Evaluation of safety and efficacy of p53MVA vaccine combined with pembrolizumab in patients with advanced solid cancers.

BACKGROUND: Vaccination of cancer patients with p53-expressing modified vaccinia Ankara virus (p53MVA) has shown in our previous studies to activate p53-reactive T cells in peripheral blood but without immediate clinical benefit. We hypothesized that the immunological responses to p53MVA vaccine may require additional immune checkpoint blockade to achieve clinically beneficial levels. We therefore conducted a phase I trial evaluating the combination of p53MVA and pembrolizumab (anti-PD-1) in patients with advanced solid tumors. PATIENTS AND METHODS: Eleven patients with advanced breast, pancreatic, hepatocellular, or head and neck cancer received up to 3 triweekly vaccines in combination with pembrolizumab given concurrently and thereafter, alone at 3-week intervals until disease progression. The patients were assessed for toxicity and clinical response. Correlative studies analyzed p53-reactive T cells and profile of immune function gene expression. RESULTS: We observed clinical responses in 3/11 patients who remained with stable disease for 30, 32, and 49 weeks. Two of these patients showed increased frequencies and persistence of p53-reactive CD8+ T cells and elevation of expression of multiple immune response genes. Borderline or undetectable p53-specific T cell responses in 7/11 patients were related to no immediate clinical benefit. The first study patient had a grade 5 fatal myocarditis. After the study was amended for enhanced cardiac monitoring, no additional cardiac toxicities were noted. CONCLUSION: We have shown that the combination of p53MVA vaccine with pembrolizumab is feasible, safe, and may offer clinical benefit in select group of patients that should be identified through further studies.

CD56dimCD57+NKG2C+ NK cell expansion is associated with reduced leukemia relapse after reduced intensity HCT.

We have recently described a specialized subset of human natural killer (NK) cells with a CD56(dim)CD57(+)NKG2C(+) phenotype that expand specifically in response to cytomegalovirus (CMV) reactivation in hematopoietic cell transplant (HCT) recipients and exhibit properties characteristic of adaptive immunity. We hypothesize that these cells mediate relapse protection and improve post-HCT outcomes. In 674 allogeneic HCT recipients, we found that those who reactivated CMV had lower leukemia relapse (26% (17-35%), P=0.05) and superior disease-free survival (DFS) (55% (45-65%) P=0.04) 1 year after reduced intensity conditioning (RIC) compared with CMV seronegative recipients who experienced higher relapse rates (35% (27-43%)) and lower DFS (46% (38-54%)). This protective effect was independent of age and graft-vs-host disease and was not observed in recipients who received myeloablative regimens. Analysis of the reconstituting NK cells demonstrated that CMV reactivation is associated with both higher frequencies and greater absolute numbers of CD56(dim)CD57(+)NKG2C(+) NK cells, particularly after RIC HCT. Furthermore, expansion of these cells at 6 months posttransplant independently trended toward a lower 2-year relapse risk. Together, our data suggest that the protective effect of CMV reactivation on posttransplant relapse is in part driven by adaptive NK cell responses.

Real-time assessment of relapse risk based on the WT1 marker in acute leukemia and myelodysplastic syndrome patients after hematopoietic cell transplantation.

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (alloHCT) for acute leukemia and myelodysplastic syndrome (MDS). Wilms' tumor Ag (WT1) is overexpressed in the majority of acute leukemia and MDS patients and has been proposed as a universal diagnostic marker for detection of impending relapse. Comprehensive studies have shown that WT1 transcript levels have predictive value in acute leukemia patients in CR after chemotherapy. However, the focus of this study is the period after alloHCT for predicting relapse onset. We analyzed the accumulation of WT1 mRNA transcripts in PB of 82 leukemia and MDS patients and defined specific molecular ratios for relapse prediction. The extensively validated WT1/c-ABL ratio was used to normalize increases in WT1 transcript levels. The observed lead time of crossing or exceeding set WT1 levels is presented along with linear interpolation to estimate the calculated day the WT1 thresholds were crossed. The WT1/c-ABL transcript ratio of 50 or above yielded 100% specificity and 75% sensitivity reliably predicting future relapse with an observed average of 29.4 days (s.d.=19.8) and a calculated average of 63 days (s.d.=29.3) lead time before morphologic confirmation. A lower ratio of 20 or above gave lower specificity, but higher sensitivity (84.8% and 87.5%, respectively) identified more patients who relapsed, at earlier times, providing an earlier warning with actual average lead time of 49.1 days (s.d.=30.8) and calculated average of 78 days (s.d.=28.8). WT1 transcript levels serve as a diagnostic relapse test with greater sensitivity than the morphologic approach used in the clinic as a readout.

Programmed death-1 receptor and interleukin-10 in liver transplant recipients at high risk for late cytomegalovirus disease.

Despite significant advances in antiviral treatment, solid organ transplant (SOT) recipients remain at heightened risk for developing late cytomegalovirus (CMV) disease. Elevated inhibitory immune signaling suggests a state of immune impairment in SOT recipients, who do not control CMV infection and develop severe clinical symptoms after discontinuation of antiviral prophylaxis. We longitudinally monitored the negative immune modulator programmed death (PD)-1 receptor on both CD4 and CD8 T cells, co-expressing the CD137 surface marker of recent activation, in a liver transplant cohort. Liver recipients who progressed to CMV disease expressed elevated levels of PD-1 on CD137(+) CD4 and CD8 T cells, following stimulation with either full-length peptide libraries or CMV lysate. This novel approach, applicable to a multitude of human leukocyte antigen types, enhances the usefulness of the PD-1 measurements as a clinical strategy to predict late CMV disease. In parallel, we detected an increased level of the immunosuppressive cytokine interleukin (IL)-10, in plasma of liver recipients diagnosed with CMV disease. CMV-specific T cells were still functional when both PD-1 and IL-10 were upregulated; however they showed a marked proliferation deficit, which may limit their ability to contain viremia and lead to CMV disease. Our preliminary observations support further investigation of dual monitoring of PD-1 and IL-10, as potential immune markers of CMV disease.

A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector.

A therapeutic CMV vaccine incorporating an antigenic repertoire capable of eliciting a cellular immune response has yet to be successfully implemented for patients who already have acquired an infection. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV. The novelty of this vaccine is the fusion of two adjacent exons from the immediate-early region of CMV, their successful expression in MVA, and robust immunogenicity in both primary and memory response models. Evaluation of the immunogenicity of the viral vaccine in mouse models shows that it can stimulate primary immunity against all three antigens in both the CD4(+) and CD8(+) T cell subsets. Evaluation of human PBMC from healthy CMV-positive donors or patients within 6 months of receiving hematopoietic cell transplant shows robust stimulation of existing CMV-specific CD4(+) and CD8(+) T cell subsets.

Kinase-deficient CMVpp65 triggers a CMVpp65 specific T-cell immune response in HLA-A*0201.Kb transgenic mice after DNA immunization.

CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) activity that could affect vaccine safety. A mutated form of CMVpp65 substituting asparagine for lysine at the adenosine triphosphate (ATP)-binding site (CMVpp65mII) is kinase-deficient. Using DNA immunizations in a transgenic human leucocyte antigen (HLA)A*0201.Kb mouse model, the mutated CMVpp65 induced cytotoxic T lymphocytes (CTL) immunity similarly to native CMVpp65. Murine CTL lines generated from these immunizations killed human cells either after sensitization with CMVpp65-specific peptides or after infection with either CMV-Towne strain or rvac-pp65. It is proposed that CMVpp65mII be evaluated in candidate vaccines for CMV.

Human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy maintain activated CD8+ T cell subsets as a strong adaptive immune response to cytomegalovirus.

CD8(+) T lymphocyte function specific for human cytomegalovirus (CMV) was evaluated in 14 patients infected with human immunodeficiency virus (HIV) receiving highly active antiretroviral therapy (HAART) and 26 CMV-seropositive donors without HIV infection. Fifty-seven percent of the HIV-infected group had CMV-specific cytolytic activity in freshly isolated peripheral blood mononuclear cells (PBMC) against targets expressing CMV pp65. Both interferon (IFN)-gamma secretion by CD8(+) T cells and the frequency of human leukocyte antigen (HLA)-tetramer-positive T cells in HLA-A*0201-positive HIV-infected subjects correlated with CMV-specific cytolysis. In contrast, PBMC from healthy CMV-seropositive donors did not have either measurable CMV-specific cytolysis or secretion of IFN-gamma without in vitro stimulation. The T helper response to CMV antigens was vigorous in healthy CMV-seropositive donors but low in the cohort of HIV-infected patients. Potent CD8(+) cytotoxic T lymphocyte responses to CMV in HIV-infected patients receiving HAART is the converse of what is found in healthy CMV-seropositive subjects and may be the predominant adaptive immune response against CMV in HIV-infected patients.

Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries.

The pp65(495-503) cytotoxic T-lymphocyte (CTL) epitope from cytomegalovirus (CMV) is universally recognized among CMV+ individuals who express an allele of the human leukocyte antigen A (HLA-A*0201). The relative binding affinity of the epitope to HLA-A*0201 is moderate, and its increased activity might prove beneficial in its use as a CTL epitope vaccine. A new approach to enhance the activity of T-cell epitopes is the use of positional scanning synthetic combinatorial libraries (PS-SCLs). Using a nonamer PS-SCL, the pp65(495-503) epitope was modified after screening a CMV-specific T-cell clone (TCC) (3-3F4) from which the native peptide sequence was derived. Two peptides with amino acid substitutions at P1, P3, P7, and P8 are between 10(3) and 10(4) more active than the native epitope. Although the native CTL epitope terminates as a free acid, both tetrasubstituted peptides only function as CTL epitopes when the carboxyl terminus is amidated. Selective substitution of the native sequence based on PS-SCL screening results identified 3 amidated monosubstituted and disubstituted peptides that are better recognized than the native epitope by TCCs from a cohort expressing HLA-A*0201. In vitro stimulation of peripheral blood mononuclear cells with each of the peptide epitope analogs stimulated memory CTLs, which recognized CMV-infected targets among a high percentage of CMV+ individuals. Binding studies of peptide analogs with HLA-Ig (immunoglobulin) dimers and 2 different TCCs correlated with in vitro lysis results. These data suggest that increasing the activity of CTL epitopes while maintaining broad recognition is possible, which holds promise for vaccine development in infectious disease and cancer.

Infrequent occurrence of natural mutations in the pp65(495-503) epitope sequence presented by the HLA A*0201 allele among human cytomegalovirus isolates.

To determine if mutations of an immunodominant HLA-restricted cytomegalovirus (CMV) peptide sequence occur in nature, the sequence corresponding to the HLA A*0201-specific peptide CMVpp65(495-503) was determined in 50 human CMV isolates. Rare mutations were detected; 6 of 50 were silent mutations at the amino terminus of the peptide, while 3 of 50 were mutations of the native methionine residue to isoleucine (M499I). The observed M499I mutation in three isolates decreased cytolytic targeting.

Population coverage by HLA class-I restricted cytotoxic T-lymphocyte epitopes.

Vaccination using cytotoxic T-lymphocyte (CTL) epitopes has become a widely used immunization strategy, especially because their structure makes them an attractive alternative to the delivery of whole proteins as immunogens. Nonetheless, their use is limited, in particular because of their specificity, being recognized only by cognate HLA alleles. The potential for immunizing a substantial portion of an ethnically diverse population using a modest number of peptides has been aided by the identification of HLA supertypes. However, the derivation of epitopes is often guided by methods that do not guarantee cross-reactivity, so we consider the feasibility of providing vaccine coverage to a multi-ethnic population under different assumptions. In particular, two large datasets are used to estimate the number of peptides needed to provide > or =90% group-specific coverage of a multiethnic population, when specificity is assumed to be either to a single serologic or molecular type. These assumptions are evaluated utilizing a clinically important epitope repertoire derived from two human cytomegalovirus proteins, and data on the in vitro memory response elicited by these peptides is presented. In summary, our combined theoretical and empiric studies suggest that 90% coverage of some ethnic groups is attainable with 11 uniquely defined HLA-restricted CTL epitopes. The derivation of four or more additional CTL epitopes is needed to attain 90% coverage of Blacks or Asians, the minimally covered groups. Ninety percent coverage of all major ethnic groups in a multi-ethnic population appears feasible without relying on cross-reactivity, but may require two to three times more CTL epitopes than estimated for serologic data, homogenous populations, or HLA alleles grouped as supertypes.

Site-directed mutation in a conserved kinase domain of human cytomegalovirus-pp65 with preservation of cytotoxic T lymphocyte targeting.

The major target of human cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) is the tegument protein CMVpp65. However, this protein has protein kinase (PK) activity, and the unknown effects on cell replication of an exogenous PK in healthy cells could limit the use of CMVpp65 as a vaccine, especially in children. In this report we show that a point mutation converting lysine to asparagine at the invariant lysine (K436), an essential site for phosphotransfer, abolishes the threonine kinase activity. The mutant CMVpp65 maintains its immunologic target characteristics, including antibody and CTL reactivity. This kinase-deficient CMVpp65 is a candidate for evaluation in future CMV vaccine development.

Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response.

CTL play a pivotal role in the immune response during viral infections. In this study, the HLA class II restricted T(H) requirement for optimal in vivo induction of HLA class I restricted CTL responses has been investigated. Towards this goal, transgenic mice expressing both HLA class I (A*0201 or A2.1) and class II (DRB1*0101 or DR1) molecules have been derived. Immunization of these mice with an HLA A*0201-restricted and CMV-specific CTL epitope (pp65(495-503)), and either of three different tetanus toxin-derived MHC class II-binding T(H) epitopes, resulted in a vigorous CTL response. CTL specific for the pp65(495-503) epitope were dramatically enhanced in mice expressing both the HLA-DR1 and HLA-A*0201 transgenes. Notably, preinjection of three TT peptides (TT(639-652), TT(830-843), and TT(947-967)) increased the capability of HLA A*0201/DR1 Tg mice to respond to subsequent immunization with the T(H) + CTL peptide mixture. These results indicate that the use of HLA A*0201/DR1 Tg mice constitute a versatile model system (in lieu of immunizing humans) for the study of both HLA class I and class II restricted T-cell responses. These studies provide a rational model for the design and assessment of new minimal-epitope vaccines based on their in vivo induction of a pathogen-specific CTL response.

Targeting of human p53-overexpressing tumor cells by an HLA A*0201-restricted murine T-cell receptor expressed in Jurkat T cells.

A potent anti-human (hu) p53 CD8+ CTL response develops in HLA A*0201 transgenic (Tg) mice after immunization with peptides corresponding to HLA A*0201 motifs from hu p53. Mice immunized with the hu P53(149-157) peptide develop a CTL response that is of moderately high affinity and is capable of recognizing hu tumor cells expressing mutated p53. In this report, the mRNAs encoding the predominantly expressed T-cell receptor (TCR) sequences were molecularly cloned from a murine (mu) CTL clone derived from immunized Tg mice, which recognized endogenously processed hu p53 restricted by HLA A*0201. The separate A and B chain TCR cDNAs were transfected in the corresponding TCR A- and B- Jurkat-CD3- mutant T-cell lines, and each rescued CD3 surface expression. Both TCR chains were simultaneously introduced into Jurkat-CD3+ cells, and the transfected Jurkat cells recognized hu T2 cells sensitized with the p53(149-157) CTL epitope but not T2 cells sensitized with a nonspecific CTL epitope. Breast, pancreatic, and sarcoma tumor cell lines, which overexpress endogenous mutated p53, were recognized in the presence of anti-CD28 costimulation, only if they also expressed HLA A*0201. Normal hu fibroblasts established from skin cultures were not recognized. These results represent the first time that a p53-specific TCR capable of recognizing hu cancer cells was heterologously expressed in a naive recipient cell, converting that cell to one recognizing hu tumor cells with mutated p53. This TCR represents a candidate molecule for a genetic strategy in combating hu cancer by an adoptive immunotherapy approach, which uses the strong xenorecognition of hu p53 in mice.

Immunotherapy of bladder cancer targeting P53.

PURPOSE: Superficial bladder cancer is often responsive to immunotherapy with bacillus Calmette-Guerin (BCG). However, some tumors progress despite BCG treatment, and most of these have mutations in the p53 tumor suppressor gene resulting in its over-expression. Overexpressed p53 is therefore a potential target for immunotherapy. The objective of this study was to demonstrate whether human bladder cancer xenografts in SCID mice could be eliminated by cytotoxic T cells (CTL) which recognize over-expressed p53. MATERIALS AND METHODS: Murine CTL which are specific for human p53 were previously generated in our laboratory by peptide immunization of HLA A2.1 transgenic mice. These CTL recognize and lyse human tumor cell lines which over-express p53 in the context of HLA A2.1. The p53 over-expressing HLA A2.1+ human bladder cancer cell line J82 was used to establish subcutaneous or intravesicular tumors in SCID mice. The mice were then administered tail vein injections of 5 x 10(7) p53-specific CTL, control CTL, or phosphate buffered saline (PBS). RESULTS: The subcutaneous tumor mean volume at 5 weeks for the p53-specific CTL treatment group was significantly lower than for both the control CTL or the PBS group (32 mm.3 versus 185 mm.3, p = 0.04 and 32 mm.3 versus 418 mm.3, p = 0.0001). In the mice with intravesicular tumors, a reduction to nonpalpable tumor size in vivo was seen with specific CTL therapy (14% palpable) versus control CTL treatment (86% palpable), the final tumor volume at necropsy was 127 mm.3 versus 246 mm.3 (N.S.). CONCLUSION: The overall response of the human bladder tumors in the SCID mouse model suggests the possibility of targeting p53 in patients with bladder cancer.

An orthotopic in vivo model of human pancreatic cancer.

BACKGROUND: Pancreatic cancer is a highly lethal disease that frequently presents in advanced stages. For most patients, treatment with great clinical efficacy does not exist. Relevant in vivo models to test novel therapies are highly desirable. METHODS: The human pancreatic ductal adenocarcinoma cell line Panc-1 was injected intraperitoneally into SCID mice. The pattern of the resulting peripancreatic as well as metastatic disease was examined. Survival experiments after chemotherapy with gemcitabine or doxorubicin, and after immunotherapy with p53-specific cytotoxic T lymphocytes were performed. RESULTS: All animals developed isolated pancreatic tumor implants within 48 hours after injection. After the formation of invasive pancreatic tumor nodules, peripancreatic and portal adenopathy developed, causing biliary obstruction. All tumor-bearing animals died of disease within 5 to 12 weeks. Survival after gemcitabine treatment and after p53-CTL injection was significantly prolonged, with some animals remaining tumor-free. Doxorubicin treatment did not yield extended survival, but led to significant toxicity. CONCLUSION: Intraperitoneal injection of Panc-1 cells into SCID mice produces a quasi-orthotopic tumor development model that shares many characteristics with human pancreatic cancer. The ease of cell injection, avoidance of cumbersome surgical intervention with its resulting mortality, and the reliable development of obstructive jaundice as a dependent comorbid factor render this a useful model for in vivo testing of novel therapeutic approaches to pancreatic cancer. Our initial therapeutic studies demonstrate that in vitro antitumor efficacy against Panc-1 cancer cells does not necessarily predict the in vivo response, highlighting the preclinical experimental value of this model.

Targeting p53 for adoptive T-cell immunotherapy.

p53 gene mutations occur in most human cancers and result in an altered protein product that accumulates within the cell. Although the observed endogenous human CTL response to p53 is weak, high-affinity, human p53-specific CTLs have been generated from HLA A2.1 transgenic mice immunized with human CTL epitope peptides. In this study, we examine the ability of HLA A2.1-restricted and human p53-specific CTLs from HLA A2.1 transgenic mice to suppress the growth of p53-overexpressing human tumors in severe combined immunodeficient (SCID) mice. In vitro, murine p53(149-157)-specific CTLs selectively lysed the p53-overexpressing pancreatic carcinoma cell line Panc-1 but did not recognize HLA A2.1- tumor cells or HLA A2.1+ normal human fibroblasts. Furthermore, in vivo, the growth of established human tumor xenografts in SCID mice was significantly reduced and survival was prolonged after the administration of p53-specific CTLs but not after the administration of control CTLs or PBS alone. Following treatment with p53(149-157)-specific CTLs, regressing Panc-1 tumors were infiltrated by the CD8+ CTLs, as demonstrated by immunohistochemistry. These findings suggest that p53(149-157)-specific and HLA A2.1-restricted murine CTLs suppress the growth of established Panc-1 tumors following adoptive transfer into SCID hosts and prolong their survival.

An HLA-restricted, p53 specific immune response from HLA transgenic p53 knockout mice.

BACKGROUND: p53 is over-expressed in most human malignancies and is therefore an attractive target for immunotherapy. Unfortunately, a human cytotoxic T cell response to p53 is difficult to generate. p53 knockout transgenic mice may provide a model to circumvent immunologic tolerance to p53 and develop high-affinity p53-specific cytotoxic T lymphocytes (CTL). METHODS: p53 knockout, HLA A2.1 transgenic mice were generated and immunized with the immunodominant wild-type p53 nonamer peptide epitope p53149-157. Two weeks later splenocytes were harvested and stimulated in vitro with acid-treated, p53 peptide-pulsed syngeneic blast cells. Cultures were restimulated weekly with acid-treated, p53 peptide-pulsed Jurkat cells transfected with the HLA A2.1 gene. Peptide-specific cytotoxic activity was measured by chromium release assay, and the resulting CD8+ effectors were cloned via limiting dilution. RESULTS: P53 peptide-specific CTL were generated against p53149-157. Clones generated from the p53149-157 cell line demonstrated high affinity and specificity for p53149-157 when presented by HLA A2.1+ antigen-presenting cells. The p53149-157 CTL killed only cells overexpressing p53 cells that were HLA A2.1+ and did not kill cells with normal levels of p53 expression or those that were HLA A2.1-. CONCLUSION: HLA transgenic mice not previously exposed to the p53 protein provide a useful model for generating high-affinity p53-specific CTL.

Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection.

The development of a protective cellular immune response against human cytomegalovirus (HCMV) is the most important determinant of recovery from HCMV infection after allogeneic bone marrow transplantation (BMT). The ultimate aim of our study is to develop an antigen-specific and peptide-based vaccine strategy against HCMV in the setting of BMT. Toward this end we have studied the cellular immune response against the immunodominant matrix protein pp65 of HCMV. Using an HLA A*0201-restricted T-cell clone reactive against pp65 from peripheral blood from a seropositive individual, we have mapped the position of the cytolytic T lymphocyte (CTL) epitope from HCMV pp65 to an 84-amino acid segment. Of the four peptides which best fit the HLA A*0201 motif in that region, one nonamer sensitized an autologous Epstein-Barr virus immortalized lymphocyte cell line for lysis. In vitro immunization of PBMC from HLA A*0201 and HCMV seropositive volunteers using the defined nonamer peptide stimulated significant recognition of HCMV infected or peptide-sensitized fibroblasts. Similarly, HLA A*0201 transgenic mice immunized with the nonamer peptide developed CTL that recognize both the immunizing peptide and endogenously processed pp65 in an HLA A*0201 restricted manner. Lipid modification of the amino terminus of the nonamer peptide resulted in its ability to stimulate immune responses without the use of adjuvant. This demonstration of a vaccine function of the nonamer peptide without adjuvant suggests its potential for use in an immunization trial of BMT donors to induce protective CTLs in patients undergoing allogeneic BMT.

The use of transgenic mice to generate high affinity p53 specific cytolytic T cells.

P53 is an attractive target immunotherapy because it is overexpressed in up to one half of all malignancies, and its overexpression often correlates with a worsened prognosis. We wanted to determine the feasibility of targeting wild-type epitopes p53 on human tumor cells. HLA A2.1 transgenic mice were immunized with the immunodominant wild-type p53 peptide epitopes, p53(149-157) and p53(264-272), along with a pan-DR helper epitope peptide in incomplete Freund's adjuvant (IFA). Twelve days later, splenocytes were harvested and stimulated with syngeneic blast cells that had been acid-treated to remove endogenous peptide and p53 peptide-pulsed. The responding cells were subsequently restimulated weekly with acid washed, peptide-pulsed Jurkat cells transfected with HLA A2.1. Peptide specific activity was tested in a chromium release assay. The resulting cytotoxic T cells (CTL) were cloned by limiting dilution. Peptide specific CTL were generated against both p53(149-157) and p53(264-272. Only p53(149-157) specific CTL were able to recognize and lyse cells that overexpressed endogenous p53. CTL clones derived from the p53(149-157) cell line demonstrated high affinity and specificity for p53(149-157) when presented by HLA A2.1+ cells. The p53(149-157) specific CTL were tested for specificity against a variety of cultured human cell lines. The CTL clones only lysed cells that overexpressed p53 in the context of HLA A2.1 and did not lyse cells with normal p53 expression or cells that lacked HLA A2.1 expression. This study demonstrates the possibility of targeting tumors, which overexpress p53, and raises the possibility transferring the high affinity, p53 specific T cell receptors from the murine CTL to human T cells.

Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions.

We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus-host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology.