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ScFv Generation Services

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All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

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Creative Biolabs offers customers superior approaches for scFv generation such as the one-stop solution for developing CAR from Hybridoma or Phage Display, which manifest as mature and delicate technologies for scFv construction. Particularly, a CAR-T Library Technology on our platform makes great contribution to the selection of desired scFv, whose expression and function are correlated with T cell activation activity.

Basically, a variable region (Fv) consists of a heavy chain variable region (VH) and a light chain variable region (VL), while a single-chain variable fragment (scFv) normally consists of a VH and a VL connected together via a peptide linker. As the most critical initial step, the generation of specific scFv and optimization of its superior affinity lay down the most basic foundation of an effective and specific CAR-T therapy. Without a doubt, a superior scFv leads to an even more superior CAR-T therapy. Creative Biolabs manifests as a world leading expert in antibody engineering field and provides our clients with the best scFv with the most superior affinity in order to meet their research needs. Currently, we offer the two strategies to generate scFv.

scFv Generation Solutions

A. Generate scFv from Hybridoma Cell Lines

Fig.1 scFv production service from Hybridoma. (Creative Biolabs Original) Fig.1 Flowchart of scFv generation from Hybridoma.

  1. Immunize mice with the antigen, and then remove the spleen to get splenocytes (plasma B cells);
  2. Fuse the acquired splenocytes with the cultured myeloma cells to establish hybridoma cell lines following by the cell limited dilution and affinity screen;
  3. Convert the Fv from the discovered full monoclonal antibody into a scFv, which can target the tumor antigen our customer interested in;
  4. Clone the superior scFv with CAR components to construct a Lentivirus vector for the future steps.

B. Generate scFv from Phage Display

At Creative Biolabs, the scFv Phage Display is established originally with an immunized phage display scFv library against the interested antigen. Creative Biolabs guarantees the scFv phage library to achieve a capacity of over 1×1010 clones. The detailed procedures are depicted as the following:

  1. Design the suitable primers for cloning the antibody genes from B lymphocytes of several different species, followed by integrating the antibody genes into a phage display vector;
  2. Transform the recombinant phagemid population into Escherichia coli to produce phage-displayed scFv depending on the helper phage;
  3. Screen the scFv phage display library with the specific antigen from our customers or the recombinant antigen prepared at Creative Biolabs;
  4. Sequence the identified scFv and optimize the affinity with multiple parameters.

Fig.1 scFv production service from Phage Display. (Creative Biolabs Original) Fig.2 Flowchart of scFv generation from Phage Display.

In addition, Creative Biolabs also provides optional molecular characterization service during scFv generation, such as western blot, ELISA or other customized requests. Of note, Our world-leading CAR-T library technology make it possible to screen high-affinity and functional CARs based on T cell display and T cell activation. With the aid of this powerful technology, the selected stable clones can be applied in clinic trials immediately. Several CAR-T libraries against CD19, Her2, Her3, EGFR, FGFR1, VEGFR, etc are available to use.

Case Studies

Case Study 1

Background

Hemophilia A is a genetic disorder resulting from a deficiency in FVIII, leading to impaired blood clotting. Standard treatment involves FVIII replacement therapy, but many patients develop inhibitory antibodies against the therapeutic protein, complicating treatment. This study aimed to engineer Tregs to suppress the immune response against FVIII, thereby preventing the formation of inhibitory antibodies and enhancing the efficacy of FVIII replacement therapy.

Methodology

The study employed the following methodology to engineer antigen-specific Tregs:

1. scFv Generation:

  • Creative Biolabs generated scFvs that were highly specific to the immunogenic C2 domain of the FVIII protein.
  • These scFvs were designed with high affinity to ensure precise targeting of FVIII by the engineered Tregs.

2. CAR Treg Engineering:

  • The scFvs provided by Creative Biolabs were incorporated into CAR constructs, which included murine CD28 and CD3ζ signaling domains.
  • These CAR constructs were used to transduce Tregs, creating FVIII-specific CAR Tregs capable of targeting FVIII.

3. In Vivo and In Vitro Experiments:

  • The engineered CAR Tregs were tested in vitro for their ability to bind and respond to FVIII.
  • In vivo experiments involved adoptive transfer of the CAR Tregs into murine models of hemophilia A to evaluate their ability to suppress the formation of inhibitory antibodies against FVIII.

Results

The scFvs generated by Creative Biolabs were instrumental in achieving several key outcomes:

1. Specific Targeting:

  • The high-affinity scFvs ensured that the CAR Tregs specifically targeted and responded to FVIII, confirming the precision and effectiveness of the scFv generation process.

2. Functional Treg Activation:

  • The CAR Tregs, equipped with scFvs from Creative Biolabs, exhibited robust activation markers and proliferated in response to FVIII stimulation in vitro.
  • Despite the initial robust activation, further refinements were needed to maintain the suppressive function of these Tregs.

3. Immune Response Modulation:

  • In vivo, the CAR Tregs initially showed promise in suppressing FVIII-specific immune responses, although some challenges were encountered in maintaining long-term tolerance.
  • The study also explored alternative constructs like TCR fusion constructs (TRuCs) to achieve more controlled signaling and better suppression of the immune response.

Conclusion

The study provided valuable insights into the differential impacts of CAR and TRuC constructs on Treg function. It demonstrated that while CAR Tregs exhibited robust activation, they lost suppressive functionality and gained an effector phenotype. In contrast, TRuC Tregs, which incorporated the FVIII-specific scFvs into the TCR complex, showed controlled signaling and maintained their suppressive capacity. These findings suggest that TRuC constructs may offer a more effective approach for engineering Tregs to induce immune tolerance in hemophilia A patients. The results highlight the importance of modulating Treg activation thresholds to preserve their suppressive functions and prevent unwanted immune responses.

Role of Creative Biolabs

Creative Biolabs provided crucial scFv generation services, which were foundational to the study. These high-affinity scFvs were designed to specifically target the FVIII protein. The expertise of Creative Biolabs in generating these scFvs enabled the researchers to develop effective CAR constructs for the Tregs.

Highlights & Advantages

  • Comprehensive Screening and Optimization
    Offers extensive screening processes to ensure the highest quality scFvs, including further optimization and cDNA synthesis.
  • Extensive Molecular Characterization
    Optional services such as Western blot, ELISA, and other customized requests to thoroughly characterize and validate scFv.
  • High-Affinity scFv Production
    Commitment to generating scFvs with superior affinity, laying the foundation for effective and specific CAR-T therapies, which are crucial for the success of immunotherapy.
  • Versatile CAR-T Libraries
    Available libraries against multiple targets like CD19, Her2, Her3, EGFR, FGFR1, and VEGFR, ensuring versatile and targeted immunotherapy options.
  • Professional Expertise
    Years of experience in antibody engineering and scFv generation, guaranteeing high-quality and reliable outcomes for research and clinical applications.
  • End-to-End Solutions
    Offers comprehensive and end-to-end solutions for scFv generation, from initial immunization to the final optimization and application, ensuring seamless and efficient service delivery.

With professional service and various technology platforms at Creative Biolabs, we offer the best screening and optimization for superior affinity. Meanwhile, the customs sequences of scFv are also welcomed for further optimization, cDNA synthesis, and subsequent service. Our years of experience guarantee we can offer you our high-quality scFv generation. Creative Biolabs offers you world-class scFv construction services to greatly accelerate your research progress with much lower unpredictable risk. Please do not hesitate to contact us.

Fig.3 One-stop CAR-T therapy development process.

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