scholarly journals Ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of liposomal antigen and CpG-ODN improved PD-1 blockade immunotherapy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mona Yazdani ◽  
Zahra Gholizadeh ◽  
Amin Reza Nikpoor ◽  
Nema Mohamadian Roshan ◽  
Mahmoud Reza Jaafari ◽  
...  
Keyword(s):  
T Cell ◽  
Dendritic Cell ◽  
Low Dose ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Cpg Odn ◽  
Dc Vaccine ◽  
Pulsed Dc ◽  
Anti Cancer ◽  
Anti Tumor Activity

AbstractLack of pre-existing tumor infiltrated T cells resulting in resistance to programmed cell death protein 1 (PD-1) blockade therapies can be solved by combining with anti-cancer vaccines and CpG-ODN in increasing T cell expansion and infiltration. Therefore, we prepared an ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of either liposomal or non-liposomal gp100 antigen (2.8 µg) plus CpG-ODN (800 ng) formulations and evaluated its anti-tumor activity in combination with anti-PD-1 therapy. Our results showed a combination of liposomal peptide plus CpG-ODN pulsed DC with anti-PD-1 antibody was more efficacious, as evidenced by a significant increase in Teff/Treg TILs with a marked fourfold elevation of IFN-γ expression level in the tumor site of treated mice which reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, this combination also led to a remarkable tumor remission and prolonged survival rate in melanoma-bearing mice compared to non-liposomal peptide plus CpG-ODN or single-treated liposomal peptide formulations. Our results provide essential insights to devise combining regimens to improve the efficacy of immune checkpoint blockers even by a low dose of peptide and CpG-ODN.

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.

Synergism between CpG ODN and IL-2 Leads to Massive CD8+ T Cell Responses Against a Self-Antigen and Epitope-Specific Anti-Tumor Immunity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3239-3239
Author(s):  
Jim Kochenderfer ◽  
Chris Chien ◽  
Ronald Gress
Keyword(s):  
T Cell ◽  
Low Dose ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Absolute Number ◽  
Negative Control ◽  
Cpg Odn ◽  
Cell Responses ◽  
Peptide Stimulation

Abstract Oligodeoxynucleotides with CpG motifs (CpG ODN) enhance vaccine-elicited T cell responses and might improve the efficacy of anti-cancer vaccines. B16F1 is a poorly immunogenic murine melanoma that expresses tyrosinase related protein-2 (TRP-2), which is also expressed by normal melanocytes and is subject to self-tolerance. Amino acids 180–188 of TRP-2 (TRP-2180–188) form an immunogenic MHC class I-presented epitope. We injected mice subcutaneously with B16F1 and then administered a vaccine containing TRP-2180–188+CpG ODN in IFA (incomplete Freund’s adjuvant) on days 0, 3, 6, and 14 after tumor injection. As a negative control, we vaccinated a second group of mice with the OVA257–264 peptide+CpG ODN in IFA. TRP-2180–188+CpG ODN vaccination did not cause epitope-specific inhibition of B16F1 growth. In an attempt to increase anti-tumor efficacy, we added low-dose IL-2 on days 7–10 and 15–18 after tumor injection to the vaccination regimen described above. With the addition of IL-2, epitope-specific inhibition of tumor growth occurred (day 19 tumor size: 21 mm2 with TRP-2180–188 vaccination versus 93 mm2 with OVA257–264 vaccination, P=0.002). Because tumor growth inhibition was epitope-specific, we hypothesized that it was due to an IL-2-mediated increase in CD8+ T cell responses against TRP-2180–188. To test this hypothesis, we gave TRP-2180–188+CpG ODN vaccines and low-dose IL-2 to mice, and then we measured TRP-2180–188-specific CD8+ T cell responses by ex vivo peptide stimulation of splenocytes for six hours followed by intracellular cytokine staining for interferon-γ (IFNγ). When mice were vaccinated with TRP-2180–188+CpG ODN and given IL-2, a mean of 18.3% of CD3+CD8+ splenocytes produced IFNγ after ex vivo peptide stimulation with TRP-2180–188, but only 0.1% of CD3+CD8+ splenocytes produced IFNγ in response to the negative control peptide OVA257–264; therefore, the mean TRP-2180–188-specific CD8+ T cell response was 18.2%. The same vaccine regimen given with control PBS injections instead of IL-2 elicited a mean TRP-2180–188-specific response of only 1.0% of CD3+CD8+ splenocytes (P<0.001, IL-2 versus no IL-2). Mice that received TRP-2180–188+CpG ODN vaccinations and IL-2 had a mean absolute number of 5.6x106 TRP-2180–188-specific CD3+CD8+ splenocytes. Mice vaccinated identically but not receiving IL-2 had only 0.1x106 TRP-2180–188-specific CD3+CD8+ splenocytes (P<0.001 IL-2 versus no IL-2). Vaccines containing TRP-2180–188 without CpG ODN given with low-dose IL-2 elicited responses in which a mean of 2.8% of CD3+CD8+ splenocytes and an absolute number of 0.5x106 CD3+CD8+ splenocytes were specific for TRP-2180–188 (P<0.001 for percentage and absolute number, IL-2+CpG ODN versus IL-2 without CpG ODN). Tumor-bearing mice generated TRP-2180–188-specific CD8+ T cell responses only when vaccinated with TRP-2180–188 despite the presence of B16F1 tumors expressing the TRP-2 protein. TRP-2180–188+CpG ODN vaccines protected mice from B16F1 challenge when B16F1 was injected 5 days after the last dose of vaccine only when IL-2 was also administered. When we vaccinated mice prophylactically, survival was higher in TRP-2180–188-vaccinated mice than in OVA257–264-vaccinated mice (P=0.0005), and 3/13 TRP-2180–188-vaccinated mice developed vitiligo that was consistent with autoimmunity against melanocytes. This is the first report of synergism between IL-2 and CpG ODN. This synergism causes a striking increase in vaccine-elicited CD8+ T cell responses and leads to self-epitope-specific anti-tumor immunity.

scholarly journals Carthamus tinctoriusEnhances the Antitumor Activity of Dendritic Cell Vaccines via Polarization toward Th1 Cytokines and Increase of Cytotoxic T Lymphocytes

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Jia-Ming Chang ◽  
Le-Mei Hung ◽  
Yau-Jan Chyan ◽  
Chun-Ming Cheng ◽  
Rey-Yuh Wu
Keyword(s):  
Dendritic Cell ◽  
T Lymphocytes ◽  
Antitumor Activity ◽  
Cytotoxic T Lymphocytes ◽  
Tumor Antigens ◽  
Ex Vivo ◽  
Cytokine Secretion ◽  
Tumor Lysate ◽  
Dc Vaccine ◽  
Pulsed Dc

Carthamus tinctorius(CT), also named safflower, is a traditional Chinese medicine widely used to improve blood circulation. CT also has been studied for its antitumor activity in certain cancers. To investigate the effects of CT on the dendritic cell (DC)-based vaccine in cancer treatment, cytokine secretion of mouse splenic T lymphocytes and the maturation of DCs in response to CT were analyzed. To assess the antitumor activity of CT extract on mouse CD117+(c-kit)-derived DCs pulsed with JC mammal tumor antigens, the JC tumor was challenged by the CT-treated DC vaccinein vivo. CT stimulated IFN-γand IL-10 secretion of splenic T lymphocytes and enhanced the maturation of DCs by enhancing immunological molecule expression. When DC vaccine was pulsed with tumor antigens along with CT extract, the levels of TNF-αand IL-1βwere dramatically increased with a dose-dependent response and more immunologic and co-stimulatory molecules were expressed on the DC surface. In addition, CT-treated tumor lysate-pulsed DC vaccine reduced the tumor weight in tumor-bearing mice by 15.3% more than tumor lysate-pulsed DC vaccine without CT treatment. CT polarized cytokine secretion toward the Th1 pathway and also increased the population of cytotoxic T lymphocytesex vivo. In conclusion, CT activates DCs might promote the recognition of antigens and facilitate antigen presentation to Th1 immune responses.

scholarly journals Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD8+ T-cell responses

2020 ◽  
Vol 4 (10) ◽  
pp. 2143-2157 ◽  
Author(s):  
Alak Manna ◽  
Timothy Kellett ◽  
Sonikpreet Aulakh ◽  
Laura J. Lewis-Tuffin ◽  
Navnita Dutta ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Dependent Manner

Abstract Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

scholarly journals Dendritic cell–based immunotherapy targeting Wilms’ tumor 1 in patients with recurrent malignant glioma

2015 ◽  
Vol 123 (4) ◽  
pp. 989-997 ◽  
Author(s):  
Keiichi Sakai ◽  
Shigetaka Shimodaira ◽  
Shinya Maejima ◽  
Nobuyuki Udagawa ◽  
Kenji Sano ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Malignant Glioma ◽  
Stable Disease ◽  
Wilms Tumor ◽  
Malignant Gliomas ◽  
Tumor Lysate ◽  
Dc Vaccine ◽  
Pulsed Dc ◽  
Wilms Tumor 1 ◽  
Vaccination Therapy

OBJECT Dendritic cell (DC)-based vaccination is considered a potentially effective therapy against advanced cancer. The authors conducted a Phase I study to investigate the safety and immunomonitoring of Wilms’ tumor 1 (WT1)-pulsed DC vaccination therapy for patients with relapsed malignant glioma. METHODS WT1-pulsed and/or autologous tumor lysate-pulsed DC vaccination therapy was performed in patients with relapsed malignant gliomas. Approximately 1 × 107 to 2 × 107 pulsed DCs loaded with WT1 peptide antigen and/or tumor lysate were intradermally injected into the axillary areas with OK-432, a streptococcal preparation, at 2-week intervals for at least 5–7 sessions (1 course) during an individual chemotherapy regimen. RESULTS Ten patients (3 men, 7 women; age range 24–64 years [median 39 years]) with the following tumors were enrolled: glioblastoma (6), anaplastic astrocytoma (2), anaplastic oligoastrocytoma (1), and anaplastic oligodendroglioma (1). Modified WT1 peptide–pulsed DC vaccine was administered to 7 patients, tumor lysate-pulsed DC vaccine to 2 patients, and both tumor lysate–pulsed and WT1-pulsed DC vaccine to 1 patient. The clinical response was stable disease in 5 patients with WT1-pulsed DC vaccination. In 2 of 5 patients with stable disease, neurological findings improved, and MR images showed tumor shrinkage. No serious adverse events occurred except Grade 1–2 erythema at the injection sites. WT1 tetramer analysis detected WT1-reactive cytotoxic T cells after vaccination in patients treated with WT1-pulsed therapy. Positivity for skin reaction at the injection sites was 80% (8 of 10 patients) after the first session, and positivity remained for these 8 patients after the final session. CONCLUSIONS This study of WT1-pulsed DC vaccination therapy demonstrated safety, immunogenicity, and feasibility in the management of relapsed malignant gliomas.

scholarly journals 595 Inhibition of integrin αvβ8 in combination with low dose radiation induces antitumor effect in advanced immune checkpoint blockade refractory tumor model

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A625-A625
Author(s):  
Natalia Reszka-Blanco ◽  
Megan Krumpoch ◽  
Michaela Mentzer ◽  
Vinod Yadav Yadav ◽  
Brianna Bannister ◽  
...  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Dendritic Cell ◽  
Tumor Immunity ◽  
Immune Checkpoint ◽  
Low Dose ◽  
Immune Checkpoint Blockade ◽  
Low Dose Radiation ◽  
Dose Radiation ◽  
Ct26 Model

BackgroundIntegrin αvβ8 activates TGFβ in immune cells. αvβ8 inhibitors have been shown to potentiate immune checkpoint blockade (ICB) in preclinical models [1]. Radioimmunotherapy (RIT) induces immunogenic cell death and antigen presentation, however it concurrently activates immunosuppressive pathways. Interestingly, αvβ8 immunosuppressive activity was implicated in radiotherapy resistance [2]. We have explored whether antagonizing αvβ8 overcomes the suppressive effect of TGFβ and restores anti-tumor immunity in advanced ICB and RIT resistant tumors.MethodsEfficacy was evaluated after combination treatment with low dose radiation, αvβ8 (clone C6D4) and PD-1 (clone J43) mAb in an advanced CT26 colon cancer syngeneic mouse model. Mice were treated at tumor volume of >120 mm3 and euthanized at 2,000 mm3. Flow cytometry and transcriptomic analysis were used to assess the mechanism of action. Tumor volumes are presented as mean±SEM. Statistics were performed by one-way ANOVA, or log-rank test. Bone marrow derived dendritic cell (BMdDC) cultures were isolated from C57BL/6 mice.ResultsCell death, including radiation-induced apoptosis, induced immunoregulatory and maturation program in a population of ex vivo cultured BMdDC, recently described as mregDC/DC3 [3,4]. mregDC/DC3 signature was associated with increased αvβ8 expression, suggesting a role of this integrin in inducing an immunosuppressive phenotype.A CT26 model was established to mimic the progression of late-stage tumors and was unresponsive to radiation, ICB and RIT. In CT26 implanted mice, αvβ8 is expressed on tumor stoma, and is not detectable on cancer cells. Addition of αvβ8 mAb to RIT markedly increased tumor regression (P=0.0067) and survival (P<0.0001). There were 8/10 complete responders with addition of αvβ8 mAb relative to 3/10 in RIT alone. Improved efficacy correlated with enhanced T cell activation and improved DC functionality. Consistent with a recent report in a less advanced CT26 model [5], αvβ8 mAb + radiation resulted in similar efficacy as conventional RIT although the effect was modest in more advanced tumors (Figure 1, A, B).Abstract 595 Figure 1Complete response (CR) with improved survival when αvβ8 inhibition is added to RIT in CT26 syngeneic model of colorectal cancer in an advanced, ICB and RIT unresponsive stage. (A) Effect of combination therapy with low dose radiation (small animal radiation research platform (SARRP) at 5 Gray (Gy) on the day of staging (day 10)), PD-1 mAb (10 mg/kg twice weekly for 2 weeks) and αvβ8 mAb (7 mg/kg three times weekly for 3 weeks) measured by tumor burden. 5Gy+PD-1 and 5Gy+αvβ8 has a minimal effect on tumor growth inhibition showing slight improvement relative to radiation alone (5Gy+IgG). Addition of αvβ8 antagonism (5Gy+αvβ8+PD-1) improves anti-tumor responses leading to CR in 8 of 10 mice. (B) Kaplan-Meier Curve presenting time to progression. 5Gy+IgG improved survival over monotherapy with either αvβ8 or PD1 mAb. 5Gy+αvβ8+PD-1 resulted in a profound improvement of the survival over all other treatment conditionsConclusionsInhibition of αvβ8 in combination with RIT eradicated an advanced tumor, unresponsive to the respective monotherapies or conventional RIT. The anti-tumor effect was driven by enhancement of adaptive immunity, improvement of DC function and reduced tumor tolerance. These data provide evidence that αvβ8 inhibition enhances RIT and may be effective against ICB refractory tumors.ReferencesReszka-Blanco NJ,Yadav V, Krumpoch M, Cappellucci L, Cui D, Dowling JE, et al., Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer. AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1559.Jin S, Lee WC, Aust D, Pilarsky C, Cordes N, β8 integrin mediates pancreatic cancer cell radiochemoresistance. Mol Cancer Res. 2019; 17(10): 2126–2138.Maier B, Leader AM, Chen ST, Tung N, Chang C, LeBerichel J, et al., A conserved dendritic-cell regulatory program limits antitumour immunity. Nature. 2020; 580 (7802): 257–262.Garris CS, Arlauckas SP, Kohler RH, Trefny MP, Garren S, Piot C, Engblom C, et al., Successful anti-PD-1 cancer immunotherapy requires T cell-dendritic cell crosstalk involving the cytokines IFN-γ and IL-12. Immunity. 2018; 49(6): 1148–1161.Dodagatta-Marri E, Ma H-Y, Liang B, Li J, Meyer DS, Chen S-Y, et al., Integrin αvβ8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy. Cell Report. 2021; 36(1): 109309Ethics ApprovalAll animal work was approved by the site Institutional Animal Care and Use Committee and was performed in conformance with the Guide for the Care and Use of Laboratory Animals within an AAALAC-accredited program. Humane euthanasia criteria were predetermined on the basis of body weight and defined clinical observations.

scholarly journals A Correlation Between Differentiation Phenotypes of Infused T Cells and Anti-Cancer Immunotherapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Hao Ren ◽  
Kunkun Cao ◽  
Mingjun Wang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Immunity ◽  
Ex Vivo ◽  
T Cell Therapy ◽  
Production Methods ◽  
Anti Cancer ◽  
Status Differentiation ◽  
Differentiation Status

T-cell therapy, usually with ex-vivo expansion, is very promising to treat cancer. Differentiation status of infused T cells is a crucial parameter for their persistence and antitumor immunity. Key phenotypic molecules are effective and efficient to analyze differentiation status. Differentiation status is crucial for T cell exhaustion, in-vivo lifespan, antitumor immunity, and even antitumor pharmacological interventions. Strategies including cytokines, Akt, Wnt and Notch signaling, epigenetics, and metabolites have been developed to produce less differentiated T cells. Clinical trials have shown better clinical outcomes from infusion of T cells with less differentiated phenotypes. CD27+, CCR7+ and CD62L+ have been the most clinically relevant phenotypic molecules, while Tscm and Tcm the most clinically relevant subtypes. Currently, CD27+, CD62L+ and CCR7+ are recommended in the differentiation phenotype to evaluate strategies of enhancing stemness. Future studies may discover highly clinically relevant differentiation phenotypes for specific T-cell production methods or specific subtypes of cancer patients, with the advantages of precision medicine.

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 Induction of the IL-9 gene by HTLV-I Tax stimulates the spontaneous proliferation of primary adult T-cell leukemia cells by a paracrine mechanism

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5163-5172 ◽  
Author(s):  
Jing Chen ◽  
Mike Petrus ◽  
Bonita R. Bryant ◽  
Vinh Phuc Nguyen ◽  
Mindy Stamer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Proximal Promoter ◽  
Type I ◽  
Dependent Manner ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Adult T Cell Leukemia ◽  
Spontaneous Proliferation

AbstractThe etiologic agent of adult T-cell leukemia (ATL) is human T cell lymphotropic virus type I (HTLV-I). The HTLV-I protein Tax alters gene expression, including those of cytokines and their receptors, which plays an important role in early stages of ATL. Here we demonstrate that expression of interleukin-9 (IL-9) is activated by Tax via an NF-κB motif in its proximal promoter, whereas IL-9 receptor-α (IL-9Rα) expression is not induced by Tax. However, supporting a role for IL-9/IL-9Rα in ATL, a neutralizing monoclonal antibody directed toward IL-9Rα inhibited ex vivo spontaneous proliferation of primary ATL cells from several patients. Fluorescence-activated cell sorter analysis of freshly isolated peripheral blood mononuclear cells from these patients revealed high level expression of IL-9Rα on their CD14-expressing monocytes. Furthermore, purified T cells or monocytes alone from these patients did not proliferate ex vivo, whereas mixtures of these cell types manifested significant proliferation through a contact-dependent manner. Taken together, our data suggest that primary ATL cells, via IL-9, support the action of IL-9Rα/CD14-expressing monocytes, which subsequently support the ex vivo spontaneous proliferation of malignant T cells. In summary, these data support a role for IL-9 and its receptor in ATL by a paracrine mechanism.

Dendritic Cells (DC) Generated from AML Blasts Express Leukemia Associated Antigens Eliciting Specific Cytotoxic T Cell Responses in the Autologous Host after DC Vaccination.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1812-1812 ◽  
Author(s):  
Li Li ◽  
Peter Reinhardt ◽  
Iwona Hus ◽  
Jacek Rolinski ◽  
Anna Dmoszynska ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mrna Expression ◽  
Cytotoxic T Cell ◽  
Fish Analysis ◽  
Specific Response ◽  
Proteinase 3 ◽  
Cpg Odn ◽  
Dc Vaccine ◽  
Significant Difference

Abstract Several groups including ours demonstrated the generation of DC from AML blasts (AML-DC). FISH analysis has been employed to assess the origin of AML-DC from AML blasts. For clinical application, this approach is not feasible because of the restriction of AML-DC in number. Therefore we established an alternative system to prove the origin of the AML-DC, using quantitative real-time PCR to test the mRNA expression of the leukemia associated antigens (LAAs) preferentially expressed antigen in melanoma (PRAME), proteinase 3, the receptor for hyaluronic acid mediated motility (RHAMM/CD168) and the Wilms tumor gene 1 (WT-1). An elevated PCR signal for PRAME was detected in 7/12 AML-DC preparations when compared with AML blasts, for RHAMM/CD168 in 6/12 AML-DC preparations, but only in 2/12 respectively 1/12 AML-DC for WT-1 and proteinase 3. All preparations showed a strong expression of at least one of the LAAs examined. The stronger PCR signals after DC generation for PRAME and RHAMM/CD168 characterize these two LAAs as favourable target structures for immunotherapies. AML-DC positive for RHAMM/CD168 mRNA tested also positive for the protein as demonstrated by immunocytochemistry. In PRAME mRNA positive AML-DC, the described PRAME derived decamer epitope peptide ALYVDSLFFL was recognized by specific T cells as proven by chromium-51 release assay, thus proving that the mRNA assessment for RHAMM/CD168 and PRAME has an immunological significance. For five patients, AML-DC were generated under good manufacturing practice (GMP) conditions. 5x10E6 AML-DC were injected s.c. in the vicinity of inguinal lymph nodes four times at a biweekly interval. No severe adverse effects were observed after DC vaccination. One patient with AML FAB M4 required blood transfusions and remained in stable condition for several months, but eventually died from pneumonia 13 months after the DC vaccinations. A 70 year-old women with a secondary AML received a complete course of AML-DC vaccinations. During the period of 4 vaccinations, the blast level dropped from 8% in the PB to 0% and no side effects were noted. Two patients died from cranial hemorrhage after the first vaccination due to thrombocytopenia caused by the AML. One patient is still under DC vaccination. ELISPOT analysis of the first two patients revealed a significant increase in interferon gamma and granzyme B releasing CD8+ T cells recognizing a leukemic blast lysate as well as specifically RHAMM derived peptides, when compared to the initial T cell frequency. Potentiation of such an AML-DC vaccine might become feasible by the addition of adjuvants such as CPG-rich oligodeoxyribonucleotides (CPG-ODN) or lipopolysaccharides (LPS). We therefore investigated the presence of receptors for such adjuvants, i.e Toll-like receptors (TLRs) on AML-DC. Quantitative mRNA expression of TLR-2, 4 and 9 in AML-DC and DC generated of monocytes from healthy volunteers did not display any significant difference. In summary, DC could be generated from AML blasts and preserved or even upregulated LAA expression. DC vaccination was well tolerated and resulted in an enhanced and specific response of cytotoxic T cells. The adequate expression of TLRs renders potentiation of the AML-DC vaccine described here by e.g. CPG-ODN possible.

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