The identification of a novel antibody for CD133 using human antibody phage display

Glumac, Paige M.; Forster, Colleen L.; Zhou, Hong; Murugan, Paari; Gupta, Shilpa; LeBeau, Aaron M.

doi:10.1002/pros.23656

Cited by 10 publications

(12 citation statements)

References 32 publications

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“…This scFv was designated 5G7. Library construction and screening procedures were similar to those reported for other targets [ 9 , 10 ]. The original scFv 5G7 obtained from the screen had the light chain Fv domain C-terminus linked via a 20-residue Gly/Ser-rich linker to the N-terminus of the heavy chain Fv domain.…”

Section: Resultsmentioning

confidence: 99%

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Structural Characterization of a Minimal Antibody against Human APOBEC3B

Tang

Demir

Kurniawan

et al. 2021

Viruses

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APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.

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Section: Resultsmentioning

confidence: 99%

“…A scFv library derived from mouse spleen DNA [ 7 ] was screened against A3Bctd-QMΔloop3 [ 8 ] in the phage display format as described [ 9 , 10 ]. A3Bctd-QMΔloop3 protein was conjugated to biotin (1:2.5 ratio, EZ-Link NHS-PEG4-Biotin, ThermoFisher Scientific, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Structural Characterization of a Minimal Antibody against Human APOBEC3B

Tang

Demir

Kurniawan

et al. 2021

Viruses

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“…All parental cell lines were authenticated by short-tandem repeat profiling prior to manipulation. Parental CWR-R1 and CWR-R1-EnzR cells were lentivirally transduced to express CD133 as described previously (32). Expression of CD133 in transduced cell lines (CWR-R1 CD133 and CWR-R1-EnzR CD133 ) was confirmed via qPCR and Western blot and compared with CD133-negative parental cell lines.…”

Section: Cell Culturementioning

confidence: 99%

“…The heavily glycosylated pentaspan transmembrane protein, CD133, has often been described as an antigen on the surface of both stem cells and cancer stem cells (31). In a previous study, we used human antibody phage display to identify a novel single-chain variable fragment (scFv) antibody for CD133, termed HA10 (32). Herein, we show that CD133 is highly overexpressed at the mRNA and protein level in a multitude of patients possessing AVPC with an ARnegative, neuroendocrine (NE) marker-positive (AR À /NE þ ) phenotype.…”

Section: Introductionmentioning

confidence: 99%

Exploitation of CD133 for the Targeted Imaging of Lethal Prostate Cancer

Glumac

Gallant

Shapovalova

et al. 2020

Clinical Cancer Research

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Purpose: Aggressive variant prostate cancer (AVPC) is a nonandrogen receptor-driven form of disease that arises in men in whom standard-of-care therapies have failed. Therapeutic options for AVPC are limited, and the development of novel therapeutics is significantly hindered by the inability to accurately quantify patient response to therapy by imaging. Imaging modalities that accurately and sensitively detect the bone and visceral metastases associated with AVPC do not exist.Experimental Design: This study investigated the transmembrane protein CD133 as a targetable cell surface antigen in AVPC. We evaluated the expression of CD133 by microarray and IHC analysis. The imaging potential of the CD133-targeted IgG (HA10 IgG) was evaluated in preclinical prostate cancer models using two different imaging modalities: near-infrared and PET imaging.Results: Evaluation of the patient data demonstrated that CD133 is overexpressed in a specific phenotype of AVPC that is androgen receptor indifferent and neuroendocrine differentiated. In addition, HA10 IgG was selective for CD133-expressing tumors in all preclinical imaging studies. PET imaging with [ 89 Zr]Zr-HA10 IgG revealed a mean %ID/g of 24.30 AE 3.19 in CD133positive metastatic lesions as compared with 11.82 AE 0.57 in CD133-negative lesions after 72 hours (P ¼ 0.0069). Ex vivo biodistribution showed similar trends as signals were increased by nearly 3-fold in CD133-positive tumors (P < 0.0001).Conclusions: To our knowledge, this is the first study to define CD133 as a targetable marker of AVPC. Similarly, we have developed a novel imaging agent, which is selective for CD133expressing tumors, resulting in a noninvasive PET imaging approach to more effectively detect and monitor AVPC.

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“…Traditionally, the discovery of mAbs derived from hyperimmunized animal models has relied on immortalization of the antibody‐secreting cell (ASC) via hybridoma technology (Akagi, Nakajima, Tanaka, & Kurihara, ; Basalp & Yucel, ; Y. Chen et al, ; Kohler & Milstein, ; Li et al, ), viral immortalization of the B cell directly (Kwakkenbos et al, ; Traggiai et al, ), or phage and yeast display of antibody fragments derived from cloning of the antigen‐experienced B cell mRNA repertoires (Chan, Lim, MacAry, & Hanson, ; Dong, Bo, Zhang, Feng, & Liu, ; Feldhaus & Siegel, ; Glumac et al, ; Grzeschik et al, ; Scholler, ). Although these methods are well validated for both drug discovery and reagent antibody generation, commercial‐scale application requires significant laboratory infrastructure, large‐scale plate‐based aseptic liquid handling automation platforms, and lengthy workflows dependent on mitotic doubling times to generate antibodies with commercial value.…”

Section: Antibody Discoverymentioning

confidence: 99%

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring

Jorgolli

Nevill²,

Winters

et al. 2019

Biotech & Bioengineering

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The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high‐paced workflows necessary to support modern large molecule drug discovery. A high‐level aspiration is a true integration of “lab‐on‐a‐chip” methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light‐induced electrokinetics with micro‐ and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single‐cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low‐throughput bioprocess workflows in biopharma and life science research.

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The identification of a novel antibody for CD133 using human antibody phage display

Cited by 10 publications

References 32 publications

Structural Characterization of a Minimal Antibody against Human APOBEC3B

Structural Characterization of a Minimal Antibody against Human APOBEC3B

Exploitation of CD133 for the Targeted Imaging of Lethal Prostate Cancer

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring

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