Rabbit Immune Repertoires as Sources for Therapeutic Monoclonal Antibodies: The Impact of Kappa Allotype-correlated Variation in Cysteine Content on Antibody Libraries Selected by Phage Display

Popkov, Mikhail; Mage, Rose G.; Alexander, Cornelius B.; Thundivalappil, Sujatha; Barbas, Carlos F.; Rader, Christoph

doi:10.1016/s0022-2836(02)01232-9

Cited by 95 publications

(110 citation statements)

References 35 publications

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“…Certain proteomically identified V H genes (2, 5, 7) did not yield specific binders by phage panning. This is not surprising, because it is well established that recombinant rabbit antibody fragments are particularly difficult to express in bacteria (27). Panning phage display of libraries using synthetic V H genes from the BSA library also showed significant enrichment after two rounds.…”

Section: Resultsmentioning

confidence: 83%

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Wine

Boutz

Lavinder

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

128

156

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We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V H CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigenspecific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V H clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.antibody proteomics | antibody repertoire | serum immunoprofiling | B-cell response | humoral response T he first Nobel Prize in Medicine was awarded to Emil von Behring, who in collaboration with Kitasato Shibasaburo and Paul Ehrlich discovered serum antitoxins (1, 2). Remarkably, after more than 100 y of intense research in immunology, little is known about the clonality, relative concentrations, and binding properties of the monoclonal antibodies that constitute the antigen-specific Ig pool in serum.At steady state, circulating antibodies are produced by terminally differentiated B lymphocytes (plasma cells) within the bone marrow, and thus cannot be accessed in living individuals (3). Although recent single B-cell cloning methods (4, 5) have led to the identification of peripheral antigen-specific B memory and/or antibody-secreting cells (plasmablasts), it is generally unknown whether the Igs encoded by peripheral blood B cells correspond to the antibodies present in circulation and especially whether they are present at physiologically relevant levels (i.e., at serum concentrations above K D corresponding to >1 μg/mL for an average affinity of individual antibodies of 5 nM)...

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Section: Resultsmentioning

confidence: 83%

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Wine

Boutz

Lavinder

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

128

156

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“…Consequently, rabbit mAbs generated by phage display have become promising reagents for therapeutic applications in humans. Based on this consideration, we previously compared antibody repertoires from rabbits with different immunoglobulin allotypes and identified the b9 allotype as superior for the generation and selection of chimeric rabbit/human Fab libraries by phage display (Popkov et al, 2003). Chimeric rabbit/human Fab are composed of rabbit variable domains and human constant domains (Fig.…”

Section: Introductionmentioning

confidence: 99%

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

Höfer

Tangkeangsirisin

Kennedy

et al. 2007

Journal of Immunological Methods

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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.

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“…The selection and purification of Fab 2S08b with specificity for Tie-2 was described in ref. 26. In ELISA, Fab VR05 bound to human and mouse VEGF-R2 and did not react with mouse or human VEGF-R1.…”

Section: Chimeric Rabbit͞human Fabs Vr05 and 2s08b Bind To Human Andmentioning

confidence: 94%

“…Specific oligonucleotide primers (26,29,30) were used to amplify heavy chain variable domain and light chain variable domain gene segments from purified phagemid DNA of Fab VR05 and Fab 2S08b. Light chain variable domain segments of VR05 and 2S08b were amplified with ompseqgtg and RKB9Jo-BL.…”

Section: Analysis Of Vegf-r2 and Tie-2 Binding In Elisamentioning

confidence: 99%

“…In addition, Fab VR05 was able to block VEGF binding to human and mouse VEGF-R2 (27). Fab 2S08b bound to human and mouse Tie-2 and did not react with human Tie-1 (26). Flow cytometry analysis revealed that the VEGF-R2 epitope, recognized by VR05, is displayed on the surface of human (HUVEC) and mouse (MS1) cells and is an accessible target for antiangiogenic therapy.…”

Section: Chimeric Rabbit͞human Fabs Vr05 and 2s08b Bind To Human Andmentioning

confidence: 99%

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Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesisin vivo

Jendreyko

Popkov

Rader

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The endothelial cell receptor-tyrosine kinases, VEGF receptor 2 (VEGF-R2) and Tie-2, and their ligands, vascular endothelial growth factor (VEGF) and angiopoietins 1 and 2, respectively, play key roles in tumor angiogenesis. Several studies suggest that the VEGF receptor pathway and the Tie-2 pathway are independent and essential mediators of angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects on tumor growth. In this study, a human melanoma xenograft model (M21) was used to analyze the effects of simultaneous intradiabody depletion of vascular endothelial growth receptor-R2 and Tie-2 on tumor angiogenesis and tumor xenograft growth. The intradiabodies were expressed from recombinant adenovirus delivered through subtumoral injection. Blockade of both VEGF-R2 and Tie-2 pathways simultaneously or the VEGF receptor pathway alone resulted in a significant inhibition of tumor growth and tumor angiogenesis (92.2% and 74.4%, respectively). In addition, immunohistochemical staining of intradiabody-treated tumors demonstrated a decreased number of tumor-associated blood vessels versus control treatment. Previous studies with intrabodies had demonstrated that the Tie-2 receptor pathway was essential for tumor growth. The simultaneous blockade of the VEGF and Tie-2 pathways resulted in effective inhibition of tumor growth and demonstrated the potential of simultaneous targeting of multiple pathways as a therapeutic strategy

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Rabbit Immune Repertoires as Sources for Therapeutic Monoclonal Antibodies: The Impact of Kappa Allotype-correlated Variation in Cysteine Content on Antibody Libraries Selected by Phage Display

Cited by 95 publications

References 35 publications

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

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

Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesisin vivo

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