Methods of diagnosing and prognosing hodgkin's lymphoma
Inventors
Migone, Thi-Sau • Klein, Jerry L. • Oki, Yasuhiro • Younes, Anas
Assignees
GlaxoSmithKline Intellectual Property Development Ltd • The University of Texas System
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Publication Number
US-8211649-B2
Publication Date
2012-07-03
Expiration Date
Abstract
The present invention relates to nucleic acid molecules encoding Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides, including soluble forms of the extracellular domain. Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to antibodies or portions thereof that specifically bind Neutrokine-alpha and/or Neutrokine-alphaSV and diagnostic and therapeutic methods using these antibodies. Also provided are diagnostic methods for detecting immune system-related disorders and therapeutic methods for treating immune system-related disorders using the compositions of the invention.
Core Innovation
The patent describes Neutrokine-alpha (TALL-1/THANK/BAFF/zTNF4/TNFSF13B; BLyS) and a splice variant, Neutrokine-alphaSV, and provides nucleic acid and amino-acid sequence information. It describes soluble extracellular-domain forms, derived fragments and epitopes, and antibodies and antibody-binding portions that bind Neutrokine-alpha and Neutrokine-alphaSV.
It also describes Neutrokine-alpha/Neutrokine-alphaSV agonist/antagonist screening assays using membrane preparations with labeled ligand. Candidate antagonism is indicated by reduced labeled binding, while receptor-mediated effects and second-messenger signaling distinguish agonism, including AMP guanylate cyclase, ion channel, and phosphoinositide hydrolysis assays.
The invention relates to improving manufacturing of recombinant soluble BLyS (rsBLyS) in Escherichia coli, producing high yields of properly folded, biologically active, trimeric rsBLyS using a phoA promoter-based induction system. The phosphate-responsive regulation is controlled by PhoR and PhoB within the phosphate regulon responding to inorganic phosphate (Pi), and the described material is purified and characterized for biological activity.
Claims Coverage
The independent claims cover methods for detecting or diagnosing Hodgkin's lymphoma and prognosing Hodgkin's lymphoma using a soluble Neutrokine-alpha polypeptide fragment defined by amino acids 134 to 285 of SEQ ID NO: 2. Across the claims, the main inventive features are the defined binding agent, measurement of serum expression level, comparison to a standard expression level of less than 0.5 ng/ml, and, in the prognostic claim, additional indicators associated with worse prognosis.
Serum marker based Hodgkin's lymphoma detection with binding agent
Contacting serum from a patient suspected of having Hodgkin's lymphoma with an agent that binds a soluble Neutrokine-alpha polypeptide consisting of the amino acid sequence from about amino acid 134 to about amino acid 285 of SEQ ID NO: 2; measuring the expression level of the soluble Neutrokine-alpha polypeptide in the serum; and comparing the measured expression level with a standard expression level of soluble Neutrokine-alpha polypeptide that is less than 0.5 ng/ml, wherein an increase relative to the standard is indicative of Hodgkin's lymphoma.
Prognosis using soluble Neutrokine-alpha expression plus additional indicators
Contacting serum from a patient diagnosed with Hodgkin's lymphoma with an agent that binds a soluble Neutrokine-alpha polypeptide consisting of the amino acid sequence from about amino acid 134 to about amino acid 285 of SEQ ID NO: 2; measuring the expression level of the soluble Neutrokine-alpha polypeptide in the serum; measuring at least one additional indicator selected from male gender, stage 4 disease, bulky disease >7.5 cm, high LDH, high α2-microglobulin, resistance to therapy, and shorter failure free survival; comparing the measured expression level with a standard expression level of soluble Neutrokine-alpha polypeptide that is less than 0.5 ng/ml, wherein an increase relative to the standard is indicative of Hodgkin's lymphoma and having at least one additional indicator is indicative of a worse prognosis.
Taken together, the independent claims focus on binding-defined soluble Neutrokine-alpha in patient serum, quantifying its expression level, and using the comparison to a standard to indicate Hodgkin's lymphoma. The prognostic independent claim further requires at least one additional listed indicator to indicate worse prognosis.
Stated Advantages
An increase in the serum expression level of the soluble Neutrokine-alpha polypeptide relative to a standard of less than 0.5 ng/ml is indicative of Hodgkin's lymphoma.
Having at least one additional indicator together with an increased soluble Neutrokine-alpha serum expression level is indicative of a worse prognosis.
High yields of properly folded, biologically active, trimeric rsBLyS are reported in recombinant soluble BLyS manufacturing in Escherichia coli.
Approximately 11-fold increase in small-scale production yield, about 435 mg/L, is reported.
Purified rsBLyS material is reported with purity of about 97% by RP-HPLC.
Endotoxin reduction is described for the purification output.
Biological activity is reported as similar or slightly increased in murine B-cell proliferation assays.
Documented Applications
Detecting or diagnosing Hodgkin's lymphoma by measuring soluble Neutrokine-alpha polypeptide expression in a patient's serum using an agent that binds the soluble Neutrokine-alpha polypeptide (SEQ ID NO: 2, amino acids 134 to 285).
Prognosing Hodgkin's lymphoma using the soluble Neutrokine-alpha serum expression level together with at least one additional indicator selected from male gender, stage 4 disease, bulky disease >7.5 cm, high LDH, high β2-microglobulin, resistance to therapy, and shorter failure free survival.
Manufacturing recombinant soluble BLyS (rsBLyS) in Escherichia coli using a phoA promoter-based PhoR/PhoB phosphate-responsive induction system.
Production of properly folded, biologically active, trimeric rsBLyS that is evaluated using murine B-cell proliferation assays.
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