Chimeric antigen receptors (CARs) are recombinant receptors consisting of an extracellular antigen-recognition domain—usually a single-chain variable fragment (scFv) from a monoclonal antibody (mAb)—linked to intracellular signaling and costimulatory domains from CD3ζ and CD28/4-1BB, respectively.
The scFvs are composed of a heavy-chain variable fragment connected to a light-chain variable fragment by a flexible linker. They are typically reformatted from a full-length Ig, with the linker optimized to preserve heavy- and light-chain variable region pairing.
CAR-T are the result of a transduced T lymphocyte with a ScFv fragment antibody able to recognize specific cancer antigens.
However, scFvs can present several drawbacks:
ü they do not always fold efficiently and can be prone to aggregation,
ü murine ScFv may induce immunogenicity,
ü they can’t access all the different epitopes, since scFv present flat or concave topologies paratopes.
In contrast, the variable regions of camelid-derived single-domain antibody fragments (VHHs or nanobodies):
ü are small, stable, with affinities comparable to traditional scFvs,
ü are less immunogenic than murine scFv,
ü can access epitopes different from those seen by scFvs, since they offer convex topology associated with smaller size,
ü do not require the additional folding and assembly steps that come with V-region pairing (in scFv libraries, natural VH–VL pairings are usually lost).
VHHs could therefore serve as suitable antigen recognition domains in CAR T cells, and several potentially interesting VHHs have been tested since the beginning of 2019.
These new constructs are called CAR-VHH-T and may be a potential future of CAR adoptive cell therapy.
mAbexperts is now able to provide a one one-stop-shop toolbox for cancer treatment and proceed as follow :
1. RD Biotech (https://www.rd-biotech.com) starts with the antigen coding sequence and the expression of recombinant cancer antigen in order to specifically generate the adequate camelid heavy chain-only antibodies (HCAbs) against it.
2. After immunization in llama and/or alpaca with this specific antigen, QVQ (http://www.qvquality.com) generates phage-display libraries from the immunized camelids. They generate a lead panel of monoclonal sdAbs binding with high affinity to the tumor associated antigens.
3. Following the collection of a patient's T cells by leukapheresis at Blood Transfusion Center in Besançon, the cells are genetically engineered by RD-Biotech through inactivated lentivirus particles containing the coding sequence for the VHH.
4. To ensure that the CAR-VHH-T is well activated, Diaclone (https://www.diaclone.com) launches its specific interferon gamma ELISpot test assay attesting that the CAR-VHH-T combines the specificity of an antibody for CD proteins, with the cytotoxic and memory functions of T cells.
5. Drawing on its expertise in secondary monoclonal antibodies, SYnAbs (https://www.synabs.be) has already generated an universal rat monoclonal antibody against the VHH construct. Thanks to this new available tool, mAbexperts’ partners are now able to detect and select specific transfected CAR-VHH-T populations on flow cytometry platform !
We could add that VHHs are also well suited in the generation of bi- and multispecific antibodies. Bispecific antibodies provide a possible solution in which they could bind simultaneously two or more tumor epitopes (bi-paratopic) or antigens simultaneously. Can we conclude that we should expect new developments in the near future?
Business to be continued...