Generation of Autologous Pre-B Acute Lymphocytic Leukemia (ALL) Reactive T Cell Lines Using ALL Blasts as Antigen Presenting Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2035-2035
Author(s):  
Rui-kun Zhong ◽  
Thomas A. Lane ◽  
Edward D Ball
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Acute Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Cd40 Expression ◽  
T Cell Lines ◽  
Antigen Presenting ◽  
Cytokine Combination

Abstract 2035 Poster Board II-12 Although hematologic remissions can be achieved in the majority of patients with adult acute lymphocytic leukemia (ALL) by chemotherapy, long term survival is only 30–40%. The inability of the immune system to recognize and eliminate residual malignant leukemia cells may be an important mechanism contributing to relapse. In this study, we investigated the possibility of using pre-B-ALL cells as antigen presenting cells in an in vitro culture to induce autologous ALL reactive T cells. After 7 day culture of Pre-B-ALL peripheral blood mononuclear cells (CD19+ 93±4%) in 96 well culture plates in culture medium supplemented with a cytokine combination of IL-2/IL-3/IL-4/IL-7/GM-CSF, CD80, CD86, CD83, CD54, HLA-Dr and CD40 expression was analyzed. Significant enhancement of CD80, CD86, CD83 and CD40 on ALL cells was observed (n=8, P≦0.001-0.02). Addition of lipopolysaccharide (LPS) to the cytokine combination further increased CD80, CD86 or CD40 expression by 3 of 8 ALL samples above the baseline enhancement by cytokines (Figure), while CD40 ligand (CD40L) enhanced expression of the co-stimulatory molecules in 4 of 8 cases. Autologous T cells remaining in the culture after day 7 were then expanded with high dose IL-2 and autologous ALL reactive T cell lines were identified by IFN-g release by T cells in response to autologous ALL cells. Autologous ALL reactive T cells were generated from 5 of 8 pre-B ALL samples studied. The data from 3 experiments demonstrated that although the cytokine combination plus LPS or CD40L could effectively induce ALL cell expression of co-stimulatory molecules, the presence of CD40L and LPS in the culture induced significantly greater activation of autologous ALL-reactive T cells than did the cytokine combination plus LPS alone, as assessed by average IFN-g release of 24-48 culture wells (P≦0.004). ALL-reactive T cell lines selected by high IFN-g release response showed effective elimination of autologous ALL cells in a 2 day co-culture assay with an E:T ratio of 1:1 by flow cytometry analysis. Average residual CD19+ cells was 2.0±1.4% for highly reactive T cell lines (n=17) vs 16.5±25.9% for the less reactive control T cell lines (n=9) (P<0.02). Conclusion: Autologous ALL-reactive T cells can be induced from most ALL patients in an in vitro culture with a cytokine combination. Adoptive transfer of these anti-ALL CTL to patients is a possible therapeutic approach for further study. Disclosures: No relevant conflicts of interest to declare.

scholarly journals T cells specific for alpha-beta interface regions of hemoglobin recognize the isolated subunit but not the tetramer and indicate presentation without processing

1989 ◽  
Vol 86 (17) ◽  
pp. 6729-6733 ◽  
Author(s):  
M Z Atassi ◽  
M Yoshioka ◽  
G S Bixler
Keyword(s):  
T Cells ◽  
Major Histocompatibility Complex ◽  
T Cell ◽  
Cell Lines ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Oligomeric Protein ◽  
T Cell Lines ◽  
Antigen Presenting

Processing of a protein antigen into fragments is believed to be a prerequisite for its presentation by the antigen-presenting cell to the T cell. This model would predict that, in oligomeric proteins, T cells prepared with specificity for regions that are buried within subunit association surfaces should recognize the respective regions in vitro equally well on the isolated subunit or on the oligomer. Three hemoglobin (Hb) alpha-chain synthetic peptides, corresponding to areas that are situated either completely [alpha-(31-45)] or partially [alpha-(41-45) and alpha-(81-95)] within the interface between the alpha and beta subunits of Hb, and a fourth peptide representing a completely exposed area in tetrameric Hb were used as immunogens in SJL/J (H-2s) mice. Peptide-primed T cells were passaged in vitro with the respective peptide to obtain peptide-specific T-lymphocyte lines. T-cell clones were isolated from these lines by limiting dilution. T-cell lines and clones that were specific for buried regions in the subunit association surfaces recognized the free peptide and the isolated subunit but not the Hb tetramer. On the other hand, T cells with specificity against regions that are not involved in subunit interaction and are completely exposed in the tetramer recognized the peptide, the isolated subunit, and the oligomeric protein equally well. The responses of the T-cell lines and clones were major histocompatibility complex-restricted. Since the same x-irradiated antigen-presenting cells were employed, the results could not be attributed to differences or defects in Hb processing. The findings indicate that in vitro the native (unprocessed and undissociated) oligomeric protein was the trigger of major histocompatibility complex-restricted T-cell responses.

scholarly journals CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells.

1996 ◽  
Vol 183 (1) ◽  
pp. 67-76 ◽  
Author(s):  
F S Wong ◽  
I Visintin ◽  
L Wen ◽  
R A Flavell ◽  
C A Janeway
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Nod Mice ◽  
Cd8 T Cell ◽  
Rapid Onset ◽  
Cytotoxic T Cell ◽  
Nonobese Diabetic ◽  
T Cell Lines

T cells play an important role in the pathogenesis of diabetes in the nonobese diabetic (NOD) mouse. CD8 cytotoxic T cell lines and clones were generated from the lymphocytic infiltrate in the islets of Langerhans of young (7-wk-old). NOD mice by growing them on (NOD x B6-RIP-B7-1)F1 islets. These cells proliferate specifically to NOD islets and kill NOD islets in vitro. The cells are restricted by H-2Kd, and all bear T cell antigen receptor encoded by V beta 6. When these CD8 T cell lines and clones are adoptively transferred to irradiated female NOD, young NOD-SCID, and CB17-SCID mice, diabetes occurs very rapidly, within 10 d of transfer and without CD4 T cells.

Naturally Processed Peptides Eluted from Acute Myeloid Leukemia Cells : A Potent Antigen Source for Immunotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1801-1801
Author(s):  
Stephanie Delluc ◽  
Lea Tourneur ◽  
Charlotte Boix ◽  
Anne-Sophie Michallet ◽  
Bruno Varet ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Immune Recognition ◽  
T Cell Lines ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogenous group of diseases characterized by a clonal proliferation of myeloid progenitors. Its poor prognosis with conventional chemotherapy justifies seeking for adjuvant immunotherapeutic approaches to eliminate minimal residual disease. We evaluated an immunotherapeutic strategy that bypass the need for epitope identification and the limitation due to HLA restriction. Naturally processed peptides were extracted by acid elution from AML cells at diagnosis, and loaded on mature dendritic cells (mDCs) derived from autologous monocytes obtained when the patients were in complete remission (CR). We evaluated i) the feasibility to elute naturally processed peptides from AML cells at diagnosis, ii) the capacity of mDCs loaded with eluted peptides (mDC/EP) to stimulate specific T cell lines in vitro. We showed that stimulation by mDC/EP was able to generate anti-leukemic T cells lines from PBMC of 6 AML patients in CR. CD4+ and CD8+ T cells were isolated from T cell lines of 5 patients and analyzed for their proliferation, INF-γ production and cytotoxicity in response to autologous or allogeneic AML targets, or to normal autologous PBMC. We showed that both CD4+ and CD8+ leukemia-specific T cells were generated in vitro by mDC/EP stimulations since proliferation of CD4+ T cells, IFN-γ secretion by CD4+ and CD8+ T cells and cytotoxicity mediated by CD8+ T cells were induced in response to stimulation with autologous AML cells. Furthermore, we could not detect auto-immune recognition of autologous normal PBMC, consistent with the specificity of the T cell response induced by mDC/EP. These results provide the proof of concept for using mDC/EP to vaccinate patients with poor-risk AML, and will soon be evaluated in a phse I/II clinical trial.

Expansion and Transformation of Primary Acute Lymphoblastic Leukemia Cells into Antigen-Presenting Cells using a Novel Culturing System Enables the Generation of Leukemia-Reactive T Cell Responses that Are Effective in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 303-303
Author(s):  
Bart A. Nijmeijer ◽  
Marianke L.J. Van Schie ◽  
Roel Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Primary Cells ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Antigen Presenting

Abstract Allogeneic cellular immunotherapy is generally ineffective in acute lymphoblastic leukemia (ALL). In vitro studies have suggested that this inefficacy may be the result of a lack of costimulatory molecule expression by ALL cells, resulting in the induction of T cell anergy. Activation of T cells by ALL cells that are transformed into adequate antigen-presenting cells (ALL-APC) may prevent the induction of T cell anergy and result in the generation of competent leukemia-reactive T cell responses for adoptive immunotherapy. However, in vitro modification of ALL cells was hampered by the fact that ALL cells from adult patients could not be cultured in vitro for prolonged periods of time. We have developed a novel serum-free culturing system for B-lineage ALL in which proliferation is initiated and sustained by ALL-cell derived growth factors. Long-term (>2 yrs) proliferation was induced in 12 out of 26 randomly selected primary samples from patients with ALL. The cell cultures ( Leiden cell lines) proliferated with a mean doubling time of 3.0 days (range 2.7–3.6 days). All Leiden cell lines presented the chromosomal abberations observed in the primary cells. The Leiden cell lines displayed an immune phenotype similar to the primary cells, exept for loss of CD34 expression. In vivo characteristics of Leiden cells were evaluated in NOD/scid mice. After intravenous inoculation, Leiden cell lines and primary cells showed identical homing patterns initially involving spleen and bone marrow, followed by the development of overt and progressive leukemia. A comparison of in vivo progression kinetics was performed for one of the Leiden cell lines and the corresponding primary cells. Weekly determination of leukemic cell counts in the blood of engrafted animals revealed that the cell line and the primary cells displayed similar doubling times in vivo of 6.3 and 7.7 days, respectively. To generate cells with improved antigen presentation function, Leiden cell lines were exposed to various activating agents. Stimulation with CpG containing oligonucleotides resulted in induction of CD40 in 9 out of 10 lines. Subsequent ligation of CD40 by culturing CpG-activated Leiden cells on fibroblasts expressing human CD40 ligand resulted in the induction of CD80 or CD86 in 7 of these 10 cell lines. To study the immune stimulatory properties of these Leiden ALL-APC, allogeneic HLA-identical T cells were first activated in vitro by coculturing these cells with either unmodified Leiden cells or with the corresponding Leiden ALL-APC for 3 days, and subsequently infused into groups of 6 leukemic NOD/scid mice. While T cells cocultured with unmodified Leiden cells did not expand in vivo, T cells cocultured with Leiden ALL-APC expanded after infusion in 5 out of 6 animals. This expansion coincided with a 20–75% decrease in leukemic cell numbers in the blood. In conclusion, the novel serum-free culturing system enables long-term culture and manipulation of a significant fraction of primary human ALL. These Leiden cell lines can be modified into ALL-APC that display adequate antigen presenting function, preventing the induction of T cell anergy as demonstrated in vivo in the NOD/scid mouse model.

Development of Chronic Lymphocytic Leukemia (CLL) Reactive Cytotoxic T Lymphocytes after Non-Myeloablative Hematopoietic Stem Cell Transplant Correlates with Anti-Leukemia Response.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 413-413
Author(s):  
Tetsuya Nishida ◽  
Ana Kostic ◽  
David G. Maloney ◽  
Rainer F. Storb ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Histocompatibility Antigens ◽  
Antitumor Response ◽  
Hematopoietic Stem ◽  
Minor Histocompatibility Antigens ◽  
Cell Responses ◽  
T Cell Lines

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) following non-myeloablative (NM) conditioning is a promising approach for treating patients with advanced fludarabine refractory CLL. In this setting, a graft versus leukemia (GVL) effect mediated by donor T cells is critical for tumor eradication. We have evaluated the development of alloreactive and CLL-reactive cytotoxic T lymphocyte (CTL) responses in patients after NM-HSCT to determine if the generation of detectable T cell responses was associated with an antitumor response. Seven patients with fludarabine refractory CLL were conditioned with fludarabine (30mg/m2 x 3 doses) and total body irradiation (2 Gy) prior to receiving G-CSF mobilized peripheral blood stem cells from an HLA matched donor. Peripheral blood mononuclear cells (PBMC) were obtained from the recipient pretransplant and at intervals after NM-HSCT. When chimerism showed a major proportion of donor CD3+ T cells, the postransplant PBMC were stimulated in vitro with recipient CLL cells from the pretransplant collections. CLL cells lack or express low levels of co-stimulatory and adhesion molecules, and are poor stimulators of T cells in vitro. Thus, prior to their use as stimulators and targets, the CLL cells were activated with CD40 ligand (CD40L), which upregulates costimulatory, adhesion, and MHC molecule expression, and turns CLL cells into effective antigen presenting cells. The cultures were stimulated weekly and supplemented with IL2 and IL7. After two stimulations, the T cell lines were tested for cytotoxicity against donor and recipient target cells including recipient CLL. T cell lines generated from four patients with a good antitumor response after NM-HSCT exhibited cytotoxicity against recipient CLL and EBV transformed B cells (B-LCL), but not against donor B-LCL. By contrast, T cell lines generated from three patients with persistent or progressive disease after NM-HSCT did not have cytotoxicity against recipient CLL, despite the development of GVHD in all patients. Multiparameter flow cytometry and IFN-g secretion assay of T cell lines from patients with an antitumor response showed that both CD8+ and CD4+ T cells produced INF-g in response to recipient CLL. We sorted and expanded CD8+ INF-g+ and CD4+ IFN-g+ T cells and both subsets were able to lyse CLL cells. The cytotoxicity of CD4+ and CD8+ T cells was inhibited completely by concanamycin A, suggesting perforin is the major mechanism for leukemia cell lysis. Twenty-one CD8+ T cell clones specific for distinct minor histocompatibility antigens expressed on CLL were isolated from T cell lines of the four responding patients. Multiple specificities were recognized in three of the four patients. Screening a cDNA expression library has identified the genes encoding two minor histocompatibility antigens recognized by CD8+ T cells, and their characterization is in progress. These findings suggest that the development after NM-HSCT of early, diverse, alloreactive T cell responses specific for antigens expressed by CLL may be an important predictor of outcome. The identification of the antigens recognized may facilitate the development of strategies to evoke an effective antitumor response in a larger fraction of patients.

Adenovirus Specific T-Cell Lines Devoid of Alloreactivity Against Haploidentical Recipients Can Be Obtained Using a Set of Adenovirus Hexon Peptides.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3273-3273
Author(s):  
Patrizia Comoli ◽  
Marco W. Schilham ◽  
Sabrina Basso ◽  
Tamara van Vreeswijk ◽  
Rita Maccario ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Peptide Pool ◽  
Specific Lysis ◽  
Hematopoietic Stem ◽  
Hexon Protein ◽  
T Cell Lines

Abstract Human Adenovirus (HAdV) infection/reactivation may cause life-threatening complications in recipients of hematopoietic stem cell transplantation (HSCT), the highest risk being observed in pediatric recipients of a T-cell depleted allograft from haploidentical family donor. The effectiveness of pharmacological therapy for HAdV infection is still suboptimal. It has been recently demonstrated that cell therapy may offer a unique opportunity to restore antiviral immune surveillance, leading to clearance of infection and prevention/treatment of disease. However, infusion of HAdV-specific T-cells in the haplo-HSCT cohort poses the concern that GVHD may ensue as a consequence of T-cell transfer. We have conducted scale-up experiments to validate a method of in vitro culture to expand T-cells specific for HAdV, based on stimulation of donor peripheral blood mononuclear cells (PBMC) with a pool of 5 30-mer peptides derived from HAdV5 hexon protein, for use in recipients of haplo-HSCT (Veltrop-Duits et al, Eur J Immunol36, p2410; 2006). A total of 20 T-cell lines were generated, starting from a median of 20 × 106 donor PBMC, that yielded a median of 80 × 106 cells. Most of the cell lines obtained included a majority of CD4+ T-lymphocytes, with a lower % CD8+ cells (median and range: 78, 19–94 and 18, 5–58, respectively) but 5/20 lines contained a high number of CD8+ T cells (ranging between 43% and 58%), which were CD56+ and/or TCRγδ+, and in 1 case also 44% NK cells. Eighteen of the 20 T-cell lines were HAdV-specific, since they showed a median proliferation to the HAdV hexon peptide pool and inactivated HAdV of 14615 (95%CI 8924–31532) and 11103 (95%CI 8805–30174) cpm/105 cells after subtraction of background (responders+irradiated autologous PBMC), respectively. HAdV-specific lysis >10% at a 2:1 effector to target (E:T) ratio was observed in 50% of the T-cell lines. The 2 non-specific, as well as the 3 T-cell lines with lower specific activity, included >40% CD8+ T-cells. Production of IFNγ in an ELIspot assay to HAdV hexon peptide pool above 40 SFU/105 cells was observed in 9 out of 13 tested T-cell lines. Evaluation of specific response to hexon peptides in showed a majority of responses to II42 (80%), with 50–60% responses to II50, II57, II61, and II64. Only 2 out of the 20 T-cell lines tested were prevalently alloreactive against the recipient. Of the 18 HAdV-specific lines, 1 showed higher proliferation to patient PBMC than to HAdV (13518 vs 11717 mean cpm), and would have thus been discarded as unsuitable for in vivo use, while the other 17 showed no alloreactivity (14) or alloreactivity between 10 and 23% of specific proliferation (3). None of these 18 T-cell lines showed lysis >5% against recipient PHA blasts in the cytotoxicity assay. Our data show that PBMC stimulation with HAdV hexon protein-derived 30-mer peptides is able to reproducibly induce the generation of HAdV-specific CD4+ T-cell lines with efficient in vitro antiviral response in most HLA-mismatched HSCT donors. The majority of these T-cell lines show low/undetectable alloreactivity against recipient targets, and could therefore be safely employed for adoptive treatment of HAdV complications developing after HSCT from a HLA-haploidentical donor.

scholarly journals T-Cell Growth Transformation by Herpesvirus Saimiri Is Independent of STAT3 Activation

2005 ◽  
Vol 79 (9) ◽  
pp. 5713-5720 ◽  
Author(s):  
Elke Heck ◽  
Doris Lengenfelder ◽  
Monika Schmidt ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Herpesvirus Saimiri ◽  
Binding Motif ◽  
New World Primates ◽  
Growth Transformation ◽  
T Cell Lines

ABSTRACT Herpesvirus saimiri (saimirine herpesvirus 2) (HVS), a T-lymphotropic tumor virus, induces lymphoproliferative disease in several species of New World primates. In addition, strains of HVS subgroup C are able to transform T cells of Old World primates, including humans, to permanently growing T-cell lines. In concert with the Stp oncoprotein, the tyrosine kinase-interacting protein (Tip) of HVS C488 is required for T-cell transformation in vitro and lymphoma induction in vivo. Tip was previously shown to interact with the protein tyrosine kinase Lck. Constitutive activation of signal transducers and activators of transcription (STATs) has been associated with oncogenesis and has also been detected in HVS-transformed T-cell lines. Furthermore, Tip contains a putative consensus YXPQ binding motif for the SH2 (src homology 2) domains of STAT1 and STAT3. Tip tyrosine phosphorylation at this site was required for binding of STATs and induction of STAT-dependent transcription. Here we sought to address the relevance of STAT activation for transformation of human T cells by introducing a tyrosine-to-phenylalanine mutation in the YXPQ motif of Tip of HVS C488. Unexpectedly, the recombinant virus was still able to transform human T lymphocytes, but it had lost its capability to activate STAT3 as well as STAT1. This demonstrates that growth transformation by HVS is independent of STAT3 activation.

scholarly journals Conformation-dependent recognition of a protein by T cells requires presentation without processing

1989 ◽  
Vol 259 (3) ◽  
pp. 731-735 ◽  
Author(s):  
M Z Atassi ◽  
G S Bixler ◽  
T Yokoi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Protein Antigen ◽  
Mouse Strains ◽  
Immune Recognition ◽  
Cell Recognition ◽  
T Cell Recognition ◽  
T Cell Lines

Presentation of a protein antigen to T cells is believed to follow its intracellular breakdown by the antigen-presenting cell, with the fragments constituting the trigger of immune recognition. It should then be expected that T-cell recognition of protein antigens in vitro will be independent of protein conformation. Three T-cell lines were made by passage in vitro with native lysozyme of T cells from two mouse strains (B10.BR and DBA/1) that had been primed with the same protein. These cell lines responded well to native lysozyme and very poorly to unfolded (S-sulphopropyl) lysozyme. The response of the T-cell lines to the antigen was major histocompatibility complex (MHC)-restricted. A line from B10.BR was selected for further studies. This line responded to the three surface-simulation synthetic sites of lysozyme (representing the discontinuous antigenic, i.e. antibody binding, sites) and analogues that were extended to a uniform size by a nonsense sequence. T-cell clones prepared from this line were specific to native lysozyme and did not respond to the unfolded derivative. Furthermore, several of these clones showed specificity to a given surface-simulation synthetic site. The exquisite dependency of the recognition by the clones on the conformation of the protein antigen and their ability to recognize the surface-simulation synthetic sites indicate that the native (unprocessed) protein was the trigger of MHC-restricted T-cell recognition.

858 A bispecific antibody targeting CD40 and EpCAM induces superior anti-tumor effects compared to the combination of the monospecific antibodies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A911-A911
Author(s):  
Peter Ellmark ◽  
Karin Hägerbrand ◽  
Mattias Levin ◽  
Laura Von Schantz ◽  
Adnan Deronic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Tumor Target ◽  
T Cell Infiltration ◽  
Antigen Presenting ◽  
Monospecific Antibodies

BackgroundAlligator has developed a new concept, Neo-X’, to enable antigen presenting cells to efficiently enhance priming of neoantigen-specific T cells, which may be the missing aspect in tumors that lack T cell infiltration. We hypothesize that binding of the CD40 x EpCAM bsAb (4224) to CD40 on DCs and EpCAM on tumor exosomes or tumor debris leads to i) activation of the DC, ii) uptake of the tumor material, iii) cross-presentation of tumor-derived neoantigen (present in exosomes or debris) and iiii) priming of tumor neoantigen-specific T cells, resulting in an increased quantity and/or quality of the tumor-targeting T cell pool. CD40 crosslinking by engagement with a tumor antigen on a tumor cell is required to achieve a functional agonistic effect, and subsequent DC activation will therefore only be achieved in the presence of tumor antigens.Methods4224 evaluated in vitro using human monocyte-derived DC, co-cultured with cells expressing EpCAM. In addition the functional effects were evaluated using tumor cell lines and B-cell lines expressing CD40. In vivo, the anti-tumor efficacy of the CD40 x EpCAM bsAb was determined in human CD40 transgenic mice bearing MB49 bladder carcinoma tumors transfected with human EpCAM or controls.ResultsIn vitro, we have demonstrated that the CD40 x EpCAM bsAb induces tumor target dependent activation of dendritic cells, as analyzed by flow cytometry measuring HLA-DR and CD86 expression on the DC and by measuring IL-12p40 levels in the supernatant. Further, the ability of bsAbs within the Neo-X’ concept to mediate co-localization of tumor debris and CD40 expressing antigen presenting cells depends on the receptor density of the tumor target. In vivo, 4224 displayed a potent, EpCAM-dependent anti-tumor effect with significantly reduced tumor growth and improved survival compared to an equivalent dose of the combination of the monospecific CD40 Ab and EpCAM targeting antibody. The tumor-localizing property of 4224 also shows potential for improved safety compared to CD40 monospecific antibodies. A biodistribution analysis demonstrated that the bispecific 4224 in the RUBY-format displayed similar half-life as the monospecific CD40 mAb in mice.ConclusionsIn conclusion, the Neo-X’ concept, by targeting CD40 and a tumor specific antigen, has the potential to mediate an expansion of the tumor-specific T cell repertoire, resulting in increased T cell infiltration and potent anti-tumor effects.

Cd8+ T-Cells Specifically Cytotoxic to Renal Cell Carcinoma (RCC) Cells Can Be Isolated from Patients with Regressing Metastatic Kidney Cancer after Allogeneic Nonmyeloablative Hematopoietic Cell Transplantation (NMHCT).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4984-4984
Author(s):  
Yoshiyuki Takahashi ◽  
Othon Mena ◽  
Ramaprasad Srinivasan ◽  
Takehito Igarashi ◽  
Andreas Lundqvist ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cell ◽  
Cell Clones ◽  
Grade Ii ◽  
T Cell Lines ◽  
Limiting Dilution

Abstract Graft-vs-tumor (GVT) effects following NMHCT induce disease regression in a subset of patients (pts) with advanced metastatic kidney cancer. At present, little is known about the antigens serving as tumor targets in responding pts. In an effort to characterize GVT effectors and their tumor antigens, we generated RCC cell lines to use as targets in cytotoxicity assays in three pts (one non-responder and two responders) undergoing a cyclophosphamide/fludarabine-based NMHCT from HLA matched siblings. Peripheral blood lymphocytes (PBL) were collected from pts at multiple time-points after transplantation and were expanded in-vitro with either irradiated patient (pre-transplant) PBL/EBV-LCL or autologous tumor cells. RCC pt #1 developed grade II skin GVHD on day 22 but did not have an objective tumor response and died from progressive tumor on day 203. In a Cr51 release assay, minor histocompatibility antigen specific (mHa) T-cell lines generated using pre-transplant pt PBL/EBV-LCL stimulators lysed 98% of pt CD40-ligand stimulated B cells (CD40L-B) but did not kill pt RCC cells. RCC pt #2 developed grade II skin GVHD on Day 51 and was noted to have regression of lung metastasis on day 183. Using PBL obtained during tumor regression, mHa- reactive T-cell lines were generated (pre-transplant pt PBL/EBV-LCL used as stimulators) that lysed 76% and 12% of pt EBV-LCL and autologous RCC tumor cells at a 20:1 E:T ratio. Following limiting dilution cloning, 6 CD8+ T cell clones were expanded that killed pt EBV-LCL (but not donor) including one MHC class-1 restricted T cell clone that lysed both pt EBV-LCL and pt tumor cells. RCC pt #3 developed grade III skin GVHD on day 120, had regression of lung metastasis on day 160, and survives more than 4 years after transplantation. PBL collected from this pt 40 months after transplant contained CD8+ T-cell populations that secreted IFN-g (0.9% by intracellular cytokine staining) when co-cultured with pt RCC cells but not after co-culture with pt CD40L-B cells. CD3+/CD8+ CTL were expanded from these PBL using pt RCC cells as stimulators; in vitro, these CTL killed the pt’s RCC cells but did not kill pt fibroblasts or pt EBV-LCL (Figure A). Following co-culture with tumor, intracellular IFN-g staining combined with TCR Vb antibodies revealed 3 tumor reactive CD8+ T-cell populations (TCR Vb7+, TCRVb5.1+, and TCRVb non-staining); 25.2% of these CTL were TCRVb7+. IFN-g secretion by TCR Vb7+ T cells was blocked when tumor cells were pre-incubated with mAbs to CD8, pan MHC class I and HLA-A11 (Figure B) consistent with recognition of an HLA-11 restricted tumor antigen. Following limiting dilution cloning, several CD8+ T-cell clones (both TCR Vb7+ and TCR Vb non-staining) with RCC-specific cytotoxicity were identified. We conclude that donor T-cells with both broad alloreactivity and tumor specificity play a role in mediating GVT effects in RCC pts having disease regression following NMHCT. Figure Figure

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