scholarly journals P07.02 High-affinity TCRs specific for cancer testis antigens as a therapy for multiple myeloma and solid tumors

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A49.1-A49
Author(s):  
MAJ de Rooij ◽  
DM van der Steen ◽  
D Remst ◽  
A Wouters ◽  
M van der Meent ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cross Reactivity ◽  
Tumor Cell Lines ◽  
High Affinity ◽  
Cancer Testis Antigens ◽  
Efficient Recognition ◽  
Cancer Testis

BackgroundCancer Testis Antigens (CTAs) are highly expressed in multiple different tumor types, but silent in normal tissue, except the testis. This tumor-restricted expression pattern makes them an ideal target for adoptive T-cell therapy. However, the responsiveness in clinical setting may be hampered because high-affinity T cells against self-antigens presented in the context of self-HLA are deleted in the thymus by negative selection. In this study, we aim to identify high-affinity T cell receptors (TCRs) specific for CTAs from the allogeneic-HLA repertoire.Materials and MethodsIn this study, HLA class I binding peptides derived from different CTA genes were identified by HLA-peptide elution experiments and subsequent mass spectrometric analysis. From the identified peptides HLA tetramers were generated to isolate peptide specific CD8+ T cells from healthy allogeneic donors. Efficacy and safety of the TCRs was determined by various different stimulation assays. The most potent TCRs were sequenced, analyzed and transduced into peripheral CD8+ and CD4+ T cells to confirm CTA specific cytokine production and cytotoxicity.ResultsMAGE and CTAG peptides were eluted from multiple myelomas, EBV-transformed lymphoblastic cells, acute myeloid leukemia and ovarium carcinomas. We selected TCRs recognizing 3 different MAGE-A1 peptides in the context of HLA-A*02:01, HLA-A*03:01 and HLA-B*07:02. Furthermore, we selected TCRs specific for MAGE-A3 in the context of HLA-B*35:01 and HLA-A*01:01; TCRs specific for MAGE-A9 in the context of HLA-A*01:01 and TCRs specific for CTAG1 in the context of HLA-A*02:01. The selected T-cell clones demonstrated efficient recognition of MAGE-A1, MAGE-A3 or CTAG1 positive multiple myeloma and solid tumor cell lines without detectable cross-reactivity.ConclusionsWe identified multiple different TCRs from the allogeneic-HLA repertoire specific for CTA genes. These TCRs demonstrate efficient recognition and killing of CTA positive multiple myeloma and solid tumor cell lines and did not show any cross-reactivity. The peptides recognized by the TCRs are presented in different HLA alleles. Since, 71% of the world population contains one of these HLA-alleles, a large percentage suffering from a MAGE or CTAG positive tumor could potentially be treated with the identified TCRs by TCR-gene therapy.Disclosure InformationM.A.J. de Rooij: None. D.M. van der Steen: None. D. Remst: None. A. Wouters: None. M. van der Meent: None. R.S. Hagedoorn: None. M.G.D. Kester: None. P.A. van Veelen: None. F.J.H. Falkenburg: None. M.H.M. Heemskerk: None.

Generation of a HuTCR mouse platform-derived MAGE-A1-directed high-affinity TCR with superior potency versus human-derived TCRs.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14515-e14515
Author(s):  
Ioannis Gavvovidis ◽  
Matthias Leisegang ◽  
Jenifer Oduro ◽  
Matthias Obenaus ◽  
Eugen Leo ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Cell Lines ◽  
Target Cells ◽  
Tumor Cell Lines ◽  
Functional Avidity ◽  
High Affinity ◽  
Selective Expression ◽  
Tumor Types ◽  
Cancer Testis

e14515 Background: As cancer-testis antigens are self-antigens, T cells expressing high-affinity TCRs against such antigens are suppressed via negative thymic selection. Therefore, patient- or donor-derived TCRs are typically of low affinity and result in a reduced antitumor effect. Using our proprietary HuTCR platform, which consists of mouse lines carrying the full human TCR α/β loci in combination with common human HLA alleles, we have isolated high-affinity TCRs specific for the cancer-testis antigen MAGE-A1 and compared them to human-derived MAGE-A1-specific TCRs that are currently reported to be in clinical development. Furthermore, we validated MAGE-A1 as a potential cancer therapy target by using immunohistochemistry to evaluate expression in several major tumor types and healthy tissue. Methods: Using scRNAseq, TCRs were isolated from HuTCR mice. Human-derived MAGE-A1-specific TCR sequences were obtained from publicly available databases. All TCRs were expressed in primary human T cells as verified using peptide-MHC-multimer staining. Functional avidity of the TCRs was analyzed by coculture with T2 target cells loaded with titrated amounts of epitope peptides and measuring cytokine concentration by ELISA. Reactivity of TCRs to endogenously processed MAGE-A1 protein was assessed by co-culture with a panel of tumor cell lines varying in MAGE-A1 and/or MHC-class-I expression. MAGE-A1 expression on protein level was evaluated by immunohistochemistry. Results: Immunization of HuTCR mice with the antigen resulted in robust CD8+ T cell responses and several TCR clonotypes were identified by scRNAseq, with the majority of clonotypes being specific to the MAGE-A1-derived peptide KVLEYVIKV and TCR affinities ranging from 0.3 nM to 3 nM. By comparison, human-derived TCRs exhibited generally lower functional avidity from 3 nM to 60 nM. In addition, HuTCR-mouse-derived TCRs were more sensitive in recognition of tumor cell lines expressing low MAGE-A1 and/or HLA-A2. Immunohistochemical analysis of MAGE-A1 expression in healthy tissues demonstrated highly selective expression of MAGE-A1 in testis, only. Screening for expression confirmed that a significant proportion of several major cancer types expresses MAGE-A1 as reported by various other groups [reviewed in Curr Opin Cell Biol. 2015 December; 37: 1–8]. Conclusions: The HuTCR mouse platform allows for the generation of high-affinity MAGE-A1-specific TCRs with increased anti-tumor efficacy as compared to human-derived TCRs against the same cancer antigen. In addition, it was confirmed that MAGE-A1 has a highly selective expression pattern in healthy tissues (testis, only), but shows distinct expression in several major human tumor types.

CD8+ T Cells Specific for Cancer-Testis Antigens Are Found in Many Patients with Multiple Myeloma and Correlate with Disease Burden.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2460-2460
Author(s):  
Oliver C. Goodyear ◽  
Karen Piper ◽  
Julie Arrazi ◽  
Naeem Khan ◽  
Premini Mahendra ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Specific Immune Response ◽  
Functional Responses ◽  
Cancer Testis Antigens ◽  
Cancer Testis

Abstract Proteins from the family known as ‘cancer-testis antigens’ (CTAg) are expressed in some cases of multiple myeloma and subsets of acute myeloid leukaemia. CTAg can stimulate CD8+ T cell responses in patients with melanoma but there are no reports of CTAg-specific immune response in patients with haematological malignancy. Such information is critical to assess whether or not these antigens act as targets for tumour-specific immunity or if they could be used as targets for immunotherapy. We have used twelve peptide epitopes from a range of cancer-testis antigens which have been previously defined as epitopes for CD8+ T cells. These were used to screen for tumour-specific T-cells in blood of patients with multiple myeloma at various stages of their disease. The IFNγ cytokine secretion assay was used to detect functional responses and magnetic selection was employed to increase the sensitivity of detection. FACS analysis was used to quantitate the frequency of responding cells. 37 patients were screened with an age range of between 45 and 88 years. Blood samples were taken at monthly intervals and the percentage of CD8+ T cells responding to each peptide was calculated. 13 patients responded to 1 or more of the peptides with a range between 0.01% and 0.7% of the total CD8+ T cell pool. The frequency of the tumour-specific response fluctuated during treatment in individual patients. Analysis of the CTAg-specific immune response in relation to disease course revealed that the immune response was generally correlated with tumour burden as revealed by the paraprotein level. CTAg HLA-peptide tetramers incorporating peptides from LAGE-1 and MAGE-2 were able to directly visualize CTAg-reactive T cells in PBMC. CTAg-specific CD8+ T cells may have been primed and expanded by expression of CTAg on tumour cells or following ‘cross presentation’ through dendritic cells. In conclusion, T cells specific for cancer-testis antigens are present in the blood of a subset of patients with multiple myeloma. The clinical significance of this observation is currently being addressed.

scholarly journals Local Activator and T Cell Engager (αBCMA x αPD-L1 x αCD3) with Enhanced Tumor Killing and Minimal Cytokine Release for the Treatment of Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Melissa Vrohlings ◽  
Stephanie Jungmichel ◽  
Jan Müller ◽  
David Senn ◽  
Thomas Schleier ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Cytokine Release ◽  
Private Company

BCMA-targeting bispecific T-cell engagers in clinical development have demonstrated encouraging preclinical efficacy. The most advanced of these is AMG 420, which showed significantly improved response rates in relapsed/refractory multiple myeloma (MM) patients. Nevertheless, median duration until relapse is currently limited to approximately 12 months, highlighting the need for new drugs with novel MoA. Recently, we reported on a Local Activator and T cell Engager (LocATE) antibody that targets BCMA and selectively blocks programmed death-ligand 1 (PD-L1) on malignant cells (ASCO, June 2019). LocATE induced superior T cell activation and cancer cell killing, in vitro and ex vivo, compared to a BCMAxCD3 BiTE alone or in combination with a PD-L1 inhibitor. Here, we sought to further characterize the novel MoA of our LocATE. To assess the therapeutic potential of the LocATE, we first investigated whether potent cytotoxicity is uncoupled from high levels of cytokine release. We evaluated three LocATE molecules with different PD-L1 affinities (low, medium, high). Using BCMA-expressing MM cell lines (U-266, MM.1S, RPMI-8226 and H929) with distinct PD-L1 surface expression levels (3 - 53%), we determined the cytokine profile (IL-2, IL-6, IFN-γ, TNF-α) and target cell lysis induced by each candidate in the presence of CD3-positive human T cells. All three candidates exhibited comparable killing potency, however, low-affinity PD-L1 LocATE antibodies induced significantly less cytokine release (up to 10-fold) than its higher PD-L1 affinity counterparts across all cell lines investigated. Notably, using the low-affinity PD-L1 LocATE, we observed a 2-fold increase in tumor cell killing compared to bispecific BCMAxCD3 targeting controls in cell lines expressing high PD-L1 levels (53%), underlining the contribution of PD-L1 inhibition. Accordingly, phenotypic profiling of effector cells showed that the LocATE more potently induced dose-dependent upregulation of the activation markers CD69, CD25 and HLA-DR compared to bispecific controls. Importantly, cytotoxic activity, T cell activation and cytokine release were not induced when BCMA-negative cells expressing high levels of PD-L1 were treated with LocATE, underlining the BCMA-selective killing mechanism. Since the superior efficacy of LocATE was found to correlate with the expression level of PD-L1 on MM cell lines and upregulation of PD-L1/PD-1 has been reported as one of the major myeloma cell escape mechanisms during treatment with BiTEs, we subsequently investigated the efficacy of LocATE using primary bone marrow samples and peripheral blood mononuclear cell (PBMCs) obtained from MM patients. Six bone marrow mononuclear cell (BMMC) and eight PBMC samples from MM donors of different disease stages were characterized for PD-1/PD-L1 expression levels; analysis of T cell frequency and level of activation/exhaustion was performed based on CD4, CD8, CD25, CD69, Tim-3, Lag-3 and PD-1 marker expression. Using patient samples with high frequencies of PD-1 expressing T cells prior to treatment, LocATE induced superior MM tumor cell lysis and T cell activation compared to BCMAxCD3 bispecific antibodies. No activity was observed on healthy cells, underlining the safe and selective killing mechanism through tumor-local reactivation of exhausted T cells. Collectively, these findings demonstrate that the simultaneous T cell redirection and tumor-specific checkpoint inhibition with the LocATE leads to an improved therapeutic index with robust tumor cell killing and low levels of cytokine release. Capable of counteracting adaptive immune resistance caused by increased PD-1/PD-L1 signaling, the LocATE antibody has the prospect to significantly improve survival for multiple myeloma patients. Disclosures Vrohlings: CDR-Life: Current Employment. Jungmichel:CDR-Life: Current Employment, Other: current option holder. Senn:CDR-Life: Current Employment. Schleier:CDR-Life: Current Employment, Current equity holder in private company. Scheifele:CDR-Life: Current Employment, Current equity holder in private company. Wendelspiess:CDR-Life: Current Employment. Leisner:CDR-Life: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Jaeger:CDR Life AG: Consultancy, Research Funding; Miltenyi: Consultancy, Honoraria; Karyopharm: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria; F. Hoffmann-La Roche: Honoraria, Research Funding. Borras:CDR-Life: Current Employment, Current equity holder in private company.

Personalized neoantigen/cancer testis antigen nanovaccine (PVAC) mobilize specific therapeutic immunity for high-risk resected gastric cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 4535-4535 ◽  
Author(s):  
Qin Liu ◽  
Hanqing Qian ◽  
Jie Shao ◽  
Qiuping Xu ◽  
Huizi Sha ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
T Cell ◽  
Cancer Patients ◽  
Stage Iiib ◽  
Cell Responses ◽  
Cancer Testis Antigens ◽  
Ct Antigens ◽  
Gastric Cancer Patients ◽  
Cancer Testis

4535 Background: 35% of stage IIIB/C Gastric cancer patients will recurrent after D2 gastrectomy within one year. Mutation-derived epitopes (neoantigens) has been demonstrated to induce tumor cell specific immune responses controlling the tumor growth. Nanovaccine can increase antigen presentation efficiency and elicit potent antitumor T cell responses with robust therapeutic efficacy. We hypothesized that vaccination with neoantigens/cancer testis (CT) antigens could expand pre-existing and induce antigen-specific T-cells populations, favouring of tumor control enhancement. Here, we report the first-in-human application of this concept in gastric cancer. Methods: Patient-specific mutation-containing neoantigens were selected on the basis of tumour-specific mutations whole-exome sequencing (WES) and RNA sequencing. Cancer testis antigens were obtained according to immunohistochemical staining and HLA-binding affinity prediction. PVAC is an amphiphiles nanovaccine loaded with multiple personalized neoantigens/cancer testis antigens designed to induce antigen specific T cells and associated antitumor responses. PVAC will be administrated to stage IIIB/IIIC gastric carcinoma after six cycles of adjuvant chemotherapy (S-1/Oxaliplatin or S-1/docetaxel). Each patient received PVAC by subcutaneous injection on Days 1, 4, 8, 15, 22, 43, 64, 85, 169, administrated with the adjuvant montanide ISA 51 VG. Safety, immunogenicity and clinical efficacy will be evaluated. Results: 25 stage IIIB or IIIC gastric cancer patients were enrolled in this study. Mean age was 54.3 years old (range: 34-70), and ECOG performance scores were 0 or 1. Repeated dosing has been well tolerated with mild local discomfort and no DLTs. Three patients were observed grade 2 local skin reactions in the injection sites. No SAEs related to PVAC have been observed. Among median follow up time of 12.6 months (range: 8.5-25.0 months), only two patients had local recurrence at 24.0 months and 10.5 months after surgery, respectivelt. The rest 23 patients remain disease free on study. Neoantigen specific T cell responses have been detected by IFN-γ Elispot from PBMCs. Conclusions: PVAC is a multiple neoantigen/CT antigens nanovaccine that personalizes tumor specific antigens and the individual patient’s capacity to respond. Addition of PVAC may prolong progression-free survival (PFS) after the standard of care chemotherapy. Clinical trial information: ChiCTR1800017319 .

Multiple Myeloma as Target for γδ T Cell Mediated Immunotherapy - Expression of the Putative Vγ9Vδ2-TCR Ligand F1-ATPase on Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3391-3391
Author(s):  
Volker Kunzmann ◽  
Judith Engert ◽  
Brigitte Kimmel ◽  
Martin Wilhelm ◽  
Hermann Einsele
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Surface Expression ◽  
Tumor Cell Lines ◽  
Myeloma Cells ◽  
F1 Atpase ◽  
Vγ9vδ2 T Cells

Abstract Activated Vγ9Vδ2 T cells, the major γδ T lymphocyte subset in humans, show cytolytic activity against various tumor cells. However, tumor antigens recognized by the TCR remained unkown so far. Recently, the ectopic surface expression of the F1-ATPase, normally expressed on the internal membrane of mitochondria, was implicated in tumor recognition of Vγ9Vδ2 T cells (Scotet E. et al., Immunity2005; 22:71–80). Surface expression of the a chain of the F1-ATPase (recognized by monoclonal antibody 7H10) strongly correlates with susceptibility of tumor cells against Vγ9Vδ2 T cell lysis. Different functions have been attributed to the ectopic expression of the F1-ATPase on the cell surface, including an immunoregulatory role induced by cell stress, receptor for angiostatin or regulation of lipoprotein transport through high-affinity apolipoprotein A-I binding. In this study we evaluated the surface expression of this F1-ATPase on hematopoetic tumor cell lines and on primary tumor cells from hematological malignancies. As already shown, the a subunit of F1-ATPase was clearly detected on several tumor cell lines which are consistently killed by activated Vγ9Vδ2 T cells (Daudi, K562, RPMI 8226), whereas the known Vγ9Vδ2 T cell resistant tumor cell lines (Raji, Jurkat) did not express detectable levels of the F1-ATPase. Analysis of 42 primary hematopoetic tumor cells (21 myeloma, 17 AML, 4 B-NHL) revealed frequent expression of F1-ATPase on primary myeloma cells (14/19 positive), whereas primary AML blasts (3/17 positive) and primary NHL cells (1/4 positive) expressed the putative Vγ9Vδ2-TCR ligand F1-ATPase less frequently. To further evaluate the functional role of F1-ATPase expression in Vγ9Vδ2 T cell mediated recognition of myeloma cells, cytotoxicity assays were performed. The mAb against the a subunit of F1-ATPase significantly decreased in vitro lysis of myeloma cells lines and primary myeloma cells by activated Vγ9Vδ2 T cells. These results suggests Vγ9Vδ2 TCR-dependent interactions between myeloma cells and Vγ9Vδ2 T cells and indicate that multiple myeloma should be considered as a major target for γδ T-cell mediated immunotherapy.

Alloreactivity as a Source of High Avidity T Cell Clones Specific for Cancer Testis-Antigens Associated with Multiple Myeloma: Prospects for Adoptive Immunotherapy with Primary T Cells Redirected toward Multiple Myeloma by T Cell Receptor Gene Transfer.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3712-3712
Author(s):  
Holger Kronig ◽  
Kathrin Hofer ◽  
Julia Neudorfer ◽  
Christian Peschel ◽  
Helga Bernhard
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
High Avidity ◽  
Peptide Epitopes ◽  
Ct Antigen ◽  
Ct Antigens ◽  
Cancer Testis

Abstract Cancer testis (CT)-antigens belong to a class of tumor antigens that are aberrantly expressed in a variety of hematological malignancies including multiple myeloma. Owing to their restricted gene expression, CT-antigens represent potential target antigens for immunotherapeutical approaches such as vaccination and adoptive T cell transfer. As the CT-antigens are self antigens, the majority of CT-antigen-specific autologous T cells display a low avidity T cell receptor (TCR), which often results in a weak tumor recognition efficiency. Our group has been focusing on the isolation of highly avid T cells against CT-antigens that are expressed in multiple myeloma, in particular MAGE-C1, MAGE-C2, and NY-ESO-1. The experimental approach was based on the stimulation of allo-restricted cytotoxic T cells, because highly avid T cells recognizing peptide epitopes in context with foreign HLA-alleles are not depleted in the thymus. HLA-A2-negative T cells were stimulated with HLA-A2-positive allogeneic dendritic cells that had been exogenously loaded with HLA-A2-binding peptides derived from NY-ESO-1, MAGE-C1 or MAGE-C2. Using this technique we were able to isolate allo-HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones with peptide-dominant binding against known and novel peptide epitopes derived from NY-ESO-1, MAGE-C1 and MAGE-C2. The expanded peptide-specific CTL clones lysed HLA-A2-positive myeloma cell lines expressing NY-ESO-1, MAGE-C1 and MAGE-C2, respectively. Of note, the MAGE-C1-specific T cells crossreacted with the corresponding MAGE-C2 peptide due to the existing sequence homology between MAGE-C1 and MAGE-C2. Current experiments focus on redirecting primary T cells toward myeloma cells by retroviral gene transfer of CT-antigen-specific TCRs. The establishment of a set of high avidity TCRs specific for CT-antigens facilitates the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of multiple myeloma.

Epigenetic Manipulation of Cancer Testis Antigen (CTA) Expression: A Strategy for Manipulating the Graft-Versus Leukaemia Response in Patients Allografted for Haematological Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 600-600 ◽  
Author(s):  
Tatjana Stankovic ◽  
Andrew McLarnon ◽  
Angelo Agathanggelou ◽  
Oliver Goodyear ◽  
Charles Craddock ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
T Cell Responses ◽  
Interferon Γ ◽  
Haematological Malignancies ◽  
Cell Responses ◽  
Cancer Testis Antigens ◽  
Reduced Intensity ◽  
Cancer Testis

Abstract An immunologically mediated graft-versus-tumor (GVT) underlies the curative effect of reduced intensity allografts in acute myeloid leukaemia (AML) and other haematologic malignancies. Cancer testis antigens (CTA) represent a family of immunodominant proteins that are variably expressed in haematological malignancies and represent a potential target of a GVT response. Importantly a number of CTA genes demonstrate promoter hypermethylation in solid tumours which can be reversed using demethylating agents such as azacitidine. We have therefore examined whether donor T cell responses to CTAs are observed in patients allografted for AML and multiple myeloma (MM) and whether epigenetic therapies can be used to manipulate T cell mediating killing of haemopoietic targets. We screened 37 patients with AML and 8 patients with MM, who had not received demethylating agent treatment, for T cell responses to 25 peptides derived from 10 CTA genes, including BAGE, LAGE, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-C2, RAGE-1, by interferon-γ cytokine secretion assay (IFN-γ CSA). We found CTA specific T cells in 11.1% of patients (5 of 45) with frequencies ranging from 0.0005% to 0.2% (median 0.024%). Subsequently, we studied expression of 15 cancer-testis antigens (CTA) at the RNA and protein levels in AML, MM and Hodgkins’ lymphoma cell lines before and after exposure to demethylating agent 5-aza-2′-deoxycytidine (Aza) and histone deacetylase inhibitor sodium valproate (VPA), as single agents and in combination. We found that expression of CTAs HAGE, SACA3, SPANXB, LAGE, XAGE, MAGEA3 could be induced in a dose dependent manner by Aza alone but not with VPA alone. We also observed that expression induced by Aza treatment was increased further by combination treatment with VPA. Induction of CTAs was confirmed in vitro in primary AML cells and in vivo in 2/6 patients on VPA/Aza trial. Furthermore, using interferon-γ ELISA assay we observed that Aza-induced expression of the CTA MAGEA3 in MM cell lines was accompanied by increased recognition by MAGEA3-specific T cells. These studies confirm the importance of members of the CTA family as targets of a T cell mediated immune response. Our data demonstrate that the expression of these putative immunodominant antigens in haematological malignancies in disease such as AML and myeloma, can be significantly up-regulated by epigenetic therapies with functional increases in target cell killing and support the use of adjunctive epigenetic therapies after allogeneic transplantation with the aim of augmenting a GVL response. A Phase II clinical trial combining post transplant azacitidine with a reduced intensity allograft is currently ongoing in patients with AML.

MAGE-A3 Specific T-Cell Receptor Adoptive Cell Transfer of Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1843-1843 ◽  
Author(s):  
Jeesun Park ◽  
Shi Zhong ◽  
Michelle Krogsgaard ◽  
Amitabha Mazumder
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Fold Increase ◽  
Cell Receptor ◽  
Tumor Cell Lines ◽  
Peptide Concentration

Abstract Abstract 1843 Background: Multiple myeloma (MM) is a cancer of plasma cells and the second most common blood cancer. Current treatment strategies such as high dose chemotherapy, autologous stem cell rescue, and allogeneic transplantation have improved response rates and increased survival. However, these treatments often include high procedure-related morbidity and mortality and can only be applied to a small minority of myeloma patients. Therefore, safe broadly applicable immunologic strategies for myeloma, such as Adoptive Cell Therapy (ACT) are urgently needed. Methods: In this study we focused on aHLA-A*0201-restricted cancer testis antigen MAGE-A3:112–120, which is widely expressed in many forms of cancers such as metastatic melanoma, non-small cell lung cancer and MM, but not expressed in most normal tissues. To develop a system of effective strategies for T-cell therapy of multiple myeloma, we employed T-cell engineering technology using a MAGE-A3specific T-cell receptor (TCR)obtained from Dr. Steven Rosenberg at the National Cancer Institute. MAGE-A3 specific TCR was sub-cloned into a lentiviral vector and tranduced into purified CD8+ T-cells from human peripheral blood mononucleocytes (hPBMCs). To test the effector functionality of the MAGE-A3 specific TCR, the MAGE-A3 TCR-transduced CD8+ T-cells were subjected to cytokine release and chromium release assays after being co-cultured with MAGE-A3 peptide-loaded T2 cells, and U266 (MAGE-A3+/HLA-A*0201+), MM1.r (MAGE-A3+/HLA-A*0201-), KAS6 (MAGE-A3-/HLA-A*0201+), and KMS11(MAGE-A3-/HLA-A*0201-) MM tumor cell lines. Results: We observedcytokine production of INF-g and IL-2 in the MAGE-A3 TCR-transduced CD8+ T-cells generally in a dose-dependent manner to the MAGE-A3 peptide-loaded T2 cells. For example, the difference of INF-g secretion bythe MAGE-A3 TCR-transduced CD8+ T-cells wasa 10-fold increase from 0.001 uM to 0.02 uM of the loaded MAGE-A3 peptide. IL-2 secretion was also increasedby 7-fold from 0.001 uM to 0.1 uM of the MAGE-A3 peptide concentration. At 10uM of the peptide concentration, there was a 29-fold increase of the IL-2 production as compared to the 0.001 uM peptide concentration. Between 10uM and 100 uMof the peptide concentration, there was a decrease in IL-2 secretion by 2-fold, which is commonly observed at high peptide concentrations presumably due to cytotoxicity. Specific lysis of tumor cells by the MAGE-A3 TCR-transduced CD8+ T-cellswas observed in all four MM tumor cell lines, and we detected higher percentage of cell lysisin U266 (38%) and MM1.r (51%) cell lines as compared to the KAS6 (11%) and KMS11(21%) cell lines. Conclusions: Our findings suggest that the MAGE-A3 TCR-engineered CD 8+ T-cells are able to specifically recognize MAGE-A3 antigen, produce IL-2 and IFN-g, and destroy MM tumor cells loaded with the MAGE-A3 antigen. This potentially could further translate into effective MAGE-A3 specific targeted tumor rejection in vivo. We also plan to transduce the MAGE-A3 TCR into hematopoietic stem cells to and test the effector function of those cells against MM tumor cells and eventually against MM patient samples. Disclosures: No relevant conflicts of interest to declare.

Constitutive Expression of CD40L by CAR-Modified Tumor Targeted T Cells Enhances Anti-Tumor Efficacy Both in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4120-4120 ◽  
Author(s):  
Kevin J. Curran ◽  
Beatrijs Seinstra ◽  
Yan Nikhamin ◽  
Raymond Yeh ◽  
Yelena Usachenko ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Inflammatory Cytokines ◽  
Cd40 Ligand ◽  
Tumor Cell Lines ◽  
Pro Inflammatory Cytokines

Abstract Abstract 4120 T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). Recent reports have demonstrated the effectiveness of CAR modified T cells in patients with relapsed or refractory malignancies. However, CAR modified T cells have yet to demonstrate the ability to recruit an endogenous anti-tumor response which would greatly enhance their therapeutic benefit. To overcome these limitations we have developed a bi-cistronic gamma-retroviral vector allowing for constitutive co-expression of a CD19-specific CAR (19–28z) and human CD40 ligand (CD40L; CD154). The CD40 ligand/CD40 system has been demonstrated to activate dendritic cells (DCs) and alter the phenotype of B cells (upregulation of co-stimulatory and adhesion molecules and secretion of pro-inflammatory cytokines) with subsequent stimulation of CD8+ T cell activation and proliferation. We now demonstrate T cells genetically modified to constitutively express CD40L undergo enhanced proliferation and up-regulated secretion of pro-inflammatory cytokines including GM-CSF and INF-g. Furthermore, T cells modified to constitutively express CD40L, upon co-culture, will alter the phenotype of CD40+ B cell tumor cell lines by enhancing the expression co-stimulatory molecules (CD80/CD86), adhesion molecules (CD54/CD58/CD70) and death receptors (CD95; Fas). These findings were similarly evident in primary patient tumor samples (e.g. CLL cells) when co-cultured with autologous T cells modified to constitutively express CD40L. We further demonstrate maturation of monocyte derived DCs with subsequent secretion of IL-12 following co-culture with autologous T cells modified to constitutively express CD40L. T cells transduced with the bi-cistronic 19–28z/CD40L vector showed enhanced in vitro cytotoxicity against a panel of CD19+ tumor cell lines. Furthermore, infusion of 19–28z/CD40L modified T cells enhances the survival of CD19+ tumor bearing immunodeficient mice (SCID/Beige) when compared to mice treated with T cells modified to express the anti-CD19 19–28z CAR alone. We conclude that further genetic modification of CAR targeted T cells to constitutively express the co-stimulatory CD40L may enhance the anti-tumor efficacy of this adoptive T cell therapy. Our data suggests this enhanced T cell efficacy may be due to both autocrine and paracrine mediated mechanisms. Disclosures: No relevant conflicts of interest to declare.

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