Design of soluble recombinant T cell receptors for antigen targeting and T cell inhibition.

The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric alphabeta TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax(11-19) epitope bound to HLA A2-HuD(87-95) (K(D) 120 microm by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K(D) 2.5 nm) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.

Epidermal growth factor receptor VIII peptide vaccination is efficacious against established intracerebral tumors.

PURPOSE: The epidermal growth factor receptor (EGFR) is often amplified and structurally rearranged in malignant gliomas and other tumors such as breast and lung, with the most common mutation being EGFRvIII. In the study described here, we tested in mouse models a vaccine consisting of a peptide encompassing the tumor-specific mutated segment of EGFRvIII (PEP-3) conjugated to keyhole limpet hemocyanin [KLH (PEP-3-KLH)]. EXPERIMENTAL DESIGN: C57BL/6J or C3H mice were vaccinated with PEP-3-KLH and subsequently challenged either s.c. or intracerebrally with a syngeneic melanoma cell line stably transfected with a murine homologue of EGFRvIII. Control mice were vaccinated with KLH. To test its effect on established tumors, C3H mice were also challenged intracerebrally and subsequently vaccinated with PEP-3-KLH. RESULTS: S.c. tumors developed in all of the C57BL/6J mice vaccinated with KLH in Freund's adjuvant, and there were no long-term survivors. Palpable tumors never developed in 70% of the PEP-3-KLH-vaccinated mice. In the C57BL/6J mice receiving the PEP-3-KLH vaccine, the tumors that did develop were significantly smaller than those in the control group (P < 0.05). PEP-3-KLH vaccination did not result in significant cytotoxic responses in standard cytotoxicity assays; however, antibody titers against PEP-3 were enhanced. The passive transfer of sera from the immunized mice to nonimmunized mice protected 31% of the mice from tumor development (P < 0.05). In vivo depletion studies showed that the effector cell population was natural killer and CD8+ T cells, and in vitro assays showed that macrophages could lyse target tumor cells with serum from the PEP-3-KLH-vaccinated mice. Peptide vaccination was also sufficiently potent to have marked efficacy against intracerebral tumors, resulting in a >173% increase in median survival time, with 80% of the C3H mice achieving long-term survival (P = 0.014). In addition, C3H mice with established intracerebral tumor that received a single treatment of PEP-3-KLH showed a 26% increase in median survival time, with 40% long-term survival (P = 0.007). CONCLUSIONS: Vaccination with an EGFRvIII-specific peptide is efficacious against both s.c. and established intracerebral tumors. The therapeutic effect of peptide vaccination may be mediated, in part, by antibody-dependent cellular cytotoxicity.

Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells.

Virus-specific CD8(+) T-cell responses play a pivotal role in limiting viral replication. Alterations in these responses, such as decreased cytolytic function, inappropriate maturation, and limited proliferative ability could reduce their ability to control viral replication. Here, we report on the capacity of HIV-specific CD8(+) T cells to secrete cytokines and proliferate in response to HIV antigen stimulation. We find that a large proportion of HIV-specific CD8(+) T cells that produce cytokines in response to cognate antigen are unable to divide and die during a 48-hour in vitro culture. This lack of proliferative ability of HIV-specific CD8(+) T cells is defined by surface expression of CD57 but not by absence of CD28 or CCR7. This inability to proliferate in response to antigen cannot be overcome by exogenous interleukin-2 (IL-2) or IL-15. Furthermore, CD57 expression on CD8(+) T cells, CD4(+) T cells, and NK cells is a general marker of proliferative inability, a history of more cell divisions, and short telomeres. We suggest, therefore, that the increase in CD57(+) HIV-specific CD8(+) T cells results from chronic antigen stimulation that is a hallmark of HIV infection. Thus, our studies define a phenotype associated with replicative senescence in HIV-specific CD8(+) T cells, which may have broad implications to other conditions associated with chronic antigenic stimulation.

Generation of anti-idiotypic reagents in the EGFRvIII tumor-associated antigen system.

The use of anti-idiotype (anti-id) vaccines for immunotherapy of human cancers is attractive, as immunization with true anti-id reagents (Ab2 beta) has been shown to induce both cellular and humoral immunity, frequently when the original antigen does not, or when a state of anergy to the self-expressed tumor-associated antigen exists. The aim of this study was to investigate the potential of an anti-id vaccine approach to the glioma-associated antigen epidermal growth factor receptor variant III (EGFRvIII) for human clinical trials. By using conventional methodology, seven rat mAbs specific for the binding site of the murine anti-EGFRvIII-specific mAb Y10, as defined by the ability to inhibit the binding of mAb Y10 to EGFRvIII expressed on cells or as purified protein, were generated, and a subset (3/7) was found to be true Ab2 beta, as defined by the ability to induce the formation of antibody directed against EGFRvIII in two species (mouse and rabbit) when used as immunogen. The ability of these three Ab2 beta to elicit a protective anti-tumor response when used as a vaccine in the syngeneic, subcutaneous C57Bl/6-B16mseEGFRvIII tumor model was investigated. Following vaccination with one Ab2 beta mAb (2C7), 6/20 mice failed to develop tumor upon challenge, and 3/20 mice with outgrowing tumors exhibited dramatic regression of incipient tumors. Vaccination with a second mAb (5G8) resulted in one tumor-free survivor and one tumor regressor; vaccination with the third Ab2 beta mAb (7D3) did not confer protection, but did significantly increase the latency period until tumor outgrowth in all vaccinated recipients. The ability of Ab2 beta mAb 2C7 to induce an anti-EGFRvIII response in non-human primates was investigated by using the saponin adjuvant approved for human clinical trial, QS-21. Three of three macaques produced anti-EGFRvIII titers, as detected on EGFRvIII-expressing cells by both ELISA and fluorescence-activated cytometric analysis, following six immunizations with Ab2 beta mAb 2C7 and QS-21. The results obtained confirm that an anti-id response in the EGFRvIII antigen system can be induced in rodents, rabbits, and non-human primates, and it may prove a useful adjunct to immunotherapeutic approaches to EGFRvIII-positive gliomas, breast carcinomas, and non-small-cell lung tumors.

Dendritic cells pulsed with a tumor-specific peptide induce long-lasting immunity and are effective against murine intracerebral melanoma.

OBJECTIVE: Dendritic cells (DCs) are specialized cells of the immune system that are capable of generating potent immune responses that are active even within the "immunologically privileged" central nervous system. However, immune responses generated by DCs have also been demonstrated to produce clinically significant autoimmunity. Targeting the epidermal growth factor receptor variant III (EGFRvIII), which is a mutation specific to tumor tissue, could eliminate this risk. The purpose of this study was to demonstrate that DC-based immunizations directed solely against this tumor-specific antigen, which is commonly found on tumors that originate within or metastasize to the brain, could be efficacious. METHODS: C3H mice were vaccinated with DCs mixed with a keyhole limpet hemocyanin conjugate of the tumor-specific peptide, PEP-3, which spans the EGFRvIII mutation, or the random-sequence peptide, PEP-1, and were intracerebrally challenged with a syngeneic melanoma expressing a murine homologue of EGFRvIII. RESULTS: Systemic immunization with DCs mixed with PEP-3-keyhole limpet hemocyanin generated antigen-specific immunity. Among mice challenged with intracerebral tumors, this resulted in an approximately 600% increase in the median survival time (>300 d, P < 0.0016), relative to control values. Sixty-three percent of mice treated with DCs mixed with the tumor-specific peptide survived in the long term and 100% survived rechallenge with tumor, indicating that antitumor immunological memory was also induced. CONCLUSION: In a murine melanoma model, immunization with DCs mixed with tumor-specific peptide results in an antigen-specific immunological response that recognizes the EGFRvIII mutation, has potent antitumor efficacy against intracerebral tumors that express EGFRvIII, and results in long-lasting antitumor immunity.