Chimeric antigen receptors (CAR)

1. Chimeric antigen receptors (CAR) 主讲人：徐赞美 指导老师：吴祖泽 段海峰

2. Chimeric antigen receptors (CAR) Chimeric antigen receptors (CAR) are recombinant receptors that provide both antigen-binding and T-cell–activating functions. Chimeric antigen receptors (CAR) are recombinant receptors for antigen, which, in a single molecule, redirect the specificity and function of T lymphocytes and other immune cells

3. contests 1.The history of CARs 2. The structure of CARs 3. CARs ﹠TCRs and BsAb ﹠ CAR 4.Three generations of CARs 5.The transducing of CARs 6.Conclusion & future perspective

4. The history of CARs • The birth of CAR technology occurred 25 years ago when it was shown that antibody variable light (V L ) or heavy (V H ) gene segments can transfer specificity for native antigen. • It was Eshhar who realized the Trans- lational potential of such non-HLA-restricted T cell recognition.

5. The history of T-cell potency in cancer therapy • T cells are the most potent cells of the immune system; however, they fail in the immunosurveillance of tumors. • The discovery of monoclonal antibodies (mAbs) represented a milestone in the history of medicine • previous concepts for improving antibody functional activities, such as drug conjugates and bispecific antibodies (BsAbs), have become a reality and have entered clinical trials. Retargeting T-cell functions with antibody-based approaches the T-cell receptor (TCR) –defined specificity chimeric antigen receptors (CARs).

6. Bispecific antibodies • BsAbs are reagents that combine the specificities of two antibodies in a single molecule. • Using quadroma technology, two different hybridoma cells are fused to raise a cell that can simultaneously produce the two parental heavy (H) and light (L) chains that join spontaneously by Fc pairing forming heterodimers • It should also be pointed out that the effector activity of nonlymphoid cells and natural killer (NK) cells has been exploited in retargeting approaches

7. (A) IgG-based BsAbs • At present, at least 40 different ways to generate BsAbs have been described (B) FAb-based BsAbs. (C) Fv-based BsAbs (D) scFv-based BsAbs with a protein spacer into the linker (E) Variants of scFv–Fc

8. Main structure of chimeric antigen receptors

9. The Antigen targeting domain • The spacer/hinge domain • The transmembrane domain • The signalling domain Antigen targeting by CAR molecules most commonlyinvolves the use of scFv. However, several alternative targetingmoieties may also serve this purpose. These include ligands, peptides, chimeric ligands, receptor derivatives and single domain antibodies some reports have suggested that different hinge regions might critically control surface expression levels, construct stability and antigen binding affinity, which directly influence the efficiency of CAR-redirected effector functions the targeting and signaling properties of CARs, focusing on their effects on T-cell specificity, potency, and safety.

10. A direct comparison of the advantages andlimitations of BsAb and CAR approaches

11. CARs and TCRs have their respective advantages and disadvantages • Although the flexibility and “dynamic range” of CARs is attractive, current CARs are limited to recognizing cell surface antigens • CARs, however, do not require antigen processing and presentation by HLA and are therefore more broadly applicable to HLA-diverse patient populations • In this regard, CARs provide a broader range of functional effects than transduced T-cell receptors (TCR), wherein strength of signaling, which is for the most part determined by the TCR’s affinity for antigen, is the principal determinant of T-cell fate. • CARs ﹠TCRs more secure , better targeted , more persistent

12. Three generations of CARs first-generation CARs: including activating receptors such as CD8/CD3-ζ fusion receptors and T-bodies ; second-generation CARs: providing dual signaling to direct combined activating and costimulatory signals; third-generation CARs :comprising more complex structures with 3 or more signaling domains. mAb, monoclonal antibody.

13. The first generation CARs • most commonly employ either intracellular sequences derived from CD3ζ or from the γ subunit of the high affinity receptor for IgE, FcεR1. • It has been demonstrated that the cellular response of CAR-redirected T cells against antigen-expressing cells depends on several interacting factors, such as expression of the CAR on the effector cell, kinetics of binding of scFv, antigen density and accessibility of the epitope on the target cell • The major limit of first-generation CARs is the poor persistence of the gene in modified T cells in vivo

14. The second generation CARs • costimulation (signal 2) was supplied by modified CAR constructs in which the affinity/binding domain was directly fused to CD28 • In addition to CD28, other costimulatory molecules, such as CD137 (4-1BB) and CD27, have been studied • the key advantage conferred by the use of second generation CARs was the induction of IL-2 secretion and T cell proliferation upon CAR cross-linking.

15. The third generation of CARs • A third generation of CARs in which a second costimulatory molecule is fused intra cellularly with the costimulatory signals, therefore, generating triple-signaling CARs, is under development • Third-generation CARs seem to have improved proliferation, cytokine secretion and a better persistence in circulation • Unfortunately, this last generation of CARs may also be dangerous and the activation can be too strong leading to cytokine storm and eventually to death

16. The transducing of CARs • There are different methods to transduce effector cells with CARs • the first is using viral vectors such as retroviruses and lentiviruses, which both stably integrate the CAR sequence into the host cell genome and allow permanent expression of these molecules • Other methods that allow expression of CARs are based on transposons or RNA transfection.

17. Conclusion & future perspective • Antibody-based T-cell retargeting has several important advantages compared with T-cell-based therapies; • in particular the joining of two functionalities in a unique molecule enables a HLA-independent recognition • However, clinical use of CARs and BsAbs is still limited by the potential immunogenicity of the antibody and possible off-target toxicity

18. CARs, having T-cell killer characteristics, are very powerful and for this reason should be directed only toward cancer cells. A good on-target and a reduction of adverse off-target effects. • the clinical safety of CARs has not yet bee demonstrated • optimization of engineering processes and cell manufacturing, expansion and persistence of effector cells still needs further improvement. • Successful clinical applications for CARs can emerge only after these problems have been solved.

19. The end !