substituted forms that are poorly recognized by IgG isotypes of A194-01. [Figure 15]Figure 15 shows the differential competition of A194-01 IgG and engineered variants and / or derivatives of A194-01 for binding to FIND25 and P30B9 IgM to ManLAM. A194 IgG does not compete with P30B9 or FIND25 for ManLAM, but the multimeric forms do compete, consistent with the broader epitope specificity of these forms. As shown above, A194 IgG does not compete significantly with FIND25 for PILAM. [Figure 16] Figure 16 is a comparison of the analysis of the effect of mannose capping on the reactivity of CS-40, A194-01 and P30B9 mAbs. Antibody binding specificity was measured by ELISA against specific glycoconjugates with different mannose substitutions. Antibody titrations are shown in Figure 16A and the structures of mannose-containing glycan antigens are shown in Figure 16B. [Figure 17] FIG. 17A is a homologous scFv-IgG. In this example, both the IgG and scFv domains are derived from the same antibody. This results in increased valency (tetravalent versus bivalent) but does not directly modify target specificity. FIG. 17B is a heterologous scFv-IgG. In addition to the increase in valency, broadened specificity is also introduced, which may allow recognition of different epitopes in a single antigen molecule. FIG. 17C is a heterologous scFv-IgM. In this formulation, different scFvs are combined with IgM constructs. An example is the linkage of A194-01 scFv with P30B9 IgM. In addition to the increase in valency, in this case additional epitope specificity will be introduced, which may allow multivalent recognition of different epitopes that may not be recognized by the homologous scFv-IgM, resulting in increased affinity. [Figure 18A]Figure 18 is a mapping of epitopes recognized by the new mAbs. The epitope specificity of P95C1 was compared with that of two previously described mAbs, A194-01 and P30B9, and two new mAbs, P61H5 and P83A8, which recognize epitopes related to the two previously described mAbs. Figure 18A is the reactivity of LAM-specific mAbs to LAM precursor molecules. P30B9 and P61H5 were more specific to ManLAM than to PILAM, while A194-01, P83A8 and P95C1 recognized both forms of LAM. P95C1 also bound efficiently to LM and PIM6. The weak reactivity of other mAbs to LM and PIM6 is at least in part due to contamination of these materials with ManLAM. [Figure 18B] FIG. 18B shows the reactivity of synthetic LAM-derived glycoconjugates. [Figure 18C] FIG. 18C shows that, in contrast to previously known mAbs, P95C1 was the only antibody that did not recognize any of the polyarabinose structures, but reacted specifically with two polymannose structures, YB-BSA-05 and YB-BSA-11, that resemble structures present in PIM6 and within the mannan domain at the base of LM and LAM. [Figure 19] Figure 19 shows the effect of isotype switching on the binding of P95C1 and P30B9 to ManLAM and PI-LAM. In P95C1, the IgM, IgA and IgG isotypes all have equivalent binding activity to both ManLAM and PILAM, unlike P30B9, which reacts exclusively with ManLAM in IgM and IgA form only, and not as IgG. [Figure 20]FIG. 20: Western blot analysis of cross-reactivity of P95C1 with LAM and additional M.tb glycolipids. FIG. 20(A): Purified LAM-associated glycolipids were resolved on a 12% SDS-PAGE gel followed by oxidation and staining of sugar molecules with periodic acid-Schiff stain to reveal material containing reactive glycans. FIG. 20(B): Parallel blots were probed with mAbs A194 IgG1, P30B9 IgM, and P95C1 IgM, followed by alkaline phosphatase conjugated anti-human IgG and IgM secondary antibodies and treatment with bcip / nbt chromogenic substrate. A194-01 cross-reacts with ManLAM from M.tb and PILAM from M.smeg. P30B9 is specific for ManLAM of M.tb. P95C1 recognizes LAM and both the LM and PIM6 species isolated from Mycobacterium tuberculosis (M.tb). The weak staining by A194-01 of bands in LM and PIM6 that co-migrate with LAM is apparently due to minor contamination of these samples with LAM. [Figure 21] FIG. 21 is an alignment of amino acid sequences in the A194 heavy and light chain variable region sequences and their comparison with their closest germline sequences. In the top alignment, the first amino acid sequence from the top (A194-VH) is the A194 heavy chain variable region sequence without the CDR3 sequence (SEQ ID NO: 23). The heavy chain variable region sequence without the CDR3 is SEQ ID NO: 21. In the top alignment, the second amino acid sequence (germline Homsap IGHV3-20*01) is SEQ ID NO: 22. In the top alignment, the third amino acid sequence is the CDR3 of the A194 heavy chain variable region and is SEQ ID NO: 23. In the bottom alignment, the first amino acid sequence from the top (A-194-Vk) is the A194 light chain variable region without the CDR3 sequence (SEQ ID NO: 26). The light chain variable region sequence without the CDR3 is SEQ ID NO: 24. In the bottom alignment, the second amino acid sequence (germline Homsap IGKV3-15*01) is SEQ ID NO: 25. In the bottom alignment, the third sequence is the CDR3 of the A194 light chain variable region, SEQ ID NO: 26. [Figure 22]22 shows the amino acid sequences in the P30B9-IgM heavy and light chain variable region sequences and their comparison with their closest germline. In the top alignment, the first amino acid sequence from the top (P30B9-Vh) is the P30B9-IgM heavy chain variable region sequence without the CDR3 sequence (SEQ ID NO: 29). The heavy chain variable region sequence without the CDR3 is SEQ ID NO: 27. The second amino acid sequence (Homsap IGHV4-34*01F) is SEQ ID NO: 28. The third amino acid sequence is the CDR3 of the P30B9-IgM heavy chain variable region and is SEQ ID NO: 29. In the bottom alignment, the first amino acid sequence from the top (P30B9-Vk) is the P30B9 light chain variable region without the CDR3 sequence (SEQ ID NO: 32). The light chain variable region sequence without the CDR3 is SEQ ID NO: 30. In the bottom alignment, the second amino acid sequence (germline Homsap IGKV1-5*03) is SEQ ID NO: 31. In the bottom alignment, the third sequence is the CDR3 of the P30B9 light chain variable region, SEQ ID NO: 32. [Diagram 23] FIG. 23 is an alignment of amino acid sequences in P95C1-IgM heavy and light chain variable region sequences and their comparison with their closest germline sequences. In the top alignment, the first amino acid sequence from the top (P95C1-VH) is the P95C1 heavy chain variable region sequence without the CDR3 sequence (SEQ ID NO: 18). The heavy chain variable region sequence without the CDR3 is SEQ ID NO: 33. In the top alignment, the second amino acid sequence (germline Homsap IGHV4-4*02) is SEQ ID NO: 34. In the top alignment, the third amino acid sequence is the CDR3 of the P95C1-gM heavy chain variable region and is SEQ ID NO: 18. In the bottom alignment, the first amino acid sequence from the top (P95C1-Vk) is the P95C1 light chain variable region without the CDR3 sequence (SEQ ID NO: 15). The light chain variable region sequence without the CDR3 is SEQ ID NO: 36. In the bottom alignment, the second amino acid sequence (germline Homsap IGKV4-1*01F) is SEQ ID NO: 37. In the bottom alignment, the third sequence is the CDR3 of the P95C1 light chain variable region, SEQ ID NO: 15. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description A.Definition Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] Anti-LAM antibodies, as disclosed herein, may take one of numerous forms in the art. Antibodies are defined in part by the antigen to which they bind, and thus an "anti-LAM antibody" is any antibody that specifically binds to at least one epitope of lipoarabinomannan (LAM), as described herein. It is understood in the art that an antibody is a glycoprotein, or an antigen-binding portion thereof, that includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The variable regions of both the heavy and light chains include framework regions (FWR) and complementarity determining regions (CDRs). While the four FWR regions are relatively conserved, the CDR regions (CDR1, CDR2 and CDR3) represent hypervariable regions and are arranged from the NH2-terminus to the COOH-terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens, while the constant regions, depending on the isotype, may mediate the binding of the immunoglobulin to host tissues or factors.

[0043] Anti-PIM6 / LAM antibodies, as disclosed herein, may take one of numerous forms in the art. An "anti-PIM6 / LAM antibody" is any antibody that specifically binds to at least one epitope shared by phosphatidylinositol mannoside 6 (PIM6) and LAM, as described herein. A human mAb specific for an epitope shared by LAM and PIM6, as described herein, is P95C1, which specifically binds to at least one polymannose structure in PIM6 and within the PIM6-associated mannan domain of LM and LAM. P95C1 binds to both LAM and PIM6, as it recognizes a common (shared) epitope, and is therefore referred to herein as an "anti-PIM6 / LAM antibody" or "anti-PIM6 / LAM monoclonal antibody," "human anti-PIM6 / LAM antibody" or "human anti-PIM6 / LAM monoclonal antibody."

[0044] It is known in the art that monoclonal and other antibodies can be manipulated to produce other antibodies or chimeric molecules which retain the specificity of the original antibody, using techniques of recombinant DNA technology. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or CDRs, of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin.

[0045] The term "antibody" (Ab), as used herein, is used in the broadest sense and may specifically include any immunoglobulin, whether natural or partially or wholly synthetically produced, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Thus, the term "antibody", as used in any context in this application, is meant to include, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE), as well as biologically relevant fragments or specific binding members thereof, including, but not limited to, Fab, F(ab')2, scFv (single chain or related entities) and (scFv)2.

[0046] The term "antibody fragment" as used herein may include antibody fragments obtained using techniques readily understood and available to those skilled in the art, as outlined herein. Thus, the term "antibody" refers to any polypeptide or protein that includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. They may be derived from natural sources or may be partially or wholly synthetically produced. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies, and linear antibodies. In particular, as used herein, a "single-chain Fv" ("sFv" or "scFv") is an antibody fragment that includes a VH and a VL antibody domain connected in a single polypeptide chain. The sFv polypeptide may further include a linker, such as a flexible polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.

[0047] The term "monoclonal antibody" or "mAb" as used herein may refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0048] The terms "variant," "derivative," and / or "variant and / or derivative," as used herein, may refer to antibodies, antibody fragments, recombinant antibodies, and proteins, protein fragments, and polypeptides obtained from natural sources or partially or wholly synthetically produced, so long as the aforementioned compounds are structurally similar, i.e., retain a degree of identity with the original unmodified antibody such that there is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity, and / or may be functionally similar to the original unmodified anti-LAM and anti-PIM6 / LAM antibodies regardless of structural identity, i.e., retain the ability to specifically bind to at least one epitope of LAM or a shared PIM6 / LAM epitope, respectively. For example, such variants and / or derivatives may include anti-LAM or anti-PIM6 / LAM antibodies with variant Fc domains, chimeric antibodies, fusion proteins, bispecific antibodies, or other recombinant antibodies. Such variant and / or derivative antibodies may not necessarily have greater binding specificity for one or more epitopes of LAM, or PIM6, and / or may be capable of binding to additional LAM or PIM6 epitopes.

[0049] The term "biological sample" refers to a sample obtained from an organism (e.g., a patient) or a component (e.g., a cell) of an organism. A sample may be any biological tissue, cell, or bodily fluid. A sample may be a "clinical sample" that is a sample derived from a subject, e.g., a human patient. Such samples include, but are not limited to, saliva, sputum, blood, blood cells (e.g., white cells), amniotic fluid, plasma, semen, bone marrow, and tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells derived therefrom. Biological samples may also include sections of tissue, such as frozen sections taken for histological purposes. Biological samples may also be referred to as "patient samples." Biological samples may also include substantially purified or isolated proteins, membrane preparations, or cell culture fluids.

[0050] The term "effective amount" or "therapeutically effective amount" as used herein may refer to an amount of a compound or agent capable of producing a medically desirable result in a treated subject. Therapeutic methods may be performed in vivo or ex vivo, alone or in combination with other drugs or therapeutic agents. A therapeutically effective amount may be administered in one or more administrations, applications or doses, and is not intended to be limited to a particular formulation or route of administration.

[0051] The term "antigen-binding fragment" or "Fab" as used herein may refer to the region on an antibody that binds to an antigen. One skilled in the art will understand that a Fab consists of one constant domain and one variable domain from each of the heavy and light chains of an antibody.

[0052] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" may refer to the binding of an antibody to an epitope on a given antigen and not to other antigens. Typically, an antibody (i) binds to an epitope of about 10% or more of the antigen, as measured, for example, by surface plasmon resonance (SPR) techniques on a BIACORE® 2000 surface plasmon resonance instrument using a given antigen, e.g., a LAM epitope, as the analyte and the antibody as the ligand, or by Scatchard analysis of the binding of the antibody to antigen-positive cells. -6 Less than M, e.g., about 10-7 Less than M, 10 -8 M, 10 -9 M or 10 -10 M or lower equilibrium dissociation constant (K D ) and (ii) binds to a given antigen with an affinity that is at least two-fold higher than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein).

[0053] The term "conservative sequence modification" or "conservative substitution" as used herein may refer to an amino acid modification that does not significantly affect or alter the binding properties of an antibody having the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is when an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be substituted with other amino acid residues from the same side chain family and the altered antibodies can be tested for retained function using the functional assays described herein.

[0054] The term "identity" as used herein may refer to the presence of a structure shared between two compositions. In the context of a protein, the term "identity" may refer to the amount of overlap (e.g., expressed as a percentage) between two or more amino acid and / or peptide sequences. In the context of a nucleic acid, the term may refer to the amount of overlap (e.g., expressed as a percentage) between two or more nucleic acid sequences. As used herein, the percent (%) identity between two sequences is equal to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (which must be introduced for optimal alignment of the two sequences) (i.e., % identity = number of identical positions / total number of positions x 100). The comparison of sequences and the determination of the percent identity between two sequences may be performed using a mathematical algorithm. Such identity is well described in the art through local alignment tools and / or algorithms, which may include pairwise alignment, multiple sequence alignment, structural alignment, and / or phylogenetic analysis. Specific examples are given below. Percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Further, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, the protein sequences of the present invention can further be used as "query sequences" to perform searches against public databases, e.g., to identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program (score=50, wordlength=3) to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0055] The terms "co-administration," "co-administered," and "in combination with," as used herein, may refer to the administration of at least two agents or therapeutics to a subject. In some embodiments, the co-administration of two or more agents / therapeutics is simultaneous. In other embodiments, a first agent / therapeutic is administered prior to a second agent / therapeutic. One of skill in the art will appreciate that the formulations and / or routes of administration of the various agents / therapeutics used may vary.

[0056] The term "carrier" as used herein may include pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Physiologically acceptable carriers are often aqueous pH buffered solutions. Examples of physiologically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as, but not limited to, ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as, but not limited to, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as, but not limited to, polyvinylpyrrolidone; amino acids such as, but not limited to, glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as, but not limited to, glucose, mannose, or dextrins; chelating agents such as, but not limited to, EDTA; sugar alcohols such as, but not limited to, mannitol or sorbitol; salt-forming counterions such as, but not limited to, sodium; and / or non-ionic surfactants such as, but not limited to, TWEEN; polyethylene glycol (PEG), and PLURONICS.

[0057] The term "treating" or "treatment" of a disease refers to carrying out a protocol that may include administering one or more agents to a patient (human or otherwise) in an effort to alleviate the signs or symptoms of the disease. Relief can occur before the appearance of the signs or symptoms of the disease, as well as after their appearance. Thus, "treating" or "treatment" of a disease includes "preventing" or "prevention" of a disease. The term "prevent" or "preventing" refers to prophylactic and / or preventative treatment, the purpose of which is to prevent or delay the targeted pathological condition or disorder. For example, in the case of infection with virulent strains of the Mycobacterium tuberculosis complex, "preventing" or "prevention" may be performed in the context of an advanced course of treatment to prevent or stop infection with virulent strains of the Mycobacterium tuberculosis complex, for example through vaccination or passive administration of protective antibodies. Such "preventing" or "prevention" may also be performed in the case of latent infection with Mycobacterium tuberculosis, where the aim is to prevent active infection and / or eliminate said latent infection from the patient. Furthermore, "treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and in particular includes protocols that have only a marginal effect on the patient.

[0058] The terms "patient," "subject," and "individual" are used interchangeably herein and may refer to a biological system to which a therapeutic agent may be administered. A biological system may include, for example, an individual cell, a set of cells (e.g., a cell culture), an organ, a tissue, or a multicellular organism. A "patient," "subject," or "individual" may refer to a human patient, subject, or individual or a non-human patient, subject, or individual.

[0059] The term "epitope" as used herein may refer to the region of an antigen to which an antibody or T cell binds. An "antigen" refers to a substance that elicits an immunological response or binds to the products of that response.

[0060] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors capable of driving the expression of a gene to which they are operatively linked are referred to herein as "expression vectors."

[0061] As used herein, "protein" and "polypeptide" are used interchangeably to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, such as glycosylation or phosphorylation.

[0062] The term "labeled," in reference to an antibody, nucleic acid, peptide, polypeptide, cell, or probe, is intended to encompass direct labeling of the antibody, nucleic acid, peptide, polypeptide, cell, or probe by coupling (i.e., physically linking) a detectable substance to the antibody, nucleic acid, peptide, polypeptide, cell, or probe.

[0063] The term "purified" or "isolated" peptide, polypeptide, or protein as used herein refers to a peptide, polypeptide, or protein that is separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide / protein may constitute at least 10% by dry weight of a purified preparation (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%). Purity may be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The isolated polypeptide / protein (e.g., anti-LAM antibody) described in the present invention may be produced by recombinant DNA technology.

[0064] B. Mycobacterium tuberculosis Tuberculosis (TB) remains one of the deadliest infectious diseases in global history, currently infecting approximately one-third of the world's population. In 2013, an estimated 9 million people developed TB and an estimated 15 million died from the disease. Although antibiotic treatments are currently available, these require long-term treatment and have increased susceptibility due to the emergence of multidrug-resistant (MDR-TB) strains, which are currently responsible for approximately 3.5% of recent infections. These strains have become much more difficult to treat and have significantly reduced cure rates. In addition, extensively drug-resistant TB (XDR-TB) strains have spread, which are even more expensive and difficult to treat than MDR-TB strains and have now been reported in 100 countries worldwide.

[0065] There is a long-established paradigm that immunity to TB depends exclusively on cellular defense mechanisms. However, studies in the HIV field emphasize the remarkable ability of the human humoral immune system to produce diverse antibodies with remarkable neutralizing breadth and potency, and the present invention emphasizes the ability of the humoral immune system to produce high affinity antibodies that recognize multiple LAM epitopes. This suggests that many of the past difficulties in demonstrating a key role in antibody-mediated defense against TB may be due to limitations in the quality and source of antibodies used in past studies, and that application of the method of the present invention to generate more highly evolved antibodies from chronically infected human patients may illustrate the key role of humoral responses in immunity to TB.

[0066] Some embodiments of the invention are directed to methods for the in vitro culture of memory B cells from infected humans and the molecular cloning of IgG heavy (H) and light (L) chain variable regions from single cells. These methods may be utilized to produce human monoclonal antibodies against the major surface antigen LAM. The invention relates to such antibodies with unique epitope specificity and binding properties, and engineered derivatives of these antibodies, as well as immunodiagnostic and immunotherapeutic uses of these antibodies.

[0067] C. Lipoarabinomannan (LAM) One prominent antigen target of the antibodies of the present invention is the surface glycolipid lipoarabinomannan (LAM), a major component of the cell wall of Mycobacterium tuberculosis complex members. The present invention identifies previously unappreciated heterogeneity in the antigenic structure of LAM and the humoral immune response to LAM in response to infection and immunity. The structure of LAM is detailed in Khoo et al., "Variation in Mannose-capped Terminal Arabinan Motifs of Lipoarabinomannan from Clinical Isolates of Mycobacterium tuberculosis and Mycobacterium avium Complex," Journal of Biological Chemistry Vol276, No.6, Feb9, 2001 (incorporated herein by reference in its entirety). The structure of LAM is a complex that exhibits four distinct structural domains; a phosphatidylinositol lipid anchor (mannosyl-phosphatidyl-myo-inositol), an α(1→6)-linked D-mannan backbone with terminal α(1→2)-Manp-linked side chains, a D-arabinan chain with multiple tetra- / hexa-arabinofuranoside branches, and an overall tripartite structure with various capping motifs. LAM consists of a heterogeneous population of molecules that can be resolved into multiple isoforms with distinct biological properties. This heterogeneity is due to mannan and arabino chains of various lengths, different branching patterns, various numbers of such branches, and modification of the arabino side chains by mannose capping, MTX addition, and acylation with fatty acids, succinic acid and lactic acid.

[0068] Virulent strains of the Mycobacterium tuberculosis complex are extensively capped with mono-, di-, and tri-α(1→2)-D-Manp sugar units, whereas rapidly growing non-pathogenic strains such as M. smegmatis have uncapped ends or phosphatidyl-myo-inositol caps (PILAMs). It has been estimated that 40–70% of the non-reducing ends of LAMs from pathogenic strains of the Mycobacterium tuberculosis complex are capped with mannose, and analysis of the relative abundance of different cap motifs in the virulent MT103 clinical strain showed that dimannosyl units are the predominant structural motif (75–80%), while mannosyl and trimannosyl motifs are present in lower concentrations (10–13%). This extensive capping may present a unique marker in differentiating virulent strains of the Mycobacterium tuberculosis complex from non-virulent / non-pathogenic strains such as M. smegmatis, and may further provide a promising antigenic target for therapeutic use of the anti-LAM antibodies of the present invention. Furthermore, some of the terminal mannose sugars in ManLAM found in M. tuberculosis strains are further modified by the α(1→4) addition of the unique structure 5-deoxy-5-methyl-thio-pentofuranose (MTX), which affects the immunoreactivity to different mAbs sensitive to the capping motif, such as A194-01 and P30B9; MTX addition enhances the reactivity with A194-01 and reduces the reactivity to P30B9. This substitution is present at low abundance and may represent a unique marker for identifying M. tuberculosis and potentially from other virulent members of the Mycobacterium tuberculosis complex, such as M. bovis and M. africanum, and may further provide a promising antigenic target for therapeutic use of the anti-LAM antibodies of the present invention.

[0069] The secreted form of LAM is an important target in immunodiagnostic assays for infection with virulent members of the M. tuberculosis complex. Furthermore, a large body of evidence indicates that LAM is a key mediator of several functions that promote productive infection and virulence. LAM is involved in maintaining cell wall integrity and resistance to β-lactam antibiotics. Reduced expression of LAM on the bacterial surface correlates with defective macrophage invasion, inhibition of phagosome-lysosome fusion, attenuation in macrophages, and enhanced susceptibility to adaptive immunity, and binding of the terminal mannosyl units of ManLAM to mannose receptors on the macrophage surface has been described as a key step in the phagocyte uptake of mycobacteria. Without wishing to be bound by theory, it is believed that ManLAM interacts with C-type lectins on dendritic cells, such as dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3), predatory nonintegrin (DC-SIGN), macrophage mannose receptor (MMR), and dectin-2. Once inside the macrophage, LAM is thought to inhibit phagosome-lysosome fusion, which leads to the destruction of the bacteria, thereby allowing the bacteria to persist inside the macrophage.

[0070] LAM is also secreted from the surface of bacteria, and extracellular LAM binds to dendritic cell-surface receptors, including DC-SIGN and Dectin-2. These interactions are thought to suppress dendritic cell function and interfere with the host immune system, thereby contributing to immune evasion. Since LAM is present in relatively large amounts during active infection, it can be detected in the blood and urine of infected patients, for example, by one or more anti-LAM antibodies of the invention. These may be used, for example, in diagnostic kits and methods related to said diagnostic kits.

[0071] D. Anti-LAM and anti-PIM6 / LAM antibodies The anti-LAM antibodies of the invention may include isolated, cultured, or engineered variants and / or derivatives of human monoclonal antibodies that recognize at least one epitope on lipoarabinomannan (LAM). Anti-PIM6 / LAM antibodies as described herein (e.g., P95C1) specifically bind to at least one polymannose structure in PIM6 and within the PIM6 cross-reactive mannan domain of LAM. Anti-LAM and anti-PIM6 / LAM antibodies of the invention may be purified according to methods known in the art. Such methods may include, for example, but are not limited to, affinity chromatography, ion exchange chromatography, immobilized metal chelate chromatography, thiophilic adsorption, physiochemical fractionation, or other antigen-specific affinity methods, including, for example, protein A, G, and L antibody binding ligands. Such purified antibodies may or may not have different structural characteristics than unpurified human monoclonal antibodies. For example, during purification, changes in conformational epitopes in human monoclonal antibodies may occur. The antibodies may be bound to additional molecules that are removed during purification. Thus, such purified antibodies may or may not have different functional activities. The anti-LAM and anti-PIM6 / LAM antibodies of the present invention may have some structural modifications. For example, the anti-LAM and anti-PIM6 / LAM antibodies of the present invention may be chemically modified, such as glycosylated, PEGylated, or otherwise, in a manner that affects stability, function, bioavailability, epitope recognition, or other functional activity. The anti-LAM and anti-PIM6 / LAM antibodies of the present invention may be modified variants and / or derivatives of the antibodies described below, which may or may not have functional or structural equivalents. Thus, such variants and / or derivatives are further contemplated within the scope of the present invention, as long as they are derived or modified, at least in part, from an isolated human monoclonal anti-LAM or anti-PIM6 / LAM antibody and / or recognize at least one epitope on LAM.

[0072] 1.A194-01 In some embodiments, the present invention is directed to human monoclonal antibody A194-01, such as its variants and / or derivatives. A194-01 is specific to LAM. A194-01 has very high binding activity to LAM, for example, the IgG isotype of A194-01 shows 50% of the maximum binding activity of the antibody at a concentration of about 20ng / ml, and therefore can show high affinity to LAM. Although A194-01 was initially isolated and purified as an IgG, A194-01 can exist as several isotypes, as well as modified and recombinant isotypes, including but not limited to IgG, IgA, IgM, monovalent single-chain Fv (scFv) fragments, Fab proteins, bivalent scFv fragments, single-chain scFv fragments (monomers), and dimeric scFv proteins in which two scFv monomers are linked together, where the individual variable light and variable heavy regions are linked, for example, by a flexible linker (Figure 1A). Some specific modified variants and / or derivatives of A194-01 include, but are not limited to, the following. One modified variant and / or derivative of A194-01 includes tetravalent scFv-IgG formed by linking A194-01 scFv antigen to the N-terminus of A194-01 IgG (FIG. 1B, FIG. 17), which can increase binding affinity and expand the range of epitopes recognized (examples of this are shown in FIG. 14 and 15). The tetravalent scFv-IgG may include leader-VH-VL-IgG or leader-VL-VH-IgG. Those skilled in the art will understand that modified scFv-IgG variants and / or derivatives may have more than just tetravalency. Another engineered variant and / or derivative of A194-01 includes a pentavalent IgM created by converting a dimeric A194-01 IgG to a human IgM-containing domain, such that the pentavalent IgM contains 10 binding sites (FIG. 1B). Those skilled in the art will appreciate that further combinations of A194-01 antigenic fragments, particularly antibody fragments exhibiting complementarity determining regions (CDRs) specific for A194-01, are possible and are contemplated within the scope of the present invention.

[0073] The IgG isotype of A194-01 recognizes a unique composite epitope expressed in unmodified Ara4 and Ara6 side chains as well as in side chains with a single mannose. Although A194-01 does not recognize side chains with di- or trimannose substitutions, A194-01 does react with such structures when they are further modified with MSX substituents. Thus, the IgG isotype of A194-01 binds with high affinity to PILAM and ManLAM, and thus binds strongly to uncapped versions of both Ara4 and Ara6 structures, somewhat less strongly to MSX-substituted Ara4 / Ara6 structures capped with a single mannose, but weakly, if at all, to di- and tri-substituted ManLAM (Figure 4). Without wishing to be bound by theory, the markedly different effects of mannose versus MSX attachment to the terminal mannose of monomannosylated Ara4 structures may reflect the difference between the α(1→2) linkage of mannose and the α(1→4) linkage of MSX substitution. Engineered variants and / or derivatives of A194-01, such as those with higher binding valency, may exhibit broader epitope specificity than A194-01 IgG isotype (Figure 14) and may further exhibit enhanced affinity for LAM (Figure 15). For example, tetravalent scFv-IgG modified A194-01 and modified IgA and IgM isotypes not only bind both Ara4 and Ara6 structures with higher affinity than A194-01 IgG isotype, but also recognize dimannose- and trimannose-capped structures, which IgG isotypes bind weakly (Figure 14). Since pathogenic species of the Mycobacterium tuberculosis complex mainly exhibit dimannose-capped structures, these engineered variants and / or derivatives of A194-01, such as scFv-IgG and IgM isotypes, may prove to be particularly useful for diagnostic kits and methods, as well as therapeutic uses.

[0074] Further modified variants and / or derivatives of A194-01 include antibodies in which the IgG1 Fc domain has been converted to a more opsogenic IgG3, i.e., by creating a multimeric version, by replacing the IgG1 constant domain with dimeric IgA or pentameric or hexameric IgM. Without wishing to be bound by theory, this may significantly enhance the avidity of anti-LAM antibodies by increasing the mobility and range of bivalent and multivalent binding that contributes to affinity (Figure 1). This is of potential clinical importance as the treatment would be particularly useful in cases of exposure or infection with MDR or X-MDR strains of Mycobacterium tuberculosis, which cannot be effectively treated with conventional antibiotics.

[0075] Table 1. Complementarity determining regions (CDRs) of A194-01 Light chain CDR1-RSIRSA (SEQ ID NO: 1) CDR2-GAS (SEQ ID NO:2) CDR3 - QQYDFWYTF (SEQ ID NO: 3) Heavy chain CDR1 - GFNFEDFG (SEQ ID NO: 4) CDR2-ISWNGANI (SEQ ID NO:5) CDR3-IDWYRDDYYKMDV (SEQ ID NO: 6)

[0076] Those skilled in the art will understand that CDRs are important for diversity of antigen specificity. Those skilled in the art will further understand that CDR3 is the most variable of the CDR regions and therefore has the greatest importance, and diversity in the CDR3 region of the variable heavy chain is sufficient for most antibody specificity. Thus, in some embodiments, anti-LAM antibodies have variable light chain CDR1, CDR2, and CDR3 regions (shown in SEQ ID NOs: 1, 2, and 3, respectively). In some embodiments, anti-LAM antibodies have variable light chain CDR1, CDR2, and CDR3 regions (shown in SEQ ID NOs: 1, 2, and 3, respectively) with conservative sequence modifications. In some embodiments, anti-LAM antibodies have variable light chain CDR1, CDR2, and CDR3 regions with up to 95% identity with SEQ ID NOs: 1, 2, and 3, respectively. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region with up to 90% identity to each of SEQ ID NOs: 1, 2, and 3. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region with up to 85% identity to each of SEQ ID NOs: 1, 2, and 3. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region with up to 80% identity to each of SEQ ID NOs: 1, 2, and 3. In some embodiments, the anti-LAM antibody has a variable light chain CDR3 region as shown in SEQ ID NO: 3. In some embodiments, the anti-LAM antibody has a variable light chain CDR3 region as shown in SEQ ID NO: 3 with conservative sequence modifications. In other embodiments, the anti-LAM antibody has a variable light chain CDR3 region with up to 95% identity to SEQ ID NO: 3. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 90% identity to SEQ ID NO: 3. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 85% identity to SEQ ID NO: 3. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 80% identity to SEQ ID NO: 3.

[0077] In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions as shown in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions as shown in SEQ ID NOs: 4, 5, and 6, respectively, with conservative sequence modifications. In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 95% identity with SEQ ID NOs: 4, 5, and 6, respectively. In other embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 90% identity with SEQ ID NOs: 4, 5, and 6, respectively. In other embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 85% identity with SEQ ID NOs: 4, 5, and 6, respectively. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR1, CDR2, and CDR3 region with up to 80% identity to each of SEQ ID NOs: 4, 5, and 6. In some embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region as shown in SEQ ID NO: 6. In some embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region as shown in SEQ ID NO: 6 with conservative sequence modifications. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 95% identity to SEQ ID NO: 6. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 90% identity to SEQ ID NO: 6. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 85% identity to SEQ ID NO: 6. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 80% identity to SEQ ID NO: 6.

[0078] In the experiments described herein, A194-01 antibody was expressed by transfection of H and L chain vectors into Expi293 cells and cultured in standard Expi293 serum-free medium for several days. Secreted antibody was purified from culture supernatant by affinity chromatography on columns conjugated with protein A or protein G ligands. Bound antibody was released from the ligand by treatment with low pH buffer (0.2 M glycine-HCl, pH 2.5) and neutralized with 1 / 50 volume of 2 M Tris buffer (pH 8.8). The buffer was exchanged with PBS by dialysis or by several rounds of concentration with centrifugal filters (Amicon Ultra centrifugal filters, 30K mw limit).

[0079] The amino acid (aa) and nucleic acid (nt) sequences for the A194 heavy and light chain sequences are as follows: A194 heavy chain nt sequence: TIFF2024174883000002.tif126170A194 Heavy chain aa sequence: TIFF2024174883000003.tif49170A194 Light chain nt sequence (kappa): TIFF2024174883000004.tif68170A194 Light chain aa sequence (κ): TIFF2024174883000005.tif25170

[0080] 2.P30B9 In some embodiments, the present invention is directed to recombinant human monoclonal antibody P30B9, including its variants and / or derivatives. P30B9 is specific for LAM. Although P30B9 was initially isolated and purified as an IgM, P30B9 may exist in several isotypes, as well as modified recombinant isotypes, including but not limited to IgM, IgG, IgA, and antigenic fragments thereof, including but not limited to monovalent single chain Fv (scFv) fragments, Fab proteins, bivalent scFv fragments, single chain scFv fragments (monomers) (separate variable light and variable heavy regions are linked, for example, by a flexible linker), and dimeric scFv proteins in which two scFv monomers are linked to each other.

[0081] The IgM isotype of P30B9 binds most avidly to dimannose-substituted Ara4 and Ara6 LAM epitopes with the Manp-α(1→2)-Manp-(1→5)-Araf structure (Figures 4, 16 and 18), but can also recognize other Manp-α-substituted structures (e.g., structures 2, 4 and 59 in Figure 8) with lower affinity. The preferential recognition of P30B9 to dimannose-capped LAM has potential clinical implications, since the dimannose cap has been reported to be the dominant LAM modification for virulent strains of the Mycobacterium tuberculosis complex. Without wishing to be bound by theory, it is believed that the terminal mannosyl units mediate the binding of LAM from virulent strains of the Mycobacterium tuberculosis complex to human macrophages and other immune cells, resulting in the perturbation of immune function and the establishment of stable infection. Without wishing to be bound by theory, it is believed that binding of the mannose cap to the mannose receptor limits phagosome-lysosome (PL) fusion and promotes bacterial survival in infected macrophages. The specificity of P30B9 for dimannose-capped LAM is demonstrated by the specificity of this mAb for glycoconjugates with this structure and in the fact that the IgM isotype of P30B9 specifically binds to LAM from either Mycobacterium tuberculosis but not to LAM from Mycobacterium smegmatis or Mycobacterium leprae, which do not have the dimannose-capped LAM epitope. This is in contrast to the IgG isotype of A194-01, which binds to PILAM, uncapped Ara4 / Ara6 residues, and monomannose-capped LAM epitopes, all of which are shared in Mycobacterium smegmatis and Mycobacterium leprae.Similar to the IgM isotype of P30B9, the IgM isotype of A194-01 can bind to dimannose- and trimannose-capped LAM epitopes, likely due to enhanced avidity (Figure 14).

[0082] Thus, the IgM isotype of P30B9 may serve as an important immunodiagnostic reagent for detecting infection with virulent members of the Mycobacterium tuberculosis complex and distinguishing said virulent members from other non-pathogenic mycobacterial species because it is specific for dimannose-capped LAM. Furthermore, the IgM isotype of the P30B9 antibody as well as engineered variants and / or derivatives of A194-01 may have immunotherapeutic activity to limit infection and pathogenesis of virulent members of the Mycobacterium tuberculosis complex, may be suitable for use in treatment, either in combination with traditional antibiotics, additional antibodies or alone, or may be used as passive immunotherapeutic agents. The IgM isotype of P30B9 specifically binds with high affinity to ManLAM derived from Mycobacterium tuberculosis (Fig. 2A, B), but not to PILAM derived from Mycobacterium smegmatis (Fig. 2C).

[0083] The modified variants and / or derivatives of P30B9 may include, for example, P30B9 expressed in IgA isotypes, including dimeric IgA1 and IgA2. While not wishing to be bound by theory, it is believed that multivalency is required for the function of P30B9, as this antibody is isolated as an IgM and shows no activity when expressed as an IgG. The present invention shows that P30B9 is active in modified IgA isotypes, including dimeric IgA1 and IgA2. This was tested by transferring the P30B9 VH domain into IgA1 and IgA2 vectors. IgA1 differs from IgA2 by the presence of a 16 amino acid insertion consisting of eight amino acid repeats enriched in proline, serine, and threonine, and modified with 3-6 O-linked oligosaccharides [Figure 5]. The binding activity of the modified IgA form of P30B9 to ManLAM was compared to that of the IgG and IgM forms. While the IgM form had the highest activity, both IgA forms were able to bind ManLAM, the IgA2 form showed weaker activity than the IgA1 form, and the IgG form was inactive in ELISA against ManLAM (Figure 6).

[0084] Table 2. Complementarity determining regions (CDRs) of P30B9 Light chain CDR1 - QSINSN (SEQ ID NO: 7) CDR2-KAS (SEQ ID NO: 8) CDR3 - QQYKAFKTF (SEQ ID NO: 9) Heavy chain CDR1-GGSFSGYY (SEQ ID NO: 10) CDR2-FDLGGSITHSRGT (SEQ ID NO: 11) CDR3-RGLAMGGTKEFDS (SEQ ID NO: 12)

[0085] Those skilled in the art will understand that CDRs are important for diversity of antigen specificity. Those skilled in the art will further understand that CDR3 is the most variable of the CDR regions and therefore has the greatest importance, and that diversity in the CDR3 region of the variable heavy chain is sufficient for most antibody specificity. Thus, in some embodiments, anti-LAM antibodies have CDR1, CDR2, and CDR3 regions of the variable light chain (shown in SEQ ID NOs: 7, 8, and 9, respectively). In some embodiments, anti-LAM antibodies have CDR1, CDR2, and CDR3 regions of the variable light chain (shown in SEQ ID NOs: 7, 8, and 9, respectively) with conservative sequence modifications. In some embodiments, anti-LAM antibodies have CDR1, CDR2, and CDR3 regions of the variable light chain (shown in SEQ ID NOs: 7, 8, and 9, respectively) with conservative sequence modifications. Each of In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region that has up to 95% identity with each of SEQ ID NOs: 7, 8, and 9. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region that has up to 90% identity with each of SEQ ID NOs: 7, 8, and 9. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region that has up to 85% identity with each of SEQ ID NOs: 7, 8, and 9. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region that has up to 80% identity with each of SEQ ID NOs: 7, 8, and 9. In some embodiments, the anti-LAM antibody has a variable light chain CDR3 region as set forth in SEQ ID NO: 9. In some embodiments, the anti-LAM antibody has a variable light chain CDR3 region as set forth in SEQ ID NO: 9 with conservative sequence modifications. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 95% identity to SEQ ID NO: 9. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 90% identity to SEQ ID NO: 9. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 85% identity to SEQ ID NO: 9. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 80% identity to SEQ ID NO: 9.

[0086] In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions as shown in each of SEQ ID NOs: 10, 11, and 12. In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions as shown in each of SEQ ID NOs: 10, 11, and 12 with conservative sequence modifications. In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 95% identity with each of SEQ ID NOs: 10, 11, and 12. In other embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 90% identity with each of SEQ ID NOs: 10, 11, and 12. In other embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 85% identity with each of SEQ ID NOs: 10, 11, and 12. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR1, CDR2, and CDR3 region with up to 80% identity to each of SEQ ID NOs: 10, 11, and 12. In some embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region as set forth in SEQ ID NO: 12. In some embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region as set forth in SEQ ID NO: 12 with conservative sequence modifications. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 95% identity to SEQ ID NO: 12. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 90% identity to SEQ ID NO: 12. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 85% identity to SEQ ID NO: 12. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 80% identity to SEQ ID NO: 12.

[0087] In the experiments described herein, P30B9 antibody was expressed by transfection of H and L chain vectors into Expi293 cells and cultured in standard Expi293 serum-free medium for several days. Secreted antibody was purified from culture supernatant by affinity chromatography on a column conjugated with protein L ligand. Bound antibody was released from the ligand by treatment with low pH buffer (0.2 M glycine-HCl, pH 2.5) and neutralized with 1 / 50 volume of 2 M Tris buffer (pH 8.8). The buffer was exchanged with PBS by dialysis or by several rounds of concentration with centrifugal filters (Amicon Ultra centrifugal filters, 30K mw limit).

[0088] The amino acid sequences for the P30B9 heavy and light chain sequences and their comparison to their closest germlines are shown in Figure 22. The amino acid and nucleotide sequences for P30B9, including the CDR3 region, are reproduced below. P30B9-heavy chain variable region: TIFF2024174883000006.tif26170P30B9-Light chain variable region: TIFF2024174883000007.tif20170P30B9-heavy chain DNA sequence: TIFF2024174883000008.tif50170P30B9-Light Chain: TIFF2024174883000009.tif41170

[0089] 3.P95C1 In some embodiments, the present invention is directed to recombinant human monoclonal antibody P95C1, such as its variants and / or derivatives. P95C1 is specific for an epitope shared by LAM, LM and PIM6. Although P95C1 was initially isolated and purified as IgM, P95C1 is active when expressed in other isotypes, including but not limited to IgG and IgA forms.

[0090] Those skilled in the art will understand that CDRs are important for diversity of antigen specificity. Those skilled in the art will further understand that CDR3 is the most variable of the CDR regions and therefore has the greatest importance, and diversity in the CDR3 region of the variable heavy chain is sufficient for most antibody specificity. Thus, in some embodiments, anti-LAM antibodies have variable light chain CDR1, CDR2, and CDR3 regions (represented by SEQ ID NOs: 13, 14, and 15, respectively). In some embodiments, anti-LAM antibodies have variable light chain CDR1, CDR2, and CDR3 regions (represented by SEQ ID NOs: 13, 14, and 15, respectively) with conservative sequence modifications. In some embodiments, anti-LAM antibodies have variable light chain CDR1, CDR2, and CDR3 regions with up to 95% identity with each of SEQ ID NOs: 13, 14, and 15, respectively. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region with up to 90% identity with each of SEQ ID NOs: 13, 14, and 15. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region with up to 85% identity with each of SEQ ID NOs: 13, 14, and 15. In other embodiments, the anti-LAM antibody has a variable light chain CDR1, CDR2, and CDR3 region with up to 80% identity with each of SEQ ID NOs: 13, 14, and 15. In some embodiments, the anti-LAM antibody has a variable light chain CDR3 region as shown in SEQ ID NO: 15. In some embodiments, the anti-LAM antibody has a variable light chain CDR3 region as shown in SEQ ID NO: 15 with conservative sequence modifications. In other embodiments, the anti-LAM antibody has a variable light chain CDR3 region with up to 95% identity with SEQ ID NO: 15. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 90% identity to SEQ ID NO: 15. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 85% identity to SEQ ID NO: 15. In other embodiments, the anti-LAM antibody has a CDR3 region of the variable light chain that has up to 80% identity to SEQ ID NO: 15.

[0091] In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions as shown in each of SEQ ID NOs: 16, 17, and 18. In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions as shown in each of SEQ ID NOs: 16, 17, and 18 with conservative sequence modifications. In some embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 95% identity with each of SEQ ID NOs: 16, 17, and 18. In other embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 90% identity with each of SEQ ID NOs: 16, 17, and 18. In other embodiments, the anti-LAM antibody has variable heavy chain CDR1, CDR2, and CDR3 regions with up to 85% identity with each of SEQ ID NOs: 16, 17, and 18. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR1, CDR2, and CDR3 region with up to 80% identity to each of SEQ ID NOs: 16, 17, and 18. In some embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region as set forth in SEQ ID NO: 18. In some embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region as set forth in SEQ ID NO: 18 with conservative sequence modifications. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 95% identity to SEQ ID NO: 18. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 90% identity to SEQ ID NO: 18. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 85% identity to SEQ ID NO: 18. In other embodiments, the anti-LAM antibody has a variable heavy chain CDR3 region with up to 80% identity to SEQ ID NO: 18.

[0092] Table 3. Complementarity determining regions (CDRs) of P95C1 Light chain CDR1: QNVLDSANNRNY (SEQ ID NO: 13) CDR2: WAS (SEQ ID NO: 14) CDR3: TQYHRLPHT (SEQ ID NO: 15) Heavy chain CDR1: GGSINTNNW (SEQ ID NO: 16) CDR2: IHRHGDT (SEQ ID NO: 17) CDR3: CPLGYCSGDDCHRVA (SEQ ID NO: 18)

[0093] P95C1 IgM / κ antibodies were first identified in the supernatants of BCL6 / Bcl-xL-transduced memory B cells and cloned from these cells into an IgM / κ expression vector using standard RT-PCR protocols. Antibodies were expressed by transfection of H and L chain vectors into Expi293 cells and cultured in standard Expi293 serum-free medium for several days. Secreted antibodies were purified from the culture supernatants by affinity chromatography on a column conjugated with protein L ligand. Bound antibodies were released from the ligand by treatment with a low pH buffer (0.2 M glycine-HCl, pH 2.5) and neutralized with 1 / 50 volume of 2 M Tris buffer (pH 8.8). The buffer was exchanged with PBS by dialysis or by several rounds of concentration in a centrifugal filter (Amicon Ultra centrifugal filter, 30K mw limit).

[0094] The amino acid sequences for the P95C1 heavy and light chains and their comparison with their closest germline are shown in Figure 23. The amino acid and nucleotide sequences for P95C1, including the CDR3 region, are reproduced below. P95C1-heavy chain variable region: TIFF2024174883000010.tif31170P95C1-Light chain variable region: TIFF2024174883000011.tif24170P95C1-heavy chain: TIFF2024174883000012.tif45170P95C1-Light Chain: TIFF2024174883000013.tif45170

[0095] E. Further variants and / or derivatives Considering the CDR regions of A194-01, P30B9, and P95C1, those skilled in the art will understand that numerous modified variants and / or derivatives of the anti-LAM antibodies disclosed herein can be constructed.For example, the anti-LAM antibodies of the present invention can be modified into chimeric, humanized, and chimeric / humanized antibodies that exhibit affinity for one or more LAM epitopes.Antibodies can also be modified into bispecific antibodies, or a single antibody construct can be modified to bind to multiple LAM epitopes.

[0096] As described herein, the anti-LAM antibodies of the present invention may be designed as a homologous scFv-IgG construct or a heterologous scFv-IgG construct. The homologous scFv-IgG construct of A194-01 is detailed in the present application (Figure 17A). One non-limiting example of a heterologous scFv-IgG construct would be when the VH and VL chains of P30B9 are linked to the A194-01 IgG by a linker (Figure 17B), although other VH / VL chains can be used, such as other anti-LAM antibodies, such as mouse anti-LAM antibodies. This may allow recognition of different epitopes in a single antigen molecule, enhancing multivalent binding and resulting in increased affinity. Alternatively, the heterologous scFv-IgG construct may result in a bispecific antibody when the additional VH / VL chains target antigens other than LAM.

[0097] The anti-LAM antibodies of the present invention may also be modified to create scFv-IgM constructs, including both homologous and heterologous scFv-IgM constructs. A non-limiting example of a homologous scFv-IgM would be where the P30B9 VH / VL chains are linked to P30B9 IgM. In this construct, all binding sites would have the same epitope specificity. A non-limiting example of a heterologous scFv-IgM construct would be where the A194-01 scFv is linked to P30B9 IgM, as opposed to the IgG constant domain [non-limiting [Figure 17C]. Such modified mutant and / or derivative constructs would retain the IgM-dependent recognition of the dimannose epitope of the parent P30B9 mAb, and add the additional binding specificity of the A194-01 scFv. This would allow recognition of a unique epitope array, which could result in increased affinity, which may be useful for improving point-of-care antigen detection assays.

[0098] F. Diagnostic Kits and Methods One embodiment of the present invention relates to diagnostic kits and methods for the detection and / or quantification of LAM and / or PIM6 in a sample. As described herein, anti-LAM antibodies A194-01 and P30B9, as well as anti-PIM6 / LAM antibody P95C1, such as modified variants and / or derivatives thereof, can be effective in detecting and / or quantifying the amount of LAM and / or PIM6 present in a sample. LAM or PIM6 can be from any source, such as from Mycobacterium tuberculosis or Mycobacterium smegmatis, or from serum or urine samples from patients, such as patients infected with virulent strains of the Mycobacterium tuberculosis complex. LAM can be, for example, PILAM, ManLAM, or uncapped / unmodified AraLAM from other mycobacterial strains, such as M. leprae. These strains differ in the nature and extent of capping that occurs, whereby different antibody combinations will have different specificities for the different forms, allowing some level of differentiation or typing. In particular, the IgM and modified IgA1 isotypes of P30B9, as well as the modified IgM and scFv-IgG isotypes of A194-01, will be well suited to detect and / or quantify dimannose-substituted ManLAM in samples from TB patients, which may in some circumstances comprise 80% of said LAM, and the various isotypes of P30B9 will be particularly useful in detecting and / or quantifying LAMs with dimannose-substituted Ara6 residues, as described herein, particularly prevalent on LAMs from Mycobacterium tuberculosis. As the P95C1 epitope is highly conserved in all species of LAM, this antibody will be suitable for detecting and / or quantifying various types of LAM in a sample when coupled with a secondary antibody of appropriate specificity.The IgG isotype of A194-01 binds very effectively to various forms of LAM, especially unsubstituted LAM, monomannosylated LAM, and PILAM, making it useful in detecting and / or quantifying LAM from various strains of mycobacteria. The modified IgM and scFv-IgG isotypes are also quite effective in detecting and / or quantifying the amount of unsubstituted LAM, monomannosylated LAM, and PILAM, and can additionally bind di- and trimannose-substituted LAM. This confers on the modified variants and / or derivatives of A194-01 greater epitope recognition than the IgG isotype of A194-01 or the IgM isotype of P30B9, but at the expense of specificity for only LAM epitopes specific to virulent strains of Mycobacterium tuberculosis. In some embodiments, quantification of the specificity for LAM and / or PIM6 is achieved by comparing the signal intensities of serially diluted control samples with known concentrations of LAM and / or PIM6 in various direct binding or antigen capture assays.

[0099] Since the IgG isotype of A194-01, the IgM / IgA isotype of P30B9, and the various isotypes of P95C1 bind to different LAM epitopes that are variably expressed in different strains of Mycobacterium tuberculosis, these particular isotypes can be used to distinguish the origin of the LAM source; dimannose-substituted LAMs, especially the dimannose-substituted Ara6 residues, comprise the majority of LAM residues in virulent strains of Mycobacterium tuberculosis, while the unsubstituted LAM / PILAM residues comprise the majority of LAM residues in rapidly growing, non-virulent strains such as Mycobacterium smegmatis. For example, a sample containing LAM that binds only A194-01 IgG and not P30B9 IgM is likely to have originated from a virulent strain of Mycobacterium tuberculosis, while a sample that binds both P30B0 IgM and A194-01 IgG is likely to have originated from a virulent strain of Mycobacterium tuberculosis or a mycobacterial species that introduces a similar capping motif.

[0100] Since the IgM / IgA isotype of P30B9 is specific for dimannose-substituted ManLAM, which is the dominant form in virulent strains of Mycobacterium tuberculosis as detailed herein, said isotype of P30B9 is an ideal candidate for diagnostic kits and methods used to diagnose patients as infected with virulent strains of Mycobacterium tuberculosis complex. Furthermore, modified IgM and scFv-IgG variants and / or derivatives of A194-01 may be suitable for use as they also recognize dimannose-substituted ManLAM epitopes and trimannose-substituted ManLAM epitopes. Such patients may have a progressive or active infection, or the infection may be latent. The strain may be multi-drug resistant (MDR) or extensively drug resistant (XDR). Specifically for patients with latent infections, changes in serum or urinary LAM concentrations may be particularly important, as an increase in concentration may indicate a change to active infection. Alternatively, a decrease in concentration in an individual with active infection may indicate that treatment is effective and should be continued, or an increase in concentration during treatment may indicate that the current treatment is not effective and should be eliminated, changed and / or modified.

[0101] A method for diagnosing an infection may comprise contacting a biological sample from said patient, such as blood, plasma, urine, sputum or other body fluid, with at least one anti-LAM antibody and / or at least one anti-PIM6 / LAM antibody of the invention, in particular an anti-LAM antibody recognizing a dimannose-substituted ManLAM and an anti-PIM6 / LAM antibody recognizing at least one polymannose structure in the PIM6 mannan domain, including, for example, the IgM and IgA isotypes of P30B9, the modified IgA, IgM and scFv-IgG isotypes of A194-01, and the various isotypes (IgG, IgM, IgA) of P95C1.

[0102] The antibody used as the detection reagent may be conjugated to a reporter molecule, such as those known in the art. The antibody may be part of a kit, for example conjugated to a substrate or part of a sandwich assay. The kit may include a first anti-LAM or anti-PIM6 / LAM capture antibody, a second anti-LAM or anti-PIM6 / LAM detector (detection) antibody that is conjugated to a reporter molecule, and a support to which the capture anti-LAM or anti-PIM6 / LAM antibody binds. The first and second anti-LAM antibodies may bind to the same LAM epitope in multiple copies on a single LAM molecule, or preferably, they may bind to different epitopes present on a single LAM molecule. The LAM and PIM6 epitopes may be any of those described herein. The kit may include a third capture or detector (detection) antibody that binds to a non-competing site on the first and second antibodies. This may increase the number of molecules captured and the number of detector molecules bound and the intensity of the corresponding signal.

[0103] The kit may include instructions for use and may further include various reagents, solvents, diluents, and / or pharma- ceutically acceptable preservatives. Such an assay was performed for the sensitivity of different biotinylated anti-LAM monoclonal antibodies [Figure 7]. In this assay, mouse anti-LAM antibody CS-35 was used to capture ManLAM from solution. This antibody was chosen for its broad specificity. CS-35 (250 ng / well) was used to capture ManLAM from solutions with different concentrations, and then different biotinylated monoclonal antibodies were used to probe for the presence of ManLAM in the capture wells. Using a cutoff of 3xSD of background, the most sensitive probe was A194-01 IgM, which gave a strong signal (1.8 OD) for the highest dilution of ManLAM (0.016 ng / well). This was superior to the two FIND mouse antibodies previously considered to be the most useful probes in this type of assay.

[0104] G. Therapeutic Compositions, Methods, Vaccines, and Vectors One embodiment of the present invention is directed to pharmaceutical compositions comprising at least one anti-LAM or anti-PIM6 / LAM antibody of the present invention, and methods of their use in treating patients in need thereof. The patient may have a latent or active infection with a virulent strain of Mycobacterium tuberculosis, and particularly usefully, the strain may be multi-drug resistant (MDR) or extensively drug resistant (XDR) to traditional treatments / antibiotics. The anti-LAM and anti-PIM6 / LAM antibodies utilized in these compositions and methods may be any anti-LAM or anti-PIM6 / LAM antibody of the present invention, but particularly usefully, may be an anti-LAM antibody that recognizes various isotypes of dimannose-capped ManLAM, particularly dimannose-capped Ara6 residues, such as P30B9 IgM or IgA1 / IgA2 isotypes and pentavalent A194-01 IgM or tetravalent scFv-IgG isotypes and P95C1.

[0105] A pharma- ceutically acceptable anti-LAM and / or anti-PIM6 / LAM antibody composition suitable for administration to a patient will contain an effective amount of anti-LAM or anti-PIM6 / LAM antibody or antibody in a formulation that retains biological activity and promotes maximum stability during storage within an acceptable temperature range. The pharmaceutical composition may also include a pharma- ceutically acceptable diluent, a pharma- ceutically acceptable carrier and / or a pharma- ceutically acceptable excipient, or any solvent as is commonly used to formulate pharmaceutical compositions for animal or human administration, depending on the formulation desired. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution, and Hank's solution. The amount of excipient useful in the pharmaceutical composition or formulation of the present invention is an amount that serves to distribute the antibody evenly throughout the composition so that it can be evenly dispersed when it needs to be delivered to a subject in need of it. It may serve to dilute the antibody to a concentration that provides the desired beneficial symptomatic or curative results while minimizing any adverse side effects that may result from too high a concentration. It may also have a preservative effect. Thus, in an antibody with high physiological activity, more of the excipient will be utilized, whereas in any active ingredient exhibiting lower physiological activity, less of the excipient will be utilized.

[0106] Pharmaceutically acceptable anti-LAM antibody and / or anti-PIM6 / LAM antibody compositions may be in liquid or solid form. Solid formulations are generally lyophilized and put into solution prior to administration, either in single or multiple doses. The formulation should not be exposed to extremes of temperature or pH to avoid thermal denaturation. Thus, it is essential that the antibody compositions of the present invention be formulated within a biologically relevant pH range. There is a need for solutions that are buffered to maintain a suitable pH range during storage, especially in liquid formulations that are stored for longer periods between formulation and administration. To date, both liquid and solid formulations require storage at lower temperatures (usually 2-8°C) to retain stability for longer periods. Formulated antibody compositions, especially liquid formulations, may contain bacteriostatic agents, including but not limited to benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben, at effective concentrations (usually <1% w / v) to prevent or minimize proteolytic degradation during storage. Bacteriostatic agents may be contraindicated for some patients. Thus, the lyophilized formulation may be reconstituted with a solution either containing or not containing such components. Additional components may be added to either the buffer or the solid antibody formulation, such as, but not limited to, sugars as cryoprotectants (including but not necessarily limited to polyhydroxyhydrocarbons such as sorbitol, mannitol, glycerol and dulcitol and / or disaccharides such as sucrose, lactose, maltose or trehalose) and, optionally, suitable salts (including but not limited to, NaCl, KCl or LiCl). Such antibody formulations, particularly liquid formulations selected for long-term storage, will depend on a useful range of total osmolality to promote long-term stability at temperatures between 2-8°C or higher while also making the formulation useful for parenteral injection. A useful range of total osmolality (total number of molecules in solution) is about 200 mOs / L to about 800 mOs / L. It will be apparent that the amount of cryoprotectant, such as sucrose or sorbitol, will depend on the amount of salt in the formulation to maintain the total osmolality of the solution within the appropriate range.Thus, salt-free formulations may contain about 5% to about 25% sucrose, with a preferred range of sucrose being about 7% to about 15%, with a particularly preferred sucrose concentration in the salt-free formulation being 10% to 12%. Alternatively, salt-free sorbitol-based formulations may contain sorbitol in the range of about 3% to about 12%, with a preferred range of about 4% to 7%, with a particularly preferred range being about 5% to about 6% sorbitol in the salt-free formulation. Of course, salt-free formulations will allow for an increased range of each cryoprotectant to maintain effective osmolality levels. These formulations may also contain divalent cations (such as, but not limited to, MgCl2, CaCl2, and MnCl2); and non-32 ionic surfactants (such as, but not limited to, polysorbate 80 (TWEEN80®), polysorbate 60 (TWEEN60®), polysorbate 40 (TWEEN40®), and polysorbate 20 (TWEEN20®), polyoxyethylene alkyl ethers such as, but not limited to, Brij58®, Brij35®, and others such as Triton X100®, Triton X114®, NP40®, Span85, and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121)). Any combination of such components, including the possible inclusion of a bacteriostatic agent, may be useful in loading the antibody-containing formulations of the invention. The antibody compositions of the present invention may also be "chemical derivatives," which refers to antibodies containing additional chemical moieties that are not normally part of an immunoglobulin molecule (e.g., pegylation). Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively, the moieties may attenuate undesirable side effects or reduce the toxicity of the base molecule.

[0107] Specific embodiments include PLGA microspheres as discussed herein and further known in the art, as well as non-degradable vehicles based on polymers including poly(ethylene-co-vinyl acetate; PEVAc). In addition, sustained release and localized delivery of antibody-based therapeutics is reviewed in Grainger, et al., 2004, Expert Opin. Biol. Ther. 4(7):1029-1044), which is incorporated herein by reference in its entirety. Suitable microcapsules capable of encapsulating antibodies may also include hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules prepared by coacervation techniques or interfacial polymerization. See PCT Publication WO 99 / 24061, entitled "Method for Producing IGF-1 Sustained-Release Formulations," in which proteins are encapsulated in PLGA microspheres, which reference is incorporated herein by reference in its entirety. Additionally, microemulsion or colloidal drug delivery systems, such as liposomes and albumin microspheres, may also be used. Other preferred sustained release compositions use bioadhesives to retain the antibody at the site of administration. As mentioned above, sustained release formulations may include biodegradable polymers into which the antibody is internalized, which may result in non-immediate release. Non-injectable devices may be described herein as "implants," "drug depot implants," "depot implants," "non-injectable depots," or any such similar term. Common depot implants may include, but are not limited to, solid biodegradable and non-biodegradable polymeric devices (such as expanded polymers or coaxial rod-like devices), as well as numerous pump systems also known in the art. Injectable devices are split into bolus injections (release and dissipation of drug after injection), and sustained or depot injections provide a storage reservoir at the injection site, allowing for sustained release of the biologic over time. Depot implants may be surgically anchored to the delivery site to provide a suitable reservoir for sustained release of the antibody over time.Such devices will be capable of delivering the formulation in amounts as therapeutically or prophylactically required for treatment over a preselected period of time. The depot implant may also provide protection to the formulation from degradation by body processes (such as proteases) over the duration of the treatment. As known in the art, the term "sustained release" refers to the gradual (continuous or discontinuous) release of such agents from a block polymer matrix over an extended period of time. Regardless of the specific device, the sustained release of anti-LAM antibody and / or anti-PIM6 / LAM antibody compositions will provide a localized biologically effective concentration of the antibody. The sustained release of the biological agent will be for a period of a day, several days, a week or more, depending on the formulation, but most likely for a month or more, or up to about six months. Natural or synthetic polymers known in the art are useful as depot implants due to properties such as versatile degradation kinetics, safety, and biocompatibility. These copolymers can be engineered to improve the pharmacokinetics of the active ingredient, shield the agent from enzymatic attack, and degrade over time at the site of attachment or injection. Those skilled in the art will appreciate that there is ample teaching in the art to manipulate the properties of these copolymers, such as their respective production processes, the catalysts used, and the final molecular weight of the sustained release depot implant or injection. Natural polymers include, but are not limited to, proteins (e.g., collagen, albumin, or gelatin); polysaccharides (cellulose, starch, alginic acid, chitin, chitosan, cyclodextrin, dextran, hyaluronic acid), and lipids.Biodegradable synthetic polymers include, but are not limited to, various polyesters, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-556), polylactic acid ([PLA]; U.S. Pat. No. 3,773,919 and European Patent No. 058,481), polylactic-polyglycolic acid (PLGA), e.g., polylactic-co-glycolide (see, e.g., U.S. Pat. Nos. 4,767,628 and 5,654,008), polyglycolide (PG), polyethylene glycol (PEG) conjugates of poly(α-hydroxy acids), polyorthoesters, polyaspirin, polyphosphagen, vinylpyrrolidone, polyvinyl alcohol (PVA), P VA-g-PLGA, PEGT-PBT copolymer (PolyActive), methacrylic acid, poly(N-isopropylacrylamide), PEO-PPO-PEO (pluronics), PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, polyorthoester (POE), or any combination thereof (as described above) (see, e.g., U.S. Pat. No. 6,991,654 and U.S. Patent Application Publication No. 20050187631, each of which is incorporated by reference in its entirety), hydrogels (e.g., Langer et al., J. Am. Soc. 2004, 144:1311-1315, 2004), and the like). See, et al., 1981, J. Biomed. Mater. Res. 15:167-277; Langer, 1982, Chem. Tech. 12:98-105, non-degradable ethylene-vinyl acetate (e.g., ethylene-vinyl acetate disks and poly(ethylene-co-vinyl acetate)), degradable lactic acid-glycolic acid copolymers such as Lupron Depot™, poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acid gels (see, e.g., U.S. Pat. No. 4,636,524), alginic acid suspensions, polyorthoesters (POE), and the like. Polylactic acid (PLA) and its copolymers with glycolide (PLGA) are well known in the art, with the commercialization of Lupron Depot™ approved in 1989 as the first parenteral sustained release formulation using a PLA polymer.Further examples of products using PLA and PLGA as excipients to achieve sustained release of active ingredients include Amidox (PLA; periodontal disease), Nutropin Depot (PLGA; with hGH), and Trelstar Depot (PLGA; prostate cancer). Other synthetic polymers included, but were not limited to, poly(c-caprolactone), poly 3-hydroxybutyric acid, poly(β-malic acid) and poly(dioxanone); polyanhydrides, polyurethanes (see WO 2005 / 013936), polyamides, cyclodestrans, polyorthoesters, n-vinyl alcohol, polyethylene oxide / polyethylene terephthalate, polyphosphates, polyphosphonates, polyorthoesters, polycyanoacrylates, polyethylene glycols, polydihydropyrans, and polyacetals. Non-biodegradable devices include, but are not limited to, various cellulose derivatives (carboxymethylcellulose, cellulose acetate, cellulose acetate propionate, ethylcellulose, hydroxypropylmethylcellulose), silicone-based implants (polydimethylsiloxane), acrylic polymers, (polymethacrylate, polymethylmethacrylate, polyhydroxy(ethylmethylacrylate), as well as polyethylene-co-(vinyl acetate), poloxamers, polyvinylpyrrolidones, poloxamines, polypropylene, polyamides, polyacetals, polyesters, polyethylene-chlorotrifluoroethylene, polytetrafluoroethylene (PTFE or "Teflon™"), styrene-butadiene rubber, polyethylene, polypropylene, polyphenylene oxide-polystyrene, poly-a-chloro-p-xylene, polymethylpentene, polysulfone and other related biostable polymers. Suitable carriers for sustained release depot formulations include, but are not limited to, microspheres, films, capsules, particles, gels, coatings, matrices, wafers, pills or other pharmaceutical delivery compositions. Examples of such sustained release formulations are described above.See also U.S. Patent No. 6,953,593; U.S. Patent No. 6,946,146; U.S. Patent No. 6,656,508; U.S. Patent No. 6,541,033; and U.S. Patent No. 6,451,346 (the contents of each of which are incorporated herein by reference). The dosage form must be capable of delivering the formulation in an amount and concentration as therapeutically required for treatment over a preselected period of time, and must provide the formulation with sufficient protection from degradation by body processes over the duration of the treatment. For example, the dosage form may be surrounded by an exterior made of a material that has properties to protect against degradation from metabolic processes and the risk of, for example, leakage, cracking, breakage, or distortion. This may prevent the dosage form contents from expelling in an uncontrolled manner under stresses that may be experienced during use due to physical forces exerted on the drug release device, i.e., in a convective drug delivery device, physical forces associated with the pressure generated inside the reservoir, for example, as a result of normal joint and other movements by the subject. The drug reservoir or other means for holding or containing the drug should also be made of a material that avoids unintended reactions with the active agent, and is preferably biocompatible (e.g., substantially non-reactive with the subject's body or bodily fluids when the dosage form is implanted). In general, each biological agent is administered to an individual for at least 12 hours to at least one week, and most likely via an implant designed to deliver the agent for at least 10, 20, 30, 100 days or at least 4 months, or at least 6 months or more, as appropriate. The anti-LAM antibody and / or anti-PIM6 / LAM antibody may be delivered at a relatively low volumetric rate, for example, about 0.001 ml / day to 1 ml / day, to minimize tissue damage or trauma near the site where the agent is released. The formulation may be released at a low dose, for example, at a rate of about 0.01 μg / hour or 0.1 μg / hour, 0.25 μg / hour, 1 μg / hour, generally up to about 200 μg / hour, depending on the particular biological agent, or the formulation is delivered at a low volumetric rate, for example, at a volumetric rate of about 0.001 ml / day to about 1 ml / day, for example 0.01 μg / day, up to about 20 mg / day.The dosage will depend on several factors, such as the potency, bioavailability, and toxicity of the active ingredient (eg, an IgG antibody) used, as well as the requirements of the subject.

[0108] In the in vivo treatment of human and non-human patients, the patient is administered or provided with a pharmaceutical formulation comprising at least one anti-LAM antibody and / or at least one anti-PIM6 / LAM antibody of the invention. When used for in vivo treatment, the anti-LAM or anti-PIM6 / LAM antibody of the invention is administered to the patient in a therapeutically effective amount (i.e., an amount that eliminates or reduces the total bacterial load). The antibody is administered to the human patient according to known methods, for example, intravenous administration, for example, as a bolus or by continuous infusion over time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibody may be administered parenterally, possibly at the target cell site, or intravenously. In some embodiments, the antibody is administered by intravenous or subcutaneous administration. The therapeutic composition of the invention may be administered to the patient or subject systemically, parenterally, or locally. The above parameters for evaluating successful treatment and improvement in the disease are easily measurable by routine procedures familiar to the physician.

[0109] For parenteral administration, anti-LAM and anti-PIM6 / LAM antibodies may be formulated in unit dose injection form (solution, suspension, emulsion) in association with a pharma- ceutically acceptable parenteral vehicle. Examples of such vehicles include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles include, but are not limited to, fixed oils and ethyl oleate. Liposomes may be used as carriers. The vehicle may contain small amounts of additives, such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0110] The anti-LAM and anti-PIM6 / LAM antibodies of the present invention may be administered to a host in any manner, method and / or combination available in the art in an amount sufficient to provide therapeutic treatment against infection with virulent strains of the Mycobacterium tuberculosis complex. These compositions may be provided to an individual by various routes known in the art, particularly parenteral routes, such as, but not limited to, intravenous (IV), intramuscular (IM); or subcutaneous (SC) administration, where IV administration is the standard in the art for administering therapeutic antibodies. These compositions may be administered as split or multiple doses (i.e., administration of the antibody at staggered times by maintaining the sterility of the formulation throughout the treatment regime).

[0111] The dose and administration schedule will depend on various factors, such as the nature of the infection, e.g., its therapeutic index, the patient, and the patient's medical history, which are readily determined by the physician. In general, a therapeutically effective amount of the antibody is administered to the patient. In some embodiments, the amount of antibody administered is within the range of about 0.01 mg / kg to about 1000 mg / kg (patient body weight), and any range therebetween. Depending on the type and severity of the infection, about 0.1 mg / kg to about 50 mg / kg body weight (e.g., about 0.1 to 15 mg / kg / dose) of the antibody is an initial candidate dose for administration to the patient, e.g., by one or more divided doses or by continuous infusion. The progress of this treatment is easily monitored by conventional methods and assays and based on criteria known to the physician or other skilled in the art. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to physicians.

[0112] These antibodies may also be administered via a gene vector that expresses the paired heavy and light chains of a given antibody. This may include a plasmid or a viral vector, such as an adenovirus or adeno-associated virus (AAV) vector, that efficiently expresses these genes. These vectors may be delivered by injection into muscle tissue and, depending on the dose, may secrete relatively large amounts of secreted antibody into the circulation for a relatively long period of time.

[0113] Other treatment regimens may be combined with administration of the anti-LAM and / or anti-PIM6 / LAM antibodies of the present invention, for example, with another anti-LAM antibody, for example, but not limited to, an anti-LAM antibody known in the art, for example, a murine anti-LAM antibody or its humanized version, or a pharmaceutical compound, for example, but not limited to, an antibiotic. Suitable antibiotics for co-administration with the anti-LAM and / or anti-PIM6 / LAM antibodies of the present invention include, but are not limited to, isoniazid, rifampin, rifapentine, ethambutol, pyrazinamide, bedaquiline, capreomycin, cycloserine, dexamethasone, kanamycin, and tinocordin. Combined administration includes co-administration using a split formulation or a single pharmaceutical formulation, and sequential administration in any order, where preferably there is a period during which both (or all) active agents simultaneously exert their biological activity. Such combined treatment may result in a synergistic therapeutic effect. The above parameters for assessing successful treatment and improvement in disease are readily measurable by routine procedures familiar to physicians.

[0114] According to another embodiment, the present invention provides a passive vaccine or pharmaceutical composition comprising at least one anti-LAM and / or anti-PIM6 / LAM antibody of the present invention and a pharma- ceutically acceptable carrier. According to one embodiment, the vaccine or pharmaceutical composition is a composition comprising at least one antibody as described herein and a pharma- ceutically acceptable carrier. The vaccine may comprise multiple antibodies having the properties described herein in any combination, and may further comprise other anti-LAM antibodies, such as those of the present invention and those known in the art, such as a murine anti-LAM antibody or a humanized version thereof. The passive vaccine may also comprise one or more pharma- ceutically acceptable preservatives, carriers, and / or excipients as known in the art.

[0115] According to another embodiment, the present invention encompasses an active vaccine or pharmaceutical composition comprising administering to a patient at least one LAM or PIM6 epitope of an antigen. The particular epitope to be utilized may be determined by testing the therapeutic activity of the antibodies described in this patent in an appropriate animal model for TB infection and / or pathogenesis. The model species may be mouse, or guinea pig, or rabbit, or primate. For example, if A194-01 is most protective, a vaccine having a form of the A194-01 epitope would be used, whereas if P30B9 is protective, a dimannose-substituted Ara6 residue may be most effective in generating an appropriate humoral response. The active vaccine may include one or more adjuvants known in the art, such as alum, aluminum hydroxide, aluminum phosphate, paraffin oil, and cytokines, such as IL-1, IL-2, IL-12. An active vaccine may contain one or more pharma- ceutically acceptable preservatives, carriers, and / or excipients known in the art.

[0116] In some embodiments, the present invention is directed to a recombinant vector, e.g., a plasmid comprising a nucleic acid encoding an immunoglobulin heavy chain (Ig VH) of an anti-LAM or anti-PIM6 / LAM antibody, and a second nucleic acid encoding an immunoglobulin light chain (Ig VL). In other embodiments, the first nucleic acid and the second nucleic acid are present in two different recombinant vectors. According to another embodiment, the present invention encompasses a method of treating tuberculosis infection in an individual, comprising administering to said individual a first nucleic acid encoding an immunoglobulin heavy chain (Ig VH) of an anti-LAM or anti-PIM6 / LAM antibody, and a second nucleic acid encoding an immunoglobulin light chain (Ig VL) of an anti-LAM or anti-PIM6 / LAM antibody, each of the nucleic acids being operably linked to a promoter region. The first nucleic acid and the second nucleic acid may be present in the same recombinant vector or in two different recombinant vectors. The recombinant vector may be a non-replicating viral vector, e.g., an adeno-associated virus (AAV), or may be a plasmid. In certain embodiments, the present invention is directed to cells transformed with one or more of the vectors disclosed herein.

[0117] The above-described antibodies and antibody compositions, vaccine compositions, and vectors may be administered for the prophylactic and therapeutic treatment of infection with virulent strains of the Mycobacterium tuberculosis complex.

[0118] H. Equivalents When a range of values ​​is given, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly indicates otherwise, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges (which may be independently included in that smaller range) are also encompassed within the invention, provided that any limit is specifically excluded in the stated range. Where the stated range includes one or both of those limits, ranges excluding either or both of those included limits are also included in the invention.

[0119] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can also be used in the practice or testing of this invention, the preferred method and material are described herein.All publications mentioned herein are incorporated herein by reference in their entirety.

[0120] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise.

[0121] The term "about" refers to a range of values ​​that would not be considered substantially different from the baseline value by one of ordinary skill in the art. For example, the term "about" may refer to values ​​that fall within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value, as well as intervening values ​​between such stated values.

[0122] The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0123] Each of the applications and patents cited in this document, and each of the documents or references cited in each of the applications and patents (including the prosecution of each issued patent; "documents cited in the applications"), patient or non-patient literature, and each of the PCT and foreign applications or patents corresponding to and / or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the documents cited in the applications, are hereby expressly incorporated herein by reference in their entirety. More generally, documents or references are cited in this text either in the reference list preceding the claims; or in the text itself; and each of these documents or references ("references cited herein"), as well as each document or reference cited within each of the references cited herein (including any manufacturer's specifications, instructions, etc.), are hereby expressly incorporated herein by reference.

[0124] The following non-limiting examples serve to further illustrate the invention. EXAMPLES

[0125] Example 1 - Using the methods described herein, memory B cells were cultured in vitro, immunoglobulin variable region genes were molecularly cloned, and several novel human monoclonal antibodies (mAbs) specific for LAM were isolated. Those skilled in the art will appreciate that these methods described herein can be adapted to selectively identify rare antibodies with very high affinity that may be present in as few as 1 out of 100,000 memory B cells circulating in a patient's blood.

[0126] Monoclonal antibodies Mouse monoclonal antibodies: Hybridoma cell lines producing LAM-specific mouse monoclonal antibodies CS-35 and CS-40 from Dr. Delphi Chatterjee's lab were recloned to homogenity, and the antibodies were purified by protein A chromatography.

[0127] Antibodies 906.41, 906.7, 908.1 and 922.5 were provided by Dr. John Spencer, and FIND25 and FIND170 were provided by Tobias Broger at FIND.

[0128] antigen Mycobacterium tuberculosis-derived H37Rv lipoarabinomannan (LAM) (NR-14848) and Mycobacterium smegmatis-derived LAM (NR-14860) were obtained from Colorado State University through BEI resources. Glycoconjugates derived from LAM were synthesized in the Lowary lab.

[0129] ELISA assay Man-LAM (H37Rv) and PI-LAM (from Mycobacterium smegmatis) were diluted in CBC buffer (7.5 mM sodium carbonate, 17.4 mM sodium bicarbonate, pH 9.0) and plated at a concentration of 100 ng / well in 96-well ELISA plates. After overnight incubation of the plates at 4°C, the wells were washed with PBS, pH 7.4, containing 0.05% TWEEN-20 (PBST) and then blocked with 1% BSA (Sigma) in PBS buffer. The PBST-washed plates were incubated with plasma from individuals infected with Mycobacterium tuberculosis and control plasma (diluted in RPMI medium containing 2% FBS) for 1 h at 37°C. Plates washed with PBST were then incubated for 1 h with a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-human IgG (gamma specific) (Millipore), or IgM (mu specific) (Millipore), or IgA (alpha specific). After a PBST wash, color was developed with 50 μL of DEA buffer. OD was measured at 405 nm in a spectrophotometer. Titers were defined as the reciprocal dilution resulting in an OD after subtraction of the background OD obtained on BSA-coated plates and were determined by exponential interpolation.

[0130] plasma titration The two purified antigens were obtained from the BEI Repository and plated on 96-well ELISA plates at a concentration of 2 μg / ml overnight at 4°C, and then the plates were blocked with 1% BSA in 1x PBS. The plasma was tested for LAM-specific titers by incubating serially diluted samples for 1 h at 37°C, followed by washing the plates three times with PBS + 0.1% Tween 20. Bound antibodies were detected with a mixture of alkaline phosphatase-conjugated goat anti-human kappa and goat anti-human lambda at a 1:1,000 dilution in 1% BSA in PBS, and the signal was developed by adding alkaline phosphate substrate to the DEA buffer. After 30 min, reactivity was measured at OD405.

[0131] Human subjects Patients with active infection with Mycobacterium tuberculosis were enrolled at the Lattimore practice of the Global Tuberculosis Institute. Active infection was defined by culture-proven tuberculosis disease or a diagnosis of clinical tuberculosis. This group included patients with a recent diagnosis of tuberculosis and those in their second month of treatment. Uninfected patients were healthy volunteers who were HIV-seronegative, had a negative tuberculin skin test, had no history of Bacillus Calmette-Guerin (BCG) vaccination, and had a negative interferon-gamma release assay (IGRA) (Quantiferon Gold In-Tube, Cellestis Inc, Valencia, CA). Written informed consent was obtained from participants, and the study was approved by the Rutgers University Institutional Review Board. TIFF2024174883000014.tif61170

[0132] Table 4. Demographics of human subjects 1. Cultivation and isolation of A194-01 (IgG isotype) Human monoclonal anti-LAM antibody A194-01 (isotype IgG) was isolated from cultured memory B cells obtained from TB-infected patient TB-194. A key component of the in vitro culture system is the presence of suitable support cells that can provide stimulation via the ligand CD40L of CD40, a member of the TNF receptor superfamily that is expressed on the surface of B cells and plays an essential role in mediating T cell-dependent immunoglobulin class switching and memory B cell development. Memory B cells were plated on a feeder layer of CD40L-expressing MS40L low cells. These cells express low levels of CD40L and have previously been shown to efficiently support the replication of memory B cells and their maturation into plasma cells (Luo, X., et al., 2003). al., Blood, 2009.113(7). These cells were generated by first infecting mouse stromal MS5 cells, which provide the B lineage growth factor IL-7, with the human CD40L transducing virus FUW-CD40L obtained from Origene (Rockville, MD). Memory B cells were isolated using the MACS Human Memory B Cell Isolation Kit from Miltenyi (catalog no. 130-093-546). Non-B cells were isolated by staining for the cell surface markers CD2, CD3, CD14, CD16, CD36, CD43, CD56, CD66b, and CD86b. and glycophorin A. To further remove naive B cells, memory B cell subpopulations were positively selected using magnetic beads coupled to antibodies against the cell surface marker CD27, a marker for memory B cells that is expressed at low levels on plasma cells but not on naive B cells. In the presence of support cells expressing CD40L, these conditions support the replication of memory B cells and their differentiation into plasmablasts, which secrete Ig into the culture supernatant at relatively high titers.

[0133] Cultures were refed at weekly intervals by replacing half of the culture supernatant with fresh medium. After 2-3 weeks, there were enough B cells to produce approximately 1-5 μg / ml of secreted antibody. Assuming the presence of 100-1,000 different clones in each well, this corresponded to an average concentration of 1-10 ng / mL Ig / B cell clone. As this concentration was rather low, the method biased towards antibodies with relatively high affinity for the target antigen. Approximately 80,000 memory B cells were purified from this patient's blood and cultured in 96 wells of a 96-well culture plate at an initial density of approximately 800 cells / well.

[0134] The culture supernatants were screened for the presence of antibodies against LAM from Mycobacterium tuberculosis by ELISA. LAM was coated at a concentration of 2 μg / mL in 50 μL / well of bicarbonate coating buffer on 96-well ELISA plates and incubated overnight at 4° C. The plates were washed four times with PBST (0.1% TWEEN20 in 1×PBS) and blocked with 200 μL of 2% skim milk in 1×PBS for 1 h at 37° C. 100 μl of culture supernatant was added to the corresponding wells of the ELISA plate containing LAM and incubated for 1 h at 37° C. After an additional washing step, AP-conjugated mouse anti-human Fab antibody was added to detect bound human antibody. After 30 min of incubation at 37° C., 100 μL of AP substrate in DEA buffer was added to the ELISA wells and reactivity was measured colorimetrically by measuring absorbance at 405 nm.

[0135] A positive signal (OD of approximately 1 at 1 hr) was detected in only one well out of the 96 wells, indicating the rarity of these cells in this sample. Cells from the positive cells were recultured in 10 wells of a 96 well plate at a density of 5-10 cells / well and rescreened for activity against LAM. This resulted in approximately 6 positive wells (OD of approximately 1 at 1 hr), again consistent with the low frequency of LAM-reactive cells, suggesting that the original positive wells contained only a single LAM-positive B cell clone. Cells from several positive subclones were lysed and used to isolate the variable regions of the heavy and light chains, which were then cloned into heavy and light chain expression vectors. A total of 10 diverse VH and 9 VL sequences were isolated from these wells, which were then tested for activity by transfecting individual combinations into 293 cells. Of the 90 combinations tested, only a single combination of heavy chain (p9045-IgG1-VH) and light chain (p9044-Vk) resulted in a positive signal for LAM. Antibodies were expressed by co-transfection of the corresponding heavy and light chain plasmids in Expi-292 cells as described by the manufacturer and grown in serum-free medium. Antibodies were purified by affinity chromatography on either Protein A beads (for IgG) or Protein L beads (for IgG) and eluted with low pH buffer. Purified antibodies were concentrated and characterized by SS-PAGE for size and purity.

[0136] 2. Isolation and culture of the IgM isotype of P30B9 Human monoclonal anti-LAM antibody P30B9, isotype IgM, was isolated from cultured memory B cells obtained from TB-infected patient TB-314. PBMCs were isolated from the blood of patient TB-314 by centrifugation on a Ficoll density gradient, and approximately 30,000 memory B cells were purified as described above using the MACS Human Memory B Cell Isolation Kit from Miltenyi. Purified memory B cells were cultured for 14 days by plating 400 cells / well on a monolayer of MS40-L cells grown in 96-well plates in the presence of IL-21 (100 ng / mL), IL-10 (100 ng / mL), IL-2 (10 ng / mL), IL-4 (2 ng / mL), and CpG (1 μM), and cell supernatants were screened by ELISA for binding to H37Rv ManLAM. ManLAM was coated at a concentration of 2 μg / mL in 50 μL / well of bicarbonate coating buffer on 96-well ELISA plates and incubated overnight at 4°C. Plates were washed 4 times with PBST (0.1% TWEEN20 in 1×PBS) and blocked with 100 μL of 1% BSA in 1×PBS for 1 h at 37°C. 50 μl of culture supernatant or diluted antibody was added to the corresponding wells of the ELISA plate containing LAM and incubated for 1 h at 37°C. After an additional washing step, AP-conjugated goat anti-human IgG (H+L) antibody was added to detect bound human antibody. After 30 min of incubation at 37°C, 50 μL of AP substrate in DEA buffer was added to the ELISA wells and reactivity was measured by measuring the yellow color at 405 nm. When probed with secondary goat anti-human IgG, IgA, IgM, kappa chain reagents, only one well out of 78 wells produced a positive signal. After expansion, the wells are transduced with BCL6 and Bcl-xL linked by a self-cleaving porcine teschovirus-1 (P2A) peptide sequence followed by an IRES-driven GFP reporter gene. These two genes stabilize long-term replication in memory B cells and allow the culture of cells even after selection of antigen-positive cells by BCR engagement.Retroviral vectors were pseudotyped with the envelope glycoprotein of Gibbon Ape Leukemia Virus (GaLV) in which the R peptide was deleted from the C-terminal TM domain. Successful transduction of primary B cells led to the expression of BCL-6, Bcl-xL and the marker protein GFP. Viral titers were determined by counting GFP-positive 293T cells under a fluorescent microscope. Activated B cells were transduced with retroviral vectors in the presence of polybrene / retronectin.

[0137] After further expansion, the transduced cells were passaged at limiting dilution in the presence of IL-21 (100 ng / mL) and IL-2 (10 ng / mL). Well B9 on plate 30 (P30B9) was selected based on its strong LAM binding activity and microscopic demonstration of the presence of a single clone. Supernatants from P30B9 bound exclusively to wells coated with H37Rv-LAM, but not to wells coated with LAM or α-crystallin from Mycobacterium smegmatis. Cells from this well were lysed and RNA was isolated using the RNeasy mini kit (Qiagen), followed by cDNA synthesis with oligo(dT) using the superscript III cDNA synthesis system (Invitrogen). The antibody heavy and light chain variable regions were amplified using Smith-Tiller primers and cloned into human heavy and light chain expression vectors. The heavy chain variable region was first cloned into a standard IgG vector. However, when combined with a light chain sequence cloned into a human kappa chain expression vector, no LAM binding activity was detected. At that point, ManLAM-reactive antibodies produced in the first stably transduced polyclonal wells were reprobed with isotype-specific reagents and found to be exclusively IgM. The P30B9 VH sequence was then cloned into an IgM heavy chain constant region expression vector, and good binding activity was obtained when cotransfected with the corresponding kappa chain.

[0138] 3. Characterization of epitope specificity of A194-01 IgG and P30B9 IgM and mouse anti-LAM antibodies against LAM To define the epitopes recognized by A.A194-01 IgG and P30B9 IgM, the binding activity of the antibodies was compared to that of several mouse LAM-specific monoclonal antibodies (CS-35, CS-40, FIND25, FIND170, and the 900 series of monoclonal antibodies represented by 908.1) against a series of 25 glycoconjugates conjugated to bovine serum albumin with synthetic glycans representing different structures present in LAM (Figure 4A). These ranged in size from 4 to 26 carbohydrate rings and represented a range of structural motifs known to be present in various mycobacterial LAMs, including several polyarabinose structures, both uncapped and capped with phosphoinositol, α(1→2)-linked mono-, di-, and tri-Manp mannose structures, and a 5-deoxy-5-methylthiopentofuranosyl (MTX) motif, as well as a variety of capped Ara4 and Ara6 structures.

[0139] Six distinct reactivity patterns were obtained with this antigen panel for these monoclonal antibodies (Figure 4B). The relative affinity of the monoclonal antibodies for these antigens was indicated by the titration characteristics; high affinity reactions retained high reactivity at intermediate dilutions, while low affinity was indicated by a rapid drop in reactivity. The most extensive pattern was seen with the mouse mAb CS-35, which reacted with moderate affinity with LAM from Mycobacterium tuberculosis and LAM from Mycobacterium smegmatis and recognized both capped and uncapped structures with basic Ara4 and Ara6 motifs, consistent with the known specificity of this mAb for the β-D-Araf-(1→2)-α-D-Araf-(1→5)-α-D-Araf-(1→5)-α-D-Araf motif.

[0140] The human monoclonal anti-LAM antibody A194-01 IgG also recognized a large fraction of these structures, often with the strongest affinity. A109-01 IgG bound avidly to all uncapped Ara4 and Ara6 structures and phosphoinositol-capped Ara4 structures, and less avidly to a subset of mannose-capped structures. A109-01 IgG bound well to monomannose-capped structures and very weakly to di- and trimannose structures, although reactivity with the latter structures was enhanced when MTX substitution was present. Four of the 900 series of mouse monoclonal antibodies (represented by 908.1) reacted with relatively weak affinity to all uncapped Ara4 and Ara6 structures, but did not react with any of the capped structures. Two mouse monoclonal antibodies from FIND (also called FIND25, KI25) bound strongly to all Ara6 structures, independent of the presence or absence of capping, but did not recognize any Ara4 structures. CS-40, known to react specifically with ManLAM, reacted weakly with LAM from Mycobacterium tuberculosis and bound preferentially to monomannose-capped Ara4 and Ara6 structures.

[0141] The human monoclonal anti-LAM antibody P30B9 IgM reacted strongly and with high specificity with ManLAM from Mycobacterium tuberculosis and with dimannose-capped Ara4 and Ara6 structures, and showed significantly weaker activity with other mannose-containing structures. Visualization of activity of this residue was dependent on the assay conditions and was seen in some assay formats (e.g., Figures 4b, 8) but not others (e.g., Figures 16, 18). Without wishing to be bound by theory, the relative specificity of P30B9 IgM for dimannose-capped structures is potentially clinically relevant, since terminal mannosyl units are known to mediate binding of lipoarabinomannan from virulent strains of the Mycobacterium tuberculosis complex to human macrophages, and furthermore, the dimannose cap is known to be the dominant modification of LAM from Mycobacterium tuberculosis.

[0142] Similar results were obtained when the epitope specificity of A104-01 IgG and P30B9 IgM was further mapped in a microarray assay against a larger panel of carbohydrate antigens. This panel included several additional polymannose structures that were recognized by P30B9 IgM but not by any of the other antibodies tested (Figure 8). This was consistent with P30B9 IgM's preference for dimannose-capped Ara4 and Ara6 structures, particularly (but not necessarily) those with Man-α(1→2)-Man-α(1→5) linked to a terminal arabinose. P30B9 IgM also reacted strongly to a pentamannose structure with Man-α(1→2)-Man-α(1→6) (59.AS-3-71) but only weakly to a similar structure with Man-α(1→3)-Man-α(1→6) (50.YB-BSA-18). Despite its preference for α(1→2) linkages, P30B9 IgM did not react with the tetramannose structure AS-2-91, which has a Man-α(1→2) linkage with an additional mannose linkage α(1→6) to the second mannose. Without wishing to be bound by theory, this suggests that the specificity of the IgM isotype of P30B9 may require that both sugars of the dimannose motif do not contain any additional substitutions.

[0143] B. More precise titration to map the precise specificity of these monoclonal antibodies against LAM-derived glycans demonstrated the important role of the terminal β-D-Araf-(1→2)-α-D-Araf-(1→5) disaccharide in the antibody recognition of the Ara4 structure. The Ara4 structure consists of a β-D-Araf-(1→2)-α-D-Araf-(1→5)-α-D-Araf-(1→5)-α-D-Araf tetrasaccharide, while the Ara6 structure has an additional β-D-Araf-(1→2)-α-D-Araf-(1→3) disaccharide branch at the second sugar. Three of the monoclonal antibodies bound to both the Ara4 and Ara6 structures, independent of mannose capping. All three monoclonal antibodies bound to the Ara4 structure (YB-8-099) and to YB-BSA-03, which corresponds to the Ara4 structure with four additional α-D-Araf-(1→5) sugars at the reducing end (Figure 9A). However, none of the monoclonal antibodies bound to a related octasaccharide (MJ-LZ-2) with a terminal β-D-Araf-(1→2)-α-D-Araf-(1→3) disaccharide, which corresponds to the lower branch of the Ara6 structure. This indicated that the higher branch of the Ara6 structure with β-D-Araf-(1→2)-α-D-Araf-(1→5) linkages was recognized by these monoclonal antibodies, whereas the lower branch with β-D-Araf-(1→2)-α-D-Araf-(1→3) disaccharide was not recognized.

[0144] The role of the terminal β-D-Araf-(1→2) bond in antibody recognition was tested by probing the reactivity of these monoclonal antibodies and the Ara6-dependent FIND25 antibody to three related polyα-D-Araf-(1→5) structures with truncated forms of the terminal disaccharide (Figure 9B). All three structures also had internal α-D-Araf-(1→3) branches. YB-BSA-07 terminated in a linear α-D-Araf-(1→5) structure and showed complete lack of reactivity with all of the anti-LAM antibodies. YB-BSA-09 had additional α-D-Araf sugars attached via (1→3) branches at the penultimate sugar of two longer branches, resembling the structure of the Ara6 branch. This structure was poorly recognized by the IgG isotypes of A194-01 and CS-35 at the higher concentrations tested. YB-BSA-10 contained a terminal β-D-Araf-(1→2) sugar on each of the branches forming two complete Ara6 structures at the non-reducing end of the polysaccharide. This structure was recognized by all of the monoclonal antibodies with relative binding strengths consistent with their affinity for the native LAM antigen. These assays demonstrated that the terminal β-D-Araf-(1→2)-α-D-Araf-(1→5) disaccharide was a critical component of all of the available arabinose-reactive LAM-specific monoclonal antibodies.

[0145] C. A crucial difference between pathogenic strains of the Mycobacterium tuberculosis complex, such as Mycobacterium tuberculosis and Mycobacterium bovis, and rapidly growing non-pathogenic strains, such as Mycobacterium smegmatis, is the presence of mannose-capped termini in the pathogenic strains. If so, monoclonal antibodies specific for different mannosylated structures may be useful for structural studies and to determine the functional contribution of these modifications. The activity of two of the monoclonal antibodies characterized in this study, CS-35 and FIND25 / 170, was not affected at all by the presence or absence of the mannose cap. On the other hand, the binding of the 900 series of monoclonal antibodies was completely inhibited by any type of mannosylation (Figure 4).

[0146] On the other hand, CS-40 bound weakly to unmodified Ara4 glycans (YB-8-099) but strongly to Ara4 (YB-8-101) and Ara6 (YB-8-149) structures with a single mannose cap. In this experiment, a modified CS-40 in which the mouse heavy chain domain was replaced with a human IgG1 constant sequence was used because it resulted in more sensitive detection of binding compared to the native mouse antibody used in Figure 4. The weak reactivity of CS-40 with uncapped arabinofuranose structures was reflected in its weak reactivity with M. smegmatis LAM compared to M. tuberculosis (M.tb) LAM. Attachment of an α(1→4)-linked MSX sugar to the terminal mannose (i.e., YB-8-141 and YB-8-149) had no effect on binding affinity, whereas attachment of a second α(1→2)-linked mannose sugar to create a dimannose cap (YB-8-111 and YB-8-125) completely abrogated CS-40 reactivity (Figure 16).

[0147] A194-01 had a more complex reactivity pattern: A194-01 bound strongly to uncapped arabinofuranosyl side chains and monomannose-capped Ara4 (YB-8-101) and Ara6 (YB-8-123) structures, whereas the mAb reacted weakly with dimannose-capped Ara4 (YB-8-111), even weaker with trimannose-capped Ara4 (YB-8-113), and barely with dimannose-capped Ara6 (YB-8-125). As seen in CS-40, MTX substitutions (YB-8-141, YB-8-149) for monomannose structures did not inhibit A104-01 binding, and of particular interest, MSX addition significantly improved the recognition of dimannose- and trimannose-capped Ara4 structures (YB-8-133, YB-8-143). In agreement with the high selectivity of P30B9 for ManLAM, the mAb specifically bound to dimannose-capped Ara4 (YB-111) and Ara6 (YB-8-125) structures. In contrast to the favorable or beneficial effect of MSX substitution on CS-40 and A194-01 binding, this substitution resulted in a complete lack of reactivity of P30B9, since the addition of an additional mannose resulted in the formation of trimannose-capped structures. These results suggested that the different monoclonal antibodies recognize different regions and structural features of the LAM structure, with some binding exclusively to arabinofuranose side chains and others binding to the capping motif with different levels of specificity.

[0148] The relative binding specificity and affinity of Ara6-reactive antibodies were compared for representative glycoconjugates (Figure 11). The overall pattern was consistent with that obtained for the native antigens PILAM and ManLAM (Figure 3) and in preliminary titrations against glycoconjugates (Figure 4). Human A194-01 IgG had higher relative affinity for all uncapped structures and for the MSX-substituted Ara6 monomannose structure (YB-8-149), reacted with equivalent affinity to Ara6 structures with a single mannose cap, but did not recognize structures with dimannose or trimannose caps. FIND25 bound to all structures with the standard Ara6 structure (either in capped or uncapped form) with similar or slightly higher affinity than CS-35, but did not bind to two structures in which one of the branches was extended at the non-reducing end away from the branch point (YB-BSA-06 and YB-BSA-08). 908.1 bound with weaker affinity to all of the uncapped structures, including the latter two, but did not recognize any of the mannose-capped structures 4.

[0149] Competitive test involving anti-LAM monoclonal antibody A, overview The ability of individual antibodies to compete for the binding of biotinylated probe mAb to LAM was titrated by ELISA. Representative competition curves for four anti-LAM antibodies A194-01, CS-35, FIND25 and P30B9 are shown in FIG. 16. As expected, all biotinylated antibodies were competed by excess amounts of their unlabeled versions. Mouse anti-LAM antibody 908.6 competed weakly, if at all, against the other antibodies. This was due in part to the weak affinity of this antibody, but also reflects the binding restriction of 908.6 to the uncapped structure and suggests that the capped structure was the dominant target in ManLAM recognized by CS-35 and FIND25.

[0150] CS-35 completely competed for all of the binding of the probe antibodies, consistent with its broad reactivity, but its competition with biotinylated A194-01 was much less potent than A194-01 alone, consistent with the lower affinity of CS-35 for LAM. CS-35 completely competed for biotinylated FIND25, whereas FIND25 only partially competed for labeled CS-35 (maximum competition of about 74%) and was even less effective for A194-01 (about 50%). Without wishing to be bound by theory, this result likely reflects the presence of the Ara4 structure recognized by A194-01 and CS-35, but not by FIND25, which binds exclusively to the Ara6 motif. The fact that FIND25 competed for the majority of CS-35 binding suggested that the Ara6 structure is more prevalent than the Ara4 structure. Despite its high affinity, A194-01 competed only against itself and not against either CS-35 or FIND25, further suggesting that the targets in LAMs recognized by the latter two antibodies consisted primarily of structures not recognized by A194-01 (e.g., dimannose- and trimannose-capped structures). In contrast to this result, A194-01 did completely and efficiently compete for binding of FIND25 to nonmannosylated PILAM, consistent with a role for the Ara6 structure in efficient mannose capping in the lack of competition in ManLAM.

[0151] Competition studies using antibody P30B9 further supported the conclusion that the majority of Ara6 structures in ManLAM are dimannose-capped and that the majority of the dimannose caps are located on Ara6 structures. P30B9 competed for approximately 70% of the binding of FIND25 and approximately 80% of the binding of CS-35 to ManLAM, confirming that the majority of structures recognized by these mouse mAbs are also recognized by P30B9. Binding of P30B9 to ManLAM was efficiently competed by itself and by both CS-35 and FIND25. The level of competition with P30B9 by FIND25 was close to 100%, indicating that essentially all of the dimannose-dependent P30B9 binding sites are located on Ara6 sites and very little on Ara4 structures. As expected, consistent with poor recognition of dimannose-capped structures by these antibodies, A194-01 competed very weakly for binding of P30B9 to ManLAM, and 908.7 did not compete at all. The inability of the latter antibody to efficiently compete for P30B9 binding confirmed that this effect requires binding of the competing mAb to the same branch as the probe mAb, and that binding to heterologous epitopes located on adjacent branches of the same molecule does not result in effective competition.

[0152] B. Relative A194-01 IgG and P30B9 IgM Affinity by Competition Assay Mapping the reactivity of individual monoclonal anti-LAM antibodies, including A194-01 IgG isotype and P30B9 IgM isotype, to specific glycan structures allowed characterization of the distribution of said specific glycan structures in LAM by antibody competition studies (Figure 10). In these competition assays, it was assumed that in the native molecule, two epitopes must potentially (but not necessarily) be in close proximity to each other on the same or adjacent arabinan branches, since one antibody competes for binding of a second (biotinylated if they are from the same species). This model was supported by the asymmetric competition patterns, where, for example, biotinylated IgG A194-01, which binds both uncapped, monomannosylated, and MSX-substituted Ara4 and Ara6 structures, was efficiently competed by itself and by engineered variants and / or derivatives of A194-01, but only partially by the mouse monoclonal antibody FIND25, which binds only to Ara6 structures. On the other hand, the mouse monoclonal antibody CS-35, which binds to all Ara4 and Ara6 structures, results in more complete competition, but is less efficient, probably due to its relatively low affinity.

[0153] The results of these assays showed some surprising and unexpected properties. For example, the IgM isotype of P30B9, which binds to all dimannose-capped ManLAM structures, was itself strongly and completely competed for by CS-35 and FIND170. Without wishing to be bound by theory, the efficient competition of P30B9 by the mouse monoclonal anti-LAM antibody FIND25 suggests that the dimannose-capped structures in native LAMs are primarily localized to the Ara6 structures recognized by the FIND antibody and are not clearly expressed on the Ara4 structures. This highlights the importance of being able to target and specifically bind to the dimannose-capped Ara6 residue, as dimannose capping appears to be the dominant form of LAM found in virulent strains of the Mycobacterium tuberculosis complex. The highly efficient competition of biotinylated FIND25 by engineered variants of A194-01, the IgM isotype, is further evidence for increased recognition of mannosylated structures by the IgM isotype of A194-01.

[0154] C. Competitive study of A194-01 IgG and P30B9 IgM and mouse anti-LAM antibodies against ManLAM and PILAM The distribution of the various structural forms in LAM was analyzed using binding competition assays between different anti-LAM monoclonal antibodies. The IgG isotype of A194-01 recognized both unmodified Ara4 and Ara6 side chains or chains with a single mannose cap, but did not bind to side chains with either dimannose or trimannose capping motifs. The two FIND mouse antibodies reacted with all forms of Ara6 but not with any Ara4 structures. P30B9 IgM was relatively specific for the dimannose-capped Ara4 and Ara6 structures.

[0155] Consistent with the broadening in reactivity to A194-01 constructs with increased valency, these constructs showed enhanced potency in antibody competition activity. When tested for the ability to compete for binding of biotinylated A194-01 IgG to ManLAM, decameric A194-01 IgM and tetrameric scFv-IgG variants competed more efficiently than the A194-01 IgG isotype itself (FIG. 11), thus demonstrating increased potential therapeutic and diagnostic utility, whereas monomeric Fab and scFv forms did not compete as effectively (FIG. 1). Dimeric scFvs of engineered variants and / or derivatives of A194-01 competed equally well with the A194-01 IgG isotype.

[0156] When the epitope specificity of the engineered variants and / or derivatives of A194-01 was compared to that of A194-01 IgG, it was observed that they had broader reactivity (Figure 14). On the one hand, the IgG isotypes did not specifically bind to the dimannose (YB-8-123, YB-8-125) and trimannose (YB-BSA-113, YB-BSA-13) substituted structures, whereas IgM recognized these structures and the scFv-IgG form had enhanced activity against some of these structures as well. This potentially suggests enhanced utility of these engineered forms of A194-01 in therapeutic and diagnostic applications, since dimannose capping, especially dimannose capped Ara6, is the dominant LAM motif in virulent Mycobacterium tuberculosis.

[0157] Unlabeled A194-01 IgG competed for binding of biotinylated A194-01 IgG to either Mycobacterium tuberculosis-derived LAM (ManLAM) (Fig. S12A) or Mycobacterium smegmatis-derived LAM (PILAM) (Fig. S12D), whereas mouse monoclonal antibodies FIND170 and P30B9 were unable to compete for binding of A194-01 to either antigen (Fig. S12A,D). This was consistent with the dominant recognition of the Ara4 structure recognized by the A194-01 IgG isotype, but not either FIND170 specific for Ara6, or P30B9 IgM, which relied on the dimannose capping residue. Similarly, A194-01 did not compete with the binding of either biotinylated FIND25 (Figure S6B) or P30B9 IgM (Figure S6C) to ManLAM, consistent with different epitope specificities for these antibodies. In contrast to the inability of A194-01 IgG to compete for binding of FIND25 to ManLAM, A194-01 IgG potently competed for approximately 90% of the binding of FIND25 to PILAM (Figure S6E), consistent with the absence of mannose capping in known PILAMs and the high affinity of A194-01 for the uncapped Ara4 and Ara6 structures.

[0158] In contrast to the inefficient competition by A194-01 IgG, P30B9 IgM competed for approximately 80% of the binding of FIND25, and FIND170 competed for the binding of P30B9 almost completely (Fig. 12B, C). This strongly suggested that the majority of Ara6 structures recognized by the FIND mouse antibody had a dimannose cap, and thus the majority of the dimannose-capped structures recognized by P30B9 IgM and P30B9 are present on Ara6 structures. This suggests that dimannose-capped Ara6 is the dominant immunological motif in the LAM motif from virulent Mycobacterium tuberculosis.

[0159] D. Further Competitive Testing Further competition studies were performed to shed light on the fact that LAM is a complex antigen with uncertain heterogeneity. The determination of the different epitope specificities of LAM-reactive monoclonal antibodies allowed the use of binding competition assays to investigate the distribution of various epitopes in native LAM. The ability of various antibodies to compete for the binding of biotinylated probe monoclonal antibodies to LAM and synthetic glycoconjugates was titrated by ELISA. Typical competition curves for the three monoclonal anti-LAM antibodies A194-01 IgG, CS-35, and FIND25 are shown in Figure 13A. The biotinylated probe monoclonal antibodies were competed by their unlabeled versions when present in sufficient excess. The mouse monoclonal antibody 908.6 competed weakly, if at all, against the other antibodies. This was due in part to the weak affinity of this antibody, but also reflected binding limitations to the uncapped structure of 908.6, and further suggested that the mannose-capped structure is the dominant target in ManLAM recognized by CS-35 and FIND25. CS-35 competed for binding of biotinylated A194-01 IgG, consistent with its broad reactivity, but less efficiently than A194-01 IgG alone, consistent with the higher affinity of A104-01 IgG for LAM. CS-35 also fully competed for biotinylated FIND25, while FIND25 only partially competed for labeled CS-35 (maximum competition of about 74%) and was even less effective for A194-01 IgG (about 50%). This result likely reflects the presence of Ara4 structures recognized by A194-01 IgG and CS-35, but not by FIND25, which binds exclusively to structures with an Ara6 backbone. Despite its overall high affinity for LAM, A194-01 IgG competed only with itself and not with either CS-35 or FIND25. This suggested that the sites in LAM recognized by mouse monoclonal antibodies are dominated by dimannose- and trimannose-capped structures that are not recognized by A194-01 IgG.

[0160] Additional competition studies using P30B9 IgM, which specifically binds to dimannose-capped ManLAM, further supported the clinically relevant conclusion that the majority of Ara6 structures in ManLAM are dimannose-capped and that the majority of the dimannose cap is present on the Ara6 structures in ManLAM from Mycobacterium tuberculosis. P30B9 IgM competed with approximately 80% of FIND25 binding to ManLAM (FIG. 13B), consistent with the majority of Ara6 structures recognized by FIND25 also being recognized by P30B9 IgM. P30B9 IgM did not compete for FIND25 binding to PILAM, consistent with the absence of the P30B9 dimannose epitope in PILAM due to the lack of mannosylation in PILAM. Furthermore, A194-01 IgG did not compete for binding of FIND25 to ManLAM, which was further consistent with the fact that the majority of dimannose-capped Ara6 structures were not recognized by A194-01 IgG. Confirming the role of mannosylation in this effect, A194-01 IgG competed very efficiently for binding of FIND25 to PILAM, which was consistent with the high affinity of A194-01 for PILAM and the absence of mannose capping in this antigen.

[0161] Competition data for the binding of biotinylated P30B9 IgM further supported this conclusion. Binding of biotinylated P30B9 IgM was most efficiently competed by itself and with equal efficiency by CS-35 and FIND25, but was slightly less complete by A194-01 IgG (Figure 13C). The level of competition by FIND25 was close to 100%, indicating that essentially all of the dimannose-dependent P30B9 IgM binding sites are located on the Ara6 structure. Consistent with this interpretation, CS-35 also competed for the binding of P30B9 to dimannose-capped Ara4 (YB-8-111) and dimannose-capped Ara6 (YB-8-125), whereas FIND170 competed only for the latter antigen and A194-01 IgG did not compete for either. The general similarity between the competition curves for ManLAM and the homogeneous YB-8-125 glycoconjugate indicated that the competition outcome correlated with the presence or absence of relevant epitopes on single carbohydrate side chains, and that indirect steric effects due to antibody binding to more distant heterologous sites played little, if any, role in the competition.

[0162] One surprising and unexpected result is the complete lack of competition between A194-01 IgG versus CS-35 and FIND25. The ability of CS-35 to compete for A194-01 IgG binding to ManLAM is expected based on the recognition of all A194-01 IgG targets by CS-35, and the less efficient competition by CS-35 than by A194-01 IgG itself is consistent with the relative affinity of these antibodies for ManLAM (Figure 3). Similarly, the incomplete competition of A194-01 IgG binding by FIND25 may be explained by the presence of the Ara4 target recognized by the former antibody but not the latter antibody.

[0163] The efficient and complete competition of biotinylated P30B9 IgM binding by both CS-35 and FIND170 suggests that the dimannose cap recognized by P30B9 IgM was almost exclusively present on the Ara6 structure recognized by the FIND mAb, which is of clinical and diagnostic significance. This was supported by the relatively efficient competition of FIND25 binding by P30B9 IgM, which blocked approximately 80% of the binding activity of FIND25 to ManLAM but had no effect on PILAM. This strongly suggests that approximately 80% of the Ara6 sites in ManLAM recognized by FIND25 have dimannose caps, and that essentially all of the dimannose caps are present on the Ara6 and not on the Ara4 structures. These results, combined with the inability of A194-01 IgG to compete with CS-35 or FIND25, indicate that the dimannose-substituted Ara6 is the dominant immunogenic structure for ManLAM from Mycobacterium tuberculosis and thus represents a highly important antigenic target.

[0164] Studies of LAM-specific antibody responses in patient plasma indicate that, independent of mapping, the response is dominated by IgG2 isotypes specific for linear Ara4 / Ara6 structures. The efficient competition of P30B9 IgM by IgG monoclonal anti-LAM antibodies specific for arabinofuranose-dependent epitopes, such as CS-35 and FIND25, suggests that a dominant IgG2 response to such epitopes in patient plasma would also compete for any dimannose-dependent ManLAM-specific antibodies, which may be produced at lower titers. Thus, even if the latter class of antibodies may have more effective antibacterial activity, it is likely that their effectiveness may be limited by competition for binding by dominant non-functional IgG2 antibodies specific for arabinose-dependent epitopes present in patient serum. Without wishing to be bound by theory, a dominant humoral response may in fact protect bacteria against the potential effects of rarer antibodies, e.g. multivalent antibodies such as P30B9 IgM, or engineered variants and / or derivatives of A194-01, e.g. pentavalent IgM isotypes or tetravalent scFv-IgG, which may provide immune regulation against infection and pathogenicity.

[0165] 5. Effect of valency on the binding of A194-01 The finding that the reactivity of dimannose-reactive P30B9 was dependent on its IgM isotype suggested that multivalency may contribute to the affinity of the antibody to LAM. Without wishing to be bound by theory, this suggested that a single LAM molecule may have multiple antibody binding sites or epitopes, consistent with the known branched structure and complexity of LAM, and that antibodies with higher valency may bind to more sites than bivalent antibodies, resulting in higher affinity. The effect of antibody valency on binding efficiency to LAM was tested for various engineered mutant and / or derivative forms and / or isotypes of human monoclonal antibody A194-01 in a binding competition assay using biotinylated A194-01 IgG as the target. The antibody formats included monovalent single-chain scFvs in which the VH and VL regions were linked by a flexible peptide linker, monovalent Fab proteins, dimeric scFv proteins in which the two scFv domains were linked by a flexible linker, native dimeric IgG (Figure 1A), and two higher valency forms, the tetravalent A194-01 scFv-IgG, and the pentavalent (decavalent for the binding site) IgM isotype (Figure 1B). Converting the intact bivalent IgG to a monovalent Fab resulted in a very large reduction in binding activity, with a >100-fold increase in concentration required to compete for 50% of the binding activity of biotinylated A194-01 IgG compared to IgG on its own (Figure 1C). The single chains also competed inefficiently, with a 33-fold reduction in activity. On the other hand, the scFv dimers competed with similar efficiency to the IgG isotype of A194-01. Without wishing to be bound by theory, this suggested that efficient binding of the bivalent form of A194-01 to LAM required attachment of both binding sites to adjacent targets in a single molecule of antigen. The higher valency form competed more efficiently on a molar basis. This could simply be due to the presence of additional binding sites, but may also reflect increased affinity of the higher valency form.

[0166] The specificity of both the A194-01 tetrameric scFv-IgG variants and / or derivatives and the A194-01 decameric IgM isotype was compared to that of the A194-01 IgG isotype against the synthetic glycoconjugate panel described above (Figure 14). This showed that both the modified scFv-IgG variants and the modified IgM isotypes had a broader reactivity with some of the glycans that the IgG isotypes only poorly or not at all recognized. Enhanced binding was seen with some of the dimannose capped structures (YB-8-111, YB-8-125, YB-8-133) and trimannose capped structures (YB-8-113, YB-8-143, YB-BSA-13), with the A194-01 modified IgM isotype having the broadest reactivity panel. Given the significance of the dimannose capping, this has significant diagnostic and therapeutic potential. Interestingly, enhanced reactivity was also seen in the A194-01 IgM isotype, which has an arabinose structure lacking the terminal β-D-Araf-(1→2) bond (YB-BSA-09), which is not recognized by any of the monoclonal anti-LAM antibodies tested. Without wishing to be bound by theory, this suggested that increased valency would result in enhanced avidity of the A194-01 IgM isotype for its basic arabinose-containing epitope until the inhibitory effect of the terminal substitution was overcome.

[0167] The reactivity properties of A194-01 IgG and the modified IgM and scFv-IgG forms of A194-01 were further analyzed by reciprocal binding competition experiments against either ManLAM or PILAM. While all three forms cross-competed for binding of biotinylated antibodies to PILAM and A194-01 IgG to ManLAM, only the higher valency scFv-IgG and IgM forms of A194-01 efficiently competed for binding of modified antibodies to ManLAM. These differences in competitive activity were consistent with the broader binding properties of the modified forms, suggesting that they bind to sites on LAM that are not recognized by the IgG form.

[0168] This conclusion was further supported by reciprocal competition studies in which different isotypes of A194-01 competed with the binding of biotinylated FIND25 IgG and P30B9 IgM to ManLAM. While A194-01 IgG did not compete with the binding of P30B9 IgM and FIND25 to ManLAM, both the scFv-IgG and IgM forms of A194-01 almost completely competed with the binding of these two monoclonal antibodies (Figure 15A,B). As expected, all three forms of A194-01 competed with the binding of FIND25 to PILAM (Figure 15C), consistent with the absence of mannosylation in PILAM. This enhanced activity suggested that these modifications would enhance the potential usefulness of this antibody in immunodiagnostic applications and may further enhance the efficacy of these reagents for immunotherapeutic purposes.

[0169] Example 2 - Isolation and characterization of novel human monoclonal antibodies specific for Mycobacterium tuberculosis (M.tb) glycolipids - Isolation of the first mAb specific for an epitope shared by LAM and PIM6 The P95C1 antibody heavy and light chains were isolated from a single B cell clone from a patient with latent tuberculosis infection (LTBI) by screening for reactivity with ManLAM. P95C1 is an IgM isotype antibody that binds to both ManLAM and PILAM. This was shown by glycoconjugate binding studies showing that P95C1 does not bind to any molecules expressing various arabinose side chains uncapped or capped with various mannose structures. The only structures recognized were two polymannose structures with structural motifs present in the mannan base conserved between PIM6 and various LAMs (Figures 18(A), 18(B), 18(C)). This LAM-PIM6 cross-reaction was confirmed by Western blot assays showing that A194-01 and P30B9 bind only to LAM, while P95C1 also reacts with the LAM precursor glycolipid molecules LM and PIM6 (Figures 20(A), 20(B)).

[0170] While P95C1, like P30B9, was naturally expressed as an IgM, in contrast to P30B9 it retained reactivity when converted to either the IgA or IgG isotype (Figure 19). This may be a reflection of the nature of the epitope or ots position within the mannan region of the LAM molecule, or it may be related to a greater number of mutations within the P95C1 variable region that match the more mature antibody sequence. The P95C1 heavy and light chain variable regions have 19 and 13 amino acid point mutations, respectively, from their closest germline antibody sequences.

[0171] Recently, it has been shown that antibodies made by individuals with latent disease are functionally superior to those with active TB in promoting phagolysosomal fusion, inflammasome activation, and macrophage killing of internalized mycobacteria (Lu et al. 2016). It is therefore intriguing that P95C1 was isolated from an LTBI patient and harbors more mutations within its variable region than P30B9 and two other LAM-specific mAbs isolated from the same LTBI patient with different ManLAM epitope specificities.

[0172] Little is known about the nature of the human humoral immune response to Mycobacterium tuberculosis (M.tb) infection. Although it is well known that M.tb surface glycolipids contribute to the inhibition of macrophage and dendritic cell activity, there is conflicting information about whether mannose-capped lipoarabinomannan (ManLAM) or phosphatidylinositol mannoside 6 (PIM6) are the major immunosuppressive surface components of M.tb. This issue is further complicated by the common contamination of purified preparations of PIM6 with ManLAM and vice versa. One way to address this issue is to test the ability of antibodies specific for these two modifiers to block these inhibitory activities. However, this has not been possible due to the absence of well-characterized antibodies specific for these two antigens. To date, no antibodies recognizing PIM6 have been reported, and the present invention describes the first high-affinity mAb that recognizes PIM6. The PIMs (PIM2 and PIM4) are precursors to ManLAM, and some structural relationship exists between the mannan domain of ManLAM and the polymannose structure of PIM6.

[0173] Antibodies can exert their functions in two ways: 1) directly by blocking host cell entry and neutralizing bacterial products, and 2) indirectly by Fc-mediated complement and cell activation mechanisms through Fc receptors. Antibody-mediated effector functions are primarily affected by antibody isotype. Recent studies have demonstrated human isotype-dependent inhibitory antibody responses to M.tb, and demonstrated that IgA (but not IgG) antibodies specific for different M.tb surface antigens can block M.tb uptake by lung epithelial cells independent of IgA Fc receptor expression. To test the effect of P95C1 isotype on LAM binding, the constant region of P95C1-IgM heavy chain was replaced with CH-IgA and CH-IgG to generate P95C1-IgA and P95C1-IgG. The binding affinities of P95C1 isotypes (IgM, IgA, IgG) to ManLAM and PILAM were comparable (FIG. 19).

[0174] Other embodiments Any improvements may be made in some or all of the antibodies, compositions, kits and methods. All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference. Any and all examples, or use of exemplary terms provided herein (e.g., "such as") are intended to illustrate the invention and do not place limitations on the scope of the invention unless specifically required. Any statements herein regarding the nature or benefits of the invention or preferred embodiments are not intended to be limiting, and the appended claims should not be considered as being limited by such statements. More generally, no language herein should be construed to indicate any unclaimed element as essential to the practice of the invention. The present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-mentioned elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly prohibited by context.

Claims

1. 1. A human monoclonal anti-lipoarabinomannan (anti-LAM) antibody, or antigen-binding portion thereof, that specifically binds to a LAM epitope comprising an Ara4 structure, an Ara6 structure, or a combination thereof, characterized in that the anti-LAM antibody comprises a CDR1 variable light region having at least 80% identity with SEQ ID NO:1 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:2 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:26 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:4 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:5 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:6 or SEQ ID NO:23 or an antigenic fragment thereof.

2. 2. The human monoclonal anti-LAM antibody or antigen-binding portion thereof according to claim 1, characterized in that the antibody comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:23, and a light chain variable region comprising the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:

26.

3. 2. The human monoclonal anti-LAM antibody or antigen-binding portion thereof according to claim 1, wherein the anti-LAM antibody is an scFv-IgG, and an IgGa or IgM antibody.

4. A human monoclonal anti-LAM antibody or antigen-binding portion thereof, characterized in that it specifically binds to a LAM epitope comprising at least one of a mannose-capped Ara4 structure and a mannose-capped Ara6 structure.

5. 5. The human monoclonal anti-LAM antibody or antigen-binding portion thereof according to claim 4, characterized in that the anti-LAM antibody comprises a CDR1 variable light region having at least 80% identity with SEQ ID NO:7 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:8 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:9 or SEQ ID NO:32 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:10 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:11 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:12 or SEQ ID NO:29 or an antigenic fragment thereof.

6. 6. The human monoclonal anti-LAM antibody or antigen-binding portion thereof according to claim 5, characterized in that the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:43 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

44.

7. 6. The human monoclonal anti-LAM antibody, or antigen-binding portion thereof, of claim 5, wherein said anti-LAM antibody is an IgM or IgA antibody.

8. 1. A human monoclonal anti-LAM antibody, or antigen-binding portion thereof, that specifically binds to a LAM epitope comprising an α-Manp(1→2) linkage structure attached at the non-reducing end of Ara4 or Ara6, characterized in that said anti-LAM antibody comprises a CDR1 variable light region having at least 80% identity with SEQ ID NO:7 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:8 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:9 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:10 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:11 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:12 or an antigenic fragment thereof.

9. 9. The human monoclonal anti-LAM antibody, or antigen-binding portion thereof, of claim 8, wherein said anti-LAM antibody is an IgM or IgA antibody.

10. A human monoclonal anti-PIM6 / LAM antibody, or an antigen-binding portion thereof, characterized in that it specifically binds to an epitope present in LAM and PIM6, which comprises at least one polymannose structure.

11. A human monoclonal anti-PIM6 / LAM antibody, or an antigen-binding portion thereof, according to claim 10, characterized in that the epitope is present within the PIM6 mannan domain and is also present in mycobacterial lipomannan (LM).

12. 11. The human monoclonal anti-PIM6 / LAM antibody, or antigen-binding portion thereof, of claim 10, characterized in that the anti-PIM6 / LAM antibody comprises a CDR1 variable light region having at least 80% identity with SEQ ID NO: 13 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO: 14 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO: 15 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO: 16 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO: 17 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO: 18 or an antigenic fragment thereof.

13. A human monoclonal anti-PIM6 / LAM antibody, or antigen-binding portion thereof, according to claim 10, characterized in that the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

48.

14. 13. The human monoclonal anti-PIM6 / LAM antibody or antigen-binding portion thereof according to claim 12, wherein said anti-PIM6 / LAM antibody is an IgM antibody.

15. 13. The human monoclonal anti-PIM6 / LAM antibody or antigen-binding portion thereof according to claim 12, wherein said anti-PIM6 / LAM antibody is an IgA or IgG antibody.

16. A kit for detecting at least one LAM epitope comprising: (a) at least a first anti-LAM antibody that specifically binds to a LAM epitope; (b) a support to which the at least a first anti-LAM antibody is attached; (c) a detection antibody labeled with a reporter molecule that specifically binds to LAM or to the at least first anti-LAM antibody; and (d) Buffer solution wherein said at least a first anti-LAM antibody is a human monoclonal anti-LAM antibody according to claim 1, 5 or 8.

17. 17. The kit of claim 16, wherein the detection antibody is a second anti-LAM antibody that specifically binds to LAM.

18. 18. The kit of claim 17, wherein at least one of the first and second anti-LAM antibodies is a scFv-IgG or IgM antibody and comprises a CDR1 variable light region having at least 80% identity with SEQ ID NO:1 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:2 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:26 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:4 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:5 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:6 or SEQ ID NO:23 or an antigenic fragment thereof.

19. 20. The kit of claim 18, wherein at least one of the first anti-LAM antibody and the second anti-LAM antibody comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:23, and a light chain variable region comprising the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:

26.

20. 1. A method for diagnosing an active tuberculosis infection in an individual, comprising: (a) obtaining a sample from an individual containing or suspected of containing LAM; (b) treating the sample to expose LAM epitopes in the individual; (c) contacting the sample with at least a first antibody that specifically binds to a first epitope on the LAM; (d) contacting the sample with a detection antibody that specifically binds to LAM or to the at least first antibody; (e) detecting binding of the at least a first antibody to the first epitope on LAM; (f) diagnosing the patient as having an active tuberculosis infection; wherein the binding of the at least a first antibody to the first epitope on LAM is indicative of an active tuberculosis infection; and The method, wherein said at least a first antibody is a human monoclonal anti-LAM antibody according to claim 1, 5 or 8, or a human monoclonal anti-PIM6 / LAM antibody according to claim 12.

21. 21. The method of claim 20, wherein the detection antibody is an anti-LAM antibody that specifically binds to LAM.

22. 22. The method of claim 21, wherein the at least first antibody and the detection antibody each comprise a CDR1 variable light region having at least 80% identity with SEQ ID NO:1 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:2 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:26 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:4 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:5 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:6 or SEQ ID NO:23 or an antigenic fragment thereof.

23. 22. The method of claim 21, wherein at least one of the primary antibody and the detection antibody is an scFv-IgG or IgM antibody and comprises a CDR1 region having a variable light region having at least 80% identity with SEQ ID NO:1 or an antigenic fragment thereof, a CDR2 variable light region having at least 80% identity with SEQ ID NO:2 or an antigenic fragment thereof, a CDR3 variable light region having at least 80% identity with SEQ ID NO:3 or SEQ ID NO:26 or an antigenic fragment thereof, a CDR1 variable heavy region having at least 80% identity with SEQ ID NO:4 or an antigenic fragment thereof, a CDR2 variable heavy region having at least 80% identity with SEQ ID NO:5 or an antigenic fragment thereof, and a CDR3 variable heavy region having at least 80% identity with SEQ ID NO:6 or SEQ ID NO:23 or an antigenic fragment thereof.

24. 21. The method of claim 20, wherein the individual is a human.

25. A method for treating tuberculosis infection in an individual, comprising administering to the individual a therapeutically effective amount of at least one human monoclonal anti-LAM antibody of claim 1, 5 or 8, or a human monoclonal anti-PIM6 / LAM antibody of claim 12.

26. 26. The method of claim 25, further comprising administering to the individual a therapeutically effective amount of at least one antibiotic.

27. 26. The method of claim 25, wherein the tuberculosis infection is a multi-drug resistant (MDR-TB) tuberculosis infection.

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