Antibodies that specifically bind to peptides associated with primary immunodeficiency disorders, namely Wiscott-Aldrich syndrome and X-linked agammaglobulinemia.

Abstract

The present disclosure provides antibodies that bind to peptides associated with the primary immunodeficiency disorders (PIDDs) Wiskott-Aldrich syndrome (WAS) and X-linked agammaglobulinemia (XLA), which can be used in peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) assays for applications such as clinical diagnosis and newborn screening for WAS and XLA, among other uses.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 004,415, filed Apr. 2, 2020, which is hereby incorporated by reference in its entirety as if fully set forth herein.

[0002] Statement Regarding Federally Sponsored Research or Development This invention was made with government support under Grant No. AI123135 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Statement Regarding the Sequence Listing The sequence listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is 2GV8205_ST25.txt. The text file is 59 KB, was created on Apr. 2, 2021, and was electronically submitted via EFS - Web.

[0004] Field of the Disclosure The present disclosure provides antibodies that bind to peptides associated with primary immunodeficiency diseases (PIDD), such as Wiskott - Aldrich syndrome and X - linked agammaglobulinemia (XLA). This antibody can be used, among other applications, for the clinical diagnosis and neonatal screening of WAS and XLA.

Background Art

[0005] Many diseases have available effective treatments. However, in many of these diseases, once symptoms appear, the disease is already life - threatening or has caused irreversible damage. Examples of such diseases include primary immunodeficiency diseases (PIDD).

[0006] Primary immunodeficiency disorders (PIDDs), also known as congenital immunodeficiency disorders (IEIs), are a group of more than 416 rare genetic disorders in which components of the immune system are missing or not functioning properly. Examples of PIDDs include Wiscott-Aldrich syndrome (WAS) and X-linked agammaglobulinemia (XLA). Early detection of PIDDs is crucial for controlling and preventing potentially life-threatening infections and chronic sequelae.

[0007] If a diagnosis could be made before clinical symptoms appear, the management of PIDD would be significantly enhanced. Neonatal screening (NBS) is a standard, public, preventative, and mandatory screening test for the 4 million infants born in the United States each year. NBS typically involves a blood test performed 24 to 48 hours after birth. The screening uses a few drops of blood from the newborn's heel, collected on filter paper. The paper containing the dried blood spot (DBS) can be stored until the test is performed.

[0008] To perform an NBS assessment, a punch of dried blood is taken from a DBS (Deep Blood Surgery) and clinical tests are performed to detect the presence of specific substances in the blood (called markers or biomarkers) that indicate disorders that are not apparent at birth but can cause serious health problems later in life. The disorders screened for vary by state, but most states screen for phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell anemia. NBS has proven to be highly effective in improving patient outcomes, preventing long-term disability in affected individuals, and reducing healthcare costs.

[0009] International application number PCT / US2019 / 054856 describes the development of a multiplexed assay that may be used to screen newborns for severe combined immunodeficiency (SCID), Wiscott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson's disease (WD). This assay may significantly improve outcomes for affected individuals by reliably diagnosing these disorders before the clinical manifestations, which are often devastating and fatal, appear. The assay may detect the presence or absence of markers associated with these disorders using dried blood spots (DBS), which are already routinely collected as part of existing neonatal screening (NBS) procedures. The multiplexed assay described in PCT / US2019 / 054856 utilizes peptide immunoaffinity enrichment combined with selective reaction monitoring mass spectrometry (immuno-SRM). [Overview of the project]

[0010] This disclosure provides antibodies that specifically bind to signature peptides associated with primary immunodeficiency disorders (PIDDs), namely Wiscott-Aldrich syndrome (WAS) and X-linked agammaglobulinemia (XLA).

[0011] Certain embodiments include an antibody or antigen-binding fragment that binds to the WASp 289 signature peptide of WAS (SEQ ID NO: 1), and comprises: a heavy chain variable (VH) domain including CDRH1 of SEQ ID NO: 3, CDRH2 of SEQ ID NO: 4, and CDRH3 of SEQ ID NO: 5, and a light chain variable (VL) domain including CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8. In certain embodiments, the antibody or antigen-binding fragment that binds to the WASp 289 signature peptide includes a VH domain including SEQ ID NO: 15. In certain embodiments, the antibody or antigen-binding fragment that binds to the WASp 289 signature peptide includes a VL domain including SEQ ID NO: 16. In certain embodiments, the antibody or antigen-binding fragment that binds to the WASp 289 signature peptide includes a heavy chain including SEQ ID NO: 17. In certain embodiments, the antibody or antigen-binding fragment that binds to the WASp 289 signature peptide includes a light chain including SEQ ID NO: 18. In certain embodiments, the antibody or its antigen-binding fragment comprises a recombinant antibody or its antigen-binding fragment.

[0012] Certain embodiments include an antibody or antigen-binding fragment that binds to the BTK545 signature peptide of XLA (SEQ ID NO: 2), and includes: a heavy chain variable (VH) domain including CDRH1 of SEQ ID NO: 9, CDRH2 of SEQ ID NO: 10, and CDRH3 of SEQ ID NO: 11, and a light chain variable (VL) domain including CDRL1 of SEQ ID NO: 12, CDRL2 of SEQ ID NO: 13, and CDRL3 of SEQ ID NO: 14. In certain embodiments, the antibody or antigen-binding fragment that binds to the BTK545 signature peptide includes a VH domain including SEQ ID NO: 23. In certain embodiments, the antibody or antigen-binding fragment that binds to the BTK545 signature peptide includes a VL domain including SEQ ID NO: 24. In certain embodiments, the antibody or antigen-binding fragment that binds to the BTK545 signature peptide includes a heavy chain including SEQ ID NO: 25. In certain embodiments, the antibody or antigen-binding fragment that binds to the BTK545 signature peptide includes a light chain including SEQ ID NO: 26. In certain embodiments, the antibody or antigen-binding fragment includes a recombinant antibody or its antigen-binding fragment.

[0013] In certain embodiments, the antibodies of this disclosure may be used in assays for the clinical diagnosis of WAS and XLA. A particular embodiment provides the use of the antibodies of this disclosure to screen neonates for WAS and XLA in an assay utilizing dried blood spots (DBS) as samples. In certain embodiments, the antibodies of this disclosure may be used in peptide immunoaffinity enrichment in combination with a selective reaction monitoring mass spectrometry (immuno-SRM) assay. In certain embodiments, the assay may utilize oral swabs, peripheral blood mononuclear cells (PBMCs), and leukocytes (WBCs) as samples. This current assay, including the antibodies, can significantly improve outcomes for affected individuals by reliably diagnosing these disorders before the appearance of clinically significant and often fatal symptoms. The antibodies may be used in multiplex immuno-SRM assays as part of a screening panel for other diseases.

[0014] In certain embodiments, the antibodies of this disclosure may be used in other methods, including immunoassays. [Brief explanation of the drawing]

[0015] [Figure 1-1] Protein targets and peptide sequences used for peptide immunoaffinity enrichment in combination with selective reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose Wiscott-Aldrich syndrome (WAS) and X-linked agammaglobulinemia (XLA). Total mass, parental ion mass, and daughter ion mass are also shown. ++ indicates a doubly charged parental ion. The ion type of the daughter ion is in parentheses. [Figure 1-2] Protein targets and peptide sequences used for peptide immunoaffinity enrichment in combination with selective reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose Wiscott-Aldrich syndrome (WAS) and X-linked agammaglobulinemia (XLA). Total mass, parental ion mass, and daughter ion mass are also shown. ++ indicates a doubly charged parental ion. The ion type of the daughter ion is in parentheses. [Figure 2A] Figures 2A and 2B. Response curves of peptides measured by multiplex immunosuppression monitoring (MRM) assays using polyclonal antibodies for WASp289 and BTK545. When multiple parent ions are monitored in a single mass spectrometry (MS) run, this type of analysis is known as multiple reaction monitoring (MRM). Using MRM analysis, it is possible to monitor multiple proteins and multiple regions of proteins (signature peptides) in a single mass spectrometry run. The response curve plots the heavy:light peak area ratio as a function of heavy peptide concentration, measured in a background matrix of digested proteins extracted from dried blood spots (DBS). This curve can be used to determine the linear range and sensitivity of the assay. Each data point is plotted as a gray box, and the linear regression is plotted as a line. Regression fitting parameters are reported in the corner of each plot. The weighting of each plot is 1 / x. (Figure 2A) WASp 289; (Figure 2B) BTK545. [Figure 2B] Figures 2A and 2B. Response curves of peptides measured by multiplex immunosuppression monitoring (MRM) assays using polyclonal antibodies for WASp289 and BTK545. When multiple parent ions are monitored in a single mass spectrometry (MS) run, this type of analysis is known as multiple reaction monitoring (MRM). Using MRM analysis, it is possible to monitor multiple proteins and multiple regions of proteins (signature peptides) in a single mass spectrometry run. The response curve plots the heavy:light peak area ratio as a function of heavy peptide concentration, measured in a background matrix of digested proteins extracted from dried blood spots (DBS). This curve can be used to determine the linear range and sensitivity of the assay. Each data point is plotted as a gray box, and the linear regression is plotted as a line. Regression fitting parameters are reported in the corner of each plot. The weighting of each plot is 1 / x. (Figure 2A) WASp 289; (Figure 2B) BTK545. [Figure 3A] MRM traces of internal standard (left panel) and endogenous (right panel) signature peptides: (Figure 3A) WASp 289. Polyclonal antibodies constituting WASp 289 were used. [Figure 3B] MRM traces of internal standard (left panel) and endogenous (right panel) signature peptides: (Figure 3B) BTK 545. Polyclonal antibodies conjugating to BTK545 were used. [Figure 4A] Signature peptide concentrations in normal controls (n=40) and patients (Pt). (Figure 4A) WASp 289; (Figure 4B) BTK 545 (WAS: n=11, BTK: n=26). ****p<0.0001, *p<0.05. Polyclonal antibodies conjugating to WASp289 and BTK545 were used. [Figure 4B]Signature peptide concentrations in normal controls (n=40) and patients. (Figure 4A) WASp 289; (Figure 4B) BTK 545 (WAS: n=11, BTK: n=26). ****p<0.0001, *p<0.05. Polyclonal antibodies conjugating to WASp289 and BTK545 were used. [Figure 5-1] ATP7B 1056 signature peptide concentrations in normal controls and patients from a blinded cohort study using clinical diagnosis and genotyping. [Figure 5-2] ATP7B 1056 signature peptide concentrations in normal controls and patients from a blinded cohort study using clinical diagnosis and genotyping. [Figure 5-3] ATP7B 1056 signature peptide concentrations in normal controls and patients from a blinded cohort study using clinical diagnosis and genotyping. [Figure 6-1] Quantification of signature peptides in normal controls from a blinded cohort study. Polyclonal antibodies conjugating to WASp289 and BTK545 were used. [Figure 6-2] Quantification of signature peptides in normal controls from a blinded cohort study. Polyclonal antibodies conjugating to WASp289 and BTK545 were used. [Figure 7-1] Concentrations of signature peptides in patients from a blinded cohort study using clinical diagnosis and genotyping. Polyclonal antibodies conjugating to WASp289 and BTK545 were used. [Figure 7-2] Concentrations of signature peptides in patients from a blinded cohort study using clinical diagnosis and genotyping. Polyclonal antibodies conjugating to WASp289 and BTK545 were used. [Figure 7-3] Concentrations of signature peptides in patients from a blinded cohort study using clinical diagnosis and genotyping. Polyclonal antibodies conjugating to WASp289 and BTK545 were used. [Figure 8A]Figures 8A and 8B. Receiver operating characteristic (ROC) plots showing the diagnostic capability of immuno-SRM for PIDD. (Figure 8A) ROC plots for WASp 289 and BTK545. True positive and false positive rates are plotted against an increasing stringent cutoff value. Identical lines indicate studies where patient-control indistinguishable. (Figure 8B) Area under the curve (AUC) and p-values ​​for the peptides shown in Figure 8A. Polyclonal antibodies conjugating to WASp 289 and BTK545 were used. [Figure 8B] Figures 8A and 8B. Receiver operating characteristic (ROC) plots showing the diagnostic capability of immuno-SRM for PIDD. (Figure 8A) ROC plots for WASp 289 and BTK545. True positive and false positive rates are plotted against an increasing stringent cutoff value. Identical lines indicate studies where patient-control indistinguishable. (Figure 8B) Area under the curve (AUC) and p-values ​​for the peptides shown in Figure 8A. Polyclonal antibodies conjugating to WASp 289 and BTK545 were used. [Figure 9] Signature peptide levels of neonatal DBS obtained from a neonatal screening laboratory in Washington State. Polyclonal antibodies conjugating WASp 289 and BTK545 were used. [Figure 10-1] The ratio of ATP7B peptide to signature peptide for patient diagnosis in a blinded cohort study. Polyclonal antibodies conjugating WASp 289 and BTK545 were used. [Figure 10-2] The ratio of ATP7B peptide to signature peptide for patient diagnosis in a blinded cohort study. Polyclonal antibodies conjugating WASp 289 and BTK545 were used. [Figure 11A] Figures 11A and 11B. Measured peptide differences between monoclonal antibodies (BTK 545, n=55; WASp 289, n=50) and polyclonal antibodies (n=40) in normal control dried blood spots. [Figure 11B]Figures 11A and 11B. Measured peptide differences between monoclonal antibodies (BTK 545, n=55; WASp 289, n=50) and polyclonal antibodies (n=40) in normal control dried blood spots. [Figure 12] Normal control (n=50) range for WASp 289 monoclonal antibody. [Figure 13] Normal control range (n=55) for BTK545 monoclonal antibody and BTK545 levels in XLA patients (n=8). [Figure 14-1] An exemplary sequence of this disclosure. [Figure 14-2] An exemplary sequence of this disclosure. [Figure 14-3] An exemplary sequence of this disclosure. [Figure 14-4] An exemplary sequence of this disclosure. [Figure 14-5] An exemplary sequence of this disclosure. [Figure 14-6] An exemplary sequence of this disclosure. [Figure 14-7] An exemplary sequence of this disclosure. [Modes for carrying out the invention]

[0016] Detailed explanation Many diseases have effective treatments available. However, in many of these diseases, once symptoms appear, the disease is already fatal or has caused irreversible damage. A good example of this is primary immunodeficiency disease (PIDD).

[0017] Primary immunodeficiency disorders (PIDDs), also known as inborn errors of immunity (IEIs), are a group of more than 416 rare genetic disorders in which components of the immune system are missing or malfunctioning. While individual PIDDs are rare, the overall incidence is estimated to be about 1 in 1200 (Tangye et al. Journal of Clinical Immunology, 2020. In Press; McCusker, C., J. Upton, and R. Warrington, Primary immunoficiency. Allergy, asthema, and clinical immunology: Official journal of the Canadian Society of Allergy and Clinical Immunology, 2018.14(Suppl 2): ​​p.61-61; Kobrynski et al. J Clin Immunol, 2014.34(8):p.954-61). Once properly diagnosed and treated, patients are often able to lead relatively normal lives (Kaveri et al. Clin Exp Immunol, 2011. 164 Suppl 2: p. 2-5; Raje, N. and C. Dinakar, Immunology and allergy clinics of North America, 2015. 35(4): p. 599-623).Depending on the severity of the condition, curative treatment with hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT), or gene therapy is also possible (Raje, N. and C. Dinakar, Immunology and allergy clinics of North America, 2015.35(4):p.599-623; Aydin et al. J Clin Immunol, 2015.35(2):p.189-98; Gaspar et al. Blood, 2009.114(17):p.3524-32; Moratto et al., Blood, 2011.118(6):p.1675-84; Parta et al., Journal of Clinical Immunology, 2017.37(6):p.548-558; Staal et al. Frontiers in Pediatrics, 2019.7(443); Ferrua et al., Lancet Haematol, 2019.6(5):p.e239-e253). Nearly ubiquitous, early detection of PIDD is crucial for the management and prevention of potentially life-threatening infections and chronic sequelae (Grunebaum et al., JAMA, 2006.295(5):p.508-18; Kanariou et al. Curr Opin Hematol, 2018.25(1):p.7-12).

[0018] Early intervention is limited by the difficulty of clinically diagnosing PIDD and the lack of simple population screening tools. Laboratory evaluation is usually triggered by evidence of recurrent and / or chronic infection. Following clinical evaluation, clinical tests required to confirm the diagnosis often include technically demanding analyses such as immunocytocyte subset analysis, protein expression, and / or enzyme activity of the patient's leukocytes (Bonilla et al., Journal of Allergy and Clinical Immunology, 2015.136(5):p.1186-1205.e78).

[0019] WAS is an immunodeficiency disorder characterized by a decrease in platelet count and size. WAS is caused by mutations in the WAS gene, which produces the WAS protein (WASp), and is often considered part of a disease spectrum that includes two other disorders: X-linked thrombocytopenia and severe congenital neutropenia. These conditions have overlapping signs and symptoms and share the same genetic cause.

[0020] The decrease in platelet count and size associated with WAS leads to a reduced ability to form blood clots. This results in symptoms such as easy bruising and prolonged bleeding following minor injuries, which can be life-threatening in some cases. Individuals with WAS are also more susceptible to infections, autoimmune disorders (e.g., eczema), and certain cancers (such as lymphoma). Once diagnosed, treatments for WAS become available. Exemplary treatments include immunoglobulin infusions, antibiotics, and stem cell transplantation. Gene therapy is also being explored as a treatment option for WAS.

[0021] XLA is a hereditary immunodeficiency disorder that prevents B cells from developing normally. XLA is caused by a mutation in a gene called Bruton's tyrosine kinase (BTK). In XLA, the body is unable to produce the antibodies necessary to defend against bacteria, viruses, and other foreign substances. Children with XLA are usually healthy for the first one to two months of life because they are protected by maternal antibodies acquired before birth. However, after this time, the maternal antibodies are eliminated from the body, and affected children develop recurrent infections. These recurrent infections can lead to organ damage. Once diagnosed, treatment for XLA is available in the form of antibody infusions and antibiotics.

[0022] Since the majority of congenital PIDDs result in a decrease or deficiency of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool, particularly in neonates, which could lead to the early diagnosis and appropriate treatment of PIDD.

[0023] To perform neonatal screening (NBS) assessment, clinical testing is performed on punches of dried blood spots (DBS) to detect the presence or absence of specific substances (called markers or biomarkers) in the blood. The disorders screened for vary by state, but in most states, phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell anemia are screened for. NBS has proven to be very effective in improving patient outcomes, preventing long-term disability in affected individuals, and reducing healthcare costs. Unfortunately, detection is often limited by very low protein concentrations in blood cells and the limited amount of blood present in DBS.

[0024] International application number PCT / US2019 / 054856 describes a robust assay and method that enables the identification of patients affected by WAS and XLA from complex biological samples such as DBS. In this assay, the target peptide is purified and concentrated from DBS using an anti-peptide antibody, and then quantified by mass spectrometry. The target peptide, known as a signature peptide, is a stoichiometric surrogate for a protein deficient or missing in individuals affected by WAS or XLA. Peptide immunoaffinity enrichment, an assay combined with selective reaction monitoring mass spectrometry (immuno-SRM), allows for highly reproducible quantification of proteins present in blood at low picomolar concentrations (Collins et al., Frontiers in Immunology, 2018.9(2756)).

[0025] Signature peptide markers and antibodies that bind to them have also been developed to diagnose specific factors for other PIDDs, such as X-linked chronic granulomatous disease (X-GGD), X-linked lymphoproliferative syndrome (XLP1; SH2D1A deficiency), familial hemophagocytic lymphohistiocytosis 2 (FHL2), ataxia (AT), unclassified immunodeficiency (CVID; B-cell dysfunction), adenosine deaminase (ADA) deficiency, and cytokine deficiency 8 (DOCK8) deficiency, as well as to detect platelet cell-specific markers (CD42) and natural killer cells (CD56) using immuno-SRM (PCT / US2021 / 020679; Collins et al., Frontiers in Immunology, 2020.11(464)).

[0026] This disclosure provides antibodies and their antigen-binding fragments that can be used in immuno-SRM assays for reliably diagnosing WAS and XLA, for example, in neonates. Antipeptide antibodies and their antigen-binding fragments can be further used in multiplex immuno-SRM assays as part of a screening panel for other diseases.

[0027] The following aspects of the present disclosure are described in more detail herein: (i) collection and processing of biological samples; (ii) peptide markers of WAS and XLA; (iii) antibodies conjugated to peptide markers of WAS and XLA; (iv) variants; (v) immunoconjugates of anti-peptide antibodies; (vi) production of recombinant proteins of the present disclosure; (vii) methods of use; (viii) kits; (ix) exemplary embodiments; (x) experimental examples; (xi) final paragraph.

[0028] (i) Collection and processing of biological samples. In certain embodiments, biological samples that may be used in the immuno-SRM assay of this disclosure include samples derived from blood or cells. In certain embodiments, the sample used in the assay is DBS. In certain embodiments, whole blood derived from the subject may be prepared by placing the blood on a filter paper card and drying the blood.

[0029] In certain embodiments, whole blood derived from the subject may be collected in any anticoagulant. In certain embodiments, whole blood derived from the subject may be collected in heparin. DBS may be prepared by pipetting 50–100 μL (e.g., 70 μL) of blood / spot onto a filter paper card (e.g., Protein Saver® 903® Card, Whatman Inc, Piscataway, NJ) and drying at room temperature. In certain embodiments, the blood is dried overnight on the filter paper card. DBS may be stored, for example, in a sealed plastic bag at -80°C until use. In certain embodiments, whole DBS may be used in the immuno-SRM assay of this disclosure. In certain embodiments, one or more 3 mm punches derived from DBS may be used in the immuno-SRM assay of this disclosure. In certain embodiments, DBS may be solubilized with 0.1% Triton X-100 in 50 mM ammonium bicarbonate.

[0030] In certain embodiments, the sample used in the immuno-SRM assay of this disclosure includes cells obtained from an oral swab or mucosal sample. In certain embodiments, mucosal samples include samples from the oral cavity, nose, genitals, and rectum (Espinosa-de Aquino et al. (2017) Methods in Ecology and Evolution 8:370-378). In certain embodiments, the oral swab sample includes cells derived from the cheek or mouth. In certain embodiments, intraoral swab samples may be obtained from subjects according to the protocols described below: CHLA. (2016, April 4). Buccal Swab Collection Procedure. CHLA-Clinical Pathology; (2016, July 27). Buccal DNA Collection Instructions. Pathway Genomics; (2017, Dec 14). Instruction for Buccal Swab Sample Collection. Otogenetics; PDXL PDXL. (2017, Nov 28). Buccal Swab collection procedure-PersonalizedDx Labs [Video]. YouTube. On World Wide Web at youtu.be / 3ftvHkfM71o?t=146; and Centers of Disease Control and Prevention (CDC). (2020, July 8). Interim Guidelines for collecting, handling, and testing clinical specimens for Covid-19. On World Wide Web at cdc.gov / coronavirus / 2019-ncov / lab / guidelines-clinical-specimens.html.

[0031] In certain embodiments, an intraoral swab sample may be obtained from a subject using the following protocol: Before sample collection, the patient refrains from smoking, eating, drinking alcohol, chewing gum, or brushing their teeth for at least 30 minutes. Carefully remove the swab from its packaging, ensuring the tip does not touch any objects or surfaces. Insert the swab into the buccal cavity on one side of the mouth, between the cheek, teeth, and upper gums. Press the tip of the swab into the inside of one cheek and rub in a circular motion, up, down, left, and right. Rotate the handle during rubbing to cover the entire tip with cheek cells. Ensure the tip does not touch the teeth, gums, or lips during the collection process. Avoid over-saturating the swab with saliva. After collection, remove the swab from the mouth without touching the teeth, gums, or lips. Allow the swab to air dry at room temperature for at least 30 minutes. The swab, with the handle removed, may be stored in a cryogenic vial. This procedure may be repeated with a second swab in the opposite cheek. Intraoral swab samples may be stored at 2-8°C for up to 72 hours after collection, or in a freezer at -80°C or below for periods longer than 72 hours. In certain embodiments, cell collection using an intraoral swab may take at least 30 seconds. In certain embodiments, cell collection with an intraoral swab may be collected from the largest mucosal surface. In certain embodiments, one to five intraoral swab samples may be collected for each subject. In certain embodiments, intraoral swab samples may be air-dried on a sterile surface for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or longer. In certain embodiments, the subject may rinse their mouth with clean water before sample collection. In certain embodiments, the sample collection area may be moistened with saline solution using a separate swab. In certain embodiments, intraoral swab samples may be stored at 25°C, 20°C, 15°C, 10°C, 5°C, 0°C, -5°C, -10°C, -15°C, -20°C, or below. In certain embodiments, intraoral swab samples may be stored at -20°C for 1 to 2 weeks. In certain embodiments, buccal samples may be collected from water and / or mouthwash rinse instead of a swab (Michalczyk et al. (2004) Bio Techniques 37(2):262-269).

[0032] In certain embodiments, cells derived from oral swab samples may be solubilized with 0.1% Triton X-100 in 50 mM ammonium bicarbonate. In certain embodiments, proteins may be isolated from oral swab samples according to the protocol described by Espinosa-de Aquino et al. (2017). In certain embodiments, cells derived from oral swab samples may be extracted with a suitable buffer such as TRIzol (Thermo Fisher Scientific, Waltham, MA), and the supernatant after nucleic acid precipitation may be used for protein extraction. In certain embodiments, proteins may be precipitated with acetone, the protein pellet may be resuspended in a suitable buffer (e.g., guanidine hydrochloride in 95% ethanol supplemented with 2.5% glycerol), the pellet may be dispersed by sonication, the pellet may be washed by centrifugation, the pellet may be dried, or the pellet may be solubilized in a suitable buffer (e.g., PBS and sodium dodecyl sulfate). In certain embodiments, the solubilized pellets may be heated to 100°C and then centrifuged to obtain the supernatant for use.

[0033] In certain embodiments, the sample used in the method of the present disclosure includes peripheral blood mononuclear cells (PBMCs). PBMCs are derived from peripheral blood and from hematopoietic stem cells (HSCs) present in the bone marrow. PBMCs are blood cells with round nuclei and may include many types of cells, such as monocytes, lymphocytes (T cells, B cells, NK cells, etc.), dendritic cells, and stem cells. PBMCs can be isolated by any technique known in the art, including density centrifugation (e.g., using a Ficol-Pack). Density gradient centrifugation separates cells by cell density. In certain embodiments, whole blood or a buffy coat layer may be layered on or below the density medium without centrifugation after mixing the two layers. In certain embodiments, the PBMCs appear as a thin white layer at the interface between the plasma and the density gradient medium. In certain embodiments, Vacutaener® blood collection tubes containing Ficol-Hypak and gel plugs for separating the Ficol solution from the blood being collected may be used (Cell Preparation Tube CPT®, BD Biosciences, San Jose, CA; Puleo et al. (2017) Bio-protocol 7(2):e2103). In certain embodiments, SepMate® tubes (STEMCELL® Technologies, Vancouver, CA) designed with inserts to prevent mixing of the density gradient medium and the sample before centrifugation may be used (Kerfoot et al., Proteomics Clin Appl, 2012.6(7-8):394-402; Grievink et al., Biopreserv Biobank. 2016 Oct;14(5):410-415; Corkum et al. (2015) BMC Immunol. 16:48; Jia et al. (2018) Biopreserv Biobank 16(2):82-91). In certain embodiments, PBMCs may be isolated by leukocyte apheresis. A leukocyte apheresis machine is an automated device that collects whole blood from a donor, separates the target PBMC fraction using high-speed centrifugation, and simultaneously returns the remaining blood components, such as plasma, red blood cells, and granulocytes, to the donor.In certain embodiments, the isolated PBMC may be solubilized with 0.1% Triton X-100 in 50 mM ammonium bicarbonate.

[0034] In certain embodiments, the sample used in the immuno-SRM assay of this disclosure includes leukocytes (WBCs). WBCs are part of the immune system and protect the body from infections and foreign invaders. In certain embodiments, WBCs include granulocytes (polymorphonuclear cells), lymphocytes (mononuclear cells), and monocytes (mononuclear cells). In certain embodiments, WBCs include lymphocytes and monocytes but do not include granulocytes. WBCs can be isolated using any technique known in the art, e.g., density gradient centrifugation (Boyum (1968) Isolation of mononuclear cells and granulocytes from human blood).By isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1 g (Scand. J. Clin. Lab Invest. Suppl. 97:77; Boyum (1977) Lymphology, 10(2):71-76); by leukocyte lysis by osmotic shock (Morgensen and Cantrell (1977) Pharm Therap. 1:369-383); by RosetteSep (trademark) (STEMCELL (trademark) Technologies, Vancouver, CA), for example, by binding unwanted cells to erythrocytes via antibodies and removal by density gradient separation (Beeton and Chandy (2007) J Vis Exp. (8):326); by magnetic beads for cell enrichment or depletion (Brocks et al.) B and / or NK cells may be isolated and optionally enriched by complement-mediated lysis for enrichment (Faguet and Agee (1993) J Imm Meth 165(2):217) and panning to remove unwanted cells, including enrichment or depletion of cells by adhesion to antibody-coated plates (Brousso et al (1997) Immunol Let 59(2):85). For a review of WBC isolation and enrichment protocols, see Dagur and McCoy (2015) Curr Protoc Cytom, 73:5.1.1-5.1.16.

[0035] (ii) Peptide markers for WAS and XLA. There are many theoretical proteolytic peptides derived from target proteins, which can be potential candidates for monoclonal antibody production. In certain embodiments, the best potential candidate peptides were selected after screening their characteristics by MS / MS. These signature peptides with the highest sensitivity and specificity were selected, and corresponding monoclonal antibodies were developed and validated using clinical samples.

[0036] Typically, one or two signature proteotypic peptides, specific to the protein of interest and consistently observed in MS experiments, are selected to stoichiometrically represent the protein of interest (Mallick et al. Nat Biotechnol 2007;25:125-131). Signature peptides may be selected by detection in previous MS experiments, the use of computational tools to predict the peptide most likely to be observable by MS, or a combination of both. In certain embodiments, trypsin peptides of 5–22 amino acids with moderate hydrophobicity may be selected. Extremely hydrophilic and extremely hydrophobic peptides may have reduced stability due to variations in retention time and loss to the surface in HPLC. In certain embodiments, methionine residues (oxidized), N-terminal glutamine (cyclized), glycine or proline following asparagine (tendency toward deamidation), and dibasic ends (e.g., adjacent lysine or arginine residues such as KK, KR, RR, RK, which may alter digestion efficiency) may be undesirable (Whiteaker and Paulovich Clin Lab Med. 2011;31(3):385-396). Shorter peptides and peptides containing proline residues may be better targets for SRM (Lange et al. Molecular Systems Biology 2008;4:222).

[0037] In certain embodiments, the peptide is a signature peptide that can serve as a biomarker to diagnose whether an individual has WAS and / or XLA. Deletion or absence of the signature peptide biomarker indicates that the individual has WAS and / or XLA. In certain embodiments, the peptide comprises a portion of WASp and / or BTK. In certain embodiments, the peptide comprises Sequence IDs 1 and 2 in Table 1.

[0038] [Table 1]

[0039] (iii) Antibodies conjugating peptide markers of WAS and XLA. Antibodies conjugating signature peptide biomarkers of the present disclosure and their antigen-binding fragments are provided. In certain embodiments, anti-peptide antibodies and their antigen-binding fragments produced against peptides of proteins that are reduced or absent in WAS and XLA may be used in immuno-SRM methods to reliably diagnose WAS and XLA. In certain embodiments, the antibodies and their antigen-binding fragments of the present disclosure include recombinant antibodies and their antigen-binding fragments.

[0040] In certain embodiments, the antibodies and antigen-binding fragments of the present disclosure include a complementarity-determining region (CDR), a variable heavy domain (VH), a variable light domain (VL), a heavy chain, and a light chain, as shown in Tables 2 and 3.

[0041] [Table 2]

[0042] [Table 3-1] [Table 3-2] [Table 3-3]

[0043] In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-WASp 289 VH domain having a leader sequence encoded by SEQ ID NO: 31. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-WASp 289 VL domain having a leader sequence encoded by SEQ ID NO: 32. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-WASp 289 heavy chain having a leader sequence encoded by SEQ ID NO: 33. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-WASp 289 light chain having a leader sequence encoded by SEQ ID NO: 34. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-BTK 545 VH domain having a leader sequence encoded by SEQ ID NO: 35. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-BTK 545 VL domain having a leader sequence encoded by SEQ ID NO: 36. In specific embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-BTK 545 heavy chain having a leader sequence encoded by SEQ ID NO: 37. In certain embodiments, the exemplary antibody or its antigen-binding fragment comprises an anti-BTK 545 light chain having a leader sequence encoded by SEQ ID NO: 38.

[0044] In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-WASp 289 VH domain that does not have a leader sequence encoded by SEQ ID NO: 62. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-WASp 289 VL domain that does not have a leader sequence encoded by SEQ ID NO: 63. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-WASp 289 heavy chain that does not have a leader sequence encoded by SEQ ID NO: 64. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-WASp 289 light chain that does not have a leader sequence encoded by SEQ ID NO: 65. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-BTK 545 VH domain that does not have a leader sequence encoded by SEQ ID NO: 66. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-BTK 545 VL domain that does not have a leader sequence encoded by SEQ ID NO: 67. In certain embodiments, the exemplary antibody or its antigen-binding fragment includes an anti-BTK 545 heavy chain that does not have a leader sequence encoded by SEQ ID NO: 68. In certain embodiments, the exemplary antibody or its antigen-binding fragment comprises an anti-BTK 545 light chain that does not have a leader sequence encoded by SEQ ID NO: 69.

[0045] Antibodies contain polypeptide ligands substantially encoded by an immunoglobulin gene(s) or fragment thereof, whether naturally occurring or partially or entirely synthetically produced. Antibodies bind specifically (or selectively) to and recognize an epitope (e.g., an antigen). Antibodies may contain any protein having a binding domain homologous or largely homologous to the immunoglobulin binding domain. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class, including any human classes such as IgG, IgM, IgA, IgD, and IgE. Recognized immunoglobulin genes include kappa and lambda light chain constant region genes, alpha, gamma, delta, epsilon, and mu heavy chain constant region genes, and numerous immunoglobulin variable region genes. The “Fc” portion of an antibody refers to that portion of an immunoglobulin heavy chain that contains one or more heavy chain constant region domains, CH1, CH2, and CH3, but does not contain the heavy chain variable region.

[0046] An intact antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (VH or VH as used herein). H Each light chain consists of a light chain variable region (VL or VL in this specification) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. LThe antibody consists of a constant region of the heavy and light chains (abbreviated as VH and VL). The constant region of the light chain contains one domain CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0047] Antibody fragments include any derivative or portion of an antibody that is less than full length. In certain embodiments, the antibody fragment retains at least a substantial portion of the specific binding ability of the full-length antibody as a binding partner. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, single-strand variable fragment (scFv), Fv, dsFv diabodies, and Fd fragments, as well as any biologically viable fragment of immunoglobulins that specifically bind to the epitopes described herein. Antibodies or antibody fragments include all or part of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, minibodies, and linear antibodies.

[0048] A single-stranded variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of an immunoglobulin, linked to a short linker peptide. An Fv fragment contains the VL and VH domains of a single arm of an antibody. Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they may be linked by a synthetic linker, for example, using recombination, which allows them to be constructed as a single protein chain, where the VL and VH regions pair to form a monovalent molecule (single-stranded Fv(scFv)). For additional information regarding Fv and scFv, see, for example, Bird, et al., Science 242(1988)423-426; Huston, et al., Proc. Natl. Acad. Sci. USA 85(1988)5879-5883; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore (eds.), Springer-Verlag, New York), (1994)269-315; WO1993 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0049] The Fab fragment is a monovalent antibody fragment containing the VL, VH, CL, and CH1 domains. The F(ab')2 fragment is a bivalent fragment containing two Fab fragments linked by disulfide crosslinking at the hinge region. For consideration of Fab and F(ab')2 fragments with extended in vivo half-lives, see U.S. Patent No. 5,869,046. The diabody contains two epitope-binding sites, which may be bivalent. See, for example, European Patent No. 0404097; WO1993 / 01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90(1993) 6444-6448. Dual-affinity retargeting antibodies (DART®; based on the Diabody format but featuring a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)) may also be used. Antibody fragments may also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9 (2003) 129-134.

[0050] Antibody fragments can be produced by any means. For example, antibody fragments may be produced enzymatically or chemically by fragmentation of an intact antibody, or recombinantly from a gene encoding a partial antibody sequence. Alternatively, antibody fragments may be produced entirely or partially synthetically. Antibody fragments may include single-chain antibody fragments. In another embodiment, this fragment may include multiple chains linked together, for example, by disulfide bonds. This fragment may also include multimolecular complexes. Functional antibody fragments typically contain at least 50 amino acids, and more typically at least 200 amino acids.

[0051] In certain embodiments, recombinant immunoglobulins may be produced. See Cabilly, U.S. Patent No. 4,816,567, and Queen et al., Proc Natl Acad Sci USA, 86:10029-10033 (1989).

[0052] An “isolated” antibody is one that has been separated from its natural environment. In certain embodiments, antibodies are purified to a purity of 95% or greater than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase high-performance liquid chromatography (HPLC)). For a review of methods for assessing antibody purity, see, for example, Flatman et al. (2007) Chromatogr. B 848:79-87.

[0053] As shown, in certain embodiments, the binding domain of an engineered antibody or antigen-binding fragment may be bound via a linker. The linker is an amino acid sequence that can provide plasticity and room for higher-order structural transfer between the binding domain of the engineered antibody or the antigen-binding fragment. Any suitable linker may be used. Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct15;65(10):1357-1369. Depending on the presentation of the desired functional domain to the target, the linker may be plastic, fixed, or semi-fixed. Commonly used plastic linkers include Gly-Ser linkers such as GGSGGGSGGSG (SEQ ID NO: 39), GGSGGGSGSG (SEQ ID NO: 40), and GGSGGGSG (SEQ ID NO: 41). Additional examples include: GGGGSGGGGS (SEQ ID NO: 42); GGGSGGGS (SEQ ID NO: 43); and GGSGGS (SEQ ID NO: 44). Linkers containing one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence, may also be used.

[0054] Those skilled in the art will understand that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used for antibody expression. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation.

[0055] Monoclonal antibodies contain antibodies obtained from a substantially homogeneous population of antibodies; that is, individual antibodies within the population are identical and / or bind to the same epitope, except for any variants that may arise during the production of the monoclonal antibody (such variants are generally present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. This type of antibody is produced by daughter cells of a single antibody-producing hybridoma. Monoclonal antibodies typically exhibit a single binding affinity to any epitope they bind to.

[0056] The modifying factor “monoclonal” indicates a characteristic of antibodies as being obtained from a homogeneous population of antibodies, and should not be interpreted as requiring antibody production by any particular method. Monoclonal antibodies recognize only one type of antigen. Monoclonal antibodies as used herein include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain originates from a particular species or is identical or homologous to a corresponding sequence of an antibody belonging to a particular antibody class or subclass, while the rest of the chain(s) originates from another species or is identical or homologous to a corresponding sequence of an antibody belonging to another antibody class or subclass, as well as a fragment of such an antibody. Techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane, “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane, “Using Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1999; Tickle et al. JALA: Journal of the Association for Laboratory Automation. 2009; 14(5): 303-307; Babcook et al. Proc. Natl. Acad. Sci. USA 1996; 93: 7843-7848; and U.S. Patent No. 5,627,052.

[0057] A "human antibody" is an antibody that is produced by a human or human cell, or contains an amino acid sequence that corresponds to the amino acid sequence of an antibody derived from the human antibody repertoire or a non-human source that utilizes other human antibody coding sequences.

[0058] The "Human Consensus Framework" refers to the Human Immunoglobulin V L or V H This framework represents the most commonly present amino acid residues in the selection of framework sequences. Generally, it is used in human immunoglobulin V.L or V H Sequence selection is performed from a subgroup of variable domain sequences. The sequence subgroup may be a subgroup such as Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In certain embodiments, V L In this case, the subgroup is subgroup Kappa I, as described by Kabat et al. (mentioned above). In certain embodiments, V H In this case, the subgroup is subgroup III, as described by Kabat et al. (mentioned above).

[0059] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from a non-human CDR and amino acid residues derived from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR corresponds to that of a non-human antibody, and all or substantially all of the FR corresponds to that of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0060] Humanized antibodies and methods for producing them have been reviewed, for example, in Almagro and Fransson, Front Biosci. 13:1619-1633, 2008, and further, for example, Riechmann et al., Nature 332:323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033, 1989; U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods Further descriptions are found in 36:25-34,2005 (describes SDR(a-CDR) grafting); Padlan, Mol.Immunol.28:489-498,1991 (describes "resurfacing"); Dall'Acqua et al., Methods 36:43-60,2005 (describes "FR shuffling"); and Osbourn et al., Methods 36:61-68,2005 and Klimka et al., Br.J.Cancer,83:252-260,2000 (describes the "guided selection" approach to FR shuffling). EP-B-0239400 further describes "CDR grafting," in which one or more CDRs of the initial antibody are placed within the framework of a non-antibody sequence, e.g., another antibody.

[0061] Human framework regions that can be used for humanization include the following: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296, 1993); framework regions derived from consensus sequences of human antibodies of certain subgroups of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285, 1992; and Presta et al., J. Immunol., 151:2623, 1993); human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684, 1997; and Rosok et al., J. Biol. Chem. 271:22611-22618, 1996).

[0062] In certain embodiments, "affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and a peptide). The affinity of molecule X for its partner Y is generally represented by the dissociation constant (K D ) or the association constant (K A ). Affinity can be measured by common methods known in the art. In certain embodiments, K D can be characterized using a radio-labeled antigen binding assay (RIA) or a surface plasmon resonance assay.

[0063] In certain embodiments, "binding" means that the binding domain of an antibody has a dissociation constant (K -8 ) of 10 D M or less, and in certain embodiments 10-5 M~10 -13 M, in a particular embodiment, 10 -5 M~10 -10 M, in a particular embodiment, 10 -5 M~10 -7 M, in a particular embodiment, 10 -8 M~10 -13 M, or 10 in a particular embodiment. -9 M~10 -13 This means that it associates with its target peptide with a dissociation constant (KD) of M. This term may also be used to indicate that the binding domain does not bind to other biomolecules present (for example, it is 10 -4 M or more, or 10 in a specific embodiment. -4 A dissociation constant (K) such that M ~ 1M D (It binds to other biomolecules that have )

[0064] In a particular embodiment, "binding" means that the binding domain of the antibody is 10 7 M -1 The affinity constants (i.e., association constants, K) mentioned above. A ), in a specific embodiment, 10 5 M -1 ~10 13 M -1 In a specific embodiment, 10 5 M -1 ~10 10 M -1 In a specific embodiment, 10 5 M -1 ~10 8 M -1 In a specific embodiment, 10 7 M -1 ~10 13 M -1 , or 10 in a particular embodiment 7 M -1 ~10 8 M -1 This means that it associates with the target peptide with an affinity constant of 10. This term may also be used to indicate that it does not bind to other biomolecules in which the binding domain exists (for example, it is 10). 4 M -1 The following aggregation constants (K A), in a specific embodiment, 10 4 M -1 ~1M -1 It binds to other biomolecules with an association constant of .

[0065] An antibody that binds to the same epitope as the antibody disclosed herein refers to an antibody that blocks the binding of the antibody disclosed herein to each peptide by 50% or more in a competitive assay, and conversely, an antibody disclosed herein blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. In an exemplary competitive assay, the immobilized WASp 289 or BTK 545 peptide is incubated in a solution containing a first labeled antibody that binds to the WASp 289 or BTK 545 peptide and a second unlabeled antibody that has been tested for its ability to compete with anti-WASp 289 or anti-BTK 545, respectively. As a control, the immobilized WASp 289 or BTK 545 peptide is incubated in a solution containing a first labeled antibody that binds to the WASp 289 or BTK 545 peptide but does not bind to the second unlabeled antibody. After incubation under conditions that allow binding of anti-WASp 289 or anti-BTK 545 to their respective peptides, excess unbound antibody is removed, and the amount of labeling associated with the immobilized WASp 289 or BTK 545 peptide is measured. If the amount of labeling associated with the immobilized WASp 289 or BTK 545 peptide is significantly reduced in the test sample compared to the control sample, it indicates that the secondary antibody is competing with anti-WASp 289 or anti-BTK 545 for binding to the WASp 289 or BTK 545 peptide, respectively. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbour Laboratory, Cold Spring Harbour, NY).

[0066] (iv) Variants. This includes variants of the antibodies described herein. Antibody variants may include variants having one or more conserved amino acid substitutions or one or more non-conservative substitutions that do not adversely affect protein binding.

[0067] In certain embodiments, conservative amino acid substitutions may not substantially alter the structural features of the reference sequence (e.g., the substituted amino acid must not disrupt the antibody / peptide bond). Examples of secondary and tertiary structures of polypeptides recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature, 354:105 (1991).

[0068] Naturally occurring amino acids are generally classified into the following conserved substitution families: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6: (large aliphatic, nonpolar residues): isoleucine ( Group 7 (non-charged): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp), and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, non-polar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0069] When making such modifications, the hydroxyl index of amino acids may be taken into consideration. The importance of the hydroxyl amino acid index in conferring interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982, J.Mol.Biol.157(1), 105-32). Each amino acid is assigned a hydroxyl index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982). These values ​​are as follows: Ile(+4.5);Val(+4.2);Leu(+3.8);Phe(+2.8);Cys(+2.5);Met(+1.9);Ala(+1.8);Gly(-0.4);Thr(-0.7);Ser(-0.8);Trp(-0.9);Tyr(-1.3);Pro(-1.6);His(-3.2);Glutamate(-3.5);Gln(-3.5);Aspartic acid(-3.5);Asn(-3.5);Lys(-3.9);and Arg(-4.5).

[0070] It is known in the art that certain amino acids may be substituted with other amino acids having similar hydroxyl index or scores, and that this may still result in proteins with similar biological activity, i.e., still obtainable proteins that are biologically and functionally equivalent. When making such modifications, substitutions of amino acids with a hydroxyl index of ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more preferred. It is also understood in the art that similar amino acid substitutions can be effectively carried out based on hydrophilicity.

[0071] As detailed in U.S. Patent No. 4,544,101, the following hydrophilic values ​​are assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Aspartic acid (+3.0±1); Glutamic acid (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that amino acids may be substituted with other amino acids having similar hydrophilic values, and that biologically equivalent, and especially immunologically equivalent, proteins can still be obtained. In such changes, substitutions of amino acids with a hydrophilicity value of ±2 or less are preferred, those with a value of ±1 or less are particularly preferred, and those with a value of ±0.5 or less are even more preferred.

[0072] As outlined above, amino acid substitutions may be based on the relative similarity of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc.

[0073] In a particular embodiment, V L The region is disclosed V L V may originate from or be obtained based thereon. L Compared to the above, this may include one or more insertions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more deletions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more amino or acid substitutions (e.g., conservative amino acid substitutions), or combinations of the above changes. Insertions, deletions, or substitutions may include the amino terminus, carboxyl terminus, or both of these terms in this region. L Any of the regions may be used, provided that each CDR contains zero change or at most one, two, or three changes, and the modified V L These are the conditions under which an antibody containing a region can specifically bind to its target epitope with the same affinity as the wild-type binding domain.

[0074] In a particular embodiment, V H The region is disclosed V H V may be derived from or obtained based thereon and disclosed herein H Compared to the above, this may include one or more insertions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more deletions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or combinations of the above changes. The insertions, deletions, or substitutions may be at the amino terminus, carboxyl terminus, or both of these in this region. H The modified V may be anywhere in the region, provided that each CDR contains zero changes, or at most one, two, or three changes. H These are the conditions under which an antibody containing a region can specifically bind to its target epitope with the same affinity as the wild-type binding domain.

[0075] In certain embodiments, the variant comprises or is a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with respect to the antibody sequence disclosed herein. In certain embodiments, the variant comprises a light chain variable region (V L ) and / or heavy chain variable region (V H ), or both, contain or be a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity, where each CDR contains zero changes or zero changes, or up to 1, 2, or 3 changes, from the antibody disclosed herein, or the fragment or derivative thereof that specifically binds to the WASp or BTK signature peptide.

[0076] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0077] In certain embodiments, it may be desirable to produce cysteine-modified antibodies, such as "thioMAb," in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites on the antibody and can be used to conjugate the antibody to other parts, as further described below. Cysteine-modified antibodies may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0078] In certain embodiments, modified antibodies may have one or more amino acids replaced by non-amino acid components, or amino acids associated with a functional group, or a functional group otherwise associated with an amino acid. Modified amino acids may be, for example, glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid moiety, or an approach conjugated with an organic derivatizer. Amino acids may be, for example, co-translated during recombinant production, or post-translationally modified (e.g., N-linked glycosylation at an NXS / T motif during expression in mammalian cells), or modified by synthetic means. Modified amino acids may be within the sequence or at the end of the sequence. Modifications also include nitrated constructs.

[0079] In certain embodiments, the variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the reference sequence. In certain embodiments, the glycosylation variants include more or fewer N-linked glycosylation sites than the reference sequence. The N-linked glycosylation sites are characterized by the sequence:Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. Substitutions of amino acid residues to create this sequence provide a potential new site for the addition of N-linked glycans. Alternatively, substitutions to eliminate this sequence remove an existing N-linked glycan. Rearrangements of N-linked carbohydrate chains are also provided in which one or more N-linked glycosylation sites (e.g., naturally occurring sites) are eliminated and one or more new N-linked sites are created. Additional antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted or substituted with another amino acid (e.g., serine) compared to the reference sequence. These cysteine ​​variants may be useful when antibodies need to be refolded into a biologically active conformation, such as after isolation of insoluble inclusion bodies. These cysteine ​​variants generally have fewer cysteine ​​residues than the reference sequence and are even in number to minimize interactions caused by unpaired cysteines.

[0080] PEGylation is a process in which polyethylene glycol (PEG) polymer chains are covalently bonded to other molecules, such as proteins. Several methods for PEGylating proteins have been reported in the literature. For example, N-hydroxysuccinimide (NHS)-PEG has been used to PEGylate free amine groups of lysine residues and the N-terminus of proteins. PEG with aldehyde groups has been used to PEGylate (PEGylate) the amino terminus of proteins in the presence of reducing agents. PEG with maleimide functional groups has been used to selectively PEGylate free thiol groups of cysteine ​​residues in proteins. Site-specific PEGylation of acetylphenylalanine residues may also be performed.

[0081] "Sequence identity %" refers to the relationship between two or more sequences determined by comparing them. In the art, "identity" also means the degree of sequence relevance between protein, nucleic acid, or gene sequences, determined by the matching of such sequence strings. "Identity" (often called "similarity") can be readily calculated by known methods, including, but not limited to, those described below: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Develeux, J., eds.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to give the best match between sequences tested. Methods for determining identity and similarity have been systematized into publicly available computer programs. Sequence alignment and percentage identity calculations can be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of sequences may be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989)), with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Related programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J.Mol.Biol.215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput.Methods Genome Res.,[Proc.Int.Symp.](1994), Meeting Date 1992, 111-20. Editor: Suhai, Sandor. Publisher: Plenum, New York, NY. In the context of this disclosure, when sequence analysis software is used for analysis, it will be understood that the results of the analysis are based on the “default values” of the referenced program. As used herein, “default values” means any set of values ​​or parameters that are originally loaded into the software when it is first initialized.

[0082] (v) Immune complex of an anti-peptide antibody. The disclosed antibody and its antigen-binding fragment may be an immune complex. In certain embodiments, the immune complex is an antibody conjugated to one or more heterologous molecules, including a label.

[0083] The label may include affinity tags. Examples of affinity tags include His tag (SEQ ID NO: 45), Flag tag (SEQ ID NOs: 46-48), Xpress tag (SEQ ID NO: 49), Avi tag (SEQ ID NO: 50), calmodulin-binding peptide (CBP) tag (SEQ ID NO: 51), polyglutamic acid tag (SEQ ID NO: 52), HA tag (SEQ ID NOs: 53-55), Myc tag (SEQ ID NO: 56), Strep tag (this is the original STREP® tag (SEQ ID NO: 57), STREP® tag II (SEQ ID NO: 58) (IBA Institut fur Bioanalytik, Germany); see, for example, US Patent No. 7,981,632), Softag 1 (SEQ ID NO: 59), Softag 3 (SEQ ID NO: 60), and V5 tag (SEQ ID NO: 61).

[0084] The label may include a detection portion. The detection portion that can be conjugated to the antibody or antigen-binding fragment of this disclosure may include: colored particles; gold nanoparticles; colloids; chemiluminescent tags; radioisotopes; fluorescent tags, for example, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), Rhodamine Red® (e.g., Thermo Fisher Scientific, Waltham, MA), cyanine fluorophores, Texas Red® (registered trademark) (Molecular Probes, Inc., Eugene, OR), phycoerythrin (PE), R-phycoerythrin, allophycocyanin (APC), phycocyanin, phycoerythrocyanin, DyLight® (registered trademark) (Thermo Fisher Scientific, Waltham, MA), Alexa Fluor® (registered trademark) (Molecular Fluorescent proteins such as Probes, Inc., Eugene, OR, Atto dyes, or GFP; as well as enzyme reporters, including, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, luciferase, and β-galactosidase. In certain embodiments, enzyme reporter labeling is used in combination with colorimetric, fluorescence-generating, or chemiluminescent substrates for visualization.

[0085] The conjugation or labeling of the antibodies or antigen-binding fragments of this disclosure to heterologous molecules may be carried out using any method known in the art, including a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionic acid (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (such as dimethyladipimidate HCl), active esters (such as disuccinimidylsberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies (WO94 / 11026).

[0086] In certain embodiments, the immune complex is a crosslinking agent reagent, such as N-(β-maleimidopropyloxy)succinimide (BMPS), N-ε-maleimidocaproyl-oxysuccinimide (EMCS), N-γ-maleimidobutyryl-oxysuccinimide (GMBS), 1,6-hexane-bis-vinyl sulfone (HBVS), m-maleimidobenzoyl-N-hydroxysuccinimide (MBS), 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), succinimidyl 3-(bromoacetamide)propionate (SBAP), succinimidyliodoacetate These may be prepared using tate (SIA), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-((beta-maleimidopropionamide)hexanoate (SMPH), sulfo-EMCS, sulfo-GMBS, N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester (sulfo-KMUS), sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoic acid (SVSB) (these may be commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL)).

[0087] In certain embodiments, the linker may be a “cleavable linker.” For example, an acid-unstable linker, a peptidase-sensitive linker, a photo-unstable linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0088] (vi) Production of recombinant proteins of the disclosure. Polypeptides (e.g., antibodies or parts of antibodies) of the disclosure can be produced by any means known in the art. In certain embodiments, polypeptides are produced using recombinant DNA technology. Nucleic acid sequences encoding polypeptides may be prepared and assembled into complete coding sequences by standard molecular cloning techniques (e.g., genomic library screening, polymerase chain reaction (PCR), primer-assisted ligation, yeast and bacterial scFv libraries, site-directed mutagenesis). The resulting coding regions may be inserted into expression vectors and used to transform suitable expression cell lines.

[0089] The term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence) that codes for a polypeptide. This definition includes various sequence polymorphisms, mutations, and / or sequence variants, such changes that do not substantially affect the function of the coded polypeptide. The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term may further include all introns and other DNA sequences spliced ​​from mRNA transcripts, along with variants arising from alternative splicing sites. Gene sequences that code for a polypeptide may be DNA or RNA that directs polypeptide expression. These nucleic acid sequences may be DNA strand sequences transcribed to RNA or RNA sequences translated to polypeptides. Nucleic acid sequences include both full-length nucleic acid sequences and non-full-length sequences derived from full-length polypeptides. The sequence may also include degenerate codons of one or more native sequences that can be introduced to provide codon preference in a particular cell type.

[0090] "Encoding" refers to the properties of a particular sequence of nucleotides in a gene, such as complementary DNA (cDNA) or messenger RNA (mRNA), that function as a template for the synthesis of other macromolecules, such as a defined sequence of amino acids. Therefore, a gene codes for a polypeptide if the transcription and translation of the mRNA corresponding to that gene produce a polypeptide in a cell or other biological system. A "polypeptide-coding gene sequence" includes all nucleotide sequences that code for the same amino acid sequence or an amino acid sequence, which are degenerate versions of each other and substantially similar in form and function.

[0091] Polynucleotide gene sequences encoding polypeptides can be operably linked to relevant regulatory sequences. For example, a functional link may exist between a regulatory sequence and an exogenous nucleic acid sequence, resulting in the expression of the exogenous nucleic acid sequence. In another example, if a first nucleic acid sequence is functionally related to a second nucleic acid sequence, the first nucleic acid sequence can be operably linked to the second nucleic acid sequence. For example, if a promoter influences the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence.

[0092] In the exemplary nucleic acid constructs (polynucleotides) used in this disclosure, a promoter is operably ligated to a nucleic acid encoding an antibody of this disclosure or a polypeptide derived from an antibody of this disclosure, i.e., the promoter and nucleic acid sequence are arranged to promote transcription of mRNA from the DNA encoding the polynucleotide. This promoter may be of genomic origin or synthetically generated. This promoter may or may not be associated with an enhancer, which may be naturally associated with a particular promoter or with a different promoter. Promoters that enable constitutive or inductive expression may be used, where expression may be controlled depending on the target host, desired expression level, properties of the target host, etc.

[0093] Optionally, the signal sequence may be located at the 5' end of the polynucleotide for proper targeting of the polypeptide to a cellular location or for secretion from the cell.

[0094] Termination regions can be utilized at the 3' end of a polynucleotide for proper transcription termination. In certain embodiments, termination regions may include polyadenylation signals. A wide variety of termination regions can be used without adversely affecting expression.

[0095] This disclosure further provides vectors comprising polynucleotides of this disclosure. As used herein, the term “vector” refers to any of a number of nucleic acids into which a desired sequence can be inserted, for example, by restriction and ligation, for transport between different genetic environments or for expression in a host cell. Nucleic acid vectors may be DNA or RNA. Examples of vectors include plasmids, phages, phagemids, bacterial genomes, and viral genomes. Cloning vectors are vectors that are replicable in a host cell and are further characterized by one or more endonuclease restriction sites, in which the vector may be cleaved in a deterministic manner, to which a desired DNA sequence is ligated, resulting in a new recombinant vector that retains the ability to replicate in a host cell. Certain vectors may autonomously replicate in the host cell into which they are introduced. Other vectors, upon introduction into a host cell, are integrated into the host cell’s genome and thereby replicate together with the host genome. Expression vectors may express polypeptides of this disclosure; i.e., the vector sequence includes regulatory sequences necessary for the transcription and translation of the polypeptide, including promoters, operators, transcription termination sites, ribosome binding sites, etc.

[0096] Vector host systems include systems such as bacterial, mammalian, yeast, insect, or plant cell systems, either in vivo (e.g., animals) or in vitro (e.g., bacteria or cell culture). The selection of an appropriate host is considered to be within the scope of those skilled in the art from the teachings herein. In certain embodiments, the host cell is a bacterium, e.g., E. coli.

[0097] Host cells are genetically engineered (infected, transduced, transformed, or transfected) using the vectors of this disclosure. In certain embodiments, the host cells contain the vectors comprising the polynucleotides of this disclosure. The engineered host cells may be cultured in conventional nutrient media appropriately modified for promoter activation, transformant selection, or polynucleotide amplification. Culture conditions such as temperature and pH are those previously used with host cells selected for expression and will be apparent to those skilled in the art.

[0098] (vi) Method of Use. The antibodies and antigen-binding fragments of this disclosure may be used for immunoaffinity enrichment of the signature peptides described herein. The enriched signature peptides may then be detected by SRM assays for the diagnosis of WAS and XLA. Signature peptides include WASp 289 and BTK 545.

[0099] In certain embodiments, signature peptide enrichment involves contacting a mixture of peptide fragments derived from a digested biological sample with one or more conjugated antibodies of the present disclosure that recognize the signature peptide and their antigen-conjugated fragments. In certain embodiments, the biological sample may be a DBS, an oral swab, a PBMC, or a WBC.

[0100] In certain embodiments, antibodies containing SEQ ID NOs: 3-8 and 15-22 are used to concentrate the WASp peptide containing SEQ ID NO: 1.

[0101] In certain embodiments, antibodies containing SEQ ID NOs. 9-14 and 23-30 are used to concentrate the BTK peptide containing SEQ ID NO. 2.

[0102] The enrichment of the desired peptide target prior to SRM can be achieved by any means known in the art. Many enrichment procedures are available, including immunoadsorption-based depletion of abundant protein species from the sample, precipitation, chromatography, electrophoresis, solvent partitioning, immunoprecipitation, immunoelectrophoresis, and immunochromatography. In certain embodiments, the SISCAPA method may be used for specific antibody-based capture of individual trypsin peptides from the digest of the sample. Anderson et al., J. Proteome Research 2004;3:235-244; U.S. Patent No. 7,632,686.

[0103] In certain embodiments, antibodies that bind to peptide markers, such as the antibodies disclosed herein, may be attached to a solid support. Certain embodiments utilize affinity columns in which the antibody is covalently bound to the chromatographic medium. In certain embodiments, POROS (Applied Biosystems, Foster City, CA) nanocolumns may be used in SISCAPA enrichment, characterized by high binding capacity, relatively high concentrations of antibody enabling rapid enrichment of target peptides, and the ability to prepare columns with various functionalization groups. Alternatively, the antibody may be attached to beads, magnetic beads, or other solid particles. One means of attachment is the binding of the antibody to a protein coated on the beads. For example, particles coated with Protein G provide antibody binding in a preferred orientation. Other attachment means may be used, such as directly coating the beads with the antibody. Magnetic particles can be utilized with a variety of chemicals that enable binding to the antibody. Enrichment with antibody attached to particles allows for parallel processing of samples. Magnetic particle processing is automated in 96-well plates for the SISCAPA enrichment step, where the sample is eluted on the plate for analysis by mass spectrometry. Other specific embodiments utilize a novel bead trap device developed to perform the bead processing step along a nanoflow chromatography system. Anderson et al. Mol Cell Proteomics 2009;8(5):995-1005. This minimizes peptide loss to the container between the elution and analysis steps. Peptide concentration can also be performed by immobilizing an anti-peptide antibody on a pipette tip. Nelson et al. Anal Chem. 1995;67(7):1153-1158. After separating the antibody-bound peptide from the free peptide, the bound peptide can be eluted. Any elution method may be used. One elution method that has proven efficient is 5% acetic acid / 3% acetonitrile. Other elution methods, including other acids and other concentrations of acetic acid, may be used to be efficient for specific peptides.

[0104] In certain embodiments, WAS and / or XLA may be screened using anti-WASp 289 and / or anti-BTK 545 antibodies disclosed in Tables 2 and 3, which bind to their respective signature peptides.

[0105] The antibodies and antigen-binding fragments of this disclosure can be used to detect levels of the WASp 289 peptide, BTK 545 peptide, and / or their corresponding proteins in a biological sample. In certain embodiments, this method involves contacting a biological sample with the anti-WASp 289 and / or anti-BTK 545 antibody or its antigen-binding fragment under conditions acceptable for the binding of the anti-WASp 289 and / or anti-BTK 545 antibody or its antigen-binding fragment to their respective peptides or proteins, and detecting whether a complex is formed between the anti-WASp 289 antibody or its antigen-binding fragment and the WASp 289 peptide or its corresponding protein, or between the anti-BTK 545 antibody or its antigen-binding fragment and the BTK 545 peptide or its corresponding protein. Such a method may be in vitro or in vivo. In certain embodiments, the biological sample includes whole blood, DBS, serum, plasma, other blood fractions, cells, tissues, and test samples derived from the subject.

[0106] In a specific embodiment, antibodies containing SEQ ID NOs: 3-8 and 15-22 are used to detect the WASp peptide containing SEQ ID NO: 1.

[0107] In a specific embodiment, antibodies containing SEQ ID NOs. 9-14 and 23-30 are used to detect the BTK peptide containing SEQ ID NO. 2.

[0108] In certain embodiments, detecting levels of WASp 289 peptide, BTK 545 peptide, and / or their corresponding proteins using anti-WASp 289 and / or anti-BTK 545 antibodies or their antigen-binding fragments may be performed as part of an assay known to those skilled in the art, such as immunoassays, ELISA (enzyme-linked immunosorbent assay), Western blotting, dot blotting, radioimmunology assays (RIA); sandwich assays; flow cytometry; fluorescence in situ hybridization (FISH); immunohistochemical staining; immunoelectrophoresis; immunoprecipitation; and immunofluorescence.

[0109] Embodiments disclosed herein may also utilize liquid chromatography and / or mass spectrometry. In certain embodiments, one or more LC purification steps are performed prior to SRM-MS. A mixture of concentrated peptides (mobile phase) passes through a material-packed column (stationary phase) to separate the peptides based on the weight and affinity of the column to the mobile and stationary phases. Conventional LC analysis relies on chemical interactions between sample components and column packing materials, where laminar flow of the sample passing through the column is the basis for separating the analyte of interest from the test sample. Those skilled in the art will understand that separation in such columns is a diffusion process. A variety of column packing materials are available for chromatographic separation of samples, and the selection of an appropriate separation protocol is an empirical process that depends on the characteristics of the sample, the analyte of interest, any interfering substances present and their properties, etc. Various packing chemistry may be used as needed (e.g., structure, polarity, solubility of the compound to be purified). In certain embodiments, the column may be polar, ion exchange (both cationic and anionic), hydrophobic interaction, phenyl, C-2, C-8, C-18 column, polar coating on a porous polymer, or other commercially available. During chromatography, the separation of materials is affected by variables such as the choice of eluent (also known as the “mobile phase”), the choice of gradient elution, and gradient conditions, temperature, etc. In certain embodiments, the analyte may be purified by applying the sample to the column under conditions in which the analyte of interest is reversibly retained by the column packing material, and one or more other materials are not retained. In these embodiments, a first mobile phase condition may be used in which the analyte of interest is retained by the column, and then, once the unretained material has been washed, a second mobile phase condition may be used to remove the retained material from the column. Alternatively, the analyte may be purified by applying the sample to the column under mobile phase conditions in which the analyte of interest elutes at a different rate compared to one or more other materials. As described above, such a procedure may concentrate the amount of one or more analytes of interest compared to one or more other components of the sample. In certain embodiments, the LC is a microflow LC (micro LC).In microflow LC, chromatographic separation is performed using flow rates in the low microliters per minute range. In certain embodiments, the LC is nanoflow LC (nanoLC). In nanoflow LC (nanoLC), chromatographic separation is performed using a flow rate of 300 nanoliters per minute. This slower flow rate results in high analytical sensitivity, due to the high concentration efficiency obtained by this type of chromatography (Cutillas, Current Nanoscience, 2005;1:65-71).

[0110] A mass spectrometer is equipped with a vapor phase ion spectrometer that measures parameters that can be converted into the mass-charge (m / z) ratio of vapor phase ions. Mass spectrometry refers to the detection of vapor phase ions using a mass spectrometer. A mass spectrometer typically includes an ion source and a mass spectrometer. Examples of mass spectrometers include time-of-flight (TOF), magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer, and hybrids thereof. A laser desorption mass spectrometer is equipped with a mass spectrometer that uses laser energy as a means of desorbing, volatilizing, and ionizing an analyte. A tandem mass spectrometer is equipped with any mass spectrometer capable of performing two consecutive steps of m / z-based identification or measurement of ions, including ions in an ion mixture. This phrase includes a mass spectrometer equipped with two mass spectrometers capable of performing two consecutive steps of m / z-based identification or spatial tandem measurement of ions. This phrase further includes a mass spectrometer equipped with a single mass spectrometer capable of performing two consecutive steps of m / z-based identification or temporal tandem measurement of ions. Therefore, this phrase explicitly includes Qq-TOF mass spectrometers, ion trap mass spectrometers, ion trap-TOF mass spectrometers, TOF-TOF mass spectrometers, Fourier transform ion cyclotron resonance mass spectrometers, electrostatic sector-magnetic sector mass spectrometers, triple quadrupole mass spectrometers, and combinations thereof.

[0111] Ionization in mass spectrometry involves the process of ionizing the analyte in a sample. Such analytes may become charged molecules used for further analysis. For example, sample ionization can be performed by electrospray ionization (ESI), laser spray ionization (LSI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atomic impact (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, and particle beam ionization. Those skilled in the art will understand that the choice of ionization method can be determined based on the analyte being measured, the type of sample, the type of detector, the choice between positive and negative modes, and so on.

[0112] A mass spectrometer comprises components that take ionized masses, separate them based on their m / z ratio, output them to a detector where they are detected, and later converted to a digital output. Suitable mass spectrometers for determining the m / z ratio include quadrupole mass spectrometers, time-of-flight (TOF) mass spectrometers, magnetic or electrostatic sector mass spectrometers, and ion trap (e.g., ion cyclotron resonance) mass spectrometers.

[0113] A selected reaction monitoring (SRM)-MS assay targets a predetermined set of peptides for a given protein of interest. SRM is a tandem mass spectrometry mode in which a specific mass of ions (parental or precursor ions) is selected in the first stage of tandem mass spectrometry, and the ionic products of the fragmentation reaction of the precursor ions are selected in the second mass spectrometry stage for detection. A specific pair of m / z values ​​associated with the selected precursor ions and fragment ions is called a transition. For each signature peptide, a fragment ion is identified that provides the optimal signal intensity and distinguishes the targeted peptide from other species present in the sample. Optimized transitions contribute to an effective SRM assay. Several such transitions (precursor / fragment ion pairs) are monitored over time, and a series of chromatographic traces are generated with the retention time and signal intensity of a particular transition as coordinates. SRM-MS analysis of signature peptides is typically performed using a triple quadrupole mass spectrometer (QQQ-MS), an instrument capable of selectively separating precursor ions corresponding to the m / z of the signature peptide and selectively monitoring peptide-specific fragment ions. In SRM analysis, specificity relies on multiple mass spectrometers (mass filters). The first quadrupole is for selecting the desired parent or precursor ion. The third quadrupole is for monitoring (one or more) fragment ions. Fragment ions are generated by dissociation induced by collision in the second quadrupole. High selectivity is possible with two levels of mass selection because co-eluting background ions are filtered very effectively. Unlike conventional tandem mass spectrometry (MS / MS) experiments that investigate all analytes in a sample, SRM analysis selectively targets (filters) specific analytes, which means an order of magnitude or even a two-order-of-magnitude improvement in sensitivity compared to conventional "full-scan" techniques. Furthermore, SRM provides a linear response over a wide dynamic range of up to five orders of magnitude. This enables the detection of small amounts of protein in highly complex mixtures.Therefore, SRM is a highly specialized detection / monitoring method with minimal background interference. When monitoring multiple parent ions in a single MS run, this type of analysis is known as multiple reaction monitoring (MRM). Using MRM analysis, it is possible to monitor multiple proteins and multiple regions of proteins (signature peptides) in a single mass spectrometry run. Selected reaction monitoring / multiple reaction monitoring mass spectrometry (SRM / MRM-MS) is described, for example, in U.S. Patent No. 8,383,417, WO2013 / 106603, and U.S. Patent Application Publication No. 2013 / 105684.

[0114] In certain embodiments, the following parameters may be used to specify an LC-SRM-MS assay of a protein under a particular LC-SRM-MS system: (1) enrichment of the trypsin peptide of a given protein; (2) retention time (RT) of the peptide on the LC column; (3) m / z value of the peptide precursor ion; (4) declustering potential used to ionize the precursor ion; (5) m / z value of the fragment ion generated from the peptide precursor ion; and (6) collision energy (CE) used to fragment the peptide precursor ion, optimized for the specific peptide. RT includes the elapsed time from injection to elution of the analyte. Declustering potential (DP) includes the potential for dissolving and dissociating ion clusters. This is also known as the "fragment voltage" or "ion transfer capillary offset voltage," depending on the manufacturer. Collision energy (CE) includes the amount of energy the precursor ion receives when accelerated into the collision cell.

[0115] To facilitate the accurate quantification of peptides by the methods disclosed herein, a set of isotopically labeled synthetic versions of the peptide of interest may be added to the sample in known amounts for use as an internal standard. Since the isotopically labeled peptides have the same physical and chemical properties as the corresponding surrogate peptides, they co-elute from the chromatographic column and are readily identifiable in the resulting mass spectrum. (Gerber et al. Proc. Natl. Asso. Sci. 2003;100:6940-6945; Kirkpatrick et al. Methods 2005;35:265-273). Isotopes capable of labeling amino acids of a given peptide include: 13 C, 2 H, 15 N, 17 O, 18 O, and 34 S is one example. In certain embodiments, the peptide is 13 C and / or 15 It is labeled with a heavy isotope of N. The addition of the labeled standard may be performed before or after proteolytic digestion. In certain embodiments, the labeled internal standard peptide is added after proteolytic digestion. Methods for synthesizing isotope-labeled peptides will be known to those skilled in the art. Thus, in certain embodiments, the experimental sample contains the internal standard peptide. In certain embodiments, the internal standard peptide contains a reference signature peptide. In certain embodiments, the signature peptide concentration may be determined by combining: (i) a ratio calculated by comparing the peak area of ​​the signature peptide with the peak area of ​​its corresponding reference signature peptide obtained from an LC-MRM-MS assay, and (ii) a known concentration of the reference signature peptide. Peptides selected as reference standards and suitable for quantification are sometimes called quantum peptides (Q-peptides). Q-peptides possess all the characteristics of proteotype peptides but also impose restrictions on the residues that can constitute the reference peptide in order to eliminate artificial modifications and / or incomplete cleavage. Holman et al.Bioanalysis 2012;4(14):1763-1786.

[0116] The absolute quantitative level of a given protein(s) can be determined by an SRM / MRM methodology, thereby comparing the SRM / MRM signature peak area of ​​individual peptides from a given protein in a single biological sample to the SRM / MRM signature peak area of ​​a known amount of "spiked" internal standard. In certain embodiments, the internal standard is a synthetic version of the same exact peptide containing one or more amino acid residues labeled with one or more heavy isotopes. Such isotope-labeled internal standards are synthesized to produce a predictable and consistent SRM / MRM signature peak that is distinct and separate from the native peptide signature peak and can be used as a comparator peak by mass spectrometry. Thus, when the internal standard is spiked in a protein preparation derived from a biological sample in a known amount and analyzed by mass spectrometry, the signature peak area of ​​the native peptide is compared to the signature peak area of ​​the internal standard peptide, and this numerical comparison indicates either the absolute molar concentration and / or absolute weight of the native peptide present in the original protein preparation from the biological sample. Absolute quantification data for fragment peptides are presented according to the amount of protein analyzed for each sample. Absolute quantification may be performed across many peptides, and therefore proteins, simultaneously in a single sample, and / or across many samples to gain insight into the absolute amounts of protein in individual biological samples and across the entire cohort of individual samples.

[0117] Another strategy for the absolute quantification of peptides is equimolar quantification using an equalizer peptide. This methodology involves chemically synthesizing the isotope-labeled Q-peptide of interest as a dipeptide. The typical amino acid sequence is located at the N-terminus of the Q-peptide and is called the equalizer peptide. After solubilization and proteolytic digestion, the amount of Q-peptide can be precisely determined by reference to a single light-labeled peptide. Then, appropriate amounts of each standard peptide are added to the sample of interest (either pre-digestion or pre-proteolysis) to facilitate absolute quantification. Holzmann et al. Anal. Chem. 2009;81:10254-10261. For absolute quantification, a quantitative concatemer (QconCAT) protein may also be used. Beynon et al. Nat. Methods 2005;2:587-589; Johnson et al. J. Am. Soc. Mass Spectrom. 2009;20:2211-2220; Ding et al. J. Proteome Res. 2011;10:3652-3659; Caroll et al. Molecular & Cellular Proteomics 2011;Sep 19:mcp-M111. In this strategy, recombinant artificial proteins, which are affinity-tagged linkages of standard peptides derived from several proteins of interest, are heterologously produced in Escherichia coli grown in stable isotope-enriched medium. The QconCAT protein is then affinity-purified and co-digested with the sample to produce a stoichiometric mixture of all the "heavy" Q peptides that comprise it, followed by analysis of proteolytic peptides and internal standards derived from the native protein. A variation of the QconCAT approach called peptide-concatenated standards (PCS) uses contiguous regions between Q peptides within artificial protein sequences that reflect the endogenous environment. (Kito et al. J. Proteome Res. 2007;6:792-800). Other specific embodiments use protein standards for absolute quantification (PSAQ). (Brun et al. Mol. Cell. Proteomics 2007;6:2139-2149).PSAQ uses recombinant proteins, but rather than peptides linked from several proteins, the entire protein to be quantified is expressed in a stable, isotopically labeled form. One or more PSAQs may then be added to the sample pre-digestion to facilitate quantification.

[0118] Certain embodiments utilize unlabeled strategies for protein quantification, such as intensity-based measurements (America and Cordewener, Proteomics 2008;8:731-749) or spectral counting (Lundgren et al. Expert Rev. Proteomics 2010;7:39-53).

[0119] To obtain the relative quantitative level of a given peptide, the signature peak area (or peak height, if the peak is sufficiently resolved) derived from mass spectrometry of individual or multiple peptides from a given protein in one biological sample may be compared to the signature peak area determined for the same peptide or peptides derived from the same protein in one or more additional and different biological samples using the same SRM / MRM method. In this way, the amount of a particular peptide(s) derived from a given protein is determined by comparing it with the same peptide or peptides derived from the same protein across two or more biological samples under the same experimental conditions. Furthermore, the relative quantification of a given peptide(s) derived from a single protein in a single sample can be determined by comparing the signature peak area of ​​that peptide in that given protein with the signature peak area of ​​other and different peptides(s) derived from different proteins in the same protein preparation derived from the biological sample using the SRM / MRM method. In this way, the amount of a particular peptide derived from a given protein, and therefore the amount of the given protein, is determined by comparing it with other proteins in the same sample. These approaches quantify individual peptides (or more) derived from a given protein relative to the amount of other peptides (or more) derived from the same or different proteins between and within a sample, where the amounts determined by signature peak area are relative to each other, regardless of the absolute weight-to-volume or weight-to-weight amount of peptides in the protein preparation derived from this biological sample. Relative quantitative data regarding individual signature peak areas between different samples may be normalized to the amount of protein analyzed per sample. Relative quantification may be performed simultaneously across many peptides in a single sample and / or across many samples to gain insight into relative protein amounts.

[0120] Signature peptide levels may be expressed in units of concentration (e.g., pmol / L). In certain embodiments, the average concentration of the signature peptide in test samples derived from subjects being screened for WAS and / or XLA may be compared to the average concentration of the corresponding peptide in normal control samples. In certain embodiments, normal control samples may be derived from one or more normal control subjects or from a population of normal control subjects. In certain embodiments, normal control subjects may be subjects that do not have or are not known to have WAS and / or XLA. In certain embodiments, normal control subjects may be subjects that do not have gene mutations associated with WAS and / or XLA.

[0121] In certain embodiments, the average concentrations of the WASp 289 signature peptide in DBS derived from a normal control population include concentrations in the ranges of 7000 pmol / L to 30000 pmol / L, 7500 pmol / L to 28000 pmol / L, and 8000 pmol / L to 26000 pmol / L. In certain embodiments, the average concentrations of the WASp 289 signature peptide in DBS derived from a normal control population include 7000 pmol / L, 7000 pmol / L, 7100 pmol / L, 7200 pmol / L, 7300 pmol / L, 7400 pmol / L, 7500 pmol / L, 7600 pmol / L, 7700 pmol / L, 7800 pmol / L, 7900 pmol / L, 8000 pmol / L, and 810 0pmol / L, 8200pmol / L, 8300pmol / L, 8400pmol / L, 8500pmol / L, 8600pmol / L, 8700pmol / L, 880 0pmol / L, 8900pmol / L, 9000pmol / L, 9100pmol / L, 9200pmol / L, 9300pmol / L, 9400pmol / L, 9500 pmol / L, 9600pmol / L, 9700pmol / L, 9800pmol / L, 9900pmol / L, 10000pmol / L, 11000pmol / L, 12 000pmol / L, 13000pmol / L, 14000pmol / L, 15000pmol / L, 16000pmol / L, 17000pmol / L, 18000pmo Includes concentrations of 1 / L, 19000 pmol / L, 20000 pmol / L, 21000 pmol / L, 22000 pmol / L, 23000 pmol / L, 24000 pmol / L, 25000 pmol / L, 26000 pmol / L, 27000 pmol / L, 28000 pmol / L, 29000 pmol / L, and 30000 pmol / L or higher.

[0122] In certain embodiments, the average concentrations of the BTK 545 signature peptide in DBS derived from a normal control population include concentrations in the ranges of 400 pmol / L to 2000 pmol / L, 500 pmol / L to 1800 pmol / L, and 600 pmol / L to 1500 pmol / L. The average concentrations of the 545 signature peptides are 400 pmol / L, 450 pmol / L, 500 pmol / L, 550 pmol / L, 600 pmol / L, 650 pmol / L, 700 pmol / L, 750 pmol / L, 800 pmol / L, 850 pmol / L, 900 pmol / L, 950 pmol / L, 1000 pmol / L, 1050 pmol / L, 1100 pmol / L, 1150 pmol / L, and 1200 pmol / L. mol / L, 1250pmol / L, 1300pmol / L, 1350pmol / L, 1400pmol / L, 1450pmol / L, 1500pmol / L, 1550pmol / L, 1600pmol / L, 1 Concentrations include 650pmol / L, 1700pmol / L, 1750pmol / L, 1800pmol / L, 1850pmol / L, 1900pmol / L, 1950pmol / L, 2000pmol / L or higher.

[0123] In certain embodiments, a predetermined cutoff value is used as a threshold for a given signature peptide. A concentration of the given signature peptide above the threshold indicates that the assayed DBS originated from an individual free from WAS and / or XLA. A concentration of the given signature peptide below the threshold, or absent, indicates that the assayed DBS originated from an individual free from WAS and / or XLA. In certain embodiments, this threshold may be determined by analyzing a population of normal controls and calculating the standard deviation (SD) of the concentration of the given signature peptide in this population. The threshold may be set at a specific SD from the mean concentration of the given signature peptide. In certain embodiments, the threshold is an SD of -1SD, -1.1SD, -1.2SD, -1.3SD, -1.4SD, -1.5SD, -1.6SD, -1.7SD, -1.8SD, -1.9SD, -2.0SD, -2.1SD, -2.2SD, -2.3SD, -2.4SD, -2.5SD, -2.6SD, -2.7SD, -2.8SD, -2.9SD, -3.0SD, or higher, from the mean concentration of a given signature peptide. In certain embodiments, for the diagnosis or screening of WAS and / or XLA, the threshold may be determined by analysis of a population of normal controls and calculation of the standard deviation (SD) of the ratio of the concentration of a given signature peptide to the endogenous concentration of ATP7B (Jung et al., J. Proteome Res. 2017;16: 862-871) in this population. The peptide concentration cutoff for each PIDD can be set to a specific standard deviation (SD) derived from the average concentration of each signature peptide or the ratio of the concentration of a particular signature peptide to the endogenous concentration of ATP7B.

[0124] In certain embodiments, the threshold concentration of the signature peptide includes -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD or higher, from the mean concentration of the corresponding signature peptide in a normal control population.

[0125] In certain embodiments, the threshold concentration of WASp 289 peptide is 3600 pmol / L or less, 3550 pmol / L or less, 3500 pmol / L or less, 3490 pmol / L or less, 3480 pmol / L or less, 3470 pmol / L or less, 3460 pmol / L or less, 3450 pmol / L or less, 3440 pmol / L or less, 3430 pmol / L or less, 3420 pmol / L or less, 3410 pmol / L or less, and 3400 pmol / L. This includes concentrations of 1 / L or less, 3300 pmol / L or less, 3200 pmol / L or less, 3100 pmol / L or less, 3000 pmol / L or less, 2900 pmol / L or less, 2800 pmol / L or less, 2700 pmol / L or less, 2600 pmol / L or less, 2500 pmol / L or less, 2300 pmol / L or less, 2200 pmol / L or less, 2100 pmol / L or less, and 2000 pmol / L or less. In certain embodiments, the threshold concentration of WASp 289 peptide includes 3384.4 pmol / L.

[0126] In certain embodiments, the threshold concentration of BTK 545 peptide is 350 pmol / L or less, 345 pmol / L or less, 340 pmol / L or less, 335 pmol / L or less, 330 pmol / L or less, 325 pmol / L or less, 320 pmol / L or less, 315 pmol / L or less, 310 pmol / L or less, 300 pmol / L or less, 290 pmol / L or less, 280 pmol / L or less, 270 pmol / L or less, 260 pmol / L or less, and 250 pmol / L. This includes concentrations of 1 / L or less, 240 pmol / L or less, 230 pmol / L or less, 220 pmol / L or less, 210 pmol / L or less, 200 pmol / L or less, 190 pmol / L or less, 180 pmol / L or less, 170 pmol / L or less, 160 pmol / L or less, 150 pmol / L or less, 140 pmol / L or less, 130 pmol / L or less, 120 pmol / L or less, 110 pmol / L or less, and 100 pmol / L or less. In certain embodiments, the threshold concentration of BTK 545 peptide includes 311.4 pmol / L or less.

[0127] One or more standard peptides can be synthesized by any method known in the art. Such synthetic peptides may further contain amino acids having one or more native modifications. Such native modifications may include deamination, amination, oxidation, and hydroxylation of glutamine and asparagine.

[0128] The methods disclosed herein include the treatment of subjects (e.g., humans) based on the results of WAS and / or XLA screening using the compositions and methods disclosed herein. The treatment of subjects includes the delivery of a therapeutically effective amount. A therapeutically effective amount includes an effective amount, an amount that provides prophylactic treatment and / or therapeutic treatment.

[0129] An "effective dose" is the amount of a composition required to produce a desired physiological change in a subject. For example, an effective dose may provide relief, elimination, or cure of symptoms of WAS and / or XLA. Effective doses are often administered for research purposes. The effective doses disclosed herein may produce statistically significant effects in animal models or in vitro assays related to the evaluation of disease development, progression, and / or resolution.

[0130] Certain embodiments may include administering the composition as a “preventive measure.” A preventive measure is administered to subjects who do not exhibit signs or symptoms of WAS and / or XLA, or who exhibit only early signs or symptoms of WAS and / or XLA, so as to reduce or mitigate the risk of developing symptoms of the disorder or the negative effects of the disorder. Thus, the preventive measure functions as a preventive measure against symptoms or negative effects of WAS and / or XLA.

[0131] In certain embodiments, prophylactic measures may prevent, delay, or reduce the onset of WAS and / or XLA. In certain embodiments, prophylactic measures may be taken before, simultaneously with, or after other prophylactic measures, such as the use of antibiotics. In certain embodiments, prophylactic measures may prevent or reduce the severity of symptoms or complications associated with WAS and / or XLA.

[0132] Symptoms and complications of WAS may include: bleeding; eczema; bloody diarrhea; and recurrent infections. Symptoms and complications of XLA may include: infection; diarrhea; growth retardation; joint disorders; nephritis; red blood cell breakdown; and inflammation of the skin and muscles.

[0133] "Therapeutic treatment" includes treatments administered to subjects exhibiting symptoms or signs of WAS and / or XLA, with the aim of reducing or eliminating the symptoms or signs of WAS and / or XLA. In certain embodiments, therapeutic treatment may provide immune function to subjects diagnosed with WAS and / or XLA. In certain embodiments, therapeutic treatment may reduce, control, or eliminate symptoms and complications of WAS and / or XLA, such as those described herein.

[0134] Effective doses, prophylactic measures, and therapeutic measures do not need to be mutually exclusive, and in certain embodiments, the administered dosage may achieve multiple treatment types.

[0135] In certain embodiments, the therapeutically effective dose provides immune system function to subjects diagnosed with WAS and / or XLA. Accordingly, in certain embodiments, methods of treatment disclosed herein include stem cell transplantation, immunoglobulin infusion, antibiotic infusion, and / or gene therapy for disorders such as WAS and / or XLA.

[0136] The precise dosage and administration schedule for a specific subject may be determined by a physician, veterinarian, or researcher, taking into account parameters such as the target, body weight, severity of the condition, previous or concurrent therapeutic interventions, the subject's sudden illness, and physical and physiological factors including the route of administration.

[0137] (viii) Kits. Kits comprising the antibodies of the present disclosure and their antigen-binding fragments are also provided. The kit may comprise a lancet for puncturing blood, a filter card for collecting blood droplets, an oral swab, a blood collection tube, a solution for solubilizing the DBS, and appropriate buffers and enzymes for digesting the marker proteins in the DBS. The kit may further comprise one or more containers comprising anti-peptide antibodies and their antigen-binding fragments for assessing the absence or reduction of WASp 289 and / or BTK545 peptides, or their corresponding proteins, and / or reagents or supplies. In certain embodiments, the kit comprises one or more containers comprising anti-WASp 289 and / or anti-BTK 545 antibodies. These antibodies may be immobilized on a solid support such as a column or beads. The kit may further comprise an elution buffer for releasing the peptides from the antibodies. In certain embodiments, the kit may comprise one or more labeled reference peptides for performing absolute quantification of the signature peptides. In certain embodiments, the kit may also comprise some or all of the essential laboratory and / or medical supplies necessary for the effective use of the kit, such as gauze, sterile adhesive strips, gloves, and tubing. Any modifications may be made to the contents of any kit described herein.

[0138] The components of the kit may be prepared for storage and later use. Relating to such containers may be notices in the form prescribed by the government agency regulating the manufacture, use, or sale of the kit, which, where applicable, reflect approval by the agency of manufacture, use, or sale.

[0139] If necessary, the kit may further include instructions for using the kit in this manner. In various embodiments, these instructions may include appropriate instructions for interpreting the results related to the use of the kit; appropriate disposal of related waste; etc. These instructions may be in the form of printed instructions provided within the kit, or they may be printed as part of the kit itself. The instructions may be in the form of sheets, brochures, booklets, CD-ROMs, or computer-readable devices, or they may provide instructions to instructions located remotely, such as on a website.

[0140] The following exemplary embodiments and examples are included for the purpose of demonstrating the particular nature of this disclosure. Those skilled in the art should recognize that many modifications may be made to the specific embodiments disclosed herein in light of this disclosure, and similar or comparable results can still be obtained without departing from the spirit and scope of this disclosure.

[0141] (ix) Exemplary embodiment. 1. An antibody or its antigen-binding fragment, which is as follows: (A) Heavy chain variable (VH) domains comprising CDRH1 having the sequence shown in SEQ ID NO: 3, CDRH2 having the sequence shown in SEQ ID NO: 4, and CDRH3 having the sequence shown in SEQ ID NO: 5, and light chain variable (VL) domains comprising CDRL1 having the sequence shown in SEQ ID NO: 6, CDRL2 having the sequence shown in SEQ ID NO: 7, and CDRL3 having the sequence shown in SEQ ID NO: 8 (B) A VH domain comprising CDRH1 having the sequence shown in SEQ ID NO: 9, CDRH2 having the sequence shown in SEQ ID NO: 10, and CDRH3 having the sequence shown in SEQ ID NO: 11, and a VL domain comprising CDRL1 having the sequence shown in SEQ ID NO: 12, CDRL2 having the sequence shown in SEQ ID NO: 13, and CDRL3 having the sequence shown in SEQ ID NO: 14, The antibody or its antigen-binding fragment, comprising the antibody or its antigen-binding fragment.

[0142] 2. An antibody or antigen-binding fragment of Embodiment 1(A), wherein: a VH domain having the sequence shown in SEQ ID NO: 15; a heavy chain having the sequence shown in SEQ ID NO: 17; a VL domain having the sequence shown in SEQ ID NO: 16; or a light chain having the sequence shown in SEQ ID NO: 18. The antibody or its antigen-binding fragment, comprising one or more of the following.

[0143] 3. The antibody or antigen-binding fragment according to Embodiment 1 or 2, wherein the VH domain has the sequence shown in Sequence ID No. 15 and the VL domain has the sequence shown in Sequence ID No. 16.

[0144] 4. The antibody or antigen-binding fragment according to any one of Embodiments 1 to 3, wherein the heavy chain has the sequence shown in SEQ ID NO: 17 and the light chain has the sequence shown in SEQ ID NO: 18.

[0145] 5. The antibody or antigen-binding fragment of Embodiment 1(B), comprising one or more of the following: a VH domain having the sequence shown in SEQ ID NO: 23; a heavy chain having the sequence shown in SEQ ID NO: 25; a VL domain having the sequence shown in SEQ ID NO: 24; or a light chain having the sequence shown in SEQ ID NO: 26.

[0146] 6. The antibody or antigen-binding fragment according to Embodiment 1 or 5, wherein the VH domain has the sequence shown in Sequence ID No. 23 and the VL domain has the sequence shown in Sequence ID No. 24.

[0147] 7. The antibody or antigen-binding fragment according to any one of embodiments 1, 5, and 6, wherein the heavy chain has the sequence shown in SEQ ID NO: 25 and the light chain has the sequence shown in SEQ ID NO: 26.

[0148] 8. Use of the antibody of Embodiment 1 in a method, as follows: Obtaining biological samples derived from the subject; The process involves enzymatically digesting a protein derived from the aforementioned biological sample to produce one or more peptides; It is about concentration, A WASp signature peptide having the antibody of Embodiment 1(A) or its antigen-binding fragment; and / or The BTK signature peptide having the antibody or antigen-binding fragment of Embodiment 1(B) is to be enriched; Liquid chromatography-multi-reaction monitoring mass spectrometry (LC-MRM-MS) is performed on the concentrated peptides to determine the concentration of each signature peptide. The aforementioned use, including.

[0149] 9. The use according to Embodiment 8, wherein the biological sample is a dried blood spot (DBS), an oral swab, peripheral blood mononuclear cells (PBMCs), or white blood cells (WBCs).

[0150] 10. The use according to Embodiment 8 or 9, wherein the enzyme is trypsin.

[0151] 11. A use as described in any one of Embodiments 8 to 10, further, The concentration of each signature peptide is compared to the concentration of a corresponding predetermined threshold concentration; The aforementioned subject is: If the concentration of the WASp signature peptide is lower than the corresponding predetermined threshold concentration, or if the WASp signature peptide is absent, then it is a WAS; If the concentration of the BTK signature peptide is lower than the corresponding predetermined threshold concentration, or if the BTK signature peptide is absent, then it is XLA. To make a diagnosis, The aforementioned use, including.

[0152] 12. Use according to any one of Embodiments 8 to 11, used as part of a neonatal screening (NBS) to further screen the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell anemia.

[0153] 13. The use according to any one of Embodiments 8 to 12, wherein the use is performed in the absence of clinical symptoms of WAS and / or XLA in the subject.

[0154] 14. The use according to Embodiment 11, wherein the corresponding predetermined threshold concentration of each signature peptide is calculated from the standard deviation of the average concentration of each signature peptide in the corresponding biological sample derived from a population of normal control subjects.

[0155] 15. The use according to Embodiment 14, wherein the biological sample is a DBS, and the average concentration of the WASp signature peptide in DBS derived from a population of normal control subjects is in the range of 7,000 pmol / L to 30,000 pmol / L.

[0156] 16. Use of Embodiment 14 or 15, wherein the corresponding predetermined threshold concentration ranges from -1.75 standard deviations (SD) to -2.75 SD of the mean concentration of the WASp signature peptide in DBS derived from a population of normal control subjects.

[0157] 17. The use according to Embodiment 14, wherein the biological sample is DBS, and the average concentration of the BTK signature in DBS derived from a population of normal control subjects is in the range of 400 pmol / L to 2000 pmol / L.

[0158] 18. The use according to Embodiment 14 or 17, wherein the corresponding predetermined threshold concentration includes -1.5SD to -2.5SD of the average concentration of the BTK signature in DBS derived from a population of normal control subjects.

[0159] 19. An assay for screening Wiscott-Aldrich syndrome (WAS) and / or X-linked agammaglobulinemia (XLA) in subjects, the assay comprising: (i) The antibody or antigen-binding fragment described in Embodiment 1 and (ii) Reference signature peptides including the following: The WASp signature peptide of WAS in SEQ ID NO: 1; and / or BTK signature peptide of XLA, SEQ ID NO: 2 Assays including

[0160] 20. The assay according to Embodiment 19, wherein the reference signature peptide is isotope-labeled.

[0161] 21. The assay according to Embodiment 19 or 20, wherein the antibody or its antigen-binding fragment is attached to a magnetic bead.

[0162] 22. The assay described in any one of Embodiments 19 to 21, and a kit comprising one or more additional components selected from a filter paper card, an oral swab, a blood collection tube, a punch tool, a digestive enzyme, a digestive buffer, a solid support for an antibody or its antigen-binding fragment, and an elution buffer.

[0163] (x) Experimental Examples. Example 1. Summary. A study was conducted to evaluate whether a multiplex assay based on peptide immunoaffinity enrichment combined with selected reaction monitoring mass spectrometry (immuno-SRM) could reliably and accurately distinguish affected patients with Wiscott-Aldrich syndrome (WAS) and X-linked agammaglobulinemia (XLA) from each other and from unaffected normal control dried blood spot (DBS) samples. Blinded multiple analyses were performed (for WAS and XLA, respectively) of peptides proteolytically produced from WASp and BTK in DBS samples from 42 primary immunodeficiency disorder (PIDD) patients, 40 normal adult controls, and 62 normal neonates. The immuno-SRM assay reliably quantified target peptides in DBS, including intra-assay and inter-assay precision (13% and 22%; 17% and 43%), linearity (1.39–2000 fmol peptides), and stability (difference of less than 0.06% at 72 hours). Analysis of signature peptides revealed a statistically significant decrease (or absence) in peptide levels in affected patients compared to the control group (WAS and BTK: p=0.0001). Immuno-SRM-based quantification of proteolytic peptides derived from WASp and BTK in DBS differentiates relevant PIDD cases from controls. This approach may be used to conduct large-scale multiple neonatal screening for selective PIDD. Data in Figures 2A–11B and Tables 5–7 were prepared with monoclonal antibodies against WASp 289 and BTK 545.

[0164] Materials and Methods. Patient Samples. PIDD and normal control blood samples were obtained from the Seattle Children's Immunology Diagnostic Laboratory. Neonatal DBS samples were collected from the Washington State Newborn Screening Laboratory (Shoreline, WA) after approval by the Institutional Review Board. XLA DBS samples were collected from 20 suspected Vietnamese patients and sent by regular mail to Seattle Children's Hospital. Genotyping of these patients by Sanger sequencing has been previously reported (Segundo et al. Front Immunol. Frontiers; 2018; 9:289). In total, DBS samples were obtained from 42 PIDD patients and 40 normal controls. Normal control and PIDD patient DBS samples were prepared by pipetting 70 μL of blood / 12 mm spot onto filter paper cards (Protein Saver 903 Card, Whatman, Piscataway, NJ), drying overnight at room temperature, and storing in a sealed plastic bag at -80°C until use. Samples from affected patients were shipped from the collection site and stored at -80°C until use.

[0165] Selection of surrogate peptides and antibody production. Surrogate peptides for WASp and BTK were selected in silico by trypsin digestion and the NCBI BLAST tool. Final peptide selection was performed according to accepted key criteria for immuno-SRM development, including peptide length, lack of post-transcriptional modifications, and uniqueness of the human genome by BLAST search as described above (Kerfoot et al. Proteomics Clin Appl. 2012;6:394-402; Abbatiello et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology;2015;14:2357-2374; Hoofnagle et al. Clin. Chem. 2016;62:48-69). Peptide selection and monoclonal antibody production of the ATP7B signature peptide have been previously reported. Jung et al. 2017, see above. Next, crude peptides were empirically screened, and their suitability for detection and quantification was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0166] Monoclonal antibodies (mAbs) were successfully generated for two peptides. Briefly, the signature peptide was synthesized with an N-terminal cysteine ​​extension and conjugated to keyhole limpet hemocyanin (KHL) for immunization. Two New Zealand white rabbits were injected for each peptide. The peptide mAbs were successfully purified by affinity from 25 mL of antiserum.

[0167] Immunoscopy-SRM assay reagents. ProteaseMAX® surfactant (number V2072) and proteomics-grade trypsin (number V5113) were purchased from Promega (Madison, WI). Bovine serum albumin standard (200 mg / mL) and (3-[3-coramidopropyl)=dimethylammonio]-1-propanesulfonate) (Pierce® CHAPS, number PI28300) surfactant were obtained from Thermo Fisher Scientific (Waltham, MA). Ammonium bicarbonate (40867-50G-F) was purchased from Fluka Analytical (Munich, Germany). Acetonitrile (item number A955), water (item number W6, LCMS optima grade), formic acid (item number PI28905), and phosphate-buffered saline (PBS, item number 10010-023) were obtained from Thermo Fisher Scientific (Waltham, MA).

[0168] A heavy, stable isotope-labeled peptide was obtained from Anaspec (Fremont, CA). The stable isotope-labeled peptide was purified to over >95% by HPLC, and the C-terminal arginine or lysine was removed. 13 C and 15 Labeling with a nitrogen atom resulted in mass shifts of +8 or +10 Da, respectively. Aliquots were stored in 5% acetonitrile / 0.1% formic acid at -20°C until use.

[0169] The antibody was immobilized on 2.8 μm Dynabeads Protein G magnetic beads (number 10004D, Invitrogen, Carlsbad, CA) in a bead ratio of 1 μg antibody to 2.5 μL. Briefly, 250 μL of beads were added to a 1.5 mL Eppendorf tube (022363204 Eppendorf), washed twice with 250 μL of 1 × PBS, and then 100 μg of antibody and 1 × PBS + 0.03% CHAPS (number 28300, Thermo Scientific, Waltham, MA) were added to obtain a total volume of 250 μL. The antibody was then bound to the beads overnight while tumbling at 4°C. The next day, the antibody was immobilized on the beads by chemical crosslinking. In short, antibody beads were collected using a magnetic pull-down filter, excess PBS was discarded, and 300 μL of freshly prepared 20 mM triethanolamine, pH 8.5 (number T58300, Sigma Aldrich, St. Louis, MO) containing 20 mM DMP (dimethyl pimelimidate dihydrochloride, number D8388, Sigma Aldrich, St. Louis, MO) was added. The sample was tumbled at room temperature for 30 minutes, and the DMP in the triethanolamine was discarded. 250 μL of 150 mM monoethanolamine (number 411000, Sigma Aldrich, St. Louis, MO) was added, and the beads were tumbled twice at room temperature for 30 minutes. The antibody beads were washed twice with 250 μL of 5% acetic acid + 0.03% CHAPS (tumbling for 5 minutes each time at room temperature), and then washed again with 250 μL of 1× PBS + 0.03% CHAPS. Next, the CD3ε, WASp, and BTK antibody-conjugated beads were washed, incubated in 5% acetic acid + 3% acetonitrile (ACN), and washed with 250 μL of 1× PBS + 0.03% CHAPS. The following two steps were repeated once. All antibody-conjugated beads were washed with 250 μL of 1× PBS + 0.03% CHAPS until a neutral pH (7.0) was reached. The washed antibody-conjugated beads were then resuspended in 250 μL of 1× PBS + 0.03% CHAPS and 2.5 μL of NaN3 (52002-5G Sigma Aldrich) for antifungal properties and stored at 4°C until use.

[0170] DBS protein extraction and trypsin digestion. For each sample (blinded normal control or patient), 17 punches of 3 mm diameter were made in a single DBS spot (13 mm) containing 70 μL of blood using a standard leather punch tool. Final sample representations were WAS: n=11, XLA: n=26, and normal control (n=40). The punches were placed in 1.5 mL Eppendorf tubes, and 490 μL of 0.1% ProteaseMax™ in 50 mM ammonium bicarbonate (pH 8) was added to each tube. The tubes were vortexed for 1 hour in an Eppendorf MixMate (Eppendorf, Hamburg, Germany), and then 10 μL of each sample was aliquoted and diluted 200-fold for the Bradford assay to measure protein concentration. Disulfide bond reduction was performed with 5 mM 2 M DTT, and 490 μL of 0.1% ProteaseMax® in 50 mM ammonium bicarbonate (pH 8) was added to each tube before incubation in a 37°C water bath for 30 minutes. Next, trypsin was added at an enzyme-to-protein ratio of 1:50 (w / w), and acetonitrile was added to a final concentration of 15%. The mixture was digested by incubation overnight in a 37°C water bath, followed by centrifugation at 13,000 RPM for 10 minutes. Each supernatant was transferred to a new tube and dried using a Savant® SpeedVac® High Capacity Concentrator (Thermo Fisher Scientific, Waltham, MA). All trypsin-treated DBS digests were stored at -80°C until use.

[0171] For samples analyzed from the NBS Laboratory in Washington State, five or six 3mm punches were used for protein extraction and digestion (n=62). The procedure was the same as for previous samples, but with the following volume reductions: 150 μL of 0.1% ProteaseMax® and 0.78 μL of DTT for each addition.

[0172] Peptide immunoaffinity enrichment. DBS digests were resuspended in 1×PBS + 0.03% CHAPS to obtain a nominal protein digest concentration of 1 μg / μL. Crosslinked antibody-coated beads were added to 2 μg total mass of antibody for each target. Then, 20 μL of 1 M Tris pH 8.0 (15568-025 UltraPure, Invitrogen, Carlsbad, CA) was added. Isotope-labeled peptides were added as internal standards (IS). This suspension was incubated overnight at 4°C with tumbling to achieve peptide capture. The following day, the antibody-bead:peptide complexes were washed twice with 100 μL PBS + 0.01% CHAPS and once with 100 μL 0.01% PBS + 0.01% CHAPS. Finally, the peptides were eluted by incubation in 30 μL of 5% acetic acid / 3% ACN. The released peptides were stored at -80°C until analysis. The procedure for the samples analyzed from the Washington State NBS laboratory was the same as for previous samples, except that the volumes were reduced as follows: 58.1 μL of 1×PBS + 0.03% CHAPS, 0.59 μg pAb of each peptide, 3.13 μL of internal standard (IS), and 12.5 μL of TRIS.

[0173] Liquid chromatography-tandem mass spectrometry. Concentrated samples were analyzed at two laboratory sites, and inter-laboratory variability in data acquisition was investigated using two separate LC-MS / MS systems and instrument configurations (described below). The measured peptide concentrations were then compared for method validation. The spectra of the parent and daughter ions of the peptide have been previously reported: Kerfoot et al. Proteomics Clin Appl. 2012;6:394-402.

[0174] The instrumentation consisted of a Waters Xevo TQ-XS MS with ion keysource technology connected to a Waters M-Class gradient and loading pump (Waters, Milford, MA). The chromatography solvents were A: H2O + 0.1% formic acid (FA) and B: ACN + 0.1% FA. First, the peptide mixture was loaded onto an M-Class Trap Symmetry 300 μm × 50 mM C18 column (100 Å, 5 μm) using a constant flow rate of 20 μL / min for 3 minutes with a 98:2 A:B ratio. Subsequently, the flow was reversed and the peptides were separated using a gradient flow across a 150 μm × 100 mm BEH C18 ikey (130 Å, 1.7 μm). The gradient programming method is shown in Table 4. The peptide monitored at this location included WASp 289.

[0175] [Table 4]

[0176] Transition and collision energy (CE) parameters were obtained from linear regression of previously optimized values ​​in Skyline and values ​​generated using Waters IntelliStart technique to identify the strongest fragments at ionization. SRM transitions were acquired at unit / unit resolution with both Q1 and Q3 quadrupoles, with residence times of 5 milliseconds, pause times of 3 milliseconds between mass ranges, and a cycle time of 1.5 seconds. All samples were run in a blinded manner.

[0177] Method performance evaluation. Response curves were performed to determine the linearity and sensitivity of the assay in a DBS background matrix. Punches from a standard control DBS (4 punches per sample) were extracted three times using extraction buffer (ProteaseMax®, ammonium bicarbonate). The extracted proteins were digested with trypsin, and the digests were pooled to create a common background matrix. Heavily stable isotope standards were spiked into the digests and serially diluted to create samples with various peptide amounts (2000, 200, 12.5, 4.17, 1.39, 0.69 fmol). Two micrograms of each antibody, covalently bound to magnetic protein G beads, were added to the background matrix and incubated overnight. The antibody beads were washed with PBS, and the eluate was analyzed by SRM.

[0178] Intra-assay and inter-assay precision was characterized by measuring endogenous (light) peptide signals over five separate days, and the accuracy and precision of each assay were evaluated. Each sample was analyzed with five complete process replications per day (including punch, extraction, decomposition, concentration, and mass spectrometry).

[0179] Finally, stability was assessed by comparing endogenous (light) peptides detected by DBS after being stored at room temperature for 1 and 3 days with peptides detected by DBS at -80°C in a sealed container. Each sample was processed through the three processes described above. Percentage differences were calculated at each time point.

[0180] Data analysis. All SRM data were analyzed and plotted using Skyline (MacCoss Lab Software, open source, Seattle, WA). MacLean et al. Bioinformatics. 2010;26:966-968. Endogenous target peptide concentrations were quantified by comparing the peak area of ​​the signature peptide with its IS added at a known concentration (100 fmol). Statistical analysis was performed using Graphpad Prism (San Diego, CA). Receiver operating characteristic (ROC) curves were constructed using Graphpad Prism and 95% confidence intervals.

[0181] Results. Peptide selection and antibody development. The selected peptide sequences, molecular weights, parent ions, and daughter ions are listed in Figure 1. The fragmentation pattern of the target peptide has been previously reported (Kerfoot et al. Proteomics Clin Appl. 2012;6:394-402). The antibody (Pacific Immunology, Ramona, CA) was generated against the peptide and was chosen for use in human samples due to its ability to successfully capture the target sequence and the absence of background signal caused by co-purified peptide contaminants.

[0182] Method performance evaluation. The analytical performance index is reported in Table 5. Overall, the linear response ranged from 1.39 to 2000 fmol of peptides (Figures 2A and 2B). The median coefficient of variation (CV) for all points on the response curve was 11%. The limit of quantification (LLOQ) was defined by the lowest point yielding CVs of 13 and 22%. The LLOQ ranged from 0.69 to 1.39 fmol. For the two peptides, the mean intra-assay (i.e., diurnal) variability was 13–22%, while the inter-assay (i.e., inter-day) variability was 17–43%.

[0183] [Table 5]

[0184] Finally, stability was evaluated by comparing the endogenous (light) peptides detected by DBS stored at room temperature for 1 and 3 days with the peptides detected by DBS stored at -80°C in a sealed container. The results are shown in Table 5. These peptides had endogenous signals exceeding LLOQ and showed little variation over time. Representative multiple reaction monitoring (MRM) chromatograms for each peptide are shown in Figures 3A and 3B.

[0185] Peptide concentrations. After analysis, normal controls were not blinded to define a normal range for comparison with affected patients. The mean peptide concentrations from normal controls were as follows (mean ± SD): WASp 289 = 10326.98 ± 4513.13 pmol / L, and BTK 545 = 1038.44 ± 465.77 pmol / L. Analysis of signature peptides revealed statistically significant (p<0.05~0.0001) decreases in the patient peptide levels compared to the control group in each case (Figure 4A, Figure 4B). The majority of affected patients had significantly reduced or absent peptide levels. For each patient, the concentration of ATP7B 1056 was also determined using a previously developed immuno-SRM methodology (Jung et al. 2017, J Proteome Res 16:862-871). These protein concentrations serve as quality control (QC) measurements, and the consistency between samples is used to assess the reproducibility of digestion and the process (Figure 5).

[0186] The peptide concentration cutoffs for each PIDD diagnosis were set at -1.75SD (WASp 289) and -2SD (BTK 545). Using these ranges, two false-positive indications occurred in normal controls. NC4 was shown to be due to WASp 289. The NC signature peptide value is shown in Figure 6. The cutoffs for reliably identifying PIDD are shown in Table 6.

[0187] [Table 6]

[0188] Using these cutoffs, specific PIDD diagnoses were predicted for each patient. The predicted diagnoses showed good agreement with clinical or genetic diagnoses, as shown in Figure 7. All cases of WAS and BTK confirmed molecularly were also diagnosed by immuno-SRM analysis. Two patients clinically diagnosed with agammaglobulinemia, patients 10 and 13, had normal levels of BTK protein according to immuno-SRM. Molecularly, no BTK mutations were found in these patients (Segundo et al. Front Immunol. Frontiers;2018;9:289). Interestingly, patient 12 with agammaglobulinemia had low levels of BTK protein, but no mutations were found in the coding region of BTK. For each signature peptide used, area under the curve (AUC) analysis of the ROC plot revealed a region with p-values ​​in the range of 0.930 (WASp 289) to 0.999 (BTK 545) within the <0.0001 range (Figures 8A, 8B). Overall, there was agreement between the clinical diagnosis and the results of the immuno-SRM assay in 97.6% of cases. Interesting outliers and cases of discrepancy are discussed further below.

[0189] Figure 9 shows the signature peptide concentrations of NBS laboratory (neonatal) samples. Each DBS sample showed a significant measured peptide concentration above the previously established diagnostic cutoff for PIDD, indicating a disease-free state.

[0190] Immuno-SRM has been demonstrated as a highly sensitive and specific proteomics screening method for the multiple detection of patients with two life-threatening PIDDs (i.e., WAS and XLA) derived from DBS. The results clearly distinguish PIDD patients from normal controls, and low levels of endogenous peptides in the intracellular proteins WASp and BTK correlate with the target diseases (WAS and XLA, respectively). These diagnoses can be made in a single run, with a total run time of 6.67 minutes per disease target. The disclosed results also demonstrate peptide stability in DBS, with minimal concentration fluctuations after 72 hours of storage at room temperature (Table 5).

[0191] The immuno-SRM platform reliably detected endogenous peptides from normal control BBSs in this highly multiplexed manner. Normal control DBSs (N=40) were not blinded and were used to define the normal range and potential positive screening cutoffs (Figure 6). In clinical laboratories, the reference range for diagnostic tests is determined by the normal distribution of the general population. The initial cutoff for screening tests is typically conservative, aiming to detect all true positives without producing an overly high screening positive rate compared to the disease incidence (Table 6). However, these cutoffs are continuously validated and adjusted according to population-based studies. Considering these parameters, the definition of a positive screening result was a range 1.75–2 standard deviations (SD) lower than the mean peptide in this example. The selected cutoff resulted in one false-positive normal control, which was screened as a WAS patient using WASP289 (Figure 6). In the case of NC4, re-screening indicated a WASp level within the normal range. These preliminary cutoffs are not static and will become more clearly defined as more normal control and patient samples are screened.

[0192] Using these cutoffs, all molecularly confirmed WAS and BTK patients covering a wide range of mutations were clearly identified (Figure 7). Hypothetically, peptide concentrations are reduced in the majority of BTK and WAS cases, regardless of genotype (Qasim et al. Br.J. Haematol. 2001;113:861-865; Jin et al. Blood. American Society of Hematology; 2004;104:4010-4019; Futatani et al. British Journal of Haematology. 2001;114(1):141-9). Therefore, these peptides provide biomarkers for diagnosis and screening.

[0193] ROC curves were constructed to assess the diagnostic capability of immuno-SRM analysis. These plots correlate the true positive rate with the false positive rate, with increasingly strict cutoff values. A lower diagnostic cutoff increases the test's ability to record true positives, but this process also increases the likelihood of false positives. Therefore, screening tests that maintain a high true positive rate and a low false positive rate will result in a graph close to the y-axis and a large AUC (Figures 8A and 8B). These values ​​demonstrate the high diagnostic accuracy of immuno-SRM analysis of PIDD's signature peptide.

[0194] QC monitoring of digestion and process performance is included in the current immuno-SRM multiplex in the form of ATP7B signature peptide measurement. Since not all detected metabolites are useful NBS targets, metabolite ratio calculations and secondary metabolite analysis are used to improve the sensitivity and specificity of NBS for specific diseases, such as the C3:C2 ratio and 2-methylcitrate analysis in methylmalonic aciduria (Lindner et al. J. Inherit. Metab. Dis. 2nd ed. 2008;31:379-385). Furthermore, target ratios can account for inter-sample variability caused by numerous factors, including sample collection quality, storage, extraction and digestion efficiency, and blood characteristics (Razavi et al. Bioanalysis. Future Science Ltd London, UK; 2016;8:1597-1609). Here, ATP7B concentrations were found to be fairly consistent across the screened samples (Figure 6). The absence of ATP7B can be helpful in warning of improperly processed or handled specimens. As an initial experiment, each PIDD peptide was compared as a ratio to the endogenous concentration of ATP7B in the same sample. Predictions based on peptide concentrations showed perfect agreement with clinical diagnoses, demonstrating that the ratios of immuno-SRM and ATP7B are effective and complementary tools for PIDD diagnosis (Figure 10 and Table 7). These types of ratios are useful in clinical immuno-SRM screening when the selected peptide is ubiquitous across a large cohort of samples and has proven to be a remarkably invariant signal.

[0195] [Table 7]

[0196] It is noteworthy that normal levels of BTK were found in two clinically defined agammaglobulinemia patients (samples 10 and 13) who lacked BTK mutations detected by Sanger sequencing (Figure 7). Therefore, these patients are likely to be without XLA, but may have other autosomal agammaglobulinemia types, although more extensive genetic testing has not been performed. Another patient (sample 12) had reduced levels of BTK protein, but no identifiable mutations in BTK. This suggests that a mutation may have been missed during sequencing of the coding region and intron-exon junctions, or that this patient may carry a BTK mutation affecting a regulatory element, polyadenylation signal, or intron region. These cases highlight the clinical utility of immuno-SRM.

[0197] Furthermore, two samples were obtained from the same WAS patient before and after bone marrow transplantation (BMT) (sample numbers 29 and 30 in Figure 7, respectively). Immuno-SRM analysis before BMT confirmed the patient had WAS. After BMT, the patient was identified as normal. This case highlights the ability of immuno-SRM to track the course of BMT treatment and confirm successful immune system rearrangement. A similar principle may be applicable to patients with monogenic disorders undergoing gene therapy.

[0198] Overall, the analysis demonstrates that the disclosed assay has a broad linear range and acceptable precision for determining the concentration of the target peptide in DBS (Table 5).

[0199] The feasibility of utilizing immuno-SRM analysis in the context of NBS was tested using randomly selected samples provided by the NBS Laboratory in Washington State. Due to limited sample availability and to test the usefulness of signature peptide analysis from smaller samples, the amount of DBS used was reduced from one whole spot to five or six 3 mm punches. The peptides of interest were easily concentrated and analyzed with minimal changes to sample handling. All signature peptide concentrations were higher than the predefined cutoff obtained from the analysis of known normal controls (Figure 9). Therefore, these patients are designated as normal. The ability to reliably perform this analysis with a significantly reduced sample input makes immuno-SRM analysis even more suitable for conversion to NBS. This high-throughput multiplexing method can effectively reduce the execution time per disease and is suitable for NBS, where the normal execution time for current automated methods is less than 3 minutes (Rashed et al. Clin.Chem. 1997; 43: 1129-1141; Khalid et al. J Med Screen. SAGE Publications, Sage UK: London, England; 2008; 15: 112-117). The success in predicting BTK patients using DBS sent at ambient temperature via conventional mail from Vietnam also highlights the potential utility of diagnostic testing in resource-poor environments where DBS collection and dispatch are economical.

[0200] Example 2. This study describes results obtained from monoclonal antibodies described herein that bind to the WASp 289 and BTK 545 signature peptides. Monoclonal antibodies are preferred over polyclonal antibodies for use in clinical, diagnostic, and neonatal screening assays due to their reproducibility and consistency. Monoclonal antibodies function comparably to polyclonal antibodies in immuno-SRM assays in their ability to concentrate signature peptides from dried blood spots, generate a normal control range, and distinguish patients from controls. Monoclonal and polyclonal antibodies of WASp 289 and BTK 545 were found to generate a normal control range with comparable standard deviations (Figures 11A, 11B, and Table 8). In these populations, the use of monoclonal antibody reagents was found to result in a moderate increase in mean peptide concentration. This is likely due to the homogeneity of the reagents compared to polyclonal antibodies, which may result in greater nonspecific binding or a reduced population of peptide-binding epitopes.

[0201] [Table 8]

[0202] The target peptide was enriched from normal control DBS using a monoclonal antipeptide antibody against WASp 289. The target signature peptide was present at a mean concentration of 14905.9 ± 4608.6 pmol / L (Figure 12).

[0203] Using a monoclonal antipeptide antibody against BTK 545, the target peptide was enriched from normal control BBS. The target signature peptide was present at a mean concentration of 1267.8 ± 478.2 pmol / L. Analysis of XLA patients (n=8) using this marker revealed that all clinically confirmed cases of XLA were diagnosed by immuno-SRM analysis of BTK 545 (Figure 13 and Table 9).

[0204] [Table 9]

[0205] The amino acid alteration in patient 3, p.R544G, eliminates the trypsin cleavage site immediately preceding the BTK 545 biomarker peptide. This alteration blocks the release of the BTK 545 peptide by trypsin digestion and, similarly, leads to the absence of wild-type endogenous BTK545 due to differences in the molecular weight of the additional amino acids. The signature peptide containing the mutation site is undetectable by MS due to the mass shift associated with the amino acid alteration. This alters the total peptide mass, making the mutated peptide undetectable by the mass spectrometer and eliminating the wild-type biomarker signal. Therefore, BTK 545 levels cannot be detected in patient 3. Thus, since BTK 545 contains the site of the point mutation in patient 3, cases that might be missed with the aforementioned signature peptide BTK 407 (PCT / US2019 / 054856) could potentially be identified using BTK 545. This suggests that using multiple signature peptides may increase the likelihood of identifying patients, especially in cases where there are known mutations that cause false negatives by immuno-SRM.

[0206] Based on monoclonal antibody analysis, the WASp 289 cutoff was set to 3384.4 pmol / L (-2.5 SD), and the BTK 545 cutoff was set to 311.4 pmol / L (-2 SD) (Table 10).

[0207] [Table 10]

[0208] While NBS is one of the most successful public health initiatives in modern times, it relies on the detection of accumulated metabolites resulting from downstream enzyme deficiencies. However, many genetic disorders, including PIDD, are characterized by protein deficiencies or reductions, limiting the scope of current NBS methods (Qasim et al. Br.J. Haematol. 2001;113:861-865; Jin et al. Blood. American Society of Hematology; 2004;104:4010-4019). By enabling the detection of PIDD-related peptides from DBS, immuno-SRM fills this gap in current coverage and allows for the expansion of NBS to treatable diseases for which current metabolite biomarkers are lacking. Immuno-SRM rapidly provides quantified evidence of protein deficiency and can be performed concurrently with initial screening and molecular analysis from DBS without further invasive procedures. Quantification of these signature peptides lays the foundation for immuno-SRM as a highly multiplexable screening and diagnostic tool for a variety of congenital disorders.

[0209] Predictive Example 1. Immuno-SRM assay of WASp and / or BTK signature peptides for the diagnosis of Wiscott-Aldrich syndrome (WAS) and X-linked agammaglobulinemia (XLA). Corresponding samples from DBS (as described above), oral swab samples, peripheral blood mononuclear cells (PBMCs), or leukocytes (WBCs) from patients with WAS or XLA, or suspected patients with WAS or XLA, and normal controls are analyzed by immuno-SRM using the signature peptides WASp 289, BTK 545, or combinations thereof, as well as their associated monoclonal antibodies as described in Example 2 and elsewhere herein.

[0210] Intraoral swab samples. The regulatory body's review board approves the intraoral swab sample protocol, and all subjects obtain written informed consent. Routine control intraoral swab samples are obtained from commercial suppliers. All intraoral swab samples are stored in the laboratory at -20°C or -80°C. Blinded samples are labeled with an ID provided by the sender, and identified and consenting patient samples are assigned a lab ID upon receipt. Peel Pouches, Copan Diagnostics 502CS01 Nylon Flocked Dry Swabs are obtained from Fisher Scientific (Chicago, IL; catalog number 23-600-951). 2 mL Cryogenic Storage Vials Internal Thread are obtained from Fisher Scientific (Chicago, IL; catalog number 12-567-501). Intraoral swab sample collection may follow the protocol described in CHLA (April 4, 2016). Oral swab collection procedure. CHLA - Clinical Pathology; (July 27, 2016). Explanation of oral DNA collection.Pathway Genomics; (2017, Dec 14). Instruction for Buccal Swab Sample Collection. Otogenetics; PDXL PDXL. (2017, Nov 28). Buccal Swab collection procedure - PersonalizedDx Labs [Video]. YouTube. On World Wide Web at youtube / 3ftvHkfM71o?t=146; and Centers of Disease Control and Prevention (CDC). (2020, July 8). Interim Guidelines for collecting, handling, and testing clinical specimens for Covid-19. On World Wide Web at cdc.gov / coronavirus / 2019-ncov / lab / guidelines-clinical-specimens.html. The tip of the oral swab containing cells may be clipped to a tube for solubilization and digestion, as previously described for DBS.

[0211] Peripheral blood mononuclear cells (PBMCs) and leukocytes (WBCs). PBMCs and WBCs are collected using protocols known in the art, such as those described in Kerfoot et al., Proteomics Clin Appl, 2012.6(7-8):394-402; Grievink et al. (2016) Biopreserv Biobank 14(5):410-415; Corkum et al. (2015) BMCImmunol.16:48; and Jia et al. (2018) Biopreserv Biobank 16(2):82-91; and Zhou et al. (2012) Clinical and Vaccine Immunology 19(7):1065-1074. Isolated PBMCs or WBCs are solubilized, and cell-derived proteins can be digested as previously described for DBS.

[0212] Immuno-SRM diagnosis will be compared to clinical diagnosis. Where possible, genetic and therapeutic information for the WAS and / or BTK genes will be obtained for each patient. Immuno-SRM assays may be multiplexed with other signature peptides of other diseases. These studies demonstrate that immuno-SRM assays utilizing the monoclonal antibodies described herein can be used to detect disclosed WASp and / or BTK signature peptides in biological samples including DBS, intraoral swab samples, PBMCs, or WBCs, and to diagnose whether a subject has WAS and / or XLA based on the detection level of the signature peptides.

[0213] (xi) Final paragraph. The exact amino acid sequence boundary of a given CDR or FR can be easily determined using one of several well-known schemes, including those described below: Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol 273:927-948 (Chothia numbering scheme); Maccallum et al. (1996) J Mol Biol 262:732-745 (Contact numbering scheme); Martin et al. (1989) Proc. Natl. Acad. Sci., 86:9268-9272 (AbM numbering scheme); Lefranc MP et al. (2003) Dev Comp Immunol 27(1):55-77 (IMGT numbering scheme); and Honegger and Pluckthun (2001) J Mol Biol 309(3):657-670 ("Aho" numbering scheme). The boundaries of a particular CDR or FR may differ depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the length of the sequence of the most common antibody region, with insertions, e.g., the insertion that was in "30a", and deletions found in some antibodies. In the two schemes, certain insertions and deletions ("indels") are located in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. In certain embodiments, the antibody CDR sequences disclosed herein are numbered by Kabat.

[0214] The nucleic acid and amino acid sequences provided herein are indicated using abbreviations of nucleotide bases and amino acid residues, as defined in Section 1.822 of the U.S. Patent Law Enforcement Regulations, and are listed in Tables 1 and 3 of WIPO Standard ST.25 (1998), Appendix 2. Although only one strand of each nucleic acid sequence is shown, it is understood that complementary strands are included in appropriate embodiments.

[0215] Except as expressly provided herein, the coding sequences of proteins disclosed herein and the protein sequences of coding sequences disclosed herein can be readily derived by those skilled in the art.

[0216] Each embodiment disclosed herein may include, essentially consist of, or consist of, the specific described elements, steps, components, or constituents. Therefore, the terms “include” or “including” should be interpreted as “include, consist of, or essentially consist of.” The transitional terms “comprise” or “comprises” mean to have, but not be limited to, an unspecified set of elements, steps, components, or constituents, even in large quantities. The transitional phrase “consists of” excludes any elements, steps, components, or constituents not specified. The transitional phrase “essentially consists of” limits the scope of the embodiment to specific elements, steps, components, or constituents, and those that do not substantially affect the embodiment. Significant effects may result in a statistically significant reduction in the ability of the antibodies or antigen-binding fragments disclosed herein to bind to their homologous peptide biomarkers.

[0217] Unless otherwise indicated, all figures used in this specification and the claims, such as quantities of components, molecular weights and other properties, and reaction conditions, should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties to be obtained by the present invention. At the very least, each numerical parameter should be interpreted in light of at least the reported number of significant figures and by applying common rounding techniques, not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims. If further clarification is needed, the term “approximately” has a meaning reasonably attributable to a person skilled in the art when used in conjunction with a stated number or range, i.e., when indicating a value that is somewhat more or somewhat less than the stated value or range (up to ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value).

[0218] Although the numerical ranges and parameters representing the broad scope of the present invention are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, the numerical values ​​inherently contain certain errors that inevitably arise from the standard deviation found in each test measurement.

[0219] In the context describing the present invention (particularly in the following claims), the terms “a,” “an,” “the,” and similar references should be construed to cover both singular and plural forms unless otherwise stated herein or unless clearly contradicted by the context. The enumeration of value ranges herein is intended merely as a simple way to refer individually to each individual value within that range. Unless otherwise stated herein, individual values ​​are incorporated herein as if they were individually stated herein. All methods described herein may be performed in any appropriate order unless otherwise stated herein or unless clearly contradicted by the context. Any use of any examples or illustrative language provided herein (e.g., “etc.”) is intended merely to better illustrate the present invention and does not otherwise limit the claimed scope of the present invention. Nothing herein should be construed as indicating an unclaimed element essential to the practice of the present invention.

[0220] The classification of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Members of each group may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. If such inclusion or removal occurs, this specification shall be deemed to include the modified groups and thus satisfy all written descriptions of Markush groups used in the appended claims.

[0221] Specific embodiments of the invention are described herein, including the best mode known to the inventor for carrying out the invention. Of course, variations of these described embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventor expects those skilled in the art to appropriately use such variations, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, any combination of the above elements in all possible variations thereof is included in the invention unless otherwise indicated herein or clearly contradicted by context.

[0222] Furthermore, throughout this specification, numerous references have been made to patents, printed publications, magazine articles, and other written text (references herein). Each of the referenced materials is hereby incorporated by reference in its entirety for its disclosed teachings.

[0223] It should be understood that the embodiments of the invention disclosed herein are merely illustrative of the principles of the invention. Other modifications that may be used are within the scope of the invention. Accordingly, by way of example and not limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely shown and described.

[0224] The details shown herein are for purposes of example only, for an exemplary discussion of the preferred embodiments of the invention, and are presented to provide what is considered to be the most useful and readily understood description of the principles and conceptual aspects of the various embodiments of the invention. In this regard, no attempt has been made to show details of the invention more specific than is necessary for a fundamental understanding of the invention, and the description with the drawings and / or examples has made apparent to those skilled in the art how some forms of the invention may be actually embodied.

[0225] The definitions and descriptions used in this disclosure are intended to control any future structures unless they are explicitly and expressly altered in the examples, or unless the application of the meaning renders any structure meaningless or essentially meaningless. Where the construction of a term renders a term meaningless or essentially meaningless, the definition should be obtained from a dictionary known to those skilled in the art, such as Webster's Dictionary, 3rd Edition, or Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. A heavy chain variable (VH) domain comprising CDRH1 having the sequence shown in SEQ ID NO: 3, CDRH2 having the sequence shown in SEQ ID NO: 4, and CDRH3 having the sequence shown in SEQ ID NO: 5, Light chain variable (VL) domains including CDRL1 having the sequence shown in SEQ ID NO: 6, CDRL2 having the sequence shown in SEQ ID NO: 7, and CDRL3 having the sequence shown in SEQ ID NO: 8 An antibody or its antigen-binding fragment that binds to the WASp 289 signature peptide, including [the specified substance].

2. A VH domain having the sequence shown in SEQ ID NO: 15; a heavy chain having the sequence shown in SEQ ID NO: 17; a VL domain having the sequence shown in SEQ ID NO: 16; or a light chain having the sequence shown in SEQ ID NO: 18 An antibody or antigen-binding fragment according to claim 1, comprising one or more of the following.

3. The VH domain has the sequence shown in Sequence ID 15, and the VL domain has the sequence shown in Sequence ID 16, or The heavy chain has the sequence shown in Sequence ID 17, and the light chain has the sequence shown in Sequence ID 18. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A process of digesting proteins derived from biological samples obtained from the subject with enzymes to produce one or more peptides, A step of concentrating the WASp signature peptide with the antibody or antigen-binding fragment thereof according to claim 1, The process involves performing liquid chromatography-multi-reaction monitoring mass spectrometry (LC-MRM-MS) on the concentrated WASp signature peptide to determine the concentration of the WASp signature peptide. A method for providing data for the diagnosis of primary immunodeficiency disorders (PIDDs), including [specific data].

5. The process further includes comparing the concentration of the WASp signature peptide with a predetermined threshold concentration. The method according to claim 4, wherein if the concentration of the WASp signature peptide is lower than the predetermined threshold concentration, or if the WASp signature peptide is not present, the subject is suspected to have Wiscott-Aldrich syndrome (WAS).

6. The method according to claim 5, wherein a predetermined threshold concentration of the WASp signature peptide is calculated from the standard deviation of the mean concentration of the WASp signature peptide in biological samples derived from a population of normal control subjects.

7. The method according to claim 6, wherein the biological sample is DBS, and the average concentration of the WASp signature peptide in DBS derived from a population of normal control subjects includes concentrations in the range of 7,000 pmol / L to 30,000 pmol / L.

8. A step of concentrating the BTK signature peptide with an antibody that binds to BTK545 or its antigen-binding fragment, The process involves performing liquid chromatography-multi-reaction monitoring mass spectrometry (LC-MRM-MS) on the concentrated BTK peptide to determine the concentration of the BTK signature peptide. The method according to any one of claims 4 to 7, further comprising:

9. An antibody or its antigen-binding fragment that binds to BTK545, A VH domain comprising CDRH1 having the sequence shown in SEQ ID NO: 9, CDRH2 having the sequence shown in SEQ ID NO: 10, and CDRH3 having the sequence shown in SEQ ID NO: 11, and A VL domain comprising CDRL1 having the sequence shown in SEQ ID NO: 12, CDRL2 having the sequence shown in SEQ ID NO: 13, and CDRL3 having the sequence shown in SEQ ID NO:

14. The method according to claim 8, including the method described in claim 8.

10. An antibody or its antigen-binding fragment that binds to BTK545, (a) A VH domain having the sequence shown in Sequence ID No. 23, and a VL domain having the sequence shown in Sequence ID No. 24, or (b) A heavy chain having the sequence shown in Sequence ID No. 25, and a light chain having the sequence shown in Sequence ID No. 26 The method according to claim 9, including the method described in claim 9.

11. The biological sample is a dried blood spot (DBS), an oral swab, peripheral blood mononuclear cells (PBMCs), or white blood cells (WBCs), and / or The method according to any one of claims 4 to 10, wherein the enzyme is trypsin.

12. The process further includes comparing the concentration of the BTK signature peptide with a predetermined threshold concentration. The method according to any one of claims 8 to 11, wherein if the concentration of the BTK signature peptide is lower than the predetermined threshold concentration, or if the BTK signature peptide is not present, the subject is suspected to be suffering from X-linked agammaglobulinemia (XLA).

13. The method according to any one of claims 4 to 12, performed as part of a neonatal screening (NBS) to further screen the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.

14. The method according to any one of claims 4 to 13, performed in a state in which the subject is free from clinical symptoms of WAS and / or XLA.

15. The method according to claim 12, wherein the predetermined threshold concentration of the BTK signature peptide is calculated from the standard deviation of the mean concentration of the BTK signature peptide in biological samples derived from a population of normal control subjects.

16. The method according to claim 15, wherein the average concentration of the BTK signature in DBS derived from a normal control population includes concentrations in the range of 400 pmol / L to 2000 pmol / L.

17. A kit for assays for screening Wiscott-Aldrich syndrome (WAS) in subjects, (i) The antibody or antigen-binding fragment thereof according to claim 1, and (ii) A reference signature peptide containing the WASp signature peptide of WAS of Sequence ID No.

1. Assay kit including

18. (iii) an antibody or antigen-binding fragment thereof that binds to BTK545, and (iv) Reference signature peptide containing the BTK signature peptide of XLA of SEQ ID NO: 2 The assay kit according to claim 17, further comprising an assay kit for screening Wiscott-Aldrich syndrome (WAS) and X-linked agammaglobulinemia (XLA) in subjects.

19. The assay kit according to claim 17 or 18, wherein the reference signature peptide is isotope-labeled, and / or the antibody or its antigen-binding fragment is attached to a magnetic bead.

20. The assay kit according to any one of claims 17 to 19, further comprising one or more additional components selected from a filter paper card, an oral cotton swab, a blood collection tube, a punch tool, a digestive enzyme, a digestive buffer, a solid support for an antibody or its antigen-binding fragment, and an elution buffer.

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