of the Max - MDC

Molecular Cell Biology
and Gene Therapy
Wolfgang Uckert
Antigen specificity of T cells is determined by their T cell receptor (TCR) and can be redirected by the transfer of
TCRα/β genes. TCR gene-modified T cells can elicit effector functions against any target antigen. The adoptive
transfer of TCR-redirected T cells into cancer patients has already led to first clinical success.
The focus of our group lies on the adoptive transfer of TCR-redirected T cells for the therapy of cancer and viral
diseases. We address questions related to: (i) generation of T cells with new antigen specificity by TCR gene transfer,
(ii) safety aspects of TCR-redirected T cells with respect to the recognition of self-antigens, (iii) adoptive transfer of
TCR-redirected T cells in mice as a prerequisite for the application of those cells in human diseases, and (iv) optimization
of TCR transfer vectors.
Redirection of T cell antigen specificity by TCR
gene transfer
Boris Engels, Haike Gelfort, Matthias Leisegang, Daniel
Sommermeyer, Lilian Stärck in collaboration with Helga
Bernhard, Antonio Pezzutto, Dolores Schendel
Using RACE-PCR we have molecularly cloned genes of
α/βTCRs recognizing tumor-associated antigens (HER2,
NY-ESO-1, RCC 53, WT-1, Melan-A), virus-specific antigens
(EBV-EBNA3a, EBV-LMP2a), and model antigens (C2, LCMVgp33,
ovalbumin). Using retrovirus vectors, TCR genes were
transferred into T cell lines and primary T cells of mouse and
human origin, respectively. TCRα- and β-chains were
expressed on the cell surface and functionality of transgenic
TCRs was demonstrated by antigen recognition, cytokine
secretion and tumor cell lysis.
To increase the avidity of TCR-redirected T cells, we introduced
different modifications into the TCR genes (codon
optimization, additional disulfide bond, murinization of constant
regions of human TCR chains) and demonstrated exemplary
for the NY-ESO-1 TCR that antigen recognition can be
considerably improved by TCR modification (Figure 1).
Designer T cells by TCR replacement
Daniel Sommermeyer in collaboration with Helga
Bernhard, Thomas Blankenstein
The expression of two TCRs in T cells (one endogenous and
one transgenic) could impair their function or cause
unwanted effects e.g. by the formation of mixed TCR heterodimers.
Using five different TCRs and four different T
cells, either mouse or human, we showed that some TCRs
are strong – in terms of cell surface expression – and
replace weak TCRs on the cell surface, resulting in exchange
of antigen specificity. Two strong TCRs are co-expressed. A
mouse TCR replaces human TCRs on human T cells. Even
though it is poorly understood why some TCRα/β combinations
are preferentially expressed on T cells, these data suggest
that designer T cells with exclusive tumor reactivity can
be generated by T cell engineering.
A new safeguard eliminates TCR gene-modified
auto-reactive T cells after adoptive transfer
Eliesa Kieback in collaboration with Jehad Charo, Thomas
Blankenstein
By transfer of TCRα/β genes, antigen specificity of T cells
can be redirected to target any antigen. However, this
immunotherapy bears the risk of auto-reactive side-effects
when the TCR recognizes antigens on self-tissue. We introduced
a new safeguard that is based on a TCR intrinsic
depletion mechanism to eliminate auto-reactive TCR-redirected
T cells. By introducing a 10 amino acid tag of the c-
myc protein into murine (OT-I, P14) and human (gp100) TCR
sequences, tag-modified TCRs maintained equal properties
compared to the wild-type receptor concerning antigen
binding and triggering of effector function. Tag-modified
TCR-redirected T cells could be depleted in vivo with a tagspecific
antibody by antibody-mediated complement lysis
and antibody-dependent cell-mediated cytotoxicity. More
important, in vivo depletion of adoptively transferred T cells
rescued mice from lethal autoimmune diabetes (Figure 2).
This new safeguard allows the termination of adoptive therapy
in case of severe side-effects.
Optimization of retroviral TCR gene transfer
Simone Reuß, Petra Biese, Matthias Leisegang
TCR-redirected T cells are exclusively generated by retrovirus-mediated
gene transfer. Therefore, packaging cell
Cancer Research 137