Transcript Chimeric antigen receptors (CAR)

Chimeric antigen receptors
(CAR)
主讲人：徐赞美
指导老师：吴祖泽
段海峰
Chimeric antigen receptors (CAR)
Chimeric antigen receptors (CAR) are recombinant
receptors that provide both antigen-binding and Tcell–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
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
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 Translational potential of such non-HLArestricted T cell recognition.
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).
• 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
• 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 BsAbs
with a protein spacer
into the linker
(E) Variants of scFv–Fc
Main structure of chimeric antigen receptors
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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.
A direct comparison of the advantages and
limitations of BsAb and CAR approaches
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
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.
• 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
• 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.
• 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 triplesignaling 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
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.
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
• 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.