Chimeric rabbit/human Fab and IgG specific for members of the Nogo-66 receptor family selected for species cross-reactivity with an improved phage display vector

Abstract

NgR1, NgR2, and NgR3 which constitute the Nogo-66 receptor family are primarily expressed by neurons in the central nervous system (CNS) and believed to limit axonal growth and sprouting following CNS injury. In an attempt to define the expression and decipher the function of individual members of the Nogo-66 receptor family, we previously reported the generation of selective rabbit polyclonal antibodies. Here we exploit the same immune repertoires by phage display technology to generate rabbit monoclonal antibodies (mAbs) with nanomolar affinity to epitopes that are specific for NgR1 and NgR2, respectively, but at the same time conserved between mouse, rat, and human orthologs. Employing phage display vector pC3C, a newly designed phagemid optimized for the generation and selection of Fab libraries with human constant domains, rabbit mAbs were selected from chimeric rabbit/human Fab libraries, characterized in terms of specificity, affinity, and amino acid sequence, and finally converted to chimeric rabbit/human IgG. Using immunofluorescence microscopy and immunoprecipitation, we demonstrate strong and specific recognition of cell surface bound Nogo-66 receptor family members by chimeric rabbit/human IgG. The rabbit mAbs reported here together with their amino acid sequences constitute a defined panel of species cross-reactive reagents in infinite supply which will aid investigations toward a functional role of the Nogo-66 receptor family in and beyond the CNS.

Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

“…After several washes with distilled water, the wells were blocked with 200 AL 3% (w/v) bovine serum albumin in PBS for 2 h at 37jC. To generate a standard curve, a recombinant fusion protein consisting of the extracellular region of human ROR1 (amino acids 24-403) fused to the COOH terminus of the Fc fragment of human IgG1 (Fc-ROR1) was cloned into mammalian expression vector pCEP4 (Invitrogen), expressed in transiently transfected HEK 293F cells, and purified by Protein A affinity chromatography as described (27). Two-fold serial dilutions of Fc-ROR1 were made in duplicate from 500 to 7.8 ng/mL.…”

The Nogo-66 Receptor NgR1 Is Required Only for the Acute Growth Cone-Collapsing But Not the Chronic Growth-Inhibitory Actions of Myelin Inhibitors

“…Neurite length of eGFP-positive neurons on feeder cells was quantified as described above. NgR1 expression in eGFP-positive neurons was monitored by anti-NgR1 immunofluorescence (Hofer et al, 2007). As a comparison, we also assayed for MAG-mediated neurite outgrowth inhibition of cortical neurons obtained from three litters of E18.5 mouse embryos, resulting in a total of wild-type (n ϭ 12), NgR1 ϩ /Ϫ (n ϭ 12), and NgR1 Ϫ/Ϫ (n ϭ 13) cultures.…”

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display

“…(Note: The antisense primers for V and V H align to J and J H germlines, respectively, whereas the antisense primer for V aligns to the C encoding sequence). Human C -pelB and C -pelB encoding sequences required for the V -C -V H and V -C -V H cassette assembly, respectively, were amplified from pC 37 and pC 36 . V -C -V H and V -C -V H cassettes were assembled in one fusion step based on 3-fragment overlap extension PCR, digested with SfiI, and cloned into pC3C as described.…”

“…V -C -V H and V -C -V H cassettes were assembled in one fusion step based on 3-fragment overlap extension PCR, digested with SfiI, and cloned into pC3C as described. 37 Transformation of Escherichia coli strain XL1-Blue (Stratagene) by electroporation yielded 9.8 ϫ 10 7 and 1.6 ϫ 10 8 independent transformants for the and phagemid libraries, respectively. Randomly picked independent transformants from each library were analyzed for Fab expression by enzyme-linked immunoabsorbent assay (ELISA) and for sequence diversity by DNA fingerprinting by using AluI as described.…”

“…37 With the use of 293fectin, 300 g of PIGG-JML-1 plasmid was transiently transfected into 3 ϫ 10 8 HEK 293F cells and kept in 300 mL of FreeStyle serum-free medium in a 500-mL spinner flask on a stirring platform at 75 rpm (CELLSPIN System; Integra) in a humidified atmosphere containing 8% CO 2 at 37°C. After 4 days, the medium was collected after centrifugation, replaced for an additional 3 to 4 days, and collected again.…”