Electrocardiographic substrates of arrhythmias in patients with end-stage and chronic kidney diseases: a case-control study.

BACKGROUND: Cardiovascular disease (CVD) is the most common cause of death in patients with renal diseases. Cardiac arrhythmia and sudden cardiac death are particularly important, and the burden is higher in patients on hemodialysis. The aim of this study is to compare specific ECG changes as markers of arrhythmias in patients with CKD and patients with end-stage renal disease (ESRD); all without clinically manifest heart disease, with normal control subjects. RESULTS: Seventy-five ESRD patients on regular hemodialysis, 75 patients with stage 3-5 CKD and 40 healthy control subjects were included. All candidates were subjected to thorough clinical evaluation and laboratory tests including serum creatinine, glomerular filtration rate calculation, serum potassium, magnesium, calcium, phosphorus, iron, parathyroid hormone, and total iron binding capacity (TIBC). Resting twelve-lead ECG was done to calculate P wave dispersion (P-WD), corrected QT interval, QTc dispersion, Tpeak-Tend interval (Tp-e), and Tp-e/QT. Patients with ESRD had a significantly higher QTc dispersion (p < 0.001) and P-WD (p = 0.001) when compared to the other 2 groups. In the ESRD group, males had a significantly higher P-WD (p = 0.045), insignificantly higher QTc dispersion (p = 0.445), and insignificantly lower Tp-e/QT ratio (p = 0.252) as compared to females. Multivariate linear regression analysis for ESRD patients showed that serum creatinine (ß = 0.279, p = 0.012) and transferrin saturation (ß = - 0.333, p = 0.003) were independent predictors of increased QTc dispersion while ejection fraction (ß = 0.320, p = 0.002), hypertension (ß = - 0.319, p = 0.002), hemoglobin level (ß = - 0.345, p = 0.001), male gender (ß = - 0.274, p = 0.009) and TIBC (ß = - 0.220, p = 0.030) were independent predictors of increased P wave dispersion. In the CKD group, TIBC (ß = - 0.285, p = 0.013) was an independent predictor of QTc dispersion while serum calcium (ß = 0.320, p = 0.002) and male gender (ß = - 0.274, p = 0.009) were independent predictors of Tp-e/QT ratio. CONCLUSIONS: Patients with stage 3-5 CKD and those with ESRD on regular hemodialysis exhibit significant ECG changes that are considered substrates for ventricular as well as supraventricular arrhythmias. Those changes were more evident in patients on hemodialysis.

Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers.

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers. SIGNIFICANCE: TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients.This article is highlighted in the In This Issue feature, p. 983.

Chemerin level and the relation to insulin resistance in chronic kidney disease.

Chemerin has been associated with different components of the metabolic syndrome, including hypertension, hyperlipidemia, and insulin resistance (IR). The aim of this study was to evaluate serum chemerin level in chronic kidney disease (CKD) patients and its relation to IR. This study was conducted on 80 participants who were classified into three groups: Group I (30 CKD patients with mean age 53 ± 12 years), Group II (30 patients with end-stage renal disease on regular hemodialysis with mean age 48 ± 14.8 years) and Group III having 20 healthy age-and sex-matched controls. Serum chemerin level, fasting blood sugar, fasting insulin, HOMA-IR index calculation, urea, creatinine, estimated glomerular filtration rate, total cholesterol, and triglyceride were measured. Body composition was assessed by dual-energy X-ray absorptiometry. In Groups I and II, we found a significantly higher mean chemerin level compared to healthy controls (P <0.001), a highly significant positive correlation between mean chemerin level and the HOMA-IR index [r = 0.56, P <0.001/(r = 0.53, P <0.001)], and a highly significant negative correlation between mean chemerin level and GFR (r = -0.51, P <0.001/r = -0.46, P <0.001). In Group I, there was also a highly significant positive correlation between mean chemerin and systolic blood pressure (r = 0.31, P <0.05), diastolic blood pressure (r = 0.39, P <0.05 and creatinine (r = 0.34, P <0.05). Chemerin might be considered a uremic IR adipokine marker in CKD Stages 3, 4, and 5.

MPN10 score and survival of molecularly annotated myeloproliferative neoplasm patients.

JAK2, CALR, MPL and triple-negative mutational status has a direct impact on symptom severity and disease burden assessed by MPN10 score in myeloproliferative neoplasms (MPNs). Among 93 patients; median MPN10 score was 48 (5-76) in JAK2 mutants versus 25 (4-80) in JAK2 negative (p < .001); 22.5 (4-65) in CALR mutants versus 35 (5-80) in CALR negative (p < .050) and 21 (10-48) in triple negative versus 40 (4-80) in JAK2/CALR/MPL mutants (p < .001). At three years, progression free and overall survival of JAK2-positive versus JAK2-negative patients were 62% versus 100% (p < .001); 85% versus 100% (p = .011) and were 100% versus 78% (p = .067); 100% versus 92% (p = .197) in CALR-positive versus CALR-negative patients and 100% versus 75% (p = .004); 100% versus 90% (p = .015) in triple negative versus mutant patients, respectively. MPN10 score in association with driver gene mutations can be used as a predictor of survival in MPN patients.

Durable Complete Response from Metastatic Melanoma after Transfer of Autologous T Cells Recognizing 10 Mutated Tumor Antigens.

Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669-78. ©2016 AACR.

Isolation and Characterization of an HLA-DPB1*04: 01-restricted MAGE-A3 T-Cell Receptor for Cancer Immunotherapy.

Long-term tumor regressions have been observed in patients following the adoptive transfer of autologous tumor-infiltrating lymphocytes or genetically modified T cells expressing MHC class I-restricted T-cell receptors (TCRs), but clinical trials have not evaluated responses to genetically modified T cells expressing antitumor MHC class II-restricted TCRs. As studies carried out in a murine tumor model system have demonstrated that the adoptive transfer of CD4 T cells could lead to the regression of established tumors, we plan to test the hypothesis that CD4 T cells can also induce tumor regressions in cancer patients. In this study, 2 MAGE-A3-specific TCRs were isolated from a regulatory T-cell clone (6F9) and an effector clone (R12C9), generated from the peripheral blood of 2 melanoma patients after MAGE-A3 vaccination. The results indicated that T cells transduced with 6F9 TCR mediated stronger effector functions than R12C9 TCR. The 6F9 TCR specifically recognized MAGE-A3 and the closely related MAGE-A6 gene product, but not other members of the MAGE-A family in the context of HLA-DPB1*04:01. To test the feasibility of a potential clinical trial using this TCR, a clinical-scale procedure was developed to obtain a large number of purified CD4 T cells transduced with 6F9 TCR. Because HLA-DPB1*04:01 is present in ∼60% of the Caucasian population and MAGE-A3 is frequently expressed in a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients.

Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes.

Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen-specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC-binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients' peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.

A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: long-term follow-up and correlates with response.

PURPOSE: Although adoptive cell therapy can be highly effective for the treatment of patients with melanoma, the application of this approach to the treatment of other solid tumors has been limited. The observation that the cancer germline (CG) antigen NY-ESO-1 is expressed in 70% to 80% and in approximately 25% of patients with synovial cell sarcoma and melanoma, respectively, prompted us to perform this first-in-man clinical trial using the adoptive transfer of autologous peripheral blood mononuclear cells that were retrovirally transduced with an NY-ESO-1-reactive T-cell receptor (TCR) to heavily pretreated patients bearing these metastatic cancers. EXPERIMENTAL DESIGN: HLA-*0201 patients with metastatic synovial cell sarcoma or melanoma refractory to standard treatments and whose cancers expressed NY-ESO-1 received autologous TCR-transduced T cells following a lymphodepleting preparative chemotherapy. Response rates using Response Evaluation Criteria in Solid Tumors (RECIST), as well as immunologic correlates of response, are presented in this report. RESULTS: Eleven of 18 patients with NY-ESO-1(+) synovial cell sarcomas (61%) and 11 of 20 patients with NY-ESO-1(+) melanomas (55%) who received autologous T cells transduced with an NY-ESO-1-reactive TCR demonstrated objective clinical responses. The estimated overall 3- and 5-year survival rates for patients with synovial cell sarcoma were 38% and 14%, respectively, whereas the corresponding estimated survival rates for patients with melanoma were both 33%. CONCLUSIONS: The adoptive transfer of autologous T cells transduced with a retrovirus encoding a TCR against an HLA-A*0201 restricted NY-ESO-1 epitope can be an effective therapy for some patients bearing synovial cell sarcomas and melanomas that are refractory to other treatments.

Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions.

PURPOSE: Cancer immunotherapy with adoptive transfer of tumor-infiltrating lymphocytes (TIL) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear. EXPERIMENTAL DESIGN: Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised nonsynonymous mutation sequences identified by whole-exome sequencing of autologous tumors. RESULTS: Screening of an autologous melanoma cDNA library using a conventional approach led to the identification of previously undescribed nonmutated targets recognized by TIL 2359 or TIL 2591. In contrast, screening of tandem minigene libraries encoding tumor-specific mutations resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation. CONCLUSIONS: These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate-mutated antigens derived from genes essential for the carcinogenesis.

In palindromic rheumatism, hand joint involvement and positive anti-CCP antibodies predict RA development after 1 year of follow-up.

This study aimed to determine the frequency of rheumatoid factor (RF) and cyclic citrullinated peptide (CCP) antibodies in a cohort of patients with palindromic rheumatism (PR) and to find determinants for progression to rheumatoid arthritis (RA). All new cases of PR (n=90) were included prospectively and followed up for 1 year, and a comparison group of RA cases (n=70) was also included. At study entry in all patients in both groups, RF and anti-CCP antibodies were tested, and the findings were compared and correlated. In the PR group at presentation, RF was positive in 30 patients (33.3%) and, in the RA group, in 45 patients (64.3%). Anti-CCP antibodies were positive in 35 patients (38.9%) with PR and in 58 patients (82.9%) with RA. In the PR group, positive correlations were observed between RF and C-reactive protein (CRP) (p=0.036), while anti-CCP positively correlated with disease duration (p=0.015) and CRP (p<0.001). At 1-year follow-up, 25 cases (27.5%) had progressed to RA, 3 (3.3%) cases had developed systemic lupus, 43 cases had responded to hydroxychloroquine with complete remission, five cases had developed other rheumatic diseases, and 14 cases had progressed to undifferentiated arthritis. After regression analysis, the involvement of hand joints and positive anti-CCP were the only predictors that determined progression into RA within a year (p<0.001 and p=0.02, respectively). Early hand joint involvement and positive anti-CCP at disease onset are good predictors for progression to RA in this domain.

Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.

Substantial regressions of metastatic lesions have been observed in up to 70% of patients with melanoma who received adoptively transferred autologous tumor-infiltrating lymphocytes (TILs) in phase 2 clinical trials. In addition, 40% of patients treated in a recent trial experienced complete regressions of all measurable lesions for at least 5 years following TIL treatment. To evaluate the potential association between the ability of TILs to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, we developed a new screening approach involving mining whole-exome sequence data to identify mutated proteins expressed in patient tumors. We then synthesized and evaluated candidate mutated T cell epitopes that were identified using a major histocompatibility complex-binding algorithm for recognition by TILs. Using this approach, we identified mutated antigens expressed on autologous tumor cells that were recognized by three bulk TIL lines from three individuals with melanoma that were associated with objective tumor regressions following adoptive transfer. This simplified approach for identifying mutated antigens recognized by T cells avoids the need to generate and laboriously screen cDNA libraries from tumors and may represent a generally applicable method for identifying mutated antigens expressed in a variety of tumor types.

Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression.

Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.

Characterization of T-cell receptors directed against HLA-A*01-restricted and C*07-restricted epitopes of MAGE-A3 and MAGE-A12.

The ability of T cells that have been genetically engineered to express T-cell receptors (TCRs) directed against tumor antigens to mediate tumor regression has been demonstrated in several clinical trials. These TCRs have primarily targeted HLA-A*0201-restricted TCRs, as approximately 50% of whites, who represent the predominant population of patients who develop melanomas, expresses this HLA class I allele. These therapies could be extended to additional patients through the use of TCRs that target epitopes that are presented by additional class I alleles that are prevalent in this population such as HLA-C*07 and HLA-A*01, which are expressed by approximately 50% and 30% of the patient population respectively. Therefore, 2 TCRs that recognize an epitope of MAGE-A12 in the context of HLA-C*07 and 2 TCRs that recognize an epitope of MAGE-A3 in the context of HLA-A*01 were isolated from tumor-reactive T-cell clones and cloned in a recombinant retroviral expression vector. Comparative studies indicated that one of the 2 MAGE-A3-reactive TCRs and one of the 2 MAGE-A12-reactive TCRs were superior to the additional TCRs in conferring transduced peripheral blood mononuclear cells with the capacity to recognize a broad array of antigen and MHC-positive target cells. These results provide support for the use of these TCRs in cancer adoptive immunotherapy trials.

An antigenic peptide produced by reverse splicing and double asparagine deamidation.

A variety of unconventional translational and posttranslational mechanisms contribute to the production of antigenic peptides, thereby increasing the diversity of the peptide repertoire presented by MHC class I molecules. Here, we describe a class I-restricted peptide that combines several posttranslational modifications. It is derived from tyrosinase and recognized by tumor-infiltrating lymphocytes isolated from a melanoma patient. This unusual antigenic peptide is made of two noncontiguous tyrosinase fragments that are spliced together in the reverse order. In addition, it contains two aspartate residues that replace the asparagines encoded in the tyrosinase sequence. We confirmed that this peptide is naturally presented at the surface of melanoma cells, and we showed that its processing sequentially requires translation of tyrosinase into the endoplasmic reticulum and its retrotranslocation into the cytosol, where deglycosylation of the two asparagines by peptide-N-glycanase turns them into aspartates by deamidation. This process is followed by cleavage and splicing of the appropriate fragments by the standard proteasome and additional transport of the resulting peptide into the endoplasmic reticulum through the transporter associated with antigen processing (TAP).

Irradiation enhances human T-cell function by upregulating CD70 expression on antigen-presenting cells in vitro.

In addition to the direct killing of tumor cells, radiation therapy can alter the balance of immune cells in vivo due to the differential radiosensitivity of different cell types. The addition of adjuvant radiation therapy before adoptive cell transfer therapy has been shown to enhance antitumor responses in both mouse models and clinical trials. This study examines the effects of in vitro irradiation on the phenotype and function of human antigen-presenting cells. The results indicated that irradiation upregulated CD70 expression on both B cells and mature dendritic cells (DCs). Expression of CD70 on mature DCs was enhanced in a dose-dependent manner, whereas under the same conditions, no significant upregulation of CD80, CD86, or CD40 was observed. The levels of expression of CD70 induced on mature DC by irradiation correlated highly with the ability of those cells to stimulate T-cell proliferation and interferon-γ production. Furthermore, significant reductions in T-cell proliferation and interferon-γ production were seen when CD70 expression on DCs was partially reduced using shRNA, as well as when DCs were incubated with a blocking anti-CD70 antibody. Radiation therapy may therefore enhance T-cell activation in vivo through the CD27 pathway by virtue of its ability to upregulate the expression of CD70 on antigen-presenting cells.

Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.

PURPOSE: Adoptive immunotherapy using tumor-infiltrating lymphocytes represents an effective cancer treatment for patients with metastatic melanoma. The NY-ESO-1 cancer/testis antigen, which is expressed in 80% of patients with synovial cell sarcoma and approximately 25% of patients with melanoma and common epithelial tumors, represents an attractive target for immune-based therapies. The current trial was carried out to evaluate the ability of adoptively transferred autologous T cells transduced with a T-cell receptor (TCR) directed against NY-ESO-1 to mediate tumor regression in patients with metastatic melanoma and synovial cell sarcoma. PATIENTS AND METHODS: A clinical trial was performed in patients with metastatic melanoma or metastatic synovial cell sarcoma refractory to all standard treatments. Patients with NY-ESO-1-positive tumors were treated with autologous TCR-transduced T cells plus 720,000 iU/kg of interleukin-2 to tolerance after preparative chemotherapy. Objective clinical responses were evaluated using Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS: Objective clinical responses were observed in four of six patients with synovial cell sarcoma and five of 11 patients with melanoma bearing tumors expressing NY-ESO-1. Two of 11 patients with melanoma demonstrated complete regressions that persisted after 1 year. A partial response lasting 18 months was observed in one patient with synovial cell sarcoma. CONCLUSION: These observations indicate that TCR-based gene therapies directed against NY-ESO-1 represent a new and effective therapeutic approach for patients with melanoma and synovial cell sarcoma. To our knowledge, this represents the first demonstration of the successful treatment of a nonmelanoma tumor using TCR-transduced T cells.

Recognition of NY-ESO-1+ tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression.

The therapeutic use of T cell receptor (TCR)-transduced peripheral blood lymphocytes (PBL) targeting tumor-associated antigens is emerging as a promising investigational treatment for patients with cancer. Initial response rates to therapy were low, suggesting the need to improve the function of TCR-transduced PBL. We constructed standard bicistronic retroviral vectors using an internal promoter or internal ribosomal entry site element as well as vectors incorporating coding sequences for 2A linker peptides between coding sequences for alpha and beta chains targeting the cancer-testis (CT) antigen, NY-ESO-1. Incorporation of coding sequences for 2A linker peptides in the bicistronic TCR expression cassette resulted in up to a fourfold increase in TCR expression and a significant improvement in effector function as measured by interferon-gamma release following co-culture with peptide-pulsed targets and NY-ESO-1+ tumors. We also sought to enhance reactivity of TCR-transduced PBL against tumor targets by modulation of tumor antigen expression on target cells. Induction of NY-ESO-1 expression on tumor targets using the demethylating agent 5-aza-2'-deoxycytidine (alone or in combination with the histone deacetylase inhibitor depsipeptide) resulted in enhanced interferon-gamma secretion by the TCR-transduced PBL on culture with treated targets. Taken together, these results indicate that design of TCR-based vectors incorporating 2A linker peptides improves TCR expression and effector function of transduced PBL. Furthermore, induction of CT antigen expression through treatment of tumor targets with chromatin-remodeling agents may augment TCR-based immunotherapy targeting these antigens. These results have relevance for TCR-based gene therapies targeting common epithelial malignancies.

Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions.

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.

Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond.

Adoptive transfer of genetically T-cell receptor (TCR)-modified lymphocytes has been recently reported to cause objective cancer regression. However, a major limitation to this approach is the mispairing of the introduced chains with the endogenous TCR subunits, which leads to reduced TCR surface expression and, subsequently, to their lower biological activity. We here show that it is possible to improve TCR gene transfer by adding a single cysteine on each receptor chain to promote the formation of an additional interchain disulfide bond. We show that cysteine-modified receptors were more highly expressed on the surface of human lymphocytes compared with their wild-type counterparts and able to mediate higher levels of cytokine secretion and specific lysis when cocultured with specific tumor cell lines. Furthermore, cysteine-modified receptors retained their enhanced function in CD4(+) lymphocytes. We also show that this approach can be employed to enhance the function of humanized and native murine receptors in human cells. Preferential pairing of cysteine-modified receptor chains accounts for these observations, which could have significant implications for the improvement of TCR gene therapy.