Generation and Characterization of a Recombinant Human CCR5-specific Antibody

Schmitz, Peter; Sutton, Jorun K.; Rader, Christoph; Elia, Marikka; Barbas, Carlos F.

doi:10.1074/jbc.m002765200

Cited by 63 publications

(41 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…55 The intrabody binding motif partially overlaps with the gp120 binding and fusion motif. [56][57][58][59][60] To further improve cellular efficacy of the intrabody, a KDEL endoplasmic retention signal has been included, which provides entrapment of newly synthesized and recycled CCR5 as well and maintains cellular compartmentalization of the CCR5 intrabody.…”

Section: Ccr5 Intrabody-mediated Protection and Enrichment Of T Cellsmentioning

confidence: 99%

See 1 more Smart Citation

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

et al. 2006

View full text Add to dashboard Cite

CCR5 is the chemokine co-receptor for R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates most often associated with primary infection. We have developed an HIV-1 self-inactivating vector, CAD-R5, containing a CCR5 single-chain antibody (intrabody) gene, which when expressed in T-cell lines and primary CD4 + T cells disrupts CCR5 cell surface expression and provides protection from R5-tropic isolate exposure. Furthermore, CAD-R5 intrabody expression in primary CD4 + T cells supports significant growth and enrichment over time during HIV-1-pulsed dendritic cell-T-cell interactions. These results indicate that CCR5 intrabody-expressing CD4 + T cells are refractory against this highly efficient primary route of infection. CD34 + cells transduced with the CAD-R5 vector gave rise to CD4 + and CD8 + thymocytes in non-obese diabetic (NOD)/ severely combined-immunodeficient (SCID)-human thymus/ liver (hu thy/liv) mice, suggesting that CCR5 intrabody expression can be maintained throughout differentiation without obvious cellular effects. CD4 + T cells isolated from NOD/SCID-hu thy/liv mice were resistant to R5-tropic HIV-1 challenge demonstrating the maintenance of protection. Our findings demonstrate delivery of anti-HIV-1 activity through CCR5 intrabodies in primary CD4 + T cells and CD34 + cell-derived T-cell progeny. Thus, gene delivery strategies that provide a selective survival and growth advantage for T effector cells may provide a therapeutic benefit for HIV-1-infected individuals who have failed conventional therapies.

show abstract

Section: Ccr5 Intrabody-mediated Protection and Enrichment Of T Cellsmentioning

confidence: 99%

“…81 The humanized CCR5 intrabody, ST6/34, was generated and cloned as described previously. 55 The CAD-R5 CCR5 intrabody vector was constructed from CAD by replacing the BamHI MND promoter fragment with an MND promoter and the intrabody fragment.…”

Section: Generation Of the Cad And Cad-r5 Hiv-1 Sin Vectorsmentioning

confidence: 99%

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

et al. 2006

View full text Add to dashboard Cite

show abstract

“…Hybridoma screening (see under "Materials and Methods") resulted in the selection of seven VEGF neutralizing antibodies (375, 435, 509, 511, 534, 567, and 578) that potently blocked binding of VEGF to hVEGFR2, and eight TNF-␣ neutralizing antibodies (1,6,15,19,34,35,42, and 43) that potently inhibited TNF-␣-induced apoptosis in the murine L929 cell line. Sequence analysis of the rabbit monoclonal antibodies showed that 7 germ lines (out of 65 functional VL-gene segments) were represented in the selected binders (Table 1).…”

Section: Selection Of Rabbit Monoclonal Antibodies From Hybridomas-mentioning

confidence: 99%

Generic Approach for the Generation of Stable Humanized Single-chain Fv Fragments from Rabbit Monoclonal Antibodies

Borras¹,

Gunde²,

Tietz³

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Despite their favorable pharmacokinetic properties, singlechain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 ؋ 10 ؊9 to 1.5 ؋ 10Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 ؋ 10 ؊10 to <1.8 ؋ 10 ؊12 M. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86°C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.Because of their favorable pharmacokinetic properties, single-chain Fv (scFv) 3 antibody fragments represent an attractive format for therapeutic applications (1, 2). scFvs are often derived from monoclonal antibodies isolated from animal or human lymphocytes. As an alternative to hybridoma screening, in vitro display technologies, e.g. phage and ribosome display, enable the selection of high affinity-binding variable domains from natural or synthetic genetic libraries. Despite the successful use of in vitro randomization and selection systems, generation of antibodies by immunization and subsequent screening of full-size antibodies (e.g. hybridoma supernatants) includes conceptual advantages. For example, in contrast to in vitro display systems, in vivo methods are less prone to preferential selection of well expressed clones, which in many cases results in loss of potentially interesting antibodies. Moreover, in vivo methods are preferred in particular for addressing complex antigens, such as integral membrane proteins that are notoriously difficult to purify. However, reducing a full-length monoclonal antibody to the scFv format frequently is challenging particularly due to solubility and stability problems, which often impair expression and purification. Therefore, technologies to humanize and stabilize the scFv format following isolation of a monoclonal antibody remain critical for the generation of scFv therapeutics. Numerous approaches have been describ...

show abstract

“…Rabbit mAbs generated by phage display offer additional advantages due to the fact that phenotype and genotype are selected at the same time. Knowledge of the rabbit mAb sequence allows the ready generation of a variety of mAb formats, including scFv, Fab, and IgG, and, importantly, humanization and affinity maturation Steinberger et al, 2000;Rader, 2001). Consequently, rabbit mAbs generated by phage display have become promising reagents for therapeutic applications in humans.…”

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Generation and Characterization of a Recombinant Human CCR5-specific Antibody

Cited by 63 publications

References 20 publications

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

Generic Approach for the Generation of Stable Humanized Single-chain Fv Fragments from Rabbit Monoclonal Antibodies

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

Contact Info

Product

Resources

About