Human genetic markers associated with response to treatments that target clostridium difficile toxin b

Abstract

To provide use of a Clostridium difficile (C. difficile) toxin B (TcdB) antibody in the manufacture of a medicament for preventing recurrent Clostridium difficile infection (CDI).SOLUTION: A preventive method comprises administering a therapeutically effective amount of a TcdB antibody. It is used when the patient, prior to administration of the medicament, tests positive for at least one copy of the better response allele of one or more TcdB treatment response markers, such as a) T allele of rs2516513 single nucleotide polymorphism (SNP), b) A allele of rs113379306 SNP, and c) A allele of rs76166871 SNP.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to genetic markers on human chromosome 6 that predict a beneficial response in patients in need thereof to treatment with molecules that target Clostridium difficile (C. difficile) toxin B. The present invention also provides methods of using the genetic markers for the diagnosis and / or treatment of said patients.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 508,066, filed May 18, 2017, currently pending, and International Application No. PCT / CN16 / 109900, filed December 14, 2016, currently pending. [Background technology]

[0003] 2. Background of the Invention Clostridium difficile (C. difficile) is an anaerobic, spore-forming, Gram-positive bacterium and is the most common cause of healthcare-associated infections in the United States (US) and Europe (Lessa et al., Burden of Clostridium difficile infection in the United States. N Engl J Med. 2015 372(9):825-34). The US Centers for Disease Control (CDC) has declared Clostridium difficile to be an urgent public health threat (Center for Disease Control and Prevention. Antibiotic resistance threats in the United States, 2013. Atlanta, GA: US, Department of Health and Human Services). According to the CDC, in 2011, C. difficile was estimated to have caused approximately 500,000 infections in the United States, including an estimated 453,000 cases of CDI and an additional 83,000 recurrent cases (Lessa et al., supra). CDI was also associated with approximately 29,000 deaths (Lessa et al., 2015, supra). Studies in Europe have reported similar estimates of CDI recurrence rates (Bauer et al., Clostridium difficile infection in Europe: a hospital-based survey. Lancet 2011;377:63-73).

[0004] Nearly all antibiotics, including clindamycin, cephalosporins, penicillins, and fluoroquinolones, have been associated with symptomatic disease caused by toxigenic C. difficile (Bartlett, JG, Clin Infect Dis. 46(10):1489-92 (2008); Gorbach, SL, N Engl J Med, 341(22):1690-1 (1999); Kelly et al., N Engl J Med 30(4):257-62 (1994)). C. difficile-associated disease (CDAD) includes antibiotic-associated diarrhea, colitis, and pseudomembranous colitis, and in some cases can progress to toxic megacolon, sepsis, and death. Although some patients with CDI respond to discontinuation of antibiotic therapy, most require treatment with additional agents such as metronidazole or vancomycin (reviewed in DuPont et al., Current Opinion in Infectious Diseases 21:500-507 (2008)). Existing therapies often fail to eliminate CDI or lead to recurrent disease, and therefore new methods of prevention or treatment are needed.

[0005] One of the biggest challenges in the treatment of CDI is preventing recurrence. After successful treatment of an initial CDI, recurrences occur in 15% to 35% of patients (Bouza et al., Antimicrobial therapy of Clostridium difficile-associated diarrhea. Med Clin N Am 2006;90:1141-63; McFarland LV. Renewed interest in a difficult disease: Clostridium difficile infections-epidemiology and current treatment strategies. Curr Opin Gastroenterol. 2009,25(1):24-35). The majority of these recurrences occur within 60 days of treatment of the initial episode (Bouza et al., 2006, supra; McFarland 2009, supra). After the first recurrence, 40% will experience a further recurrence, and after two recurrences, the likelihood of an additional episode of CDI increases further (Bouza et al., 2006, supra; McFarland 2009, supra).

[0006] Bezlotoxumab, a human monoclonal antibody (mAb) that binds to and neutralizes C. difficile toxin B (TcdB), was recently approved by the FDA to reduce recurrence of C. difficile infection (CDI) in patients aged 18 years or older who are receiving antibiotic treatment for CDI and who are at high risk for CDI recurrence. A phase 3 clinical trial showed that a single dose of 10 mg / kg intravenous (IV) bezlotoxumab was safe and effective in preventing CDI recurrence in patients with CDI receiving antibiotic therapy. Despite the expected beneficial impact of the availability of novel treatments for the prevention of CDI recurrence, the therapeutic efficacy of medicines targeting C. difficile toxin B can vary widely among patients suffering from C. difficile infection. To provide better and more cost-effective treatment for CDI, methods are needed to identify patients most likely to benefit from treatment with drugs that target C. difficile toxin B, such as TcdB antibodies. Summary of the Invention

[0007] SUMMARY OF THEINVENTION The present invention is based on the discovery that genetic variations, e.g., single nucleotide polymorphisms (SNPs), on human chromosome 6, and various human leukocyte antigen (HLA) alleles are significantly associated with response to treatment with Clostridium difficile toxin B ("TcdB") antibodies in patients suffering from Clostridium difficile (C. difficile) infection (CDI) and / or Clostridium difficile-associated disease (CDAD) and / or symptoms thereof. Genetic polymorphisms and variant alleles associated with response to treatment with pharmaceutical agents targeting TcdB are referred to herein as "TcdB treatment response markers."

[0008] In one embodiment of the present invention, the TcdB treatment response marker is a SNP that is the C / T polymorphism identified in the NCBI SNP database as rs2516513. The presence of the T allele is associated with better treatment response, whether present in a homozygous (T / T) or heterozygous (C / T) state. Overall, the T / T and C / T genotypes are associated with an approximately two-fold reduction in C. difficile recurrence rates compared to the entire unstratified patient population treated with bezlotoxumab for CDI. Although the T allele has a lower frequency than the C allele in most ethnic groups (minor allele), it is common in many ethnic groups, which may provide guidance to medical practitioners, health authorities and health insurance providers in selecting the appropriate population of C. difficile infected patients who may benefit from TcdB medicines, such as Tcdb antibodies.

[0009] The inventors have also identified associations between other genetic variants on chromosome 6 with beneficial responses to treatment with drugs targeting TcdB, e.g., improved C. difficile recurrence rates in CDI patients. The genetic variants are shown in Table 1 below, where PS means polymorphic site according to the SNP NCBI database. [Table 1]

[0010] The inventors herein contemplate that testing an individual for the presence of one or more of the TcdB treatment response markers in Table 1 will be useful in various pharmacogenetic products and methods, including identifying patients in CDI patients who are most likely to respond to treatment with a drug targeting TcdB, and to assist health care providers in determining whether to prescribe such a drug (e.g., a TcdB antibody) to a CDI patient or a patient who has previously suffered from CDI. For example, the inventors contemplate testing an individual for the presence of one or more of the TcdB treatment response markers in Table 1, including at least one copy (i.e., heterozygous or homozygous) of one or more of the TcdB treatment response markers listed in Table 1, i.e., the T allele for the rs2516513 SNP, the A allele for the rs113379306, the A allele for the rs76166871 SNP, the HLA-DRB1 * 07:01 Allele, HLA-DQB1 * 02:02 Alleles or HLA-DQA1 * It is envisioned that testing of subjects for the presence of the 02:01 allele will be useful in such products and methods, as well as to aid such health care providers.

[0011] SNPs and alleles in Table 1 (HLA-DRB1 * 07:01, rs2516513, rs113379306, rs76166871, HLA-DQB1 * 02:02 and HLA-DQA1 * 02:01), alone or in combination, are useful for predicting CDI recurrence. Also, SNPs and genetic variants in linkage disequilibrium (LD) with the SNPs in Table 1 can be used alone or in combination with the SNPs and alleles in Table 1.

[0012] Additionally, as described above with respect to the rs2516513 SNP, the overall frequency of the TcdB treatment response markers of the present invention within a population may show a distribution of lower frequencies in certain subgroups or ethnic groups within the overall population. See, for example, Table 1A below, which shows the HLA-DRB1 gene expression profile in exemplary ethnic groups. *07:01 Allele, HLA-DQB1 * 02:02 Alleles and HLA-DQA1 * The frequencies of the 02:01 allele are shown. The frequencies shown are for various specific subgroups and represent a representative population sampling. Frequencies in other populations not listed vary and can be obtained from Gonzalez-Galarza et al., Allele Frequency Net 2015 Update: New Features for HLA Epitopes, KIR and Disease and HLA Adverse Drug Reaction Associations. Nucleic Acid Research 2015, 28, D784-8. Such information can be useful in selecting appropriate populations of C. difficile infected patients who may benefit from TcdB medicines (e.g., TcdB antibodies). [Table 2]

[0013] As used herein, "testing a subject for the presence of one or more, at least one copy of a TcdB therapeutic response marker" means that, for use in the methods of the present invention, a subject may be tested for one, two, three, four, five or six of the Tcd therapeutic response markers of the present invention, in any combination, and in combination with linked variants of the TcdB therapeutic response markers of the present invention, as described below.

[0014] Accordingly, the present invention relates to a method for preventing recurrence of Clostridium difficile infection comprising administering a therapeutically effective amount of a therapy targeting Clostridium difficile (C. difficile) toxin B (TcdB therapy) to a patient in need thereof, where the patient has, prior to administration of the TcdB therapy, a positive test result for at least one copy (or "at least one of the copies") of one or more TcdB treatment response markers or better response alleles from linked variants (i.e., variants in high LD) of TcdB treatment response markers, where the one or more TcdB treatment response markers are selected from the group consisting of (a) the T allele of the rs2516513 single nucleotide polymorphism (SNP), (b) the A allele of the rs113379306 SNP, (c) the A allele of the rs76166871 SNP, (d) the HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 allele, (e) HLA-DQB1 of the HLA-DQB1 gene * 02:02 allele, and (f) HLA-DQA1 of the HLA-DQA1 gene * 02:01 alleles.

[0015] The invention also provides a method for determining whether a human patient is likely to respond to a pharmaceutical agent targeting Clostridium difficile (C. difficile) toxin B (TcdB), the method comprising: (a) obtaining or having obtained a biological sample from the patient; (b) determining whether a better response allele of at least one TcdB response marker or linked variant of the invention is present in the biological sample; and (c) diagnosing the patient as more likely to benefit from treatment with a TcdB pharmaceutical agent if the presence of one or more copies of the better response allele is detected in the biological sample.

[0016] In some embodiments of this aspect of the invention, the method further comprises a step (d) comprising administering to the diagnosed patient a therapeutically effective amount of a TcdB medicament.

[0017] In preferred embodiments of the methods provided herein, the therapeutic targeting TcdB is a TcdB antibody or an antigen-binding fragment thereof. In further preferred embodiments, the TcdB antibody is bezlotoxumab.

[0018] The present invention further relates to a pharmaceutical product comprising a pharmaceutical composition and prescribing information, wherein the pharmaceutical composition comprises a TcdB antibody and the prescribing information comprises a pharmacogenetic indication, wherein the pharmacogenetic indication comprises treatment of C. difficile infection or prevention of C. difficile recurrence in a C. difficile infected patient who tests positive for at least one copy of a better response allele selected from the TcdB treatment response markers or linked variants of the present invention.

[0019] The present invention also provides a kit for testing a patient for the presence or absence of at least one copy of a better response allele selected from the TcdB therapeutic response markers of the present invention, the kit comprising a set of reagents which may include oligonucleotides designed to genotype at least one of the TcdB therapeutic response markers.

[0020] The invention also includes the use of bezlotoxumab in the manufacture of a medicament for the prevention of C. difficile recurrence in a human patient, wherein the patient has a positive test result for a better response allele of a TcdB treatment response marker disclosed herein.

[0021] DETAILED DESCRIPTION OF THE PRESENT EMBODIMENT I. Definitions and Abbreviations In order that the present invention may be more readily understood, certain scientific and technical terms are specifically defined below. Unless specifically defined elsewhere herein, all other scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the present invention belongs when used in a similar context as used herein.

[0022] As used in this specification, including the appended claims, the singular terms "a," "an," and "the" include their plural counterparts unless the context clearly indicates otherwise.

[0023] "About" used to modify a numerically defined parameter (e.g., the dosage of a therapeutic agent described herein or the length of treatment time) means that the parameter can vary by 10% above or below the numerical value recited for that parameter.

[0024] "Actoxumab" (alternatively referred to herein as "ACT" and also known as MK3415) is a fully human IgG1 monoclonal antibody (mAb) that specifically binds to the receptor binding domain of C. difficile toxin A. Actoxumab comprises light chain CDRs (CDRL1, CDRL2, and CDRL3) consisting of the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and heavy chain CDRs (CDRH1, CDRH2, and CDRH3) consisting of the amino acid sequences set forth in SEQ ID NO: 10, 11, and 12, respectively. Actoxumab comprises a V domain comprising the amino acid sequence set forth in SEQ ID NO: 9. L region and the amino acid sequence set forth in SEQ ID NO: 13. H and a region.

[0025] "Actoxumab / bezlotoxumab" (alternatively referred to herein as "ACT+BEZ" and also known as MK3415A) refers to the combination of actoxumab and bezlotoxumab antibodies, and / or the use of such a combination for the treatment of patients with CDI, including prevention of recurrence of CDI.

[0026] An "allele" is a particular form of a gene or other genetic locus, distinguished from other forms by its particular substitution of a nucleotide sequence, and the term allele also includes any alternative polymorphism of a gene at that locus.

[0027] "Beneficial outcome" means the desired clinical result of treatment with a pharmaceutical agent that targets TcdB (e.g., a TcdB antibody), including, but not limited to, alleviation of one or more disease symptoms, reduction in the extent of disease (e.g., improved C. difficile recurrence rates in the case of treatment of CDI), stabilized (i.e., not worsening) disease, slowing of disease progression, improvement or palliation of pathology, prolonged survival (compared to expected survival if not treated), recurrence-free survival, remission (partial or complete), and cure (i.e., elimination of disease).

[0028] A "better response allele" is a particular form of a gene or other genetic locus that, when present in a patient, confers an improved clinical measure (e.g., an improved measure of Clostridium difficile recurrence rate) compared to that measure in a patient in which such form of the gene or other genetic locus is not present.

[0029] "Bezlotoxumab" (alternatively referred to herein as "BEZ" and also known as MK-6072) is a fully human mAb of the IgG1 / kappa isotype subclass that binds with high affinity (Kd=19 pM) to the C-terminal ligand binding domain of C. difficile toxin B (U.S. Patent No. 8,394,819, the disclosure of which is incorporated herein by reference in its entirety). Bezlotoxumab is currently in development for the prevention of CDI recurrence in patients 18 years of age or older receiving antibiotic therapy for CDI. Bezlotoxumab is a mAb that ... 2, 3 and 4, and heavy chain CDRs (CDRH1, CDRH2 and CDRH3) consisting of the amino acid sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively. Bezlotoxumab comprises V comprising the amino acid sequence set forth in SEQ ID NO: 1. L region and the amino acid sequence set forth in SEQ ID NO:5 H and a region.

[0030] "CDI recurrence" generally refers to a new episode of CDI in a patient after a previous episode that has already resolved in the patient. More specifically, CDI recurrence in a patient is diagnosed by the occurrence of new symptoms of CDAD, e.g., a new episode of diarrhea with a positive C. difficile stool toxin test, after the resolution of a previous episode and after cessation of standard treatment therapy.

[0031] As used throughout this specification and claims, "consisting essentially of" and its derivatives, such as "consisting essentially of" or "consisting essentially of," refers to the inclusion of any recited element or group of elements, and the optional (i.e., optional) inclusion of other elements of a similar or different nature to the recited elements that do not materially alter the basic or novel characteristics of the specified administration regimen, method, or composition.

[0032] A "patient" (alternatively referred to herein as a "subject" or "individual") refers to a mammalian subject that may be infected with C. difficile. In a preferred embodiment, the patient is a human. In additional preferred embodiments, the individual is an adult, i.e., a human at least 18 years of age. The patient may be treated prophylactically or therapeutically. Prophylactic treatment provides an active pharmaceutical ingredient (e.g., a TcdB antibody) sufficient to reduce the likelihood or severity of C. difficile infection or recurrence or its effects (i.e., CDAD). Therapeutic treatment (e.g., an antibiotic) may be administered to ameliorate or prevent C. difficile infection or its effects, or to reduce the severity of C. difficile infection or CDAD.

[0033] "High risk" patients are those with one or more of the following risk factors for recurrent CDI: prior episode of CDI in the past 6 months, severe CDI at baseline [according to the Zar score described in Zar et al., A comparison of vancomycin and metronidazole for the treatment of Clostridium difficile-associated diarrhea, stratified by disease severity. Clin. Infect. Dis. 45:302-307 (2007)], age 65 years or older, CDI due to a highly virulent strain (027, 078 or 244 ribotype), immunocompromised status, and concomitant systemic antibiotics.

[0034] Those who are "in need of treatment" include those who already have a C. difficile infection, and those who are prone to infection, or anyone for whom a reduction in the likelihood of infection is desired, or individuals who are showing symptoms of CDAD. Individuals susceptible to CDI include those who have recently had a C. difficile infection, regardless of whether the individual has a positive test result for CDI at the time the particular treatment is administered. For example, patients who are in need of treatment with a TcdB targeting drug may include those who currently have CDI, or those who have recently had CDI that is undetectable at the time of treatment, such as those who have a positive test result for CDI one month prior to treatment with a TcdB drug, or 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to treatment with a TcdB drug. Those at high risk for C. difficile infection include those receiving antibiotic treatment, e.g., certain clindamycin, cephalosporins, and fluoroquinolones, as well as older patients (over 65 years), immunosuppressed patients, health care workers, and patients with long-term hospitalization.

[0035] "Isolated" is typically used to describe the purified state of a biological molecule, such as RNA, DNA, oligonucleotides, or proteins, and in this context means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other substances, such as cell debris and growth medium. In general, the term "isolated" does not contemplate the complete absence of other biological molecules or substances, or the absence of water, buffers, or salts, although these should not be present in amounts that would substantially interfere with the methods of the invention.

[0036] "Locus" means a location on a chromosome or DNA molecule that corresponds to a gene, a position associated with a physical or phenotypic characteristic, such as a polymorphic site.

[0037] A "nucleotide pair" is the set of two nucleotides (which can be the same or different) found at a polymorphic site on two copies of a chromosome from an individual.

[0038] "Oligonucleotide" refers to a nucleic acid that is typically 5-100 contiguous bases long, most frequently 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30, or 20-25 contiguous bases long. The sequence of the oligonucleotide can be designed to specifically hybridize to any of the allelic forms of the locus, and such oligonucleotides are referred to as allele-specific probes. When the locus is a PS that contains a SNP, the complementary allele of the SNP can be present at any position within the allele-specific probe. Other oligonucleotides useful in the practice of the invention specifically hybridize to a target region adjacent to the PS, with their 3' ends located 1 to about 10 or less, preferably about 5 nucleotides, from the PS. Such oligonucleotides that hybridize adjacent to the PS are useful in polymerase-mediated primer extension methods and are referred to herein as "primer extension oligonucleotides." In a preferred embodiment, the 3' end of the primer extension oligonucleotide is a deoxynucleotide that is complementary to the nucleotide located immediately adjacent to the PS.

[0039] "Pharmaceutically acceptable" refers to molecules and compositions that are "generally regarded as safe" when administered to humans, e.g., that are physiologically tolerated and do not typically cause allergic or similar undesirable reactions, such as acute gastric peristalsis, etc. In another embodiment, the term refers to molecules and compositions approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or another commonly recognized pharmacopoeia, for use in animals, and more particularly, in humans.

[0040] "Polymorphic site" or "PS" refers to a position in a locus or gene where a polymorphism is found, such as, for example, a single nucleotide polymorphism (SNP), a restriction fragment length polymorphism (RFLP), variable number of tandem repeats (VNTR), dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, insertion elements such as Alu, and deletions or insertions of one or more nucleotides. A PS usually precedes and follows a highly conserved sequence in a population of interest, and thus the location of a PS is typically defined with respect to a consensus nucleic acid sequence of 30-40 nucleotides that collectively includes the PS, which in the case of SNPs is commonly referred to as the "SNP context sequence." The location of a PS may also be identified by its location in a consensus or reference sequence. Those skilled in the art understand that the location of an individual PS may not be in the exact same position in the reference or context sequence in each individual in a population of interest due to the presence of one or more insertions or deletions in the individual compared to the consensus or reference sequence. Moreover, it is routine for one of skill in the art to design a robust, specific and highly accurate assay for detecting alternative alleles at a polymorphic site in any given individual, given the identity of the context or reference sequence in which the PS is located and the alternative alleles in the PS to be detected. Thus, the specification of the location of any PS described herein with respect to a particular position in a reference or context sequence is for convenience only, and one of skill in the art will understand that any nucleotide position specifically recited includes virtually any nucleotide position where the same PS is actually located within the same locus in any individual tested for the presence or absence of the genetic markers of the present invention using any of the genotyping methods described herein or other genotyping methods well known in the art.

[0041] "Reference SNP" or "rs" number refers to the accession number assigned to each SNP by the National Center for Biotechnology Information (NCBI).

[0042] "TcdA" means Clostridium difficile toxin A, a large clostridial toxin derived from toxigenic isolates of Clostridium difficile.

[0043] "TcdB" means Clostridium difficile toxin B, a large clostridial toxin derived from toxigenic isolates of Clostridium difficile.

[0044] "TcdA antibody" means an antibody that specifically binds to toxin A of Clostridium difficile, such as actoxumab.

[0045] "TcdB antibody" means an antibody that specifically binds to toxin B of Clostridium difficile, e.g., bezlotoxumab.

[0046] "TcdB drug" means a pharmaceutical or biological therapeutic agent that targets Clostridium difficile (C. difficile) toxin B. As defined herein, TcdB drugs include both small molecules and biological drugs (e.g., antibodies or antigen-binding fragments thereof).

[0047] "TcdB therapeutic response marker" or "TcdB drug response marker" refers to a genetic marker of the present invention that is useful for predicting a patient's response to a TcdB drug.

[0048] "Treatment" or "treating" refers to the administration of a therapeutic agent, such as a composition containing a TcdB antibody as described herein, either internally or externally, to an individual in need of the therapeutic agent. Typically, the therapeutic agent is administered in a therapeutically effective amount, which refers to an amount effective to produce one or more beneficial results. The therapeutically effective amount of a particular agent may vary depending on factors such as the condition, age and weight of the patient being treated, and the patient's sensitivity to the therapeutic agent, e.g., ability to respond. Whether a beneficial or clinical result has been achieved may be assessed by any clinical measurement typically used by a physician, physician's assistant or other skilled health care provider to assess the presence, severity or progression of a target disease, symptom or adverse effect. Typically, a therapeutically effective amount of a substance will result in at least 5%, usually at least 10%, more usually at least 20%, more usually at least 30%, preferably at least 40%, more preferably at least 50%, and most preferably at least 50% improvement in a relevant clinical scale compared to a baseline state or a state that would be expected if not treated. The clinical measure of improvement will be at least 60%, ideally at least 70%, more ideally at least 80%, and most ideally at least 90%. For example, in one embodiment where the condition or disorder is a C. difficile infection, the clinical measure of improvement is an improvement in a measure of C. difficile recurrence rate. Although embodiments of the invention (e.g., methods of treatment or articles of manufacture) may not achieve the desired clinical benefit or outcome in all patients, they should achieve it in a statistically significant number of patients as determined by any statistical test known in the art, such as logistic regression analysis based on a likelihood ratio test with 2 degrees of freedom.

[0049] The following abbreviations are used throughout this specification and have the following meanings:

[0050] ASO: Allele-specific oligonucleotide CDAD: Clostridium difficile-associated disease CDI: Clostridium difficile infection CDR: Complementarity determining region GWAS: Genome-wide association study HLA: Human leukocyte antigen LD: Linkage disequilibrium mAB: Monoclonal antibody PCR: Polymerase chain reaction PS: Polymorphic site rCDI: Recurrent CDI RFLP: Restriction fragment length polymorphism SNP: Single nucleotide polymorphism TcdA: Clostridium difficile toxin A TcdB: Clostridium difficile toxin BV H : antibody variable heavy chain region V L : Variable light chain region of antibody VNTR: Variable number of tandem repeats II. Usefulness of the TcdB therapeutic response marker of the present invention The phenotypic effects of the response markers described herein support the use of these markers in a variety of commercial applications, including, but not limited to, clinical trials of investigational or already approved TcdB pharmaceuticals in patients selected based on the presence or absence of a genetic response marker of the invention, pharmaceutical compositions and drugs comprising TcdB therapeutics (e.g., small molecules, antibodies or antigen-binding fragments thereof) to treat patients having a TcdB therapeutic response marker of the invention, diagnostic methods, and pharmacogenetic therapeutic methods, which involve tailoring a patient's drug therapy based on whether the patient has at least one TcdB therapeutic response marker of the invention.

[0051] The utility of any of the commercial applications claimed in this application does not require that the correlation between the presence of the response markers of the invention and the occurrence of a desired response to a TcdB drug or TcdB antibody be observed in 100% of individuals administered a TcdB drug or TcdB antibody, nor does it require that the diagnostic method or kit have a particular degree of specificity or sensitivity in determining the presence or absence of a response marker in each individual, nor does it require that the diagnostic methods claimed in this application be 100% accurate in predicting for all individuals whether they are likely to have a beneficial response to a TcdB drug or TcdB antibody. Thus, it is the inventors' intent in this invention that the terms "determine", "determining" and "predict" should not be construed as requiring a definite or fixed result, but rather that these terms should be interpreted to mean that the claimed method will provide an accurate result for the majority of individuals, or that the result or prediction for any given individual is more likely to be accurate than it is likely to be inaccurate.

[0052] Preferably, the accuracy of the results provided by the diagnostic methods of the invention is that which a person skilled in the art or a regulatory agency would deem appropriate for the particular application for which the method is used.Similarly, the usefulness of the claimed drugs and methods of treatment does not require that they produce the claimed or desired effect in all individuals; all that is required is that a clinical practitioner, applying their professional judgment consistent with all applicable criteria, judges that the likelihood of achieving the claimed effect of treating a given individual according to the claimed method or with the claimed drug is sufficiently high to justify prescribing the treatment or drug.

[0053] A. Testing the TcdB therapeutic response markers of the present invention The presence or absence of the TcdB therapeutic response markers of the present invention can be detected by any of a variety of genotyping or sequencing techniques commonly used in the art. Techniques that can be used to genotype an individual at the genomic location of the TcdB therapeutic response markers of the present invention include, but are not limited to, sequencing and next-generation sequencing techniques, RNA sequencing, mass spectrometry, comparative genomic hybridization, SNP-based arrays, restriction fragment length polymorphism, Sanger sequencing, chemical sequencing (e.g., Maxam and Gilbert), denaturing high performance liquid chromatography, DNA hybridization techniques and PCR. Those skilled in the art can select any of the many techniques available to determine the genotype of an individual for one or more TcdB therapeutic response markers. These techniques allow not only the detection of the presence or absence of a particular TcdB therapeutic response marker of the present invention, but also the determination of whether an individual is heterozygous or homozygous at that site.

[0054] Typical genotyping or sequencing techniques may use one or more oligonucleotides complementary to the region containing or adjacent to the PS of interest, although hybridization methods and alternative methods for detecting mutant or polymorphic sites may be used. The sequence of the oligonucleotide used to genotype a particular PS of interest is typically designed based on the context sequence for the PS.

[0055] The location of the polymorphic sites identified above in each individual is present in a reference coding or genomic DNA sequence surrounding the PS of interest, or in one of the context sequences or their complementary sequences shown in Table 2 below. Table 2 shows the context nucleic acid sequences for the SNPs determined to be TcdB treatment response markers according to the present invention. Three HLA alleles, namely HLA-DRB1 * 07:01, HLA-DQB1 * 02:02 and HLA-DQA1 *02:01 has also been shown herein to be a TcdB therapeutic response marker according to the present invention. [Table 3]

[0056] As recognized by those skilled in the art, the nucleic acid sample containing each PS can be a complementary double-stranded molecule, and therefore, the reference to each site on the sense strand also refers to the corresponding site on the complementary antisense strand.Similarly, the reference to each genotype obtained for PS on both copies of one strand of chromosome is equivalent to the complementary genotype obtained for the same PS on both copies of the other strand.For example, the C / C genotype for rs2516513 PS on the sense strand of gene is equivalent to the G / G genotype for that PS on antisense strand.

[0057] The context sequences and their complementary sequences listed herein can be used to design probes and primers for genotyping TcdB therapeutic response markers in nucleic acid samples obtained from human subjects of interest using any of a variety of methods well known in the art that allow for determining whether an individual has at least one copy of a better response allele of one or more TcdB therapeutic response markers identified herein. The nucleic acid molecules used in such methods generally include RNA, genomic DNA, or cDNA derived from RNA.

[0058] Typically, genotyping methods involve assaying a nucleic acid sample prepared from a biological sample obtained from an individual to determine the identity of the nucleotide or nucleotide pair present at one or more polymorphic sites of interest. Nucleic acid samples can be prepared from virtually any biological sample. For example, convenient samples include whole blood serum, semen, saliva, tears, feces, urine, sweat, buccal material, skin and hair. Non-malignant somatic cells are preferred because they allow the determination of the identity of both alleles present at the PS of interest without contamination by tumor-specific mutations.

[0059] Nucleic acid samples can be prepared for analysis using any technique known to those skilled in the art.Preferably, such techniques result in the isolation of genomic DNA that is sufficiently pure to determine the genotype of the desired polymorphic site in the nucleic acid molecule.In order to enhance the sensitivity and specificity of the determination, it is often desirable to amplify the target region containing the PS to be genotyped from the nucleic acid sample.Nucleic acid isolation and amplification techniques can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York) (2001).

[0060] Any amplification technique known to those skilled in the art may be used in the practice of the present invention, including, but not limited to, polymerase chain reaction (PCR) techniques. PCR may be performed using materials and methods known to those skilled in the art (see generally, PCR Technology: Principals and Applications for DNA Amplification (H.A. Erlich, ed., Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., Academic Press, San Diego, Calif., 1990); Matilla et al., Nucleic Acids Res. 19:4967 (1991); Eckert et al., PCR Methods and Applications 1:17 (1991); PCR (McPherson et al., eds., IRL Press, Oxford); and U.S. Patent No. 4,683,202). Other suitable amplification methods include ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989) and Landegren et al., Science 241:1077 (1988)), transcriptional amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)), self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990)), isothermal methods (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-6 (1992)), and nucleic acid-based sequence amplification (NASBA).

[0061] The amplified target region is assayed to determine the identity of at least one of the alleles present in the PS within the target region.It will be easily understood by those skilled in the art that if both alleles of a locus are represented within the amplified target, only one allele will be detected in the PS of an individual who is homozygous at that PS, whereas if an individual is heterozygous for that PS, two different alleles will be detected.

[0062] The identity of an allele can be determined directly, a process known as positive typing, or it can be determined presumptively, a process called negative typing. For example, if the SNP is known to be guanine or cytosine in a reference population, the PS can be determined in a positive manner to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine if the individual is heterozygous at that site. Alternatively, the PS can be determined in a negative manner to be not guanine (and thus cytosine / cytosine) or not cytosine (and thus guanine / guanine). In either case, if it is determined that at least one copy of the better response allele is present as set forth in Table 1, the determination is considered a positive test result for the better response allele in the methods, uses, products or kits described herein.

[0063] Identification of alleles or allele pairs (e.g., two nucleotides in the case of SNPs) in PS in a nucleic acid sample obtained from an individual can be accomplished using any technique known to those of skill in the art. Preferred techniques allow for rapid and accurate assay of multiple PSs with minimal sample processing. Some examples of suitable techniques include, but are not limited to, direct DNA sequencing of the amplified target region (with or without amplification), capillary electrophoresis, hybridization of allele-specific probes, single-strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, temperature gradient electrophoresis, mismatch detection, nucleic acid arrays, primer-specific extension, protein detection, imputation, and other techniques well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York) (2001); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, New York) (1997); Orita et al., Proc. Nat. Acad. Sci. USA 86, 2766-2770 (1989); Humphries et al., in Molecular Diagnosis of Genetic Diseases, pp. 32 1-340, 1996; Wartell et al., Nucl. Acids Res. 18: 2699-706 (1990); Hsu et al. (1994) Carcinogenesis 15: 1657-1662; Sheffield et al., Proc. Natl. Acad. Sci. USA 86: 232-6 (1989); Winter et al., Proc. Natl. Acad. Sci. USA 82: 7575 (1985); Myers et al. (1985) Nature 313: 495; Rosenbaum and Reissner (1987) Biophys Chem. 265: 12753; Modrich, Ann. Rev. Genet. 25: 229-53 (1991); Howie et al., Fast and accurate genotype imputation in genome-wide association studies through pre-phasing. Nature Genetics 44(8):955-959 (2012); U.S. Patent No. 6,300,063; U.S. Patent No. 5,837,832; U.S. Patent No. 5,459,039; and HuSNP Mapping Assay, reagent kit and user manual, Affymetrix Part No. 90094 (Affymetrix, Santa Clara, Calif.).

[0064] In some embodiments, the identity of alleles in PS is determined by using polymerase-mediated primer extension method.Some such methods are described in patent and scientific literature, including "Genetic Bit Analysis" method (WO 92 / 15712) and ligase / polymerase-mediated genetic bit analysis (US Pat. No. 5,679,524).Related methods are disclosed in WO 91 / 02087, WO 90 / 09455, WO 95 / 17676 and US Pat. Nos. 5,302,509 and 5,945,283.The extension primer containing the complement of polymorphism can be detected by mass spectrometry, as described in US Pat. No. 5,605,798.

[0065] Another primer extension method uses allele-specific PCR (Ruano, G. et al., Nucl. Acids Res. 17:8392 (1989); Ruano, G. et al., Nucl. Acids Res. 19:6877-82 (1991); WO 93 / 22456; Turki et al., J. Gun. Invest. 95:1635-41 (1995)).

[0066] Yet another primer extension method for identifying and analyzing polymorphisms uses single-base extension (SBE) of a fluorescently labeled primer coupled with fluorescence resonance energy transfer (FRET) between the label of the added base and the label of the primer. Typically, methods such as those described in Chen et al., Proc. Nat. Acad. Sci. 94:10756-61 (1997) use a locus-specific oligonucleotide primer labeled with 5-carboxyfluorescein (FAM) on the 5' end. The labeled primer is designed so that its 3' end is immediately adjacent to the polymorphic site of interest. The labeled primer is hybridized to the locus and single-base extension of the labeled primer is performed using fluorescently labeled dideoxyribonucleotides (ddNTPs) by dye-terminator sequencing, except that no deoxyribonucleotides are present. Enhancement of the fluorescence of the added ddNTPs in response to excitation at the wavelength of the labeled primer is used to infer the identity of the added nucleotide.

[0067] An example of a genotyping assay is the TaqMan® SNP Genotyping Assay from Thermo Fisher Scientific, Waltham, Massachusetts, USA, or an assay with similar reliability, accuracy and specificity.In one embodiment of such an assay, two allele-specific probes are used to target a specific PS.In this case, each probe has a different fluorescent label and quenching molecule attached to it.Also, two allele-specific primers are used.Upon DNA strand extension, Taq DNA polymerase cleaves the fluorescent label, and the cleavage results in detectable fluorescence emission.

[0068] In all of the above methods, the accuracy and specificity of an assay designed to detect the identity of an allele at any PS is typically validated by performing the assay on a DNA sample in which the identity of the allele at that PS is known. Preferably, a sample representative of each possible allele is included in the validation process. For diploid loci (e.g., on autosomes), validation samples typically include a sample that is homozygous for the major allele at the PS, a sample that is homozygous for the minor allele at the PS, and a sample that is heterozygous at the PS. These validation samples are typically also included as controls when performing the assay on a test sample (i.e., a sample in which the identity of the allele at the PS is unknown). The specificity of an assay can also be confirmed by comparing the assay results for a test sample with the results obtained for the same sample using a different type of assay, for example, by determining the sequence of the amplified target region believed to contain the PS of interest and comparing the determined sequence to art-recognized context sequences (e.g., context sequences described herein).

[0069] The length of the context sequence required to ensure that the appropriate genomic location is assayed will vary depending on the uniqueness of the sequence in the target region (e.g., one or more highly homologous sequences may exist in other genomic regions). One of skill in the art can readily determine the appropriate length of the context sequence for any PS using known techniques (e.g., by BLASTing the context sequence against publicly available sequence databases). For amplification target regions that provide a first level of specificity, examination of about 30-60 bases of context sequence on each side of the PS in a known sample is typically sufficient to ensure that the assay design is specific to the PS of interest. In some cases, a validated assay may not provide a clear result for a test sample. This is usually caused by the sample containing insufficient purity or amount of DNA, and a clear result is usually obtained by repurifying or reisolating the DNA sample or by assaying the sample with a different type of assay.

[0070] Several assay techniques can be used to detect PS characterized by insertion / deletion mutations. Insertion / deletion mutants can be detected by Sanger sequencing using dideoxynucleoside triphosphates. In some embodiments, commercially available software packages, such as Mutation Surveyor® software available from SoftGenetics LLC, State College, Pennsylvania, USA, can detect homozygous and heterozygous insertion / deletion mutants. Also, the fragment analysis method disclosed in Hjelm et al., The Journal of Molecular Diagnostics 12(5), pp 607-610 (2010) can be used to characterize insertion and deletion mutants.

[0071] Furthermore, when performing any of the methods described herein that require the determination of the presence or absence of a TcdB treatment response marker, such a determination can be made by consulting a data repository that contains sufficient information about the genetic makeup of the patient to determine whether the patient has the marker. Preferably, the data repository lists whether the TcdB treatment response marker is present and absent in the individual. The data repository can include the individual's medical history, a medical data card, a computer-accessible file (e.g., a flat ASCII file), or other electronic or non-electronic media on which the appropriate information or genetic data can be stored. As used herein, a medical data card is a portable storage device, such as a magnetic data card, a smart card (which has an on-board processing device and is sold by vendors such as Siemens (Munich Germany)), or a flash memory card. When the data repository is a computer-accessible file, such a file can be located on a variety of media, including a server, a client, a hard disk, a CD, a DVD, a personal digital assistant, such as a smartphone, a tape, a zip disk, a computer's internal ROM (read-only memory), or the Internet or World Wide Web. Other media for storing computer-accessible files will be apparent to those skilled in the art.

[0072] The present invention also provides methods for identifying TcdB treatment response markers by determining whether an individual has alleles (e.g., specific nucleotide sequences) at different loci that are in high linkage disequilibrium (LD) with better response alleles for the rs2516513 SNP, rs113379306 SNP, rs76166871 SNP, or one of the other TcdB treatment response markers identified in Table 1 above. It is envisioned that tests for car can be determined. Two particular alleles at different loci on the same chromosome are said to be in LD if the presence of one of the alleles at one locus tends to predict the presence of the other allele at the other locus. Such variants are referred to herein as linked or proxy variants, and can be any type of variant (e.g., SNP, insertion or deletion variants) that are in high LD with the allele of interest for better response.

[0073] Linked variants are readily identified by determining the degree of linkage disequilibrium (LD) between the better response alleles and candidate linked alleles, for example, of the rs2516513 SNP, rs113379306 SNP, or rs76166871 SNP. Candidate linked variants can be alleles of currently known polymorphisms. Other candidate linked variants can be readily identified by the skilled artisan using any technique well known in the art for discovering polymorphisms.

[0074] The degree of LD between the better response allele and the candidate linked variant in one of the TcdB treatment response markers (e.g., rs2516513 SNP, rs113379306 SNP, and / or rs76166871 SNP) can be determined using any LD measurement known in the art. LD patterns in genomic regions are easily determined empirically in appropriately selected samples using a variety of techniques known in the art for determining whether any two alleles (e.g., between nucleotides in different PSs) are in linkage disequilibrium (see, e.g., GENETIC DATA ANALYSIS II, Weir, Sineuer Associates, Inc. Publishers, Sunderland, MA 1996). Those skilled in the art can easily select which method for determining LD is most suitable for a particular population sample size and genomic region. One of the most frequently used measures of linkage disequilibrium is r 2which is calculated using the formula described by Devlin et al. (Genomics, 29(2):311-22 (1995)). 2 is a measure of how well an allele X at a first locus predicts the presence of an allele Y at a second locus on the same chromosome. Only when the prediction is perfect (e.g., X if and only if Y) does the measure reach 1.0.

[0075] In one embodiment, the locus of the linked variant is a TcdB treatment response marker of interest, such as rs2516513 SNP, rs113379306 SNP, rs76166871 SNP, HLA-DRB1 * 07:01 Allele, HLA-DQB1 * 02:02 Alleles or HLA-DQA1 * 02:01 allele PS. Other linked variants are present within a genomic region of about 100 kilobases, more preferably about 10 kb, extending across either of the PS of the 02:01 allele. Other linked variants have LD with the better response alleles of at least 0.5, more preferably at least 0.75, even more preferably at least 0.85 or at least 0.90, even more preferably at least 0.95, and most preferably 1.0, when measured in an appropriate reference population. 2 This r 2 The reference population used for the measurement can be the general population, a population using TcdB medicines, a population diagnosed with CDAD and / or CDI, or a population of members who self-identify as belonging to the same ethnic group, such as Caucasian, African American, Hispanic, Latino, Native American, etc., or any combination of these categories. Preferably, the reference population reflects the genetic diversity of the population of patients treated with TcdB medicines. For example, in Figures 5A, 5B and 5C, several r2 values ​​can be observed for SNPs rs2516513, rs113379306 and rs76166871. It is also known that there are several HLA alleles in high LD with each other, for example, HLA-DRB1 * 07:01 and HLA-DQA1* 02:01 is in almost complete LD state with each other in the GWAS data (r 2 =0.98).

[0076] In some embodiments, the health care provider determines whether a TcdB treatment response marker in Table 1 (e.g., rs2516513 SNP, rs113379306 SNP, rs76166871 SNP, HLA-DRB1 * 07:01 Allele, HLA-DQB1 * 02:02 Alleles or HLA-DQA1 * Whether a patient has a TcdB treatment response marker as described herein is determined by ordering a diagnostic test designed to determine whether the patient has at least one copy of one or more better response alleles (02:01 alleles). Preferably, the test determines the identity, i.e., genotype, of both the response allele and the alternative allele in this PS. In some embodiments, the testing laboratory prepares a nucleic acid sample from a biological sample (e.g., a blood sample or buccal swab) obtained from the patient. In some embodiments, a blood sample from the patient is collected by a health care provider, such as a physician, or a physician's staff, or by a technician at a diagnostic laboratory. In some embodiments, the patient is provided with a kit for collecting a buccal swab from the inside of the cheek, which the patient then submits to a health care provider's staff or sends directly to a diagnostic laboratory.

[0077] In some embodiments, the testing laboratory does not know the identity (who) of the individuals whose samples it is testing. That is, samples received by the laboratory are anonymized in some manner before being sent to the laboratory. For example, the sample is identifiable only by a number or some other code ("sample ID"), and the results of the diagnostic method may be reported to the sponsor using the sample ID.

[0078] In some embodiments, after the test results are obtained, the testing laboratory generates a test report that indicates whether the better response allele is present or absent at the genotyping polymorphic site, and preferably indicates whether the patient is heterozygous or homozygous for the better response allele. In some embodiments, the test report is a document that is generated by the testing laboratory and sent to the patient or the patient's health care provider as a hard copy or by email. In other embodiments, the test report is generated by a computer program and displayed on a video monitor in the health care provider's place of business. The test report may include a verbal communication of the test results directly to the patient or the patient's health care provider, such as a physician or an authorized employee in the health care provider's place of business. Similarly, the test report may include a record of the test results that the health care provider makes in the patient's file.

[0079] In one embodiment, if the patient exhibits a positive test result for at least one copy of the better response allele, the test report further indicates that the patient exhibits a positive test result for the TcdB therapeutic response marker, whereas if the individual exhibits a negative test result for the better response allele, the test report further indicates that the patient exhibits a negative test result for the TcdB therapeutic response marker.

[0080] Typically, an individual is tested for the presence of a TcdB therapeutic response marker prior to the initiation of therapy with a TcdB pharmaceutical, e.g., a TcdB antibody, although it is anticipated that such testing may occur at any time after the first dose of the TcdB pharmaceutical is administered to the individual. For example, a healthcare provider (e.g., a physician or physician's assistant) may be concerned that the patient is not responding adequately and may wish to test the individual to determine whether continued treatment with the TcdB pharmaceutical is warranted. In some embodiments, the healthcare provider may determine whether the individual should be tested for a TcdB therapeutic response marker. For example, the healthcare provider may consider whether to prescribe the patient a medication that is appropriate for patients who test positive for a TcdB therapeutic response marker.

[0081] In determining how to utilize the TcdB treatment response marker test results in the treatment of any individual patient, the healthcare provider can also take into account other relevant circumstances, such as whether the patient is considered to be at "high risk" for CDI recurrence, the patient's age, weight, sex, genetic background and race, and input the combination of these factors and the genetic marker test results into a model that helps guide the healthcare provider in selecting a therapy and / or a treatment regimen with that therapy.

[0082] Detection of the presence or absence of any of the TcdB treatment response markers can be performed using a kit specifically designed for this purpose. In one embodiment, the kit of the present invention detects, for example, rs2516513 SNP, rs113379306 SNP, rs76166871 SNP, HLA-DRB1 * 07:01 Allele, HLA-DQB1 * 02:02 Alleles or HLA-DQA1 * The 02:01 allele comprises a set of oligonucleotides designed to identify each of the alleles in the PS.

[0083] Thus, the present invention relates to a kit for testing a patient for the presence or absence of at least one copy of a better response allele of one or more TcdB treatment response markers, wherein said one or more TcdB treatment response markers are: a) the T allele of the rs2516513 single nucleotide polymorphism (SNP), b) the A allele of the rs113379306 SNP, c) the A allele of the rs76166871 SNP, d) the HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 allele, e) HLA-DQB1 of the HLA-DQB1 gene * 02:02 allele, and f) HLA-DQA1 of the HLA-DQA1 gene *The kit comprises one or more sets of oligonucleotides designed to genotype at least one of the TcdB treatment response markers, the set being selected from the group consisting of the 02:01 allele or a linked variant of the TcdB treatment response marker.

[0084] In one embodiment of this aspect of the invention, the patient has been diagnosed with or is suspected of having a C. difficile infection or is exhibiting symptoms of CDAD, hi another embodiment, the patient is at high risk for rCDI.

[0085] In a first embodiment of this aspect of the invention, the TcdB treatment response marker is the rs2516513 SNP and the better response allele is the T allele.

[0086] In a second embodiment, the TcdB treatment response marker is the rs113379306 SNP and the better response allele is the A allele.

[0087] In a third embodiment, the TcdB treatment response marker is the rs76166871 SNP and the better response allele is the A allele.

[0088] In a fourth embodiment, the TcdB treatment response marker is the HLA-DRB1 gene and the better response allele is HLA-DRB1 * 07:01 allele.

[0089] In a fifth embodiment, the TcdB treatment response marker is the HLA-DQB1 gene and the better response allele is HLA-DQB1 * 02:02 allele.

[0090] In a sixth embodiment, the TcdB treatment response marker is The HLA-DQA1 gene, and the allele associated with a better response is HLA-DQA1 * 02:01 allele.

[0091] In a seventh embodiment, the kit comprises at least two sets of oligonucleotides designed to genotype at least two of the TcdB treatment response markers.

[0092] In a subembodiment of the seventh embodiment, the two TcdB treatment response markers are the T allele of the rs2516513 SNP and the HLA-DRB1 allele of the HLA-DRB1 gene. * Includes the 07:01 allele.

[0093] In preferred subembodiments of any of the above embodiments, the oligonucleotide is an allele-specific oligonucleotide probe.

[0094] As stated above, one aspect of the invention relates to a kit for testing a patient for the presence or absence of at least one copy of a better response allele of one or more of the TcdB therapeutic response markers. In a subembodiment of any of the above embodiments, the kit comprises one set of oligonucleotides designed to genotype one of the TcdB therapeutic response markers. In other subembodiments, the kit comprises two, three, four, five, six or more sets of oligonucleotides designed to genotype multiple TcdB therapeutic response markers of the invention.

[0095] In some embodiments, the oligonucleotides in the kit are allele-specific probes or allele-specific primers. In other embodiments, the kit includes primer extension oligonucleotides. In yet other embodiments, the set of oligonucleotides is a combination of allele-specific probes, allele-specific primers and primer extension oligonucleotides. The kit may include oligonucleotides designed to detect the presence of other genetic markers associated with response to TcdB drugs.

[0096] The oligonucleotide in the kit of the present invention must be capable of specifically hybridizing to the target region of the polynucleotide.Specific hybridization as used herein means that the oligonucleotide forms an antiparallel duplex structure with the target region under certain hybridization conditions, but does not form such a structure with non-target regions when incubated with the polynucleotide under the same hybridization conditions.In some embodiments, the target region contains the PS of interest, while in other embodiments, the target region is located adjacent to the PS, 1-10 nucleotides away.

[0097] The composition and length of each oligonucleotide in the kit will depend on the nature of the genomic region containing the PS and the type of assay to be performed using the oligonucleotide, and is readily determined by one of skill in the art.

[0098] For example, the polynucleotides used in the assay may constitute an amplification product, and therefore the specificity required of the oligonucleotides is for hybridization to target regions in the amplification product, not in the genome or cDNA isolated from the individual. As another example, when a kit is designed to simultaneously genotype two or more polymorphic sites, the melting temperatures of the oligonucleotides for each PS in the kit will typically be within a narrow range, preferably less than about 5°C, more preferably less than about 2°C.

[0099] In some embodiments, each oligonucleotide in the kit is a perfect complement of its target region. An oligonucleotide is said to be a "perfect" or "perfect" complement of another nucleic acid molecule if each nucleotide of one of the molecules is complementary to the nucleotide at the corresponding position of the other molecule. For detecting polymorphisms, perfectly complementary oligonucleotides are preferred, but deviations from perfect complementarity are envisioned. However, such deviations must not prevent the molecule from specifically hybridizing to the target region. For example, an oligonucleotide primer can have a non-complementary fragment at its 5' end, with the remainder of the primer being perfectly complementary to the target region. Alternatively, non-complementary nucleotides may be interspersed within the probe or primer, but the resulting probe or primer must still be able to specifically hybridize to the target region.

[0100] In some preferred embodiments, each oligonucleotide in the kit specifically hybridizes to its target region under stringent hybridization conditions. Stringent hybridization conditions are sequence-dependent and vary depending on the situation. In general, stringent hybridization conditions are selected to be about 5°C lower than the thermal melting point (Tm) of specific sequence at a given ionic strength and pH. Tm is the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Target sequences are generally present in excess, so that at Tm, 50% of the probes are occupied at equilibrium.

[0101] Typically, stringent conditions include a salt concentration of at least about 0.01-1.0 M sodium ion (or other salt) at pH 7.0-8.3, and a temperature of at least about 25° C. for short oligonucleotide probes (e.g., 10-50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents, such as formamide. For example, conditions of 5×SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and temperatures of 25-30° C. are suitable for allele-specific probe hybridization. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), chapters 7, 9, and 11, and NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH, Haymes et al., IRL Press, Washington, DC, 1985.

[0102] A non-limiting example of stringent hybridization conditions includes hybridization in 4x sodium chloride / sodium citrate (SSC) at about 65-70°C (alternatively, hybridization in 4x SSC + 50% formamide at about 42-50°C), followed by one or more washes in 1x SSC at about 65-70°C. A non-limiting example of highly stringent hybridization conditions includes hybridization in 1x SSC at about 65-70°C (alternatively, hybridization in 1x SSC + 50% formamide at about 42-50°C), followed by one or more washes in 0.3x SSC at about 65-70°C. A non-limiting example of low stringency hybridization conditions includes hybridization in 4xSSC at about 50-60°C (or hybridization in 6xSSC+50% formamide at about 40-45°C), followed by one or more washes in 2xSSC at about 50-60°C. Stringency conditions ranging from moderate to above (e.g., 65-70°C or 42-50°C) are also intended to be included in the present invention. SSPE (1xSSPE is 0.15M NaCl, 10mM NaH2PO4 and 1.25mM EDTA, pH 7.4) can be used in place of SSC (1xSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, with washes being performed for 15 minutes each after hybridization is complete.

[0103] The hybridization temperature for hybrids expected to be less than 50 base pairs in length is the melting temperature (T m ), where Tm is determined according to the following formula: For hybrids less than 18 base pairs in length, T m (°C) = 2 (number of A+T bases) + 4 (number of G+C bases). For hybrids with a length of 18 to 49 base pairs, T m (℃)=81.5+16.6(log 10[Na+]) + 0.41(% G+C) - (600 / N), where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] = 0.165M for 1x SSC).

[0104] The oligonucleotides in the kits of the present invention may include ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives of any phosphorylation state, and other functionally equivalent derivatives. Alternatively, the oligonucleotides may have a non-phosphate-containing backbone, which may include linkages such as carboxymethyl, acetamide, carbamate, polyamide (peptide nucleic acid (PNA)), and the like (Varma, in Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Meyers, ed., pp. 6 17-20, VCH Publishers, Inc., 1995). The oligonucleotides may be produced by chemical synthesis using any suitable method known in the art, or may be derived from biological samples, for example, by restriction digestion. The oligonucleotides may contain detectable labels according to any technique known in the art, including the use of radioactive labels, fluorescent labels, enzyme labels, proteins, haptens, antibodies, sequence tags, and the like. The oligonucleotides in the kits may be manufactured and sold as analyte-specific reagents (ASRs), or may be components of approved diagnostic devices.

[0105] In some embodiments, the set of oligonucleotides in the kit has different labels that allow simultaneous determination of the identity of alleles at two or more polymorphic sites. The oligonucleotides can also comprise ordered arrays of oligonucleotides immobilized on solid surfaces such as microchips, silica beads (e.g., BeadArray technology from Illumina, San Diego, CA) or glass slides (see, e.g., WO 98 / 20020 and WO 98 / 20019). Kits that include such immobilized oligonucleotides can be designed to perform a variety of polymorphism detection assays, including, but not limited to, probe hybridization and polymerase extension assays.

[0106] Kits of the invention may also contain other reagents, such as hybridization buffers (e.g., if oligonucleotides are to be used as allele-specific probes) or dideoxynucleotide triphosphates (ddNTPs; e.g., if alleles at a polymorphic site are to be detected by primer extension). Kits designed for use in polymerase-mediated genotyping assays may also contain reaction buffers and polymerases optimized for the polymerase-mediated assay to be performed.

[0107] The kits of the invention may also contain reagents for detecting when specific hybridization or when specific polymerase-mediated extension has occurred. Such detection reagents may include biotin- or fluorescently-tagged oligonucleotides or ddNTPs and / or enzyme-labeled antibodies, as well as one or more nucleic acid sequences that generate a detectable signal upon enzymatic action. The substrate may include:

[0108] It will be understood by those skilled in the art that the sets of reagents and oligonucleotides for performing the assay will be provided in separate containers arranged within the kit container, as appropriate to maintain biological or chemical activity and enable appropriate use in the assay.

[0109] In another embodiment, each of the oligonucleotides and all other reagents in the kit comprises at least one or more of the PSs in Table 1 (e.g., rs2516513 SNP, rs113379306 SNP, rs76166871 SNP, HLA-DRB1 SNP in the HLA-DRB1 gene). * 07:01 Alleles, HLA-DQB1 of the HLA-DQB1 gene * 02:02 HLA-DQA1 allele or HLA-DQA1 gene * The kits have been quality tested for optimal performance in assays designed to determine genotypes for the 02:01 allele. In some embodiments, the kits include an instruction manual describing how to use the determined genotype to assign the presence or absence of a response marker to a test nucleic acid sample.

[0110] In some preferred embodiments, the set of oligonucleotides in the kit is allele-specific oligonucleotide.The term allele-specific oligonucleotide (ASO) used herein refers to the oligonucleotide that can specifically hybridize with one of the alleles of PS in the target region containing PS under sufficiently stringent conditions, but does not hybridize with the same region containing different alleles.As those skilled in the art will understand, allele specificity depends on various stringency conditions that can be easily optimized, including salt and formamide concentration and temperature (for both hybridization and washing steps).

[0111] Examples of hybridization and washing conditions typically used for ASO probes and primers are described in Kogan et al., “Genetic Prediction of Hemophilia A” in PCR Protocols, A Guide to Methods and Applications , Academic Press, 1990, and Ruaflo et al., Proc. Natl. Acad. Sci. USA 87:6296-300 (1990).

[0112] Typically, ASO is fully complementary to one allele, but contains a single mismatch to the other allele.In ASO probe, the single mismatch is preferably located at the center position of the oligonucleotide probe, so that it aligns with the polymorphic site in the target region (e.g., about 7th or 8th position in 15-mer, 8th or 9th position in 16-mer, and 10th or 11th position in 20-mer).In ASO primer, the single mismatch is located at the 3'-end nucleotide, or preferably the second nucleotide from the 3'-end.ASO probes and primers that hybridize to coding or non-coding strands are included in the present invention.

[0113] In some embodiments, the kit comprises a pair of allele-specific oligonucleotides for each PS to be assayed, one member of the pair specific for one allele (e.g., the better-response allele) and the other member specific for the other allele. In such embodiments, the oligonucleotides in the pair may have different lengths or different detectable labels, allowing the user of the kit to determine the genotype of the PS to be assayed.

[0114] In yet other preferred embodiments, the oligonucleotides in the kit are primer extension oligonucleotides. The termination mixture for polymerase-mediated extension from any of these oligonucleotides is selected to terminate extension of the oligonucleotide at or one base after the PS of interest, depending on the alternative nucleotides present in the PS.

[0115] In another embodiment, the kit comprises a pair of allele-specific oligonucleotide probes for genotyping at least one of the polymorphic sites in Table 1. In one embodiment, one ASO probe in the pair comprises a nucleotide sequence of at least 15 nucleotides that is identical or fully complementary to the better responding allele of the context sequence shown in Table 2, and the other ASO probe in the pair comprises a nucleotide sequence of at least 15 nucleotides that is identical or fully complementary to the other allele of the context sequence shown in Table 2.

[0116] In another embodiment, the kit comprises a pair of ASO probes, comprising a first ASO probe and a second ASO probe, for genotyping rs2516513 SNP, rs113379306 SNP or rs76166871 SNP. In one embodiment, the first ASO probe in the pair comprises a nucleotide sequence of at least 15 nucleotides that is identical or completely complementary to the better-response allele of rs2516513 SNP, rs113379306 SNP or rs76166871 SNP, and the second ASO probe in the pair comprises a nucleotide sequence of at least 15 nucleotides that is identical or completely complementary to the other allele of rs2516513 SNP, rs113379306 SNP or rs76166871 SNP.

[0117] In another embodiment, the kit comprises two or more pairs of ASO probes. In a particular embodiment, the kit comprises a first pair of ASO probes for genotyping the rs2516513 SNP and a second pair of ASO probes for genotyping the rs113379306 SNP. In another embodiment, the kit comprises a first pair of ASO probes for genotyping the rs2516513 SNP and a second pair of ASO probes for genotyping the rs76166871 SNP. In another embodiment, the kit comprises a first pair of ASO probes for genotyping the rs113379306 SNP and a second pair of ASO probes for genotyping the rs76166871 SNP. In yet another embodiment, the kit comprises a first pair of ASO probes for genotyping the rs2516513 SNP, a second pair of ASO probes for genotyping the rs113379306 SNP, and a third pair of ASO probes for genotyping the rs76166871 SNP.

[0118] In another embodiment, the kit comprises HLA-DRB1 * The method comprises a pair of allele-specific oligonucleotide probes for genotyping the 07:01 allele. In one embodiment, one ASO probe in the pair is a probe for the HLA-DRB1 allele of the better response. * The other ASO probe in the pair comprises a nucleotide sequence of at least 15 nucleotides that is identical to or completely complementary to a different allele of the HLA-DRB1 gene, e.g., an allele of the HLA-DRB1 gene that is common in the test population, or (2) a nucleotide sequence of at least 15 nucleotides that is identical to or completely complementary to a different allele of the HLA-DRB1 gene, e.g., an allele of the HLA-DRB1 gene that is common in the test population, under high stringency conditions. *In another embodiment, the kit includes a nucleotide sequence of at least 15 nucleotides that does not hybridize to the 07:01 allele but binds to two or more different alleles of the HLA-DRB1 gene (e.g., a consensus oligonucleotide sequence). * In another embodiment, the cocktail comprises an ASO probe that is identical or fully complementary to the 07:01 allele and that binds to two or more different alleles of the HLA-DRB1 gene. In another embodiment, one ASO probe in the pair is a probe that binds to the HLA-DRB1 gene with a better response. * The ASO probe in the pair contains a nucleotide sequence of at least 15 nucleotides that is identical to or completely complementary to the 07:01 allele, and the other ASO probe in the pair contains a nucleotide sequence of at least 15 nucleotides that is a control sequence, i.e., a positive or negative control.

[0119] In another embodiment, the kit comprises HLA-DQB1 * The method comprises a pair of allele-specific oligonucleotide probes for genotyping the 02:02 allele. In one embodiment, one ASO probe in the pair is a probe for the better responding HLA-DQB1 * The other ASO probe in the pair comprises a nucleotide sequence of at least 15 nucleotides that is identical or completely complementary to a different allele of the HLA-DQB1 gene, e.g., an allele of the HLA-DQB1 gene that is common in the test population, or (2) that is identical or completely complementary to a different allele of the HLA-DQB1 gene, e.g., an allele of the HLA-DQB1 gene that is common in the test population, under high stringency conditions. * In another embodiment, the kit includes a nucleotide sequence of at least 15 nucleotides that does not hybridize to the 02:02 allele but binds to two or more different alleles of the HLA-DQB1 gene (e.g., a consensus oligonucleotide sequence). *In another embodiment, the cocktail comprises one ASO probe that is identical or fully complementary to the 02:02 allele and two or more ASO probes that bind to two or more different alleles of the HLA-DRB1 gene. In another embodiment, one ASO probe in the pair is a probe that is identical to or fully complementary to the 02:02 allele of the HLA-DRB1 gene, and binds to two or more different alleles of the HLA-DRB1 gene. * 02:02 allele, and the other ASO probe in the pair contains a nucleotide sequence of at least 15 nucleotides that is identical to or completely complementary to the allele, and the other ASO probe in the pair contains a nucleotide sequence of at least 15 nucleotides that is a control sequence, i.e., a positive or negative control.

[0120] In another embodiment, the kit comprises HLA-DQA1 * The method comprises a pair of allele-specific oligonucleotide probes for genotyping the 02:01 allele. In one embodiment, one ASO probe in the pair is a probe for the better response HLA-DQA1 * The other ASO probe in the pair is either (1) identical to or completely complementary to a different allele of the HLA-DQA1 gene, e.g., an allele of the HLA-DQA1 gene that is common in the test population, or (2) is identical to or completely complementary to a different allele of the HLA-DQA1 gene, e.g., an allele of the HLA-DQA1 gene that is common in the test population, under high stringency conditions. * In another embodiment, the kit includes a nucleotide sequence of at least 15 nucleotides that does not hybridize to the 02:01 allele but binds to two or more different alleles of the HLA-DQA1 gene (e.g., a consensus oligonucleotide sequence). * In another embodiment, the cocktail comprises one ASO probe that is identical or fully complementary to the 02:02 allele and two or more ASO probes that bind to two or more different alleles of the HLA-DQA1 gene. In another embodiment, one ASO probe in the pair is a probe that is identical to or fully complementary to the 02:02 allele of the HLA-DQA1 gene with a better response. *02:01 A nucleotide sequence of at least 15 nucleotides that is identical to or perfectly complementary to an allele In one embodiment, the ASO probe in the pair contains a nucleotide sequence, and the other ASO probe in the pair contains a control sequence, i.e., a nucleotide sequence of at least 15 nucleotides that is a positive or negative control.

[0121] In further particular embodiments of any of the above kits, HLA-DRB1 * 07:01 Allele, HLA-DQB1 * 02:02 Alleles or HLA-DQA1 * ASO probes binding to the 02:01 allele were associated with better responses to HLA-DRB1 * HLA-DRB1, which is not identical or completely complementary to the 07:01 allele * Highly homologous to the 07:01 allele or HLA-DRB1 * The probe comprises a nucleotide sequence of at least 15 nucleotides that is highly homologous to the complement of the 07:01 allele. "Highly homologous" means that the probe has sufficient homology to the target sequence to specifically bind, i.e., hybridize, to the target sequence under conditions of high stringency.

[0122] In some embodiments of any of the above kits of the invention, the kit includes a kit for detecting rs2516513 SNP, rs113379306 SNP, rs76166871 SNP, HLA-DRB1 * 07:01 Allele, HLA-DQB1 * 02:02 Alleles or HLA-DQA1 * In another embodiment, the kit comprises one set of ASO probes for detecting the 02:01 allele, hi other embodiments, the kit comprises two, three, four, five, six or more sets of ASO probes for detecting two or more of the TcdB therapeutic response markers or linked variants of the TcdB therapeutic response markers of the invention.

[0123] B. Pharmaceutical Compositions, Medicaments, and Treatment Regimens In one aspect, the present invention provides a method for preventing recurrence of Clostridium difficile infection comprising administering a therapeutically effective amount of a therapy targeting Clostridium difficile (C. difficile) toxin B (TcdB therapy or TcdB medicament) to a patient in need thereof, where the patient has tested positive for at least one copy of a better response allele from one or more TcdB treatment response markers or linked variants prior to administration of the TcdB therapy, where the one or more TcdB treatment response markers are selected from the group consisting of (a) the T allele of the rs2516513 single nucleotide polymorphism (SNP), (b) the A allele of the rs113379306 SNP, (c) the A allele of the rs76166871 SNP, (d) the HLA-DRB1 allele of the HLA-DRB1 gene, and (e) the HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 allele, (e) HLA-DQB1 of the HLA-DQB1 gene * 02:02 allele, and (f) HLA-DQA1 of the HLA-DQA1 gene * 02:01 alleles.

[0124] In a first embodiment (embodiment E1) of the method, the therapy targeting TcdB is a TcdB antibody or antigen-binding fragment thereof.

[0125] In a second embodiment (embodiment E2) of the method, the therapy that targets TcdB is a small molecule drug.

[0126] In a third embodiment (embodiment E3) of the method, the therapy targeting TcdB is a TcdB antibody or antigen-binding fragment thereof, which comprises three heavy chain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain CDRs (CDRL1, CDRL2 and CDRL3), where: a) CDRH1 consists of the amino acid sequence set forth in SEQ ID NO:7 or is a variant of SEQ ID NO:7 with one or two conservative amino acid substitutions; b) CDRH2 consists of the amino acid sequence set forth in SEQ ID NO:8 or is a variant of SEQ ID NO:8 with one or two conservative amino acid substitutions; c) CDRH3 consists of the amino acid sequence set forth in SEQ ID NO:9 or is a variant of SEQ ID NO:9 with one or two conservative amino acid substitutions; d) CDRL1 consists of the amino acid sequence set forth in SEQ ID NO:2 or is a variant of SEQ ID NO:2 with one or two conservative amino acid substitutions; e) CDRL2 consists of the amino acid sequence set forth in SEQ ID NO:3 or is a variant of SEQ ID NO:3 with one or two conservative amino acid substitutions; and f) CDRL3 consists of the amino acid sequence set forth in SEQ ID NO:4 or is a variant of SEQ ID NO:4 having one or two conservative amino acid substitutions.

[0127] In a fourth embodiment (embodiment E4) of the method, the therapy targeting TcdB is a TcdB antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1.

[0128] In a fifth embodiment (Embodiment E5) of the method, the therapy targeting TcdB is bezlotoxumab.

[0129] In a sixth embodiment (embodiment E6) of the method, the TcdB-targeting therapy is as described in any one of embodiments E1-E5, and the patient is a human patient who has tested positive for CDI prior to administration of the TcdB therapy.

[0130] In a seventh embodiment of the method (embodiment E7), the TcdB-targeting therapy is as described in any one of embodiments E1-E5, and the patient is a human patient suspected of having CDI or exhibiting symptoms of CDAD, or was suspected of having CDI or exhibiting symptoms of CDAD, prior to administration of the TcdB therapy.

[0131] In an eighth embodiment (embodiment E8) of the method, the therapy targeting TcdB is as described in any one of embodiments E1 to E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response markers are: (a) the T allele of the rs2516513 SNP; (b) the A allele of the rs113379306 SNP; (c) the A allele of the rs76166871 SNP; (d) the HLA-DRB1 allele of the HLA-DRB1 gene; * 07:01 allele, (e) HLA-DQB1 of the HLA-DQB1 gene * 02:02 allele, and (f) HLA-DQA1 of the HLA-DQA1 gene * 02:01 allele.

[0132] In a ninth embodiment (embodiment E9) of the method, the TcdB targeting therapy is as described in any one of embodiments E1-E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response marker comprises the T allele or linked variant of the rs2516513 SNP.

[0133] In a tenth embodiment (embodiment E10) of the method, the therapy targeting TcdB is as described in any one of embodiments E1-E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response marker comprises the A allele or linked variant of the rs113379306 SNP.

[0134] In an eleventh embodiment (embodiment E11) of the method, the therapy targeting TcdB is as described in any one of embodiments E1-E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response marker comprises the A allele or a linked variant of the rs76166871 SNP.

[0135] In a twelfth embodiment (embodiment E12) of the method, the therapy targeting TcdB is as described in any one of embodiments E1 to E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response marker is HLA-DRB1 of the HLA-DRB1 gene. * 07:01 Includes alleles or linked variants.

[0136] In a thirteenth embodiment (embodiment E13) of the method, the therapy targeting TcdB is as described in any one of embodiments E1 to E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response marker is HLA-DQB1 of the HLA-DQB1 gene. * 02:02 Includes alleles or linked variants.

[0137] In a fourteenth embodiment (embodiment E14) of the method, the therapy targeting TcdB is as described in any one of embodiments E1 to E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response marker is HLA-DQA1 of the HLA-DQA1 gene. * 02:01 Includes alleles or linked variants.

[0138] In a fifteenth embodiment (embodiment E15) of the method, the therapy targeting TcdB is as described in any one of embodiments E1 to E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response markers are the T allele of the rs2516513 SNP and the HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 Contains both alleles.

[0139] In a sixteenth embodiment (embodiment E16) of the method, the TcdB-targeting therapy is as described in any one of embodiments E1 to E5, the patient is as described in embodiment E6 or E7, and the TcdB therapy response marker comprises two or more of the TcdB therapy response markers or linked variants listed in Table 1.

[0140] In another embodiment of any of the above methods and embodiments (e.g., embodiments E1-E16), the method further comprises treating the patient with an antibiotic that is effective against a Clostridium difficile (C. difficile) infection.

[0141] In another embodiment of any of the above methods and embodiments (eg, embodiments E1-E16), the patient is a high-risk patient.

[0142] In some embodiments, the antibiotic is selected from the group consisting of vancomycin, metronidazole, and fidaxomicin.

[0143] In a subembodiment of any of the above embodiments, the patient tests positive for one copy of a better response allele of one or more TcdB treatment response markers or linked variants of the invention. In an alternative subembodiment, the patient tests positive for two copies of a better response allele of one or more TcdB treatment response markers or linked variants of the invention.

[0144] The present invention further provides a method for determining whether a human patient is likely to respond to a pharmaceutical agent targeting Clostridium difficile (C. difficile) toxin B (TcdB), the method comprising: (a) obtaining or having obtained a biological sample from the patient; and (b) determining whether at least one TcdB response marker or linked variant better response allele is present in the biological sample, the TcdB response marker being one of: (i) the T allele of the rs2516513 single nucleotide polymorphism (SNP); (ii) the A allele of the rs113379306 SNP; (iii) the A allele of the rs76166871 SNP; (iv) the HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 Allele, (v) HLA-DQB1 of the HLA-DQB1 gene * 02:02 allele, and (vi) HLA-DQA1 of the HLA-DQA1 gene * 02:01 alleles, and (c) diagnosing the patient as susceptible to treatment with a TcdB pharmaceutical if the presence of the better response allele is detected in the biological sample.

[0145] In some embodiments of the invention described above, the method further comprises administering to the diagnosed patient a therapeutically effective amount of a TcdB medicament. The method includes a step (d) comprising:

[0146] In one embodiment of the method, the TcdB pharmaceutical is a TcdB antibody or an antigen-binding fragment thereof. In a particular embodiment, the TcdB antibody or an antigen-binding fragment thereof is bezlotoxumab.

[0147] In additional embodiments, the method further comprises administering to the patient an antibiotic effective against Clostridium difficile infection, hi certain embodiments, the antibiotic is selected from the group consisting of vancomycin, metronidazole, and fidaxomicin.

[0148] In some embodiments of any of the foregoing methods, in step (b), the T allele of the rs2516513 SNP or the HLA-DRB1 allele of the HLA-DRB1 gene * 07:01 allele is detected, and in step (c), the T allele of the rs2516513 SNP or the HLA-DRB1 allele of the HLA-DRB1 gene is detected. * If the 07:01 allele was detected, or if the T allele of the rs2516513 SNP or the HLA-DRB1 gene was detected, * If both 07:01 alleles are detected, the patient is diagnosed as susceptible to treatment with TcdB antibodies.

[0149] In an embodiment of the method of determining the above, the patient is a human diagnosed with a Clostridium difficile infection or exhibiting symptoms of Clostridium difficile-associated disease. In a particular embodiment, the patient is an adult.

[0150] In an embodiment of the above determination method, step (b) comprises sending the biological sample to a diagnostic laboratory to determine whether a better response allele of the TcdB therapeutic response marker is present in the sample.

[0151] The present invention also relates to a pharmaceutical product comprising a pharmaceutical composition and prescription information, wherein the pharmaceutical composition comprises a TcdB antibody, and the prescription information comprises a pharmacogenetic indication, wherein the pharmacogenetic indication is selected from the group consisting of (a) a T allele of the rs2516513 single nucleotide polymorphism (SNP), (b) an A allele of the rs113379306 SNP, (c) an A allele of the rs76166871 SNP, and (d) an HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 allele, (e) HLA-DQB1 of the HLA-DQB1 gene * 02:02 allele, and (f) HLA-DQA1 of the HLA-DQA1 gene * The present invention relates to a method for treating a Clostridium difficile infection or preventing a Clostridium difficile recurrence in a C. difficile infected patient who has a positive test result for at least one copy of a better response allele selected from a TcdB treatment response marker or linked variant selected from the group consisting of 02:01 alleles.

[0152] In an embodiment of this aspect of the invention, the TcdB antibody response marker is the T allele of the rs2516513 SNP and / or the HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 allele.

[0153] In another embodiment of the pharmaceutical product of the present invention, the pharmacogenetic indication further comprises treatment of a C. difficile infection with an antibiotic, hi a particular embodiment, the antibiotic is selected from the group consisting of vancomycin, metronidazole and fidaxomicin.

[0154] In some embodiments of any of the agents of the invention, the TcdB antibody is bezlotoxumab.

[0155] The present invention also relates to a method for identifying a nucleic acid molecule comprising the steps of: (a) the T allele of the rs2516513 SNP; (b) the A allele of the rs113379306 SNP; (c) the A allele of the rs76166871 SNP; and (d) the HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 allele, (e) HLA-DQB1 of the HLA-DQB1 gene * 02:02 allele, and (f) HLA-DQA1 of the HLA-DQA1 gene * The present invention provides use of bezlotoxumab in preventing C. difficile recurrence in a subgroup of patients who have tested positive for a better responding allele of a TcdB treatment response marker selected from the group consisting of the 02:01 allele, or a linked variant of said TcdB treatment response marker.

[0156] The present invention further provides the use of bezlotoxumab in the manufacture of a medicament for the prevention of C. difficile recurrence in a human patient, wherein the patient has (a) a T allele of the rs2516513 SNP, (b) an A allele of the rs113379306 SNP, (c) an A allele of the rs76166871 SNP, and (d) an HLA-DRB1 allele of the HLA-DRB1 gene. * 07:01 allele, (e) HLA-DQB1 of the HLA-DQB1 gene * 02:02 allele, and (f) HLA-DQA1 of the HLA-DQA1 gene * 02:01 allele, or a better responding allele of a linked variant of said TcdB treatment response marker.

[0157] In some embodiments of the use, the better response allele of the TcdB antibody response marker is the T allele of the rs2516513 SNP or the HLA-DRB1 allele of the HLA-DRB1 gene. *In a further embodiment of the use, the patient has the T allele of the rs2516513 SNP and the HLA-DRB1 allele of the HLA-DRB1 gene. * Positive test results for both 07:01 alleles.

[0158] In some embodiments, the TcdB pharmaceutical used in the pharmaceutical compositions, medicaments, methods and uses of the invention can be any known TcdB pharmaceutical, such as an antibody. In one embodiment, the TcdB pharmaceutical is a TcdB antibody. In some preferred embodiments, the TcdB antibody is bezlotoxumab. In additional embodiments, the TcdB antibody is an antibody or antigen-binding fragment thereof that specifically binds to TcdB, which comprises light chain CDRs (CDRL1, CDRL2 and CDRL3) consisting of the amino acid sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively, and heavy chain CDRs (CDRH1, CDRH2 and CDRH3) consisting of the amino acid sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively. In more particular embodiments, the TcdB antibody is an antibody or antigen-binding fragment thereof that specifically binds to TcdB, which comprises a V-type CDR comprising the amino acid sequence set forth in SEQ ID NO: 1, L region and the amino acid sequence set forth in SEQ ID NO:5 H and a region.

[0159] In some embodiments of the methods and uses of the invention, the TcdB antibody is administered in combination with an antibiotic. In more particular embodiments, the TcdB antibody is administered in combination with an antibiotic selected from the group consisting of vancomycin, metronidazole and fidaxomicin. The TcdB antibody and the antibiotic may be administered in the same or separate dosage forms.

[0160] Disorders that may be treated with the pharmaceutical compositions, medicaments, kits, methods and uses of the invention according to the invention are generally those that are susceptible to treatment with TcdB medicaments, i.e., TcdB antibodies achieve a clinically measurable beneficial result in a group of patients with the disease. Exemplary diseases and conditions that are susceptible to treatment with TcdB medicaments include antibiotic-associated diarrhea, colitis and pseudomembranous colitis, and / or Clostridium difficile (C. difficile) infection.

[0161] In certain embodiments, the present invention provides pharmaceutical compositions, medicaments, kits, methods and uses for treating C. difficile infection, including instructions for administering a therapeutically effective amount of a TcdB antibody to a patient in need of such treatment. In certain embodiments, the disease or condition being treated is C. difficile infection. In other embodiments, the patient is exhibiting symptoms of CDAD.

[0162] In a preferred embodiment, the TcdB antibody response markers of the present invention are used in conjunction with any TcdB antibody monotherapy or combination therapy treatment regimen comprising a TcdB antibody and an antibiotic to treat asthma, including allergic asthma. In a specific embodiment, the antibiotic is selected from the group consisting of metronidazole, vancomycin and fidaxomicin.

[0163] The dosage and administration regimen of other substances used in the combination therapy of the present invention for the treatment of disorders that are sensitive to the treatment with TcdB antibody is determined by the attending physician, taking into account the approved dosage and administration regimen in the package insert and the age, sex and general health of the patient.Substances administered in the combination therapy can be administered simultaneously (i.e., in the same composition, or in separate compositions administered one after the other) or sequentially.This is particularly useful when the components of the combination are administered in different administration schedules (e.g., one component is administered once a day and another component is administered once a week) or when their preferred pharmaceutical compositions are different (e.g., one is a tablet and the other is a capsule or infusion).Therefore, a kit containing separate dosage forms is advantageous.

[0164] When performing a combination therapy selected to treat a patient based on the presence or absence of a TcdB antibody response marker in the patient, the combined therapeutic agents, or pharmaceutical compositions or compositions containing those therapeutic agents, can be administered in any order, such as sequentially, together, simultaneously, etc. The amounts of the various therapeutic agents in such a combination therapy can be different amounts (different dosage amounts) or the same amounts (same dosage amounts). In some embodiments, the combined agents are administered at a dose that would normally be used if such agents were used in a monotherapy to treat the patient's disease or condition, while in other embodiments, the agents are administered at a dose that is lower than the dose that would normally be used if such agents were used in a monotherapy to treat the disease or condition.

[0165] The inventors also contemplate herein that the TcdB therapeutic response markers described herein may be used to obtain regulatory approval to market novel TcdB drugs or TcdB antibodies for pharmacogenetic indications (i.e., indications that include a disease component and a TcdB therapeutic marker component). The disease component is a disease that is susceptible to treatment with a TcdB drug, e.g., CDI, and the genetic marker component is a patient who tests positive for at least one copy of one of the better response alleles described in Table 1 (e.g., for at least one copy of the T allele of the rs2516513 SNP). Similarly, the inventors contemplate that the TcdB therapeutic response markers ... It is contemplated herein that antibodies will be useful in obtaining approval for such pharmacogenetic indications.

[0166] Obtaining approval for a pharmacogenetic indication may involve measuring the frequency of the desired response to the drug in two separate patient groups treated with the drug. Each individual in one of the groups has a disease and genetic profile that places the individual within the proposed pharmacogenetic indication. Individuals in the other group may be randomly selected without regard to whether they have the genetic marker component of the proposed pharmacogenetic indication. Alternatively, individuals are assigned to the other group in a manner that provides a "control" group in which the ratio of individuals satisfying and not satisfying the genetic marker component is similar to that observed in the general population or in a population of patients with the disease component of the proposed pharmacogenetic indication. The drug for which approval is sought could be administered to the two groups in a prospective clinical trial. Alternatively, a retrospective pharmacogenetic analysis of patients already treated with the drug could be performed. Those skilled in the art may readily determine alternative study designs for testing individual pharmacogenetic indications of interest.

[0167] The drug for which a pharmacogenetic indication is sought could be evaluated with other therapeutically active substances, such as another drug with therapeutic efficacy against the disease or condition in the proposed pharmacogenetic indication, or a substance intended to reduce the frequency of adverse effects caused by the drug. In some embodiments, the pharmacogenetic indication for which regulatory approval is sought may include other markers (genetic markers or biomarkers) or predictors of response to the drug.

[0168] The pharmacogenetic study could be designed in consultation with the regulatory agency or government representatives that require approval before marketing the pharmacogenetic drug in each country. Preferably, the regulatory agency is authorized by the government of a major developed country, such as Australia, Canada, China, the European Union member states, Japan, Korea, Taiwan, etc. Most preferably, the regulatory agency is authorized by the government of the United States, and the type of application for approval to be submitted will depend on the legal requirements set forth in the latest edition of the Food, Drug, and Cosmetic Act applicable to the drug, and may also include other considerations such as the cost of submitting to the regulatory agency and the marketing strategy of the drug. For example, if a pharmaceutical formulation in the drug has already been approved for the disease component of the proposed pharmacogenetic indication, the application could be a paper NDA, or an application filed under Section 351(k) of the Public Health Services Act, a supplemental NDA or BLA, or an abbreviated NDA or BLA, whereas if the pharmaceutical formulation has not been approved before, the application could require to be a full NDA or BLA. These terms have the meanings applied to them by those of ordinary skill in the pharmaceutical arts or as defined in the Drug Price Competition and Patent Term Restoration Act of 1984 or the Biologics Price Competition and Innovation Act of 2009 (BPCIA) of the Patient Protection and Affordable Care Act (2010).

[0169] One desired outcome of a pharmacogenetic clinical trial using the TcdB therapeutic response markers of the present invention is approval to market a drug product that includes (1) a TcdB pharmaceutical composition and (2) prescribing information that includes the pharmacogenetic indication for which the pharmaceutical composition is recommended. The prescribing information is typically found in a product insert (often referred to as a package insert) or label for the drug.

[0170] As mentioned above, a pharmacogenetic indication has two components: a disease component and a TcdB therapeutic response marker component. Thus, the prescribing information will describe a genetically defined patient group for which the drug has shown efficacy against one or more diseases, symptoms, or medical conditions. In some embodiments, the prescribing information indicates how to identify individuals in the genetically defined group. For example, in some embodiments, the prescribing information indicates that the drug is indicated for individuals who test positive for a better response allele of a TcdB antibody response marker described herein. Alternatively, the prescribing information may indicate that the drug is contraindicated for individuals who test negative for a better response allele of a TcdB antibody response marker described herein. In other embodiments, the approved label may indicate that the TcdB medication is indicated for preventing recurrence of CDI in patients with a particular genotype of a TcdB therapeutic response marker, for example, patients who are homozygous (T / T) or heterozygous (C / T) at the rs2516513 SNP, or patients who have a better response allele of another TcdB therapeutic response marker described herein. In some preferred embodiments, the prescribing information includes the name of at least one approved diagnostic test used to detect the presence or absence of a genetic marker component required for the pharmacogenetic indication. As noted above, the pharmacogenetic indication in the pharmacogenetic drug of the invention may include additional markers or predictors of response to the TcdB pharmaceutical composition, and / or a requirement to use the drug in combination with one or more other therapeutically active agents (e.g., antibiotics). The prescribing information may include information regarding recommended dosages and treatment regimens.

[0171] In a preferred pharmacogenetic pharmaceutical of the invention, the pharmaceutical composition comprises a TcdB pharmaceutical. In a preferred embodiment of this aspect of the invention, the TcdB pharmaceutical is a TcdB antibody. In another preferred embodiment, the TcdB antibody is bezlotoxumab. In a further embodiment, the TcdB antibody is an antibody or antigen-binding fragment thereof that specifically binds to TcdB, which comprises light chain CDRs (CDRL1, CDRL2 and CDRL3) consisting of the amino acid sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively, and heavy chain CDRs (CDRH1, CDRH2 and CDRH3) consisting of the amino acid sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively. In a further particular embodiment, the TcdB antibody is an antibody or antigen-binding fragment thereof that specifically binds to TcdB, which comprises a V-type CDR comprising the amino acid sequence set forth in SEQ ID NO: 1, L region and the amino acid sequence set forth in SEQ ID NO:5 H and a region. A preferred pharmacogenetic indication of the pharmaceutical agent of the present invention includes at least one copy of one of the better response alleles for the TcdB antibody response markers listed in Table 1, and the use of the pharmaceutical composition for the treatment of patients suffering from C. difficile infection. In a preferred embodiment, the patient tests positive for at least one copy of the T allele for the rs2516513 SNP. In some embodiments, the prescribing information states that the TcdB antibody pharmaceutical composition is indicated in combination with an antibiotic (e.g., vancomycin, metronidazole, and fidaxomicin) for the treatment of patients suffering from C. difficile infection.

[0172] All analytical and mathematical operations involved in carrying out the methods and uses described herein or using the kits, compositions and medicines described herein can be performed by a computer. For example, the computer can execute a computer program that assigns the presence or absence of a better response allele of the TcdB antibody response marker to an individual based on genotype data entered by a testing laboratory employee or a treating medical provider. The same computer or a different computer can also output a predicted response to TcdB antibody therapy based on the response marker assignment. Data regarding the presence or absence of a better response allele of the TcdB antibody response marker in an individual can be stored as part of a relational database (e.g., in the case of an Oracle database or a set of ASCII flat files) that contains other clinical and / or genetic data about the individual. These data can be stored on the computer's hard drive or can be stored, for example, on a CD ROM or on one or more other storage devices accessible by the computer. For example, the data can be stored on one or more databases connected to the computer via a network.

[0173] All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing methodologies and materials that might be used in connection with the present invention.

[0174] Although various embodiments of the present invention are described herein with reference to the accompanying drawings, it should be understood that the present invention is not limited to those precise embodiments, and that various changes and modifications may be made therein by those skilled in the art without departing from the scope and spirit of the present invention as defined in the claims. [Brief description of the drawings]

[0175] [Figure 1]FIG. 1 is a diagrammatic representation of the study designs MODIFY I (PN001) and MODIFY II (PN002) used in measuring efficacy in patients treated with a TcdB antibody, namely bezlotoxumab. [Diagram 2] FIG. 2 shows an analysis of the percentage of subjects (PN001+PN002, FAS population) with CDI recurrence after treatment with actoxumab / bezlotoxumab (MK3415A), bezlotoxumab (MK-6072) or placebo. [Diagram 3] Figure 3 shows an overview of CDI recurrence by high-risk subgroup (PN001+PN002, FAS population). [Figure 4] FIG. 4 shows the percentage and number of subjects with CDI recurrence in the various treatment arms of PN001 and PN002, and those used in the GWAS analysis. [Diagram 5] FIG. 5A shows examples of SNPs in linkage disequilibrium (LD) with rs2516513, FIG. 5B shows examples of SNPs in LD with rs113379306, and FIG. 5C shows examples of SNPs in LD with rs76166871. [Figure 6] Figure 6 shows imputed HLA data from GWAS data in xMHC pooled from PN001 and PN002 (N=1001). 219 HLA alleles were imputed to three class I and four class II HLA genes, of which 112 were common with MAF>0.01. [Figure 7] FIG. 7 shows a "Manhattan plot" identifying p-values ​​of genetic variants associated with treatment response and CDI recurrence rate. [Figure 8] FIG. 8 shows the QQ plot of the recurrent CDI GWAS analysis. [Figure 9]Figure 9 shows a region plot of p-values ​​of genetic variants around rs2516513 showing association with treatment response and CDI recurrence. Diamond dots represent the target SNP rs2516513. Grey scale represents LD information of SNP with respect to rs11209026. Curve represents recombination rate (cM / Mb according to HapMap data). [Figure 10] rCDI stratified by genotype and risk category. Figure 10 shows a significant reduction in the rate of rCDI occurring in BEZ-treated participants carrying the T allele of rs2516513 (p-value=3.04E-05, -21.5% risk difference, compared to -10.7% overall risk difference without genotype stratification) and the HLA-DRB1*07:01 allele (p-value=1.95E-05, -32.3% risk difference, compared to -10.5% overall risk difference without genotype stratification) compared to PBO recipients. [Figure 11] rCDI stratified by genotype and rCDI risk category. Figure 11 shows that the reduced risk of rCDI in BEZ-treated participants carrying the T allele of rs2516513 and HLA-DRB1*07:01 compared with PBO recipients varied according to risk factor.

[0176] Example 1 Clinical Trial of Actoxumab / Bezlotoxumab and Bezlotoxumab in Subjects with CDI To identify genetic contributions to treatment response, we used a genome-wide association study (GWAS) approach to perform pharmacogenetic (PGx) analyses on C. difficile-infected subjects undergoing clinical trials using an antibody against C. difficile toxin B to assess whether the mean treatment difference varied among patient subgroups defined by genetic variants. The clinical trial designs of the two clinical trials, designated MODIFY I (PN001) and MODIFY II (PN002), are shown in Figure 1.

[0177] PN001 was an adaptive design phase 3 trial. Subjects with confirmed CDI and receiving standard treatment for CDI (metronidazole, vancomycin, or fidaxomicin) were randomized in a 1:1:1:1 ratio to one of four treatment arms (bezlotoxumab, actoxumab, actoxumab / bezlotoxumab, or placebo). At a prespecified interim analysis, the actoxumab arm was discontinued. This decision was due to low efficacy and an observed increase in the number of deaths and SAEs compared with placebo. Overall, a total of 1412 patients with confirmed CDI were randomized and received study drug (390 in the bezlotoxumab arm, 235 in the actoxumab arm, 387 in the actoxumab / bezlotoxumab arm, and 400 in the placebo arm). PN001 met its primary efficacy objective, demonstrating that treatment with bezlotoxumab significantly reduced the proportion of subjects with CDI recurrence over 12 weeks compared with treatment with placebo (one-sided p=0.0003). CDI recurrence rates were 17.4% in the bezlotoxumab group and 27.6% in the placebo group. In the actoxumab / bezlotoxumab group, CDI recurrence rate was 15.9%, significantly better than placebo (one-sided p<0.0001) but not significantly better than bezlotoxumab (p=0.2997). Thus, the addition of actoxumab / bezlotoxumab to treatment (i.e., actoxumab / bezlotoxumab) did not confer an efficacy benefit over bezlotoxumab alone. The reduction in CDI recurrence rates in the bezlotoxumab group compared to the placebo group was also observed in prespecified subgroups of subjects with a high risk of recurrence and / or CDI-related adverse events (i.e., patients aged 65 years or older, with a history of CDI in the past 6 months prior to the baseline episode, with clinically severe CDI, 027 ribotype, epidemic strain, or immunocompromised status).For the key secondary endpoint of overall cure (also known as sustained clinical response), a numerical difference was observed in favor of the bezlotoxumab-treated group (60.1%) compared to the placebo group (55.2%), but did not achieve statistical significance (p=0.0861).Actoxumab / bezlotoxumab showed no superiority over bezlotoxumab for the overall cure endpoint (one-sided p=0.6532) (58.7% for actoxumab / bezlotoxumab and 60.1% for bezlotoxumab). PN002 was identical in design and conduct to PN001 with three exceptions: it was a traditional clinical trial of fixed design with three treatment arms (bezlotoxumab, actoxumab / bezlotoxumab, and placebo), and had an extended 9-month follow-up period in a subset of subjects (~300) to evaluate CDI recurrence and colonization with toxigenic C. difficile. All other design features were identical between PN001 and PN002. A total of 1167 patients with confirmed CDI were randomized to receive study medication (396 in the bezlotoxumab group, 390 in the actoxumab / bezlotoxumab group, and 381 in the placebo group). PN002 also met its primary efficacy objective, demonstrating that treatment with bezlotoxumab significantly reduced the proportion of subjects with CDI recurrence over 12 weeks compared with treatment with placebo (one-sided p=0.0003). CDI recurrence rates were 15.7% and 25.7% in the bezlotoxumab and placebo groups, respectively [-9.9 (95% CI: -15.5, -4.3)]. The CDI recurrence rate in the actoxumab / bezlotoxumab group was 14.9%, significantly better than the placebo group (one-sided p<0.0001), but not significantly better than the bezlotoxumab group (p=0.3718). Consistent with PN001, a reduction in the CDI recurrence rate in the bezlotoxumab group compared with the placebo group was observed in all prespecified subject subgroups. With regard to the key secondary endpoint of overall cure, bezlotoxumab demonstrated superiority over placebo (66.8% in the bezlotoxumab group and 52.1% in the placebo group; one-sided p<0.0001 [14.6 (95% CI: 7.7, 21.4)]).When actoxumab / bezlotoxumab was compared with bezlotoxumab alone in achieving overall cure, the planned one-sided p value designed to show that actoxumab / bezlotoxumab was superior to bezlotoxumab was 0.9969, and the one-sided p value to assess whether bezlotoxumab was superior to actoxumab / bezlotoxumab was 0.0031.

[0178] According to the Integrated Statistical Analysis Plan (ISAP), data from the two phase 3 studies were pooled to obtain a pooled statistical analysis of efficacy and safety. From the pooled data set (PN001+PN002), the CDI recurrence rate was 16.5% in bezlotoxumab and 26.6% in placebo, with an adjusted difference in CDI recurrence rate between the bezlotoxumab and placebo groups of 10.0% (95% CI: -14.0% to -6.0%, one-sided p<0.0001) (shown in Figure 2).

[0179] In prespecified subject subgroups (i.e., patients aged 65 years or older, with a history of CDI in the past 6 months prior to the baseline episode, clinically severe CDI, 027 ribotype, epidemic strain, or immunocompromised status), bezlotoxumab reduced the rate of CDI recurrence compared with placebo (Figure 3).

[0180] For example, the CDI recurrence rate in patients aged 65 years or older in the bezlotoxumab group was 15.4% compared with 31.4% in the placebo group (difference: -16.0 [95% CI, -21.7, -10.2]). In patients with a history of CDI in the past 6 months before the baseline episode, the CDI recurrence rate was 25.0% in the bezlotoxumab group compared with 41.1% in the placebo group (difference: -16.1 [95% CI, -24.7, -7.3]). Results from the pooled dataset for the secondary endpoint of overall cure provide strong supporting evidence that treatment with bezlotoxumab significantly increases the proportion of subjects achieving overall cure over 12 weeks compared with placebo. Overall cure rates were 63.5% in the bezlotoxumab group and 53.7% in the placebo group. The estimated adjusted difference between the bezlotoxumab and placebo groups was 9.7% (95% CI: 4.8% to 14.5%, one-sided p<0.0001).

[0181] In conclusion, the phase 3 trials demonstrated that treatment with bezlotoxumab was superior to placebo in preventing CDI recurrence (primary endpoint) over 12 weeks in patients receiving standard of care for CDI. In both phase 3 trials, bezlotoxumab was effective in the entire study population and in all subgroups of patients considered to be at high risk for CDI recurrence and / or CDI-related adverse events. Superior efficacy of bezlotoxumab alone compared with placebo was also demonstrated in PN002 and in the pooled PN001+PN002 dataset for the secondary endpoint of overall global cure. Bezlotoxumab was well tolerated and showed a safety profile similar to placebo.

[0182] Example 2 Genome-wide association study to identify SNPs associated with response to treatment including TcdB antibodies and antibioticsA genome-wide association study was performed to assess whether genetic variants across the human genome were associated with treatment differences across patients randomized to the various study arms. Because analysis of clinical trials showed that treatment benefit was primarily from bezlotoxumab and not from actoxumab, subjects from treatment arms containing bezlotoxumab (bezlotoxumab alone and bezlotoxumab + actoxumab) were pooled and compared with placebo (no monoclonal antibody administered; 0.9% NaCl only). The pooled dataset for the GWAS analysis was based on approximately 897 patients from both MODIFY I and II (PN001 and PN002; also referred to herein as the "PGx dataset"). Subjects from the actoxumab arm alone were not used in the GWAS analysis. Only subjects who provided proper consent for genetic analysis were included in the GWAS analysis. A summary of CDI recurrence rates in each of the subjects and subject groups used in the GWAS analysis is shown in Figure 4.

[0183] Genotype and clinical data DNA was extracted from the peripheral blood of consenting subjects using standard methods. Genetic data was generated from DNA samples using the UK Biobank Axiom® Array (Affymetrix, Santa Clara) and the manufacturer's recommended reagents and conditions. The array interrogates approximately 800,000 polymorphic sites across the genome. Appropriate genetic quality control procedures and imputation methods were applied to the data set.

[0184] Example 3 statistical methodsTwo pivotal studies, MODIFY I and II (PN001 and PN002), were conducted to evaluate bezlotoxumab (MK-6072), actoxumab (MK-3415), the combination of bezlotoxumab and actoxumab (MK-3415A), and placebo for the prevention of C. difficile recurrence in patients receiving standard-of-care antibiotics. The primary clinical endpoint of interest was rCDI, defined as the occurrence of a new episode of diarrhea (≥3 loose stools within 24 hours) associated with a positive local or central laboratory stool test for toxigenic C. difficile after clinical resolution of the first CDI episode. The primary efficacy endpoint was the proportion of patients with CDI recurrence assessed by week 12. Initial efficacy results from those two studies showed that bezlotoxumab was superior to placebo in reducing CDI recurrence rates (p<0.0001), and actoxumab alone showed no efficacy compared to placebo (p=0.3182).

[0185] Using UK Biobank arrays, commercially available Affymetrix Axiom r Genetic data were generated on the platform. The Axiom array has approximately 836,000 genetic variants. Genotype imputation on genotyped samples from both PN001 and PN002 studies using 1000 Genomes Phase 3 reference data and Impute2 software (Howie et al., Fast and accurate genotype imputation in genome-wide association studies through pre-phasing. Nature Genetics 44(8):955-959(2012)) was performed after genetic quality control (QC) and prior to genetic analysis. The primary genetic variant set used as input to the analysis described below included both assayed and imputed SNPs. The study objective was to perform genome-wide single variant analysis to identify genetic variants associated with treatment response as defined by reduced risk of CDI recurrence.

[0186] Genetic principal components (PCs) were calculated using EIGENSOFT (Price et al., (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38:904-909), and the first five PCs were used as covariates in the statistical model to control for confounding due to population stratification in the sample, which included patients from multiple ethnic groups. For the analysis of CDI recurrence endpoints, logistic regression based on likelihood ratio tests (LRTs) was used to evaluate the combined prognostic and treatment-related association of each genetic variant on response. The top five PCs and associated covariates, including HOSPSTR (hospitalization flag, inpatient or outpatient) and SOCSTR (standard of care (SOC) flag, fidaxomycin, metronidazole or vancomycin), were included in the model, and genotypes were coded to detect additive genetic effects. For each genetic variant, genotypes were numerically coded for individual patients as 0, 1 or 2, depending on the copy number of the minor allele. For the study or comparison, treatment was recorded numerically as 0, 1, and 2 depending on whether the patient received placebo + SOC therapy, monotherapy (actotoxumab or bezlotoxumab alone, MK-3415 or MK-6072, respectively) + SOC therapy, or combination therapy (actotoxumab + bezlotoxumab MK-3415A) + SOC therapy. The full model was as follows:

number

[0187] The comparison models were as follows:

number

[0188] Differences in -2 × log(LR) were compared to a chi-square distribution with 2 degrees of freedom. Due to limited power in SNPs with low MAF, only common SNPs with minor allele frequency (MAF) ≥ 0.01 were tested in this analysis. Bonferroni was used for multiplicity correction. Statistical significance was declared using a criterion of p-value threshold 5E-8. ​​This means that SNPs with p-values ​​less than 5E-8 are considered genome-wide statistically significant.

[0189] In addition to traditional GWAS analysis, HLA association analysis in xMHC (extended major histocompatibility complex) was performed. HLA alleles at three class I loci (HLA-A, HLA-B and HLA-C) and four class II loci (HLA-DRB1, HLA-DQA1, HLA-DQB1 and HLA-DPB1) were imputed using HIBAG (Zheng et al., HIBAG-HLA Genotype Imputation with Attribute Bagging, The Pharmacogenomics Journal, doi:10.1038 / tpj.2013.18). Best-guess imputed HLA types were used by setting the call rate threshold at 0.5, which means that imputed genotypes were set as missing if their imputation posterior probability was less than 0.5. Multi-allelic HLA types were converted to bi-allelic HLA alleles for each unique HLA allele. By assuming an additive genetic model, bi-allelic HLA alleles were then scored as 0, 1, and 2 with respect to their number of minor allele carriers. For example, if an HLA allele is HLA-A * If 11:01, the genotype is X / X, HLA-A * 11:01 / X and HLA-A * 11:01 / HLA-A *11:01 would be assigned as 0, 1 and 2, respectively. A total of 219 HLA alleles from three class I and four class II HLA genes were imputed by HIBAG, of which 112 were common with MAF>1% and were used in the analysis. The statistical analysis method for HLA alleles was exactly the same as described above for GWAS SNPs. Bonferroni was used for multiplicity correction, and the statistically significant p-value threshold was set at 4.46E-04 (0.05 / 112) for HLA association analysis.

[0190] Example 4 Evaluation of GWAS results for CDI recurrence when bezlotoxumab-containing arms are compared with placebo The original GWAS data were QCed and further imputed using Impute2 software. For subject filtering, subjects with genotypes below 97% of loci, problematic gender confirmation (F>0.8 for male and F<0.2 for female), duplications or at least second-degree consanguinity by confirming all pairwise IBD (Identity by Descent) values, and large heterozygosity (more than 3 standard deviations from the mean) were excluded. For SNP filtering (after sample filtering), SNPs with call rates below 97% and non-chromosomal SNPs were excluded. After QC and imputation, 1,001 subjects and 7,570,264 variants were available for analysis. As actoxumab showed no efficacy compared to placebo (p=0.3182), 104 subjects who received actoxumab alone were further excluded in the analysis. Based on the definition, rCDI patients had to first reach clinical cure [defined as 14 days or less of SOC therapy and absence of diarrhea (2 or less loose stools per 24 hours) for 2 consecutive days after completion of SOC therapy for the first CDI episode], therefore, 193 patients who did not show an initial clinical cure were not included in the analysis. The MAF or HLA allele frequency distribution of imputed HLA data from GWAS data in pooled xMHC from both PN001 and PN002 (N=1,001) is visualized in Figure 6.

[0191] Genome-wide association results were summarized in Manhattan plots (Figure 7) and QQ plots (Figure 8). GWAS analysis confirmed that three SNPs were associated with rCDI endpoints. In this case, rs2516513 (6:31447588) was located in the xMHC, and rs113379306 (6:17333351) and rs76166871 (6:17329940) were located outside the xMHC, close to the xMHC. HLA association analysis also identified three HLA alleles that were similarly associated with rCDI endpoints. Detailed association results of these six markers are summarized in Table 3. Similar results were obtained when the GWAS data were analyzed with the addition of subset data including patients who did not achieve clinical cure and patients of European ancestry as sensitivity analyses (results not shown). Topographic plots of rs2516513 are shown in Figure 9. [Table 4]

[0192] Additional conditional analyses were performed to identify relatively independent signals for the six SNPs and HLA alleles. Table 4 shows the pairwise linkage disequilibrium (LD) information of statistically significant SNPs and HLA alleles from the GWAS and HLA association analysis for the six markers. The rs76166871 and rs113379306 SNPs were in almost complete LD with each other (LD r 2 =0.99) and HLA-DRB1 * 07:01 and HLA-DQA1 * 02:01 is almost perfect LD (LD r 2 =0.98), whereas the rs2516513 and rs113379306 SNPs were completely independent of each other (LD r 2 =0). HLA-DRB1 * 07:01 and HLA-DQB1 * The 02:02 allele was in moderate LD (LD r 2= 0.68). The only unique statistically significant finding from the conditional analysis was the rs2516513 SNP based on the GWAS results, since the p-value for rs76166871 was not significant (p>0.05) when conditional on rs2516513. * 07:01 and HLA-DQB1 * 02:02 has an LD correlation r of 0.68 2 HLA-DRB1 * 07:01 is more significant in single marker analysis. * 07:01 HLA-DQB1 when combined with rs2516513 * 02:02 It could explain more of the variation. The top two signals, rs2516513 and HLA-DRB1 * The results of the conditional analysis during 07:01 are shown in Table 5, which shows that they were relatively independent of each other. [Table 5]

[0193] The SNP association region plot of rs2516513 (6:31447588, assayed) in the xMHC region is shown in Figure 9. rs2516513 is an intergenic and downstream variant near the HCP5 gene. Several SNPs around the rs2516513 SNP also showed association with rCDI endpoints, mainly due to high LD with this SNP. * The genetic effects for the 07:01 marker are summarized in Tables 6 and 7, respectively. The risk difference for rCDI development in the subpopulation of HLA-DRB1 carriers (bezlotoxumab + bezlotoxumab / actoxumab combination minus placebo) was -21.50%, approximately double the rate from the overall population (-10.70%). *The risk difference for rCDI development in the 07:01 carrier subpopulation was -32.30%, which is approximately three times the rate from the overall population (-10.70%). These results demonstrate the relevance of markers identified from GWAS and HLA association analysis for predicting subjects who will show improved benefit from bezlotoxumab treatment. Combinations of SNPs and HLA alleles may also provide utility in predicting subjects who will show improved benefit from bezlotoxumab treatment. As an example, Table 8 shows that the risk difference for rCDI development in the 07:01 carrier subpopulation was -32.30%, which is approximately three times the rate from the overall population (-10.70%). * Subjects carrying either the 07:01 allele, as well as the rs2516513 T allele and HLA-DRB1 * 07:01 Shows the genetic effects in subjects who carry both alleles. [Table 6] TIFF2025061120000010.tif99159TIFF2025061120000011.tif78165

[0194] Example 5 rCDI stratified by genotype and risk category The rCDI rate of the PGx population was compared with the high / low risk subgroups of rCDI stratified by SNP rs2516513. High risk of rCDI was defined by having one or more of the following six factors: history of one or more episodes of CDI in the past 6 months, severe CDI at baseline [according to Zar score], age 65 years or older, CDI caused by a highly virulent strain (027, 078 or 244 ribotype), immunocompromised status, or concurrent administration of systemic antibiotics. Low risk of rCDI was defined as not having any of the high risk factors of rCDI.

[0195] Compared with PBO recipients, the T allele of rs2516513 (p-value=3.04E-05 with a risk difference of -21.5%, compared with an overall risk difference of -10.7% without genotype stratification) and HLA-DRB1 *A significant reduction in rCDI rates was found in bezlotoxumab ("BEZ")-treated participants carrying the 07:01 allele (-32.3% risk difference with p-value = 1.95E-05, compared to -10.5% overall risk difference without genotype stratification) (see Figure 10). * The effect size of the 07:01 allele was more pronounced in BEZ-treated participants with a high risk of rCDI (risk difference vs. PBO -24.6%, p-value = 5.69E-05; and risk difference vs. PBO -33.2%, p-value = 9.81E-05, respectively).In CC homozygous patients, rCDI rates were similar in both treatment arms and in patients at high and low risk of rCDI.

[0196] Trends in the effects of these alleles were also observed in participants in the low-risk subgroup, although they were not statistically significant (T allele: risk difference for PBO -10.6%, p-value = 0.15; and HLA-DRB1 * 07:01 allele: risk difference vs. PBO -28.3%, p-value = 0.085. This may be due to the small number of participants in these subgroups. Figure 11 shows the association between the T allele of rs2516513 and HLA-DRB1 in patients with BRAF compared to PBO recipients. * The reduction in risk of rCDI in BEZ-treated participants carrying 07:01 varied according to risk factors. Results from rs2516513 genotypes indicate that participants carrying the T allele showed a strong trend towards benefit from BEZ treatment, regardless of rCDI risk category. Meanwhile, 47% of participants carrying the CC genotype, who were at high risk for rCDI, saw limited benefit from BEZ treatment. HLA-DRB1 * High-risk participants with the 07:01 genotype benefited from BEZ treatment, regardless of genetic stratification, but the association was not statistically significant in the low-risk subgroup (p-value = 0.085). In the low-risk subgroup, approximately 9% of participants carrying the T allele and HLA-DRB1 *Approximately 4% of people carrying the 07:01 allele may have benefited from BEZ treatment.

Claims

1. 1. A method of preventing recurrence of C. difficile infection comprising administering a therapeutically effective amount of a therapy targeting Clostridium difficile (C. difficile) toxin B (TcdB therapy) to a patient in need thereof, the patient having a positive test result for at least one copy of a better response allele from one or more TcdB therapy response markers prior to administration of the TcdB therapy, the one or more TcdB therapy response markers being selected from the group consisting of: a) the T allele of the rs2516513 single nucleotide polymorphism (SNP); b) the A allele of the rs113379306 SNP; c) the A allele of the rs76166871 SNP; d) the A allele of the HLA-DRB1 gene; ＊ 07:01 allele, e) HLA-DQB1 of the HLA-DQB1 gene ＊ 02:02 allele, f) HLA-DQA1 of the HLA-DQA1 gene ＊ 02:01 allele; and g) a linked variant of one or more of the TcdB therapeutic response markers set forth in a)-f).

2. 2. The method of claim 1, wherein the therapy targeting TcdB is a TcdB antibody or an antigen-binding fragment thereof.

3. a) CDRH1 consists of the amino acid sequence set forth in SEQ ID NO:7 or is a variant of SEQ ID NO:7 with one or two conservative amino acid substitutions; b) CDRH2 consists of the amino acid sequence set forth in SEQ ID NO:8 or is a variant of SEQ ID NO:8 with one or two conservative amino acid substitutions; c) CDRH3 consists of the amino acid sequence set forth in SEQ ID NO:9 or is a variant of SEQ ID NO:9 with one or two conservative amino acid substitutions; d) CDRL1 consists of the amino acid sequence set forth in SEQ ID NO:2 or is a variant of SEQ ID NO:2 with one or two conservative amino acid substitutions; e) CDRL2 consists of the amino acid sequence set forth in SEQ ID NO:3 or is a variant of SEQ ID NO:3 with one or two conservative amino acid substitutions; and f) The method according to claim 2, wherein CDRL3 consists of the amino acid sequence set forth in SEQ ID NO: 4 or is a variant of SEQ ID NO: 4 having one or two conservative amino acid substitutions.

4. The method of claim 2 or 3, wherein the Tcdb antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

1.

5. The method of any one of claims 2 to 4, wherein the TcdB therapy is bezlotoxumab.

6. The patient is administered the T allele of the rs2516513 SNP and the HLA-DRB1 allele of the HLA-DRB1 gene prior to administration of the TcdB antibody. ＊ The method of any one of claims 2 to 5, which shows a positive test result for at least one copy of the 07:01 allele.

7. The patient is administered the T allele of the rs2516513 SNP and the HLA-DRB1 allele of the HLA-DRB1 gene prior to administration of the TcdB antibody. ＊ The method of any one of claims 2 to 6, which shows a positive test result for at least one copy of the 07:01 allele.

8. 20. The method of any one of the preceding claims, wherein the method further comprises treating the patient with an antibiotic effective against C. difficile infection.

9. 9. The method of claim 8, wherein the antibiotic is selected from the group consisting of vancomycin, metronidazole and fidaxomicin.

10. 1. A method of determining whether a human patient is likely to respond to a pharmaceutical agent targeting C. difficile toxin B (TcdB), comprising: (a) obtaining or having obtained a biological sample from the patient; and (b) determining whether a better response allele of at least one TcdB response marker is present in the biological sample, wherein the TcdB response marker is: i. the T allele of the rs2516513 single nucleotide polymorphism (SNP); ii. the A allele of the rs113379306 SNP; iii. the A allele of the rs76166871 SNP; iv. the HLA-DRB1 allele of the HLA-DRB1 gene. ＊ 07:01 allele, v. HLA-DQB1 of the HLA-DQB1 gene ＊ vi. HLA-DQA1 of the HLA-DQA1 gene ＊ (c) diagnosing the patient as susceptible to treatment with a TcdB pharmaceutical if the presence of the better response allele is detected in the biological sample.

11. 11. The method of claim 10, further comprising a step (d) comprising administering to the diagnosed patient a therapeutically effective amount of a TcdB medication.

12. 12. The method of claim 11, wherein the TcdB pharmaceutical is a TcdB antibody or an antigen-binding fragment thereof.

13. 13. The method of claim 12, wherein step (d) further comprises administering to the patient an antibiotic effective against Clostridium difficile infection.

14. 14. The method of claim 13, wherein the antibiotic is selected from the group consisting of vancomycin, metronidazole and fidaxomicin.

15. In step (b), the T allele of the rs2516513 SNP or the HLA-DRB1 allele of the HLA-DRB1 gene ＊ 07:01 allele is detected, and in step (c), the T allele of the rs2516513 SNP or the HLA-DRB1 allele of the HLA-DRB1 gene is detected. ＊ If the 07:01 allele was detected, or if the rs2516513 SNP and the HLA-DRB1 gene were A-DRB1 ＊ The method of any one of claims 10 to 14, wherein the patient is diagnosed as susceptible to treatment with a TcdB antibody if both 07:01 alleles are detected.

16. The method of any one of claims 12 to 15, wherein the TcdB antibody is bezlotoxumab.

17. 17. The method of any one of claims 10 to 16, wherein the patient is a human diagnosed with a Clostridium difficile infection or exhibiting symptoms of Clostridium difficile associated disease.

18. 18. The method of any one of claims 10 to 17, wherein step (b) comprises sending the biological sample to a diagnostic laboratory to determine whether a better response allele of a TcdB therapeutic response marker is present in the sample.

19. The method of any one of claims 1 to 18, wherein the patient has an increased risk of recurrent CDI.

20. 1. A pharmaceutical product comprising a pharmaceutical composition and prescribing information, wherein the pharmaceutical composition comprises a TcdB antibody, and the prescribing information comprises a pharmacogenetic indication, the pharmacogenetic indication being selected from the group consisting of: a) the T allele of the rs2516513 single nucleotide polymorphism (SNP); b) the A allele of the rs113379306 SNP; c) the A allele of the rs76166871 SNP; d) the HLA-DRB1 allele of the HLA-DRB1 gene. ＊ 07:01 allele, e) HLA-DQB1 of the HLA-DQB1 gene ＊ 02:02 allele, f) HLA-DQA1 of the HLA-DQA1 gene ＊ 02:01 allele; g) treatment of C. difficile infection or prevention of C. difficile recurrence in a C. difficile infected patient who tests positive for at least one copy of a better response allele of one or more TcdB treatment response markers selected from the group consisting of: a) 02:01 allele; and b) linked variants of one or more TcdB treatment response markers as set forth in a)-f).

21. The TcdB antibody response marker is the T allele of the rs2516513 SNP or the HLA-DRB1 gene. ＊ 21. The pharmaceutical product of claim 20, which is the 07:01 allele.

22. 22. The pharmaceutical product of claim 20 or 21, wherein the pharmacogenetic indication further comprises treatment of a C. difficile infection with an antibiotic.

23. 23. The pharmaceutical product of claim 22, wherein the antibiotic is selected from the group consisting of vancomycin, metronidazole and fidaxomicin.

24. The pharmaceutical product of any one of claims 20 to 23, wherein the TcdB antibody is bezlotoxumab.

25. a) the T allele of the rs2516513 single nucleotide polymorphism (SNP); b) the A allele of the rs113379306 SNP; c) the A allele of the rs76166871 SNP; d) the HLA-DRB1 allele of the HLA-DRB1 gene. ＊ 07:01 allele, e) HLA-DQB1 of the HLA-DQB1 gene ＊ 02:02 allele, and f) HLA-DQA1 of the HLA-DQA1 gene ＊ A kit for testing a patient for the presence or absence of at least one copy of a better response allele of one or more TcdB therapeutic response markers selected from the group consisting of the 02:01 allele or a linked variant of a TcdB therapeutic response marker, the kit comprising a set of oligonucleotides designed to genotype at least one of the TcdB therapeutic response markers.

26. 26. The kit of claim 25, wherein the TcdB therapeutic response marker is the rs2516513 SNP.

27. 27. The kit of claim 25 or 26, wherein the oligonucleotide is an allele-specific oligonucleotide probe.

28. a) the T allele of the rs2516513 single nucleotide polymorphism (SNP); b) the A allele of the rs113379306 SNP; c) the A allele of the rs76166871 SNP; d) the HLA-DRB1 allele of the HLA-DRB1 gene. ＊ 07:01 allele, e) HLA-DQB1 of the HLA-DQB1 gene ＊ 02:02 allele, f) HLA-DQA1 of the HLA-DQA1 gene ＊ and g) a) a linked variant of one or more of the TcdB treatment response markers described in a)-f).

29. 2. Use of bezlotoxumab in the manufacture of a medicament for the prevention of C. difficile recurrence in a human patient, the patient having a positive test result for one or more TcdB treatment response markers, or better response alleles of one or more linked variants of the TcdB treatment response markers, the TcdB treatment response markers being selected from the group consisting of: a) the T allele of the rs2516513 single nucleotide polymorphism (SNP); b) the A allele of the rs113379306 SNP; c) the A allele of the rs76166871 SNP; d) the HLA-DRB1 allele of the HLA-DRB1 gene. ＊ 07:01 allele, e) HLA-DQB1 of the HLA-DQB1 gene ＊ 02:02 allele, and f) HLA-DQA1 of the HLA-DQA1 gene ＊ 02:01 alleles.

30. The better response allele of the TcdB treatment response marker is the T allele of the rs2516513 SNP or the HLA-DRB1 allele of the HLA-DRB1 gene. ＊ 30. The use according to claim 29, which is the 07:01 allele.

31. 31. The use according to claim 29 or 30, wherein the patient has a high risk of CDI recurrence.

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