Quantitative Assessment of the Impact of Ex Vivo Manipulation on the In Vivo Functionality of Human T Cells in RAG2−/− γc−/− mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 450-450
Author(s):  
Rozemarijn S. van Rijn ◽  
Elles R. Simonetti ◽  
Gert Storm ◽  
Mark Bonyhadi ◽  
Anton Hagenbeek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Critical Parameters ◽  
In Vivo Evaluation ◽  
Human T Cells ◽  
The Impact

Abstract T cells retrovirally modified to express therapeutic genes encoding cytokines, exogenous TCRs or suicide molecules represent a novel class of immune therapeutics of great potency. However, recent clinical trials using retrovirally-modified T cells have indicated that T cells exhibit a diminished reactivity upon ex vivo manipulation. In addition, virus-specific memory T cells seem to be lost during gene transfer. In a BNML rat model we have shown that the culture procedure is one of the critical parameters. To preserve T cell reactivity, reliable models are required which permit readout of human T cell activity. We recently developed a huPBMC-RAG2−/−γc−/− mouse model for xenogeneic graft-versus-host disease (xGVHD), in which iv injection of 15 x 106 human T cells into RAG2−/−γc−/− mice consistently leads to high level engraftment and lethal xGVHD within 3 weeks in 80% of mice (van Rijn et al, Blood 2003). We have now used this model to analyze in vivo functionality of human T cells following different ex vivo culture procedures. For this, we cultured human T cells for 7 days with either of the two currently available clinically applicable stimulation conditions: 1) via CD3 and 2) via CD3/CD28. In addition, we included CD3/CD28/4-1BB stimulation to explore the effect of extensive costimulation. Mice were injected with escalating doses T cells. HuCD45+ cells in peripheral blood were measured by FACS. Lethal xGVHD occurred at only 6 times (90.106) the dose of fresh cells for CD3-stimulated T cells and 3 times for CD3/28- or CD3/28/4-1BB-stimulated cells. About 20% of surviving mice developed chronic xGVHD, independent of culture method. While lethal xGVHD was always associated with very high levels of engraftment (up to 95%) engraftment levels in chronic mice ranged from 1–75%. To compare the impact of the different culture conditions on in vivo T cell function, we analyzed engraftment potential. The fraction of huCD45+ cells was plotted against the time and the areas under the curves were compared. Based on a total of 68 mice, statistical analysis showed a 2-fold improvement of engraftment potential for C28-costimulated human T cells compared to CD3-stimulated cells (P<0.0001). Additional ligation of 4-1BB did not increase engraftment potential. In addition, different T cell subsets (naïve, memory, effector) were monitored based on the combined expression of CD45RA, CD27 and CCR7. For all primary T cells and variably cultured T cells, a strikingly similar pattern was observed in vivo. After 3 weeks mainly effector and memory effector T cells (both CD4+ and CD8+) could be detected, suggesting a (xeno-)antigen-driven survival and expansion. This was a very consistent observation independent of donor, culture condition, engraftment level or severity of disease. In conclusion, in vitro costimulation preserves in vivo functionality of human T cells and should therefore be included in future clinical protocols for ex vivo manipulation of T cells. These data show the feasibility to use the huPBMC-RAG2−/−γc−/− model for in vivo evaluation of in vitro effects on human T cells. This model is the most sensitive to date for in vivo evaluation of human T cells and will be a promising new tool for the study of human T cells in, for instance, autoimmune disease, cancer and infectious diseases like AIDS.

Naive and Ex Vivo Activated Human T Cells Generate Consistent Engraftment and Lethal Graft-Versus-Host Disease (GvHD) in NOD SCID β 2M Null Mice: A New Xenogeneic Model for GvHD.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3106-3106
Author(s):  
Bruno Nervi ◽  
Michael P. Rettig ◽  
Julie K. Ritchey ◽  
Gerhard Bauer ◽  
Jon Walker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
Activation Markers ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Human T Cells ◽  
The Impact

Abstract GvHD remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. The human GvHD pathophysiology includes recipient tissue destruction and proinflammatory cytokine production associated with the conditioning regimen; donor T cells become allo-activated, proliferate, and mediate tissue injury in various organs, including the liver, skin, and gut. Modern therapeutic strategies to control GvHD while maintaining the beneficial graft-versus-leukemia effects require ex vivo T cell stimulation and expansion. Multiple studies have demonstrated that these ex vivo expanded T cells exhibit decreased survival and function in vivo, including reduced alloreactivity and GvHD potential. Unfortunately no in vivo models exist to consistently examine the impact of ex vivo manipulation of human T cells (HuT) on T cell function. Naive HuT were compared to HuT activated using CD3/28 beads (XcyteTMDynabeads) with 50 U/ml IL-2 for 4 days (Act). We initially evaluated the HuT engraftment and GvHD potential of naive and Act in RAG2γ null mice (n=22) conditioned with clodronate liposomes on day −1 and 350cGy on day 0, as previously described by others. We injected 107 and 1.5x107 naive or Act HuT intravenously (iv). All mice exhibited low HuT engraftment and no lethal GvHD. NOD SCIDβ 2M null mice (β 2M) were next conditioned with 250cGy on day −1 (n=34), or 300cGy on day 0 (n=21). 107 naive vs Act HuT were injected retroorbitaly (ro). Lower HuT doses or iv injection resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. 60% of the mice conditioned with 250cGy that received naive HuT developed lethal GvHD, in comparison to 75% of mice that received 300cGy and nave HuT, and 100% of mice that received 300cGy and Act HuT. Table 1 250cGy 300cGy Naive (n=34) Naive (n=8) Activated (n=13) *p<0.02 PB engraftment (%HuT) 20%±15 33%±21 59%±19 Lethal GvHD 60% 75% 100% All mice receiving 300cGy had well preserved CD4/CD8 ratios (1–1.5). Tissue infiltration was greatest in mice that had received 300cGy and Act HuT (spleen, liver, lung, kidney: 50–70%). Of interest, serum levels of hu IFNγ dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=270 ug/ml and day 15=36,000 ug/ml) compared to mice that did not develop lethal GvHD (day 10=40 ug/ml and day 17=1,020 ug/ml)(p<0.05). Interestingly, the up-regulation of the activation markers CD25 and CD30 in HuT, and IFNγ production predicted lethal GvHD in β 2M null mice. In summary, we developed a xenogeneic model of lethal GvHD where naive or ex vivo Act HuT injected ro in sublethaly irradiated β 2M not only engraft, expand in vivo, but also infiltrate and damage different mouse target organs. HuT are allo-activated against mouse antigens and damage the target tissues, sharing the major characteristics of human GvHD and causing the death of mice. This model will allow us to study the effects of specific ex vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on the outcome of GvHD, to determine improved therapeutic interventions.

Development of human NKG2D-CD3ε chimeric antigen receptor (CAR) for T-cell-mediated cancer immunotherapy.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 150-150
Author(s):  
Sergei Kusmartsev ◽  
Johaness Vieweg ◽  
Victor Prima
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Adaptor Protein ◽  
T Cell Subsets ◽  
Human T Cells

150 Background: NKG2D is a lectin-like type 2 transmembrane receptor that expressed by natural killer cells and some T cell subsets. Stimulation of NKG2D receptor with specific agonistic ligands produces activating signals through signaling adaptor protein DAP10 leading to the enhanced cytokine production, proliferation, and cytotoxicity against tumor cells. There is strong evidence that NKG2D ligands are expressed in many human tumors, including melanoma, leukemia, myeloma, glioma, and carcinomas of the prostate, breast, lung, and colon. Recent studies also demonstrated that T cells bearing chimeric antigen receptor (CAR) NKG2D linked to CD3ζ (zeta) chain produce marked in vitro and in vivo anti-tumor effects. The aim of current study was to determine whether human T cells bearing chimeric antigen receptor (CAR) NKGD2 linked to CD3ε (epsilon) chain could be activated by the NKG2D-specific stimulation and able to kill human cancer cells. Given the important role of CD3ε in activation and survival of T cells, we hypothesized that NKG2D-CDε-bearing T cells could exert strong in vitro and in vivo anti-tumor effects. Methods: NKG2D CAR was produced by linking human NKG2D to DAP10 and the cytoplasmic portion of the CD3ε chain. Original full-length human cDNA clones were obtained from NIH Mammalian Gene Collection (MGC). Functional domain analysis and oligonucleotide design in the in-Fusion system of DNA cloning (Clontech) was used to generate the retroviral expression constructs. Results: Human PBMC-derived T cells were retrovirally transduced with newly generated NKG2D-CD3ε CAR DNA construct. These NKG2D CAR-expressing human T cells responded to NKG2D-specific activation by producing IFN-γ and exhibited significant cellular cytotoxicity against human tumor cells in vitro. In vivo studies demonstrated that NKG2D-CD3ε-bearing cells are capable of inhibiting growth of DU-145 human prostate cancer in the immunodeficient mice. Conclusions: Collectively, our data indicate the feasibility of developing chimeric antigen receptor NKG2D-CD3ε for T cells and suggest that adoptive transfer of T cells bearing NKG2D-CD3ε CAR could be potentially effective for immunotherapy of cancer patients.

scholarly journals Brief Ex Vivo Incubation with Fas Ligand Selectively Depletes Alloreactive T Cells and Antigen Presenting Cells from Stem Cell Grafts

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2033-2033
Author(s):  
Hilit Levy-Barazany ◽  
Liat Pinkas ◽  
Galina Rodionov ◽  
Nitzan Marelly ◽  
Michal Tzadok ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Fas Ligand ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Ifn Γ ◽  
Antigen Presenting

Abstract Graft versus host disease (GvHD) proceeds to be the Achilles' heel of hematopoietic stem cell transplantation, with clinicians continue facing a classic conflict: too much GvHD and the patient is at risk for transplant-related mortality and decreased quality of life; too little GvHD and the patient is at increased risk of relapse of their malignant disease. T cells and antigen presenting cells (APCs) are major components of the hematopoietic G-CSF mobilized peripheral blood cells (PBCs) graft. While GvHD is T cell mediated, the APCs are required for the initiation and maintenance of the GvHD. To reduce the risk for GvHD, grafts are sometimes depleted of their T cells, however, while preventing GvHD, the critically important attributes of graft versus leukemia (GvL) effect and engraftment are reduced significantly. Novel strategies that aim to abrogate or ameliorate GvHD, while preserving engraftment and GvL are of great need. A short incubation (2hr) of G-CSF mobilized PBCs with multimeric Fas ligand (i.e. ApoGraft) selectively induces apoptosis in T cell subsets and APCs (Panels A and B), but not in CD34+ progenitor cells (data not shown). FasL treatment preferentially induces apoptosis in mature T cell subsets which express high levels of Fas (CD95), such as T stem cell memory (TSCM), T central memory (TCM), and T effector memory (TEM) cells, as well as the pro-inflammatory T cell subtypes TH1 and TH17 cells, while no apoptotic signal is detected in the non-expressing CD95 naïve T cells (Panel A). The expression of T cells and APCs activation markers; CD25 and HLA-DR, respectively, is significantly reduced following apoptotic challenge in vitro (Panel C), as well as in transplanted mice (data not shown). Furthermore, upon an activation stimulus with anti CD3/CD28 beads in vitro, ApoGraft derived T cells secrete lower levels of IFN-γ, than G-CSF mobilized PBCs derived T cells (Panel D). To gain deeper understanding of the kinetics of GvHD development in vivo, NSG mice were transplanted with ApoGraft or G-CSF mobilized PBCs. Homing, expansion and differentiation of human leukocytes subtypes within the mice bone marrow, spleen and blood, were monitored 3, 7 and 14 days post transplantation. Decreased levels of T and B cells infiltration and expansion were detected in the spleen (Panels E and F), suggesting reduced formation of allo-reactive T cell clones. Reduced proliferation of these cells was associated with lower levels of IFN-γ secreted to the plasma (Panel H) and was in correlation with reduced GvHD and prolonged survival of the ApoGraft transplanted mice (Panel G). Importantly, we have previously demonstrated both in-vitro and in-vivo that ApoGraft has similar GvL and stem cell engraftment capabilities, compared to control G-CSF mobilized PBCs (data not shown). In conclusion, in contrast to conventional T- cell depletion methods, ApoGraft, an ex-vivo FasL-treated graft, affects both the T-cells and APCs, leading to reduced GvHD, while maintaining GvL and engraftment potential (Panel I). ApoGraft is currently being evaluated in a Phase I/II clinical trial (NCT02828878) in subjects with hematologic malignancies undergoing matched related allo-HSCT. Figure. Figure. Disclosures Levy-Barazany: Cellect Biotherapeutics Ltd: Employment. Pinkas:Cellect Biotherapeutics Ltd: Employment. Rodionov:Cellect Biotherapeutics Ltd: Employment. Marelly:Cellect Biotherapeutics Ltd: Employment. Tzadok:Cellect Biotherapeutics Ltd: Employment. Bakimer:Cellect Biotherapeutics Ltd: Employment. Yarkoni:Cellect Biotherapeutics Ltd: Employment. Peled:Cellect Biotherapeutics Ltd: Consultancy. Zuckerman:Cellect Biotherapeutics Ltd: Consultancy.

627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A663-A663
Author(s):  
Keegan Cooke ◽  
Juan Estrada ◽  
Jinghui Zhan ◽  
Jonathan Werner ◽  
Fei Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Mouse Models ◽  
T Cell Activation ◽  
Phase 1 ◽  
Phase 1 Study ◽  
Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

696 Brentuximab vedotin, a CD30-directed antibody-drug conjugate, selectively depletes activated Tregs in vitro and in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A738-A738
Author(s):  
Bryan Grogan ◽  
Reice James ◽  
Michelle Ulrich ◽  
Shyra Gardai ◽  
Ryan Heiser ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Efflux Pump ◽  
Apoptotic Cell ◽  
T Cell Subsets ◽  
Memory Cells ◽  
Antibody Drug Conjugate ◽  
Selective Depletion

BackgroundRegulatory T cells (Tregs) play an important role in maintaining immune homeostasis, preventing excessive inflammation in normal tissues. In cancer, Tregs hamper anti-tumor immunosurveillance and facilitate immune evasion. Selective targeting of intratumoral Tregs is a potentially promising treatment approach. Orthogonal evaluation of tumor-infiltrating lymphocytes (TILs) in solid tumors in mice and humans have identified CCR8, and several tumor necrosis family receptors (TNFRs), including TNFSFR8 (CD30), as receptors differentially upregulated on intratumoral Tregs compared to normal tissue Tregs and other intratumoral T cells, making these intriguing therapeutic targets.Brentuximab vedotin (BV) is approved for classical Hodgkin lymphoma (cHL) across multiple lines of therapy including frontline use in stage III/IV cHL in combination with doxorubicin, vinblastine, and dacarbazine. BV is also approved for certain CD30-expressing T-cell lymphomas. BV is comprised of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE).The activity of BV in lymphomas is thought to primarily result from tumor directed intracellular MMAE release, leading to mitotic arrest and apoptotic cell death.The role CD30 plays in normal immune function is unclear, with both costimulatory and proapoptotic roles described. CD30 is transiently upregulated following activation of memory T cells and expression has been linked to highly activated/suppressive IRF4+ effector Tregs.MethodsHere we evaluated the activity of BV on CD30-expressing T cell subsets in vitro and in vivo.ResultsTreatment of enriched T cell subsets with clinically relevant concentrations of BV drove selective depletion of CD30-expressing Tregs > CD30-expressingCD4+ T memory cells, with minimal effects on CD30-expressing CD8+ T memory cells. In a humanized xeno-GVHD model, treatment with BV selectively depleted Tregs resulting in accelerated wasting and robust T cell expansion. The observed differential activity on Tregs is likely attributable to significant increases in CD30 expression and reduced efflux pump activity relative to other T cell subsets. Interestingly, blockade of CD25 signaling prevents CD30 expression on T cell subsets without impacting proliferation, suggesting a link between CD25, the high affinity IL-2 receptor, and CD30 expression.ConclusionsTogether, these data suggest that BV may have an immunomodulatory effect through selective depletion of highly suppressive CD30-expressing Tregs.AcknowledgementsThe authors would like to thank Michael Harrison, PharmD for their assistance in abstract preparation.Ethics ApprovalAnimals studies were approved by and conducted in accordance with Seattle Genetics Institutional Care and Use Committee protocol #SGE-024.

scholarly journals IMMU-31. QUANTITATIVE EVALUATION OF ROUTES OF ADMINISTRATION OF IMMUNOTHERAPEUTIC T CELLS TO ORTHOTOPIC GLIOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii111-ii111
Author(s):  
Lan Hoang-Minh ◽  
Angelie Rivera-Rodriguez ◽  
Fernanda Pohl-Guimarães ◽  
Seth Currlin ◽  
Christina Von Roemeling ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Ex Vivo ◽  
Adoptive T Cell Therapy ◽  
Whole Body ◽  
T Cell Therapy ◽  
Clinical Responses

Abstract SIGNIFICANCE Adoptive T cell therapy (ACT) has emerged as the most effective treatment against advanced malignant melanoma, eliciting remarkable objective clinical responses in up to 75% of patients with refractory metastatic disease, including within the central nervous system. Immunologic surrogate endpoints correlating with treatment outcome have been identified in these patients, with clinical responses being dependent on the migration of transferred T cells to sites of tumor growth. OBJECTIVE We investigated the biodistribution of intravenously or intraventricularly administered T cells in a murine model of glioblastoma at whole body, organ, and cellular levels. METHODS gp100-specific T cells were isolated from the spleens of pmel DsRed transgenic C57BL/6 mice and injected intravenously or intraventricularly, after in vitro expansion and activation, in murine KR158B-Luc-gp100 glioma-bearing mice. To determine transferred T cell spatial distribution, the brain, lymph nodes, heart, lungs, spleen, liver, and kidneys of mice were processed for 3D imaging using light-sheet and multiphoton imaging. ACT T cell quantification in various organs was performed ex vivo using flow cytometry, 2D optical imaging (IVIS), and magnetic particle imaging (MPI) after ferucarbotran nanoparticle transfection of T cells. T cell biodistribution was also assessed in vivo using MPI. RESULTS Following T cell intravenous injection, the spleen, liver, and lungs accounted for more than 90% of transferred T cells; the proportion of DsRed T cells in the brains was found to be very low, hovering below 1%. In contrast, most ACT T cells persisted in the tumor-bearing brains following intraventricular injections. ACT T cells mostly concentrated at the periphery of tumor masses and in proximity to blood vessels. CONCLUSIONS The success of ACT immunotherapy for brain tumors requires optimization of delivery route, dosing regimen, and enhancement of tumor-specific lymphocyte trafficking and effector functions to achieve maximal penetration and persistence at sites of invasive tumor growth.

scholarly journals PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1959-1959
Author(s):  
Jeong A Park ◽  
Hong fen Guo ◽  
Hong Xu ◽  
Nai-Kong V. Cheung
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
Activated T Cells ◽  
The Impact ◽  
Anti Tumor Activity

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

scholarly journals Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Jennifer A. Juno ◽  
Kathleen M. Wragg ◽  
Anne B. Kristensen ◽  
Wen Shi Lee ◽  
Kevin J. Selva ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Seminal Plasma ◽  
Target Cells ◽  
Ccr5 Expression ◽  
Ccr5 Receptor ◽  
The Impact ◽  
Hiv 1

ABSTRACT Sexual HIV-1 transmission occurs primarily in the presence of semen. Although data from macaque studies suggest that CCR5+ CD4+ T cells are initial targets for HIV-1 infection, the impact of semen on T cell CCR5 expression and ligand production remains inconclusive. To determine if semen modulates the lymphocyte CCR5 receptor/ligand axis, primary human T cell CCR5 expression and natural killer (NK) cell anti-HIV-1 antibody-dependent beta chemokine production was assessed following seminal plasma (SP) exposure. Purified T cells produce sufficient quantities of RANTES to result in a significant decline in CCR5bright T cell frequency following 16 h of SP exposure (P = 0.03). Meanwhile, NK cells retain the capacity to produce limited amounts of MIP-1α/MIP-1β in response to anti-HIV-1 antibody-dependent stimulation (median, 9.5% MIP-1α+ and/or MIP-1β+), despite the immunosuppressive nature of SP. Although these in vitro experiments suggest that SP-induced CCR5 ligand production results in the loss of surface CCR5 expression on CD4+ T cells, the in vivo implications are unclear. We therefore vaginally exposed five pigtail macaques to SP and found that such exposure resulted in an increase in CCR5+ HIV-1 target cells in three of the animals. The in vivo data support a growing body of evidence suggesting that semen exposure recruits target cells to the vagina that are highly susceptible to HIV-1 infection, which has important implications for HIV-1 transmission and vaccine design. IMPORTANCE The majority of HIV-1 vaccine studies do not take into consideration the impact that semen exposure might have on the mucosal immune system. In this study, we demonstrate that seminal plasma (SP) exposure can alter CCR5 expression on T cells. Importantly, in vitro studies of T cells in culture cannot replicate the conditions under which immune cells might be recruited to the genital mucosa in vivo, leading to potentially erroneous conclusions about the impact of semen on mucosal HIV-1 susceptibility.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

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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