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 thespecificity and function of Tlymphocytes and other immune cells
3 contests 1.The history of CARs 2. The structure of CARs 3. CARs ﹠TCRs and BsAb ﹠ CAR4.Three generations of CARs5.The transducing of CARs6.Conclusion & future perspective
4 The history of CARsThe 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 medicineprevious 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 approachesthe T-cell receptor (TCR) –defined specificitychimeric 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 formingheterodimersIt should also be pointed out that the effector activity of nonlymphoid cells and natural killer (NK) cells has been exploited in retargeting approaches
7 At present, at least 40 different ways to generate BsAbs have been described (A) IgG-based BsAbs(B) FAb-based BsAbs.(C) Fv-based BsAbs(D) scFv-based BsAbswith a protein spacerinto the linker(E) Variants of scFv–Fc
9 The Antigen targeting domain The spacer/hinge domain The transmembrane domainThe signalling domainAntigen 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 antibodiessome reports have suggested that different hinge regions might critically control surface expression levels,construct stability and antigen binding affinity, which directly influencethe efficiency of CAR-redirected effector functionsthe targeting and signaling properties of CARs, focusing on their effects on T-cell specificity, potency, and safety.
10 A direct comparison of the advantages and limitations 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 antigensCARs, however, do not require antigen processing and presentation by HLA and are therefore more broadly applicable to HLA-diverse patient populationsIn 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 ﹠TCRsmore 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 cellThe 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 CD28In addition to CD28, other costimulatory molecules, such as CD137 (4-1BB) and CD27, have been studiedthe 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 developmentThird-generation CARs seem to have improved proliferation, cytokine secretion and a better persistence in circulationUnfortunately, 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 CARsthe first is using viral vectors such as retroviruses and lentiviruses, which both stably integrate the CARsequence into the host cell genome and allow permanent expression of these moleculesOther 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 recognitionHowever, clinical use of CARs and BsAbs is stilllimited by the potential immunogenicity of theantibody 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 demonstratedoptimization of engineering processes and cellmanufacturing, expansion and persistence of effector cells still needs further improvement.Successful clinical applications for CARs can emerge only after these problems have been solved.