Isolation of An Anti-CD20 Single Chain Variable Fragment From a Naive Human Phage-ScFv Library

CD20 is a non-glycosylated transmembrane protein expressed on normal B cells, malignant B cells, and plasma cells, but not their stem cell precursors. It is an ideal target for antibody therapy because it is not significantly shed or internalized, and CD20 expression is generally not lost after antibody binding. Depleted B cells can also be regenerated after antibody therapy. Rituximab, a chimeric monoclonal antibody against CD20, was the first monoclonal antibody to be approved by the FDA for lymphoma. Results from clinical trials have shown that anti-CD20 monoclonal antibodies, which can be used unchanged or as carriers for radionuclides or toxins, have significant therapeutic effects and can be administered with minimal side effects. Several studies have also shown anti-CD20 monoclonal antibodies to be useful against autoimmune diseases such as rheumatoid arthritis. Phage display is an effective tool in genomics and drug development. The ability to obtain high affinity binders for any antigen with decreased cost and time expenditure has been useful in the development of therapeutics. Single chain variable fragments (scfvs) are the smallest antibody fragments which maintain the specificity and the affinity of the parent antibody. ScFvs can be conjugated to radioisotopes, toxins, drugs, highly effective Fc region isotypes, and gene delivery vectors, which can be employed in diagnosis and therapy. Studies have shown that chimeric and humanized forms of anti-CD20 demonstrate improved clinical efficacy, as compared with murine anti-CD20 monoclonal antibody, as well as a decrease in HAMA (human anti-mouse antibody) response, which is frequently observed with murine antibodies. Although chimerization of murine antibodies through protein engineering can retain the affinity and specificity of the parental molecule, this strategy is time-consuming and does not yield the benefit of fully human antibodies. To further improve the efficacy of anti-CD20 antibodies, a na•ve human scFv phage library (Tomlinson I+J) was used in the present study to screen for human antibodies against CD20. A cell based screening strategy was employed in this study by establishing a tetracycline inducible CD20 CHO cell line, to better mimic the microenvironment where CD20 and anti-CD20 antibody interaction would occur. Panning of the phage library against the CD20 transfected cell lines yielded clones with affinity for CD20 antigen. PCR analysis showed the expected 935bp scFv band, and monoclonal ELISA showed an affinity for CD20 antigen. Periplasmic protein was extracted from the clone and subjected to dot blot analysis, showing that in its native form, Clone 3 had an affinity for CD20. A fusion gene with the CD20 extracellular domain and an in vivo biotinylation domain was constructed for the isolation and characterization of antibody fragments. A functional biotinylated CD20 fusion protein was obtained from bacterial production and western blot analysis under denaturing conditions yielded a 20kDa band as expected

DEK protein level is a biomarker of CD138positive normal and malignant plasma cells.

Overexpression of DEK oncogene is associated with increased proliferation of carcinoma cells and it is observed in several solid tumors due to the amplification of the 6p22.3 chromosomal region where DEK locates. Although the same chromosomal amplification occurs in multiple myeloma (MM), a plasma cell neoplasm, whether the expression and the copy number of the DEK gene are affected in MM remains elusive. We show that despite the increased copy number in CD138positive MM cells (4 out of 41 MM samples), DEK mRNA expression was down-regulated compared with that in CD138negative bone marrow (BM) cells of the same patients (P<0.0001). DEK protein was not detectable by immunohistochemistry (IHC) in CD138positive normal plasma cells or in malignant plasma cells of MM patients (n = 56) whereas it was widely expressed in normal and neoplastic B-cells. Stable knockdown or overexpression of DEK in CD138positive MM cell lines did not affect the proliferation and viability of the cells profoundly in the presence or absence of chemotherapeutic agent melphalan whereas knockdown of DEK moderately but significantly increased the expression level of CD138 (p<0.01). Decreased DEK expression in plasma cells suggests a potential role of this gene in plasma cell development and lack of detectable DEK protein by IHC could be used as a biomarker for normal and malignant plasma cells

DEK protein level is a biomarker of CD138(positive) normal and malignant plasma cells

Overexpression of DEK oncogene is associated with increased proliferation of carcinoma cells and it is observed in several solid tumors due to the amplification of the 6p22.3 chromosomal region where DEK locates. Although the same chromosomal amplification occurs in multiple myeloma (MM), a plasma cell neoplasm, whether the expression and the copy number of the DEK gene are affected in MM remains elusive. We show that despite the increased copy number in CD138(positive) MM cells (4 out of 41 MM samples), DEK mRNA expression was down-regulated compared with that in CD138(negative) bone marrow (BM) cells of the same patients (P< 0.0001). DEK protein was not detectable by immunohistochemistry (IHC) in CD138(positive) normal plasma cells or in malignant plasma cells of MM patients (n = 56) whereas it was widely expressed in normal and neoplastic B-cells. Stable knockdown or overexpression of DEK in CD138(positive) MM cell lines did not affect the proliferation and viability of the cells profoundly in the presence or absence of chemotherapeutic agent melphalan whereas knockdown of DEK moderately but significantly increased the expression level of CD138 (p< 0.01). Decreased DEK expression in plasma cells suggests a potential role of this gene in plasma cell development and lack of detectable DEK protein by IHC could be used as a biomarker for normal and malignant plasma cells

Agammaglobulinemia and Absent B Lineage Cells in a Patient Lacking the p85α Subunit of PI3K

Whole exome sequencing was used to determine the causative gene in patients with B cell defects of unknown etiology. A homozygous premature stop codon in exon 6 of PIK3R1 was identified in a young woman with colitis and absent B cells. The mutation results in the absence of p85α but normal expression of the p50α and p55α regulatory subunits of PI3K. Bone marrow aspirates from the patient showed <0.1% CD19(+) B cells with normal percentages of TdT(+)VpreB(+)CD19(−) B cell precursors. This developmental block is earlier than that seen in patients with defects in the B cell receptor signaling pathway or in a strain of engineered mice with a similar defect in p85α. The number and function of the patient’s T cells were normal. However, Western blot showed markedly decreased p110δ, as well as absent p85α, in patient T cells, neutrophils, and dendritic cells. The patient had normal growth and development and normal fasting glucose and insulin. Mice with p85α deficiency have insulin hypersensitivity, defective platelet function, and abnormal mast cell development. In contrast, the absence of p85α in the patient results in an early and severe defect in B cell development but minimal findings in other organ systems

Molecular and clinical features of the fresh or frozen patient (MM) samples (n = 41).

<p>Molecular and clinical features of the fresh or frozen patient (MM) samples (n = 41).</p

Molecular and clinical features of the fresh or frozen patient (MM) samples (n = 41).

<p>Molecular and clinical features of the fresh or frozen patient (MM) samples (n = 41).</p

Pathological feauters and normalized <i>DEK</i> expression levels of FFPE samples of controls (1–8) and MM patients (9–38).

<p>Pathological feauters and normalized <i>DEK</i> expression levels of FFPE samples of controls (1–8) and MM patients (9–38).</p