scholarly journals Doxorubicin-conjugated bacteriophages carrying anti-MHC class I chain-related A for targeted cancer therapy in vitro

2014 ◽  
pp. 2183
Author(s):  
Chanvit Leelayuwat ◽  
Achara Phumyen ◽  
Siriporn Chantasorn ◽  
Amonrat Jumnainsong
Keyword(s):  
Cancer Therapy ◽  
Mhc Class I ◽  
Class I ◽  
Targeted Cancer Therapy

Direct binding of peptide to empty MHC class I molecules on intact cells and in vitro

Cell ◽  
1990 ◽  
Vol 62 (3) ◽  
pp. 563-567 ◽  
Author(s):  
Ton N.M. Schumacher ◽  
Marie-Thérèse Heemels ◽  
Jacques J. Neefjes ◽  
W.Martin Kast ◽  
Cees J.M. Melief ◽  
...  
Keyword(s):  
Mhc Class I ◽  
Class I ◽  
Intact Cells ◽  
Direct Binding ◽  
Mhc Class I Molecules

ATP is required for in vitro assembly of MHC class I antigens but not to transfer of peptides acrosss the ER membrane

Cell ◽  
1991 ◽  
Vol 67 (2) ◽  
pp. 265-274 ◽  
Author(s):  
Frédéric Lévy ◽  
Reinhard Gabathuler ◽  
Roger Larsson ◽  
Sune Kvist
Keyword(s):  
Mhc Class I ◽  
Class I ◽  
Mhc Class I Antigens ◽  
In Vitro Assembly ◽  
Er Membrane

scholarly journals Immunotherapy of a murine tumor with interleukin 2. Increased sensitivity after MHC class I gene transfection.

1987 ◽  
Vol 166 (6) ◽  
pp. 1716-1733 ◽  
Author(s):  
J S Weber ◽  
G Jay ◽  
K Tanaka ◽  
S A Rosenberg
Keyword(s):  
T Cells ◽  
Mhc Class I ◽  
Interleukin 2 ◽  
Class I ◽  
High Dose ◽  
Class I Mhc ◽  
High Doses ◽  
I Gene

We have shown that two weakly immunogenic MCA sarcomas developed in our laboratory that are sensitive to high-dose IL-2 immunotherapy express class I MHC in vivo and in vitro. Two nonimmunogenic MCA sarcomas are relatively insensitive to IL-2 therapy and express minimal or no class I MHC molecules in vivo and in vitro. To study the role of MHC in the therapy of tumors with IL-2, a class I-deficient murine melanoma, B16BL6, was transfected with the Kb class I gene. Expression of class I MHC rendered B16BL6 advanced pulmonary macrometastases sensitive to IL-2 immunotherapy. 3-d micrometastases of CL8-2, a class I transfected clone of B16BL6, were significantly more sensitive to IL-2 therapy than a control nontransfected line. Expression of Iak, a class II MHC molecule, had no effect on IL-2 therapy of transfectant pulmonary micrometastases in F1 mice. By using lymphocyte subset depletion with mAbs directed against Lyt-2, therapy of class I transfectant macrometastases with high-dose IL-2 was shown to involve an Lyt-2 cell. In contrast, regression of micrometastases treated with low-dose IL-2 involved Lyt-2+ cells, but regression mediated by high doses of IL-2 did not. We hypothesize that both LAK and Lyt-2+ T cells effect IL-2-mediated elimination of micrometastases, but only Lyt-2+ T cells are involved in macrometastatic regression. Low doses of IL-2 stimulate Lyt-2+ cells to eliminate class I-expressing micrometastases, but high doses of IL-2 can recruit LAK cells to mediate regression of micrometastases independent of class I expression. Only high-dose IL-2, mediating its effect predominantly via Lyt-2+ cells, is capable of impacting on MHC class I-expressing macrometastases. Macrometastases devoid of class I MHC antigens appear to be resistant to IL-2 therapy.

Generation of Antigen Specific Cytotoxic T Lymphocytes (CTL) by Dendritic Cells (DCs) Transfected with In Vitro Transcribed (IVT) SART-1 and WT-1 mRNA.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3859-3859
Author(s):  
Anri Saito ◽  
Miwako Narita ◽  
Toshio Yano ◽  
Naoko Sato ◽  
Asuka Sekiguchi ◽  
...  
Keyword(s):  
Mhc Class I ◽  
Tumor Antigen ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Target Cells ◽  
Mhc Restriction ◽  
Specific Ctls ◽  
Specific Antigens

Abstract Transfection with tumor antigen RNA is one of the promising tools not only because of a possible sufficient amplification of tumor antigen RNA but also because of the absence of antigen peptides-associated MHC restriction. Several succeeded experiments about generation of CTLs using DCs transfeced in vitro transcribed (IVT) cancer specific antigen mRNA such as PSA, CEA, hTERT and MUC-1 have been reported in these a few years. In addition, recent reports about the simultaneous presentation of peptides in both MHC class I and class II molecules on DCs after mRNA electroporation show another superiority of mRNA transfection into DCs. In this presentation, we demonstrate successful generation of tumor antigen specific CTLs using with DCs transfected with IVT mRNA such as SART-1 and WT-1 by electroporation. This is the first report about the generation of SART-1 and WT-1 specific CTLs by using mRNA transfected DCs. [Methods] HLA-A24 positive human PB CD14+ cell-derived DCs were transfected with IVT mRNA (SART-1and WT-1) by electroporation. MRNA transfected DCs were co-cultured with autologous lymphocytes. The bulk co-cultures were re-stimulated several times with same DCs. CD8+ cells were separated and CTL activity was evaluated by 51chromium release assay. To determine whether the induced CTL cells could recognize the target cells in an HLA class I restricted manner, anti-HLA class I monoclonal antibodies were utilized to block the cytotoxicity of effectors. [Results] Electroporation of mRNA showed no effect on the surface phenotypes and antigen presenting ability of DCs. In addition to the demonstration of efficient transfection of M1 mRNA into DCs by using RT-PCR, which eliminated the amplification of transfected mRNA by the treatment with RNase before RNA extraction from the transfected cells, we identified the definite expression of WT-1 protein in the cytoplasm of DCs by using immunoblotting. CTL assay indicated that 1) DCs transfected with mRNA stimulated the generation of antigen-specific CTLs which are capable of lysing autologous DCs transfected with the same mRNA. 2) CTLs also demonstrated cytotoxic ability against cell lines such as KE-4 presenting SART-1 peptides on HLA-A24, MEGO1 presenting WT-1 peptides on MHC class I, and HLA-A24 cDNA transfected T2 which were used as target cells after co- incubation with 9 mer SART-1 peptides with strong affinity to HLA-A24. 3) Each cytotoxicities were markedly blocked after co-incubation of target cells with anti-MHC class I antibody and not inhibited with anti-MHC class II antibody. [Conclusion] Our results showed that IVT mRNA-transfected DCs which is constructed non-virally have a highly efficient ability to stimulate specific T-cell immunity against tumor. Unlike peptide- or tumor cells extract-pulsed DCs based vaccines, anti-tumor immunotherapy using the DCs transfected with antigen mRNA could be extended to a wide range of patients who have previously been excluded from clinical trials for the reason of the un-identification of tumor specific antigens, for the reason of the impossibility of obtaining sufficient tumor specimens, or for the reason of MHC restriction of the tumor specific antigens.

In Vitro Stimulation with WT1-Peptide Mix Elicits Functional Polyclonal CTL From Adult Donors and Cord Blood

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2096-2096
Author(s):  
Deepa Kolaseri Krishnadas ◽  
Mindy Stamer ◽  
Kim Dunham ◽  
Lei Bao ◽  
Kenneth Lucas
Keyword(s):  
Cord Blood ◽  
Mhc Class I ◽  
Healthy Adult ◽  
Class I ◽  
Target Cells ◽  
Cell Transplant ◽  
Blood Lymphocytes ◽  
Leukemic Relapse ◽  
Ifn Γ

Abstract Abstract 2096 Elevated expression of Wilms’ tumor antigen (WT1) in most leukemias and limited expression on normal tissues makes WT1 a potential target for cancer immunotherapy. Fighting leukemia using in vitro generated WT1-CTL has been under active study. Previous studies have focused mainly on cloning WT1-CTL or generating CTL that are limited to a single epitope. We have recently generated WT1-A*0201 peptide-specific CTL from peripheral blood lymphocytes (PBL) of healthy adult donors by enriching and expanding antigen-specific CD137+ cells. Using this method we demonstrate the generation of polyclonal WT1-CTL from healthy adult donors and from cord blood by stimulating with an overlapping pool of peptides derived from full length WT1. The WT1-peptide mix (WT1-pMix) is a mix of 110 peptides consisting 15-mers overlapped by 11 amino acids. The WT1-pMix stimulated PBL from adult donors were selected based on CD137+ expression. After three stimulations, 0.3% of the cells were positive for CD137. These cells were expanded with anti-CD3 and IL-2 resulting in 100–200 fold expansion. Therefore, from a typical apheresis product yielding approximately 4–10 × 109 cells, approximately 1.2 – 3 × 109 WT1-specific cells could be generated for immunotherapy. The expanded CTL displayed antigen-specific IFN-γ production in four out of five adult donors studied. They also killed antigen-specific target cells and WT1 expressing leukemia lines in a MHC restricted manner. Occasionally, these CTL did not produce IFN-γ although they demonstrated killing. WT1-specific cytotoxicity directed against partially matched leukemia lines (Molt-4 and 697) sharing MHC-class I was partially blocked with mAb against MHC-class I but not against MHC-class II and an unmatched leukemia line (BA25) remained close to negative control. Similar to functionally active WT1-CTL from healthy adult donors, polyclonal CTL were generated from cord blood using WT1-pMix with a potential specificity for several HLA backgrounds. These CTL are CD4 and CD8 positive. Three out of five donors studied produced IFN-γ and lysed autologous B blast (BB) pulsed with WT1-pMix and partially HLA matched WT1 expressing leukemia lines. Since recipients of unrelated donor cord blood transplants have no options for donor derived cellular immunotherapy in the event of leukemic relapse, WT1-specific CTL generated from cord blood mononuclear cells could be used to prevent or treat leukemic relapse in these patients. This method utilizes unmodified PBL or cord blood lymphocytes to generate clinically relevant number of CTL in 5–6 weeks following initial stimulation, potentially avoiding the need for CD8+ selection. The generation of WT1-specific CTL using an overlapping WT1-pMix enhances our ability to treat patients from several HLA backgrounds. These findings provide the foundation for further pre-clinical studies in which these cells could be isolated from donor T lymphocytes, and then infused back into patients for treatment or prevention of leukemic relapse in high risk stem cell transplant (SCT) patients. Disclosures: No relevant conflicts of interest to declare.

Non-Cognate Cytotoxic T Cells Can Be Retargeted Against CD20 Positive Tumour Cells Using [Fab' × MHC Class I/Peptide] Conjugates.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2838-2838
Author(s):  
Angela D Hamblin ◽  
Ben CR King ◽  
Ruth R French ◽  
Claude H Chan ◽  
Alison L Tutt ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Mhc Class I ◽  
Peripheral Blood ◽  
Class I ◽  
B Cell Depletion ◽  
Cytokine Release ◽  
Anti Cd20

Abstract Abstract 2838 To circumvent cytotoxic T lymphocyte (CTL) tolerance of tumour-associated antigens, the concept of redirecting CTLs against non-cognate targets has developed. One way of doing this is to use bispecific antibodies comprising anti-CD3 and anti-tumour antigen moieties. Unfortunately, this is frequently associated with unacceptable toxicity due to inflammatory cytokine release. As an alternative our approach has been to use a bivalent conjugate recognising a tumour antigen (through an antibody fragment) and a defined population of CTLs (specific for a single antigenic peptide e.g. viral epitope) through peptide presented in the context of recombinant MHC class I. We have produced a conjugate consisting of an anti-human CD20 Fab' fragment joined via a chemical crosslinker (succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate) to murine MHC class I/peptide (Kbα1-α3 domains/β2microglobulin presenting the ovalbumin-derived peptide SIINFEKL; expressed bacterially as a continuous polypeptide single chain trimer after Yu et al, J Immunol 2002). Size exclusion chromatography allowed purification of conjugates with [Fab':MHC class I/peptide] ratios of 1:1 and 2:1 (F2 and F3 respectively). In vitro both constructs were able to redirect the transgenic murine CTL line OT-1 (specific for KbSIINFEKL) to lyse human CD20+ tumour cells (lymphoblastoid Daudi cell line) at effector: target ratios of 10:1. This lysis could be blocked by the addition of 100 fold excess of either anti-CD20 F(ab')2 or the Kb/SIINFEKL-specific antibody 25D1. The constructs were also able to cause in vitro proliferation of naïve OT-1 cells (but not irrelevant CD8+ T cells) in the presence of human CD20+ cells in both thymidine incorporation and CFSE dilution assays. Using a human CD20 transgenic mouse model (Ahuja et al, J Immunol 2007) we have evaluated both constructs in vivo for their ability to redirect adoptively transferred OT-1 cells to deplete B cells from the peripheral blood. A single dose of 1 nmole F3 and 2 nmole F2 caused respectively up to 95% and 85% B cell depletion at day 7. The efficacy of lower doses suggested a dose: response relationship. As a marker of toxicity, we have measured cytokine levels at 2, 8 and 24 hours following a dose of 1 nmole F3 and compared them to those seen after administration of an [anti-CD3 × anti-CD20] bispecific F(ab')2 at a dose (0.5 nmole) which produced similar day 7 peripheral blood B cell depletion: phosphate-buffered saline was given as a negative control. Maximal cytokine release was seen at 2 hours with the levels of IL-4, IL-5, KC, IL-2 and IL-10 being lower after administration of the F3 than after the bispecific F(ab')2. However, interestingly, the F3 resulted in greater IL-12 release. Overall these data suggest that [Fab' × MHC class I/peptide] constructs have the potential to redirect non-cognate CTLs to deplete CD20+ malignant B cells from the peripheral blood and that this is associated with a lower level of cytokine release than a similarly efficacious dose of an anti-CD3-containing bispecific F(ab')2. Furthermore, the ability of [Fab' × MHC class I/peptide] constructs to cause proliferation of OT-1 cells in vitro suggests it may be possible to use a single molecule to both generate a secondary cytotoxic T cell response and subsequently to retarget it, increasing the viability of the approach if adopted in the clinic. Disclosures: No relevant conflicts of interest to declare.

Sialoadhesin-Positive Host Macrophages Play an Essential Role in Graft-Versus-Leukemia Reactivity in Mice

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4375-4386 ◽  
Author(s):  
Susanne Müerköster ◽  
Marian Rocha ◽  
Paul R. Crocker ◽  
Volker Schirrmacher ◽  
Victor Umansky
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Class I ◽  
Peptide Epitope ◽  
Peptide Epitopes ◽  
Graft Versus Leukemia

We recently established an effective immune T-cell–mediated graft-versus-leukemia (GVL) murine model system in which complete tumor remissions were achievable even in advanced metastasized cancer. We now describe that this T-cell–mediated therapy is dependent on host macrophages expressing the lymphocyte adhesion molecule sialoadhesin (Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive immunotherapy (ADI) or the treatment of these animals with anti-Sn monoclonal antibodies led to complete or partial inhibition of the immune T-cell–mediated therapeutic effect. Furthermore, Sn+ host macrophages in livers formed clusters during ADI with donor CD8 T cells. To test for a possible antigen presentation function of these macrophages, we used as an in vitro model the antigen β-galactosidase for which a dominant major histocompatibility complex (MHC) class I Ld-restricted peptide epitope is known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL). We demonstrate that purified Sn+ macrophages can process exogenous β-galactosidase and stimulate MHC class I peptide-restricted CTL responses. Thus, Sn+ macrophages, which are significantly increased in the liver after ADI, may process tumor-derived proteins via the MHC class I pathway as well as via the MHC class II pathway, as shown previously, and present respective peptide epitopes to CD8 as well as to CD4 immune T cells, respectively. The synergistic interactions observed before between immune CD4 and CD8 T cells during ADI could thus occur in the observed clusters with Sn+ host macrophages.

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