Tumors have evolved a mechanism to evade the immune system. Therefore, in order to help our immune systems identify cancerous cells, researchers have discovered a way to recombinantly express specific tumor associated antigenic determinants on the surface of T cells. These tumor associated antigenic determinants are known as CARs, because they are composed of an antigen binding region as well as a T cell signaling domain.
Chimeric Antigen Receptors (CARs) are synthetic recombinant proteins similar to mAb's. Their extracellular domain (ECD) is composed of single chain variable region (ScFv) derived from the heavy and light variable chains of a mAb. The transmembrane domain (TMD) is connected to the extracellular domain by a peptide based spacer region, allowing enhanced CAR flexibility and binding. The TMD is embedded within the T-cell membrane and acts as the main connection between the ECD and intracellular domain (ICD). The ICD is composed of two parts, the costimulatory domain (CSD) and the signaling domain (SD). The CSD is composed of various immune system enhancer proteins which increase the immune effect of the SD. The SD is derived from the T-cell itself, and induces downstream immunological cascades.
Figure 1. Diagram of a CAR. The variable heavy (Fig 1A1) and light (Fig 1A2) chains of an ScFv are connected one another by a linker region (B). The ScFv is connected via a spacer region (C) to the TMD (D). The ICD is then composed of the CSD (E) and the SD (F).
In order to generate a CAR-T cell, a ScFv region is generated from the heavy and light variable regions of a mAb already identified to bind to the TAA of interest. The ScFv region sequence is then cloned into an expression cassette which also contains the sequences for the TMD and ICD of choice. This plasmid is then transfected into a group of T cells in hopes of generating T cells which express the full length CAR (Figure 2). The finalized T cell expressing the TAA ScFV on its cell surface along with the intracellular immune signaling domain is considered a CAR-T cell. Recently, CAR-T cells have been used in therapies to treat various kinds of cancers by using TAA ScFv's to identify and kill cancerous tissue. However, these clinical trials are only the beginning of CAR-T therapy, but researchers are hopeful that many more trials will be carried out in the near future. In order to conduct these clinical trials, researchers have to measure the pharmacokinetics and pharmacodynamics of the CAR-T cells. This can be accomplished through the use of anti-idiotype mAb's.
Figure 2. CAR-T Cell Therapy Generation. In order to generate a CAR-T cell an animal is immunized with a TAA (1A) and the resulting mAb is isolated, purified (2A) and sequenced. Simultaneously, a human T cell (1B) is isolated (2A) and sequenced. Then, the mAb ScFv region sequence (3A) and the TMD/ICD (3B) are cloned into an expression cassette (4). While the plasmid is being generated, a patient (5) will go through T cell isolation (6). The resulting T cells will then be transfected with the CAR expression vector in order to generate patient specific T cells which express a TAA CAR on their cell surface (7). These T cells are then transplanted back into the patient and used as personalized cancer therapy (8).
An anti-idiotype mAb targets the antigen binding site of another antibody. It is primarily used to track the concentration of antibody drugs within a patients serum during clinical trials. The generation of an anti-idiotype mAb against a CAR-T cell would require immunization of the specific ScFv region which targets a TAA (Figure 3). However, the mouse's immune system is not able to generate mAb's which are specific enough to identify the true CAR-T levels in human serum. GenScript's MonoRab™ service can generate mAb's against the most difficult targets, even antigens non-immunogenic in mice. This is because our proprietary hetero-hybridoma methodology includes an optimized fusion partner to improve overall fusion efficiency while utilizing the rabbit's natural ability to generate high specificity, high affinity mAb's against a wide range of targets.
Figure 3. Development of anti-idiotype mAb Against CAR-T. The CAR ScFv region (1) is sequenced (2) and to generate a recombinatly expressed (3) ScFv (4). The recombinant ScFv is then used as an immunogen in GenScripts MonoRab™ service (5) to generate a mature anti-idiotype mAb against the original CAR (6).
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