Anti-CD3 Activated T Cells from Cord Blood Can Be Expanded Ex Vivo and Armed with Bispecific Antibodies To Lyse Her2/Neu + and CD20+ Targets: A Strategy for Providing Anti-Tumor/Lymphoma Effects after Cord Blood Transplant.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2751-2751
Author(s):  
Carly B. Sorenson ◽  
Thomas D. Frandsen ◽  
Suanne Dorr ◽  
Voravit Ratanatharathorn ◽  
Joseph P. Uberti ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Cell Transplant ◽  
Activated T Cells ◽  
Specific Cytotoxicity ◽  
The Mean

Abstract Our previous studies showed that anti-CD3 activated T cells (ATC) from peripheral blood mononuclear cells could be expanded in interleukin-2 (IL-2) for 14 days and armed with anti-CD3 x anti-Her2/neu (Her2Bi)[J Hemat & Stem Cell Res10:247,2001], anti-CD3 x anti-CD20 (CD20Bi)[Exp Hemat33:452,2005], or anti-CD3 x anti-EGFR [Clin Cancer Res12:183,2006] bispecific antibody (BiAb)and can kill Her2/neu, CD20, and EGFR+ tumor targets, respectively. In this study, we asked whether anti-CD3 activated cord blood T cells (CBATC) could be expanded and targeted with Her2Bi and CD20Bi to tumors or hematologic malignancies for infusions after cord blood stem cell transplant (CBSCT). CB mononuclear cells were activated with anti-CD3 (20 ng/ml) and expanded for 14 days in IL-2 (100 IU/ml). CBATC were armed with Her2Bi or CD20Bi and tested for specific cytotoxicity directed SK-BR-3, Raji, or B9C targets and cytokine secretion or IFNγ EliSpots after binding to tumor cells. Our results show the mean expansion of CBATC to be 43-fold (n=8) after 14 days of culture. By the end of culture, the proportions of CD8+ and CD4+ were 82% and 18%, respectively. The proportion of cells expressing CD19 or CD20 did not exceed 6.3%, CD56+ cells were <3.6% and CD3-CD16+CD56+ cells was <0.7%. Cells positive for CD4+CD25+ or CD8+ CD25+ were 4.2% or 7.1%, respectively (n=2). Specific cytotoxicity was optimized when CBATC were armed with 50 ng/106 cells of Her2Bi or CD20Bi (arming dose ranged from 5, 50, and 500 ng/106 cells); arming significantly increased cytotoxicity of the armed CBATC over that seen for unarmed CBATC. Cytotoxicity peaked between days 12 and 14 for both BiAbs. The ability of CD20Bi armed ATC to produce Elispots for IFNγ were tested by arming ATC the CD20Bi after 0, 8, 11, and 13 days of culture peaked on day 8. Only CD20+ targets induced Elispots and day 8 armed ATC exhibited peak numbers (2,200 Elispots(ranged from 1,700 to 1,800 on day 8)/106 armed ATC plated). At an effector/target ratio (E:T) of 25:1, the mean cytotoxicity of CBATC armed with Her2Bi or CD20Bi was 60% (n=4) and 35% (n=1), respectively. In an extended culture to day 47, mean cytotoxicity for Her2Bi-armed CBATC was 36% at an E/T of 25:1 compared to 4.35% for unarmed CBATC. Unarmed CBATC did not kill Daudi targets. Armed CBATC mediated both specific cytotoxicity and secreted IFN-γ as measured by ELISA or EliSpots. Both fresh and frozen CB could be used in the assays. In a clinical application, specific cytotoxicity of armed CBATC could be used to augment anti-tumor and anti-lymphoma effects after CBSCT.

Impaired Alloantigen Presenting Activity of Cord Blood Nucleated Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2203-2203
Author(s):  
Sandeep Chunduri ◽  
Dolores Mahmud ◽  
Javaneh Abbasian ◽  
Damiano Rondelli
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
T Cell Response ◽  
Solid Organ ◽  
Cd34 Cells

Abstract Transplantation of HLA-mismatched cord blood (CB) nucleated cells has limited risk of severe acute graft-versus-host disease and graft rejection. This may depend on naïve T cells not yet exposed to many antigens and on immature antigen-presenting cells (APC) not delivering appropriate signals to allogeneic T cells. In order to test the APC activity of human circulating CB cells in-vitro, we initially used irradiated CB mononuclear cells (MNC) or immunomagnetically selected CD34+ cells, CD133+ cells, or CD14+ monocytes to stimulate the proliferative response of incompatible blood T cells in mixed leukocyte culture (MLC). CB MNC failed to induce allogeneic T cell proliferation, while CD34+ and CD133+ progenitors or CD14+ monocytes induced potent T cell alloresponses. Nevertheless, since allogeneic T cell response was not restored after depletion of CD3+ cells in the CB, nor the add-back of irradiated CB MNC to CD34+ or CD14+ stimulators inhibited allo-T cells, a direct suppressive effect of CB MNC was excluded. Allogeneic peripheral blood cytotoxic T-lymphocyte (CTL) responses were not induced after 7 days of stimulation with irradiated CB MNC, although after 4 weekly rechallenges with CB MNC, on average a 23% lysis of antigen-specific CB PHA-blasts was observed at the highest effector:target ratio (50:1). To test the tolerogenic potential of CB MNC, T cells initially exposed to CB MNC were rechallenged in secondary MLC with CB MNC, or CD34+ cells, or monocyte-derived dendritic cells (Mo-DC) generated in liquid culture with GM-CSF and IL-4. Allogeneic T cells were still unresponsive upon rechallenge with CB MNC, but proliferated upon 3 days of restimulation with CD34+ cells or Mo-DC from the same CB. Surprisingly, the supernatant of these latter MLCs did inhibit completely a 3rd party MLC. Instead, the supernatant of blood T cells that had been activated by CB CD34+ cells or Mo-DC both in primary and secondary MLC did not. These results show an impaired allo-APC activity of CB MNC but not CB CD34+ cells, and suggest that T cells releasing immunosuppressive cytokines may be activated by CB MNC and then expanded by a second more potent stimulation with professional APC. This hypothesis could explain the sustained engraftment of HLA-mismatched CB stem cell transplants in humans. Based on these results, the in-vivo or ex-vivo downregulation of T cell alloreactivity induced by CB MNC will be tested in experimental models of stem cell, as well as solid organ transplantation.

Immunotherapy with Anti-CD3 x Anti-CMV Bispecific Antibody (CMVBi) Armed Donor Derived Activated T Cells (ATC) – A Novel Strategy against Cytomegalovirus (CMV) Post Allogeneic Stem Cell Transplantation (SCT).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1157-1157
Author(s):  
Mayur S Ramesh ◽  
Archana Thakur ◽  
Philip Pellett ◽  
Subhendu Das ◽  
Zaid S Al-Kadhimi ◽  
...  
Keyword(s):  
T Cells ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
Human Fibroblasts ◽  
Interleukin 2 ◽  
Target Cells ◽  
Normal Donor ◽  
Activated T Cells ◽  
Specific Cytotoxicity ◽  
Novel Strategy

Abstract Abstract 1157 Poster Board I-179 Introduction CMV reactivation and infection can cause profound negative outcome post allogeneic SCT. Current management strategies against CMV are suboptimal and are associated with adverse effects. Induction of anti-CMV T cell responses by vaccination has not been helpful in immunocompromised hosts. Immunotherapy with CMV specific donor-derived cytotoxic T lymphocytes (CTL) is effective after allografting, but it is expensive, labor intensive, and diffcult to replicate in most centers. Non-toxic targeted therapy is needed to improve clinical outcomes. In earlier studies, we generated anti-CD3 chemically heteroconjugated with anti-Her2/neu or anti-CD20 bispecific antibody (BiAb). Ex vivo expanded anti-CD3 activated T cells (ATC) and armed with BiAb exhibit high levels of specific cytotoxicity directed at the respective tumor antigens. We propose a novel strategy of using ATC armed ex-vivo with engineered CMVBi to target CMV antigens. We tested the strategy initially in an in vitro cell culture model using CMV-infected fibroblasts and T cells from normal human donors. We hypothesize that donor anti-CD3 ATC armed with CMVBi will target and eliminate CMV-infected target cells. Materials and methods Normal donor peripheral blood mononuclear cells (PBMC) were used to generate ATC by activation with anti-CD3 (OKT3) and interleukin 2 (IL-2). CMVBi was created by chemical heteroconjugation of OKT3 (murine IgG2a) monoclonal antibody and polyclonal anti-CMV (Cytogam®). Specific cytotoxicity was tested by 51Cr release assay using CMV-infected or uninfected human fibroblasts as target cells. Effector populations tested included CMVBi armed ATC and ATC alone; additional controls included CMVBi alone, Cytogam® alone, CMVBi armed, and unarmed PBMC. Cytotoxicity was assessed for CMVBi and an irrelevant BiAb at arming doses ranging from 1 to 500 ng/106 ATC with effector to target ratios (E:T) ranging from 25:1 to 3.125:1. Interferon gamma (IFNγ) EliSpots were used to determine cytokine response after exposing CMV-infected and uninfected fibroblasts to unarmed and CMVBi-armed ATC. Results CMVBi arming with as little as 1 ng/106 ATC was significantly more cytotoxic for target cells than unarmed ATC. There was an incremental increase in cytotoxicity with CMVBi armed ATC as the mutiplicity of infection (MOI) was increased in target cells. At all E:T ratios (25:1, 12.5:1, 6.25:1, 3.125:1 ), ATC armed with a dose of 50 ng/106 CMVBi demonstrated markedly enhanced killing of CMV-infected targets (MOI 1) compared to unarmed ATC (see table). In the uninfected control cells, both unarmed and armed ATC lysis was barely detectable over spontaneous lysis. Immunofluorescent studies showed that CMVBi-armed ATC specifically aggregated around GFP fluorescence-marked CMV-infected fibroblasts, whereas unarmed ATC did not aggregate. Cytokine secretion analyzed by IFNγ EliSpot confirmed the superior cytotoxicity of CMVBi-armed ATC. Conclusion We used polyclonal Cytogam® to make CMVBi-armed ATC that were able to kill target cells expressing various CMV antigens with high specificity. CMVBi-armed PBMC was only minimally cytotoxic in comparision to CMVBi-armed ATC. Hence, infusion of CMVBi alone to patients is unlikely to be as effective as infusion of ATC armed ex-vivo with CMVBi. We demonstrated similar degree of cytotoxicity using different donor ATC including CMV sero-negative donors. This effect is independent of MHC mediated antigen presentation hence, overcomes CMV immune escape. This non-MHC restricted specific killing strategy could be easily adapted for the prevention and/or treatment of CMV infection/disease after allogeneic SCT using donor-derived ATC. Our current use of BiAb-armed ATC for cancer immunotherapy in humans illustrates the feasibility of adoptive immunotherapy with CMVBi-armed ATC in SCT. Disclosures Lum: Transtarget Corporation: Founder.

Transfer of Cellular and Humoral Anti-Tumor Immunity with Immune T Cells After Stem Cell Transplant (SCT) for Metastatic Breast Cancer Following “Vaccination” with Anti-CD3 x Anti-Her2/Neu Bispecific Antibody (Her2Bi) Armed Activated T Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1914-1914
Author(s):  
Archana Thakur ◽  
Zaid Al-Kadhimi ◽  
Cassara Pray ◽  
Elyse N. Tomaszewski ◽  
Ritesh Rathore ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Stem Cell ◽  
Metastatic Breast ◽  
Cell Transplant ◽  
Antibody Synthesis ◽  
Activated T Cells ◽  
Cd8 Cells ◽  
Specific Cytotoxicity

Abstract Abstract 1914 Novel therapeutic approaches are needed for women with metastatic breast cancer (BrCa). In our phase I clinical trial, infusions of anti-CD3 activated T cells (ATC) armed with anti-CD3 x anti-Her2/neu bispecific antibody (Her2Bi) induced specific cytotoxicity (SC) directed at SK-BR-3 breast cancer cells by fresh peripheral blood lymphocytes (PBL) and induced elevated serum levels of Th1 cytokines. In this study, we took advantage of armed ATC induced anti-tumor immune responses by infusing “immune” T cells collected by leukopheresis. We expanded “immune T cells” with anti-CD3 and IL-2, followed by a cryopreservation for multiple re-infusions of ATC after high dose chemotherapy (HDC) and autologous stem cell transplant (SCT). We asked whether this approach would transfer anti-tumor responses back into patients after HDC and SCT to accelerate the development of cellular and humoral anti-tumor responses. This strategy of “prime” with armed ATC and “boost” with ATC was performed in 5 evaluable patients. The “priming” portion involved 8 infusions of (2 infusions/week for 4 weeks) armed ATC given with daily low dose IL-2 (300,000 IU/m2/day) and twice weekly GM-CSF (250 μg/m2). Approximately 3 weeks after the armed ATC infusions, patients underwent a second leukopheresis for the collection and expansion of ATC. The expanded ATC from 6 patients at an effector:target ratio (E/T) of 25:1 exhibited specific cytotoxicity (SC) ranging from 3.7–25.8 (mean = 13.6%) directed at the SK-BR-3. Phenotyping of the ATC showed a mean of 50.2 % (25–74) CD4+ cells, 30.4 % (16.3-51.3) CD8+ cells, 11.6% (4.5-24.3) CD56+CD16+ cells, and 29.5% (10.4–41) CD4+CD25+ cells. A separate leukopheresis after G-CSF stimulation was performed to obtain CD34+ cells for the SCT. After HDC and PBSCT, 5 evaluable patients received multiple infusions with a mean total of 54×109 ATC (16–110 × 109) beginning day +1 after SCT. No G-CSF was given to accelerate engraftment. There were no dose-limiting side effects or delays in engraftment. One patient developed sepsis, multiple organ failure and recovered fully with supportive care and antibiotics. Phenotyping at 2 weeks after SCT showed the mean proportions of CD4+ and CD8+ cells to be 55.5 and 17.7%, respectively. Specific cytotoxicity (SC) directed at SK-BR-3 targets ranged from 4.7 to 70% at E/T of 25:1 up to 18 months post SCT but not against a negative control cell line-Daudi. Mean serum anti-SK-BR-3 antibody levels were 800 ng/ml preSCT and 1500, 1080 and 1360 ng/ml at 1, 2, and 3 months post SCT, respectively. In vitro anti-SK-BR-3 antibody synthesis was assessed using a new assay (Thakur et al, Cancer Immunol Immunother EPub, 2011) showed easily detectable levels of in vitro anti-SK-BR-3 antibody synthesis. The mean anti-SK-BR-3 antibody synthesis in the presence of CpG in pre-immunotherapy (Pre-IT), mid-IT, 1 months post-IT, pre-SCT and 1, 2, and 3 months post-SCT is summarized in the table. PBL produced anti-SK-BR-3 antibody pre-SCT and there was clear recovery of anti-SK-BR-3 antibody synthesis by PBL at 2 and 3 months after SCT. These data show infusions of immune ATC transferred cytotoxic T lymphocytes and humoral antibody activity directed at tumor antigens. These novel findings suggest that adaptive immunity was transferred into patients by ATC infusions and the stem cell product after myeloablation and SCT leading to rapid reconstitution of anti-tumor immunity.Time PointsCulture ConditionsMean±SD (n=3)Pre-ITCpG23±27Mid-ITCpG66±671M Post-ITCpG41±67Pre-SCTCpG92±101M Post-SCTCpG9±182M Post-SCTCpG45±273M Post-SCTCpG82±55 Disclosures: Lum: Transtarget Inc: Equity Ownership, Founder of Transtarget.

scholarly journals Safety and feasibility of virus-specific T cells derived from umbilical cord blood in cord blood transplant recipients

2019 ◽  
Vol 3 (14) ◽  
pp. 2057-2068 ◽  
Author(s):  
Allistair A. Abraham ◽  
Tami D. John ◽  
Michael D. Keller ◽  
C. Russell Y. Cruz ◽  
Baheyeldin Salem ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Viral Disease ◽  
Viral Reactivation ◽  
Cell Transplant ◽  
Transplant Recipients ◽  
Cell Recovery

Abstract Adoptive transfer of virus-specific T cells (VSTs) has been shown to be safe and effective in stem cell transplant recipients. However, the lack of virus-experienced T cells in donor cord blood (CB) has prevented the development of ex vivo expanded donor-derived VSTs for recipients of this stem cell source. Here we evaluated the feasibility and safety of ex vivo expansion of CB T cells from the 20% fraction of the CB unit in pediatric patients receiving a single CB transplant (CBT). In 2 clinical trials conducted at 2 separate sites, we manufactured CB-derived multivirus-specific T cells (CB-VSTs) targeting Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus (CMV) for 18 (86%) of 21 patients demonstrating feasibility. Manufacturing for 2 CB-VSTs failed to meet lot release because of insufficient cell recovery, and there was 1 sterility breach during separation of the frozen 20% fraction. Delayed engraftment was not observed in patients who received the remaining 80% fraction for the primary CBT. There was no grade 3 to 4 acute graft-versus-host disease (GVHD) associated with the infusion of CB-VSTs. None of the 7 patients who received CB-VSTs as prophylaxis developed end-organ disease from CMV, EBV, or adenovirus. In 7 patients receiving CB-VSTs for viral reactivation or infection, only 1 patient developed end-organ viral disease, which was in an immune privileged site (CMV retinitis) and occurred after steroid therapy for GVHD. Finally, we demonstrated the long-term persistence of adoptively transferred CB-VSTs using T-cell receptor-Vβ clonotype tracking, suggesting that CB-VSTs are a feasible addition to antiviral pharmacotherapy.

Enhanced Anti-Breast Cancer Cytotoxicity after Autologous Peripheral Blood Stem Cell Transplant (PBSCT) by Boosting with Multiple Infusions of Activated T Cells (ATC) Armed with Anti-CD3 x Anti-Her2 Bispecific Antibody (Her2Bi) Prior to PBSCT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2748-2748
Author(s):  
Lawrence G. Lum ◽  
Zaid Al-Kadhimi ◽  
Cassara Skuba ◽  
Voravit Ratanatharathorn ◽  
Joseph P. Uberti ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Bispecific Antibody ◽  
Stem Cell Transplant ◽  
Peripheral Blood Stem Cell ◽  
Cell Transplant ◽  
Activated T Cells ◽  
Specific Cytotoxicity

Abstract More aggressive treatment strategies are needed for women with metastatic breast cancer(mBrCa). Although peripheral blood stem cell transplant (PBSCT) permits the use of high-dose chemotherapy (HDC) that could not otherwise be given, results for PBSCT have not been encouraging. In order to boost tumor kill, we combined a protocol that targets Her2 using activated T cells (ATC) armed with anti-CD3 × anti-Her2 bispecific antibody (Her2Bi) with a second protocol that involves infusions of ATC after PBSCT to boost anti-tumor immunity. In our phase I trial using ATC with Her2Bi to treat women with mBrCA who have Her2 positve or negative disease, we found that multiple infusions of armed ATC induced cytotoxicity directed at BrCa cells in the peripheral blood mononuclear cells (PBMC) of the patients that develop in 2 weeks and last up to 4 mos (Clin Canc Res12:569,2006). Armed ATC lyse tumors that are Her2 low-expressors (0–1+). Targeting leads to specific cytotoxicity, induces cytokine/chemokine release, and proliferation of the armed ATC. In a second study, ATC were infused after PBSCT into 23 women with mBrCa leading to 70% overall survival and 50% progression free survival at 32 mos after PBSCT. Therefore, the combined strategy involved obtaining T cells by another leukopheresis after the boosting with armed ATC, expanding the immune T cells and infusing the expanded ATC after PBSCT to transfer pre-immune anti-BrCa cytotoxicity to reconstitute cytotoxicity after PBSCT. Two patients (one pt was Her2 3+ ; one pt was Her2 0–1+) underwent treatment with this treatment approach. Both patients were given 8 infusions (20 billion/infusion with a total of 160 billion) of ATC armed with Her2Bi in the first protocol and subsequently leukopheresed and ATC were produced for the second protocol. The expanded ATC at an effector:target ratio (E/T) of 25:1, exhibited cytotoxicity at BrCa tumor cells (SK-BR-3) at 71% and 75% for pts 1 and 2, respectively. The cell product for pt 1 contained 68% CD3+, 32% CD4+, 39% CD8+, 29% CD16+56+, 12% CD4+CD25+, and 15% CD8+CD25+ cells and the cell product for pt 2 contained 35% CD3+, 25% CD4+, 8.5% CD8+, 23.3% CD16+56+, 10.4% CD4+CD25+, and 4.7% CD8+CD25+ cells. The protocol involves infusing 15 doses of ATC after PBSCT with 3 doses of ATC/week for 3 weeks and then ATC once/week for six more weeks. The pt1 and pt 2 received total of 114 and 70 billion ATC, respectively. Pt1 developed anti-BrCA cytotoxicity of 9% 2 weeks after PBSCT. Pt2 exhibited anti-BrCa cytotoxicity at an E:T of 25:1 of 38% and 15% at 3 weeks and 6 months after PBSCT, respectively. Phenotyping of peripheral blood at 6 mos after PBSCT showed 61% CD3+, 36% CD4+, 19.5% CD8+, and 15.5% CD56+ cells. There was no cytotoxicity directed at Daudi cells. These data strongly suggest that transfer of pre-immune cells after PBSCT accelerate immune reconstitution of tumor specific cytotoxicity after PBSCT. The preboost strategy with targeted T cells is being combined in a proof of principle trial to assess whether enhanced cytotoxicity can be consistently enhanced after PBSCT.

Induction of Adenovirus Specific T-Cells in Response to 15-Mer Peptides in Cord Blood

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5439-5439
Author(s):  
Lianne Margreet Haveman ◽  
Maja Bulatovic ◽  
Berent Prakken ◽  
Marc Bierings
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Interleukin 2 ◽  
Haematopoietic Stem Cell ◽  
Clear Correlation ◽  
Adoptive Therapy ◽  
Activation Markers

Abstract Human adenovirus (HAdV) infections are a serious life-threat in haematopoietic stem cell transplantation (HSCT) patients, particularly in children. In recent years, umbilical cord blood transplants (UCBT) emerged as an alternative stem cell source. The advantages are the rapid availability of donor cells, the possibility of using HLA-mismatched transplants and a lower risk of graft versus host disease. However, the immune response after UCBT is often delayed, leading to a higher risk of viral infections. Because no effective antiviral medication exists for severe HAdV infection adoptively transfer of HAdV specific T-cells from cord blood may be a promising treatment. The aim of this study was to see if it was possible to induce HAdV specific T-cells in response to five recently detected 15-mer peptides ( Haveman LM, Int. Imm. 2006), a pool of these five peptides or complete inactivated HAdV (MOI 10). Mitogen Concovalin A ( ConA) was used as control. Cord blood mononuclear cells (CBMC) derived from 34 uncomplicated full term deliveries were isolated and cultured with the different antigens. Proliferation expressed as Stimulation Index (SI) was determined. A SI&gt;1.7 was seen as positive. In 14 neonates a response to in total 36 of the 5 different HAdV peptides could be seen and in 7 of them also a response to the pool of the peptides. Only in 3 neonates a response to complete HAdV could be detected (Figure 1). To induce HAdV specific T-cells CBMCs of 20 neonates were cultured with the different HAdV antigens in addition of interleukin 2 (40IU/ml) for 7 days. By using FACS analysis the upregulation of the activation markers CD25 and CD69 could be detected in response to the HAdV peptides (n=41) or to the complete HAdV (n=26). Induced HAdV specific T-cells expressed the cytotoxins IFN□, TNFα, perforin and granzyme B, produced by CD8+ T-cells, but in higher extent by CD4+ T-cells. This indicates that induced HAdV specific T-cells play an essential role in killing HAdV infections. No clear correlation could be seen between proliferation and the production of the cytotoxins. The induction of a specific immune response to HAdV peptides in cord blood is an important step towards adoptive therapy. Figure 1. Proliferation of CBMC in response to HAdV peptides, complete HAdV en ConA Figure 1. Proliferation of CBMC in response to HAdV peptides, complete HAdV en ConA

Phase I Dose Esclation of Activated T Cells (ATC) Armed with Anti-CD3 × Anti-CD20 Bispecific Antibody (CD20Bi) after Stem Cell Transplant (SCT) In Non-Hodgkin's Lymphoma (NHL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 488-488
Author(s):  
Lawrence G. Lum ◽  
Voravit Ratanatharathorn ◽  
Archana Thakur ◽  
Zaid Al-Kadhimi ◽  
Cassara Skuba ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
High Risk ◽  
Phase I ◽  
Stem Cell Transplant ◽  
Cytotoxic T Cell ◽  
Cell Transplant ◽  
Activated T Cells ◽  
Serum Cytokines ◽  
The Mean

Abstract Abstract 488 Relapse rates after autologous peripheral blood stem cell transplant (SCT) for high risk refractory or relapsed NHL patients (pts) remain unacceptable. Resistance to rituximab is a major limitation to successful treatment in pts with NHL. In preclinical studies, we showed that anti-CD3 activated T cells (ATC) armed with CD20Bi could kill rituximab-resistant CD20+ lymphoma cells (Exp Hemat 33:452). Arming ATC with CD20Bi converts each ATC into a CD20-specific cytotoxic T cell. A phase I trial was done in 15 high risk or refractory NHL pts (ages 54–67) who met eligibility criteria for SCT to determine: 1) whether multiple infusions of armed ATC (aATC) given after SCT are safe (primary endpoint); 2) whether infusions accelerate immune recovery (secondary endpoint); and 3) whether such infusions induce an anti-lymphoma effect (secondary endpoint). T cells were activated with OKT3 and expanded in IL-2 from a leukopheresis product, armed with CD20Bi, and cryopreserved for infusions after SCT. At the time of SCT, there were 6 pts in 1st or 2nd complete remission (CR), 4 pts with primary refractory disease (PRD), 4 pts in partial response, and 1 pt with progressive disease (PD). The first 3 patients received 5 × 109 aATC three times per week for 3 weeks and then once per week for 6 weeks for a total aATC dose of 75 × 109. For logistical reasons, the schedule was revised to 1 infusion/week for 4 weeks with dose levels of 10, 15, and 20 × 109 aATC per infusion for total doses of 40, 60, and 80 × 109 aATC. The median dose of CD34+ cells/kg was 4.0 × 106 (1.04-12.30 × 106) and median total infused dose of aATC was 67 × 109 (8.5-80 × 109). The median viability of ATC product was 92%. The ATC product for 15 patients contained medians of 96.5% CD3+, 67% CD4+, and 48.9% CD8+ cells. A total of 15 pts received aATC and 12 of 15 pts were evaluable for aATC toxicities (received >80% of targeted dose). The table summarizes side effects by the number of episodes by dose level for grade 1–3 scores up to 7 days after infusions using the NCI Immunotherapy Toxicity Scoring system. The median day to engraftment was 14.75 days (11-28) after SCT. Phenotyping of patient peripheral blood lymphocytes (PBL) before and after the 2nd infusion in 2 pts showed that circulating aATC peaked between 8 and 12 hrs at levels between 25 and 10% of PBL. Phenotyping of PBL showed that the mean %CD4 cells and the CD4/CD8 ratio in PBL were in the normal range up to a month after SCT. There was a >4x increase in the mean (± SD) number of IFNγ EliSpots from preSCT (30.5 ± 20.5/106 PBL) to post SCT (125.6 ± 130/106 PBL). These data show that IFNγ EliSpots after stimulation with CD20+ Daudi targets increased over the preSCT values (p < 0.008, paired-Wilcoxon test). The median cytotoxicity directed at K562 targets mediated by NK cells was 8.2, 12.7, and 13.7% at 2 weeks, 1 month, and 3 months, respectively. Serum cytokines/chemokines peaked ∼4 hrs after infusions. There was a >250x increase in IL-2 receptors, >10x increase in IL-2, IL-7, and IL-15, >100x increase in MIP-1β and IP10, and >10x increase in MIP-1α, MIG, and MICP-10. At 90 days after SCT, 9 pts were in CR and 6 pts had PD. The median survival for all patients is 20.9 months, for pts who were transplanted in CR remains undefined, and for those transplanted with PRD is 20.4 months. These findings strongly show that expanding ATC from heavily pretreated NHL pts is feasible, aATC infusions are safe, induce high levels of serum cytokines and chemokines, and may accelerate recovery of helper and cytotoxic T cell functions after SCT. These results will help design future approaches to increase the GVL effect after SCT for CD20+ malignancies. Table: Grade 1-3 Events (NCI Immunotherapy Toxicity) Dose 5 billion 10 billion 15 billion 20 billion # of Pts 3 3 3 3 # of infusions 15 4 4 4 Fever 3 10 12 12 Chills 2 7 10 8 N/V 0 7 3 4 Hypotension 3 4 1 5 Headache 0 2 2 3 Malaise 1 2 7 11 Diarrhea 0 2 0 0 Hypertension 0 1 0 0 Tachycardia 3 2 5 4 Dyspnea 1 0 0 0 Hypoxia 0 0 0 1 Disclosures: Lum: Transtarget Inc: Equity Ownership.

scholarly journals Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

2021 ◽  
Vol 67 (2) ◽  
pp. 95-101
Author(s):  
Monica Vuță ◽  
Ionela-Maria Cotoi ◽  
Ion Bogdan Mănescu ◽  
Doina Ramona Manu ◽  
Minodora Dobreanu
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Gradient Centrifugation ◽  
Intracellular Cytokine Staining ◽  
Interleukin 2 ◽  
Middle Aged

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

scholarly journals Cord-Blood-Stem-Cell-Derived Conventional Dendritic Cells Specifically Originate from CD115-Expressing Precursors

Cancers ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 181 ◽  
Author(s):  
Maud Plantinga ◽  
Colin G. de Haar ◽  
Ester Dünnebach ◽  
Denise A.M.H. van den Beemt ◽  
Kitty W.M. Bloemenkamp ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Dendritic Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Treatment Success ◽  
Gene Set Enrichment Analysis ◽  
Long Term Memory ◽  
Vaccination Strategies

Dendritic cells (DCs) are professional antigen-presenting cells which instruct both the innate and adaptive immune systems. Once mature, they have the capacity to activate and prime naïve T cells for recognition and eradication of pathogens and tumor cells. These characteristics make them excellent candidates for vaccination strategies. Most DC vaccines have been generated from ex vivo culture of monocytes (mo). The use of mo-DCs as vaccines to induce adaptive immunity against cancer has resulted in clinical responses but, overall, treatment success is limited. The application of primary DCs or DCs generated from CD34+ stem cells have been suggested to improve clinical efficacy. Cord blood (CB) is a particularly rich source of CD34+ stem cells for the generation of DCs, but the dynamics and plasticity of the specific DC lineage development are poorly understood. Using flow sorting of DC progenitors from CB cultures and subsequent RNA sequencing, we found that CB-derived DCs (CB-DCs) exclusively originate from CD115+-expressing progenitors. Gene set enrichment analysis displayed an enriched conventional DC profile within the CD115-derived DCs compared with CB mo-DCs. Functional assays demonstrated that these DCs matured and migrated upon good manufacturing practice (GMP)-grade stimulation and possessed a high capacity to activate tumor-antigen-specific T cells. In this study, we developed a culture protocol to generate conventional DCs from CB-derived stem cells in sufficient numbers for vaccination strategies. The discovery of a committed DC precursor in CB-derived stem cell cultures further enables utilization of conventional DC-based vaccines to provide powerful antitumor activity and long-term memory immunity.

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