|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
GENE THERAPY
From St Jude Children's Research Hospital, Memphis,
TN; University of Tennessee School of Medicine, Memphis, TN; George
Washington University, Washington, D.C.; Yale University School of
Medicine, New Haven, CT; and Howard Hughes Medical Institute, New
Haven, CT.
Adoptive immunotherapy using receptor-modified T lymphocytes has
shown promise in preclinical studies for the treatment of infectious
and malignant diseases. These modified T cells express chimeric
receptors that link ligand recognition and signal transduction domains
in a single gene product. Typically, a single chain Fv fragment is
genetically attached to the cytoplasmic domain of the T-cell receptor
(TCR) Cellular immunotherapy using autologous or
host-compatible antigen-specific T lymphocytes has shown significant
potential in the treatment of malignant and infectious
diseases.1-3 As an alternative to using conventional T
lymphocytes, preclinical and limited clinical studies support the
therapeutic use of receptor-modified T lymphocytes. These
receptor-modified cells express single-chain chimeric receptors that
contain both antigen recognition and signal transduction
domains.4-8 Most frequently, antigen recognition occurs
through single-chain Fv (scFv) fragments, although other recognition
domains may be used.9 Ligand engagement stimulates the T
lymphocyte through the receptor's integrated signal transduction domain, most commonly the cytoplasmic regions of the T-cell receptor (TCR) Despite their therapeutic potential, there have been only limited
efforts to develop modified receptors with improved signal transduction
characteristics. Because of data showing that the limited TCR signaling
domains present within chimeric receptors are inadequate to fully
activate T lymphocytes, such efforts may be essential to develop
clinically effective receptors.10 Although the chimeric
receptors serve as surrogates for the TCR, they differ from native TCRs
in important aspects. Only a restricted subset of the TCR's signal
transduction domains are present in the chimeric receptor. The TCR
signals through the cytoplasmic domains of the invariant An alternative is to qualitatively modify the functional
characteristics of chimeric receptors. The interaction of
receptor-modified T lymphocytes with their targets is fundamentally
different from that of a TCR with major histocompatibility complex
(MHC)-peptide. When TCR interacts with MHC-peptide, the CD4 or CD8
coreceptors are recruited to the signaling complex via their
interactions with class II or class I MHC ligand.13 These
coreceptors escort the src kinase lck to the TCR, promoting
phosphorylation of the TCR's ITAMs as well as other components of the
multiprotein signaling complex that forms around the
TCR.14 The presence of coreceptor is estimated to enhance
T-cell sensitivity to antigen by a factor of 100. When chimeric
receptors on receptor-modified T lymphocytes encounter MHC unrestricted
ligands, coreceptor will not colocalize with receptor. Indeed,
crosslinking a chimeric receptor in which the IAs
class II MHC is linked to the We hypothesized that incorporating coreceptor activity may increase the
sensitivity and potency of chimeric receptors. We therefore developed
chimeric receptors in which the src family kinase lck or the CD4
cytoplasmic tail is directly linked to the cytoplasmic tail of Construct synthesis
Transfections
Antibodies and flow cytometry
Immunoprecipitation and Western blot Chimeric receptor tyrosine phosphorylation was assayed as previously described.20 Briefly, 107 4G4 transfectants were incubated with AF6-88.5 (anti- H-2Kb) supernatant on ice for 20 minutes, washed with phosphate-buffered saline (PBS), and warmed briefly to 37°C prior to the addition of goat antimouse IgG at 37°C. Cells were incubated at 37°C for 2 to 5 minutes then lysed for 0.5 to 1 hour with buffer containing 1% Brij97 (Sigma, St Louis, MO) or 1% NP-40 and protease and phosphatase inhibitors (10 mM Na4P2O7.10H2O, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethanesulfonyl fluoride (PMSF), 10 µg/mL leupeptin, and 10 µg/mL aprotinin). Insoluble material was removed by centrifugation at 15 000g for 15 minutes. Lysate was precleared with protein A-sepharose, and immunoprecipitated with AF6-88.5 anti-Kb, H146 anti-CD3- , or anti-ZAP-70 and
protein A-sepharose (Amersham Pharmacia Biotech). Proteins were eluted
by boiling under reducing conditions in loading buffer, separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
and transferred to nitrocellulose or polyvinylidenefluoride (PVDF)
membrane. Tyrosine phosphorylation was analyzed by Western blotting
with biotinylated or nonbiotinylated 4G10 monoclonal primary antibody
(Upstate Biotechnology, Lake Placid, NY) followed by
streptavidin-horseradish peroxidase (HRP) (Amersham) or goat antimouse
HRP (Biorad, Hercules, CA). Blots were alternatively stained with
anti-CD3- (H146), lck, or ZAP-70 specific antibodies followed by
protein A-HRP or goat antimouse HRP. Detection used enhanced
chemiluminescence (Amersham). When blots were stained with more than
one antibody they were stripped by incubation in 100 mM
2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) at 50°C for
30 minutes prior to reprobing. ZAP-70 and lck blotting was performed
with polyclonal rabbit antisera produced in the laboratory of one of
the authors (D.L.).
Calcium flux We loaded 1.5 × 106 cells with 10 µg indo-1 (Molecular Probes) for 45 minutes at room temperature, washed the cells once and incubated them with 0.5 mL AF6-88.5 (anti-H-2Kb) supernatant at 4°C for 20 minutes. Cells were washed and kept at 4°C until immediately prior to analysis, at which time they were warmed to 37°C. They were then analyzed on a FACS Vantage SE flow cytometer (Becton Dickinson) at a rate of 1000 cells per second. Baseline fluorescence ratio at 530 and 295 nm was measured for 60 seconds, 50 µg goat antimouse IgG was added (Affinipure), and cells were analyzed for 7 additional minutes.Interleukin-2 production We stimulated 105 T cell hybridomas with varying dilutions of AF6-88.5 supernatant loaded onto goat antimouse IgG-coated wells in 96-well plates. After 24 hours, or the designated time after stimulation, supernatant was harvested, and interleukin-2 (IL-2) production was quantitated by bioassay as has been previously described.21 Absolute IL-2 concentrations were determined by interpolation of results with those obtained using dilutions of recombinant IL-2 (R&D Systems).CD69 up-regulation Transduced 4G4 cell lines were incubated with 50 µg/mL ovalbumin 257-264 (SIINFEKL; Hartwell Center for Bioinformatics and Biotechnology, St Jude Children's Research Hospital) for 16 hours, washed 3 times with Hanks balanced salt solution (HBSS), then irradiated with 20 000 rad. A quantity of 5 × 104 of the peptide-pulsed 4G4 cells were added to 4 × 105 splenocytes from OT-1 transgenic mice (Jackson Laboratory, Bar Harbor, ME) and incubated for 20 hours. Cells were stained with CD69- and CD8-specific antibodies in the presence of Fc block (Pharmingen) and analyzed by flow cytometry.Intracellular cytokine staining We added 5 × 104 transduced 4G4 cells to 106 OT-1 transgenic lymph node cells in the presence of 50 µg/mL ovalbumin 257-264 peptide and 10 µg/mL Brefeldin A (Epicenter Biotechnologies). After 6 hours incubation at 37°C, cells were fixed with 1% paraformaldehyde (Ted Pella) for 15 minutes at room temperature, permeabilized with 0.5% saponin (Sigma) and stained with phycoerythrin-conjugated anti-IL-2 (Pharmingen) for 30 minutes at 4°C. Cells were washed and analyzed by flow cytometry.
Expression of chimeric receptor constructs The chimeric constructs and their constituent components are shown in Figure 1. Each receptor includes the same extracellular and transmembrane domains derived from the murine H-2 Kb molecule and signaling domains derived from the cytoplamsic domains of, CD4, CD28, and/or lck. Constructs incorporating lck include all
catalytic and regulatory domains of lck except the membrane proximal
region, which is involved in lck fatty acylation and membrane
association. Receptors were subcloned into the pJZ442 Moloney
murine leukemia virus (MMLV)-driven retroviral vector upstream from an
internal ribosomal entry site linked to green fluorescent protein
(GFP). Retroviral supernatant was used to transduce the murine
surface-TCR-deficient T cell hybridoma 4G4.18,19 Transduced cells were anlayzed by flow cytometry with
H-2Kb-specific antibody. Good correlation between GFP
levels and H-2Kb expression was observed in most primary
transductants. However, receptors containing CD4 distal to the cytoplasmic domain (Kb--CD4) failed to express surface
receptor, even in the presence of high levels of GFP (Figure
2). Similarly, receptors expressing the
CD28 tail distal to showed only low levels of surface receptor (Kb--CD28). Unlike the placement of the CD4 or CD28 tail
distal to , placement of these domains proximal to the chain and
immediately distal to the transmembrane domain resulted in good surface
expression. Transductants were sorted for expression of GFP and
H-2Kb (Figure 2). Western blot analysis of receptors
immunoprecipitated with -specific antibody confirmed the predicted
receptor sizes (data not shown).
Biochemical responsiveness of chimeric receptors To determine whether provision of costimulatory or coreceptor function could enhance chimeric receptor signaling, basal and activation-induced tyrosine phosphorylation were analyzed. Phosphorylation patterns differ with the different receptors (Figure 3A). The Kb-,
Kb-CD4-, and Kb-CD28- receptors show no
significant basal phosphorylation but substantial stimulation-induced
phosphorylation. When normalized to total chain present, the
stimulation-induced phosphorylation of the Kb-CD4- and
Kb-CD28- receptors are modestly increased relative to
that of the Kb- receptor. This demonstrates that
coreceptor or costimulatory domains can modulate receptor
sensitivity.
Tyrosine phosphorylation of the receptors including the src kinase lck
cannot be compared with those receptors lacking lck. First, lck
contains tyrosine phosphorylation sites that cannot be distinguished
from sites within Whereas receptors including lck would be expected to enhance receptor
phosphorylation directly through the enzymatic activity of the lck, the
CD4 tail has no intrinsic kinase activity. The CD4 component of the
Kb-CD4- Weak lck association is also observed after stimulation of receptors
including the CD28 costimulatory tail (Kb-CD28- Subsequent to receptor crosslinking, among the earliest detectable
events in TCR signal transduction is phosphorylation and activation of
the To correlate receptor phosphorylation with downstream signaling events,
the T-cell lines were loaded with the calcium-binding fluorochrome
indo-1 and stimulation-induced calcium flux was measured by flow
cytometry. Figure 4 demonstrates that
total calcium flux is similar in cells expressing the
Kb-
Stimulation of the Kb-CD28- In contrast to cells expressing chimeric receptors containing
coreceptor or costimulatory functions independently, cells including both of these functions (Kb-CD28- Functional responsiveness of chimeric receptors To analyze the influence of the inclusion of coreceptor and costimulatory domains in chimeric receptors on T-cell function, IL-2 production was measured after receptor stimulation with plate-bound anti-H-2Kb. Figure 5 demonstrates that the addition of either CD28-tail, CD4-tail, or lck to the chain reduced by approximately 2- to 4-fold the antibody
concentration required to stimulate equivalent IL-2 production.
Furthermore, cells expressing the Kb-CD28-,
Kb-CD4-, or Kb--lck receptors produced
consistently greater total quantities of IL-2 at optimal stimulation
conditions compared with those expressing the Kb-
receptor. Including both coreceptor and costimulatory functions in a
single-chain chimeric receptor had a more substantial effect. Peak IL-2
production by cells expressing the Kb-CD28--lck
receptor was approximately 2- to 3-fold greater than those expressing
the Kb- receptor. Further, these cells showed an 8- to
12-fold increase in sensitivity based on antibody titration. Therefore,
coreceptor and costimulatory activities synergize when linked with ,
resulting in a single-chain chimeric receptor superior at transducing
stimulatory signals and inducing functional response compared with
receptors including these functions independently.
Chimeric receptor response to low-affinity ligand The above data involve receptor crosslinking with a high-affinity antibody ligand. This would be anticipated to mimic the interaction that occurs with chimeric receptors containing scFv receptor domains. However, this high-affinity interaction may not reflect all types of chimeric receptors and ligands. Chimeric receptors containing scTCR or MHC ligand recognition domains would be expected to have low affinities for ligand. Response to low-affinity ligand will be necessary if the Kb-containing receptors described here are to target alloreactive T lymphocytes. To assess for response after low-affinity engagement of the chimeric receptors OT-1 TCR transgenic T cells specific for an H-2Kb-restricted ovalbumin peptide were used to stimulate the transduced 4G4 cell lines. After binding of antigenic peptide to the extracellular (H-2Kb) domain of the chimeric receptor, the OT-1 TCR can engage and stimulate the chimeric receptor-ovalbumin peptide complex. The dissociation constant (Kd) of the transgenic TCR for this peptide-MHC is approximately 13 µM, 100- to 1000-fold lower than a typical antibody-antigen interaction.25 Only a subset of chimeric receptor molecules would be anticipated to incorporate the ovalbumin peptide, further limiting the avidity of the TCR-chimeric receptor interaction in this analysis.Initial studies measuring IL-2 production after coincubation of
peptide-pulsed 4G4 cells with OT-1 T cells demonstrated significant background due to the production of IL-2 by the OT-1 T lymphocytes. Intracytoplasmic cytokine staining was therefore used to distinguish IL-2 production by stimulator and responder cells. At analysis, transduced 4G4 cells were segregated by flow cytometry from transgenic T cells by their expression of GFP and by cell size. Response to
stimulation of chimeric receptors with anti-H-2Kb antibody
was also assayed to provide correlation with the IL-2 production study
shown in Figure 5. Figure 6A shows that
anti-H-2Kb antibody stimulates IL-2 production in a
fraction of receptor-modified 4G4 cells. The hierarchy of this
production is similar though distinct from that observed in Figure 5.
Whereas Figure 5 shows that cells expressing receptors with coreceptor
function (Kb-
Stimulation of the transduced 4G4 cells with OT-1 T cells shows a
similar pattern of responsiveness as anti-Kb stimulation.
All cell lines capable of signaling in response to antibody
crosslinking were capable of responding to low-affinity TCR and
peptide-mediated stimulation. The Kb-CD28- One caveat to this analysis is that different chimeric receptors may present the ovalbumin peptide and engage the OT-1 cells with variable efficiency. To control for this, after engaging the 4G4 cell lines, OT-1 T cells were stained for upregulation of the CD69 activation marker. OT-1 T-cell activation would indicate that an adequate density of chimeric receptor-peptide ligand is present on the 4G4 cells for effective ligand-receptor engagement. When each of the different 4G4 T-cell lines were pulsed with peptide and then coincubated with OT-1 T cells, more than 90% of OT-1 T cells up-regulated the early T-cell activation marker CD69, showing that antigen was effectively presented by all the cell lines (Figure 6B).
Current understanding of T-cell signal transduction would suggest
that the limited TCR signal transduction domains most commonly used in
single-chain chimeric receptors of receptor-modified T lymphocytes will
not provide an optimal signal. The requirements for effective signal
transduction by Sufficient induction of src kinase activity is important for the initiation of T-cell receptor signal transduction and may affect the balance between positive and negative regulatory elements. Mice deficient in the src kinase lck show severely impaired signaling through the TCR.27 Likewise, T-cell signaling is dramatically reduced in the absence of coreceptor, which ushers lck to the TCR after TCR engagement.13 Agonist ligands in the presence of coreceptor may act as antagonists in its absence.28,29 Partial phosphorylation of TCR ITAMs, possibly reflecting inadequate src kinase activity, has been associated with negative signaling.30 It would be expected that inadequate stimulation through chimeric receptors may likewise generate downmodulatory signals into receptor-modified T cells. Costimulatory function is likewise crucial for adequate T-cell activation. CD28 is the primary costimulatory receptor. It promotes T-cell proliferation and cytokine production, and inhibits apoptosis.31 Costimulatory stimulation also prevents the induction of anergy and significantly diminishes the TCR signal intensity required to activate T lymphocytes. We compared 9 related single-chain chimeric receptors transfected into
the 4G4 T cell hybridoma. 4G4 lacks surface TCR as a result of the
absence of expressed TCR Several conclusions can be made (Table
1). Most significantly, these studies
demonstrate that coreceptor and costimulatory activities are both
important for chimeric receptor signal transduction and that receptors
can be designed that incorporate these activities. Linking lck, the
tail of CD4, or the tail of CD28 to
There is significant overlap between the signaling pathways used
by the TCR and CD28. Because these receptors are functionally complementary, their signaling mechanisms may be partially distinct. Nevertheless, whether CD28 provides a qualitatively distinct signal from that transmitted by the TCR or merely provides a quantitative enhancement is not established. If signaling mechanisms are distinct then receptors with integrated coreceptor and costimulatory activities would be expected to synergize, enhancing cytokine production through
at least partially nonredundant pathways. This expectation is supported
by our finding that a receptor including TCR, coreceptor, and
costimulatory domains (Kb-CD28- It is of interest that T-cell signaling subunits can be linked in a modular fashion. There are limits, however, in receptor engineering. Thus we found that placement of CD28 or CD4-tail distal to the membrane resulted in significantly diminished or abolished receptor expression (Figure 2). A similar result with CD28-tail was observed by others.16 Although we have not examined the mechanism, it likely results from clathrin-dependent receptor uptake and degradation as a result of exposure of dileucine motifs in the receptor tails. Further studies on the mechanism of chimeric receptor internalization may permit formation of receptors with increased stability and surface expression. These studies describe novel chimeric receptors with potential therapeutic application. Figure 6 demonstrates that recognition of the extracellular class I MHC domain by antigen-specific T cells activates the genetically modified T cell. It is anticipated that similar recognition of alloreactive T cells will occur, permitting genetically modified T cells expressing these receptors to target allo-MHC-specific T cells involved in graft rejection or graft-versus-host disease. However, validation of the clinical usefulness of these receptors will require additional analyses. It is possible that some receptors uniting multiple components of the TCR signal transduction machinery may be too sensitive, resulting in unacceptable levels of nonspecific stimulation. Different therapeutic applications and therapeutic cell types may require distinct receptor types depending on the receptor affinity for ligand, ligand density, presence of costimulatory and adhesion molecules on target cells, and desired sensitivity thresholds. The results presented, however, demonstrate that by varying the constituents of chimeric receptors using a modular design it will be possible to synthesize single-chain receptors with predetermined sensitivity, potency, and function for cellular immunotherapy.
We thank Dario Vignali for helpful discussions; Richard Cross, Dick Ashmun, and Mahnaz Paktinat for their assistance with flow cytometric sorting and analysis; and Dan Dan Lu for assistance with Western blot analyses.
Submitted March 26, 2001; accepted June 7, 2001.
Supported by the American Lebanese Syrian Affiliated Charities (T.L.G., P.N.), National Institutes of Health (NIH) grant K08 AI01480 and P30 CA21765 (T.L.G.), NIH grant AI42963 (D.L.), the Arthritis Foundation (D.L.), and the Howard Hughes Medical Institute (R.A.F.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Terrence L. Geiger, Department of Pathology, St Jude Children's Research Hospital, 332 N Lauderdale St, DT-4047E, Memphis, TN 38105; e-mail: terrence.geiger{at}stjude.org.
1.
Bordignon C, Carlo-Stella C, Colombo MP, et al.
Cell therapy: achievements and perspectives.
Haematologica.
1999;84:1110-1149 2. Dazzi F, Goldman J. Donor lymphocyte infusions. Curr Opin Hematol. 1999;6:394-399[CrossRef][Medline] [Order article via Infotrieve]. 3. Riddell SR, Greenberg PD. Principles for adoptive T cell therapy of human viral diseases. Ann Rev Immunol. 1995;13:545-586[CrossRef][Medline] [Order article via Infotrieve].
4.
Mitsuyasu RT, Anton PA, Deeks SG, et al.
Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects.
Blood.
2000;96:785-793 5. Eshhar Z. Tumor-specific T-bodies: towards clinical application. Cancer Immunol Immunother. 1997;45:131-136[CrossRef][Medline] [Order article via Infotrieve]. 6. Abken H, Hombach A, Reinhold U, Ferrone S. Can combined T-cell and antibody-based immunotherapy outsmart tumor cells? Immunol Today. 1998;19:2-5[CrossRef][Medline] [Order article via Infotrieve]. 7. Abken H, Hombach A, Heuser C, Sircar R, Pohl C, Reinhold U. Chimeric T-cell receptors: highly specific tools to target cytotoxic T-lymphocytes to tumour cells. Cancer Treat Rev. 1997;23:97-112[CrossRef][Medline] [Order article via Infotrieve]. 8. Bitton N, Gorochov G, Debre P, Eshhar Z. Gene therapy approaches to HIV-infection: immunological strategies: use of T bodies and universal receptors to redirect cytolytic T-cells. Front Biosci. 1999;4:D386-D393[Medline] [Order article via Infotrieve]. 9. Willemsen RA, Weijtens ME, Ronteltap C, et al. Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR. Gene Ther. 2000;7:1369-1377[CrossRef][Medline] [Order article via Infotrieve].
10.
Brocker T.
Chimeric Fv-zeta or Fv-epsilon receptors are not sufficient to induce activation or cytokine production in peripheral T cells.
Blood.
2000;96:1999-2001 11. Clevers H, Alarcon B, Wileman T, Terhorst C. The T cell receptor/CD3 complex: a dynamic protein ensemble. Ann Rev Immunol. 1988;6:629-662[CrossRef][Medline] [Order article via Infotrieve]. 12. Weiss A. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein tyrosine kinases. Cell. 1993;73:209-212[CrossRef][Medline] [Order article via Infotrieve]. 13. Janeway CJ. The T cell receptor as a multicomponent signalling machine. Ann Rev Immunol. 1992;10:645-676[Medline] [Order article via Infotrieve]. 14. Anderson SJ, Levin SD, Perlmutter RM. Involvement of the protein tyrosine kinase p56lck in T cell signaling and thymocyte development. Adv Immunol. 1994;56:151-178[Medline] [Order article via Infotrieve].
15.
Geiger T, Leitenberg D, Flavell R.
The T-cell-receptor 16. Finney HM, Lawson AD, Bebbington CR, Weir AN. Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product. J Immunol. 1998;161:2791-2797 |
Top
Abstract
Introduction
chain.
, 
,
and 
lck, and Kb-CD28-
and 
chains.