The Use of Monoclonal Antibody Directed Chimeric Antigen Receptors to Facilitate Conventional T Cell and Treg Control of GvHD and Tissue Tolerance in Murine Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3355-3355
Author(s):  
Antonio Pierini ◽  
Bettina Iliopoulou ◽  
Heshan Peiris ◽  
Magdiel Perez Cruz ◽  
Jeanette Baker ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
T Cell ◽  
Organ Transplantation ◽  
Adoptive Transfer ◽  
Skin Grafts ◽  
Mhc I ◽  
Cell Homing ◽  
University Patents

Abstract INTRODUCTION Regulatory T cells (Treg) modulate allograft immune responses and Treg-based cellular therapies can be used for prevention of graft-versus-host disease (GvHD) following hematopoietic cell transplantation and for prevention of allograft rejection following tissue or organ transplantation. Treg adoptive transfer has limitations including that Treg do not necessarily home to sites where they are needed and can become inactivated in inflammatory milieus. METHODS We used new technologies of T cell engineering to force the expression of a chimeric antigen receptor on T cells and Treg that recognizes labeled therapeutic monoclonal antibodies (mabCAR), allowing for precise control of their localization in vivo. The mabCAR recognizes fluorescein isothiocyanate (FITC) through a FITC-specific single-chain variable fragment fused to a CD28 and TCRζ costimulatory domain. Any monoclonal antibody (mab) coupled to FITC within its Fc domain can be recognized. We tested this approach with T cells and Treg to ameliorate GvHD and induce tolerance to pancreatic islet grafts. RESULTS We first tested our mabCAR construct in conventional T cells (Tcon) which when transfected and stimulated with a FITC-mab become activated and expressed higher levels of CD44 (p=0.0003), CD25 (p=0.009) and produced more IFNγ (p=0.04). To test if mabCAR transient transfection alters Tcon homing after adoptive transfer, we injected luc+ mabCAR Tcon directed against MAdCAM1 (a gut and lymph node endothelial integrin) or SDF1 (a chemokine mainly expressed in the bone marrow) into allogeneic hosts. MAdCAM1-directed Tcon mainly homed to the gut and lymph nodes, while SDF1-directed Tcon homed to bones and spleen. SDF1-directed Tcon induced a milder GvHD (p<0.001), demonstrating that cell homing impacts GvHD severity. We then tested mabCAR Treg ability to maintain their suppressive activity in vitro and in vivo. We found that mabCAR transiently transfected into Treg have increased ability to proliferate in response to anti-CD3/CD28 stimulatory beads (p<0.01) and highly suppress Tcon proliferation when co-cultured with allogeneic irradiated splenocytes. We injected MAdCAM1 directed Treg in an allogeneic GvHD model and these mabCAR Treg prolonged survival (p=0.03), improved GvHD score (p<0.001) and mouse weight profile (p<0.001), thus demonstrating that mabCAR Treg retain regulatory functions. Finally, we tested if mabCAR Treg could induce tolerance to allogeneic pancreatic islet grafts in sublethally irradiated hosts. Luc+gfp+ mabCAR Treg homed and expanded over time (p<0.05) to the site of the allogeneic islet grafts (right kidney capsule) when FITC-anti-allogeneic MHC-I mab directed the Treg as compared to isotype mab controls (see figure). Allo-MHC-I directed mabCAR-Treg prolonged allogeneic islet graft survival in comparison to isotype-mabCAR Treg (p=0.002) allowing for production of higher insulin levels. To assess if allo-MHC-I Treg promoted antigen-specific tolerance, we performed secondary skin graft transplantation. We found that mice which received MHC-I Treg showed a significant delay in the rejection of skin grafts from mice with the same MHC mismatch as the previous islet-allografts in comparison to third-party skin grafts (p=0.02) offering strong evidence that CAR-Treg could be used to enhance antigen-specific graft protection. CONCLUSION MabCAR expression can be used to control immune cell homing after transfer in different models according to localizing mab availability. We believe that the mabCAR approach may represent a new tool for optimizing cellular therapies to modulate GvHD and for inducing tolerance in islet and organ transplantation. Figure Figure. Disclosures Pierini: Stanford University: Patents & Royalties. Iliopoulou:Stanford University: Patents & Royalties. Negrin:Stanford University: Patents & Royalties. Kim:Stanford University: Patents & Royalties. Meyer:Stanford University: Patents & Royalties.

scholarly journals H-2 restriction of virus-specific T-cell-mediated effector functions in vivo. II. Adoptive transfer of delayed-type hypersensitivity to murine lymphocytic choriomeningits virus is restriced by the K and D region of H-2.

1976 ◽  
Vol 144 (3) ◽  
pp. 776-787 ◽  
Author(s):  
R M Zinkernagel
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Gamma Globulin ◽  
Effector T Cells ◽  
Delayed Type Hypersensitivity ◽  
T Effector Cells ◽  
Immune Lymphocytes ◽  
D Region

In mice, primary footpad swelling after local infection with lymphocytic choriomeningitis virus (LCMV) and delayed-type hypersensitivity (DTH) adoptively transferred by LCMV immune lymphocytes are T-cell dependent. Nude mice do not develop primary footpad swelling, and T-cell depletion abrogates the capacity to transfer LCMV-specific DTH. Effector T cells involved in eliciting dose-dependent DTH are virus specific in that vaccinia virus-immune lymphocytes could not elicit DTH in LCMV-infected mice. The adoptive transfer of DTH is restricted to H-2K or H-2D compatible donor-recipient combinations. Distinct from the fowl-gamma-globulin DTH model, I-region compatibility is neither necessary nor alone sufficient. Whatever the mechanisms involved in this K- or D-region associated restriction in vivo, it most likely operates at the level of T-cell recognition of "altered self" coded in K or D. T cells associated with the I region (helper T cells and DTH-T cells to fowl-gamma-globulin) are specific for soluble, defined, and inert antigens. T cells associated with the K and D region (T cells cytotoxic in vitro and in vivo for acute LCMV-infected cells, DTH effector T cells, and anti-viral T cells) are specific for infectious, multiplying virus. The fact that T-cell specificity is differentially linked with the I region or with the K and D regions of H-2 may reflect the fundamental biological differences of these antigens. Although it cannot be excluded that separate functional subclasses of T-effector cells could have self-recognizers for different cell surface structures coded in I or K and D, it is more likely that the antigen parameters determine whether T cells are specific for "altered" I or "altered" K- or D-coded structures.

scholarly journals A Novel Role for IL-6 Receptor Classic Signaling: Induction of RORγt+Foxp3+ Tregs with Enhanced Suppressive Capacity

2019 ◽  
Vol 30 (8) ◽  
pp. 1439-1453 ◽  
Author(s):  
Julia Hagenstein ◽  
Simon Melderis ◽  
Anna Nosko ◽  
Matthias T. Warkotsch ◽  
Johannes V. Richter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Th17 Cells ◽  
Inflammatory Diseases ◽  
Double Positive ◽  
T Effector Cells ◽  
Suppressive Capacity

BackgroundNew therapies blocking the IL-6 receptor (IL-6R) have recently become available and are successfully being used to treat inflammatory diseases like arthritis. Whether IL-6 blockers may help patients with kidney inflammation currently remains unknown.MethodsTo learn more about the complex role of CD4+ T cell-intrinsic IL-6R signaling, we induced nephrotoxic nephritis, a mouse model for crescentic GN, in mice lacking T cell–specific IL-6Ra. We used adoptive transfer experiments and studies in reporter mice to analyze immune responses and Treg subpopulations.ResultsLack of IL-6Ra signaling in mouse CD4+ T cells impaired the generation of proinflammatory Th17 cells, but surprisingly did not ameliorate the course of GN. In contrast, renal damage was significantly reduced by restricting IL-6Ra deficiency to T effector cells and excluding Tregs. Detailed studies of Tregs revealed unaltered IL-10 production despite IL-6Ra deficiency. However, in vivo and in vitro, IL-6Ra classic signaling induced RORγt+Foxp3+ double-positive Tregs (biTregs), which carry the trafficking receptor CCR6 and have potent immunoregulatory properties. Indeed, lack of IL-6Ra significantly reduced Treg in vitro suppressive capacity. Finally, adoptive transfer of T cells containing IL-6Ra−/− Tregs resulted in severe aggravation of GN in mice.ConclusionsOur data refine the old paradigm, that IL-6 enhances Th17 responses and suppresses Tregs. We here provide evidence that T cell–intrinsic IL-6Ra classic signaling indeed induces the generation of Th17 cells but at the same time highly immunosuppressive RORγt+ biTregs. These results advocate caution and indicate that IL-6–directed therapies for GN need to be cell-type specific.

scholarly journals Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

2010 ◽  
Vol 207 (8) ◽  
pp. 1791-1804 ◽  
Author(s):  
Elizabeth D. Thompson ◽  
Hilda L. Enriquez ◽  
Yang-Xin Fu ◽  
Victor H. Engelhard
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

scholarly journals A Novel Approach for the Treatment of T Cell Malignancies: Targeting T Cell Receptor Vβ Families

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 631
Author(s):  
Jie Wang ◽  
Katarzyna Urbanska ◽  
Prannda Sharma ◽  
Reza Nejati ◽  
Lauren Shaw ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Lymphomas ◽  
Novel Approach ◽  
T Cell Malignancies

Peripheral T cell lymphomas (PTCLs) are generally chemotherapy resistant and have a poor prognosis. The lack of targeted immunotherapeutic approaches for T cell malignancies results in part from potential risks associated with targeting broadly expressed T cell markers, namely T cell depletion and clinically significant immune compromise. The knowledge that the T cell receptor (TCR) β chain in human α/β TCRs are grouped into Vβ families that can each be targeted by a monoclonal antibody can therefore be exploited for therapeutic purposes. Here, we develop a flexible approach for targeting TCR Vβ families by engineering T cells to express a chimeric CD64 protein that acts as a high affinity immune receptor (IR). We found that CD64 IR-modified T cells can be redirected with precision to T cell targets expressing selected Vβ families by combining CD64 IR-modified T cells with a monoclonal antibody directed toward a specific TCR Vβ family in vitro and in vivo. These findings provide proof of concept that TCR Vβ-family-specific T cell lysis can be achieved using this novel combination cell–antibody platform and illuminates a path toward high precision targeting of T cell malignancies without substantial immune compromise.

scholarly journals IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell–mediated rejection of systemic lymphoma in immunodeficient mice

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3508-3519 ◽  
Author(s):  
John C. Markley ◽  
Michel Sadelain
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Memory T Cells ◽  
Tumor Rejection ◽  
Effector Memory ◽  
Transfer Model ◽  
Human T Cell

Abstract The γc-cytokines are critical regulators of immunity and possess both overlapping and distinctive functions. However, comparative studies of their pleiotropic effects on human T cell–mediated tumor rejection are lacking. In a xenogeneic adoptive transfer model, we have compared the therapeutic potency of CD19-specific human primary T cells that constitutively express interleukin-2 (IL-2), IL-7, IL-15, or IL-21. We demonstrate that each cytokine enhanced the eradication of systemic CD19+ B-cell malignancies in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull mice with markedly different efficacies and through singularly distinct mechanisms. IL-7– and IL-21–transduced T cells were most efficacious in vivo, although their effector functions were not as enhanced as IL-2– and IL-15–transduced T cells. IL-7 best sustained in vitro T-cell accumulation in response to repeated antigenic stimulation, but did not promote long-term T-cell persistence in vivo. Both IL-15 and IL-21 overexpression supported long-term T-cell persistence in treated mice, however, the memory T cells found 100 days after adoptive transfer were phenotypically dissimilar, resembling central memory and effector memory T cells, respectively. These results support the use of γc-cytokines in cancer immunotherapy, and establish that there exists more than 1 human T-cell memory phenotype associated with long-term tumor immunity.

Development of a Nonhuman Primate Model for Analysis of the Adoptive Transfer of Antigen-Specific T Cell Clones.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 770-770
Author(s):  
Carolina Berger ◽  
Michael Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Transfer ◽  
Primate Model ◽  
T Cell Clones ◽  
Cell Clones

Abstract In principle, the adoptive transfer of T cell clones specific for antigens expressed by pathogens or malignant cells could be therapeutically effective and allow precise control of the specificity, function, and magnitude of T cell immunity. However, the infusion of large numbers of cultured T cells or T cell clones in clinical trials has frequently failed to eradicate tumors or provide long-term control of infection. This may be due in part to the acquisition of an effector phenotype by the T cells during in vitro culture, which reduces their ability to survive in vivo and establish an immune response of sufficient magnitude for sustained efficacy. Several approaches including the administration of cytokines such as IL15, or lymphodepletion prior to cell transfer might promote the establishment of T cell memory after T cell transfer. To facilitate the rational development of clinical trials of T cell therapy, we have employed a nonhuman primate model of adoptive T cell transfer in which culture conditions and cell doses identical to those in human studies are utilized, and designed strategies to permit rigorous analysis of the persistence, function, phenotype, and migration of transferred cells. CD8+ CTL specific for macaque CMV were detected using an overlapping peptide panel and cytokine flow cytometry, isolated as individual T cell clones by limiting dilution, and propagated to large numbers in vitro. The T cell clones were transduced to express an intracellular truncated CD19 (ΔCD19) surface marker to allow tracking and functional assessment of T cells in vivo, and enriched by immunomagnetic selection to high purity (&gt;98%) prior to transfer. The persistence of transferred ΔCD19+ T cells in the blood and their migration to the bone marrow and lymph nodes was determined by flow cytometry after staining with anti CD19, CD8, and CD3 antibodies. The infusion of ΔCD19+CD8+ CTL (3 x 108/kg) was safe and the cells remained detectable in vivo for &gt;5 months. ΔCD19+CD8+ T cells were easily detected in the blood 1 day after transfer at a level of 2.7% of CD8+ T cells and gradually declined over 56 days to a stable population of 0.15–0.2% of CD8+ T cells. At the time of transfer the ΔCD19+CD8+ T cells had an effector phenotype (CD62L− CD127−), but gradually converted to a CD62L+CD127+ memory phenotype in vivo. The infused T cells were found at high levels in lymph node and bone marrow at day 14 after transfer (1.4% and 2.5%, respectively) and the cells at these sites were predominantly CD62L+. The ΔCD19+CD62L+ T cells lacked direct lytic function and expressed low levels of granzyme B, consistent with memory T cells. Sorting of these cells from post-transfer PBMC showed that in vitro activation restored lytic activity. The transferred ΔCD19+CD62L+ T cells in post-infusion PBMC produced IFNγ and TNFα comparable to endogenous CMV-specific CD8+ CTL. These results demonstrate that a subset (5–10%) of transferred CD8+ CTL clones can persist long-term as functional memory T cells. The macaque CD8+ T cell clones are responsive to IL15 in vitro and a safe regimen for administering IL15 to macaques that boosts endogenous T cells has been identified. Studies are now in progress to determine if IL15 can enhance the efficiency with which effector and memory CD8+ T cell responses can be augmented after adoptive transfer of T cell clones.

Adoptive Transfer of Adenovirus Specific T-Cells in Children with Systemic Adenovirus Infection after Allogeneic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 80-80
Author(s):  
Tobias F. Feuchtinger ◽  
Susanne Matthes-Martin ◽  
Celine Richard ◽  
Thomas Lion ◽  
Klaus Hamprecht ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Adoptive Transfer ◽  
Cell Transfer ◽  
New Treatment ◽  
T Cell Transfer

Abstract Allogeneic stem cell transplantation (SCT) has become an increasing treatment option for a variety of malignant and non-malignant disease. During immune reconstitution the host is at significant risk for viral infections. Human adenovirus (HAdV) infection is especially in children an important and serious complication. Virus-specific T-cells are essential for the clearance of HAdV, since antiviral chemotherapy has been insufficient to date. We present a new treatment option using virus-specific donor T-cells for adoptive transfer of immunity to patients with systemic HAdV-infection. We isolated in 6 patients with systemic HAdV-infection after SCT virus-specific T-cells of the donor, according to INF-γ secretion after short in vitro stimulation with viral antigen, resulting in a combination of CD4+ and CD8+ T-cells. Between 5-50x103/kg T-cells were infused for adoptive transfer. For follow-up, the infection and the in-vivo expansion of infused T-cells were evaluated. Isolated cells showed high specificity and markedly reduced but residual alloreactivity in-vitro. In three of four evaluable patients the infused T-cells underwent an in-vivo expansion and in these three patients the viral load decreased in peripheral blood after adoptive T-cell transfer. In-vivo expansion of specific T-cells was dose-independent. T-cell infusion was well tolerated. One patient experienced GvHD°II of the skin after T-cell transfer. In conclusion specific T-cell immunotherapy as a new treatment approach for children was performed in 6 cases of systemic HAdV-infection after allogeneic SCT. Induction of a specific T-cell response through adoptive transfer has been shown feasible and effective to protect from HAdV-related complications.

Establishing T Cell Memory by Adoptive Transfer of T Cell Clones.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 866-866
Author(s):  
Carolina Berger ◽  
Michael C. Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Annexin V ◽  
T Cell Memory ◽  
T Cell Clones ◽  
Cell Clones

Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for &gt;30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

Establishing CD8+ T Cell Immunity by Adoptive Transfer of Autologous, IL-15 Expanded, Anti-Tumor CTL with a Central/Effector Memory Phenotype Can Induce Objective Clinical Responses.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 782-782 ◽  
Author(s):  
Marcus Butler ◽  
Philip Friedlander ◽  
Mary Mooney ◽  
Linda Drury ◽  
Martha Metzler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
De Novo ◽  
Effector Memory ◽  
Large Numbers ◽  
Central Effector

Abstract Abstract 782 The goal of cellular immunotherapy is to build long-lasting anti-tumor immunologic “memory” in patients and reject tumors for a lifetime. Previously, we and others demonstrated that IL-15 promotes the generation of T cells with a central memory (CM) phenotype which have the capacity to persist and establish effective anti-tumor memory in vivo. Furthermore, it has been shown that CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Based on these findings, we developed a system to generate large numbers of long-lived antigen-specific CD8+ T cells with a memory phenotype. This in vitro culture system utilizes IL-15 and a standardized, renewable artificial antigen presenting cell (aAPC) which was produced by transducing CD80, CD83, and HLA-A*0201 to the human cell line, K562. This aAPC can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory (CM/EM) phenotype, possess potent effector function, and can be maintained in vitro for >1 year without any feeder cells or cloning. We hypothesized that adoptive transfer of these CTL with a CM/EM phenotype should result in anti-tumor memory in humans even without lymphodepletion or high dose IL-2. For our “first-in-human” clinical study, we chose the melanoma antigen MART1 as a target antigen, since MART1-specific HLA-A*0201+-restricted precursor CTL are detectable in some melanoma patients and can be immunophenotyped pre-infusion. Autologous CD8+ T cells were stimulated weekly with peptide-pulsed human cell-based aAPC and expanded with low dose IL-2 and IL-15. After three weeks, polyclonal MART1 CTL were reinfused without additional lymphodepletion, chemotherapy, IL-2, or vaccination. Eight study participants have enrolled and received a total of 15 MART1 CTL infusions (31% MART1 multimer positivity, median). All but one subject received two reinfusions where the 2nd graft was produced from CD8+ T cells harvested two weeks after the 1st reinfusion. To date, ≥2×109 CTL with potent effector function and a CM/EM phenotype were successfully generated for all subjects. No dose limiting toxicities were observed at either Dose Level 1 (2×108/m2) or Dose Level 2 (2×109/m2). Clinical activity was observed with a response by RECIST criteria in 1 subject, which was confirmed by a negative PET/CT 100 days following the last CTL infusion. In addition, 1 patient experienced a mixed response, 1 had stable disease, 3 had progression, and 2 are currently on active therapy. Multimer staining showed that, immediately post infusion, the percentage of CD8+ T cells specific for MART1 temporarily increased in all subjects, with the highest (6.5%) observed in subject #7. In 4 subjects, sustained increases in the frequency of MART1 specific T cells by more than two-fold (range 2.0-10x) for ≥21 days were observed despite the fact that no exogenous cytokines or vaccination was administered. Moreover, an increase of detectable MART1 specific T cells which display a CM phenotype was observed in all evaluable subjects and was observed for ≥35 days in 6 of 8 subjects. In subject #2, the conversion of MART1 CTL immunophenotype from a naïve to a mixture of naïve/memory phenotypes was observed for more than 6 months. We identified 10 individual MART1 T cell clonotypes from peripheral CD45RA- memory T cells on day 21. Clonotypic TCR Vbeta CDR3 analysis revealed that CTL grafts contained 7 out of 10 of these clonotypes. Furthermore, 6 clonotypes persisted in the peripheral CD45RA- memory fraction on days 39, 67 and/or 132. In Subject #3, who showed a mixed clinical response, 5 individual MART1 T cell clonotypes were isolated from lung metastases. 4 out of 5 clones were included in the CTL grafts. This finding supports the possibility that infused CTL can traffic and localize to sites of disease. Intriguingly, in both subjects, we were able to identify MART1 CTL clonotypes that were not detectable in the CTL grafts but possibly emerged after CTL infusion, indicating that adoptive transfer of MART1-specific CTL may provoke a de novo antitumor response. Taken together, these results suggest that CM/EM MART1 CTL generated ex vivo using our cell-based artificial APC in the presence of IL-15 may persist in vivo and induce de novo anti-tumor responses. Further enhancement of anti-tumor activity may be achieved through vaccination, cytokine administration, and/or removal of cytokine sinks and inhibitory factors following appropriate lymphodepletion. Disclosures: No relevant conflicts of interest to declare.

Reinforcement of Cancer Immunotherapy by Adoptive Transfer of Cblb-Deficient Cytotoxic T Lymphocytes Combined with a Dendritic Cell Vaccine

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 957-957
Author(s):  
Christina Lutz-Nicoladoni ◽  
Patrizia Stoizner ◽  
Magdalena Pircher ◽  
Stephanie Wallner ◽  
Anna Maria Wolf ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Cancer Immunotherapy ◽  
Cytotoxic T Lymphocytes ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Dc Vaccine

Abstract Abstract 957 Introduction: Various approaches to induce immunological rejection of tumors including transfer of autologous tumor infiltrating lymhocytes (TIL) after ex vivo clonal expansion or application of ex vivo transduced antigen specific T cell (TCR) transgenic T cells have been elaborated. In general, adoptive T cell transfer (ATC) has been combined with lympho-depleting agents (e.g. cyclophosphamide). However, the therapeutic efficacy of these cancer immunotherapy approaches is limited due to insufficient in vivo activation, expansion and survival of transferred effector immune cells, which is mainly due to suppressive mileu signals and immune evasion mechanisms induced by TGF-β. The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is assumed to confer TGF-β resistance. Thus we performed a proof-of-concept study evaluating Cbl-b targeting as “intracellular adjuvant” strategy to improve ATC for cancer immunotherapy. Material and Methods: We first tested the in vitro sensitivity of CTL towards TGF-β mediated immuno-suppressive cues and then in vivo evaluated the anti-tumor reactivity of cblb-deficient cytotoxic T lymphocytes (CTL) in murine tumor models alone or in combination with a dendritic cell (DC) vaccine. Results: Cblb-deficient CTL are hyper-responsive to TCR/CD28-stimulation in vitro and protected from the negative cues induced by TGF-β as determined by quantification fo IFN-g secretion and quantification of their proliferative capacity. Unexpectedly, adoptive transfer of polyclonal, non TCR-transgenic cblb-deficient CD8+ CTL, however, is not sufficient to reject B16ova or EG7 tumors in vivo, which is in clear contrast to previous reports using lymphopenic animals receiving adoptively transferred TCR-transgenic T cells. Thus, we next evaluated in vivo re-activation of adoptively transferred cblb-deficient T cells by a DC vaccine (i.e. SIINFEKL-pulsed DC). In strict contrast to ATC monotherapy, this approach now markedly delays tumor outgrowth and significantly increase survival rates, which is paralleled by an increased CTL infiltration rate to the tumor site and an enrichment of ova-specific and IFN-g-secreting CTL in the draining lymph nodes. Moreover, compared to wild-type CTL, cblb-deficient mice vaccinated with the DC vaccine show an increased cytolytic activity in vivo. Conclusions: In summary, we provide experimental evidence that genetic inactivation of cblb in polyclonal, non-TCR transgenic adoptively transferred CTL might serve as a novel “adjuvant approach”, suitable to augment the effectiveness of anti-cancer immunotherapies using ATC in immune-competent recipients. Disclosures: No relevant conflicts of interest to declare.

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