Requirements for effective antitumor responses of TCR transduced T cells

Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.     

TCR gene therapy of spontaneous prostate carcinoma requires in vivo T cell activation

Analogous to the clinical use of recombinant high-affinity Abs, transfer of TCR genes may be used to create a T cell compartment specific for self-Ags to which the endogenous T cell repertoire is immune tolerant. In this study, we show in a spontaneous prostate carcinoma model that the combination of vaccination with adoptive transfer of small numbers of T cells that are genetically modified with a tumor-specific TCR results in a marked suppression of tumor development, even though both treatments are by themselves without effect. These results demonstrate the value of TCR gene transfer to target otherwise nonimmunogenic tumor-associated self-Ags provided that adoptive transfer occurs under conditions that allow in vivo expansion of the TCR-modified T cells.     

Dual T cell receptor expressing CD8+ T cells with tumor- and self-specificity can inhibit tumor growth without causing severe autoimmunity

The engineering of Ag-specific T cells by expression of TCR genes is a convenient method for adoptive T cell immunotherapy. A potential problem is the TCR gene transfer into self-reactive T cells that survived tolerance mechanisms. We have developed an experimental system with T cells that express two TCRs with defined Ag-specificities, one recognizing a tumor-specific Ag (LCMV-gp(33)), the other recognizing a self-Ag in the pancreas (OVA). By using tumor cells expressing high and low amounts of Ag and mice expressing high and low levels of self-Ag in the pancreas (RIP-OVA-Hi and RIP-OVA-Lo), we show that 1) tumor rejection requires high amount of tumor Ag, 2) severe autoimmunity requires high amount of self-Ag, and 3) if Ag expression on tumor cells is sufficient and low in the pancreas, successful adoptive T cell therapy can be obtained in the absence of severe autoimmunity. These results are shown with T cells from dual TCR transgenic mice or T cells that were redirected by TCR gene transfer. Our data demonstrate that the approach of adoptively transferring TCR redirected T cells can be effective without severe side effects, even when high numbers of T cells with self-reactivity were transferred.     

Memory generation and maintenance of CD8+ T cell function during viral persistence

During infection with viruses that establish latency, the immune system needs to maintain lifelong control of the infectious agent in the presence of persistent Ag. By using a gamma-herpesvirus (gammaHV) infection model, we demonstrate that a small number of virus-specific central-memory CD8+ T cells develop early during infection, and that virus-specific CD8+T cells maintain functional and protective capacities during chronic infection despite low-level Ag persistence. During the primary immune response, we show generation of CD8+ memory T cell precursors expressing lymphoid homing molecules (CCR7, L-selectin) and homeostatic cytokine receptors (IL-7alpha, IL-2/IL-15beta). During long-term persistent infection, central-memory cells constitute 20-50% of the virus-specific CD8+ T cell population and maintain the expression of L-selectin, CCR7, and IL-7R molecules. Functional analyses demonstrate that during viral persistence: 1) CD8+ T cells maintain TCR affinity for peptide/MHC complexes, 2) the functional avidity of CD8+ T cells measured as the capacity to produce IFN-gamma is preserved intact, and 3) virus-specific CD8+ T cells have in vivo killing capacity. Next, we demonstrate that at 8 mo post-virus inoculation, long-term CD8+ T cells are capable of mediating a protective recall response against the establishment of gammaHV68 splenic latency. These observations provide evidence that functional CD8+ memory T cells can be generated and maintained during low-load gammaHV68 persistence.     

Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines

cDNAs encoding TCR alpha- and beta-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5'RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157-165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these alpha- and beta-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4(+) and CD8(+) T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide (<20 pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-gamma, GM-CSF, IL-4, and IL-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h (51)Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.     

TCR transgenes and transgene cassettes for TCR gene therapy: status in 2008

The genetic introduction of T cell receptor genes into T cells has been developed over the past decade as a strategy to induce defined antigen-specific T cell immunity. With the potential value of TCR gene therapy well-established in murine models and the feasibility of infusion of TCR-modified autologous T cells shown in a first phase I trial, the next key step will be to transform TCR gene transfer from an experimental technique into a robust clinical strategy. In this review, we discuss the different properties of the TCR transgene and transgene cassette that can strongly affect both the efficacy and the safety of TCR gene transfer. This paper is a focussed research review based on a presentation given at the sixth annual meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany,15–16 May 2008.     

Targeting the Wilms tumor antigen 1 by TCR gene transfer: TCR variants improve tetramer binding but not the function of gene modified human T cells

We have previously described the functional activity of a human TCR specific for an HLA-A2-presented peptide derived from the Wilms tumor Ag 1 (WT1). Recent studies showed that the expression and function of human TCR was improved by the introduction of an additional disulfide bond between the alpha- and beta-chains or by the exchange of the human constant region for murine sequences. In this study, we analyzed the functional activity of WT1-TCR variants expressed in Jurkat cells and in primary T cells. The introduction of cysteine residues or murine constant sequences into the WT1-TCR did not result in a global reduction of mispairing with wild-type TCR chains. Instead, the level of mispairing was affected by the variable region sequences of the wild-type TCR chains. The analysis of freshly transduced peripheral blood T cells showed that the transfer of modified TCR constructs generated a higher frequency of Ag-responsive T cells than the transfer of the wild-type TCR. After several rounds of peptide stimulation this difference was no longer observed, as all transduced T cell populations accumulated approximately 90% of Ag-responsive T cells. Although the Ag-responsive T cells expressing the modified TCR bound the HLA-A2/WT1 tetramer more efficiently than T cells expressing the wild-type TCR, this did not improve the avidity of transduced T cells nor did it result in a measurable enhancement in IFN-gamma production and cytotoxic activity. This indicated that the enhanced tetramer binding of modified WT1-TCR variants was not associated with improved WT1-specific T cell function.     

High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens

The alpha- and beta-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-gamma), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4(+) T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.     

The fate of low affinity tumor-specific CD8+ T cells in tumor-bearing mice

A major challenge in tumor immunology is how best to activate the relatively low avidity self-specific and tumor-specific T cells that are available in the self-tolerant repertoire. To address this issue, we produced a TCR transgenic mouse expressing a class I-restricted hemagglutinin (HA)-specific TCR (clone 1 TCR) derived from a mouse that expressed HA as a self-Ag in the insulin-producing beta cells of the pancreatic islets (InsHA) mice. Upon transfer of clone 1 TCR CD8(+) T cells into InsHA mice, very few cells were activated by cross-presented HA, indicating that the cells were retained in InsHA mice because they ignored the presence of Ag, and not because they were functionally inactivated by anergy or tuning. Upon transfer into recipient mice in which HA is expressed at high concentrations as a tumor-associated Ag in spontaneously arising insulinomas (RIP-Tag2-HA mice), a high proportion of clone 1 cells were activated when they encountered cross-presented tumor Ag in the pancreatic lymph nodes. However, the activated cells exhibited very weak effector function and were soon tolerized. The few activated cells that did migrate to the tumor were unable to delay tumor progression. However, when HA-specific CD4 helper cells were cotransferred with clone 1 cells into RIP-Tag2-HA recipients and the mice were vaccinated with influenza, clone 1 cells were found to exert a significant level of effector function and could delay tumor growth. This tumor model should prove of great value in identifying protocols that can optimize the function of low avidity tumor-specific T cells.     

Functional reconstitution of class II MHC-restricted T cell immunity mediated by retroviral transfer of the alpha beta TCR complex

Transfer of the alphabeta TCR genes into T lymphocytes will provide a means to enhance Ag-specific immunity by increasing the frequency of tumor- or pathogen-specific T lymphocytes. We generated an efficient alphabeta TCR gene transfer system using two independent monocistronic retrovirus vectors harboring either of the class II MHC-restricted alpha or beta TCR genes specific for chicken OVA. The system enabled us to express the clonotypic TCR in 44% of the CD4+ T cells. The transduced cells showed a remarkable response to OVA323-339 peptide in the in vitro culture system, and the response to the Ag was comparable with those of the T lymphocytes derived from transgenic mice harboring OVA-specific TCR. Adoptive transfer of the TCR-transduced cells in mice induced the Ag-specific delayed-type hypersensitivity in response to OVA323-339 challenge. These results indicate that alphabeta TCR gene transfer into peripheral T lymphocytes can reconstitute Ag-specific immunity. We here propose that this method provides a basis for a new approach to manipulation of immune reactions and immunotherapy.     

Transfer of TCR genes into mature T cells is accompanied by the maintenance of parental T cell avidity

The adoptive transfer of tumor-specific T cells expanded in vitro can be of significant therapeutic value in select cancer patients. This strategy is limited though, as it is often difficult, if not impossible, to obtain T cells of clinical value. The transfer of TCR genes to mature T cells to generate tumor-reactive T cells provides a potential mechanism to overcome these limitations. To evaluate the feasibility of such an approach and the quality of the resulting T cells, we generated replication-deficient retroviral vectors using the well-characterized OT-1 TCR genes. After transducing murine T cells, we were able to expand large numbers of Ag-specific T cells that were functionally active against tumor cells expressing the relevant Ag. Furthermore, we found that T cells expressing retrovirally encoded TCR had avidity that was similar to that of the parental clone. This maintenance of avidity was despite variable expression of the retrovirally encoded TCR and the presence of potentially competing endogenous TCRs. These results suggest that the inherent qualities of the TCR, as dictated by the coding sequence, are the most critical parameters in the generation of high-avidity T cells.     

Increased frequency of suppressive regulatory T cells and T cell-mediated antigen loss results in murine melanoma recurrence

Therapeutic treatment of large established tumors using immunotherapy has yielded few promising results. We investigated whether adoptive transfer of tumor-specific CD8(+) T cells, together with tumor-specific CD4(+) T cells, would mediate regression of large established B16BL6-D5 melanomas in lymphopenic Rag1(-/-) recipients devoid of regulatory T cells. The combined adoptive transfer of subtherapeutic doses of both TRP1-specific TCR transgenic Rag1(-/-) CD4(+) T cells and gp100-specific TCR transgenic Rag1(-/-) CD8(+) T cells into lymphopenic recipients, who received vaccination, led to regression of large (100-400 mm(2)) melanomas. The same treatment strategy was ineffective in lymphoreplete wild-type mice. Twenty-five percent of mice (15/59) had tumors recur (15-180 d postregression). Recurrent tumors were depigmented and had decreased expression of gp100, the epitope targeted by the CD8(+) T cells. Mice with recurrent melanoma had increased CD4(+)Foxp3(+) TRP1-specific T cells compared with mice that did not show evidence of disease. Importantly, splenocytes from mice with recurrent tumor were able to suppress the in vivo therapeutic efficacy of splenocytes from tumor-free mice. These data demonstrate that large established tumors can be treated by a combination of tumor-specific CD8(+) and CD4(+) T cells. Additionally, recurrent tumors exhibited decreased Ag expression, which was accompanied by conversion of the therapeutic tumor-specific CD4(+) T cell population to a Foxp3(+)CD4(+) regulatory T cell population.     

Redundancy of direct priming and cross-priming in tumor-specific CD8+ T cell responses

Against a subset of human cancers, vigorous tumor-specific CD8+ T cell responses can develop either spontaneously or upon allogeneic transplantation. However, the parameters that determine the induction of such pronounced anti-tumor immunity remain ill defined. To dissect the conditions required for the induction of high magnitude T cell responses, we have developed a murine model system in which tumor-specific T cell responses can be monitored directly ex vivo by MHC tetramer technology. In this model, tumor challenge of naive mice with Ag-bearing tumor cells results in a massive Ag-specific T cell response, followed by CD8+ T cell-dependent tumor rejection. We have subsequently used this model to assess the contribution of direct priming and cross-priming in the induction of tumor immunity in a well-defined system. Our results indicate that direct priming of T cells and Ag cross-priming are redundant mechanisms for the induction of tumor-specific T cell immunity. Moreover, T cell responses that arise as a consequence of Ag cross-presentation can occur in the absence of CD4+ T cell help and are remarkably robust.     

Withdrawal of stimulation may initiate the transition of effector to memory CD4 cells.

The initial steps that determine development of memory in CD4 cells are unknown. To distinguish an intrinsic capacity of effectors to become memory cells from contributions of as yet undefined survival factors, we analyzed the effects of withdrawal of signals via TCR, costimulation, and cytokines from Th1 or Th2 primary effectors induced in vitro from TCR-transgenic CD4 cells. Withdrawal of stimulation caused the transition of effectors to resting populations with a memory phenotype that did not undergo division following transfer to normal syngeneic recipients. The return of effectors to rest was accompanied by acquisition of the capacity to function as memory cells in vivo as defined by extended persistence and a more rapid response to Ag in vivo than naive cells in adoptive hosts. Upon challenge with Ag, these in vitro-rested Th1 and Th2 cells were similar to long-term in vivo-rested memory cells, but distinct from in vitro-generated primary effectors and in vivo-restimulated memory effectors by their ability to resist apoptosis. Cessation of stimulation may occur when activated CD4 cells exit lymphoid tissues after priming and transition to memory may be initiated if effectors either fail to gain access to Ag in peripheral tissues where restimulation can lead to activation-induced cell death or do not receive sufficient stimuli to continue a response. Our results suggest that the first stage leading to stable CD4 memory could occur stochastically and independently of instructional processes and as such, the development of memory may be a default pathway when signals that direct responses are not received.     

Antigen-specific T cells transduced with IL-10 ameliorate experimentally induced arthritis without impairing the systemic immune response to the antigen

For the treatment of rheumatoid arthritis, efficient drug delivery methods to the inflamed joints need to be developed. Because T cells expressing an appropriate autoantigen-specific receptor can migrate to inflamed lesions, it has been reasoned that they can be employed to deliver therapeutic agents. To examine the ability and efficiency of such T cells as a vehicle, we employed an experimentally induced model of arthritis. Splenic T cells from DO11.10 TCR transgenic mice specific for OVA were transduced with murine IL-10. Adoptive transfer of the IL-10-transduced DO11.10 splenocytes ameliorated OVA-induced arthritis despite the presence of around 95% nontransduced cells. Using green fluorescent protein as a marker for selection, the number of transferred cells needed to ameliorate the disease was able to be reduced to 10(4). Preferential accumulation of the transferred T cells was observed in the inflamed joint, and the improvement in the disease was not accompanied by impairment of the systemic immune response to the Ag, suggesting that the transferred T cells exert their anti-inflammatory task locally, mainly in the joints where the Ag exists. In addition, IL-10-transduced DO11.10 T cells ameliorated methylated BSA-induced arthritis when the arthritic joint was coinjected with OVA in addition to methylated BSA. These results suggest that T cells specific for a joint-specific Ag would be useful as a therapeutic vehicle in rheumatoid arthritis for which the arthritic autoantigen is still unknown.     

Induction of central deletional T cell tolerance by gene therapy

Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag K(b) were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2K(b) resulted in lifelong expression of K(b) on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2K(b) transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with K(b) transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to K(b). Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.     

Retrogenic modeling of experimental allergic encephalomyelitis associates T cell frequency but not TCR functional affinity with pathogenicity

The properties of a self-specific T cell's TCR that determine its pathogenicity are not well understood. We developed TCR retroviral transgenic, or retrogenic, models of myelin oligodendroglial glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) to compare the pathologic potential of five H-2 Ab/MOG35-55-specific TCRs. The TCRs were cloned and retrovirally transduced into either TCRalphabeta-deficient hybridoma cells or Rag1-/- bone marrow progenitor cells. Comparison of the hybridomas, identical except for TCR sequence, revealed distinct responsiveness, or functionally determined affinity, for cognate Ag. Retrogenic mice were produced by transfer of transduced progenitor cells into Rag1-/- recipients. T cells were detected within 4 wk. Engraftment levels varied considerably among the different TCRs and showed separate variability among individual mice. T cells were predominantly naive and virtually exclusively CD4+ and CD25-. Relative responses of the retrogenic T cells to Ag paralleled those of the hybridoma cells. Induction of EAE through active immunization led to rapid and severe disease in all mice expressing MOG-specific TCR. The mice additionally developed spontaneous disease, the incidence of which varied with the individual receptors. Interestingly, spontaneous disease frequency and intensity could not be correlated with the functional affinity of the respective TCR. Instead, it was associated with engraftment level, even when measured weeks before the onset of disease symptoms. Our results demonstrate the feasibility of using retrogenic modeling to compare TCRs in the EAE system. They further suggest that affinity is not a primary determinant in spontaneous EAE development in mice expressing monotypic TCRs and that autoreactive T cell frequency is of greater significance.     

T cells from tumor-immune mice nonspecifically expanded in vitro with anti-CD3 plus IL-2 retain specific function in vitro and can eradicate disseminated leukemia in vivo.

The therapeutic efficacy of adoptive immunotherapy of cancer has been shown to positively correlate with the dose of tumor-immune T cells transferred. Therefore, the success of this therapy is critically dependent on the ability to procure large numbers of functionally active T cells. Previous studies in animal models have shown that the limited therapeutic efficacy of a small number of immune T cells can be greatly enhanced by expansion of T cells in vitro to greater numbers before transfer in vivo. Optimal regimens for T cell expansion in vitro have generally employed the use of intermittent stimulation of the TCR with specific Ag followed by exogenous IL-2. The use of IL-2 alone does not provide for requisite episodic up-regulation of IL-2R. Stimulation of the invariant CD3 portion of the TCR/CD3 complex with antibody to CD3 (anti-CD3) represents an alternative method of up-regulating IL-2R and has been used to nonspecifically induce the growth of Ag-specific T cell lines and clones long-term in vitro with maintenance of function and specificity. The current study examined whether resting T cell populations containing small numbers of memory tumor-specific T cells could be rendered more effective in tumor therapy by nonspecific expansion in vitro with anti-CD3 plus IL-2. Spleens from C57BL/6 mice previously immunized to FBL-3, a syngeneic virus-induced leukemia, were nonspecifically stimulated with anti-CD3 plus IL-2. The resultant T cells were expanded in number, were nonlytic to FBL-3 but retained the ability to become lytic upon specific stimulation by FBL-3, and were effective in specific tumor therapy. The Ag-specific anti-tumor immune function declined on a per cell basis after each cycle of anti-CD3-induced T cell expansion. However, the approach resulted in a substantial increase in total T cell number and an overall net increase in the function of the effector T cell population. Thus, stimulation of tumor-immune T cell populations with anti-CD3 plus IL-2 represents a nonspecific method for expanding the number of specific effector T cells for cancer therapy.     

Hypervariable region 1 variants act as TCR antagonists for hepatitis C virus-specific CD4+ T cells.

In various human viral infections, the appearance of mutated epitopes displaying TCR antagonistic activity has been correlated with the severity and persistence of infection. In hepatitis C virus (HCV) infection, where the virus persistence has been associated with the rapid and substantial Ag modifications occurring during replication, TCR antagonism has been evidenced in CD8+ T cell responses. However, CD4+ T cell antagonism may be another important strategy by which HCV eludes a protective response, because sustained Th responses directed against several HCV Ags are associated with a self-limited course of infection. The data reported here represent the first evidence that variants of the hypervariable region (HVR1) of the putative Envelope 2 protein of HCV can act as powerful TCR antagonists for HVR1-specific CD4+ T cells isolated from HCV-infected individuals. Using classical antagonism assays, we observed strong inhibition of cellular proliferation and cytokine production when the agonist and the antagonist ligands were simultaneously presented by the same APCs. The presence in HVR1 of conserved residues, critical for binding to HLA-DR molecules, supports the function of HVR1 variants as TCR antagonists. In conclusion, our data evidence an antagonism phenomenon, which was achieved by naturally occurring class II-restricted T cell epitopes whose mechanism was addressed in terms of the antagonist capacity to inhibit agonist-mediated TCR down-regulation and early signal transduction.