Modular peptide binders – development of a predictive technology as alternative for reagent antibodies
Current biomedical research and diagnostics critically depend on detection agents for specific recognition and quantification of protein molecules. Monoclonal antibodies have been used for this purpose over decades and facilitated numerous biological and biomedical investigations. Recently, however, it has become apparent that many commercial reagent antibodies lack specificity or do not recognize their target at all.
Thus, synthetic alternatives are needed whose complex designs are facilitated by multidisciplinary approaches incorporating experimental protein engineering with computational modeling. Here, we review the status of such an engineering endeavor based on the modular armadillo repeat protein scaffold and discuss challenges in its implementation.
Monoclonal Antibodies Generation: Updates and Protocols on Hybridoma Technology
- Since its inception in 1975, the hybridoma technology revolutionized science and medicine, facilitating discoveries in almost any field from the laboratory to the clinic. Many technological advancements have been developed since then, to create these “magical bullets.” Phage and yeast display libraries expressing the variable heavy and light domains of antibodies, single B-cell cloning from immunized animals of different species including humans or in silico approaches, all have rendered a myriad of newly developed antibodies or improved design of existing ones.
- However, still the majority of these antibodies or their recombinant versions are from hybridoma origin, a preferred methodology that trespass species barriers, due to the preservation of the natural functions of immune cells in producing the humoral response: antigen specific immunoglobulins.
- Remarkably, this methodology can be reproduced in small laboratories without the need of sophisticate equipment. In this chapter, we will describe the most recent methods utilized by our Monoclonal Antibodies Core Facility at the University of Texas-M.D. Anderson Cancer Center. During the last 10 years, the methods, techniques, and expertise implemented in our core had generated more than 350 antibodies for various applications.
Novel cell line development strategy for monoclonal antibody manufacturing using translational enhancing technology
Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4.
Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3 g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003 cells were evaluated by fed-batch culture.
The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003 cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5 g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production.
Antibodies Processed Using High Dilution Technology Distantly Change Structural Properties of IFNγ Aqueous Solution
Terahertz spectroscopy allows for the analysis of vibrations corresponding to the large-scale structural movements and collective dynamics of hydrogen-bonded water molecules. Previously, differences had been detected in the emission spectra of interferon-gamma (IFNγ) solutions surrounded by extremely diluted solutions of either IFNγ or antibodies to IFNγ without direct contact compared to a control. Here we aimed to analyse the structural properties of water in a sample of an aqueous solution of IFNγ via terahertz time-domain spectroscopy (THz-TDS). Tubes with the IFNγ solution were immersed in fluidised lactose saturated with test samples (dilutions of antibodies to IFNγ or control) and incubated at 37 °C for 1, 1.5-2, 2.5-3, or 3.5-4 h.
Fluidised lactose was chosen since it is an excipient in the manufacture of drugs based on diluted antibodies to IFNγ. After incubation, spectra were recorded within a wavenumber range of 10 to 110 cm-1 with a resolution of 4 cm-1. Lactose saturated with dilutions of antibodies to IFNγ (incubated for more than 2.5 h) changed the structural properties of an IFNγ aqueous solution without direct contact compared to the control. Terahertz spectra revealed stronger intermolecular hydrogen bonds and an increase in the relaxation time of free and weakly bound water molecules. The methodology developed on the basis of THz-TDS could potentially be applied to quality control of pharmaceuticals based on extremely diluted antibodies.
Fabrication of a Lateral Flow Assay for Rapid In-Field Detection of COVID-19 Antibodies Using Additive Manufacturing Printing Technologies
The development of lateral flow immunoassay (LFIA) using three-dimensional (3D) printing and bioprinting technologies can enhance and accelerate the optimization process of the fabrication. Therefore, the main goal of this study is to investigate methods to speed up the developing process of a LFIA as a tool for community screening.
To achieve this goal, an in-house developed robotic arm and microfluidic pumps were used to print the proteins during the development of the test. 3D printing technologies were used to design and print the housing unit for the testing strip. The proposed design was made by taking into consideration the environmental impact of this disposable medical device.
Anti-ROBO1 Reference Antibody (Asclepius Technology anti-Robo1 CAR) |
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| E24CHA370 | EnoGene | 100 μg | 225 EUR |
D-Dimer (Up-converting Phosphor Technology) |
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| abx095256-80Units | Abbexa | 80 Units | 1579.2 EUR |
Myoglobin (Up-converting Phosphor Technology) |
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| abx095254-80Units | Abbexa | 80 Units | 1144.8 EUR |
Electrophoresis baffle for Life Technology Novex |
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| HTDB02 | Shanghai WSHT Biotechnology | pcs | 2.8 EUR |
Creatine Kinase MB (Up-converting Phosphor Technology) |
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| abx095253-80Units | Abbexa | 80 Units | 1144.8 EUR |
Interleukin-6 (Up-converting Phosphor Technology) |
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| abx095258-80Units | Abbexa | 80 Units | 1914 EUR |
Procalcitonin (Up-converting Phosphor Technology) |
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| abx095257-80Units | Abbexa | 80 Units | 1429.2 EUR |
Hach DR3900 VIS Spectral Photometer With RFID Technology - EACH |
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| WAT3800 | Scientific Laboratory Supplies | EACH | 10044 EUR |
C-Reactive Protein (Up-converting Phosphor Technology) |
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| abx095259-80Units | Abbexa | 80 Units | 1144.8 EUR |
Cardiac Troponin I (cTnI) (Up-converting Phosphor Technology) |
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| abx095252-80Units | Abbexa | 80 Units | 1579.2 EUR |
Heart-fatty acid-binding protein (Up-converting Phosphor Technology) |
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| abx095255-80Units | Abbexa | 80 Units | 1914 EUR |
N-terminal Pro-B-type Natriuretic Peptide (Up-converting Phosphor Technology) |
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| abx095251-80Units | Abbexa | 80 Units | 1579.2 EUR |
Lipoprotein-associated phospholipase A2 (Up-converting Phosphor Technology) |
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| abx095250-80Units | Abbexa | 80 Units | 1863.6 EUR |
Blue screw cap (9mm) (PP: red natural rubber/clear TEF septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK100 |
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| CHSC9-10 | Scientific Laboratory Supplies | PK100 | 17.55 EUR |
Blue screw cap (9mm) (PP: red natural rubber/clear TEF septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK5000 |
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| CHSC9-10F | Scientific Laboratory Supplies | PK5000 | 787.05 EUR |
Blue screw cap (9mm) (PP: red natural rubber/clear TEF septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK1000 |
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| CHSC9-10T | Scientific Laboratory Supplies | PK1000 | 166.05 EUR |
Blue screw cap (9mm) (PP: red silicone/ivory PTFE septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK100 |
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| CHSC9-20 | Scientific Laboratory Supplies | PK100 | 20.25 EUR |
Blue screw cap (9mm) (PP: red silicone/ivory PTFE septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK5000 |
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| CHSC9-20F | Scientific Laboratory Supplies | PK5000 | 907.2 EUR |
Blue screw cap (9mm) (PP: red silicone/ivory PTFE septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK1000 |
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| CHSC9-20T | Scientific Laboratory Supplies | PK1000 | 191.7 EUR |
Blue screw cap (9mm) (PP: white silicone/blue PTFE septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK100 |
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| CHSC9-30 | Scientific Laboratory Supplies | PK100 | 21.6 EUR |
Blue screw cap (9mm) (PP: white silicone/red PTFE septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK5000 |
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| CHSC9-30F | Scientific Laboratory Supplies | PK5000 | 967.95 EUR |
Blue screw cap (9mm) (PP: white silicone/red PTFE septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK1000 |
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| CHSC9-30T | Scientific Laboratory Supplies | PK1000 | 203.85 EUR |
Blue screw cap (9mm) (PP: white silicone/blue PTFE septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK5000 |
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| CHSC9-40F | Scientific Laboratory Supplies | PK5000 | 1451.25 EUR |
Blue screw cap (9mm) (PP: white silicone/blue PTFE septa: 1.0mm thickness) with AVCS technology ; Level 1 Everyday Analysis - PK1000 |
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| CHSC9-40T | Scientific Laboratory Supplies | PK1000 | 306.45 EUR |
ReadiLinkâ„¢ Protein Biotinylation Kit *Powered by ReadiViewâ„¢ Biotin Visionization Technology* |
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| 5521 | AAT Bioquest | 1 kit | 227 EUR |
ReadiLinkâ„¢ Protein Biotinylation Kit *Powered by ReadiViewâ„¢ Biotin Visionization Technology* |
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| 5521-3Reactions | AAT Bioquest | 3 Reactions | 222 EUR |
Convenience kit: 2mL clear screw glassvial (9mm short thread) with SureStop technology and write-on patch; and blue screw cap (9mm) (PP: white silicone/red PTFE septa: 1mm thickness) with AVCS technol - PK100 |
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| 6AK592W | Scientific Laboratory Supplies | PK100 | 60.75 EUR |
Blue screw cap (9mm) (PP: white silicone/blue PTFE septa: 1.0mm thickness) - preslit; with AVCS technology ; Level 1 Everyday Analysis - PK100 |
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| CHSC9-40 | Scientific Laboratory Supplies | PK100 | 32.4 EUR |
Single B cell technologies for monoclonal antibody discovery
Monoclonal antibodies (mAbs) are often selected from antigen-specific single B cells derived from different hosts, which are notably short-lived in ex vivo culture conditions and hence, arduous to interrogate. The development of several new techniques and protocols has facilitated the isolation and retrieval of antibody-coding sequences of antigen-specific B cells by also leveraging miniaturization of reaction volumes.
Alternatively, mAbs can be generated independently of antigen-specific B cells, comprising display technologies and, more recently, artificial intelligence-driven algorithms. Consequently, a considerable variety of techniques are used, raising the demand for better consolidation. In this review, we present and discuss the major techniques available to interrogate antigen-specific single B cells to isolate antigen-specific mAbs, including their main advantages and disadvantages.