Adnectin-Based CAR Design & Construction

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Although the chimeric antigen receptor (CAR) engineered T cell therapy has achieved encouraging clinical trial results for the treatment of hematological cancers, further optimization may extend the success of this treatment to more patients and other cancer types. Currently, most CAR constructs used in clinical trials integrate single-chain variable fragments (scFv) into an extracellular antigen recognition domain. The immunogenicity of non-human scFv may lead to host rejection of CAR-T cells and impair its persistence and efficacy. The limited availability of scFv and the slow discovery of new monoclonal antibodies also limit the development of new CAR constructs.

Creative Biolabs has developed a novel CAR construct - Adnectin-based CAR. Adnectin is a kind of affinity molecule derived from the tenth type III domain of human fibronectin. It can be used as a substitute for scFv and as the antigen binding part in CAR molecule design. Compared with scFv-based CAR, T cells engineered with adnectin-based CAR have the same cell-killing activity against tumor cells in vitro. In addition, through optimal affinity regulation, adnectin-based CARs show higher selectivity for target cells. Therefore, the novel adnectin CARs design can potentially promote the development of T cell immunotherapy against cancer and other diseases.

About Adnectin

Adnectin-Based CAR Design & Construction Adnectin is derived from a single domain scaffold, the tenth type III domain (10Fn3) of human fibronectin. The 10Fn3 domain is a member of the immunoglobulin (Ig) superfamily and contains a beta sandwich protein fold, similar to the Ig variable domain, but without disulfide bonds. Therefore, the 10Fn3 domain can be modified to mimic the binding of scFv to proteins of interest other than its natural target integrin. Through mRNA, phage, or yeast display, adnectins can be effectively adapted to bind to the target of interest with high affinity and specificity.

Adnectins vs scFv

Adnectins usually have a smaller size and a more stable structure than scFv, and are monomers without disulfide bonds, which may reduce the basic level of CAR activation caused by random cross-linking or dimerization of the extracellular domain derived from scFv.
Adnectin is derived from human fibronectin and therefore has minimal immunogenicity. Due to the above characteristics, adnectin has multiple advantages over scFv, and may be suitable as a replacement antigen binding domain of CAR molecules.

Adnectin-Based CAR Design

Adnectin can be used as the antigen recognition domain in CAR constructs instead of scFv. We have designed multiple antigen-specific adnectin-CARs. Firstly, mRNA display is used to generate different adnectin clones, which target the target extracellular domain with different affinities. Then, different clones of target-specific adnectins were cloned upstream of the hinge domain to replace the scFv sequence. The adnectin-based extracellular domain is connected by the CD8α hinge and transmembrane domain, CD28 and 4-1BB costimulatory domains, and CD3ζ T cell receptor signaling domain. It is worth noting that we also provide adnectin-based first-generation and second-generation CARs.

scFv-Based CAR and Adnectin-Based CAR

Fig.2 scFv-Based CAR and Adnectin-Based CAR

One-stop Adnectin-Based CAR Development Services

With state-of-art Smart™ CAR development platform, Creative Biolabs is capable of offering CAR-T-cell development services. For the regular or custom CAR backbone construction, please refer to our related services: CAR Design & Construction. To further assess CAR biological efficacy (e.g., cytokine production, tumor killing, and CAR-T cell proliferation), our scientists can also provide comprehensive downstream services to complete your whole research. Please click: CAR Cell In Vitro Assay Service, CAR-T Preclinical In Vivo Assay for more details.

CAR Biomarker indentification & selection High affinity scFv generation CAR design & construction Virus packaging & CAR transfection CAR in vitro assay CAR Pre-clinical in vivo assay CAR clinical trial

For more detailed information, please feel free to contact us or directly sent us an inquiry.

Reference

Han, X., et al. Adnectin-based design of chimeric antigen receptor for T cell engineering. Molecular Therapy. 2017. 25(11), 2466-2476.