Case Studies

Discover cutting-edge advancements in biotechnology with Eurofins CALIXAR, where innovation meets precision in small molecule screening, antibody discovery, bioassay development, structure determination, vaccine discovery, and protein identification. Our unique and custom-built approach to research and development sets us apart, providing solutions that redefine industry standards.

Small molecule screening

Adenosine receptor A2A: a unique & custom-built approach

Eurofins CALIXAR’s Adenosine receptor A2A facilitates reliable fragment-based drug design (FBDD), structure-based drug discovery (SBDD) and antibody discovery corresponding to this specific target.

Unlike alternative methods that result in mutated and truncated versions of the Adenosine receptor A2A, Eurofins CALIXAR's approach highlights the preservation of the receptor's functional integrity. With conventional methods, the Adenosine receptor A2A, like all GPCRs, is inherently unstable and challenging to produce in a native state. Previous approaches could only lock the receptor in a specific conformation, leading to a solution that was impure, non-native (truncated and mutated), and lacking post-translational modifications. However, Eurofins CALIXAR has overcome this challenge with native full-length A2AR.

Our native A2AR were screened with 200k compounds for agonistic and antagonistic effect by Affinity Selection - Mass Spectrometry (AS-MS) and Saturation Transfer Difference - Nuclear Magnetic Resonance (STD-NMR). Epitope mapping also showed its potential binding sites.  

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References

• Enabling STD-NMR fragment screening using stabilized native GPCR : A case study of adenosine receptor - Igonet S et al. 2018
• Novel systematic detergent screening method for membrane proteins solubilization. - Desuzinges Mandon E. et al. 2017

Antibody Discovery

Purified, stabilized, and native full-length proteins without mutation and truncation play a crucial role in antibody development by serving as high-quality antigens. Their high quality ensures that the antibodies generated in response to these antigens have high specificity and affinity for the target protein, which is essential for the accuracy and reliability of subsequent antibody-based assays and applications. Besides, as antigens for immunization, the full-length and unmodified structure of the protein ensures that the antibodies recognize the natural conformation and epitopes present in the native protein. These antibodies are instrumental in various research, diagnostic, and therapeutic applications, making them essential tools in the field of life sciences and biotechnology.

Evaluated immunogenicity with purified A2AR protein

Purified A2AR protein or a receptor, can be used as antigens to initiate the generation of antibodies, which are specific to A2AR by animal immunization. Eurofins CALIXAR’s Adenosine receptor A2A, with maintained native structure, is able to bind to agonists, antagonists, as well as allosteric modulators. Wild-type, full-length A2AR  in CALX condition were evaluated with immunization of 3 mice using ELISA assay. Our purified A2AR exhibits a higher immunogenicity than mutated stabilized protein, which has 8 mutations and C-ter truncation. 

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Antibody identification with Human NaV1.7 

As an ion channel protein, Human NaV1.7 involves the expression and purification of this protein for various research and potential therapeutic purposes. This protein could be used as an antigen to immunize animals (e.g., mice or rabbits) for antibody generation, especially therapeutic antibodies designed to target Nav1.7. The purified Nav1.7 protein is also used as an immunogen to initiate an immune response in animals. 

Human NaV1.7 is purified and stabilized in native full length protein, which is non-aggregated and homogenous. Its functionality in Cy5-ProTx-II (Smartox) characterization is also determined by fluorescence polarization. Cy5-ProTx-II is a fluorescently labeled ProTx-II, a famous Nav1.7 blocker. A specific binding of Cy5-ProTX-II is observed with native Nav1.7 and abolished with denatured Nav1.7. 

Unveiling CD20's Potential for Enhanced Antibody Development

CD20, a pivotal membrane protein on B-lymphocytes, is a crucial target for monoclonal antibodies treating various diseases. This study focuses on the extraction and purification of native CD20 from different cell lines.

A cutting-edge Eurofins CALIXARene-based detergent approach improves CD20 protein yield, ensuring non-aggregated, concentration-dependent oligomerization. While specific structural insights remain elusive, purified CD20 forms confined nanodroplets that don't aggregate. Notably, purified CD20 binds rituximab and obinutuzumab, offering prospects for advanced antibody research, with potential applications for other challenging membrane proteins.

References

• Enabling STD-NMR fragment screening using stabilized native GPCR : A case study of adenosine receptor - Igonet S et al. 2018

• Novel systematic detergent screening method for membrane proteins solubilization. - Desuzinges Mandon E. et al. 2017

• Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab - Morgane A. et al. 2019

Bioassay Development

Elevating Bioassay Potential: The Role of CD20

CD20, a membrane protein prominently found on B-lymphocytes, is a vital target for monoclonal antibodies in treating various diseases. This study focused on the extraction and purification of native CD20 from different cell lines, unlocking its potential for bioassay development.

Leveraging a state-of-the-art Eurofins CALIXARene-based detergent approach, this method boosts CD20 protein yield, resulting in non-aggregated, concentration-dependent oligomerization. Although specific structural details remain elusive, purified CD20 forms confined nanodroplets that resist aggregation. Moreover, the purified CD20 demonstrates its capacity to bind rituximab and obinutuzumab, offering a promising avenue for advanced bioassay research, with the potential to extend to other challenging membrane proteins.

References

Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab - Morgane A. et al. 2019

Structure Determination

Unlock KCC2's Structural Complexity

Understanding the intricate structure of membrane proteins is pivotal, especially in neurological disorders like epilepsy, autism, and schizophrenia. By using innovative Eurofins CALIXARene detergent, researchers successfully solubilized and purified native, unaggregated KCC2, shedding light on its structure. Specific binding with an inhibitor (VU0463271) was confirmed through surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations, aligning with KCC2's functional profile.

Electron microscopy unveiled KCC2's existence as monomers and dimers in solution. Monomers displayed a structured "head" and "core," connected by a flexible "linker." Dimers formed an asymmetrical "S-shape" architecture, with disulfide bridges facilitating dimerization.

Adding a tag to the C-terminus impaired KCC2 function. These findings enhance our knowledge of KCC2's structure and function, offering exciting prospects for research in neurological diseases.

References

• Molecular architecture of potassium chloride co-transporter KCC2  - Morgane A. et al. 2017

• Novel systematic detergent screening method for membrane proteins solubilization. - Desuzinges Mandon E. et al. 2017

Vaccine Discovery

High-quality, purified proteins are pivotal in driving innovation within vaccine development. Whether producing split virus vaccines or working with native pools of antigens, these proteins underpin the structural integrity and efficacy of vaccines. From creating vaccines against influenza, leptospirosis, HIV, and Chikungunya, these stabilized antigens serve as a cornerstone for enhancing immunization strategies and ultimately advancing healthcare.

In the realm of split virus vaccines, the technique of splitting enveloped viruses into native membrane antigens has revolutionized the creation of inactivated viruses, ensuring that the structural integrity of these vital components remains intact. For influenza vaccines, the egg-based production method provides a foundation for vaccine efficacy.

Novel antigen stabilization approach for vaccines

Virus splitting is a key step for the vaccine preparation process that requires detergent use. However, it is challenging to obtain an efficient virus splitting while maintaining immunogenicity using classical detergents. Eurofins CALIXAR's innovative surfactant and detergent-based approach has revolutionized virus splitting and antigen stabilization, ensuring efficient vaccine preparation without compromising immunogenicity. This proprietary method has demonstrated success in low-dose split inactivated vaccine development for influenza and holds promise for other enveloped viruses like Arbovirus (Mandon E et al., Vaccine, 2020) and HIV (Jawhari A et al., World Vaccine Congress, Washington 2017).

Workflow used to generate and analyze fragmentation of purified egg-based NYMC X-179A (A(H1N1)) virus using triton-X100 or CALX-R compound. This includes virus production, virus fragmentation, dialysis/ultrafiltration/formaldehyde inactivation, in vitro hemagglutination (HA) and single radial immunodiffusion assays (SRID), and in vivo validation.

References

Novel Eurofins CALIXARene-based surfactant enables low dose split inactivated vaccine protection against influenza infection - Mandon E et al., Vaccine, 2020

Protein identification

PDAC Target Identification - Enhancing Cancer Research 

High-quality protein is a key in advancing various fields, from drug development to cancer research. In an important case study, our expertise in protein design and production played a pivotal role in identifying critical targets for biological research, especially in the context of pancreatic ductal adenocarcinoma (PDAC). We determined the BAG3 receptor and IFITM-2 as key players in PDAC growth and metastasis, and further developed an anti-BAG3 antibody with promising therapeutic potential, effectively reducing tumor growth and preventing metastasis in various mouse models. This case study exemplifies our commitment to providing innovative protein solutions that drive groundbreaking research and improved cancer treatments.

Our results were further validated through a complementary Co-IP approach involving IFITM2. Moreover, the RNAi approach demonstrated that silencing IFITM2 effectively abrogated BAG3-induced signaling in macrophages, leading to reduced IL-6 release, and decreased AKT and p38 phosphorylation. This study underscores the critical importance of target identification in unraveling the intricate mechanisms of pancreatic cancer growth and metastasis, paving the way for potential therapeutic interventions.

References

BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages - ROSATI E et al., Nature, 2015