WO2017204349A1

WO2017204349A1 - Methods and assays for estimating acid-fast bacteria viability, and kits therefor

Info

Publication number: WO2017204349A1
Application number: PCT/JP2017/019794
Authority: WO
Inventors: Masanori Kawasaki; Yongge Liu; Kiyonori Katsuragi
Original assignee: Otsuka Pharmaceutical Co., Ltd.
Priority date: 2016-05-27
Filing date: 2017-05-26
Publication date: 2017-11-30

Abstract

The present invention relates to assays and methods for calculating, estimating, or determining, the amount of viable acid-fast bacilli in a sample based on the result of the detection of lipoarabinomannan (LAM). The present disclosure further relates to the detection of LAM of acid-fast bacilli using an antibody, and calculating, estimating, or determining, the amount of viable acid-fast bacilli in a sample based on the result of the detection of LAM. These assays and methods may be used for the monitoring of treatments for tuberculosis, and for the development and testing of new treatments.

Description

METHODS AND ASSAYS FOR ESTIMATING ACID-FAST BACTERIA VIABILITY, AND KITS THEREFOR

This application relates generally to assays and methods for calculating, estimating, or determining, the amount of viable acid-fast bacilli in a sample. Assays and methods contemplated herein include the detection of lipoarabinomannan (LAM) of acid-fast bacilli, and calculating, estimating, or determining, the amount of viable acid-fast bacilli in a sample based on the result of the LAM detection. The present disclosure further relates to the detection of LAM of acid-fast bacilli using a binding agent such as an antibody.

Tuberculosis, caused by infection with Mycobacterium tuberculosis, ranks alongside Human Immunodeficiency Virus (HIV) as a leading cause of death worldwide. In 2014, the estimated global incidence of tuberculosis was 9.6 million cases, with 1.5 million deaths attributable to M. tuberculosis infection. This continued morbidity and mortality results from deficiencies in each of the diagnosis, treatment, and prevention, of tuberculosis. Until about 2010, tuberculosis diagnostics were principally based on traditional microbiological assays, including the microscopic analysis of sputum smears, and the culturing of clinical samples to grow M. tuberculosis. However, these traditional assays present significant drawbacks. For example, the microscopic analysis of sputum smears is of very low sensitivity, and the culturing of clinical samples to grow acid-fast bacilli requires several weeks to provide a result, due to the extremely slow growth of M. tuberculosis.

Recent advancements in tuberculosis diagnostics include the development of Xpert MTB/RIF. See, e.g., World Health Organization (“WHO Global Tuberculosis Report 2015” at apps.who.int/iris/bitstream/10665/191102/1/9789241565059_eng.pdf) and World Health Organization (“Roadmap for rolling out Xpert MTB/RIF for rapid diagnosis of TB and MDR-TB,” 2010, at www.who.int/tb/laboratory/roadmap_xpert_mtb-rif.pdf). Xpert MTB/RIF is based on the detection of DNA, and can be used to diagnose multidrug-resistant tuberculosis (by detecting rifampin resistance, an indicator of multidrug-resistant tuberculosis).

Treatment for tuberculosis typically involves the same general treatment regimen without obtaining drug susceptibility. This general treatment regimen follows the direct observed therapy-short course (DOTs), to ensure compliance and the completion of treatment. However, in addition to a lack of safe and effective tuberculosis treatments, there exists no real-time tool or assay that can be used to accurately estimate the number of viable bacteria. As a consequence, it is difficult to monitor the effectiveness of treatment. This inevitably results in good responders being treated for an unnecessarily long time (and often, with drugs that cause significant side-effects), and results in poor responders not being identified soon enough. Poor responders can either be individuals who are non-compliant (e.g., they do not take the required drugs at all, or do not take the required drugs for long enough), or may be individuals who are compliant, but nonetheless are infected with M. tuberculosis isolates that are resistant to the drugs being taken. Poor responders have the potential to continue to spread the infection to others, and to facilitate the progression of further antibiotic resistance.

In this connection, microscopic analysis of sputum smears has often been used as a detection assay. However, microscopic analysis of sputum smears is generally unable to distinguish between viable bacteria and dead bacteria, and consequently, it exhibits poor sensitivity, and poor quantitative value. Conversely, the culturing of clinical samples (to grow M. tuberculosis) provides a quantitative measure of viable bacterial number, since dead bacteria are unable to grow and be detectable by this assay. But a major drawback of this assay is the time needed to obtain a result, which is generally several weeks. As a consequence, the ability to monitor compliance with, or the effectiveness of, tuberculosis treatment still lags far behind other infections, such as hepatitis C (HCV) or HIV, in which viral load can be rapidly detected, and used effectively as a monitoring tool to determine effectiveness of treatment. See, e.g., “Recommendations for Testing, Managing, and Treating Hepatitis C,” Am. Assoc. Study Liver Dis., 2016: 1-51, available from www.hcvguidelines.org; see also World Health Organization (“Consolidated ARV Guidelines 2013,” Geneva World Health Organ., 2013, 14(7): 269, available from: www.who.int/hiv/pub/guidelines/arv2013/); and see “How Viral Load Monitoring Can Improve HIV Treatment,” Med. Sans Front., 2012.

As noted above, traditional tuberculosis detection methods and assays relied upon sputum smear microscopy, and bacterial culture. While sputum smears may be obtained and analyzed on the same day, it has very poor quantitative value, since it cannot reliably distinguish between viable and dead bacilli. Accordingly, its use for the monitoring of treatment efficacy or response is questionable. Bacterial culture overcomes this limitation, since the culturing only grows up viable bacteria (and thus does not detect dead bacteria). Hence, the growth of M. tuberculosis on agar (solid culture) is recognized as the gold standard for detecting viable M. tuberculosis; the number of viable bacteria can be quantified by counting the number of colony-forming units (CFU) observable after several weeks of growth. However, the results of such solid-culture analysis are only available after 4-8 weeks of culture. Recent improvements in liquid-culture have reduced the culture time needed before a result can be obtained, but at least 2-4 weeks is still required before a result is obtained.

One such liquid-culture method is called “Mycobacteria Growth Indicator Tube” (MGIT), which automates the culturing and detection. The time-to-detection (TTD) in MGIT has consistently been shown to correlate closely with solid-medium CFU counts. See Bark et al. (“Comparison of time to positive and colony counting in an early bactericidal activity study of anti-tuberculosis treatment,” Int. J. Tuberc. Lung Dis., 2013, 17(11): 1448-51); Bark et al. (“Time to detection of Mycobacterium tuberculosis as an alternative to quantitative cultures,” Tuberculosis, 2011, 91(3): 257-9); Diacon et al. (“Time to detection of the growth of Mycobacterium tuberculosis in MGIT 960 for determining the early bactericidal activity of anti-tuberculosis agents,” Eur. J. Clin. Microbiol. Infect. Dis., 2010, 29(12): 1561-5); and Diacon et al. (“Time to liquid culture positivity can substitute for colony counting on agar plates in early bactericidal activity studies of antituberculosis agents,” Clin. Microbiol. Infect., 2012, 18(7): 711-7). Because of the ease-of-use of MGIT versus solid-culture, MGIT-TTD has been used as a surrogate marker of viable bacterial number. However, it still takes several weeks to obtain a result, it is not a real-time read, and therefore has limited utility in monitoring response to treatment.

The MGIT assay consists of liquid broth medium that is known to yield better recovery and faster growth of mycobacteria than solid culture. The MGIT tube, in addition to the liquid medium, contains an oxygen-quenched fluorochrome embedded in silicone at the bottom of the tube. During bacterial growth within the tube, the free oxygen is utilized and is replaced with carbon dioxide. With depletion of free oxygen, the fluorochrome is no longer inhibited, resulting in fluorescence within the MGIT tube when visualized under UV light. The intensity of fluorescence is directly proportional to the extent of oxygen depletion. MGIT tubes may be incubated at 37℃ and read manually under a UV light, or entered into a MGIT 960 instrument where they are incubated and monitored for increasing fluorescence every 60 minutes. For the MGIT 960 instrument, once the fluorescence reaches a pre-specified level and automatically detected by the instrument, it will signal “culture positive.” The time to this positivity is called MGIT-time to detection (TTD). The higher the number of viable bacteria in the clinical specimen, the quicker the tube becomes culture positive, and thus the shorter the TTD. It has been determined that the MGIT-TTD is a surrogate marker of colony-forming unit (CFU) counts on solid media culture. The instrument declares a tube negative if it remains negative for six weeks (42 days). Because of the automation of the MGIT system, MGIT has become the accepted method for measuring viable bacterial number.

Nucleic acid amplification-based tests (NAAT), such as Xpert, generally provide good sensitivity and specificity for diagnosing tuberculosis infection. The Xpert MTB/RIF Assay, performed on the GeneXpert Instrument Systems, is a qualitative, nested real-time polymerase chain reaction (PCR) in vitro diagnostic test for the detection of M. tuberculosis complex DNA in raw sputum or concentrated sediments prepared from induced or expectorated sputum. In specimens where M. tuberculosis complex (MTB-complex) is detected, the Xpert MTB/RIF Assay also detects the rifampin-resistance associated mutations of the rpoB gene (which is a surrogate marker of multidrug-resistant tuberculosis). The Xpert MTB/RIF Assay is intended for use with specimens from patients for whom there is clinical suspicion of tuberculosis, and who have received no anti-tuberculosis therapy (or less than 3 days of therapy). This test is intended as an aid in the diagnosis of pulmonary tuberculosis when used in conjunction with clinical and other laboratory findings.

However, because genomic material from both live- and dead-bacteria is generally present in sputum samples from tuberculosis patients, the detection of the mycobacterial DNA does not distinguish between the existence of live and dead bacteria. Therefore, NAAT assays may provide a positive result even from samples from patients that are culture-negative. This observation has been reported in studies using traditional NAAT methods. See Hellyer et al. (“Strand displacement amplification and the polymerase chain reaction for monitoring response to treatment in patients with pulmonary tuberculosis,” J. Infect. Dis., 1996, 173: 934-41); and Thomsen et al. (“Monitoring Treatment of Patients with Pulmonary Tuberculosis: Can PCR Be Applied?,” J. Clin. Microbiol., 1999, 37(11): 3601-7).

Furthermore, newer NAAT methods, such as Xpert MTB/RIF, have also failed to detect the decrease in viable bacilli during effective tuberculosis treatment. In a study by Friedrich et al. (“Assessment of the sensitivity and specificity of Xpert MTB/RIF assay as an early sputum biomarker of response to tuberculosis treatment,” Lancet Respir. Med., 2013, 1(6): 462-70), the sputa from tuberculosis patients obtained during treatment were examined by an Xpert MTB/RIF test, using both positivity and real-time amplification cycle-threshold, and by culturing. The Xpert MTB/RIF test showed a poor correlation with the bacterial culture results. Further, in another study directly comparing the Xpert MTB/RIF assay with liquid- and solid-culture for quantification of early bactericidal activity, the Xpert MTB/RIF assay also showed a poor correlation with the bacterial culture results. See Kayigire et al. (“Direct comparison of Xpert MTB/RIF assay with liquid and solid mycobacterial culture for quantification of early bactericidal activity,” J. Clin. Microbiol., 2013, 51(6): 1894-8). Further still, Boyles et al. (“False-positive Xpert?? MTB/RIF assays in previously treated patients: Need for caution in interpreting results,” Int. J. Tuberc. Lung Dis., 2014, 18(7): 876-8) reported that false-positive Xpert MTB/RIF results were identified in patients months, and even years, after successful tuberculosis treatment (with the sputum samples from such patients being culture-negative). Accordingly, NAAT methods based on the amplification of DNA cannot reliably be used to estimate the number of viable bacteria that remain during treatment. Although the level of M. tuberculosis RNA might potentially be able to better differentiate between live and dead bacilli, because RNA has a much shorter half-life than DNA, RNA detection is much more challenging due to its instability and significant loss during sputum manipulation. See Desjardin et al. (“Measurement of Sputum Mycobacterium tuberculosis Messenger RNA as a Surrogate for Response to Chemotherapy,” American Journal of Respiratory and Critical Care Medicine, 1999, 160(1): 203); Hellyer et al. (“Quantitative analysis of mRNA as a marker of viability of Mycobacterium tuberculosis,” J. Clin. Microbiol., 1999, 37(2): 290-5); Honeyborne et al. (“Molecular bacterial load assay, a culture-free biomarker for rapid and accurate quantification of sputum Mycobacterium tuberculosis bacillary load during treatment,” J. Clin. Microbiol., 2011, 49(11): 3905-11); and Honeyborne et al. (“The molecular bacterial load assay replaces solid culture for measuring early bactericidal response to antituberculosis treatment,” J. Clin. Microbiol., 2014, 52(8): 3064-7).

In sum, a need exists for a real-time measurement assay that can estimate the number of viable bacteria in sputum specimens from tuberculosis patients. See Datta et al. (“Clinical Evaluation of Tuberculosis Viability Microscopy for Assessing Treatment Response,” Clin. Inf. Dis., 2015, 60: 1186-1195); and Lawn et al. (“Dead or Alive: Can Viability Staining Predict Response to Tuberculosis Treatment?,” Clin. Inf. Dis., 2015, 60: 1196-1198). At present, the lack of such an available assay hinders the development of new tuberculosis drugs and treatments, and negatively impacts the management of patient treatment.

LAM is a major component of the M. tuberculosis cell wall, and may constitute up to about 1.5% of the total bacterial weight. See Hunter et al. (“Structure and antigenicity of the phosphorylated lipopolysaccharide antigens from the leprosy and tubercle bacilli,” J. Biol. Chem., 1986, 261(26): 12345-51). Antibody-based immunoassays for the detection of LAM in urine have been evaluated for their ability to detect tuberculosis infection. One such immunoassay is Clearview (registered TM) TB ELISA (Inverness Medical Innovations), which used polyclonal anti-LAM antibodies. This assay exhibits very low sensitivity, but moderate specificity, for the detection of tuberculosis infection via the detection of LAM in urine. See Dheda et al. (“Clinical utility of a commercial LAM-ELISA assay for TB diagnosis in HIV-infected patients using urine and sputum samples, PLoS One, 2010, 5(3): 1-8); and Hanifa et al. (“The diagnostic accuracy of urine LAM test for tuberculosis screening in a South African correctional facility,” PLoS One, 2015, 10(5): e0127956). Another immunoassay is Determine-TB LAM Ag (registered TM) from Alere, which is an immunochromatographic test using anti-LAM polyclonal antibodies to detect LAM in urine. Its use is limited to severe AIDS patients whose CD4 counts are less than 200/mm³, because of poor sensitivity in non-HIV patients. See Minion et al. (“Diagnosing tuberculosis with urine lipoarabinomannan: Systematic review and meta-analysis,” Eur. Respir. J., 2011, 38(6): 1398-405).

Because LAM is a major component of the bacterial cell wall, LAM should be contained in both live and dead bacilli. LAM detection in sputum was expected to be useful for diagnosis, but not for estimating the number of viable bacteria (since LAM is released from dead bacilli also). The present inventors have discovered, however, that the detection of LAM in sputum unexpectedly correlates closely with the number of viable bacteria.

WO2013/129634 A1 US20160083458 A1

WHO Global Tuberculosis Report 2015 Roadmap for rolling out Xpert MTB/RIF for rapid diagnosis of TB and MDR-TB, 2010 Am. Assoc. Study Liver Dis., 2016: 1-51 World Health Organization, Consolidated ARV Guidelines 2013, Geneva World Health Organ., 2013, 14(7): 269 How Viral Load Monitoring Can Improve HIV Treatment, Med. Sans Front., 2012. Bark et al., Int. J. Tuberc. Lung Dis., 2013, 17(11): 1448-51 Bark et al., Tuberculosis, 2011, 91(3): 257-9 Diacon et al., Eur. J. Clin. Microbiol. Infect. Dis., 2010, 29(12): 1561-5 Diacon et al., Clin. Microbiol. Infect., 2012, 18(7): 711-7 Hellyer et al., J. Infect. Dis., 1996, 173: 934-41 Thomsen et al., J. Clin. Microbiol., 1999, 37(11): 3601-7 Friedrich et al., Lancet Respir. Med., 2013, 1(6): 462-70 Kayigire et al., J. Clin. Microbiol., 2013, 51(6): 1894-8 Boyles et al.. Int. J. Tuberc. Lung Dis., 2014, 18(7): 876-8 Desjardin et al., American Journal of Respiratory and Critical Care Medicine, 1999, 160(1): 203 Hellyer et al., J. Clin. Microbiol., 1999, 37(2): 290-5 Honeyborne et al., J. Clin. Microbiol., 2011, 49(11): 3905-11 Honeyborne et al., J. Clin. Microbiol., 2014, 52(8): 3064-7 Datta et al., Clin. Inf. Dis., 2015, 60: 1186-1195 Lawn et al., Clin. Inf. Dis., 2015, 60: 1196-1198 Hunter et al., J. Biol. Chem., 1986, 261(26): 12345-51 Dheda et al., PLoS One, 2010, 5(3): 1-8 Hanifa et al., PLoS One, 2015, 10(5): e0127956). Minion et al., Eur. Respir. J., 2011, 38(6): 1398-405

This application relates generally to assays and methods for calculating, estimating, or determining, the amount of viable acid-fast bacilli in a sample. Assays and methods contemplated by the present disclosure include the detection of LAM of acid-fast bacilli, such as Mycobacterium tuberculosis, and calculating, estimating, or determining, the amount of viable acid-fast bacilli in a sample based on the result of the detection of LAM. The present disclosure further relates to the detection of LAM of acid-fast bacilli using a binding agent such as an antibody, and calculating, estimating, or determining, the amount of viable acid-fast bacilli in a sample based on the result of the detection of LAM.

The present disclosure further relates to non-culture-based methods for estimating the bacterial load in a sample, to be used in place of a liquid- or a solid culture-based assay for estimating bacterial load. The bacterial load in the sample may be determined based on the amount of viable acid-fast bacilli determined as above.

The present disclosure relates to methods for evaluating the efficacy of treatment regimen for tuberculosis based on the estimation. The present disclosure further relates to treatment methods, and methods for modifying tuberculosis treatments based on the evaluations provided herein.

The present disclosure also relates to a kit for calculating, estimating, or determining, the amount of viable acid-fast bacilli, such as M. tuberculosis, in a sample. The kit may include an anti-LAM antibody.

Fig. 1 depicts the correlation of LAM concentration with MGIT-TTD in sputa obtained from tuberculosis patients prior to treatment. Fig. 2 depicts the positivity of different detection methods (LAM, MGIT and NAAT) during anti-tuberculosis treatment. It shows that the LAM positivity tracks that from MGIT, but NAAT stays positive most of in MGIT-negative samples. Graphs in Fig. 3 depict the concentration of LAM, and the MGIT-TTD, for individual patients during anti-tuberculosis treatment, and shows that a decrease in LAM closely correlates with prolongation of MGIT-TTD. X axis: treatment days, left Y axis: MGIT TTD (hour), Right Y axis: Log10 LAM (pg/mL). Open symbol: negative for MGIT culture; below detection limit for LAM. Continuation of Fig 3-1. Continuation of Fig 3-2. Continuation of Fig. 3-3. Continuation of Fig. 3-4. Continuation of Fig. 3-5. Continuation of Fig. 3-6. Continuation of Fig. 3-7. Continuation of Fig. 3-8. Continuation of Fig. 3-9.

Detailed Description of The Invention

The details of embodiments of the presently disclosed subject matter are set forth in the accompanying description below. Other features, objects, and advantages of the presently disclosed subject matter will be apparent from the specification, figures, and claims. All publications, patent applications, patents, and other references noted herein are incorporated by reference in their entirety.

In some embodiments of the present disclosure, and as described herein and in WO 2013129634A1, the entire disclosure of which is herein incorporated by reference, the anti-LAM binding agent is an antibody that specifically binds to LAM of acid-fast bacilli. In some embodiments, the antibody is able to distinguish and specifically recognize an acid-fast bacilli from other bacteria existing in vivo, and, for example, may be able to distinguish LAM of acid-fast bacilli from other LAM-like antigens of bacteria. In certain embodiments, the anti-LAM antibody can distinguish between LAM from tubercle bacilli and LAM from non-tuberculous acid-fast bacilli. LAM is one of the main lipoglycans forming cell membranes and cell walls of bacteria in the genus Mycobacterium (acid-fast bacilli) including tubercle bacilli. Ordinarily, LAM includes a mannosyl phosphatidylinositol anchor (MPI), a sugar backbone including a D-mannan core and a D-arabinan domain, and a capping motif. However, depending on the type of bacteria, there are differences in the number of residues of sugar (e.g., mannose) included within a molecule, branched structure of a sugar chain, the number of acyl groups, and the type of fatty acids forming the acyl groups.

Tubercle bacilli belong to the genus Mycobacterium of the family Mycobacteriaceae, and are a type of a bacterial group referred together with other bacteria belonging to the genus Mycobacterium as acid-fast bacilli. However, tubercle bacilli are distinguished from other acid-fast bacilli (nontuberculous acid-fast bacilli) by the fact that they can grow at 37°C but not at 28°C, and by the fact that they have a heat-resistant catalase. Four types of tubercle bacilli are known, i.e., tubercle bacillus (Mycobacterium tuberculosis, human tubercle bacillus), bovine tubercle bacillus (M. bovis, bovine tubercle bacillus, bovine bacillus), Mycobacterium africanum (M. africanum), and vole tubercle bacillus (M. microti). Among these, human tubercle bacillus (M. tuberculosis) is pathogenic for humans as a bacterium causing tuberculosis, and M. bovis and M. africanum infect humans on rare occasions. M. microti is not pathogenic for humans. Furthermore, BCG was obtained by attenuating M. bovis through successive long-term subculturing, and is used as a vaccine (attenuated live bacteria vaccine) for tuberculosis prevention.

In some embodiments, the antibody is, for example, a human antibody, a mouse antibody, a rat antibody, a domestic fowl antibody, a rabbit antibody or a goat antibody. It may also be a polyclonal or monoclonal antibody, or a variant thereof (such as an F(ab’)₂, Fab’, Fab or Fv fragment). The antibody may also be chimeric, humanized, or completely human.

Here, “CDR” is an abbreviation of “Complementarity Determining Region.” CDRs are regions that exist in a variable region of immunoglobulin, and are regions involved in specific binding of an antibody to an antigen. Among those, “a heavy chain CDR” refers to a CDR that exists in a variable region of a heavy chain of immunoglobulin, and “a light chain CDR” refers to a CDR that exists in a variable region of a light chain of immunoglobulin.

The heavy chain variable region is a region generally including heavy chain CDR1 to CDR3, and the light chain variable region is a region generally including light chain CDR1 to CDR3. Although there is no particular limitation in the arrangement order of these CDR1 to CDR3, preferably, both in the heavy chain variable region and light chain variable region, CDR1, CDR2, and CDR3 are arranged in this order in a direction from the N-terminal side to the C-terminal side continuously or through other amino acid sequences.

The heavy chain variable region and/or light chain variable region may have, as other amino acid sequences, amino acid sequences referred to as framework region sequences (hereinafter, simply referred to as “FR”). The amino acid sequence of the FR may be an amino acid sequence derived from a framework region (FR) of a heavy chain variable region or a light chain variable region of immunoglobulin, a variant thereof, or a partial modification thereof obtained by introducing a restriction enzyme recognition site at one part of the amino acid sequence derived from an FR.

In the heavy chain variable regions of immunoglobulin, for example, a region between the N-terminal of the heavy chain variable region and CDR1 described above is defined as “FR1,” a region between CDR1 and CDR2 is defined as “FR2,” a region between CDR2 and CDR3 is defined as “FR3,” and a region between CDR3 and the C-terminal of the heavy chain variable region is defined as “FR4.” Similarly, in the light chain variable region of immunoglobulin, for example, a region between the N-terminal of the light chain variable region and CDR1 is defined as “FR1,” a region between CDR1 and CDR2 is defined as “FR2,” a region between CDR2 and CDR3 is defined as “FR3,” and a region between “CDR3” and the C-terminal of the variable region is defined as “FR4.”

These FRs have a function as a linker connecting each of the above-described CDR1, CDR2, and CDR3 that are important as antigen recognition sequences, and are regions contributing to formation of three-dimensional conformation of variable regions.

In one embodiment of the invention, the antibody contains the following CDR sequences (a)-(f):
(a) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1.
(b) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2.
(c) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3.
(d) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4.
(e) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5.
(f) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6.

In another embodiment of the invention, the antibody contains the following CDR sequences (g) to (l):
(g) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14.
(h) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 15.
(i) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 16.
(j) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17.
(k) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18.
(l) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19.

In yet another embodiment of the invention, the antibody contains the following CDR sequences (m)-(r):
(m) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 24.
(n) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 25.
(o) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 26.
(p) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 27.
(q) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 28.
(r) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 29.

In some embodiments of the invention, the antibody contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In SEQ ID NO: 7, a region from the N-terminal to the 30th amino acid corresponds to “FR1” of the heavy chain variable region, an amino acid region from the 31st amino acid to the 35th amino acid corresponds to “CDR1” (SEQ ID NO: 1) of the heavy chain variable region, an amino acid region from the 36th amino acid to the 49th amino acid corresponds to “FR2,” an amino acid region from the 50th amino acid to the 65th amino acid corresponds to “CDR2” (SEQ ID NO: 2), an amino acid region from the 66th amino acid to the 96th amino acid corresponds to “FR3,” an amino acid region from the 97th amino acid to the 106th amino acid corresponds to “CDR3” (SEQ ID NO: 3), and an amino acid region from the 107th amino acid to the 119th amino acid corresponds to “FR4.”

Furthermore, in SEQ ID NO: 8, a region from the N-terminal to the 23rd amino acid corresponds to “FR1” of the light chain variable region, an amino acid region from the 24th amino acid to the 36th amino acid corresponds to “CDR1” (SEQ ID NO: 4) of the light chain variable region, an amino acid region from the 37th amino acid to the 51st amino acid corresponds to “FR2,” an amino acid region from the 52nd amino acid to the 58th amino acid corresponds to “CDR2” (SEQ ID NO: 5), an amino acid region from the 59th amino acid to the 89th amino acid corresponds to “FR3,” an amino acid region from the 90th amino acid to the 102nd amino acid corresponds to “CDR3” (SEQ ID NO: 6), and an amino acid region from the 103rd amino acid to the 112nd amino acid corresponds to “FR4.”

In other embodiments of the invention, the antibody contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21. In SEQ ID NO: 20, a region from the N-terminal to the 35th amino acid corresponds to “FR1” of the heavy chain variable region, an amino acid region from the 36th amino acid to the 40th amino acid corresponds to “CDR1” (SEQ ID NO: 14) of the heavy chain variable region, an amino acid region from the 41st amino acid to the 54th amino acid corresponds to “FR2,” an amino acid region from the 55th amino acid to the 74th amino acid corresponds to “CDR2” (SEQ ID NO: 15), an amino acid region from the 75th amino acid to the 106th amino acid corresponds to “FR3,” an amino acid region from the 107th amino acid to the 119th amino acid corresponds to “CDR3” (SEQ ID NO: 16), and an amino acid region from the 120th amino acid to the 130th amino acid corresponds to “FR4.”

Furthermore, in SEQ ID NO: 20, a region from the N-terminal to the 20th amino acid corresponds to “FR1” of the light chain variable region, an amino acid region from the 21st amino acid to the 28th amino acid corresponds to “CDR1” (SEQ ID NO: 17) of the light chain variable region, an amino acid region from the 29th amino acid to the 44th amino acid corresponds to “FR2,” an amino acid region from the 45th amino acid to the 51st amino acid corresponds to “CDR2” (SEQ ID NO: 18), an amino acid region from the 52nd amino acid to the 83rd amino acid corresponds to “FR3,” an amino acid region from the 84th amino acid to the 95th amino acid corresponds to “CDR3” (SEQ ID NO: 19), and an amino acid region from the 96th amino acid to the 116th amino acid corresponds to “FR4.”

In other embodiments of the invention, the antibody contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31. In SEQ ID NO: 30, a region from the N-terminal to the 30th amino acid corresponds to “FR1” of the heavy chain variable region, an amino acid region from the 31st amino acid to the 35th amino acid corresponds to “CDR1” (SEQ ID NO: 24) of the heavy chain variable region, an amino acid region from the 36th amino acid to the 49th amino acid corresponds to “FR2,” an amino acid region from the 50th amino acid to the 65th amino acid corresponds to “CDR2” (SEQ ID NO: 25), an amino acid region from the 66th amino acid to the 96th amino acid corresponds to “FR3,” an amino acid region from the 97th amino acid to the 108th amino acid corresponds to “CDR3” (SEQ ID NO: 26), and an amino acid region from the 109th amino acid to the 121st amino acid corresponds to “FR4.”

Furthermore, in SEQ ID NO: 54, a region from the N-terminal to the 23rd amino acid corresponds to “FR1” of the light chain variable region, an amino acid region from the 24th amino acid to the 34th amino acid corresponds to “CDR1” (SEQ ID NO: 27) of the light chain variable region, an amino acid region from the 35th amino acid to the 49th amino acid corresponds to “FR2,” an amino acid region from the 50th amino acid to the 56th amino acid corresponds to “CDR2” (SEQ ID NO: 28), an amino acid region from the 57th amino acid to the 87th amino acid corresponds to “FR3,” an amino acid region from the 88th amino acid to the 100th amino acid corresponds to “CDR3” (SEQ ID NO: 29), and an amino acid region from the 101st amino acid to the 110th amino acid corresponds to “FR4.”

The present disclosure further contemplates mutations, including additions, insertions, substitutions and/or deletions, within antibody sequences, including, for example, in a CDR sequence, within a framework region (such as in any of FR1 to FR4 of the heavy chain variable region, and/or in any of FR1 to FR4 of the light chain variable region, or in any variable or constant region). Although there is no particular limitation in the number of mutations that can be introduced, the number of introduced mutations may be set such that the amino acid sequence identity with that before mutation is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Also, the number of mutations within a heavy or light chain variable domain, or within a particular region within a heavy or light chain variable domain, such as a CDR region or a framework region, may be 1-100, 1-50, 1-30, 1-20, 1-10, or 1-5.

A framework region derived from any animal species may be used. Examples of such animal species may include, but are not particularly limited to, human, rabbit, chicken, horse, cow, goat, sheep, dog, mouse, hamster, and rat. In some embodiments, the amino acid sequences are preferably derived from rabbit, chicken, or human, and more preferably from human. It should be noted that the amino acid sequences of human-derived FR1 to FR4 are known in the art (Kabat, et al. US Department of Health AND human Services, NIH (1991), USA), and is described in, for example, a website by NCBI.

In some embodiments of the invention, the antibody has a structure in which a heavy chain variable region and a light chain variable region are connected, either directly, or indirectly, such as through a linker. The linker may be, for example, a peptide having a linker sequence formed of an amino acid sequence whose number of amino acid residues is ordinarily about 8 to 30, about 8 to 20, or about 8 to 15. Examples of preferable linker sequences include, but are not limited to, a GS linker sequence [(Gly-Gly-Gly-Ser: SEQ ID NO: 9)_n, (Gly-Gly-Gly-Gly-Ser: SEQ ID NO: 10)_n; n is the number of repeats] or the like. In some embodiments, a peptide having a sequence with 1 to 3 (n is an integer of 1 to 3) repeats of such a GS linker sequence is used as the linker. In other embodiments, a peptide having a sequence (GGGGSGGGGSGGGGS: SEQ ID NO: 11) with three repeats of the GS linker sequence, and a peptide (Example 6) having another sequence (GGGGSGGDGSGGGGS: SEQ ID NO: 23) are used as the linker.

In some embodiments, the antibody is a single-chain antibody containing the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the antibody is a single-chain antibody containing the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the antibody is a single-chain antibody containing the amino acid sequence of SEQ ID NO: 13.

Antibodies of the present invention also include an antibody that distinguishes and specifically recognizes tubercle bacillus from nontuberculous acid-fast bacilli, including an antibody that distinguishes LAM of tubercle bacillus from LAMs of nontuberculous acid-fast bacilli, and specifically binds to tubercle bacillary LAM. The tubercle bacillus that is distinguished from non-tubercle bacillary acid-fast bacilli and is specifically recognized by an antibody of the present invention may be human tubercle bacillus (M. tuberculosis) and bovine tubercle bacillus (M. bovis).

As described in WO 2013129634A1, the entire disclosure of which is herein incorporated by reference, when reaction against tubercle bacillary LAM is compared through a competition method, specific binding with respect to tubercle bacillary LAM can be said to be occurring when the required amount of nontuberculous acid-fast bacilli LAM is 10 times or more of that of tubercle bacillary LAM. Furthermore, when LAM is detected through sandwiching by an immobilized antibody and a detection antibody, the antibody of the present disclosure can be determined as having more preferable binding specificity with respect to tubercle bacillary LAM if reactivity to nontuberculous acid-fast bacilli LAM has been reduced to between 1/10 and 1/100, for example, of that to tubercle bacillary LAM.

Affinity of an antibody can be easily measured with a known technology, for example, measuring a saturation binding isotherm of ¹²⁵I labeled IgG or its fragment, or through non-linear regression analysis using homologous substitution of ¹²⁵I IgG by non-labeled IgG as described by Motilsky in Analyzing Data with GraphPad Prizm (1999), GraphPad Software Inc., San Diego, CA. Other methods known in the art may be used for the measurement, and the method may be, for example, a method described in Scatchard et al. Ann. NY Acd. Sci., 51,660 (1949).

The antibody of the present invention may be produced in accordance with, but not limited to, the phage display method (G. Smith, Science, 228, 1315 (1985)), using tubercle bacillus as an antigen. Here, examples of tubercle bacilli may include the above-described tubercle bacillus (Mycobacterium tuberculosis, human tubercle bacillus), bovine tubercle bacillus (M. bovis, bovine tubercle bacillus, bovine bacillus), Mycobacterium africanum, and vole tubercle bacillus. Tubercle bacillus (Mycobacterium tuberculosis, human tubercle bacillus), bovine tubercle bacillus (M. bovis, bovine tubercle bacillus, bovine bacillus) and Mycobacterium africanum are preferable, and bovine tubercle bacillus is more preferable. As described above, BCG is obtained by attenuating bovine tubercle bacillus (M. bovis) through successive long-term subculturing.

Furthermore, the antibody of the present disclosure includes a multivalent antibody, including a single-chain antibody as described above. Such multivalent antibodies include bivalent antibodies, trivalent antibodies, and tetravalent antibodies. Such multivalent antibodies can be produced in accordance with a known method (K. Zuberbuhler, Protein Engineering, Design & Selection, 22, 169 (2009)). Specifically, a multivalent antibody can be produced by, for example, in the case with a bivalent antibody, connecting genes of a heavy chain and a light chain of a single-chain antibody using a gene of a constant region, cloning the connected genes in a vector capable of expressing it in mammalian cells, transforming mammalian cells with the vector including the genes, and culturing the cells.

Antibodies contemplated by the present disclosure may also be produced, for example, by immunizing a nonhuman animal with an immunogen. The non-human animal may be an animal other than human, and examples thereof include mammals such as mouse, rat, hamster, guinea pig, rabbit, monkey, dog, goat, sheep, pig, horse, and cow, and birds such as chicken, duck, turkey, and quail. Mammals (small animals) such as mouse, rat, hamster, guinea pig, and rabbit are preferable, and rabbit is more preferable. In some embodiments, BCG may be used as an immunogen (immunizing antigen); and the technique for immunization is not particularly limited and a method known in the art can be appropriately selected to be used. Examples thereof include a method of administration through subcutaneous, intravenous, or intra-abdominal injection of BCG together with, if necessary, an adjuvant. Subcutaneous administration is preferable. Examples of the adjuvant may include, but are not limited to, Complete Freund’s adjuvant and Incomplete Freund’s adjuvant. It should be noted that administration of BCG is preferably performed for about 2 to 5 times with an interval of about 2 weeks after the first administration (first immunization).

Spleen cells of a non-human animal immunized in such a manner are useful as cells for producing an anti-LAM antibody. The spleen is removed from the immunized non-human animal in ten-odd days to several months after first immunization of BCG, and is used to produce and obtain the antibody. Specifically, for example, cells (antibody producing cells) prepared from a spleen removed from an immunized non-human animal are fused with myeloma cells in accordance with a known method using a polyethylene-glycol method or electrical stimulation, and culturing the cells in HAT selection medium to obtain hybridomas. Then, by screening, from among the hybridomas, a hybridoma that produces an antibody which binds to LAM using a known method such as limiting dilution analysis, a hybridoma that produces an antibody that binds to LAM can be obtained.

The antibody of the present invention described above can be used for detecting acid-fast bacilli, preferably tubercle bacillus. In other words, it is possible to determine whether or not a subject is carrying acid-fast bacilli, particularly tubercle bacillus, and to estimate the amount of viable bacilli thereof.

The present disclosure also relates to a method for estimating the amount of viable acid-fast bacilli, such as M. tuberculosis, in a sample. The sample may be, for example, a sputum sample. Such a method of estimating may be conducted, for example, through the following steps of (1)-(3):

(1) obtaining a first sample isolated from a patient diagnosed with, or suspected of having, an M. tuberculosis infection;

(2) contacting at least a part of the first sample with an antibody or antigen binding fragment thereof that binds to LAM from M. tuberculosis, and detecting the amount of LAM bound to the antibody or antigen-binding fragment thereof; and

(3) estimating the amount of viable M. tuberculosis in the sample, based on the detected amount of LAM bound to the antibody or antigen-binding fragment thereof.

The subject’s biological sample that is brought into contact with the antibody of the present invention in the step of (2) may be a biological sample in which acid-fast bacilli, particularly tubercle bacillus, exists, and examples of the biological sample may include sputum, blood (serum, plasma), lung lavage fluid, gastric juice, urine, feces, skin, and pancreatic juice, etc. The biological sample is preferably sputum, or blood, and is more preferably sputum. Here, the subject that is subjected to the assay is preferably human, however, other than human, animals such as horse, cow, goat, sheep, dog, chicken, mouse, hamster, and rat may also be used as a subject.

The conditions under which the antibody and the biological sample are brought into contact with each other is not particularly limited, as long as it is conditions under which binding between the antibody and LAM may occur. Examples of a method thereof may include incubating the antibody with the sample, or at least a part of the sample, under a temperature condition of generally 45°C or lower, preferably about 4 to 40°C, and more preferably about 25 to 40°C; and leaving or incubating the mixture for about 0.5 to 40 hours, and preferably about 1 to 20 hours. Furthermore, there is also no particular limitation in a solvent used in the binding reaction and pH thereof as long as there is no adverse effect on the reaction; and, in accordance with or conforming to a known method, a buffer (e.g., citrate buffer, phosphate buffer, tris salt buffer, acetate buffer, etc.,) can be used such that the pH becomes, for example, about 5 to 9.

The step of (2) may be conducted in a state in which the antibody of the present invention is immobilized onto a solid-phase. Such immobilizing includes both cases of the antibody of the present invention being bound to the solid carrier in a detachable manner, or in an undetachable manner. As the solid carrier used for immobilizing the antibody, various carriers commonly used in the art can be used, and examples thereof may include a wide range of articles such as sticks, beads, plates (including microplates), test tubes, and the like formed from various materials such as glass, cellulose powder, Sephadex, Sepharose, polystyrene, filter papers, carboxymethyl cellulose, nitrocellulose, ion-exchange resins, dextran, plastic films, plastic tubes, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, etc. There is no particular limitation in the immobilizing method, and both a physical bond and a chemical bond can be used depending on the various solid carriers. Examples thereof may include: chemical reactions such as a diazo method as a covalent binding method, peptide methods (acid-amide derivative method, carboxyl chloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide activated polysaccharide method, cellulose carbonate derivative method, and a method using a condensation reagent), alkylation method, carrier binding methods using a cross-linking reagent (e.g., using glutaraldehyde, hexamethylene isocyanate, or the like as a cross-linking reagent), and a carrier binding method using Ugi reaction; ionic bond methods using a carrier such as ion-exchange resins; and physical adsorption methods using, as a carrier, porous glass such as glass beads.

The antibody of the present invention may be used in a labeled state using any labeling substance. Here, examples of the labeling substance may include: enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase; fluorescent substances such as fluorescein isocyanate and rhodamine; radioactive substances such as ³²P and ¹²⁵I; coloring substances (coloration substance) such as latex including natural rubber latex and synthetic latex such as a polystyrene latex colored with metal colloids such as a gold colloid and a white colloid or pigments of red, blue, or the like; and chemiluminescence substances. Labeling of the antibody with these labeling substances can be conducted in accordance with a hitherto known method depending on the various labeling substances. A second antibody may also be used, which is labeled, and which binds to the anti-LAM antibody.

The step of (2) also encompasses detecting/assaying an immune complex (antigen-antibody bound substance) obtained through a binding reaction between the antibody and LAM. Here, detecting/assaying the immune complex (antigen-antibody bound substance) and conditions for that is not particularly limited, and a method and conditions identical to or conforming to a common immunoassay method may be used. Specifically, depending on the type of labeling substance used for labeling the antibody, various methods that are generally used for immunochemical assay can be used, such as, for example, radioisotopic immunoassay (RIA method), ELISA method, fluorescent antibody method, plaque method, spotting method, agglutination method, Ouchterlony method, etc., (e.g., cf. p.30-53 in “Hybridoma method and monoclonal antibody” published by R&D planning K.K., on March 5, 1982). For an ELISA method, a sandwich method may be used, for example.

When a solid-phase sandwich method is used, an assay target which is an acid-fast bacillus, preferably tubercle bacillus, in a test sample can be assayed, for example, in the following manner. First, a biological sample (e.g., sputum, saliva, or blood etc.,) is added as a test sample containing an assay target which is an acid-fast bacillus, preferably tubercle bacillus, to a solid-phased antibody obtained by immobilizing (including detachable immobilizing) an antibody that causes a specific antigen-antibody reaction with LAM of the assay target acid-fast bacilli, preferably tubercle bacillus, to allow an antigen-antibody reaction to occur. Next, unbound substances are removed by, for example, washing; an antibody that causes a specific antigen-antibody reaction with LAM of the assay target acid-fast bacilli, preferably tubercle bacillus, is added to allow reaction with assay-target bacteria in the antigen-antibody bound substance generated above; and an antigen-antibody bound substance (a complex of “antibody - acid-fast bacillus - antibody”, and preferably a complex of “antibody - tubercle bacillus - antibody”) generated in the reaction is detected (qualitative measurement) or an amount thereof is measured (quantitative measurement).

Assay of the antigen-antibody bound substance (the complex of “antibody - acid-fast bacillus - antibody” and preferably the complex of “antibody - tubercle bacillus - antibody”) can be conducted easily by using an antibody (labeled antibody) that is labeled with any of the labeling substances described above. To allow the assay to be conducted more easily, for example, it is possible to use an immunochromatography using an antibody labeled with a colored latex particle or the like such as a gold colloid etc. A person skilled in the art will know well about the selection of various means for these assay techniques and modifications thereof, and the present invention may be realized with any of such techniques (see “Clinical Test Method Manual” Kanehara Shuppan, 1995, etc.).

The present disclosure also encompasses methods in which samples are obtained at different times, and analyzed according to the methods herein. For example, a second sample may be isolated from the same patient between 1-8 weeks, between 1-4 weeks, or between 1-2 weeks, after a first sample is isolated from the patient. In some embodiments, the patient has undergone treatment with a treatment regimen between the time of isolation of the first and second samples. In some embodiments, the treatment regimen is continued in response to an estimation that the amount of viable acid-fast bacteria, such as M. tuberculosis, in the second sample is lower than in the first sample. In other embodiments, the treatment regimen may be discontinued in response to a determination that the amount of viable acid-fast bacteria, such as M. tuberculosis, in the second sample is not lower than in the first sample. In some embodiments, the patient is administered a different treatment after the treatment regimen is discontinued.

The present invention further provides a kit for estimating the amount of viable acid-fast bacilli, such as M. tuberculosis, in a sample. The kit may include an anti-LAM antibody, and for example, one or more reagents for detecting binding between the antibody and LAM. Furthermore, for convenience of conducting the assay, the kit may further include suitable reaction solutions, dilution solutions, rinsing solutions, reaction stop solutions, labeled activity measurement reagents, and the like.

The present disclosure also relates to a method for treating a patient with an acid-fast bacilli infection, such as M. tuberculosis. Such a treatment method may be conducted, for example, through the following steps of (a)-(d) or (a1)-(e1):

(a) obtaining a first sample isolated from a patient diagnosed with an acid-fast bacillus infection, such as M. tuberculosis, wherein the first sample is obtained before or after treatment has commenced with a treatment regimen for treating an acid-fast bacillus infection, such as M. tuberculosis;

(b) obtaining a second sample isolated from the patient later in time, wherein the patient has been treated with the treatment regimen in the time between the isolation of the first and second samples;

(c) contacting at least a part of the first and second samples with an antibody or antigen-binding fragment thereof that binds to LAM from an acid-fast bacillus infection, such as M. tuberculosis, and detecting the amount of LAM bound to the antibody or antigen-binding fragment thereof, wherein the detected amount of LAM in the first and the second samples correlates with the amount of viable acid-fast bacteria in the first and second samples, respectively; and

(d) continuing to administer the treatment regimen to the patient when a decrease in the amount of LAM is detected in the second sample as compared to the first sample; or discontinuing the administration of the treatment regimen to the patient when a decrease in the amount of LAM is not detected in the second sample as compared to the first sample, followed by administering the patient a different treatment for the acid-fast bacilli after the treatment regimen is discontinued.

(a1) obtaining a first sample isolated from a patient diagnosed with an M. tuberculosis infection, wherein said first sample is obtained before or after treatment has commenced with a treatment regimen for treating M. tuberculosis;

(b1) obtaining a second sample isolated from said patient later in time, wherein said patient has been treated with the treatment regimen in the time between the isolation of said first and second samples;

(c1) contacting at least a part of said first and second samples with an antibody or antigen-binding fragment thereof that binds to LAM from M. tuberculosis, and detecting the amount of LAM bound to said antibody or antigen-binding fragment thereof,

(d1) making a determination of the amount of viable M. tuberculosis in said first and second samples, based on the detected amount of LAM bound to said antibody or antigen-binding fragment thereof; and

(e1) continuing to administer said treatment regimen to said patient when a decrease in the amount of viable M. tuberculosis is detected in said second sample as compared to said first sample; or discontinuing the administration of said treatment regimen to said patient when a decrease in the amount of viable M. tuberculosis is not detected in said second sample as compared to said first sample, followed by administering said patient a different treatment for M. tuberculosis after said treatment regimen is discontinued.

The present disclosure further relates to a method for evaluating the effectiveness of a treatment regimen for treating a patient with an acid-fast bacilli infection, such as M. tuberculosis, said method comprising the above described steps of (a)-(c) and (d') below or the above described steps of (a1)-(d1) and (e1') below.

(d') evaluating said treatment regimen to said patient as being effective when a decrease in the amount of (LAM) is detected in said second sample as compared to said first sample, or
evaluating said treatment regimen to said patient as being ineffective, when a decrease in the amount of (LAM) is not detected in said second sample as compared to said first sample.

(e1') evaluating said treatment regimen to said patient as being effective, when a decrease in the amount of viable M. tuberculosis is detected in said second sample as compared to said first sample or
evaluating said treatment regimen to said patient as being ineffective, when a decrease in the amount of viable M. tuberculosis is not detected in said second sample as compared to said first sample.

In some embodiments, when the treatment regimen is evaluated effective, the treatment regimen may be continued to be administered. When the treatment regimen is evaluated ineffective, the treatment regimen may be discontinued and followed by a different treatment for M. tuberculosis.

In some embodiments, the sample is sputum. In some embodiments, the patient is diagnosed with, or suspected of having, a pulmonary M. tuberculosis infection. In yet further embodiments, the detecting of the amount of LAM bound to the antibody or the antigen-binding fragment thereof is measured by immunoassay.

As the antituberculosis treatment regimen described here, those that are known in the art can be used, such as rifampicin, isoniazid (isonicotinic acid hydrazide), pyrazinamide, streptomycin and a salt thereof, and ethambutol and a salt thereof. Combinations thereof are also encompassed. However, the antituberculosis medicament is not limited thereto, and includes approve or unapproved medicaments that exhibit bactericidal action (antituberculosis activity) against tubercle bacilli. Therapy for active tuberculosis is often conducted by administration of four or more types of therapeutic agents for at least six months. When tuberculosis infection is suspected, first, a smear test using Ziehl-Neelsen staining or fluorescence staining is performed. If bacteria exist by a number of 10,000 or more in 1 ml of a sputum specimen, it is considered “smear positive.” A sputum-smear-positive patient is particularly important as a source of infection, clinically and in terms of public health. Therefore, a smear positive patient requires hospital treatment in a tuberculosis ward. One index of switching to outpatient treatment is to have negative smear test results for three consecutive days. Therefore, by assaying LAM in a biological sample such as sputum urine, and blood, it is possible to monitor therapeutic agent effectiveness, and in particular, to obtain a real-time estimation of the amount of viable bacteria in response to the treatment, and to allow a more accurate determination of whether a tuberculosis patient on treatment is no longer infectious and thus can be released from airborne infection isolation in the tuberculosis ward to an outpatient setting.

The present invention also contemplates LAM binding agents other than antibodies, including, for example, an aptamer, or a fusion protein containing a binding moiety that binds to LAM.

All references cited herein are hereby incorporated by reference in their entireties, whether previously specifically incorporated or not. As used herein, the terms “a”, “an”, and “any” are each intended to include both the singular and plural forms.

Having now fully described the invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

Examples

LAM concentration in sputum as a surrogate for quantifying the amount of viable mycobacteria.

Two clinical studies were conducted in Manila, the Philippines. In the first study, it was demonstrated that LAM concentration using ELISA, in which plates were coated with bivalent modified TB-scFv (complete antibody form, SEQ ID NO: 12) and bivalent modified G3-scFv (SEQ ID NO: 22), and bivalent modified Myco-scFv (SEQ ID NO: 13) used as a detection antibody as described in WO2013129634A1 in sputa obtained from pulmonary tuberculosis patients prior to treatment inversely correlated with MGIT-TTD (an indirect measurement of bacterial load in the sputum samples). This is depicted in Figure 1. In Figure 1, the LAM concentration is log₁₀ transformed; LAM concentration has a very wide dynamic range up to1,000,000 pg/mL (log₁₀ of 6). The R² value is 0.7150 and 0.8016, for linear fit and non-linear fit, respectively. Since MGIT-TTD is an accepted surrogate measurement of the number of viable bacterial number, the results of this study show that, unexpectedly, LAM concentration is an excellent indicator of viable bacterial number prior to the start of treatment.

In the second study, LAM concentrations from sputum samples obtained from pulmonary tuberculosis patients during standard 4-drug treatment (isoniazid, rifampin, ethambutol, and pyrazinamide) were determined. Sputa were obtained weekly for the first 4 weeks during the required 6-month treatment. The same samples were examined by MGIT culture and an NAAT test (LAMP: loop-mediated isothermal amplification of nucleotide). As depicted in Figure 2, all samples were positive prior to treatment. As expected, samples progressively turned culture-negative based on the MGIT results, and by the end of the 2-month treatment, close to 90% of sputa turned MGIT culture-negative. However, the result of the LAMP analysis was positive from the majority of the samples, because of the detection of DNA from dead bacteria. Unexpectedly, the percentage of samples that became negative in the LAM assay tracked the trend that was observed for MGIT, and surprisingly, was remarkably different from the trend observed with the LAMP analysis. Further, by the end of the 2-month treatment, a similar percentage of samples became LAM and MGIT negative. These data strongly indicate that LAM concentration is an accurate surrogate measurement of the number of viable M. tuberculosis in the specimen.

Further still, the ability to estimate viable bacterial number was further confirmed at the individual patient level. LAM concentration (log₁₀ transformed; left Y-axis) and MGIT-TTD (hours; right Y-axis) are plotted for each individual patient during the treatment (X-axis) in Figure 3. As Figure 3 depicts, LAM concentration and MGIT-TTD tracked each other closely during treatment, with decreases in LAM concentration being associated with the prolongation of MGIT-TTD. Most patients responded to the treatment, as LAM concentration decreased and MGIT-TTD increased, and by the 2-month treatment, most had undetectable LAM and were MGIT culture-negative. However, one patient, indicated as a “poor responder,” had a high level of LAM and a short MGIT-TTD. These data indicate that LAM concentration can be used as a surrogate marker of MGIT-TTD, and can be used to identify non-responders during treatment, and particularly, during the early stages of treatment (for example, within 1 week, 2 weeks, 4 weeks, 8, weeks, etc.). Additionally, because LAM results were obtained in real-time, whereas the culture results required weeks of waiting, the LAM measurement was able to provide a real-time estimation of viable bacterial number. Without being limited to any particular theory, this correlation may be due to specific binding of the antibody to LAM on viable bacteria; rapid degradation of LAM once released from dead bacilli; or rapid removal of LAM released from dead bacilli by immune cells, for example.

Currently, proof-of-concept trials for tuberculosis drugs include the evaluation of early bactericidal activity (EBA) to gauge the activity of candidate compounds in a 14-day treatment period. Current EBA trials rely on laboratory quantitative bacterial counts from culture that is slow (i.e., weeks of delay prior to obtaining results), are resource demanding, and there are only a few limited sites worldwide capable of performing such studies. A surrogate marker of viable bacterial number, with real-time determination, would therefore significantly speed up the identification of the best candidate compounds and regimens. To this end, changes in LAM concentration were measured during the first 14 days of treatment, and compared with the changes in MGIT-TTD. As shown in Table 1 below, during the first 14 days of treatment, LAM exhibited an average 1.24 log₁₀ decrease. This corresponded to an increase of 227.8 hours of MGIT-TTD. As reported previously, a standard 4-drug treatment of tuberculosis patients during the first 14-days reduces the number of viable bacterial number (measured in solid medium culture as colony-forming-unit; cfu) by a log₁₀ of 1.67 log. Diacon et al. (“Early bactericidal activity of delamanid (OPC-67683) in smear-positive pulmonary tuberculosis patients,” Int. J. Tuberc. Lung Dis., 2011, 15(7): 949-54.

Claims

A method for estimating the amount of viable Mycobacterium tuberculosis (M. tuberculosis) in a sample obtained from a subject, said method comprising:
contacting at least a part of a sample isolated from the subject with an antibody or antigen-binding fragment thereof that binds to lipoarabinomannan from M. tuberculosis, and detecting the amount of lipoarabinomannan bound to said antibody or antigen-binding fragment thereof; and
estimating the amount of viable M. tuberculosis in said sample, based on the detected amount of lipoarabinomannan bound to said antibody or antigen-binding fragment thereof.
The method according to claim 1, wherein the subject is diagnosed with, or suspected of having, a tuberculosis infection.
The method according to claim 2, wherein the subject is diagnosed with, or suspected of having, a pulmonary M. tuberculosis infection.
The method of any one of claims 1-3, wherein the sample is a sputum sample.
The method of any one of claims 1-4, wherein the antibody is selected the group consisting of (A) to (C) below:
(A) An antibody containing the following CDR sequences (a)-(f):
(a) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1,
(b) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2,
(c) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3,
(d) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4,
(e) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and
(f) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) An antibody containing the following CDR sequences (g) to (l):
(g) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(h) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 15,
(i) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(j) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17,
(k) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and
(l) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and
(C) An antibody containing the following CDR sequences (m)-(r):
(m) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 24,
(n) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 25,
(o) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 26,
(p) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 27,
(q) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 28, and
(r) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 29.
The method of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof that binds to lipoarabinomannan from M. tuberculosis can distinguish between lipoarabinomannan from tubercle bacilli and lipoarabinomannan from non-tuberculous acid-fast bacilli.
A method for evaluating the effectiveness of a treatment regimen for treating M. tuberculosis, said method comprising:
contacting at least a part of a first and second samples with an antibody or antigen-binding fragment thereof that binds to lipoarabinomannan from M. tuberculosis, and detecting the amount of lipoarabinomannan bound to said antibody or antigen-binding fragment thereof, wherein the detected amount of lipoarabinomannan in the first and the second samples correlates with the amount of viable M. tuberculosis in said first and second samples, respectively,
wherein the first sample is isolated from a patient diagnosed with an M. tuberculosis infection, and is obtained before or after treatment has commenced with a treatment regimen for treating M. tuberculosis; and
wherein the second sample is isolated from said patient later in time, wherein said patient has been treated with the treatment regimen in the time between the isolation of said first and second samples;
evaluating said treatment regimen to said patient as being effective, when a decrease in the amount of lipoarabinomannan is detected in said second sample as compared to said first sample, or
evaluating said treatment regimen to said patient as being ineffective, when a decrease in the amount of lipoarabinomannan is not detected in said second sample as compared to said first sample.
The method of claim 7, wherein the patient is diagnosed with, or suspected of having, a pulmonary M. tuberculosis infection.
The method of claim 7 or 8, wherein the sample is a sputum sample.
The method of any one of claims 7-9, wherein the antibody is selected the group consisting of (A) to (C) below:
(A) An antibody containing the following CDR sequences (a)-(f):
(a) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1,
(b) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2,
(c) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3,
(d) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4,
(e) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and
(f) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) An antibody containing the following CDR sequences (g) to (l):
(g) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(h) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 15,
(i) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(j) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17,
(k) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and
(l) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and
(C) An antibody containing the following CDR sequences (m)-(r):
(m) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 24,
(n) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 25,
(o) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 26,
(p) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 27,
(q) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 28, and
(r) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 29.
The method of any one of claims 7-10, wherein the antibody or antigen-binding fragment thereof that binds to lipoarabinomannan from M. tuberculosis can distinguish between lipoarabinomannan from tubercle bacilli and lipoarabinomannan from non-tuberculous acid-fast bacilli.
A kit for estimating the amount of viable M. tuberculosis in a biological sample obtained from a subject, which comprises:
at least one antibody or antigen-binding fragment thereof that binds to lipoarabinomannan from M. tuberculosis, and
one or more reagents for detecting binding between the antibody and lipoarabinomannan.
The kit of claim 12, wherein the subject is diagnosed with, or suspected of having, a M. tuberculosis infection.
The kit of claim 13, wherein the subject is diagnosed with, or suspected of having, a pulmonary M. tuberculosis infection.
The kit of any one of claims 12-14, wherein the sample is a sputum sample.
The kit of any one of claims 12-15, wherein the antibody is selected the group consisting of (A) to (C) below:
(A) An antibody containing the following CDR sequences (a)-(f):
(a) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1,
(b) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2,
(c) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3,
(d) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4,
(e) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and
(f) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) An antibody containing the following CDR sequences (g) to (l):
(g) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(h) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 15,
(i) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(j) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17,
(k) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and
(l) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and
(C) An antibody containing the following CDR sequences (m)-(r):
(m) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 24,
(n) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 25,
(o) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 26,
(p) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 27,
(q) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 28, and
(r) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 29.
The kit of any one of claims 12-16, wherein the antibody or antigen-binding fragment thereof that binds to lipoarabinomannan from M. tuberculosis can distinguish between lipoarabinomannan from tubercle bacilli and lipoarabinomannan from non-tuberculous acid-fast bacilli.
A method for treating M. tuberculosis, said method comprising:
(a) obtaining a first sample isolated from a patient diagnosed with an M. tuberculosis infection, wherein said first sample is obtained before or after treatment has commenced with a treatment regimen for treating M. tuberculosis;
(b) obtaining a second sample isolated from said patient later in time, wherein said patient has been treated with the treatment regimen in the time between the isolation of said first and second samples;
(c) contacting at least a part of said first and second samples with an antibody or antigen-binding fragment thereof that binds to lipoarabinomannan from M. tuberculosis, and detecting the amount of lipoarabinomannan bound to said antibody or antigen-binding fragment thereof, wherein the detected amount of lipoarabinomannan in the first and the second samples correlates with the amount of viable M. tuberculosis in said first and second samples, respectively; and
(d) continuing to administer said treatment regimen to said patient when a decrease in the amount of lipoarabinomannan is detected in said second sample as compared to said first sample; or discontinuing the administration of said treatment regimen to said patient when a decrease in the amount of lipoarabinomannan is not detected in said second sample as compared to said first sample, followed by administering said patient a different treatment for M. tuberculosis after said treatment regimen is discontinued.
The method of claim 18, wherein the patient is diagnosed with, or suspected of having, a pulmonary M. tuberculosis infection.
The method of claim 18 or 19, wherein the sample is a sputum sample.
The method of any one of claims 18-20, wherein the antibody is selected the group consisting of (A) to (C) below:
(A) An antibody containing the following CDR sequences (a)-(f):
(a) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1,
(b) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2,
(c) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3,
(d) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4,
(e) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and
(f) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) An antibody containing the following CDR sequences (g) to (l):
(g) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(h) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 15,
(i) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(j) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17,
(k) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and
(l) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19; and
(C) An antibody containing the following CDR sequences (m)-(r):
(m) Heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 24,
(n) Heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 25,
(o) Heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 26,
(p) Light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 27,
(q) Light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 28, and
(r) Light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 29.
The method of any one of claims 18-21, wherein the antibody or antigen-binding fragment thereof that binds to lipoarabinomannan from M. tuberculosis can distinguish between lipoarabinomannan from tubercle bacilli and lipoarabinomannan from non-tuberculous acid-fast bacilli.