High efficiency CHO cell display-based antibody maturation.
Ontology highlight
ABSTRACT:
SUBMITTER: Luo R
PROVIDER: S-EPMC7229201 | biostudies-literature | 2020 May
REPOSITORIES: biostudies-literature
Publications
High efficiency CHO cell display-based antibody maturation.
Luo Ruiqi R Zhao Yun Y Fan Yingjun Y An Lili L Jiang Tao T Ma Shaohua S Hang Haiying H
Scientific reports 20200515 1
Previously, we developed a CHO cell display-based antibody maturation procedure in which an antibody (or other protein) gene of interest was induced to mutate by activation-induced cytidine deaminase (AID) and then form a library by simply proliferating the CHO cells in culture. In this study, we further improved the efficiency of this maturation system by reengineering AID, and optimizing the nucleic acid sequence of the target antibody gene and AID gene as well as the protocol for AID gene tra ...[more]
Similar Datasets
Project description:Antibody stability and affinity are two important features of its applications in therapy and diagnosis. Antibody display technologies such as yeast and bacterial displays have been successfully used for improving both affinity and stability. Although mammalian cell display has also been utilized for maturing antibody affinity, it has not been applied for improving antibody stability. Previously, we developed a Chinese hamster ovary (CHO) cell display platform in which activation-induced cytidine deaminase (AID) was used to induce antibody mutation, and antibody affinity was successfully matured using the platform. In the current study, we developed thermo-resistant (TR) CHO cells for the purpose of maturing both antibody stability and affinity. We cultured TR CHO cells displaying an antibody mutant library and labeled them at temperatures above 41 °C, enriching cells that displayed antibody mutants with both the highest affinities and the highest display levels. To evaluate our system, we chose three antibodies to improve their affinities and stabilities. We succeeded in simultaneously improving both affinities and stabilities of all three antibodies. Of note, we obtained an anti-TNFα antibody mutant with a Tm (dissolution temperature) value 12 °C higher and affinity 160-fold greater than the parent antibody after two rounds of cell proliferation and flow cytometric sorting. By using CHO cells with its advantages in protein folding, post-translational modifications, and code usage, this procedure is likely to be widely used in maturing antibodies and other proteins in the future.
| S-EPMC9404881 | biostudies-literature
Project description:Affinity maturation, an essential component of antibody engineering, is crucial for developing therapeutic antibodies. Cell display system coupled with somatic hypermutation (SHM) initiated by activation-induced cytidine deaminase (AID) is a commonly used technique for affinity maturation. AID introduces targeted DNA lesions into hotspots of immunoglobulin (Ig) gene loci followed by erroneous DNA repair, leading to biased mutations in the complementary determining regions. However, systems that use an in vivo mimicking mechanism often require several rounds of selection to enrich clones possessing accumulated mutations. We previously described the human ADLib® system, which features autonomous, AID-mediated diversification in Ig gene loci of a chicken B cell line DT40 and streamlines human antibody generation and optimization in one integrated platform. In this study, we further engineered DT40 capable of receiving exogenous antibody genes and examined whether the antibody could be affinity matured. The Ig genes of three representative anti-hVEGF-A antibodies originating from the human ADLib® were introduced; the resulting human IgG1 antibodies had up to 76.4-fold improvement in binding affinities (sub-picomolar KD) within just one round of optimization, owing to efficient accumulation of functional mutations. Moreover, we successfully improved the affinity of a mouse hybridoma-derived anti-hCDCP1 antibody using the engineered DT40, and the observed mutations remained effective in the post-humanized antibody as exhibited by an 8.2-fold increase of in vitro cytotoxicity without compromised physical stability. These results demonstrated the versatility of the novel B cell-based affinity maturation system as an easy-to-use antibody optimization tool regardless of the species of origin.Abbreviations: ADLib®: Autonomously diversifying library, ADLib® KI-AMP: ADLib® knock-in affinity maturation platform, AID: activation-induced cytidine deaminase, CDRs: complementary-determining regions, DIVAC: diversification activator, ECD: extracellular domain, FACS: fluorescence-activated cell sorting, FCM: flow cytometry, HC: heavy chainIg: immunoglobulin, LC: light chain, NGS: next-generation sequencing, PBD: pyrrolobenzodiazepine, SHM: somatic hypermutation, SPR: surface plasmon resonance.
| S-EPMC9542628 | biostudies-literature
Project description:Molecular evolution is an important step in the development of therapeutic antibodies. However, the current method of affinity maturation is overly costly and labor-intensive because of the repetitive mutation experiments needed to adequately explore sequence space. Here, we employed a long short term memory network (LSTM)-a widely used deep generative model-based sequence generation and prioritization procedure to efficiently discover antibody sequences with higher affinity. We applied our method to the affinity maturation of antibodies against kynurenine, which is a metabolite related to the niacin synthesis pathway. Kynurenine binding sequences were enriched through phage display panning using a kynurenine-binding oriented human synthetic Fab library. We defined binding antibodies using a sequence repertoire from the NGS data to train the LSTM model. We confirmed that likelihood of generated sequences from a trained LSTM correlated well with binding affinity. The affinity of generated sequences are over 1800-fold higher than that of the parental clone. Moreover, compared to frequency based screening using the same dataset, our machine learning approach generated sequences with greater affinity.
| S-EPMC7955064 | biostudies-literature
Project description:Mammalian display enables the selection of biophysically favorable antibodies from a large IgG antibody library displayed on the plasma membrane of mammalian cells. We constructed and validated a novel mammalian display platform utilizing the commercially available Flp-In CHO cell line as a starting point. We introduced a single copy of a landing pad for Bxb1 integrase-driven recombinase-mediated cassette exchange into the FRT site of the Flp-In CHO line to facilitate the efficient single-copy integration of an antibody display cassette into the genome of the cell line. We then proceeded to demonstrate the ability of our platform to select biophysically favorable antibodies from a library of 1 × 106 displayed antibodies designed to improve the biophysical properties of bococizumab via randomization of problematic hydrophobic surface residues of the antibody. Enrichment of bococizumab variants via fluorescence-activated cell sorting selections was followed by next generation sequencing and thorough characterization of biophysical properties of 10 bococizumab variants that subsequently allowed attribution of the mutations to the biophysical properties of the antibody variants. The mammalian displayed variants exhibited reduced aggregation propensity and polyreactivity, while critically retaining its target binding thereby demonstrating the utility of this valuable tool.
| S-EPMC10203562 | biostudies-literature
Project description:Discovery of monoclonal antibodies is most commonly performed using phage or yeast display but mammalian cells are used for production because of the complex antibody structure, including the multiple disulfide bonds and glycosylation, required for function. As this transition between host organisms is often accompanied by impaired binding, folding or expression, development pipelines include laborious plate-based screening or engineering strategies to adapt an antibody to mammalian expression. To circumvent these problems, we developed a plasmid-based Fab screening platform on Chinese hamster ovary (CHO) cells which allows for antibody selection in the production host and in the presence of the same post-translational modifications as the manufactured product. A hu4D5 variant with low affinity for the human epidermal growth factor receptor (HER2) growth factor receptor was mutagenized and this library of ~10(6) unique clones was screened to identify variants with up to 400-fold enhanced HER2 binding. After two rounds of fluorescence activated cell sorting (FACS), four unique clones exhibited improved antigen binding when expressed on the CHO surface or as purified human IgG. Three of the four clones contained free cysteines in third complementarity determining region of the antibody heavy chain, which did not impair expression or cause aggregation. The improved clones had similar yields and stabilities as hu4D5 and similar sub-nanomolar affinities as measured by equilibrium binding to target cells. The limited size of mammalian libraries restricts the utility of this approach for naïve antibody library screening, but it is a powerful approach for antibody affinity maturation or specificity enhancement and is readily generalizable to engineering other surface receptors, including T-cell receptors and chimeric antigen receptors.
| S-EPMC5892863 | biostudies-other
Project description:The induction of somatic hypermutation (SHM) in various cell lines by activation-induced cytidine deaminase (AID) has been used in protein-directed selection, especially in antibody affinity maturation. Several antibody affinity maturation systems based on mammalian cells have been developed in recent years, i.e., 293T, H1299, Raji and CHO cells. However, the efficiency of in vitro AID-induced hypermutation is low, restricting the application of such systems. In this study, we examined the role of Ig and Ek enhancers in enhancing SHM in the episomal vector pCEP4 that expresses an anti-high mobility group box 1 (HMGB1) full-length antibody. The plasmid containing the two enhancers exhibited two-fold improvement of mutation rate over pCEP4 in an AID expression H1299 cell line (H1299-AID). With the engineered episomal vector, we improved the affinity of this antibody in H1299-AID cells by 20-fold.
| S-EPMC6698961 | biostudies-literature
Project description:Improving the efficiency of solar cells through novel materials and devices is critical to realize the full potential of solar energy to meet the growing worldwide energy demands. We present here a highly efficient radial p-n junction silicon solar cell using an asymmetric nanowire structure with a shorter bottom core diameter than at the top. A maximum short circuit current density of 27.5 mA/cm(2) and an efficiency of 7.53% were realized without anti-reflection coating. Changing the silicon nanowire (SiNW) structure from conventional symmetric to asymmetric nature improves the efficiency due to increased short circuit current density. From numerical simulation and measurement of the optical characteristics, the total reflection on the sidewalls is seen to increase the light trapping path and charge carrier generation in the radial junction of the asymmetric SiNW, yielding high external quantum efficiency and short circuit current density. The proposed asymmetric structure has great potential to effectively improve the efficiency of the SiNW solar cells.
| S-EPMC4495391 | biostudies-other
Project description:Antigen preparations in the form of detergent-solubilized cell lysates could, in principle, render membrane proteins (MPs) compatible with in vitro antibody engineering technologies. To this end, detergent-solubilized cell lysates were coupled with the yeast surface display platform to affinity mature an anti-transferrin receptor (TfR) single-chain antibody (scFv). Lysates were generated from TfR-expressing HEK293 cells by solubilization with detergent-containing buffer after undergoing plasma membrane-restricted biotinylation. Lysate-resident TfR was then combined with a mutagenic anti-TfR scFv library in a competitive, dissociation rate screen, and scFvs were identified with up to 4-fold improved dissociation rates on the surface of yeast. Importantly, although the lysates contained a complex mixture of biotinylated proteins, the engineered scFvs retained their TfR binding specificity. When secreted by yeast as soluble proteins, mutant scFvs bound to cell surface TfR with 3-7-fold improvements in equilibrium binding affinity. Although a known MP antigen was targeted for purposes of this study, employing biotin tagging as a means of antigen detection makes the lysate-based approach particularly flexible. We have previously shown that yeast display can be used to identify lead antibodies using cell lysate-resident MP antigens, and combined with this work showing that antibodies can also be quantitatively engineered using cell lysates, these approaches may provide a high-throughput platform for generation and optimization of antibodies against MPs.
| S-EPMC3542525 | biostudies-literature
Project description:The elicitation of broadly and efficiently neutralizing antibodies in humans by active immunization is still a major obstacle in the development of vaccines against pathogens such as the human immunodeficiency virus (HIV), influenza virus, hepatitis C virus or cytomegalovirus. Here, we describe a mammalian cell surface display and monoclonal antibody (mAb)-mediated panning technology that allows affinity-based selection of envelope (Env) variants from libraries. To this end, we established an experimental setup featuring: 1) single and site specific integration of Env to link genotype and phenotype, 2) inducible Env expression to avoid cytotoxicity effects, 3) translational coupling of Env and enhanced green fluorescent protein expression to normalize for Env protein levels, and 4) display on HEK cells to ensure native folding and mammalian glycosylation. For proof of concept, we applied our method to a chimeric HIV-1 Env model library comprising variants with differential binding affinities to the V3-loop-directed mAbs 447-52D and HGN194. Fluorescence-activated cell sorting selectively enriched a high affinity variant up to 56- and 55-fold for 447-52D and HGN194, respectively, after only a single round of panning. Similarly, the low affinity variants for each antibody could be selectively enriched up to 237-fold. The binding profiles of membrane-bound gp145 and soluble gp140 chimeras showed identical affinity ranking, suggesting that the technology can guide the identification of Env variants with optimized antigenic properties for subsequent use as vaccine candidates. Finally, our mAb-based cellular display and selection strategy may also prove useful for the development of prophylactic vaccines against pathogens other than HIV.
| S-EPMC5627598 | biostudies-literature
Project description:Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, thus necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments. This platform enabled isolation of antibody fragments with improved digoxigenin binding and modest affinity maturation in several different ncAA contexts. Azide-functionalized fragments exhibited improved binding kinetics relative to their methionine counterparts, facile chemical modification through azide-alkyne cycloaddition, and retention of binding properties after modification. The results described here suggest new possibilities for protein engineering, including modulation of molecular recognition events by ncAAs and direct screening of libraries of chemically modified proteins.
| S-EPMC4199383 | biostudies-literature