Development of an efficient method for selection of stable cell pools for protein expression and surface display with Expi293F cells

Chen, Chuan; Wang, Zening; Sun, Zehua; Li, Wei; Dimitrov, Dimiter S.

doi:10.1002/cbf.3787

Cited by 3 publications

(2 citation statements)

References 45 publications

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“…For ALK-Fc and VH20-TCE expression, pZeo-ALK ecto-Fc and pZeo-VH20-OKT3-Fc plasmid were transfected into Expi293F cell line. The stable cell pool selection process followed the description in our previously published manuscript [ 26 ].…”

Section: Methodsmentioning

confidence: 99%

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy

Chen,

Sun,

Wang

et al. 2024

Antibodies

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The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

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Section: Methodsmentioning

confidence: 99%

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy

Chen,

Sun,

Wang

et al. 2024

Antibodies

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“…For scale production of CPB cells for ISKNV and SCRV vaccines, we developed a suspension culture system of CPB cells on cytodex 1 microcarrier in 3-L bioreactor [ 18 ]. The stability, growth rate, and virus sensitivity of cells directly influence vaccine production scales, production costs, and competitiveness [ 19 , 20 , 21 ]. At present, CPB cells below 120 passages are prone to mutation, apoptosis, and are not stable.…”

Section: Introductionmentioning

confidence: 99%

The Stability and Efficency of CPB Cells Were Acclimated for Virus Proliferation

Niu,

Ma,

Liang

et al. 2024

Vaccines

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Background: Vaccinations are still the most effective means of preventing and controlling fish viral diseases, and cells are an important substrate for the production of a viral vaccine. Therefore, the rapid-stable growth and virus sensitivity of cells are urgently needed. Methods: Chinese perch brain 100th passage (CPB p100) were acclimated in a low serum with 5% FBS L-15 for 50 passages, then transferred to 8% FBS L-15 for 150 passages. Additionally, the morphology and cell type of CPB 300th passage (CPB p300) cells were identified. We analyzed the transfection efficiency and virus sensitivity of CPB p300 cells, and then optimized the conditions of ISKNV, SCRV, and LMBV multiplication in CPB cells. Results: CPB p300 cells were more homogeneous, and the spread diameter (20–30) µm in CPB p300 cells became the dominant population. The doubling time of CPB p300 was 1.5 times shorter than that of CPB p100.However, multiplication rate of CPB p300 was 1.37 times higher than CPB p100. CPB p300 cells were susceptible to ISKNV, SCRV, and LMBV, and the optimal conditions of ISKNV, SCRV, and LMBV multiplication were simultaneous incubation, 0.6 × 105 cells/cm2 and MOI = 0.1; infection at 48 h, 0.8 × 105 cells/cm2 and MOI = 0.01; simultaneous incubation, 0.7 × 105 cells/cm2 and MOI = 0.05, respectively. The time and economic costs of ISKNV, SCRV, and LMBV multiplication in CPB p300 cells were significantly reduced. Conclusions: The acquisition of CPB p300 cells laid a good material foundation for the production of ISKNV, SCRV, and LMBV vaccines.

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Fc engineering by monoclonal mammalian cell display for improved affinity and selectivity towards FcγRs

Wang,

Kang,

Ebrahimpour

et al. 2024

Antibody Therapeutics

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Fc optimization can significantly enhance therapeutic efficacy of monoclonal antibodies. However, existing Fc engineering approaches are sub-optimal with noted limitations, such as inappropriate glycosylation, polyclonal libraries, and utilizing fragment but not full-length IgG display. Applying cell cycle arrested recombinase-mediated cassette exchange, this study constructed high-quality monoclonal Fc libraries in CHO cells, displayed full-length IgG on cell surface, and preformed ratiometric fluorescence activated cell sorting (FACS) with the antigen and individual FcγRs. Identified Fc variants were quantitatively evaluated by flow cytometry, ELISA, kinetic and steady-state binding affinity measurements, and cytotoxicity assays. An error-prone Fc library focusing on the hinge-CH2 region was constructed in CHO cells with a functional diversity of 7.5 × 106. Panels of novel Fc variants with enhanced affinity and selectivity for FcγRs were isolated. Particularly, clone 2a-10 (G236E/K288R/K290W/K320M) showed increased binding strength towards FcγRIIa-131R and 131H allotypes with kinetic dissociation constants (KD-K) of 140 nM and 220 nM, respectively, while reduced binding strength towards FcγRIIb compared to WT Fc; clone 2b-1 (K222I/V302E/L328F/K334E) had KD-K of 180 nM towards FcγRIIb; clone 3a-2 (P247L/K248E/K334I) exhibited KD-K of 190 nM and 100 nM towards FcγRIIIa-176F and 176 V allotypes, respectively, and improved potency of 2.0 ng/ml in ADCC assays. Key mutation hotspots were identified, including P247 for FcγRIIIa, K290 for FcγRIIa, and K334 for FcγRIIb bindings. Discovery of Fc variants with enhanced affinity and selectivity towards individual FcγR and the identification of novel mutation hotspots provide valuable insights for further Fc optimization and serve as a foundation for advancing antibody therapeutics development.

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Development of an efficient method for selection of stable cell pools for protein expression and surface display with Expi293F cells

Cited by 3 publications

References 45 publications

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy

The Stability and Efficency of CPB Cells Were Acclimated for Virus Proliferation

Fc engineering by monoclonal mammalian cell display for improved affinity and selectivity towards FcγRs

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